C4. Performance Evaluation Test - Test

Sansure Biotech Inc.
Six Respiratory Pathogens Nucleic Acid Diagnostic Kit

(PCR-Fluorescence Probing)
Study on reference value
Content
1. Purpose
2. Samples used for determining cut-off value
3. Methods of determining cut-off value
4. Specific test data about determining cut-off value
5. Verification and confirmation of determining cut-off value
6. Summary of determining cut-off value
Determination of cut-off value

1. Purpose
The Six Respiratory Pathogens Nucleic Acid Diagnostic Kit
(PCR-Fluorescence Probing) produced by our company (hereinafter referred
as this kit) use multiple fluorescent quantitative PCR methods to detect
whether the throat swab samples of patients with similar symptoms of
respiratory tract infection caused by unknown reason, such as fever, swelling
and pain, cough, headache and dyspnea, contain the specific targets of nucleic
acid of influenza A virus (Flu A), influenza B virus (Flu B), Respiratory
Syncytial Virus (RSV), Adenoviruses (Adv), Human rhinovirus (HRV) and
Mycoplasma Pneumonia (MP), so as to determine whether the suspected
patients are infected with the above 6 respiratory pathogens. In order to
ensure the validity of detection results of targets (targeted nucleic acid
sequences), an internal standard (a positive internal control) is added to PCR
system to monitor the validity of PCR reaction and to avoid false negative.
The following is the basis for determining the cut-off value of the target gene
and internal standard detection through experimental research and statistical
analysis, which is used as the basis for judging the results of clinical samples.
2. Samples used for determining cut-off value

1) 639 clinical throat swab samples
The samples were collected from the disease prevention and control
center in Loudi and Hunan provincial people’s hospital. Among them, 92
clinically samples were positive for influenza A virus, 92 samples were
positive for influenza B virus, 91 samples were positive for respiratory
syncytial virus, 92 samples were positive for Adenovirus, 92 samples were
positive for Human rhinovirus, 92 samples were positive for Mycoplasma
pneumoniae, and 88 samples were clinically negative. The positive and
negative results were confirmed by fluorescent PCR detection kits (Joint
detection kit for nucleic acid of influenza A and B virus by Shanghai ZJ
Bio-Tech Co., Ltd., certificate No. 20173404656; Respiratory syncytial virus
nucleic acid detection kit by Daan Gene Co., Ltd., certificate No. 20173400171;
Adenovirus nucleic acid detection kit by Puruikang biotechnology Co., Ltd.,
certificate No. 20163400197; Mycoplasma pneumoniae nucleic acid detection
kit by Snasure biotech inc., certificate No. 20173400078; Human Rhinovirus
nucleic acid detection kit by Land Medical Technology Co., Ltd., certificate No.
20153400295 ).
2) Diluted clinically positive samples for the 6 pathogens
The positive samples for influenza A virus and influenza B virus with
detection limit concentration were prepared from a clinical throat swab
sample that was determined by the Reed-Muench method and diluted with
Hanks solution. 1 positive sample for influenza A virus was determined by
Reed-Muench method and diluted with Hanks solution to the detection limit
concentration, and diluted into 48 parts; 1 positive sample for influenza B
virus was determined by Reed-Muench method and diluted with Hanks
solution To the detection limit concentration, dilute into 48 parts.
The positive samples for respiratory syncytial virus, adenovirus,
human rhinovirus, and Mycoplasma pneumoniae with detection limit
concentration were diluted from the clinically positive throat swab samples
with determined value. The positive sample for respiratory syncytial virus
with a concentration of 3.03E+07 copies/mL were diluted with normal saline
to the detection limit concentration and to 48 parts; The positive sample for
Adenovirus with a concentration of 3.77E+07 copies/mL were diluted with
Determination of cut-off value 1

normal saline to the detection limit concentration and to 48 parts; The positive
sample for Human rhinovirus with a concentration of 2.95E+07 copies/mL
were diluted with normal saline to the detection limit concentration and to 48
parts; The positive sample for Mycoplasma pneumoniae with a concentration
of 6.24E+07 copies/mL were diluted with normal saline to the detection limit
concentration and to 48 parts.
3) 2076 clinical throat swab samples (Clinical trail)
The clinical samples are all from clinical research. There were a total of
2076 throat swab samples collected for detection. Among them, 422 samples
were collected from Wuhan infectious disease hospital, 420 samples were
collected from Jiangxi provincial children’s hospital, 410 samples were
collected from the affiliated hospital of Hangzhou normal university, 414
samples were collected from Guangzhou women and children medical center,
and 410 samples were collected from Hunan provincial people’s hospital.
3. Methods of determining cut-off value

1) According to the six respiratory pathogens detected by this kit, one
clinically positive throat swab sample were selected for each pathogen, and
then were diluted by 2-10 times to determine the detection sensitivity of
clinical samples.
2) According to the detection sensitivity, 48 positive samples with
detection limit concentration were prepared, and then the Ct reference value
was statistically analyzed.
3) Before the clinical trail, 639 clinical samples were tested. According to
ROC curve analysis and the reference value of the positive samples with
detection limit concentration, the reference value of the target gene and
internal standard of this kit was drawn up.
4) The validity of the proposed reference value was verified through the
clinical trial of the clinical trial unit to determine the final reference value of
this kit.
4. Specific test data about determining cut-off value

4.1 Detection sensitivity of clinical samples
4.1.1 Influenza A virus
4.1.1.1 Choose one clinically strong positive throat swab sample for influenza
A virus
One clinically strong positive throat swab sample for influenza A virus,
whose concentration was 5.0 × 103 TCID50/mL determined by the
Reed-Muench method, was detected by the qualified kit in our company. The
results are shown in Figure 1.

Figure 1 Amplification results of the clinically strong positive throat swab sample for
influenza A virus
4.1.1.2 Serial dilution, detection sensitivity determination

Firstly, the clinically strong positive throat swab sample for influenza A
virus (5.0 × 103 TCID50/mL) was diluted with Hanks solution to 2.5×103
TCID50/mL、2.5×102 TCID50/mL、25 TCID50/mL、2.5 TCID50/mL、0.5 TCID50/mL
and 0.2T CID50/mL, and then was detected with this kit. Detection results are
shown in the following Figure 2.
Figure 2. Amplification results of 0.2~2.0 × 103 TCID50/mL influenza A virus

Table 1. Ct value of samples with 0.2~2.5 × 103 TCID50/mL influenza A virus
Concentration(TCI Target FAM Internal standard
D50/mL) ROX
2.5×103 23.20 20.77
2.5×102 27.22 24.50

25 30.75 28.06
2.5 34.69 31.02
0.5 Undetermined 36.96
0.2 Undetermined Undetermined*
*Note：Being an internal standard, it will be diluted with the dilution of
samples, so there is the detection result of negative internal.
It can be seen from the above graph that the sample with 2.5 TCID50/mL
virus was detected as positive, while 0.5 TCID50/mL was detected as negative.
Because of the difference in concentration, it is necessary to make gradient
dilution by small intervals for the sample with 2.5 TCID50/mL virus, to
determine the measurable concentration between 2.5 TCID50/mL and 0.5
TCID50/mL.
The above samples were diluted to 2.0 TCID50/mL, 1.5 TCID50/mL and 1.0
TCID50/mL and were repeatedly detected 8 times. The test results are shown
in the following Table 2 and Figures 3-5.
Table 2. Sensitivity detection
Detected positive
Diluted samples Positive rate(%)
samples/Sample size
2.0 TCID50/mL 8/8 100%
1.5 TCID50/mL 8/8 100%
1.0 TCID50/mL 7/8 87.5%
Figure 3. Amplification results of samples with 2.0 TCID50/mL influenza A virus


It can be seen from Table 1 and Figures 3-5 that, the detection rate of
samples with 2.0 TCID50/mL and 1.5 TCID50/mL virus is 100%, while the
detection rate of samples with 1.0 TCID50/mL is 87.5%. In order to ensure the
accuracy of detection, the sensitivity of this kit to detect clinical samples is
determined to be 2.0 TCID50/mL, which is consistent with the test results of
the analytical sensitivity of the registration application file CH3.5.05.
4.1.2 Influenza B virus

4.1.2.1 Choose one clinically strong positive throat swab sample for influenza
B virus
One clinically strong positive throat swab sample for influenza B virus,
whose concentration was 1.0 × 103 TCID50/mL determined by the

influenza B virus
Firstly, the clinically strong positive throat swab sample for influenza B
virus (5.0 × 103 TCID50/mL) was diluted with Hanks solution to 2.5 × 103
TCID50/mL、2.5 × 102 TCID50/mL, 25 TCID50/mL, 2.5 TCID50/mL, 0.5 TCID50/mL
and 0.2 TCID50/mL, and then was detected with this kit. Detection results are
shown in the following Figure 7.
Figure 7. Amplification results of 0.2~2.0 × 103 TCID50/mL influenza B virus

Table 3. Ct value of samples with 0.2~2.5 × 103 TCID50/mL influenza B virus
Concentration Internal standard
Target HEX
(TCID50/mL) ROX
2.5×103 22.92 21.05
2.5×102 26.55 24.58

25 30.16 28.28
2.5 33.30 31.18
It can be seen from the above graph that the sample with 2.5 TCID50/mL
virus was detected as positive, while 0.5 TCID50/mL was detected as negative.
dilution by small intervals for the sample with 2.5 TCID50/mL virus, to
determine the measurable concentration between 2.5 TCID50/mL and 0.5
TCID50/mL.
The above samples were diluted to 2.0 TCID50/mL, 1.5 TCID50/mL and 1.0
TCID50/mL and were repeatedly detected 8 times. The test results are shown
in the following Table 4 and Figures 8-10.
Detected positive
samples/Sample size
2.0 TCID50/mL 8/8 100%
1.5 TCID50/mL 8/8 100%
1.0 TCID50/mL 7/8 87.5%
Figure 8. Amplification results of samples with 2.0 TCID50/mL influenza B virus

samples with 2.0 TCID50/mL and 1.5 TCID50/mL virus is 100%, while the
detection rate of samples with 1.0 TCID50/mL is 87.5%. In order to ensure the
determined to be 2.0 TCID50/mL, which is consistent with the test results of
4.1.3 Respiratory syncytial virus

4.1.3.1 Choose one clinically strong positive throat swab sample for RSV
One clinically strong positive throat swab sample for RSV, whose
concentration was 3.03 E+07 copies/mL determined by the Reed-Muench
method, was detected by the qualified kit in our company. The results are
shown in Figure 11.

RSV
Firstly, the clinically strong positive throat swab sample for RSV
(3.03E+07 copies/mL) was diluted with Hanks solution to 2.0E+06 copies/mL,
2.0E+05 copies/mL, 2.0E+04 copies/mL, 2.0E+03 copies/mL, 200 copies/mL, 100
copies/mL, and then was detected with this kit. Detection results are shown in
the following Figure 12.
Figure 12. Amplification results of 100~2.0E+06 copies/mL RSV
Table 5. Ct value of samples with 100~2.0E+06copies/mL RSV

Target CY5
(copies/mL) ROX
2.0E+06 22.96 20.98
2.0E+05 26.34 24.24
2.0E+04 30.10 28.11
2.0E+03 32.52 30.85

200 Undetermined 36.38

It can be seen from the above graph that the sample with 2000 copies/mL
virus was detected as positive, while 200 copies/mL was detected as negative.
dilution by small intervals for the sample with 200 copies/mL virus, to
determine the measurable concentration between 200 copies/mL and 2000
copies/mL.
The above samples were diluted to 600 copies/mL, 500 copies/mL and 400
copies/mL and were repeatedly detected 8 times. The test results are shown in
the following Table 6 and Figures 13-15.
Detected positive
samples/Sample size
600 copies/mL 8/8 100%
500 copies/mL 8/8 100%
400 copies/mL 7/8 87.5%
Figure 13. Amplification results of samples with 600 copies/mL RSV


samples with 600 copies/mL and 500 copies/mL virus is 100%, while the
detection rate of samples with 400 copies/mL is 87.5%. In order to ensure the
determined to be 500 copies/mL, which is consistent with the test results of
4.1.4 Adenovirus
4.1.4.1 Choose one clinically strong positive throat swab sample for
Adenovirus
One clinically strong positive throat swab sample for Adenovirus, whose
concentration was 3.77E+07 copies/mL determined by the Reed-Muench
method, was detected by the qualified kit in our company. The results are

shown in Figure 16.
Figure 16. Amplification results of the clinically strong positive throat swab sample
for Adenovirus

Firstly, the clinically strong positive throat swab sample for Adenovirus
(3.77E+07 copies/mL) was diluted with Hanks solution to 2.0E+06 copies/mL,
2.0E+05 copies/mL, 2.0E+04 copies/mL, 2.0E+03 copies/mL, 200 copies/mL, 100
copies/mL, and then was detected with this kit. Detection results are shown in
the following Figure 17.
Figure 17. Amplification results of samples with 100~2.0E+06 copies/mL Adenovirus

Table 7. Ct value of samples with 100~2.0E+06 copies/mL Adenovirus
Target FAM
(copies/mL) ROX
2.0E+06 22.49 20.78
2.0E+05 26.30 24.27
2.0E+04 30.24 27.86

2.0E+03 33.33 30.41

200 Undetermined Undetermined
100 Undetermined Undetermined
copies/mL.
Detected positive
samples/Sample size
600 copies/mL 8/8 100%
500 copies/mL 8/8 100%
400 copies/mL 7/8 87.5%
Figure 18. Amplification results of samples with 600 copies/mL Adenovirus


4.1.5 Human rhinovirus

4.1.5.1 Choose one clinically strong positive throat swab sample for Human
rhinovirus
One clinically strong positive throat swab sample for Human rhinovirus,
whose concentration was 2.95E+07copies/mL determined by the

Human Rhinovirus

Firstly, the clinically strong positive throat swab sample for Human
Rhinovirus (2.95E+07 copies/mL) was diluted with Hanks solution to 2.0E+06
copies/mL, 2.0E+05 copies/mL, 2.0E+04 copies/mL, 2.0E+03 copies/mL, 200
copies/mL, 100 copies/mL, and then was detected with this kit. Detection
results are shown in the following Figure 22.
Figure 22. Amplification results of samples with 100~2.0E+06 copies/mL Human

Rhinovirus
Table 9. Ct value of samples with 100~2.0E+06 copies/mL Human Rhinovirus

Target HEX
(copies/mL) ROX
2.0E+06 23.22 20.41
2.0E+05 26.83 24.08

2.0E+04 30.70 27.78

2.0E+03 33.46 30.78
copies/mL.
Detected positive
samples/Sample size
600 copies/mL 8/8 100%
500 copies/mL 8/8 100%
400 copies/mL 7/8 87.5%
Figure 23. Amplification results of samples with 600 copies/mL Human rhinovirus

4.1.6 Mycoplasma pneumoniae

4.1.6.1 Choose one clinically strong positive throat swab sample for
Mycoplasma pneumoniae
One clinically strong positive throat swab sample for Mycoplasma
pneumoniae, whose concentration was 6.24E+07 copies/mL determined by the


Firstly, the clinically strong positive throat swab sample for Mycoplasma
pneumoniae (6.24E+07 copies/mL) was diluted with Hanks solution to 2.0E+06
copies/mL, 2.0E+05 copies/mL, 2.0E+04 copies/mL, 2.0E+03 copies/mL, 200
copies/mL, 100 copies/mL, and then was detected with this kit. Detection
results are shown in the following Figure 27.
Figure 27. Amplification results of samples with 100~2.0E+06 copies/mL Mycoplasma

pneumoniae
Table 1. Ct value of samples with100~2.0E+06 copies/mL Mycoplasma pneumoniae
Target CY5
(copies/mL) ROX
2.0E+06 23.72 20.70
2.0E+05 27.40 24.55

2.0E+04 30.65 27.84

2.0E+03 34.12 31.23
Mycoplasma pneumoniae was detected as positive, while 200 copies/mL was
detected as negative. Because of the difference in concentration, it is necessary
to make gradient dilution by small intervals for the sample with 200
copies/mL Mycoplasma pneumoniae, to determine the measurable
concentration between 200 copies/mL and 2000 copies/mL.
Detected positive
Diluted samples Positive rate (%)
samples/Sample size
600 copies/mL 8/8 100%
500 copies/mL 8/8 100%
400 copies/mL 7/8 87.5%
Figure 28. Amplification results of samples with 600 copies/mL Mycoplasma

pneumoniae


pneumoniae

pneumoniae
samples with 600 copies/mL and 500 copies/mL Mycoplasma pneumoniae is
100%, while the detection rate of samples with 400 copies/mL is 87.5%. In
order to ensure the accuracy of detection, the sensitivity of this kit to detect
clinical samples is determined to be 500 copies/mL, which is consistent with
the test results of the analytical sensitivity of the registration application file
CH3.5.05.
4.2 Research on reference cut-off value

According to the detection sensitivity of clinical samples, the above
mentioned clinical sample with influenza A virus was diluted with Hanks
solution to 2.0 TCID50/mL and to 48 parts (positive samples with detection

limit concentration), and the detection results are shown in Table 13:
Table 13. Ct value of targeted gene detection of 48 positive samples for
influenza A virus at the detection limit concentration
Sam FAM Samp FAM/ Samp FAM/ Sam FAM Sam FAM
ple /Ct le Ct le Ct ple /Ct ple /Ct
1 34.70 11 37.15 21 36.16 31 36.37 41 36.18
2 33.80 12 38.18 22 35.50 32 35.84 42 34.87
3 34.68 13 35.54 23 35.43 33 36.64 43 34.67
4 34.51 14 35.42 24 35.44 34 36.41 44 35.58
5 35.72 15 36.59 25 34.91 35 36.43 45 35.84
6 35.75 16 36.09 26 36.35 36 35.64 46 34.76
7 35.80 17 36.67 27 34.64 37 34.79 47 35.31
8 35.45 18 34.07 28 35.55 38 35.07 48 37.47
9 35.87 19 35.62 29 36.59 39 34.97 / /
10 34.73 20 37.81 30 35.08 40 34.25 / /
Results showed that the Ct values of 48 positive samples for influenza A

virus at the detection limit concentration were subject to normal distribution,
and were all less than 40. The upper limit (X+2.33 S) of 99% reference range
was 37.83.
mentioned clinical sample for influenza B virus was diluted with Hanks
solution to 2.0 TCID50/mL and to 48 parts (positive samples with detection
influenza B virus at the detection limit concentration
Sam HEX/ Sampl HEX/ Samp HEX/ Sam HEX Sam HEX
ple Ct e Ct le Ct ple /Ct ple /Ct
1 36.92 11 35.28 21 35.72 31 34.89 41 36.27
2 35.19 12 36.16 22 35.77 32 35.84 42 36.19
3 35.30 13 35.43 23 35.26 33 36.13 43 35.62
4 35.02 14 36.47 24 39.69 34 35.97 44 37.18
5 35.78 15 34.92 25 35.88 35 36.34 45 36.20
6 35.34 16 36.47 26 35.71 36 36.32 46 35.20
7 36.65 17 35.44 27 35.77 37 35.68 47 34.85
8 36.30 18 36.32 28 36.27 38 36.77 48 36.44
9 35.67 19 37.31 29 36.43 39 36.16 / /
10 35.00 20 37.20 30 38.38 40 34.86 / /
Results showed that the Ct values of 48 positive samples for influenza B

virus at the detection limit concentration were subject to normal distribution,
and were all less than 40. The upper limit (X+2.33 S) of 99% reference range

was 38.16.
mentioned clinical sample for RSV was diluted with Hanks solution to 500
copies/mL and to 48 parts (positive samples with detection limit
concentration), and the detection results are shown in Table 15:
Table 15. Ct value of targeted gene detection of 48 positive samples for RSV at
the detection limit concentration
Sam CY5/ Sampl CY5/C Samp CY5/ Sam CY5/ Sam CY5/
ple Ct e t le Ct ple Ct ple Ct
1 36.36 11 35.21 21 36.48 31 36.43 41 37.01
2 35.34 12 35.79 22 34.97 32 35.68 42 35.41
3 35.52 13 37.66 23 35.68 33 35.02 43 39.46
4 35.83 14 35.60 24 36.10 34 34.87 44 34.58
5 34.95 15 34.49 25 35.62 35 36.69 45 36.24
6 38.44 16 35.50 26 34.77 36 34.62 46 36.24
7 38.71 17 35.07 27 35.51 37 36.53 47 36.99
8 35.21 18 36.57 28 35.28 38 35.54 48 36.20
9 35.56 19 35.76 29 37.54 39 38.37 / /
10 34.76 20 35.10 30 36.16 40 35.20 / /
Results showed that the Ct values of 48 positive samples for RSV at the
detection limit concentration were subject to normal distribution, and were all
less than 40. The upper limit (X+2.33 S) of 99% reference range was 38.61.
mentioned clinical sample for Adenovirus was diluted with Hanks solution to
500 copies/mL and to 48 parts (positive samples with detection limit
concentration), and the detection results are shown in Table 16:
Adenovirus at the detection limit concentration
Sam FAM Sampl FAM/ Samp FAM/ Sam FAM Sam FAM
ple /Ct e Ct le Ct ple /Ct ple /Ct
1 35.92 11 37.14 21 36.01 31 37.52 41 36.85
2 36.72 12 36.53 22 36.85 32 37.01 42 36.93
3 36.21 13 38.31 23 36.10 33 36.99 43 36.38
4 36.33 14 35.80 24 36.48 34 36.83 44 36.23
5 36.38 15 36.21 25 37.05 35 36.60 45 36.76
6 36.09 16 36.01 26 36.89 36 36.59 46 36.62
7 35.66 17 37.37 27 36.91 37 36.90 47 37.06
8 36.29 18 36.45 28 37.38 38 36.73 48 36.53
9 37.20 19 38.60 29 37.37 39 36.72 / /
10 36.13 20 36.66 30 37.28 40 36.72 / /
Results showed that the Ct values of 48 positive samples for Adenovirus
at the detection limit concentration of Adenovirus were subject to normal
distribution, and were all less than 40. The upper limit (X+2.33 S) of 99%

reference range was 38.06.

mentioned clinical sample for Human rhinovirus was diluted with Hanks
solution to 500 copies/mL and to 48 parts (positive samples with detection
Human rhinovirus at the detection limit concentration
Sam HEX/ Sampl HEX/ Samp HEX/ Sam HEX Sam HEX
ple Ct e Ct le Ct ple /Ct ple /Ct
1 35.78 11 35.67 21 35.33 31 35.40 41 34.53
2 36.16 12 35.76 22 36.93 32 35.31 42 34.94
3 35.07 13 36.48 23 36.16 33 36.02 43 35.12
4 35.38 14 36.06 24 36.42 34 36.27 44 35.23
5 35.35 15 35.35 25 35.39 35 35.73 45 35.99
6 35.23 16 36.75 26 35.49 36 35.70 46 35.35
7 35.44 17 35.76 27 35.68 37 35.29 47 35.76
8 35.82 18 37.28 28 37.93 38 36.46 48 36.87
9 35.47 19 35.78 29 36.18 39 36.31 / /
10 35.58 20 36.64 30 36.07 40 35.36 / /
Results showed that the Ct values of 48 positive samples for Human

rhinovirus at the detection limit concentration were subject to normal
mentioned clinical sample for Mycoplasma pneumoniae was diluted with
Hanks solution to 500 copies/mL and to 48 parts (positive samples with
detection limit concentration), and the detection results are shown in Table 18:
Mycoplasma pneumoniae at the detection limit concentration
Sam CY5/ Samp CY5/C Samp CY5/ Sam CY5/ Sam CY5/
ple Ct le t le Ct ple Ct ple Ct
1 36.17 11 34.82 21 35.30 31 35.92 41 36.47
2 35.46 12 37.00 22 35.60 32 35.67 42 34.25
3 35.57 13 35.12 23 35.21 33 35.50 43 35.26
4 34.98 14 37.10 24 35.88 34 35.30 44 36.43
5 35.40 15 36.81 25 34.23 35 34.13 45 35.86
6 34.56 16 35.20 26 34.54 36 35.20 46 35.22
7 35.78 17 34.47 27 35.64 37 35.57 47 35.73
8 35.60 18 35.86 28 35.16 38 35.85 48 34.41
9 35.88 19 35.80 29 35.19 39 35.22 / /
10 36.18 20 35.33 30 35.17 40 35.12 / /

Results showed that the Ct values of 48 positive samples for Mycoplasma

pneumoniae at the detection limit concentration were subject to normal
4.3 Research on reference value for the detection of clinically

positive and negative samples
4.3.1 The detection of clinical negative sample
The six respiratory pathogens nucleic acid diagnostic kit that has passed
the quality inspection was used and the strict operation was conducted in
accordance with the product instructions, so as to detect 88 confirmed clinical
negative samples for influenza A, influenza B, RSV, Adenovirus, Human
rhinovirus and Mycoplasma pneumoniae. Detection results are shown in
Table 20 below.
Table 20. Detection results of 88 clinical negative samples
Samp Ct Sam Ct Sam Ct Sam Ct Sam Ct
le value ple value ple value ple value ple value
Undete HRV: Undet
1 19 Undete. 37 55 Undete. 73
. 42.57 e.
Undete Undet
2 20 Undete. 38 Undete. 56 Undete. 74
. e.
Undete Undet
. e.
Undete Adv: Undet
. 40.53 e.
Undete Adv: Undet
. 42.84 e.
Adv:
Undete 43.32; MP: Undet
6 24 Undete. 42 60 78
. Flu A: 40.19 e.
40.32
Undete HRV: Undet
. 40.19 e.
Undete HRV:
. 42.83
Undete Undet
. e.
Undete HRV: Undet
10 28 Undete. 46 Undete. 64 82
. 40.22 e.
Undete HRV: MP: Undet
11 29 Undete. 47 65 83
. 43.00 41.06 e.
Undete Undet
. e.
Undete Undet
. e.
14 Undete 32 Undete. 50 Undete. 68 Undete. 86 Undet

. e.
Undete Undet
. e.
Undete Undet
. e.
Undete
17 35 Undete. 53 Undete. 71 Undete. / /
.
Undete HRV:
18 36 54 Undete. 72 Undete. / /
. 44.92
4.3.2 The detection of clinical positive sample

positive samples for influenza A virus. Detection results are shown in Table 21
below.
Table 21. Detection results of 92 clinical positive samples for influenza A
Sam FAM/ Sam FAM/ Sam FAM/ Sam FAM/ Sam FAM/
ple Ct ple Ct ple Ct ple Ct ple Ct
1 25.62 20 32.95 39 35.31 58 26.80 77 27.68
2 30.42 21 28.69 40 35.50 59 26.89 78 35.27
3 36.64 22 28.35 41 31.38 60 32.86 79 27.49
4 30.79 23 28.75 42 31.20 61 33.78 80 24.38
5 23.95 24 30.23 43 38.26 62 31.12 81 26.56
6 32.89 25 31.02 44 35.78 63 26.73 82 31.24
7 24.11 26 26.60 45 27.02 64 24.80 83 37.66
8 32.84 27 37.75 46 28.46 65 26.63 84 24.26
9 26.15 28 24.39 47 29.14 66 31.01 85 30.73
10 26.22 29 37.73 48 28.86 67 38.10 86 31.01
11 28.26 30 28.75 49 31.35 68 28.71 87 26.40
12 27.77 31 27.16 50 33.54 69 31.09 88 30.83
13 31.11 32 26.70 51 29.02 70 33.30 89 26.47
14 31.20 33 29.26 52 29.02 71 37.41 90 35.55
15 26.38 34 26.63 53 26.97 72 29.37 91 24.45
16 32.94 35 27.14 54 36.03 73 26.64 92 33.12
17 33.23 36 28.64 55 33.13 74 35.65 / /
18 26.36 37 31.30 56 26.76 75 28.94 / /
19 28.91 38 26.84 57 31.19 76 31.07 / /
positive samples for influenza B virus. Detection results are shown in Table 22

below.
Table 22. Detection results of 92 clinical positive samples for influenza B
Sam HEX/C Sam HEX/C Sam HEX/C Sam HEX/C Sam HEX/
ple t ple t ple t ple t ple Ct
1 21.57 20 29.25 39 32.41 58 26.37 77 21.75
2 28.36 21 21.95 40 30.42 59 28.54 78 21.94
3 33.58 22 31.41 41 22.04 60 24.71 79 23.01
4 30.28 23 28.35 42 25.21 61 30.20 80 26.56
5 28.55 24 28.23 43 24.15 62 22.36 81 26.65
6 31.71 25 33.77 44 30.51 63 26.31 82 24.96
7 30.20 26 30.18 45 22.97 64 28.60 83 26.37
8 24.47 27 22.18 46 28.42 65 33.03 84 29.18
9 22.36 28 23.54 47 22.35 66 25.93 85 34.34
10 28.08 29 34.77 48 23.92 67 26.63 86 28.51
11 28.24 30 24.86 49 28.64 68 25.61 87 22.18
12 33.52 31 28.22 50 23.98 69 32.76 88 22.13
13 33.87 32 29.28 51 26.02 70 32.91 89 28.85
14 34.66 33 28.77 52 22.17 71 31.18 90 29.03
15 22.39 34 24.23 53 24.60 72 32.43 91 34.26
16 33.66 35 23.62 54 28.32 73 34.14 92 29.23
17 34.30 36 31.36 55 28.63 74 28.39 / /
18 33.73 37 30.28 56 22.93 75 22.20 / /
19 30.12 38 21.95 57 24.25 76 31.13 / /
positive samples for RSV. Detection results are shown in Table 23 below.
Table 23. Detection results of 91 clinical positive samples for RSV
Sam CY5/C Sam CY5/C Sam CY5/C Sam CY5/C Sam CY5/
1 29.10 20 33.68 39 28.92 58 23.19 77 25.29
2 28.31 21 32.73 40 22.99 59 32.07 78 24.52
3 30.78 22 24.10 41 23.74 60 32.87 79 30.40
4 28.47 23 31.12 42 22.91 61 28.79 80 23.13
5 25.37 24 28.52 43 24.22 62 34.74 81 31.56
6 34.31 25 28.85 44 33.88 63 30.51 82 28.38
7 23.48 26 22.88 45 32.63 64 24.96 83 26.55
8 26.24 27 27.20 46 33.06 65 28.46 84 32.77
9 32.08 28 22.97 47 30.37 66 23.80 85 24.28

10 33.04 29 28.68 48 32.97 67 28.69 86 23.52

11 29.58 30 23.47 49 29.38 68 26.50 87 28.63
12 32.48 31 30.66 50 26.40 69 29.95 88 24.38
13 29.15 32 25.51 51 34.48 70 30.00 89 24.80
14 26.61 33 23.32 52 29.30 71 28.62 90 33.24
15 31.03 34 29.02 53 26.63 72 34.60 91 33.29
16 29.71 35 34.61 54 32.40 73 31.00 / /
17 23.48 36 35.31 55 26.57 74 28.57 / /
18 34.90 37 27.01 56 28.43 75 28.61 / /
19 25.07 38 23.15 57 23.26 76 32.82 / /
positive samples for Adenovirus. Detection results are shown in Table 24
below.
Table 24. Detection results of 92 clinical positive samples for Adenovirus
Sam FAM/ Sam FAM/ Sam FAM/ Sam FAM/ Sam FAM/
1 36.16 20 30.31 39 27.84 58 34.81 77 37.16
2 23.41 21 30.40 40 36.30 59 30.36 78 37.38
3 25.09 22 32.32 41 37.18 60 34.34 79 35.01
4 29.85 23 36.78 42 28.11 61 38.07 80 32.47
5 28.68 24 25.89 43 32.99 62 30.19 81 32.40
6 29.48 25 32.51 44 35.51 63 34.97 82 32.81
7 29.87 26 36.51 45 36.62 64 25.99 83 37.40
8 29.68 27 25.87 46 23.83 65 36.58 84 35.10
9 27.98 28 35.28 47 37.97 66 34.85 85 34.43
10 33.90 29 28.51 48 23.50 67 32.78 86 30.19
11 31.98 30 37.39 49 30.06 68 32.31 87 36.73
12 34.91 31 36.80 50 32.38 69 38.20 88 34.74
13 29.74 32 32.69 51 36.26 70 34.43 89 38.34
14 37.36 33 32.75 52 36.32 71 36.90 90 34.57
15 32.29 34 34.21 53 38.57 72 35.32 91 30.24
16 31.90 35 38.58 54 35.29 73 36.85 92 25.67
17 34.35 36 34.25 55 37.44 74 36.97 / /
18 32.21 37 34.40 56 34.88 75 37.37 / /
19 32.37 38 30.14 57 27.89 76 27.88 / /

positive samples for Human rhinovirus. Detection results are shown in Table
25 below.
Table 25. Detection results of 92 clinical positive samples for Human
rhinovirus
Sam HEX/ Sam HEX/ Sam HEX/ Sam HEX/ Sam HEX/
1 33.57 20 24.04 39 31.28 58 30.67 77 30.32
2 28.52 21 28.21 40 28.00 59 29.08 78 28.54
3 23.91 22 26.65 41 30.83 60 32.15 79 32.35
4 30.15 23 26.70 42 32.50 61 26.25 80 29.16
5 26.77 24 30.35 43 28.15 62 28.31 81 31.84
6 28.15 25 21.72 44 33.20 63 32.55 82 22.17
7 27.52 26 33.48 45 28.73 64 31.68 83 28.10
8 24.15 27 31.55 46 27.17 65 31.51 84 28.66
9 29.76 28 22.16 47 29.66 66 29.04 85 30.30
10 29.12 29 33.89 48 30.17 67 30.51 86 30.99
11 30.28 30 24.07 49 31.32 68 29.79 87 32.95
12 26.47 31 21.42 50 29.22 69 26.20 88 25.61
13 25.76 32 28.70 51 30.23 70 29.09 89 28.56
14 26.41 33 23.63 52 34.85 71 23.82 90 29.56
15 33.23 34 28.58 53 26.11 72 30.58 91 30.83
16 30.38 35 32.13 54 30.39 73 26.25 92 30.63
17 31.03 36 22.21 55 25.91 74 30.47 / /
18 30.31 37 30.89 56 28.34 75 29.59 / /
19 28.69 38 24.40 57 26.13 76 26.34 / /
positive samples for Mycoplasma pneumoniae. Detection results are shown in
Table 26 below.
Table 26. Detection results of 92 clinical positive samples for Mycoplasma
pneumoniae
Sam CY5/C Sam CY5/C Sam CY5/C Sam CY5/C Sam CY5/
1 23.12 20 31.27 39 33.40 58 33.34 77 29.83
2 24.99 21 29.44 40 27.67 59 34.19 78 25.65
3 29.89 22 35.01 41 27.21 60 33.14 79 31.45
4 25.38 23 29.78 42 35.39 61 31.38 80 31.55
5 31.86 24 25.09 43 29.05 62 31.11 81 29.70
6 33.53 25 29.36 44 35.64 63 31.51 82 31.74
7 33.82 26 23.13 45 31.16 64 34.56 83 32.11

8 31.88 27 31.69 46 34.34 65 29.30 84 33.10

9 29.38 28 36.21 47 33.95 66 29.23 85 31.02
10 25.68 29 34.49 48 33.65 67 33.14 86 29.81
11 34.05 30 27.49 49 36.30 68 27.12 87 27.65
12 29.99 31 34.84 50 35.26 69 37.16 88 33.77
13 25.01 32 35.31 51 29.09 70 35.04 89 26.52
14 31.56 33 22.87 52 33.76 71 34.75 90 36.16
15 27.60 34 31.77 53 29.25 72 34.04 91 33.59
16 31.82 35 31.80 54 27.36 73 23.14 92 26.36
17 29.50 36 33.27 55 26.62 74 31.57 / /
18 29.57 37 27.53 56 34.62 75 33.06 / /
19 36.00 38 33.22 57 33.86 76 36.28 / /
4.3.3 ROX curve analysis

The results of the control reagents and culture method were used as the
standard to determine the negative or positive of the sample, and SPSS 19.0
software was used to perform ROC analysis on the Ct values of the above 88
negative samples and the positive samples for each pathogen, to determine
the reference Ct value of the target gene detection. For those undetected Ct
value, the value was set as 45 (The reaction cycles of PCR amplification
program is 45). The output after ROC analysis is as follows:
Result 1-1 Basic information of ROC curve analysis on influenza A virus
Result 1-2 Related indicators such as the area under the ROC curve of influenza A
virus
The coordinates of the curve
The test of results: Ct value
If larger than or equal to
Sensitivity 1－Specificity
the value, then be positivea
22.9500 1.000 1.000

24.0300 1.000 .989

24.1850 1.000 .978
24.3200 1.000 .967
24.3850 1.000 .957
24.4200 1.000 .946
24.6250 1.000 .935
25.2100 1.000 .924
25.8850 1.000 .913
26.1850 1.000 .902
26.2900 1.000 .891
26.3700 1.000 .880
26.3900 1.000 .870
26.4350 1.000 .859
26.5150 1.000 .848
26.5800 1.000 .837
26.6150 1.000 .826
26.6350 1.000 .804
26.6700 1.000 .793
26.7150 1.000 .783
26.7450 1.000 .772
26.7800 1.000 .761
26.8200 1.000 .750
26.8650 1.000 .739
26.9300 1.000 .728
26.9950 1.000 .717
27.0800 1.000 .707
27.1500 1.000 .696
27.3250 1.000 .685
27.5850 1.000 .674
27.7250 1.000 .663
28.0150 1.000 .652
28.3050 1.000 .641
28.4050 1.000 .630
28.5500 1.000 .620
28.6650 1.000 .609
28.7000 1.000 .598
28.7300 1.000 .587
28.8050 1.000 .565
28.8850 1.000 .554
28.9250 1.000 .543
28.9800 1.000 .533
29.0800 1.000 .511
29.2000 1.000 .500
29.3150 1.000 .489
29.8000 1.000 .478
30.3250 1.000 .467
30.5750 1.000 .457

30.7600 1.000 .446

30.8100 1.000 .435
30.9200 1.000 .424
31.0150 1.000 .402
31.0450 1.000 .391
31.0800 1.000 .380
31.1000 1.000 .370
31.1150 1.000 .359
31.1550 1.000 .348
31.1950 1.000 .337
31.2200 1.000 .315
31.2700 1.000 .304
31.3250 1.000 .293
31.3650 1.000 .283
32.1100 1.000 .272
32.8500 1.000 .261
32.8750 1.000 .250
32.9150 1.000 .239
32.9450 1.000 .228
33.0350 1.000 .217
33.1250 1.000 .207
33.1800 1.000 .196
33.2650 1.000 .185
33.4200 1.000 .174
33.6600 1.000 .163
34.5250 1.000 .152
35.2900 1.000 .141
35.4050 1.000 .130
35.5250 1.000 .120
35.6000 1.000 .109
35.7150 1.000 .098
35.9050 1.000 .087
36.3350 1.000 .076
37.0250 1.000 .065
37.5350 1.000 .054
37.6950 1.000 .043
37.7400 1.000 .033
37.9250 1.000 .022
38.1800 1.000 .011
39.2900 1.000 .000
42.6600 .989 .000
46.0000 .000 .000
a. The minimum cut-off value is the minimum observation
value minus one, and the maximum cut-off value is the
maximum observation value plus one. All other cut-off
values are the average of two adjacent observations.
Result 1-3 The pairing values(sensitivity, 1-specificity) corresponding to different

diagnostic cut-off values (reference values) of influenza A virus

It can be seen from the output results of the above ROC analysis: 1) The
area under the ROC curve is 1.000, indicating a well diagnostic accuracy of the
reagent; 2) The best diagnostic cut-off value is 39.29 (It is basically the same
with the results “the upper limit of the 99% reference value range is 37.83” in
the above described Section 4.2), at this time, the sensitivity (Sensitivity) is
1.000, which is 100%; the specificity is (1-0.000), which is 100%. Based on the
sensitivity testing requirements and ROC analysis results, the Ct reference
value for target gene detection of influenza A virus is tentatively proposed as
40; those with Ct ≤ 40 are considered positive; those with Ct > 40 are
considered negative. This criteria was used to determine the above mentioned
180 clinical samples. Results showed that 92 were positive samples and 88
were negative samples, which were consistent with the results of the control
reagents and sequencing method.
Result 2-1 Basic information of ROC curve analysis on influenza B virus
Result 2-2 Related indicators such as the area under the ROC curve of influenza B
virus

20.5700 1.000 1.000
21.6600 1.000 .989
21.8450 1.000 .978
21.9450 1.000 .967
21.9950 1.000 .946
22.0850 1.000 .935
22.1500 1.000 .924
22.1750 1.000 .913
22.1900 1.000 .891

22.2750 1.000 .880

22.3550 1.000 .870
22.3750 1.000 .848
22.6600 1.000 .837
22.9500 1.000 .826
22.9900 1.000 .815
23.2750 1.000 .804
23.5800 1.000 .793
23.7700 1.000 .783
23.9500 1.000 .772
24.0650 1.000 .761
24.1900 1.000 .750
24.2400 1.000 .739
24.3600 1.000 .728
24.5350 1.000 .717
24.6550 1.000 .707
24.7850 1.000 .696
24.9100 1.000 .685
25.0850 1.000 .674
25.4100 1.000 .663
25.7700 1.000 .652
25.9750 1.000 .641
26.1650 1.000 .630
26.3400 1.000 .620
26.4650 1.000 .598
26.5950 1.000 .587
26.6400 1.000 .576
27.3650 1.000 .565
28.1500 1.000 .554
28.2250 1.000 .543
28.2350 1.000 .533
28.2800 1.000 .522
28.3350 1.000 .511
28.3550 1.000 .500
28.3750 1.000 .489
28.4050 1.000 .478
28.4650 1.000 .467
28.5250 1.000 .457
28.5450 1.000 .446
28.5750 1.000 .435
28.6150 1.000 .424
28.6350 1.000 .413
28.7050 1.000 .402
28.8100 1.000 .391
28.9400 1.000 .380
29.1050 1.000 .370
29.2050 1.000 .359

29.2400 1.000 .348

29.2650 1.000 .337
29.7000 1.000 .326
30.1500 1.000 .315
30.1900 1.000 .304
30.2400 1.000 .283
30.3500 1.000 .261
30.4650 1.000 .250
30.8200 1.000 .239
31.1550 1.000 .228
31.2700 1.000 .217
31.3850 1.000 .207
31.5600 1.000 .196
32.0600 1.000 .185
32.4200 1.000 .174
32.5950 1.000 .163
32.8350 1.000 .152
32.9700 1.000 .141
33.2750 1.000 .130
33.5500 1.000 .120
33.6200 1.000 .109
33.6950 1.000 .098
33.7500 1.000 .087
33.8200 1.000 .076
34.0050 1.000 .065
34.2000 1.000 .054
34.2800 1.000 .043
34.3200 1.000 .033
34.5000 1.000 .022
34.7150 1.000 .011
39.8850 1.000 .000
46.0000 .000 .000
Result 2-3 The pairing values (sensitivity, 1-specificity) corresponding to different
diagnostic cut-off values (reference values) of influenza B virus
value for target gene detection of influenza B virus is tentatively proposed as
40; those with Ct ≤ 40 are considered positive; those with Ct > 40 are

Result 3-1 Basic information of ROC curve analysis on Respiratory syncytial virus
Result 3-2 Related indicators such as the area under the ROC curve of respiratory
syncytial virus
21.8800 1.000 1.000
22.8950 1.000 .989
22.9400 1.000 .978
22.9800 1.000 .967
23.0600 1.000 .956
23.1400 1.000 .945
23.1700 1.000 .934
23.2250 1.000 .923
23.2900 1.000 .912
23.3950 1.000 .901
23.4750 1.000 .890
23.5000 1.000 .868
23.6300 1.000 .857
23.7700 1.000 .846
23.9500 1.000 .835
24.1600 1.000 .824
24.2500 1.000 .813
24.3300 1.000 .802
24.4500 1.000 .791
24.6600 1.000 .780
24.8800 1.000 .769
25.0150 1.000 .758
25.1800 1.000 .747

25.3300 1.000 .736

25.4400 1.000 .725
25.8750 1.000 .714
26.3200 1.000 .703
26.4500 1.000 .692
26.5250 1.000 .681
26.5600 1.000 .670
26.5900 1.000 .659
26.6200 1.000 .648
26.8200 1.000 .637
27.1050 1.000 .626
27.7550 1.000 .615
28.3450 1.000 .604
28.4050 1.000 .593
28.4450 1.000 .582
28.4650 1.000 .571
28.4950 1.000 .560
28.5450 1.000 .549
28.5900 1.000 .538
28.6150 1.000 .527
28.6250 1.000 .516
28.6550 1.000 .505
28.6850 1.000 .495
28.7400 1.000 .484
28.8200 1.000 .473
28.8850 1.000 .462
28.9700 1.000 .451
29.0600 1.000 .440
29.1250 1.000 .429
29.2250 1.000 .418
29.3400 1.000 .407
29.4800 1.000 .396
29.6450 1.000 .385
29.8300 1.000 .374
29.9750 1.000 .363
30.1850 1.000 .352
30.3850 1.000 .341
30.4550 1.000 .330
30.5850 1.000 .319
30.7200 1.000 .308
30.8900 1.000 .297
31.0150 1.000 .286
31.0750 1.000 .275
31.3400 1.000 .264
31.8150 1.000 .253
32.0750 1.000 .242
32.2400 1.000 .231

32.4400 1.000 .220

32.5550 1.000 .209
32.6800 1.000 .198
32.7500 1.000 .187
32.7950 1.000 .176
32.8450 1.000 .165
32.9200 1.000 .154
33.0050 1.000 .143
33.0500 1.000 .132
33.1500 1.000 .121
33.2650 1.000 .110
33.4850 1.000 .099
33.7800 1.000 .088
34.0950 1.000 .077
34.3950 1.000 .066
34.5400 1.000 .055
34.6050 1.000 .044
34.6750 1.000 .033
34.8200 1.000 .022
35.1050 1.000 .011
40.1550 1.000 .000
46.0000 .000 .000
diagnostic cut-off values (reference values) of respiratory syncytial virus
reagent; 2) The best diagnostic cut-off value is 40.16 (It has high similarity
value for target gene detection of respiratory syncytial virus is tentatively
proposed as 40; those with Ct ≤ 40 are considered positive; those with Ct > 40
are considered negative. This criteria was used to determine the above
mentioned 180 clinical samples. Results showed that 91 were positive samples
and 88 were negative samples, which were consistent with the results of the
control reagents and sequencing method.
Result 4-1 Basic information of ROC curve analysis on Adenovirus

Result 4-2 Related indicators such as the area under the ROC curve of Adenovirus
If larger than or equal to the
value, then be positivea
22.4100 1.000 1.000
23.4550 1.000 .989
23.6650 1.000 .978
24.4600 1.000 .967
25.3800 1.000 .957
25.7700 1.000 .946
25.8800 1.000 .935
25.9400 1.000 .924
26.9150 1.000 .913
27.8600 1.000 .902
27.8850 1.000 .891
27.9350 1.000 .880
28.0450 1.000 .870
28.3100 1.000 .859
28.5950 1.000 .848
29.0800 1.000 .837
29.5800 1.000 .826
29.7100 1.000 .815
29.7950 1.000 .804
29.8600 1.000 .793
29.9650 1.000 .783
30.1000 1.000 .772
30.1650 1.000 .761
30.2150 1.000 .739
30.2750 1.000 .728
30.3350 1.000 .717
30.3800 1.000 .707
31.1500 1.000 .696
31.9400 1.000 .685
32.0950 1.000 .674
32.2500 1.000 .663
32.3000 1.000 .652
32.3150 1.000 .641
32.3450 1.000 .630

32.3750 1.000 .620

32.3900 1.000 .609
32.4350 1.000 .598
32.4900 1.000 .587
32.6000 1.000 .576
32.7200 1.000 .565
32.7650 1.000 .554
32.7950 1.000 .543
32.9000 1.000 .533
33.4450 1.000 .522
34.0550 1.000 .511
34.2300 1.000 .500
34.2950 1.000 .489
34.3450 1.000 .478
34.3750 1.000 .467
34.4150 1.000 .457
34.5000 1.000 .435
34.6550 1.000 .424
34.7750 1.000 .413
34.8300 1.000 .402
34.8650 1.000 .391
34.8950 1.000 .380
34.9400 1.000 .370
34.9900 1.000 .359
35.0550 1.000 .348
35.1900 1.000 .337
35.2850 1.000 .326
35.3050 1.000 .315
35.4150 1.000 .304
35.8350 1.000 .293
36.2100 1.000 .283
36.2800 1.000 .272
36.3100 1.000 .261
36.4150 1.000 .250
36.5450 1.000 .239
36.6000 1.000 .228
36.6750 1.000 .217
36.7550 1.000 .207
36.7900 1.000 .196
36.8250 1.000 .185
36.8750 1.000 .174
36.9350 1.000 .163
37.0650 1.000 .152
37.1700 1.000 .141
37.2700 1.000 .130
37.3650 1.000 .120
37.3750 1.000 .109

37.3850 1.000 .098

37.3950 1.000 .087
37.4200 1.000 .076
37.7050 1.000 .065
38.0200 1.000 .054
38.1350 1.000 .043
38.2700 1.000 .033
38.4550 1.000 .022
38.5750 1.000 .011
39.5550 1.000 .000
41.6850 .989 .000
43.0800 .977 .000
44.1600 .966 .000
46.0000 .000 .000
diagnostic cut-off values (reference values) of Adenovirus
value for target gene detection of Adenovirus is tentatively proposed as 40;
those with Ct ≤ 40 are considered positive; those with Ct > 40 are considered
negative. This criteria was used to determine the above mentioned 180 clinical
samples. Results showed that 92 were positive samples and 88 were negative
samples, which were consistent with the results of the control reagents and
sequencing method.
Result 5-1 Basic information of ROC curve analysis on Human rhinovirus
Result 5-2 Related indicators such as the area under the ROC curve of Human
rhinovirus


20.4200 1.000 1.000
21.5700 1.000 .989
21.9400 1.000 .978
22.1650 1.000 .967
22.1900 1.000 .957
22.9200 1.000 .946
23.7250 1.000 .935
23.8650 1.000 .924
23.9750 1.000 .913
24.0550 1.000 .902
24.1100 1.000 .891
24.2750 1.000 .880
25.0050 1.000 .870
25.6850 1.000 .859
25.8350 1.000 .848
26.0100 1.000 .837
26.1200 1.000 .826
26.1650 1.000 .815
26.2250 1.000 .804
26.2950 1.000 .783
26.3750 1.000 .772
26.4400 1.000 .761
26.5600 1.000 .750
26.6750 1.000 .739
26.7350 1.000 .728
26.9700 1.000 .717
27.3450 1.000 .707
27.7600 1.000 .696
28.0500 1.000 .685
28.1250 1.000 .674
28.1800 1.000 .652
28.2600 1.000 .641
28.3250 1.000 .630
28.4300 1.000 .620
28.5300 1.000 .609
28.5500 1.000 .598
28.5700 1.000 .587
28.6200 1.000 .576
28.6750 1.000 .565
28.6950 1.000 .554
28.7150 1.000 .543
28.8850 1.000 .533

29.0600 1.000 .522

29.0850 1.000 .511
29.1050 1.000 .500
29.1400 1.000 .489
29.1900 1.000 .478
29.3900 1.000 .467
29.5750 1.000 .457
29.6250 1.000 .446
29.7100 1.000 .435
29.7750 1.000 .424
29.9700 1.000 .413
30.1600 1.000 .402
30.2000 1.000 .391
30.2550 1.000 .380
30.2900 1.000 .370
30.3050 1.000 .359
30.3150 1.000 .348
30.3350 1.000 .337
30.3650 1.000 .326
30.3850 1.000 .315
30.4300 1.000 .304
30.4900 1.000 .293
30.5450 1.000 .283
30.6050 1.000 .272
30.6500 1.000 .261
30.7500 1.000 .250
30.8600 1.000 .228
30.9400 1.000 .217
31.0100 1.000 .207
31.1550 1.000 .196
31.3000 1.000 .185
31.4150 1.000 .174
31.5300 1.000 .163
31.6150 1.000 .152
31.7600 1.000 .141
31.9850 1.000 .130
32.1400 1.000 .120
32.2500 1.000 .109
32.4250 1.000 .098
32.5250 1.000 .087
32.7500 1.000 .076
33.0750 1.000 .065
33.2150 1.000 .054
33.3550 1.000 .043
33.5250 1.000 .033
33.7300 1.000 .022
34.3700 1.000 .011

37.5200 1.000 .000

40.2050 .989 .000
41.3950 .977 .000
42.7000 .966 .000
42.9150 .955 .000
43.9600 .943 .000
44.9600 .932 .000
46.0000 .000 .000
Result 1-3 The pairing values (sensitivity, 1-specificity) corresponding to different
diagnostic cut-off values (reference values) of Human rhinovirus
value for target gene detection of Human rhinovirus is tentatively proposed
as 40; those with Ct ≤ 40 are considered positive; those with Ct > 40 are
Result 6-1 Basic information of ROC curve analysis on Mycoplasma pneumoniae
Result 6-2 Related indicators such as the area under the ROC curve of Mycoplasma

pneumoniae

If larger than or equal
to the value, then be Sensitivity 1－Specificity
positivea
20.5700 1.000 1.000
21.6600 1.000 .989
21.8450 1.000 .978
21.9450 1.000 .967
21.9950 1.000 .946
22.0850 1.000 .935
22.1500 1.000 .924
22.1750 1.000 .913
22.1900 1.000 .891
22.2750 1.000 .880
22.3550 1.000 .870
22.3750 1.000 .848
22.6600 1.000 .837
22.9500 1.000 .826
22.9900 1.000 .815
23.2750 1.000 .804
23.5800 1.000 .793
23.7700 1.000 .783
23.9500 1.000 .772
24.0650 1.000 .761
24.1900 1.000 .750
24.2400 1.000 .739
24.3600 1.000 .728
24.5350 1.000 .717
24.6550 1.000 .707
24.7850 1.000 .696
24.9100 1.000 .685
25.0850 1.000 .674
25.4100 1.000 .663
25.7700 1.000 .652
25.9750 1.000 .641
26.1650 1.000 .630
26.3400 1.000 .620
26.4650 1.000 .598
26.5950 1.000 .587
26.6400 1.000 .576
27.3650 1.000 .565
28.1500 1.000 .554
28.2250 1.000 .543

28.2350 1.000 .533

28.2800 1.000 .522
28.3350 1.000 .511
28.3550 1.000 .500
28.3750 1.000 .489
28.4050 1.000 .478
28.4650 1.000 .467
28.5250 1.000 .457
28.5450 1.000 .446
28.5750 1.000 .435
28.6150 1.000 .424
28.6350 1.000 .413
28.7050 1.000 .402
28.8100 1.000 .391
28.9400 1.000 .380
29.1050 1.000 .370
29.2050 1.000 .359
29.2400 1.000 .348
29.2650 1.000 .337
29.7000 1.000 .326
30.1500 1.000 .315
30.1900 1.000 .304
30.2400 1.000 .283
30.3500 1.000 .261
30.4650 1.000 .250
30.8200 1.000 .239
31.1550 1.000 .228
31.2700 1.000 .217
31.3850 1.000 .207
31.5600 1.000 .196
32.0600 1.000 .185
32.4200 1.000 .174
32.5950 1.000 .163
32.8350 1.000 .152
32.9700 1.000 .141
33.2750 1.000 .130
33.5500 1.000 .120
33.6200 1.000 .109
33.6950 1.000 .098
33.7500 1.000 .087
33.8200 1.000 .076
34.0050 1.000 .065
34.2000 1.000 .054
34.2800 1.000 .043
34.3200 1.000 .033
34.5000 1.000 .022
34.7150 1.000 .011

37.4800 1.000 .000

40.6250 .989 .000
43.0300 .977 .000
46.0000 .000 .000
diagnostic cut-off values (reference values) of Mycoplasma pneumoniae
value for target gene detection of Mycoplasma pneumoniae is tentatively
proposed as 40; those with Ct ≤ 40 are considered positive; those with Ct > 40
are considered negative. This criteria was used to determine the above
mentioned 180 clinical samples. Results showed that 92 were positive samples
and 88 were negative samples, which were consistent with the results of the
control reagents and sequencing method.
4.4 Setting basis and results of reference value of internal standard
detection
The function of the internal standard is to prevent false negative results
in clinical trail. The samples used for the research of internal standard
reference value were the same as the 88 negative samples in Section 4.3 above.
The test results of internal standard are shown in Table 27 below:
Table 27. Detection results of the internal standard (ROX channel) of 88 clinical
negative samples
Internal Internal Internal Internal
Sample No.
Sample No.
Sample No.
Sample No.
standard standard standard standard

ROXCt ROXCt ROXCt ROXCt
React React React React React React React React
ion A ion B ion A ion B ion A ion B ion A ion B
No
1 23.17 20.72 23 23.99 19.82 45 23.91 67 28.21 32.85
Ct
2 23.84 26.21 24 19.34 24.18 46 23.06 35.78 68 30.18 33.29
3 18.65 22.15 25 17.61 15.73 47 33.86 31.89 69 29.25 33.16
4 28.40 21.75 26 22.16 22.30 48 33.21 33.93 70 27.35 32.41
No
5 30.01 18.55 27 30.18 32.08 49 32.87 71 27.40 31.27
Ct
6 16.70 39.67 28 21.23 31.55 50 31.08 32.13 72 25.29 30.30
7 21.73 23.25 29 21.51 26.37 51 30.20 23.97 73 28.28 28.20
8 31.04 30.54 30 31.80 17.06 52 19.73 19.79 74 35.14 23.17
9 22.21 26.37 31 29.80 25.51 53 18.84 35.45 75 33.47 22.24
10 24.20 30.41 32 23.84 25.89 54 30.52 33.44 76 20.85 23.68
11 28.54 18.04 33 29.16 28.84 55 29.05 32.77 77 24.20 22.01

No
12 31.26 31.93 34 15.81 29.09 56 26.20 31.29 78 25.47
Ct
13 32.01 27.49 35 29.73 32.91 57 22.00 30.30 79 27.30 28.85
14 22.08 32.35 36 22.67 32.97 58 27.08 28.17 80 35.46 31.48
15 22.29 21.36 37 19.95 32.48 59 24.01 25.18 81 33.65 31.61
No
16 31.24 19.19 38 27.42 32.83 60 20.98 28.83 82 32.20
Ct
17 26.47 15.94 39 25.31 34.83 61 20.96 22.11 83 30.62 23.63
18 28.57 30.63 40 20.11 35.56 62 35.67 33.66 84 28.05 23.37
19 31.26 18.15 41 35.77 36.91 63 33.18 33.38 85 25.53 20.13
20 29.77 29.87 42 35.78 37.70 64 32.07 32.05 86 26.60 34.12
21 30.43 29.78 43 33.51 32.56 65 31.13 33.33 87 33.48 32.23
22 28.81 26.43 44 23.16 23.48 66 30.30 33.86 88 21.72 22.07
The results showed that the internal standard Ct values of 88 samples

were approximately subject to normal distribution, and the upper limit of 99%
reference value range (X+2.33 S) was 40.12. Considering the complexity of
clinical trails, the internal standard reference value (the maximum detection
Ct value) is set to 40 to effectively monitor the internal standard.
5. Verification and confirmation of cut-off value

The above-mentioned proposed reference value was used to determine
the 2076 samples of clinical assessment. The positive percentage agreement
between the reagent to be evaluated and the control reagent was 99.07% (95%
CI: 98.25%~99.57%), negative percentage agreement was 97.38% (95% CI:
96.26%~98.24%) and the total percentage agreement was 98.17% (95% CI:
97.50%~98.70%); There were 2076 samples in total, including 933 positive
samples, 1077 negative samples and 66 samples with non-confirming
detection results, and the overall agreement percentage was 96.82%. The
results show that the detection results of the reagent to be evaluated were well
consistent with the sequencing control method. Through the verification of
clinical trials, it is confirmed that the above proposed reference value meets
the performance requirements of this kit in clinical application.
6. Summary of determining cut-off value

According to the above-mentioned experimental research and clinical
verification, it is confirmed that the detection reference values of this kit are as
follows:
6.1 Ct reference value of internal standard detection is 40:

In the detection results of negative samples, if Ct value of the internal
standard ≤ 40, then the result is valid; Otherwise, it’s invalid, and the cause
should be found and eliminated (such as whether there are inhibitors in the
sample or other causes from test operation, equipment or sampling method),
and repeated test should be done on the same sample.
6.2 Ct reference value of target gene detection (cut-off value) is 40:

If Ct value of detection result of the target gene in the sample to be

evaluated > 40 (Or Ct value is undetected), and Ct value of internal standard ≤
40, then the content of nucleic acid of pathogen to be evaluated in the sample
is considered to be lower than the lower detection limit of the kit. If Ct value
of internal standard > 40, then the result is invalid, and the cause should be
found and eliminated and repeated test should be conducted on the same
sample.
If Ct value of detection results of the sample to be evaluated ≤ 40, then the
sample is considered to be positive with the pathogen to be evaluated.

Accuracy Study
In accordance with the technical requirements of the Six Respiratory

Pathogens Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing), three
batches (2017001, 2017002, 2017003) of qualified reagents were continuously
produced. The overall performance evaluation of this kit was carried out on
the three fluorescent quantitative PCR machines, including ABI 7500
fluorescent quantitative PCR instrument, Life Technologies QuantStudioTM 5
and SLAN-96P automatic medical PCR analysis system. The evaluation
results of the total analytical performance of these three batches of kits are
described below.
Analytical performance evaluation on ABI 7500 PCR instrument

1. Interferences analysis
1.1 Anti-PCR inhibitor/interfering substance effect
The main purposes of extracting pathogen DNA/RNA are as follows:
enriching the concentration of target nucleic acid sequences, ensuring the
integrity of the target nucleic acid sequence, increasing the uniformity of the
PCR template solution, and removing PCR inhibitors. It is one of the factors
that determine the success of RT-PCR. For example, under the conditions of
low pH and high salt concentration, the modified superparamagnetic
nanoparticles can specifically adsorb viral nucleic acids, and then pure nucleic
acids can be enriched after the following washing steps. Theoretically, the
magnetic bead method can remove PCR inhibitors/interfering substances well
when extracting nucleic acid. But in actual operation, there are many
unforeseen unknown factors, so we need to verify this via experiments. In
addition, the composition of the PCR reaction system also determines the
influence of inhibitors/interfering substances on the PCR reaction.
Appropriate PCR buffer, pH, and the concentration of appropriate dNTPs,
enzymes and other raw materials can reduce the influence of
inhibitors/interfering substances on the PCR reaction. Therefore, it is
necessary for us to experiment to verify this point.
The lentiviral particles containing the target conserved sequence of
influenza A virus, influenza B virus, respiratory syncytial virus, and human
rhinovirus, and the cloning plasmid containing the target conserved sequence
of adenovirus and Mycoplasma pneumoniae were used as the research objects.
The lentiviral particles and cloned plasmids of each pathogen were diluted
with normal saline to a concentration of 7.0×103 TU/mL (and copies/mL), and
mixed with equal volumes to form the test sample. The test sample was
divided into several parts, and the possible PCR inhibitors/interfering
substances listed in Table 1-1 were added to each part, and the detection was
carried out according to the operating method in the manual of this kit. The
test sample without interfering substances was used as the control. The test
results of three batches of the kit on the ABI 7500 instrument are shown in
Table 1-2 to Table 1-4 and Figure 1-1 to Figure 1-6 (only the batch of 2017001 is
taken as an example for screenshot display).
Table 1-1 PCR inhibitor/interfering substance added and its concentration
Accuracy study 1
Final Final
Sample Sample
Inhibitor name concentrati Inhibitor name concentrati
No. No.
on on
Oxymetazoline
F1 100 μg/mL F9 SDS 100 μg/mL
hydrochloride
F2 Dexamethasone 50 μg/mL F10 EDTA 10 μg/mL
Cefmenoxime
F3 50 μg/mL F11 Urea 100 μg/mL
Hydrochloride
F4 Menthol 50 μg/mL F12 Heme 10 μg/mL
Purified
F5 Zanamivir 100 μg/mL F13 20 μg/mL
Mucin
F6 Ribavirin 100 μg/mL F14 FeCl3 100 μg/mL
Absolute
F7 Azithromycin 100 μg/mL F15 20%(v/v)
ethanol
Human whole
F8 NaCl 60 μg/mL F16 20%(v/v)
blood
Figure 1-1 The effect test curve of influenza A virus anti-inhibitor/interfering substance
(2017001 batch)
Figure 1-2 The effect test curve of influenza B virus anti-inhibitor/interfering substance
(2017001 batch)
Accuracy study 2
Figure 1-3 The effect test curve of respiratory syncytial virus anti-inhibitor/interfering
substance (2017001 batch)
Figure 1-4 The effect test curve of Adenovirus anti-inhibitor/interfering substance

(2017001 batch)
Figure 1-5 The effect test curve of Human rhinovirus anti-inhibitor/interfering substance
(2017001 batch)
Accuracy study 3
Figure 1-6 The effect test curve of Mycoplasma pneumoniae anti-inhibitor/interfering
Table 1-2 Statistical results of Ct value of inhibitor/interference substance removal effect

test(2017001 batch)
PCR mix A PCR mix B
FAM Ct HEX Ct CY5 Ct FAM Ct HEX Ct CY5 Ct
value value value value value value
Con 30.7 30.7 31.0 30.8 31.2 31.4 31.4 30.9 31.2 31.1 31.6 31.6
trol 6 9 3 0 3 0 2 8 3 0 2 1
30.7 30.8 30.9 30.9 30.8 31.9 31.0 31.3 31.5 31.2 31.4 31.5
F1
4 5 6 8 5 6 3 8 8 4 9 4
30.8 30.9 31.1 31.0 31.6 32.2 31.4 31.4 31.4 31.4 31.4 31.6
F2
9 3 0 8 2 6 6 5 8 4 5 2
31.0 30.7 31.3 30.6 32.0 31.6 31.7 31.5 31.4 31.2 32.2 31.9
F3
8 2 5 4 5 6 4 0 4 2 3 0
31.2 31.1 31.3 31.1 31.7 31.7 31.7 31.8 31.3 31.2 32.0 31.7
F4
1 8 1 8 5 6 0 2 4 5 0 1
31.1 31.2 31.4 31.0 31.6 31.7 31.6 31.5 31.5 31.3 31.9 31.4
F5
1 6 1 6 0 6 9 9 8 9 4 7
30.7 31.3 31.5 31.3 31.5 31.3 31.8 31.5 31.5 31.3 31.7 31.5
F6
3 0 5 6 2 0 2 4 0 3 9 6
31.1 31.1 31.0 31.0 31.7 30.8 32.0 31.5 31.3 31.2 31.7 32.0
F7
2 8 4 6 1 2 0 8 6 6 3 0
30.8 30.9 30.5 30.7 31.7 31.4 31.3 31.4 31.0 30.6 31.3 31.6
F8
9 1 7 7 6 8 5 9 8 7 3 2
31.4 31.0 31.0 30.8 31.1 31.8 31.4 31.8 31.1 31.0 31.5 31.8
F9
0 1 7 6 7 5 2 3 8 6 1 9
30.9 31.0 31.1 31.2 31.4 31.8 31.4 31.3 31.3 31.1 31.7 31.4
F10
8 1 6 1 0 4 0 6 0 7 0 8
31.2 31.0 31.3 31.4 31.2 31.8 31.3 31.4 31.1 30.9 31.4 31.3
F11
4 3 1 0 7 1 2 9 7 7 0 0
31.1 30.7 31.2 31.1 30.7 31.2 31.8 31.6 31.2 31.1 31.8 31.4
F12
1 0 8 5 5 2 2 5 4 8 8 0
30.9 31.2 30.6 31.4 31.4 31.0 31.6 31.5 31.2 31.1 31.6 31.9
F13
3 9 7 0 3 7 4 3 2 2 4 0
Accuracy study 4
30.9 31.3 31.2 30.8 31.7 31.1 31.2 31.9 31.1 31.0 31.4 31.9
F14
8 4 0 9 7 7 5 0 0 6 9 0
30.9 31.3 31.2 31.0 31.4 31.0 31.3 32.2 31.3 31.2 31.6 32.0
F15
8 1 0 5 7 6 4 6 6 3 0 4
30.9 30.9 30.6 30.6 30.7 31.4 31.2 31.3 30.9 30.5 31.6 31.5
F16
3 1 7 7 9 4 3 2 3 5 7 2
Table1-3 Statistical results of Ct value of inhibitor/interference substance removal effect

test(2017002 batch)
PCR mix A PCR mix B
Con 31.0 30.6 31.1 31.1 31.0 31.2 31.0 30.9 31.2 30.2 31.1 31.3
trol 4 4 0 9 6 5 3 9 7 6 6 8
30.3 31.0 31.2 31.5 31.2 31.5 31.1 31.0 31.2 31.2 30.9 31.2
F1
5 2 0 7 5 7 5 0 0 1 4 2
30.6 30.7 31.2 31.4 31.5 31.6 31.0 31.2 31.2 31.2 31.2 31.2
F2
5 6 9 3 6 1 8 4 5 9 7 4
30.8 30.5 31.0 31.1 31.7 31.2 31.5 31.4 31.4 31.2 31.8 31.5
F3
7 0 6 4 8 9 1 3 9 8 5 8
30.9 30.9 30.9 31.3 30.4 31.7 31.2 31.4 31.5 31.1 31.4 31.6
F4
1 9 3 5 1 4 5 3 8 5 3 5
30.7 30.6 31.1 31.4 31.6 31.6 31.3 31.2 31.4 31.6 31.3 30.9
F5
0 5 5 2 9 8 9 9 2 1 1 6
30.6 30.7 31.6 31.8 31.5 31.1 31.3 31.4 31.5 31.1 31.5 31.1
F6
2 9 6 8 5 6 0 2 0 7 6 3
30.7 30.9 31.8 31.6 31.9 31.8 31.3 31.3 31.2 31.4 31.9 31.4
F7
6 9 0 8 3 1 9 9 2 1 0 5
30.3 30.4 31.4 31.3 31.2 32.1 31.0 31.2 31.0 31.0 32.0 31.7
F8
5 4 8 6 9 3 5 5 7 6 1 1
31.0 30.6 31.3 31.5 31.2 31.5 31.1 31.1 31.1 31.0 31.7 31.6
F9
2 7 3 6 3 7 5 1 3 7 5 5
31.0 30.9 31.3 31.5 31.9 31.6 31.1 31.2 31.2 31.2 31.5 31.6
F10
8 0 4 0 1 0 7 9 7 1 2 2
30.8 30.9 30.6 31.1 31.4 32.0 31.1 31.4 31.2 31.3 32.0 31.3
F11
6 6 4 8 2 7 3 1 0 2 4 5
31.0 30.6 30.6 31.0 31.4 31.6 31.3 31.3 31.4 31.3 31.8 31.4
F12
3 8 2 1 4 5 3 7 1 7 2 7
30.9 30.9 31.3 31.3 31.1 31.6 31.2 31.6 31.2 31.5 31.4 31.3
F13
9 5 8 4 6 6 3 8 4 6 5 6
30.3 30.5 31.6 32.0 31.6 31.9 31.3 31.1 31.3 31.3 31.2 31.4
F14
0 8 4 2 7 1 5 7 8 0 8 3
30.3 30.7 31.2 32.0 31.7 32.4 31.1 31.2 31.3 31.5 31.1 31.3
F15
8 0 0 1 7 0 4 6 7 9 6 1
30.7 30.6 31.3 31.6 31.4 31.1 30.9 31.1 30.7 31.1 31.6 32.0
F16
0 2 0 8 7 6 9 1 8 2 0 0
test(2017003 batch)
Accuracy study 5
PCR mix A PCR mix B
Con 30.7 30.7 31.0 30.9 31.5 30.9 30.5 30.9 31.2 31.0 31.3 31.1
trol 9 9 7 5 9 0 8 3 7 6 3 6
307 31.0 31.3 31.1 31.5 31.4 30.7 31.1 31.2 31.1 31.2 31.2
F1
7 9 6 7 0 3 6 8 7 8 1 4
30.8 31.0 31.2 31.1 31.3 30.7 31.3 31.3 31.4 31.5 31.3 31.4
F2
0 7 0 2 9 8 4 4 2 8 1 0
31.0 31.1 31.0 31.4 31.5 31.6 31.8 31.7 31.2 31.4 31.3 31.3
F3
5 9 0 3 0 1 4 5 9 3 9 4
30.9 31.0 31.6 31.4 31.5 31.3 31.4 31.5 31.6 31.2 31.6 31.1
F4
0 8 3 0 1 8 6 9 2 3 0 7
30.9 31.2 31.5 31.5 31.8 31.5 31.4 31.8 31.6 31.4 31.5 31.3
F5
8 4 1 6 3 9 2 4 2 6 8 8
31.1 31.3 31.4 31.3 31.1 31.6 31.5 31.2 31.4 31.4 31.2 31.2
F6
4 6 3 6 1 8 0 5 6 8 7 5
31.3 31.2 31.3 31.3 31.4 31.4 31.4 31.1 31.3 31.2 31.1 31.4
F7
0 1 4 7 1 8 5 9 3 4 3 2
30.9 30.9 30.9 31.0 31.4 31.2 31.3 31.0 31.2 31.0 31.2 31.4
F8
7 1 9 2 9 6 5 9 3 0 7 8
31.2 31.3 31.0 31.1 31.2 31.5 31.2 31.3 31.3 31.0 31.2 31.3
F9
3 1 1 9 4 8 8 5 3 0 3 0
31.1 31.1 31.5 31.7 31.4 31.3 31.3 31.3 30.6 31.1 31.3 31.2
F10
4 6 1 1 8 9 7 3 1 6 1 0
31.0 31.1 31.2 31.5 31.3 31.3 31.5 31.3 31.2 31.2 31.3 31.3
F11
6 3 8 0 8 8 5 9 4 4 2 4
31.2 31.2 31.4 31.4 31.5 31.6 31.5 31.4 31.3 31.5 31.3 31.5
F12
0 4 4 5 9 5 7 8 0 1 1 7
31.1 31.3 31.9 31.8 31.8 31.5 31.4 31.4 31.3 31.4 31.3 31.7
F13
9 8 0 3 1 3 6 0 6 9 6 0
31.2 31.3 31.5 31.3 31.5 31.1 31.5 31.3 31.2 31.2 31.2 31.6
F14
3 2 9 6 5 6 8 5 2 6 9 1
31.1 31.2 31.8 31.6 31.5 31.6 31.4 31.2 30.0 31.2 31.3 30.7
F15
9 1 1 7 3 5 0 0 7 8 1 2
30.8 31.0 30.6 30.7 30.5 31.5 31.3 31.4 30.9 30.9 31.1 30.9
F16
4 1 3 9 2 0 5 7 2 1 2 8
Conclusion: It can be seen from the experimental results of the above

three batches of reagents that, after extracting nucleic acid with the
recommended sample processing reagents, the test results of various potential
PCR inhibitors/interfering substances in the samples on the ABI7500
Accuracy study 6
instrument were not significantly different from the control samples that do
not contain interfering substances. It shows that potential PCR
inhibitors/interfering substances have no interference effect on the PCR
reaction, and have no effect on the detection of respiratory pathogens in this
kit.
1.2 PCR inhibitor removal ability test
The positive throat swab samples with low concentration of influenza A
virus, influenza B virus, respiratory syncytial virus, adenovirus, Mycoplasma
pneumoniae, and human rhinovirus were used as the test samples. The test
sample was divided into several parts, and each part was added with the
possible PCR inhibitors/interfering substances listed in Table 1-1, and the
detection was carried out according to the operating method in the manual of
this kit. The magnetic bead method was used for nucleic acid extraction and
the test sample without interfering substances was used as the control. The
test results are shown in Table 1-5 to Table 1-7 and Figure 1-7 to Figure 1-12
(only the batch of 2017001 is taken as an example for screenshot display).
Figure 1-7 The test curve of influenza A virus anti-inhibitor/interfering substance

removal effect (2017001 batch)
Figure 1-8 The test curve of influenza B virus anti-inhibitor/interfering substance

Accuracy study 7
Figure 1-9 The test curve of respiratory syncytial virus anti-inhibitor/interfering
substance removal effect (2017001 batch)
Figure 1-10 The test curve of Adenovirus anti-inhibitor/interfering substance removal

effect (2017001 batch)
Figure 1-11 The test curve of Human rhinovirus anti-inhibitor/interfering substance

Accuracy study 8
Figure 1-12 The test curve of Mycoplasma pneumoniae anti-inhibitor/interfering
Table 1-5 Statistical results of Ct value of inhibitor/interfering substance removal
effect test(2017001 batch)
Influenza A Influenza B
RSV Adenovirus MP HRV
virus virus
Test Test Test Test Test Test Test Test Test Test Test Test
1 2 1 2 1 2 1 2 1 2 1 2
Contr
28.04 27.42 30.71 28.95 28.05 27.64 31.88 30.57 31.12 32.09 31.39 30.58
ol
F1 27.02 27.34 30.29 28.98 28.10 27.90 30.63 30.82 31.85 32.08 31.18 30.84
F2 27.27 27.37 29.30 29.11 28.20 28.04 31.05 31.02 31.93 31.71 31.39 30.94
F3 27.28 27.06 29.96 29.38 28.22 27.85 31.32 31.12 31.91 31.71 31.22 31.09
F4 27.44 27.35 29.96 29.19 28.57 27.82 31.45 31.24 31.72 32.58 31.19 31.10
F5 27.32 27.31 30.10 30.02 28.79 28.19 31.01 31.22 31.89 32.11 30.94 31.04
F6 27.38 27.18 30.26 29.29 26.58 27.81 31.24 31.30 31.90 32.09 30.83 31.01
F7 27.81 26.94 30.04 29.37 27.10 28.25 30.91 31.17 31.86 31.73 31.20 31.11
F8 26.78 27.66 31.27 28.47 26.34 26.59 30.60 30.53 31.90 32.40 30.24 30.38
F9 26.71 26.89 28.82 29.22 26.76 26.76 30.76 30.93 31.98 32.06 30.76 30.78
F10 27.54 27.52 29.09 29.26 27.49 26.96 30.94 31.10 32.19 32.36 30.84 30.75
F11 27.33 26.64 28.88 29.21 28.12 27.31 31.27 31.45 32.04 31.45 31.06 30.91
F12 26.34 26.64 29.21 28.16 28.11 27.06 31.31 31.41 32.23 32.28 31.07 31.16
F13 26.53 26.70 28.53 29.08 27.83 27.65 30.86 31.32 32.00 32.21 30.86 31.04
F14 25.83 26.98 28.29 28.22 28.24 26.58 31.30 31.24 32.83 32.16 31.30 31.01
F15 26.96 27.07 29.64 28.97 28.32 26.33 30.93 31.22 32.10 31.81 30.65 30.76
F16 26.82 28.50 29.68 29.67 27.37 26.27 30.42 30.66 32.19 32.38 30.90 30.73

virus virus
1 2 1 2 1 2 1 2 1 2 1 2
Contr
26.46 26.53 29.70 29.38 27.14 26.89 29.73 29.92 31.50 32.07 30.81 30.11
ol
Accuracy study 9
F1 26.66 27.13 29.61 29.65 27.20 27.45 30.09 30.02 31.53 31.67 30.18 30.15
F2 27.19 27.65 29.67 29.91 27.39 27.33 30.26 30.38 31.80 31.96 30.54 30.53
F3 27.41 27.54 29.31 29.29 27.57 27.60 30.54 30.41 31.57 31.13 30.44 30.33
F4 27.54 27.67 29.59 29.25 27.65 27.72 30.16 31.02 31.85 31.91 30.62 30.30
F5 27.44 27.63 29.51 29.61 27.93 27.65 30.27 30.27 31.57 31.75 29.85 30.63
F6 27.31 27.15 29.16 29.51 27.67 27.50 30.29 30.25 31.37 31.70 30.37 30.23
F7 27.32 27.53 28.98 29.43 27.49 27.65 30.04 30.30 31.60 31.38 30.31 30.69
F8 27.34 27.40 29.44 29.07 27.20 27.03 29.88 30.19 31.79 31.94 30.08 30.11
F9 27.26 27.48 29.73 29.47 27.53 27.23 30.19 30.24 32.35 31.43 30.00 30.37
F10 27.41 27.62 29.61 29.39 27.31 27.30 30.39 30.27 31.35 31.22 30.38 30.57
F11 27.35 27.40 29.30 28.75 27.67 27.34 30.54 30.56 31.66 32.08 30.30 30.56
F12 27.59 27.42 29.40 29.63 27.43 27.59 30.53 30.80 32.12 31.07 30.30 30.64
F13 27.72 27.56 29.71 29.57 27.75 27.83 30.31 30.44 32.04 31.83 30.35 30.36
F14 27.43 27.43 29.48 29.20 27.77 27.33 30.29 30.31 31.73 31.90 30.45 30.66
F15 27.16 27.37 29.67 29.37 27.43 27.46 29.90 30.24 31.35 31.54 30.25 29.97
F16 27.50 27.45 29.46 29.21 26.77 26.98 30.07 30.14 31.82 31.50 30.07 30.01

virus virus
1 2 1 2 1 2 1 2 1 2 1 2
Contr
26.79 26.78 29.17 29.22 26.90 27.20 29.95 30.11 31.19 30.66 31.10 30.15
ol
F1 26.85 27.08 29.39 29.41 27.16 27.35 30.48 30.31 30.48 30.73 30.41 30.64
F2 27.24 27.23 28.35 29.58 27.27 27.62 30.26 30.47 30.24 30.62 30.46 30.43
F3 27.32 27.24 29.61 29.16 27.52 27.67 30.80 30.80 30.61 30.31 30.77 30.67
F4 27.64 27.22 29.52 29.36 27.67 27.86 30.58 30.78 30.94 30.76 30.36 31.50
F5 27.29 27.36 29.46 29.83 27.91 27.83 30.67 30.60 30.84 30.35 30.51 30.41
F6 27.57 27.17 29.50 29.47 27.47 27.41 30.39 30.67 31.25 31.03 29.66 30.22
F7 26.98 27.35 29.27 30.02 27.51 27.80 30.34 30.53 30.70 30.87 30.43 30.26
F8 27.00 27.14 28.94 29.49 27.65 27.63 30.29 30.10 30.30 30.29 30.24 30.22
F9 27.23 27.17 29.23 29.39 27.12 27.76 30.58 30.63 30.56 30.85 30.14 30.32
F10 27.56 27.10 29.47 29.41 27.53 27.75 30.49 30.72 30.90 30.32 30.13 30.45
F11 27.13 27.30 30.01 29.47 27.66 27.69 30.69 30.84 30.79 30.26 30.53 30.53
F12 27.63 27.48 29.78 29.59 27.81 27.33 30.81 30.89 31.16 30.84 30.39 30.49
F13 27.18 27.57 29.48 29.83 27.82 27.56 30.56 31.00 30.86 30.35 30.29 30.61
F14 27.60 27.41 29.29 29.61 27.68 27.40 30.62 30.55 30.45 30.49 29.73 30.27
F15 27.29 27.24 29.25 29.78 27.48 27.26 30.19 30.46 30.50 30.65 30.25 30.28
F16 27.09 27.06 29.10 29.44 27.60 27.04 30.09 30.37 31.10 29.41 30.06 30.22
Conclusion: It can be seen from the above experimental results that, by

using the clinically positive throat swab sample as the test sample, the test
results of samples with different concentrations of various PCR
inhibitors/interfering substances have no significant differences after nucleic
acid extraction via the magnetic bead method. It shows that the magnetic
bead method is beneficial for removing PCR inhibitors/interfering substances.
Accuracy study 10
2. Measurement accuracy
2.1 Test of reference samples
The reference samples P1-P12 required in the technical requirements of
the six respiratory pathogens nucleic acid diagnostic kit (PCR-fluorescence
probing) were used as the test samples. Reference samples P1-P12 consist of
12 positive samples for the six inactivated respiratory pathogens with
different concentrations. Among them, P1 is influenza A/H1N1 (2009) virus,
P2 is influenza A H3 subtype virus, P3 is influenza A H5 subtype virus, P4 is
influenza A H7N9 subtype virus, and P5 is influenza B virus Victoria subtype,
P6 is influenza B virus Yamagata subtype, P7 is respiratory syncytial virus, P8
is adenovirus 3 subtype, P9 is adenovirus 4 subtype, P10 is adenovirus 7
subtype, P11 is Mycoplasma pneumoniae, and P12 is human Rhinovirus. Each
reference sample is diluted with Hanks virus preservation solution (pH
7.2-7.6) or normal saline. 3 batches of qualified kits were used to detect the
samples on the same ABI 7500 instrument, and each sample were repeatedly
tested 3 times to evaluate the measurement accuracy of this kit.
Test results are as follows:
Figure1-13 Amplification curve of reference samples P1-P12 by using PCR mix A

(2017001 batch)

(2017002 batch)
Accuracy study 11
(2017003 batch)
Figure1-16 Amplification curve of reference samples P1-P12 by using PCR mix B

(2017001 batch)

(2017002 batch)
Accuracy study 12
(2017003 batch)
Table 1-8 Test results of reference samples P1-P12(2017001 batch)
Test results PCR mix A PCR mix B
Reference STDEV
sample Test1 Test2 Test3 Test1 Test2 Test3
P1 24.52 23.37 23.73 No Ct No Ct No Ct 0.59
P2 27.16 27.21 27.06 No Ct No Ct No Ct 0.08
P3 19.80 19.83 19.88 No Ct No Ct No Ct 0.04
P4 29.66 29.68 29.67 No Ct No Ct No Ct 0.01
P5 30.32 29.96 29.80 No Ct No Ct No Ct 0.27
P6 34.02 33.72 34.06 No Ct No Ct No Ct 0.19
P7 28.23 28.36 28.31 No Ct No Ct No Ct 0.07
P8 No Ct No Ct No Ct 21.41 21.24 21.79 0.28
P9 No Ct No Ct No Ct 25.48 25.74 26.02 0.27
P10 No Ct No Ct No Ct 28.35 28.33 28.26 0.05
P11 No Ct No Ct No Ct 24.57 24.69 24.60 0.06
P12 No Ct No Ct No Ct 32.30 32.50 32.30 0.12
Reference STDEV
P1 24.48 23.63 23.62 No Ct No Ct No Ct 0.49
P2 27.15 27.21 27.07 No Ct No Ct No Ct 0.07
P3 19.82 19.62 19.49 No Ct No Ct No Ct 0.17
P4 29.87 29.57 29.59 No Ct No Ct No Ct 0.17
P5 29.90 30.28 30.13 No Ct No Ct No Ct 0.19
P6 34.15 34.15 34.36 No Ct No Ct No Ct 0.12
P7 28.27 28.19 28.39 No Ct No Ct No Ct 0.10
P8 No Ct No Ct No Ct 21.24 21.14 21.50 0.19
P9 No Ct No Ct No Ct 25.57 25.77 25.76 0.11
P10 No Ct No Ct No Ct 28.32 28.36 28.27 0.05
P11 No Ct No Ct No Ct 24.43 24.52 24.27 0.13
P12 No Ct No Ct No Ct 32.16 32.15 32.14 0.01
Accuracy study 13
Reference STDEV
P1 24.52 23.34 23.64 No Ct No Ct No Ct 0.61
P2 27.19 27.32 27.22 No Ct No Ct No Ct 0.07
P3 19.96 19.51 19.54 No Ct No Ct No Ct 0.25
P4 29.65 29.76 29.81 No Ct No Ct No Ct 0.08
P5 30.01 30.00 30.16 No Ct No Ct No Ct 0.09
P6 33.84 33.97 33.80 No Ct No Ct No Ct 0.09
P7 28.15 28.24 28.20 No Ct No Ct No Ct 0.05
P8 No Ct No Ct No Ct 21.16 21.19 20.78 0.23
P9 No Ct No Ct No Ct 25.60 25.71 25.83 0.12
P10 No Ct No Ct No Ct 28.53 28.43 28.32 0.11
P11 No Ct No Ct No Ct 24.60 24.56 24.14 0.25
P12 No Ct No Ct No Ct 31.70 32.03 31.83 0.17
Conclusion: It can be seen from the above that, the test results of reference
samples P1-P12 by using the 3 batches of reagents (2017001, 2017002, 2017003)
were all positive, and there was no non-specific amplification. The standard
deviation (STDEV) of the Ct values of each sample is between 0.01 and 0.61.
Therefore, this kit has good detection accuracy.
2.2 Test of clinical samples

The positive throat swab samples for influenza A virus, influenza B virus,
respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae and human
rhinovirus, and negative throat swab sample from people with normal
physical examination were used as test samples. 8 positive samples with each
pathogen and negative samples were tested on the same ABI 7500 instrument
with 3 batches of qualified kits to evaluate the measurement accuracy of this
kit.
Figure 1-19 The test results of samples for influenza A virus, influenza B virus and
respiratory syncytial virus by using PCR mix A (2017001 batch)
Accuracy study 14
respiratory syncytial virus by using PCR mix B (2017001 batch)
Accuracy study 15
Figure 1-25 The test results of samples for Adenovirus, Mycoplasma pneumoniae and
Human rhinovirus by using PCR mix A (2017001 batch)
Accuracy study 16
Human rhinovirus by using PCR mix B (2017001 batch)
Accuracy study 17
Figure 1-31 The test results of negative samples by using PCR mix A (2017001 batch)
Accuracy study 18
Figure 1-32 The test results of negative samples by using PCR mix B (2017001 batch)
Accuracy study 19
Table 1-11 Test results of samples for influenza A virus
Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
PCR mix A 26.77 20.97 18.84 29.08 32.12 23.92 25.64 22.78
2017001
PCR mix B No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
PCR mix A 26.75 21.06 19.41 30.17 33.47 25.21 27.02 23.86
2017002
PCR mix A 26.58 20.98 18.23 29.31 32.91 24.44 26.23 22.67
2017003
Table 1-12 Test results of samples for influenza B virus

Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
PCR mix A 29.71 24.38 23.06 30.95 26.45 32.83 20.07 28.04
2017001
PCR mix A 29.58 24.34 23.19 30.99 26.49 33.30 19.58 27.85
2017002
2017003 PCR mix A 29.62 24.53 23.02 30.86 27.10 33.89 19.85 28.00
Accuracy study 20
Table 1-13 Test results of samples for respiratory syncytial virus

Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
PCR mix A 25.76 28.20 21.00 24.57 29.01 15.00 31.38 26.06
2017001
PCR mix A 25.82 29.01 21.77 25.45 30.01 16.69 32.52 26.94
2017002
PCR mix A 25.16 28.70 21.17 25.49 29.54 15.98 32.02 26.33
2017003
Table 1-14 Test results of samples for Adenovirus

Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
PCR mix A No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
2017001
PCR mix B 22.69 26.78 31.06 32.99 23.06 21.16 19.21 27.81
2017002
PCR mix B 22.53 26.71 30.08 34.65 23.71 21.32 19.34 27.98
2017003
PCR mix B 21.58 26.79 29.84 33.70 23.28 21.39 19.44 27.86
Table 1-15 Test results of samples for Mycoplasma pneumoniae

Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
2017001
PCR mix B 23.01 26.25 21.21 29.09 31.00 24.71 26.94 30.50
2017002
PCR mix B 23.06 26.11 21.02 29.08 31.49 25.16 27.35 30.51
2017003
PCR mix B 23.48 26.71 21.34 30.09 31.96 25.18 27.77 30.60
Table 1-16 Test results of samples for Human rhinovirus

Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
2017001
PCR mix B 20.99 24.96 26.80 31.60 32.78 23.15 28.58 20.44
2017002
PCR mix B 21.31 24.50 26.25 31.81 32.81 22.71 28.11 18.67
2017003
PCR mix B 20.91 24.47 26.31 31.98 32.30 22.53 28.04 18.72
Table 1-17 Test results of negative samples (No amplification curve was observed for
Accuracy study 21
FAM, HEX and CY5, only the Ct value of internal standard was collected)
Ct value of internal standard
Batch Reaction mix
1 2 3 4 5 6 7 8
PCR mix A 27.13 27.27 27.06 26.87 27.04 26.85 27.67 27.56
2017001
PCR mix B 28.02 27.49 26.83 26.85 27.16 27.20 27.24 27.87
PCR mix A 27.97 27.46 27.37 27.52 27.88 27.51 27.44 27.78
2017002
PCR mix B 27.55 27.24 27.54 27.56 28.29 28.67 28.77 28.91
PCR mix A 27.27 27.73 27.43 27.92 27.50 27.76 27.54 27.41
2017003
PCR mix B 27.46 27.67 27.43 27.57 27.67 27.52 27.57 27.67
Conclusion: It can be seen from the above that, the test results of positive
throat swab samples for influenza A virus, influenza B virus, Respiratory
syncytial virus, Adenovirus, Mycoplasma pneumoniae and Human
rhinovirus by using the 3 batches of reagents (2017001, 2017002, 2017003)
were all positive, and there was no non-specific amplification. Negative
samples were all negative, and the test results of internal standard were all
within the range of reference value. Therefore, this kit has good detection
accuracy.
2.3 Test of national standard samples

The positive reference samples PC01~PC06 in the national reference
samples (batch number 370006-201501) for influenza A/B virus nucleic acid
detection reagent were used as test samples. Samples were tested on the same
ABI 7500 instrument with 3 batches of qualified kits to evaluate the
measurement accuracy of this kit.
Figure 1-37 Test results of PC01 and PC02 by using PCR mix A (2017001 batch)
Accuracy study 22
Figure 1-38 Test results of PC01 and PC02 by using PCR mix B (2017001 batch)
Figure 1-39 Test results of PC03—PC06 by using PCR mix A (2017001 batch)
Accuracy study 23
Figure 1-40 Test results of PC03—PC06 by using PCR mix B (2017001 batch)
Accuracy study 24
Accuracy study 25
Accuracy study 26
Accuracy study 27
Table 1-18 Test results of national reference samples PC01~PC06 (2017001 batch)
Reference
samples Test1 Test2 Test1 Test2
PC01 32.03 31.77 No Ct No Ct
PC02 31.27 30.76 No Ct No Ct
PC03 30.47 30.24 No Ct No Ct
PC04 29.39 29.34 No Ct No Ct
PC05 29.38 29.36 No Ct No Ct
PC06 29.23 29.09 No Ct No Ct
Reference
PC01 30.93 30.89 No Ct No Ct
PC02 31.30 30.72 No Ct No Ct
PC03 30.13 29.65 No Ct No Ct
PC04 30.50 30.17 No Ct No Ct
PC05 29.53 29.39 No Ct No Ct
PC06 28.79 28.74 No Ct No Ct
Reference
PC01 30.93 31.26 No Ct No Ct
PC02 31.33 31.17 No Ct No Ct
PC03 29.60 29.38 No Ct No Ct
PC04 30.08 29.73 No Ct No Ct
Accuracy study 28
PC05 29.29 29.02 No Ct No Ct
PC06 28.53 28.44 No Ct No Ct
Conclusion: It can be seen from the above that, when testing the positive
reference samples PC01~PC06 in the national reference samples of influenza
A/B virus nucleic acid detection reagent by using the 3 batches of reagents
(2017001, 2017002, 2017003), PC01 and PC02 were all positive with influenza B
virus, PC03—PC06 were all positive with influenza A virus, and there was no
non-specific amplification. The results met the requirements of national
reference sample test for influenza A/B virus nucleic acid detection reagents.
Analytical performance evaluation on Life Technologies

QuantStudioTM 5
Since the Life Technologies QuantStudioTM 5 fluorescent PCR instrument
and the ABI 7500 fluorescent PCR instrument are both ABI fluorescent PCR
instruments, and their structural principles are basically the same. So it is
believed that the reagent performance of this kit on the Life Technologies
QuantStudio 5 fluorescent PCR instrument is the same as that on the ABI 7500
instrument. Therefore, the analytical performance evaluation of this kit on the
Life Technologies QuantStudioTM 5 fluorescent PCR instrument is omitted.
Analytical performance evaluation on SLAN-96P PCR

instrument
1. Interferences analysis
1.1 Anti-PCR inhibitor/interfering substance effect
The main purposes of extracting pathogen DNA/RNA are as follows:
enriching the concentration of target nucleic acid sequences, ensuring the
integrity of the target nucleic acid sequence, increasing the uniformity of the
PCR template solution, and removing PCR inhibitors. It is one of the factors
that determine the success of RT-PCR. For example, under the conditions of
low pH and high salt concentration, the modified superparamagnetic
nanoparticles can specifically adsorb viral nucleic acids, and then pure nucleic
acids can be enriched after the following washing steps. Theoretically, the
magnetic bead method can remove PCR inhibitors/interfering substances well
when extracting nucleic acid. But in actual operation, there are many
unforeseen unknown factors, so we need to verify this via experiments. In
addition, the composition of the PCR reaction system also determines the
influence of inhibitors/interfering substances on the PCR reaction.
Appropriate PCR buffer, pH, and the concentration of appropriate dNTPs,
enzymes and other raw materials can reduce the influence of
inhibitors/interfering substances on the PCR reaction. Therefore, it is
necessary for us to experiment to verify this point.
The lentiviral particles containing the target conserved sequence of
rhinovirus, and the cloning plasmid containing the target conserved sequence
of adenovirus and Mycoplasma pneumoniae were used as the research objects.
The lentiviral particles and cloned plasmids of each pathogen were diluted
with normal saline to a concentration of 7.0 × 103 TU/mL (and copies/mL), and
Accuracy study 29
mixed with equal volumes to form the test sample. The test sample was
divided into several parts, and the possible PCR inhibitors/interfering
substances listed in Table 2-1 were added to each part, and the detection was
carried out according to the operating method in the manual of this kit. The
test sample without interfering substances was used as the control. The test
results of three batches of the kit on the SLAN-96P PCR instrument are shown
in Table 2-2 to Table 2-4 and Figure 2-1 to Figure 2-6 (only the batch of 2017001
is taken as an example for screenshot display).
Table 2-1 PCR inhibitor/interfering substance added and its concentration
Sample Final Sample Final

Inhibitor name Inhibitor name
No. concentration No. concentration
Oxymetazoline
hydrochloride
Cefmenoxime
Hydrochloride
F5 Zanamivir 100 μg/mL F13 Purified Mucin 20 μg/mL
F7 Azithromycin 100 μg/mL F15 Absolute ethanol 20%(v/v)
Human whole
blood
Figure 2-1 The test curve of influenza A virus anti-inhibitor/interfering substance effect
(2017001 batch)
Accuracy study 30
Figure 2-2 The test curve of influenza B virus anti-inhibitor/interfering substance effect
(2017001 batch)
Figure 2-3 The effect test curve of respiratory syncytial virus anti-inhibitor/interfering
Figure 2-4 The effect test curve of Adenovirus anti-inhibitor/interfering substance

(2017001 batch)
Accuracy study 31
Figure 2-5 The effect test curve of Human rhinovirus anti-inhibitor/interfering substance
(2017001 batch)
Figure 2-6 The effect test curve of Mycoplasma pneumoniae anti-inhibitor/interfering


test (2017001 batch)
PCR mix A PCR mix B

Cont 29.9 30.2 31.5 31.7 31.3 31.0 29.0 29.0 30.5 30.8 31.6 31.1
rol 6 4 7 1 3 7 4 2 0 1 6 5
30.0 30.2 31.6 31.8 31.2 31.6 28.9 28.8 30.7 30.6 31.4 31.6
F1
6 5 9 0 1 0 6 2 6 8 7 0
30.0 30.0 31.6 31.6 31.3 31.3 28.9 28.8 33.6 30.7 31.4 31.3
F2
9 8 3 0 6 9 5 8 6 9 5 9
30.1 30.0 31.2 31.4 31.1 31.2 28.9 28.7 30.7 30.6 31.8 31.2
F3
5 9 4 9 2 3 1 6 5 2 1 8
30.3 30.2 31.5 31.7 31.4 31.1 28.9 28.7 30.9 30.8 31.3 31.5
F4
3 8 1 0 5 5 0 4 7 2 6 9
30.3 30.1 31.4 31.6 30.8 31.2 28.9 28.8 30.8 30.9 31.5 31.7
F5
8 8 0 7 7 9 7 0 2 7 4 1
Accuracy study 32
30.2 30.1 31.2 31.4 30.8 31.0 28.9 28.9 30.7 30.5 31.1 31.7
F6
9 5 0 7 2 8 1 1 5 5 3 4
30.2 30.1 31.3 31.5 31.0 30.0 29.0 28.0 30.7 30.6 31.1 31.1
F7
0 0 9 2 7 8 2 5 9 9 6 8
30.1 30.1 31.2 31.5 31.4 31.3 28.9 28.9 30.8 30.8 31.4 31.3
F8
0 9 8 1 9 0 8 1 8 1 1 4
30.0 30.3 31.6 31.3 31.2 31.5 28.4 28.6 30.6 30.7 31.3 31.4
F9
3 0 1 0 9 7 5 7 1 8 5 2
30.2 30.1 31.6 31.5 31.2 31.5 29.0 28.8 30.8 30.8 31.5 31.5
F10
7 8 6 8 9 1 0 7 1 3 4 4
30.3 30.2 31.4 31.6 31.3 31.3 28.9 28.9 30.6 30.8 31.5 31.4
F11
4 8 4 3 3 5 3 8 5 1 8 8
30.4 30.4 31.3 31.5 31.7 31.4 28.9 28.5 30.8 30.7 31.5 31.4
F12
0 3 1 5 0 9 9 4 3 5 7 6
30.3 30.4 31.6 31.6 30.8 31.0 28.8 28.7 30.7 30.7 31.4 31.5
F13
1 5 1 8 8 1 0 8 7 6 3 0
30.4 30.3 31.7 31.6 30.9 31.1 28.9 29.0 30.7 30.8 31.6 31.6
F14
9 0 8 6 4 5 6 7 0 6 4 1
30.3 30.3 31.1 31.7 30.8 31.0 28.8 28.9 30.6 30.7 30.9 31.2
F15
1 0 5 2 8 1 4 1 0 4 5 3
30.4 30.1 31.6 31.4 31.2 30.9 28.8 28.8 30.7 30.8 31.5 31.3
F16
4 7 0 6 6 1 9 6 4 7 5 3

test(2017002 batch)
PCR mix A PCR mix B
Cont 28.9 29.1 31.0 30.3 30.6 30.9 28.0 28.0 31.0 31.2 31.8 31.6
rol 1 9 6 7 0 5 8 5 4 2 1 6
29.0 29.1 30.9 31.0 30.2 31.2 28.0 28.0 31.1 31.1 32.0 31.7
F1
0 0 7 4 4 1 7 5 7 3 8 1
28.7 28.8 31.1 31.2 30.6 31.2 28.0 28.0 31.2 31.0 32.3 32.6
F2
0 7 4 2 2 1 4 6 8 5 7 2
28.8 28.8 31.0 31.0 30.9 31.2 28.0 28.1 31.1 31.1 32.1 31.9
F3
9 8 6 3 2 4 4 3 8 5 3 7
29.0 28.8 30.9 30.8 31.1 31.3 28.1 28.0 30.9 31.2 32.1 31.8
F4
3 5 9 4 6 2 6 9 6 1 5 5
29.0 28.8 30.9 30.9 30.7 31.3 28.1 28.0 30.9 31.2 31.9 31.9
F5
0 3 5 5 5 1 7 8 5 0 6 9
28.8 29.0 30.8 30.8 30.8 31.4 28.1 28.1 30.9 31.2 32.2 32.3
F6
8 0 4 9 8 3 2 1 6 0 1 5
28.9 28.9 31.0 31.2 30.9 31.5 28.0 28.3 30.9 31.0 32.0 31.9
F7
0 5 5 6 5 1 4 0 6 7 3 7
28.9 28.8 30.8 30.9 31.0 31.1 27.9 28.0 31.0 31.1 32.0 31.6
F8
8 7 3 5 2 3 3 0 0 6 6 4
F9 28.8 28.8 31.0 30.9 31.3 31.2 28.0 27.9 31.1 31.0 32.2 32.2
Accuracy study 33
8 4 0 6 0 3 8 8 0 3 7 0
28.9 28.8 31.0 30.7 31.3 31.3 28.0 27.9 31.1 30.3 32.1 31.9
F10
0 7 5 8 0 9 0 9 6 2 3 1
28.7 28.9 31.0 31.0 31.0 31.0 28.1 28.0 31.1 31.1 32.1 31.9
F11
9 4 6 4 3 6 1 1 3 9 6 6
29.0 28.8 31.0 31.2 31.1 31.0 28.0 28.0 30.9 31.0 32.1 32.3
F12
3 8 9 8 7 5 6 4 2 6 9 7
28.8 28.8 31.0 31.1 30.8 31.0 28.1 28.0 31.0 31.1 31.7 31.8
F13
0 5 1 2 7 9 3 2 2 3 6 5
29.0 28.8 31.0 31.0 30.8 31.3 28.0 28.0 31.1 31.0 31.9 31.7
F14
6 0 7 5 8 7 2 2 7 2 8 5
28.9 28.8 31.1 31.2 30.7 31.6 27.9 28.0 31.0 31.1 32.1 32.1
F15
3 8 0 3 3 5 4 4 3 8 9 8
28.9 28.9 30.8 31.0 31.2 31.1 28.0 28.1 31.2 31.0 32.2 32.1
F16
3 7 9 8 4 4 0 7 0 5 6 4

test(2017003 batch)
PCR mix A PCR mix B
Cont 28.6 28.5 31.1 31.0 31.3 31.1 28.1 28.1 30.6 30.3 31.4 31.7
rol 2 5 0 2 0 7 2 0 7 8 3 3
28.5 28.5 30.2 31.1 31.1 31.4 28.1 28.1 30.4 30.4 31.8 31.6
F1
2 4 4 1 2 6 4 7 9 6 0 1
28.5 28.5 31.1 31.3 31.6 31.3 28.0 27.9 30.3 30.5 32.2 31.4
F2
5 8 7 0 2 0 4 4 0 8 5 8
28.6 28.5 31.0 31.1 31.1 31.6 28.0 28.1 30.2 30.4 31.8 31.8
F3
9 9 4 6 7 4 7 0 8 9 8 4
28.6 28.5 29.6 30.9 31.3 31.1 28.0 28.1 30.5 30.5 31.7 31.7
F4
0 8 7 4 9 2 5 8 8 7 2 8
28.6 28.6 31.0 30.9 30.7 31.0 28.2 28.1 30.5 30.5 31.4 31.6
F5
1 7 2 4 0 7 5 3 1 9 1 2
28.6 28.6 31.0 31.1 31.0 31.3 28.1 28.1 30.7 30.6 31.5 31.5
F6
8 8 8 3 5 8 5 5 7 1 4 6
28.7 28.7 30.9 31.0 31.1 31.4 28.1 28.1 30.7 30.4 31.3 31.7
F7
5 3 7 2 8 4 9 6 1 4 0 6
28.8 28.6 31.1 31.1 31.2 31.2 28.0 28.0 30.5 30.6 31.3 31.3
F8
2 4 3 3 9 5 8 9 8 2 4 2
28.4 28.6 31.1 30.9 31.2 31.1 28.1 28.1 30.5 30.5 31.8 31.4
F9
6 8 3 9 9 0 4 4 7 2 6 9
28.5 28.7 31.0 31.1 31.1 31.2 28.1 28.0 30.6 30.6 31.5 31.6
F10
0 9 3 2 4 9 5 7 3 0 8 7
28.4 28.7 31.1 31.0 31.2 31.2 28.1 28.0 30.5 30.6 31.5 31.6
F11
5 7 9 6 9 9 0 6 8 1 6 7
28.7 28.6 31.1 31.0 31.0 31.3 28.1 28.2 30.5 30.5 31.7 32.0
F12
2 9 1 8 4 0 0 5 1 3 0 5
Accuracy study 34
28.7 28.7 30.9 31.0 31.2 31.1 28.1 28.0 30.6 30.8 31.6 31.7
F13
9 9 6 5 0 0 4 8 1 4 3 3
28.7 28.6 31.1 31.2 30.9 31.3 28.1 28.2 30.6 31.2 31.6 31.6
F14
1 6 5 1 7 1 1 7 0 3 6 9
28.8 28.8 31.3 31.2 31.0 31.0 28.0 28.0 30.7 31.0 32.0 31.5
F15
7 3 3 3 8 9 6 9 3 8 3 5
28.6 28.6 31.1 30.9 31.3 30.8 28.0 28.8 30.5 30.4 32.1 31.7
F16
5 3 4 6 1 8 7 7 1 7 1 6
Conclusion: It can be seen from the experimental results of the above

three batches of reagents that, after extracting nucleic acid with the
recommended sample processing reagents, the test results of various potential
PCR inhibitors/interfering substances in the samples on the SLAN-96P
instrument were not significantly different from the control samples that do
not contain interfering substances. It shows that potential PCR
inhibitors/interfering substances have no interference effect on the PCR
reaction, and have no effect on the detection of respiratory pathogens in this
kit.
1.2 PCR inhibitor removal ability test
The positive throat swab samples with low concentration of influenza A
virus, influenza B virus, respiratory syncytial virus, adenovirus, Mycoplasma
pneumoniae, and human rhinovirus were used as the test samples. The test
sample was divided into several parts, and each part was added with the
possible PCR inhibitors/interfering substances listed in Table 2-1, and the
detection was carried out according to the operating method in the manual of
this kit. The magnetic bead method was used for nucleic acid extraction and
the test sample without interfering substances was used as the control. The
test results are shown in Table 2-5 to Table 2-7 and Figure 2-7 to Figure 2-12
(only the amplification curve of 2017001 batch is screenshot and displayed).
Figure 2-7 The test curve of influenza A virus anti-inhibitor/interfering substance

Accuracy study 35
Figure 2-8 The test curve of influenza B virus anti-inhibitor/interfering substance
Figure 2-9 The test curve of respiratory syncytial virus anti-inhibitor/interfering

Figure 2-10 The test curve of Adenovirus anti-inhibitor/interfering substance removal

effect (2017001 batch)
Accuracy study 36
Figure 2-11 The test curve of Mycoplasma pneumoniae anti-inhibitor/interfering
Figure 2-12 The test curve of Human rhinovirus anti-inhibitor/interfering substance

virus virus
1 2 1 2 1 2 1 2 1 2 1 2
Contr
28.33 28.42 30.98 31.00 30.08 30.20 29.94 29.90 32.90 32.19 31.18 31.06
ol
F1 28.28 28.39 31.00 30.92 30.44 30.21 29.89 29.90 32.49 32.40 31.00 30.91
F2 28.31 28.22 31.25 30.98 30.12 29.95 29.91 29.95 32.45 32.82 30.72 30.88
F3 28.26 28.25 31.02 31.21 30.20 30.19 29.90 29.90 32.47 32.52 30.81 30.89
F4 28.32 28.30 30.50 30.96 30.22 30.16 29.95 30.05 32.66 32.25 30.71 30.99
F5 28.33 28.23 31.04 31.05 30.30 30.15 29.97 29.96 33.03 32.36 31.02 30.91
F6 28.30 28.29 30.90 31.05 30.38 30.43 29.93 29.94 33.04 32.55 30.92 30.96
F7 28.15 28.26 31.04 30.92 30.36 30.14 29.89 29.96 33.14 32.99 30.84 30.89
F8 28.39 28.41 31.00 31.04 30.13 30.07 29.61 29.81 32.17 32.28 30.88 30.82
F9 28.34 28.35 30.90 31.18 30.13 30.27 29.82 29.95 32.44 32.26 30.72 30.92
F10 28.27 28.35 31.09 30.94 30.23 30.19 29.94 29.96 32.52 32.60 30.94 31.02
F11 28.30 28.35 31.07 31.09 30.35 30.26 29.90 30.00 32.62 32.55 30.82 31.01
F12 28.41 28.49 30.93 30.97 30.33 30.27 29.97 29.85 32.78 32.75 30.88 30.89
Accuracy study 37
F13 28.45 28.49 31.13 30.97 30.22 30.14 30.08 29.94 33.10 32.75 31.03 31.00
F14 28.38 28.37 30.94 30.95 30.39 30.30 30.6 30.03 32.61 33.33 30.89 31.15
F15 28.31 28.50 31.12 30.99 30.16 30.35 30.12 29.84 32.57 32.64 30.86 30.94
F16 28.40 28.41 31.07 31.01 30.36 30.12 30.00 29.85 32.91 31.96 30.88 30.98

virus virus
1 2 1 2 1 2 1 2 1 2 1 2
Contr
28.13 28.28 31.08 31.11 29.87 30.24 29.96 29.52 31.65 32.24 30.02 30.10
ol
F1 28.14 28.15 31.02 31.13 30.30 30.27 30.11 29.88 31.62 32.42 30.17 30.18
F2 28.26 28.24 30.89 31.15 30.17 30.10 30.10 30.09 31.94 32.64 30.36 30.18
F3 28.59 25.54 30.93 31.03 30.33 30.11 29.99 30.14 31.89 32.04 30.11 30.13
F4 28.40 28.28 31.07 30.89 30.67 30.83 29.91 30.05 32.08 32.23 30.21 30.11
F5 28.34 28.38 30.96 31.10 30.15 30.03 29.98 29.89 32.02 32.16 30.00 29.93
F6 28.18 28.32 30.87 31.07 30.37 30.14 29.94 30.14 32.63 31.61 30.12 30.14
F7 28.16 28.21 31.13 31.41 30.36 30.10 30.05 29.90 31.94 31.93 30.01 29.89
F8 28.25 28.27 31.40 31.07 30.20 30.21 29.93 29.94 31.37 31.55 30.31 30.09
F9 28.21 28.42 31.07 31.00 30.33 30.27 29.97 29.81 31.72 31.69 30.14 30.37
F10 28.59 28.55 31.18 31.10 29.45 30.49 29.96 29.95 32.04 32.43 30.29 30.10
F11 28.42 28.36 31.22 31.04 30.27 30.20 29.93 29.98 31.65 31.66 29.94 30.40
F12 28.50 28.43 31.19 32.11 30.32 30.20 29.89 30.07 31.75 32.70 30.30 30.13
F13 28.45 28.53 31.92 30.70 30.31 30.22 29.89 30.00 31.95 32.04 30.30 28.46
F14 28.33 28.56 31.13 31.07 30.58 30.69 29.93 29.91 31.78 32.08 30.10 30.27
F15 28.31 28.18 31.02 30.98 30.14 30.25 29.91 30.02 31.81 31.59 30.06 30.11
F16 28.14 28.14 31.12 31.23 30.20 30.25 30.07 30.24 32.24 31.61 30.46 30.26

effect test (2017003 batch)
virus virus
1 2 1 2 1 2 1 2 1 2 1 2
Contr
28.28 28.23 30.88 31.03 30.33 30.52 29.83 29.81 31.55 32.34 30.38 30.02
ol
F1 28.31 28.31 30.91 30.98 30.80 30.67 29.79 29.87 31.89 32.28 30.05 30.23
F2 28.21 28.16 31.05 31.22 30.83 30.86 29.83 29.83 32.46 32.40 30.31 30.09
F3 28.14 28.08 30.90 31.03 30.97 30.58 29.98 29.98 31.66 32.11 30.10 30.08
F4 28.14 28.06 31.25 31.32 30.55 30.52 29.97 29.96 32.70 31.87 30.05 30.18
F5 28.22 28.12 31.16 30.98 30.55 30.16 29.70 29.82 32.04 33.35 30.20 30.14
F6 28.16 28.18 30.92 31.26 30.79 30.67 29.63 29.82 32.08 32.88 30.21 30.12
F7 28.17 28.10 31.01 31.01 30.38 30.62 29.69 29.96 31.59 32.93 30.25 30.06
Accuracy study 38
F8 28.14 28.12 31.18 31.07 30.41 30.35 30.01 29.96 31.61 31.87 30.11 30.00
F9 28.26 29.00 30.94 31.27 30.41 30.72 29.99 30.13 31.83 31.44 29.86 30.48
F10 28.20 28.16 31.03 31.27 30.53 30.49 30.11 29.93 31.82 31.96 30.15 29.98
F11 28.19 28.14 31.38 31.17 30.37 30.46 29.96 30.06 32.52 31.93 30.14 29.63
F12 28.12 28.26 31.32 31.17 30.46 30.12 30.16 29.95 32.12 32.19 30.12 29.94
F13 28.15 28.19 31.22 31.18 30.64 30.59 29.98 29.94 32.00 32.18 30.02 30.39
F14 28.15 28.24 31.17 31.19 31.57 31.23 29.93 29.99 31.65 32.14 30.21 30.16
F15 28.17 28.19 31.30 30.92 30.81 30.50 29.57 29.71 31.94 32.66 29.93 30.13
F16 28.22 28.32 31.09 31.10 30.71 30.58 30.11 29.94 32.66 31.11 30.11 30.19
Conclusion: It can be seen from the above experimental results that, by using
the clinically positive throat swab sample as the test sample, the test results of
samples with different concentrations of various PCR inhibitors/interfering
substances have no significant differences after nucleic acid extraction via the
magnetic bead method. It shows that the magnetic bead method is beneficial
for removing PCR inhibitors/interfering substances.
2. Measurement accuracy
2.1 Test of reference samples
The reference samples P1-P12 required in the technical requirements of
the six respiratory pathogens nucleic acid diagnostic kit (PCR-fluorescence
probing) were used as the test samples. Reference samples P1-P12 consist of
12 positive samples for the six inactivated respiratory pathogens with
different concentrations. Among them, P1 is influenza A/H1N1 (2009) virus,
P2 is influenza A H3 subtype virus, P3 is influenza A H5 subtype virus, P4 is
influenza A H7N9 subtype virus, and P5 is influenza B virus Victoria subtype,
P6 is influenza B virus Yamagata subtype, P7 is respiratory syncytial virus, P8
is adenovirus 3 subtype, P9 is adenovirus 4 subtype, P10 is adenovirus 7
subtype, P11 is Mycoplasma pneumoniae, and P12 is human Rhinovirus. Each
reference sample is diluted with Hanks virus preservation solution (pH
7.2-7.6) or normal saline. 3 batches of qualified kits were used to detect the
samples on the same SLAN-96P instrument, and each sample were repeatedly
tested 3 times to evaluate the measurement accuracy of this kit.
Figure 2-13 Amplification curve of reference samples P1-P12 by using PCR mix A
(2017001 batch)
Accuracy study 39
(2017002 batch)
(2017003 batch)
Figure 2-16 Amplification curve of reference samples P1-P12 by using PCR mix B
(2017001 batch)
Accuracy study 40
(2017002 batch)
(2017003 batch)
Table 2-8 Test results of reference samples P1-P12 (2017001 batch)
Reference STDEV
P1 24.69 23.69 23.03 No Ct No Ct No Ct 0.84
P2 26.50 26.75 26.73 No Ct No Ct No Ct 0.14
P3 19.38 19.31 18.93 No Ct No Ct No Ct 0.24
P4 29.34 29.21 29.37 No Ct No Ct No Ct 0.09
P5 28.36 28.26 28.22 No Ct No Ct No Ct 0.07
P6 31.92 32.61 32.39 No Ct No Ct No Ct 0.35
P7 25.73 25.77 25.42 No Ct No Ct No Ct 0.19
P8 No Ct No Ct No Ct 20.36 19.97 20.26 0.20
P9 No Ct No Ct No Ct 24.85 25.11 24.96 0.13
P10 No Ct No Ct No Ct 27.47 27.33 27.75 0.21
P11 No Ct No Ct No Ct 21.86 22.07 22.12 0.14
P12 No Ct No Ct No Ct 30.30 30.31 30.38 0.04
Accuracy study 41
Reference STDEV
P1 24.00 23.91 24.09 No Ct No Ct No Ct 0.09
P2 27.35 27.66 27.52 No Ct No Ct No Ct 0.16
P3 20.12 19.91 19.98 No Ct No Ct No Ct 0.11
P4 29.70 29.97 29.91 No Ct No Ct No Ct 0.14
P5 27.13 27.18 27.17 No Ct No Ct No Ct 0.03
P6 31.35 31.50 31.21 No Ct No Ct No Ct 0.15
P7 24.63 24.60 24.70 No Ct No Ct No Ct 0.05
P8 No Ct No Ct No Ct 21.27 20.96 21.27 0.18
P9 No Ct No Ct No Ct 26.12 26.12 25.84 0.16
P10 No Ct No Ct No Ct 28.55 28.40 28.48 0.08
P11 No Ct No Ct No Ct 21.16 21.07 21.14 0.05
P12 No Ct No Ct No Ct 29.42 29.22 29.43 0.16

Reference STDEV
P1 24.04 24.07 23.96 No Ct No Ct No Ct 0.06
P2 27.65 27.65 27.42 No Ct No Ct No Ct 0.13
P3 20.42 20.44 20.21 No Ct No Ct No Ct 0.13
P4 30.21 30.38 29.93 No Ct No Ct No Ct 0.23
P5 27.58 27.12 27.50 No Ct No Ct No Ct 0.25
P6 31.70 31.47 31.67 No Ct No Ct No Ct 0.13
P7 25.20 25.26 25.21 No Ct No Ct No Ct 0.03
P8 No Ct No Ct No Ct 21.32 21.32 21.28 0.02
P9 No Ct No Ct No Ct 26.17 25.98 26.10 0.10
P10 No Ct No Ct No Ct 28.73 28.22 28.62 0.27
P11 No Ct No Ct No Ct 27.02 21.42 21.23 3.29
P12 No Ct No Ct No Ct 29.60 29.40 29.57 0.11
Conclusion: It can be seen from the above that, the test results of reference
samples P1-P12 by using the 3 batches of reagents (2017001, 2017002, 2017003)
were all positive, and there was no non-specific amplification. The standard
deviation (STDEV) of the Ct values of each sample is between 0.02 and 3.29.
2.2 Test of clinical samples

rhinovirus, and negative throat swab sample from people with normal
physical examination were used as test samples. 8 positive samples for each
pathogen and negative samples were tested on the same SLAN-96P
instrument with 3 batches of qualified kits to evaluate the measurement
accuracy of this kit.
Accuracy study 42
Accuracy study 43
Accuracy study 44
Accuracy study 45
Accuracy study 46
Accuracy study 47
Table 2-11 Test results of samples for influenza A virus
Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
PCR mix A 25.65 19.60 17.98 28.07 31.31 22.96 24.52 22.17
2017001
PCR mix A 25.90 20.07 17.68 28.97 31.21 23.70 25.19 21.88
2017002
2017003 PCR mix A 26.18 20.01 18.31 28.55 31.37 23.72 25.36 21.91
Accuracy study 48
Table 2-12 Test results of samples for influenza B virus

Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
PCR mix A 30.14 25.44 24.41 31.97 27.84 37.28 21.11 28.90
2017001
PCR mix A 32.39 26.90 25.68 33.72 29.07 36.19 22.40 30.27
2017002
PCR mix A 32.14 26.36 25.21 33.59 28.59 37.33 21.97 29.64
2017003
Table 2-13 Test results of samples for respiratory syncytial virus

Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
PCR mix A 24.51 27.00 19.89 23.66 27.85 14.60 30.55 24.95
2017001
PCR mix A 24.68 27.40 20.48 24.23 28.45 14.70 30.86 25.23
2017002
PCR mix A 25.23 27.52 20.38 24.25 28.30 14.76 30.75 25.31
2017003
Table 2-14 Test results of samples for Adenovirus

Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
2017001
PCR mix B 20.64 25.57 28.73 31.82 21.92 19.86 17.76 26.81
2017002
PCR mix B 20.46 25.05 28.26 31.59 21.33 19.03 17.34 26.65
2017003
PCR mix B 20.75 25.23 28.18 31.37 21.88 19.96 17.82 28.95
Table 2-15 Test results of samples for Mycoplasma pneumoniae

Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
2017001
PCR mix B 24.75 28.28 22.78 31.53 34.03 27.07 28.94 32.75
2017002
PCR mix B 21.88 25.37 20.06 28.97 30.50 24.20 26.85 29.93
2017003
PCR mix B 24.29 27.68 22.29 30.74 32.92 26.18 28.48 31.75
Accuracy study 49
Table 2-16 Test results of samples for Human rhinovirus
Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
2017001
PCR mix B 23.17 26.76 28.25 34.54 35.93 25.13 30.30 21.18
2017002
PCR mix B 23.50 26.94 28.73 34.49 36.41 25.62 30.59 21.46
2017003
PCR mix B 23.12 26.56 28.35 33.66 34.71 24.95 30.38 20.99
Table 2-17 Test results of negative samples (No amplification curve was observed for
FAM, HEX and CY5, only the Ct value of internal standard was collected)
Ct value of samples
Batch Reaction mix
1 2 3 4 5 6 7 8
PCR mix A 27.38 27.09 27.45 27.11 27.80 27.11 26.94 27.32
2017001
PCR mix B 27.49 27.14 27.26 27.24 27.09 27.13 27.07 27.37
PCR mix A 28.56 28.49 28.27 28.53 28.44 28.45 28.71 28.58
2017002
PCR mix B 28.38 28.49 28.51 28.45 28.67 28.99 28.77 28.58
PCR mix A 27.40 27.63 27.55 27.98 27.79 27.94 27.73 27.52
2017003
PCR mix B 27.33 27.54 27.74 27.79 27.94 27.89 27.77 27.34
Conclusion: It can be seen from the above that, the test results of positive
throat swab samples for influenza A virus, influenza B virus, Respiratory
syncytial virus, Adenovirus, Mycoplasma pneumoniae and Human
rhinovirus by using the 3 batches of reagents (2017001, 2017002, 2017003)
were all positive, and there was no non-specific amplification. Negative
samples were all negative, and the test results of internal standard were all
within the range of reference value. Therefore, this kit has good detection
accuracy.
2.3 Test of national standard samples

The positive reference samples PC01~PC06 in the national reference
samples (batch number 370006-201501) for influenza A/B virus nucleic acid
detection reagent were used as test samples. Samples were tested on the same
SLAN-96P instrument with 3 batches of qualified kits to evaluate the
measurement accuracy of this kit.
Accuracy study 50
Accuracy study 51
Accuracy study 52
Accuracy study 53
Accuracy study 54
Reference
PC01 28.97 29.37 No Ct No Ct
PC02 29.12 29.21 No Ct No Ct
PC03 28.16 27.96 No Ct No Ct
PC04 28.12 28.17 No Ct No Ct
PC05 28.13 28.73 No Ct No Ct
PC06 28.05 28.02 No Ct No Ct
Reference
PC01 29.15 29.15 No Ct No Ct
PC02 29.11 29.10 No Ct No Ct
PC03 28.06 27.97 No Ct No Ct
PC04 27.99 28.20 No Ct No Ct
PC05 28.08 28.09 No Ct No Ct
PC06 28.06 28.08 No Ct No Ct
Reference
PC01 29.47 29.12 No Ct No Ct
Accuracy study 55
PC02 29.18 29.13 No Ct No Ct
PC03 28.01 28.02 No Ct No Ct
PC04 27.97 28.06 No Ct No Ct
PC05 28.05 28.05 No Ct No Ct
PC06 28.13 28.18 No Ct No Ct
Conclusion: It can be seen from the above that, when testing the positive
reference samples PC01~PC06 in the national reference samples for influenza
A/B virus nucleic acid detection reagent by using the 3 batches of reagents
(2017001, 2017002, 2017003), PC01 and PC02 were all positive with influenza B
virus, PC03—PC06 were all positive with influenza A virus, and there was no
non-specific amplification. The results met the requirements of national
reference sample testing for influenza A/B virus nucleic acid detection
reagents. Therefore, this kit has good detection accuracy.
Accuracy study 56
Precision Study

described below.

Precision evaluation
Precision refers to the degree to which multiple parallel test results are
close to each other under the same conditions. The closer the test values, the
higher the precision. Precision reflects the repeatability of the kit operation.
1. Test of precision reference samples

In this experiment, 6 precision reference samples of six respiratory
pathogens nucleic acid diagnostic kits were selected for the imprecision
determination of the kit. Among the 6 reference samples, R1 and R2 are
composed of lentiviral particles containing conserved sequences of influenza
A virus and influenza B virus. The lentivirus of each pathogen is diluted with
normal saline to 5.0 × 104 TU/mL and 5.0 × 103 TU/mL. Each pathogen
lentivirus at the same concentration is mixed in equal volume to R1 and R2.
R3 and R4 are composed of lentiviral particles containing the conserved
sequence of the respiratory syncytial virus. The pathogen lentivirus is diluted
with normal saline to 2.5 × 104 TU/mL and 2.5 × 103 TU/mL to prepare R3 and
R4. R5 and R6 are consisted of cloning plasmids containing adenovirus and
Mycoplasma pneumoniae and lentiviral particles containing the target
conserved sequences of human rhinovirus. The pathogen cloning plasmids
were diluted with normal saline to 5.0 × 104 copise/mL and 5.0 × 103
copise/mL.The lentiviral particles containing the target conserved sequence of
human rhinovirus were diluted with normal saline to 5.0 × 104 TU/mL and 5.0
× 103 TU/mL. The cloning plasmid and lentiviral particles of each pathogen at
the same concentration were mixed in equal volume to R5 and R6. Each
sample was repeatedly tested 10 times, and the detection rate of reference
samples R1, R3 and R5 at medium concentration and reference samples R2,
R4 and R6 at medium concentration were counted and the coefficient of
variance of the detected Ct values of R1-R6 were counted to judge the
precision of this kit.
1.1 Imprecision within and between batches

1.1.1 Imprecision within batches
According to the above-mentioned precision measurement method, the
precision performance of the same batch of reagents under the same operator
was investigated. The test results are as follows (only the amplification curve
of the 2017001 batch of reagents was shown):
Research data of precision evaluation 1

Figure1-1 Imprecision test results of negative control within batches (2017001 batch)
Figure1-2 Imprecision test results of R1 and R2 within batches (2017001 batch)

Table 1-1 Ct value of imprecision test of reagents within the batch of 2017001
Proj R1 R2 R3 R4 R5 R6
ect FA FA FA FA
HEX HEX CY5 CY5 HEX CY5 HEX CY5
No. M M M M
22.3 24.0 25.5 27.4 25.2 29.1 23.1 24.5 25.7 26.1 27.6 28.9
1
6 2 7 8 9 2 3 3 9 9 9 0
22.0 23.6 25.5 27.6 25.2 29.3 22.9 24.4 25.6 25.6 27.2 28.4
2
2 4 9 3 2 5 3 0 3 7 1 2
22.0 23.6 25.4 27.3 25.3 29.2 22.8 24.3 25.5 25.7 27.2 28.3
3
7 7 3 0 4 3 8 4 6 7 7 4
22.0 23.7 25.5 27.4 25.3 29.0 22.7 24.2 25.4 25.8 27.2 28.5
4
7 1 7 4 2 2 1 5 5 8 9 3
21.7 23.5 25.7 27.4 25.4 28.9 22.5 24.0 25.3 25.9 27.3 28.5
5
8 0 3 8 2 7 5 9 2 4 3 9
21.9 23.5 25.8 27.6 25.3 28.7 22.7 24.2 25.5 26.0 27.4 28.5
6
8 3 9 4 5 8 5 9 2 1 2 7
22.0 23.6 25.8 27.5 25.2 28.5 22.9 24.4 25.6 26.1 27.7 28.8
7
8 7 1 7 5 2 2 0 0 6 1 2
22.3 23.9 25.5 27.4 25.4 29.2 23.0 24.4 25.6 26.0 27.5 28.5
8
2 1 4 1 3 0 2 9 9 4 0 6
22.3 24.0 26.0 27.7 25.2 29.1 23.0 24.5 25.7 25.9 27.4 28.4
9
1 0 2 4 2 7 4 1 2 2 0 4
22.2 23.7 25.4 27.2 25.4 29.2 22.9 24.2 25.5 25.9 27.4 28.5
10
1 4 4 9 3 4 1 9 5 0 1 7
0.81 0.77 0.77 0.54 0.33 0.86 0.75 0.56 0.53 0.61 0.61 0.60
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
22.8 23.9 26.4 27.8 25.0 28.7 23.4 24.7 24.9 26.2 27.2 27.5
1
6 8 9 5 8 7 1 6 6 2 6 4

22.7 23.9 26.4 27.6 24.8 28.8 23.6 24.7 25.0 26.2 27.4 27.7
2
0 2 2 9 5 9 4 5 8 4 5 5
22.8 24.0 26.4 28.1 24.7 28.3 23.6 24.7 24.9 26.4 27.5 28.0
3
1 1 5 0 2 9 2 9 9 2 7 0
22.8 24.1 26.4 28.0 24.6 28.5 23.8 24.6 24.9 26.5 27.7 27.9
4
1 7 0 6 6 0 2 3 2 5 2 6
22.3 23.4 26.6 28.2 25.0 28.1 23.2 24.2 24.7 26.4 27.4 27.7
5
5 2 0 4 4 6 7 7 8 8 5 0
22.7 23.7 26.5 27.9 25.2 28.6 23.3 24.3 25.0 26.4 27.4 27.6
6
3 4 1 5 8 8 1 5 3 8 5 9
23.0 24.2 26.4 28.1 25.1 28.3 23.4 24.7 24.9 26.5 27.6 27.6
7
2 5 0 9 8 6 7 4 0 1 2 9
23.2 24.3 26.4 28.0 24.9 28.4 23.5 24.7 25.1 26.6 27.9 27.5
8
1 7 0 4 1 0 4 4 3 2 0 9
23.2 24.5 26.6 28.1 24.9 28.5 23.4 24.6 25.0 26.0 27.0 27.4
9
5 0 2 0 5 4 5 1 4 9 3 6
23.1 24.3 26.6 27.8 25.1 28.7 23.6 24.8 25.0 26.1 27.3 27.7
10
8 6 0 1 8 6 8 7 6 7 2 7
1.23 1.36 0.34 0.63 0.82 0.79 0.73 0.79 0.41 0.69 0.89 0.61
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
22.0 23.3 25.1 26.6 24.3 27.7 22.6 23.7 24.5 25.4 26.7 27.3
1
2 3 3 9 4 2 5 3 8 3 3 6
21.7 22.8 25.1 26.6 24.0 28.1 22.5 23.8 24.3 25.1 26.3 27.1
2
3 8 0 9 2 5 1 7 9 3 9 9
22.1 23.4 25.1 26.7 24.1 28.2 22.5 23.9 24.4 25.2 26.6 27.3
3
1 3 4 8 8 3 7 4 4 0 5 4
21.7 23.0 25.2 26.7 24.1 28.2 22.3 23.5 24.2 25.4 26.7 27.4
4
2 2 4 2 8 1 6 4 9 8 3 3
21.4 22.7 25.2 26.9 23.8 28.3 22.1 23.3 24.1 25.3 26.7 27.3
5
1 3 3 5 5 2 7 3 7 7 1 5
21.7 22.8 26.3 27.1 24.2 28.3 22.2 23.3 24.2 25.3 26.7 27.3
6
5 8 8 0 2 6 1 1 2 0 6 9
21.8 23.0 25.3 27.0 24.1 28.0 22.4 23.6 24.3 25.4 26.8 27.3
7
2 8 2 0 2 9 7 7 7 0 8 8
21.6 23.0 25.3 26.9 24.4 28.1 22.2 23.4 24.0 25.2 26.7 27.4
8
8 8 1 4 9 3 1 8 9 8 2 4
21.8 23.0 25.2 26.7 24.1 27.9 22.2 23.5 24.2 25.1 26.4 27.2
9
8 3 1 6 7 5 7 9 0 6 3 2
22.0 23.2 25.4 26.9 24.3 28.1 22.1 23.3 24.1 25.2 26.4 27.3
10
1 0 3 9 1 0 8 6 2 7 8 0
0.93 0.92 1.48 0.56 0.73 0.66 0.79 0.94 0.64 0.47 0.60 0.31
CV
% % % % % % % % % % % %

1.1.2 Imprecision between batches
test results of the above three batches of reagents are summarized, the relative
error is calculated, and the inter-batch imprecision is determined.
Table 1-4 Imprecision test results among three batches of reagents

ect FA FA FA FA
No. M M M M
22.3 24.0 25.5 27.4 25.2 29.1 23.1 24.5 25.7 26.1 27.6 28.9
1
6 2 7 8 9 2 3 3 9 9 9 0
22.0 23.6 25.5 27.6 25.2 29.3 22.9 24.4 25.6 25.6 27.2 28.4
2
2 4 9 3 2 5 3 0 3 7 1 2
22.0 23.6 25.4 27.3 25.3 29.2 22.8 24.3 25.5 25.7 27.2 28.3
3
7 7 3 0 4 3 8 4 6 7 7 4
22.0 23.7 25.5 27.4 25.3 29.0 22.7 24.2 25.4 25.8 27.2 28.5
4
7 1 7 4 2 2 1 5 5 8 9 3
21.7 23.5 25.7 27.4 25.4 28.9 22.5 24.0 25.3 25.9 27.3 28.5
5
8 0 3 8 2 7 5 9 2 4 3 9
21.9 23.5 25.8 27.6 25.3 28.7 22.7 24.2 25.5 26.0 27.4 28.5
6
8 3 9 4 5 8 5 9 2 1 2 7
22.0 23.6 25.8 27.5 25.2 28.5 22.9 24.4 25.6 26.1 27.7 28.8
7
8 7 1 7 5 2 2 0 0 6 1 2
22.3 23.9 25.5 27.4 25.4 29.2 23.0 24.4 25.6 26.0 27.5 28.5
8
2 1 4 1 3 0 2 9 9 4 0 6
22.3 24.0 26.0 27.7 25.2 29.1 23.0 24.5 25.7 25.9 27.4 28.4
9
1 0 2 4 2 7 4 1 2 2 0 4
22.2 23.7 25.4 27.2 25.4 29.2 22.9 24.2 25.5 25.9 27.4 28.5
10
1 4 4 9 3 4 1 9 5 0 1 7
22.8 23.9 26.4 27.8 25.0 28.7 23.4 24.7 24.9 26.2 27.2 27.5
11
6 8 9 5 8 7 1 6 6 2 6 4
22.7 23.9 26.4 27.6 24.8 28.8 23.6 24.7 25.0 26.2 27.4 27.7
12
0 2 2 9 5 9 4 5 8 4 5 5
22.8 24.0 26.4 28.1 24.7 28.3 23.6 24.7 24.9 26.4 27.5 28.0
13
1 1 5 0 2 9 2 9 9 2 7 0
22.8 24.1 26.4 28.0 24.6 28.5 23.8 24.6 24.9 26.5 27.7 27.9
14
1 7 0 6 6 0 2 3 2 5 2 6
22.3 23.4 26.6 28.2 25.0 28.1 23.2 24.2 24.7 26.4 27.4 27.7
15
5 2 0 4 4 6 7 7 8 8 5 0
22.7 23.7 26.5 27.9 25.2 28.6 23.3 24.3 25.0 26.4 27.4 27.6
16
3 4 1 5 8 8 1 5 3 8 5 9
23.0 24.2 26.4 28.1 25.1 28.3 23.4 24.7 24.9 26.5 27.6 27.6
17
2 5 0 9 8 6 7 4 0 1 2 9
23.2 24.3 26.4 28.0 24.9 28.4 23.5 24.7 25.1 26.6 27.9 27.5
18
1 7 0 4 1 0 4 4 3 2 0 9
23.2 24.5 26.6 28.1 24.9 28.5 23.4 24.6 25.0 26.0 27.0 27.4
19
5 0 2 0 5 4 5 1 4 9 3 6
20 23.1 24.3 26.6 27.8 25.1 28.7 23.6 24.8 25.0 26.1 27.3 27.7

8 6 0 1 8 6 8 7 6 7 2 7
22.0 23.3 25.1 26.6 24.3 27.7 22.6 23.7 24.5 25.4 26.7 27.3
21
2 3 3 9 4 2 5 3 8 3 3 6
21.7 22.8 25.1 26.6 24.0 28.1 22.5 23.8 24.3 25.1 26.3 27.1
22
3 8 0 9 2 5 1 7 9 3 9 9
22.1 23.4 25.1 26.7 24.1 28.2 22.5 23.9 24.4 25.2 26.6 27.3
23
1 3 4 8 8 3 7 4 4 0 5 4
21.7 23.0 25.2 26.7 24.1 28.2 22.3 23.5 24.2 25.4 26.7 27.4
24
2 2 4 2 8 1 6 4 9 8 3 3
21.4 22.7 25.2 26.9 23.8 28.3 22.1 23.3 24.1 25.3 26.7 27.3
25
1 3 3 5 5 2 7 3 7 7 1 5
21.7 22.8 26.3 27.1 24.2 28.3 22.2 23.3 24.2 25.3 26.7 27.3
26
5 8 8 0 2 6 1 1 2 0 6 9
21.8 23.0 25.3 27.0 24.1 28.0 22.4 23.6 24.3 25.4 26.8 27.3
27
2 8 2 0 2 9 7 7 7 0 8 8
21.6 23.0 25.3 26.9 24.4 28.1 22.2 23.4 24.0 25.2 26.7 27.4
28
8 8 1 4 9 3 1 8 9 8 2 4
21.8 23.0 25.2 26.7 24.1 27.9 22.2 23.5 24.2 25.1 26.4 27.2
29
8 3 1 6 7 5 7 9 0 6 3 2
22.0 23.2 25.4 26.9 24.3 28.1 22.1 23.3 24.1 25.2 26.4 27.3
30
1 0 3 9 1 0 8 6 2 7 8 0
2.29 2.07 2.11 1.82 2.05 1.55 2.23 2.04 2.22 1.83 1.58 1.95
CV
% % % % % % % % % % % %
Conclusion: It can be seen from the above results that, the positive
detection rate of the three batches of kits for normal negative control are all
0% and the positive detection rate of the positive control samples R1-R6 are all
100%. For the precision reference samples R1-R6, the CV of detected Ct values
within the three batches of kits range from 0.31% to 1.48%, the CV of detected
Ct values between batches range from 1.55% to 2.29%, and both of the values
are less than 5%. Therefore, it can be considered that this kit has good
detection precision within and between batches.
1.2 Intra-day and inter-day imprecision

1.2.1 Intra-day imprecision
precision performance of the same batch of reagents in the same day under
different operator was investigated. In the same day, the first test results are
shown in Figure 1-1 to Figure 1-4 in the intra-batch precision experiment; the
precision detection test results under different operators are shown in Figure
1-5 to Figure 1-8 (only the amplification curve of the 2017001 batch of reagents
was shown):

Figure 1-5 Intra-day imprecision test results of negative control (2017001 batch)
Figure 1-6 Intra-day imprecision test results of R1 and R2 (2017001 batch)

Table 1-5 Ct value of intra-day imprecision test of reagents within the batch of 2017001
ect FA FA FA FA
No. M M M M
22.3 24.0 25.5 27.4 25.2 29.1 23.1 24.5 25.7 26.1 27.6 28.9
1
6 2 7 8 9 2 3 3 9 9 9 0
22.0 23.6 25.5 27.6 25.2 29.3 22.9 24.4 25.6 25.6 27.2 28.4
2
2 4 9 3 2 5 3 0 3 7 1 2
22.0 23.6 25.4 27.3 25.3 29.2 22.8 24.3 25.5 25.7 27.2 28.3
3
7 7 3 0 4 3 8 4 6 7 7 4
22.0 23.7 25.5 27.4 25.3 29.0 22.7 24.2 25.4 25.8 27.2 28.5
4
7 1 7 4 2 2 1 5 5 8 9 3
21.7 23.5 25.7 27.4 25.4 28.9 22.5 24.0 25.3 25.9 27.3 28.5
5
8 0 3 8 2 7 5 9 2 4 3 9
21.9 23.5 25.8 27.6 25.3 28.7 22.7 24.2 25.5 26.0 27.4 28.5
6
8 3 9 4 5 8 5 9 2 1 2 7
22.0 23.6 25.8 27.5 25.2 28.5 22.9 24.4 25.6 26.1 27.7 28.8
7
8 7 1 7 5 2 2 0 0 6 1 2
22.3 23.9 25.5 27.4 25.4 29.2 23.0 24.4 25.6 26.0 27.5 28.5
8
2 1 4 1 3 0 2 9 9 4 0 6
22.3 24.0 26.0 27.7 25.2 29.1 23.0 24.5 25.7 25.9 27.4 28.4
9
1 0 2 4 2 7 4 1 2 2 0 4
22.2 23.7 25.4 27.2 25.4 29.2 22.9 24.2 25.5 25.9 27.4 28.5
10
1 4 4 9 3 4 1 9 5 0 1 7
22.3 22.9 25.4 26.2 25.1 28.4 23.0 23.5 25.4 25.3 26.2 28.0
11
5 6 3 5 1 9 4 2 6 8 1 8
22.2 22.8 25.5 26.4 25.0 28.8 22.9 23.4 25.3 25.5 26.2 28.0
12
7 1 6 3 3 2 7 4 8 1 5 2
22.2 22.8 25.7 26.6 24.8 29.0 22.8 23.3 25.3 25.7 26.3 28.2
13
1 4 1 4 8 9 6 2 1 3 9 6
22.0 22.4 25.8 26.7 24.9 29.0 22.5 23.1 24.9 26.1 26.8 28.5
14
3 3 9 4 6 8 1 1 7 8 0 3

21.6 22.3 25.7 26.5 24.8 28.9 22.4 22.8 24.8 26.0 26.5 28.3
15
6 6 6 5 0 3 2 7 4 2 3 0
22.0 22.5 25.7 26.5 25.0 28.7 22.4 23.0 25.0 25.8 26.3 28.2
16
7 9 1 3 8 6 7 8 2 9 8 3
22.2 22.7 25.8 26.8 25.1 28.6 22.9 23.3 25.1 25.8 26.4 28.2
17
3 2 7 2 8 7 9 3 8 0 1 1
22.4 23.0 25.5 26.4 25.3 28.8 22.7 23.3 25.1 25.5 25.9 28.1
18
0 7 3 7 4 0 7 4 1 2 2 6
22.1 22.6 25.4 26.3 25.2 28.5 22.7 23.2 25.1 25.3 26.0 27.8
19
7 5 7 8 2 5 8 2 9 8 7 8
22.1 22.8 25.4 26.4 25.2 28.9 22.6 23.1 25.0 25.6 26.3 28.1
20
7 8 8 5 4 2 0 2 5 9 2 7
0.86 2.39 0.70 1.93 0.71 0.88 0.92 2.52 1.08 0.96 2.22 0.92
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
22.8 23.9 26.4 27.8 25.0 28.7 23.4 24.7 24.9 26.2 27.2 27.5
1
6 8 9 5 8 7 1 6 6 2 6 4
22.7 23.9 26.4 27.6 24.8 28.8 23.6 24.7 25.0 26.2 27.4 27.7
2
0 2 2 9 5 9 4 5 8 4 5 5
22.8 24.0 26.4 28.1 24.7 28.3 23.6 24.7 24.9 26.4 27.5 28.0
3
1 1 5 0 2 9 2 9 9 2 7 0
22.8 24.1 26.4 28.0 24.6 28.5 23.8 24.6 24.9 26.5 27.7 27.9
4
1 7 0 6 6 0 2 3 2 5 2 6
22.3 23.4 26.6 28.2 25.0 28.1 23.2 24.2 24.7 26.4 27.4 27.7
5
5 2 0 4 4 6 7 7 8 8 5 0
22.7 23.7 26.5 27.9 25.2 28.6 23.3 24.3 25.0 26.4 27.4 27.6
6
3 4 1 5 8 8 1 5 3 8 5 9
23.0 24.2 26.4 28.1 25.1 28.3 23.4 24.7 24.9 26.5 27.6 27.6
7
2 5 0 9 8 6 7 4 0 1 2 9
23.2 24.3 26.4 28.0 24.9 28.4 23.5 24.7 25.1 26.6 27.9 27.5
8
1 7 0 4 1 0 4 4 3 2 0 9
23.2 24.5 26.6 28.1 24.9 28.5 23.4 24.6 25.0 26.0 27.0 27.4
9
5 0 2 0 5 4 5 1 4 9 3 6
23.1 24.3 26.6 27.8 25.1 28.7 23.6 24.8 25.0 26.1 27.3 27.7
10
8 6 0 1 8 6 8 7 6 7 2 7
22.3 23.4 26.7 27.7 25.5 28.6 23.2 24.0 25.3 26.3 27.2 28.4
11
6 3 0 6 9 9 0 3 6 3 4 7
22.7 23.7 26.7 27.6 25.5 28.9 23.3 24.1 25.2 26.2 27.4 28.6
12
8 5 4 0 5 8 1 8 8 6 5 8
22.6 23.7 27.0 28.0 25.4 28.6 23.5 24.3 25.3 26.1 27.3 28.5
13
2 7 0 3 6 8 9 6 9 9 6 2
22.7 23.7 26.8 28.0 25.3 28.8 23.4 24.2 25.2 26.3 27.4 28.5
14
0 0 9 2 2 9 4 9 6 0 1 1

22.4 23.5 26.9 28.1 25.5 28.2 22.8 23.6 25.1 26.5 27.5 28.6
15
9 1 6 8 5 6 5 3 0 2 9 2
22.8 23.5 26.8 28.0 25.2 28.8 23.1 24.0 25.4 26.7 27.5 28.5
16
3 3 5 3 8 3 4 7 5 1 7 4
23.0 24.0 27.0 28.2 25.6 28.6 23.2 24.2 25.4 27.1 27.9 28.8
17
4 0 3 3 1 2 9 3 6 8 6 2
23.1 24.1 27.0 28.0 25.5 28.7 23.2 24.1 25.3 26.7 27.7 28.5
18
3 1 2 0 1 4 3 7 4 4 4 6
23.2 24.2 26.8 27.9 25.6 29.1 23.0 23.9 25.0 25.6 26.8 28.1
19
1 4 7 0 6 8 4 7 8 6 3 1
22.9 24.1 26.8 27.8 25.7 28.8 23.4 24.3 25.4 25.6 26.8 28.4
20
7 4 1 7 5 7 5 0 3 7 1 7
1.20 1.37 0.85 0.64 1.30 0.90 1.00 1.38 0.81 1.31 1.11 1.61
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
22.0 23.3 25.1 26.6 24.3 27.7 22.6 23.7 24.5 25.4 26.7 27.3
1
2 3 3 9 4 2 5 3 8 3 3 6
21.7 22.8 25.1 26.6 24.0 28.1 22.5 23.8 24.3 25.1 26.3 27.1
2
3 8 0 9 2 5 1 7 9 3 9 9
22.1 23.4 25.1 26.7 24.1 28.2 22.5 23.9 24.4 25.2 26.6 27.3
3
1 3 4 8 8 3 7 4 4 0 5 4
21.7 23.0 25.2 26.7 24.1 28.2 22.3 23.5 24.2 25.4 26.7 27.4
4
2 2 4 2 8 1 6 4 9 8 3 3
21.4 22.7 25.2 26.9 23.8 28.3 22.1 23.3 24.1 25.3 26.7 27.3
5
1 3 3 5 5 2 7 3 7 7 1 5
21.7 22.8 26.3 27.1 24.2 28.3 22.2 23.3 24.2 25.3 26.7 27.3
6
5 8 8 0 2 6 1 1 2 0 6 9
21.8 23.0 25.3 27.0 24.1 28.0 22.4 23.6 24.3 25.4 26.8 27.3
7
2 8 2 0 2 9 7 7 7 0 8 8
21.6 23.0 25.3 26.9 24.4 28.1 22.2 23.4 24.0 25.2 26.7 27.4
8
8 8 1 4 9 3 1 8 9 8 2 4
21.8 23.0 25.2 26.7 24.1 27.9 22.2 23.5 24.2 25.1 26.4 27.2
9
8 3 1 6 7 5 7 9 0 6 3 2
22.0 23.2 25.4 26.9 24.3 28.1 22.1 23.3 24.1 25.2 26.4 27.3
10
1 0 3 9 1 0 8 6 2 7 8 0
21.7 23.2 25.1 26.5 24.6 27.8 22.0 23.2 24.6 24.9 26.3 27.7
11
3 0 7 7 8 4 5 3 2 0 7 1
21.7 23.1 25.2 26.9 24.7 27.9 22.0 23.1 24.5 24.9 26.3 27.8
12
0 0 7 0 4 2 9 8 0 7 9 6
21.7 23.2 25.3 26.7 24.7 28.0 22.0 23.2 24.6 24.8 26.3 27.6
13
2 0 4 4 2 7 1 9 0 2 4 7
21.5 22.9 25.3 26.8 24.4 28.0 21.5 22.8 24.2 26.4 27.1 28.0
14
9 3 9 7 7 6 3 7 5 4 1 0

21.1 22.7 25.4 26.9 24.0 28.0 21.6 22.8 24.2 26.2 26.8 27.8
15
6 8 4 7 6 8 6 4 4 4 7 8
21.3 22.9 25.4 26.9 24.3 27.9 21.9 23.2 24.5 26.1 26.6 27.6
16
8 4 0 4 1 5 7 1 7 4 7 1
21.7 23.0 25.4 27.0 24.2 27.7 22.1 23.3 24.6 26.1 26.7 27.6
17
6 7 9 1 5 8 4 5 8 3 1 7
21.8 23.2 25.5 27.0 24.2 27.9 21.8 23.1 24.4 26.1 26.7 27.7
18
7 1 3 0 5 2 8 1 8 7 7 0
21.8 23.0 25.2 26.6 24.3 27.8 21.8 23.0 24.4 26.0 26.6 27.7
19
4 9 4 8 2 3 7 6 7 5 9 8
21.9 23.1 25.4 27.0 24.2 27.9 22.0 23.1 24.5 26.0 26.7 27.8
20
0 4 8 2 6 7 4 3 2 9 5 2
1.04 0.76 1.07 0.55 0.95 0.62 1.30 1.29 0.74 1.99 0.75 0.88
CV
% % % % % % % % % % % %
1.2.2 Inter-day imprecision

in different days was investigated. The first test results in the first day are
shown in Figure 1-1 to Figure 1-4; the precision detection test results
performed by the same operators on alternate days are shown in Figure 1-9 to
Figure 1-12 (only the amplification curve of the 2017001 batch of reagents was
shown):
Figure 1-9 Inter-day imprecision test results of negative control (2017001 batch)
Figure 1-10 Inter-day imprecision test results of R1 and R2 (2017001 batch)

Table 1-8 Ct value of inter-day imprecision test of reagents within the batch of 2017001
ect FA FA FA FA
No. M M M M
22.3 24.0 25.5 27.4 25.2 29.1 23.1 24.5 25.7 26.1 27.6 28.9
1
6 2 7 8 9 2 3 3 9 9 9 0
22.0 23.6 25.5 27.6 25.2 29.3 22.9 24.4 25.6 25.6 27.2 28.4
2
2 4 9 3 2 5 3 0 3 7 1 2
22.0 23.6 25.4 27.3 25.3 29.2 22.8 24.3 25.5 25.7 27.2 28.3
3
7 7 3 0 4 3 8 4 6 7 7 4
22.0 23.7 25.5 27.4 25.3 29.0 22.7 24.2 25.4 25.8 27.2 28.5
4
7 1 7 4 2 2 1 5 5 8 9 3
21.7 23.5 25.7 27.4 25.4 28.9 22.5 24.0 25.3 25.9 27.3 28.5
5
8 0 3 8 2 7 5 9 2 4 3 9
21.9 23.5 25.8 27.6 25.3 28.7 22.7 24.2 25.5 26.0 27.4 28.5
6
8 3 9 4 5 8 5 9 2 1 2 7
22.0 23.6 25.8 27.5 25.2 28.5 22.9 24.4 25.6 26.1 27.7 28.8
7
8 7 1 7 5 2 2 0 0 6 1 2

22.3 23.9 25.5 27.4 25.4 29.2 23.0 24.4 25.6 26.0 27.5 28.5
8
2 1 4 1 3 0 2 9 9 4 0 6
22.3 24.0 26.0 27.7 25.2 29.1 23.0 24.5 25.7 25.9 27.4 28.4
9
1 0 2 4 2 7 4 1 2 2 0 4
22.2 23.7 25.4 27.2 25.4 29.2 22.9 24.2 25.5 25.9 27.4 28.5
10
1 4 4 9 3 4 1 9 5 0 1 7
22.9 23.4 26.7 27.0 25.3 28.5 23.1 23.4 25.2 26.3 26.8 28.5
11
4 9 7 5 7 3 1 9 0 2 6 9
23.0 23.4 26.5 26.9 25.2 28.5 23.1 23.4 25.2 28.4 26.9 28.4
12
0 8 5 3 5 9 2 5 4 8 7 7
23.0 23.3 26.8 27.0 25.1 28.4 23.3 23.6 25.3 26.6 26.9 28.4
13
4 3 9 8 6 6 4 5 1 6 1 5
23.1 23.3 26.8 27.0 25.3 28.4 22.9 23.2 24.9 26.8 27.0 28.6
14
6 0 7 4 8 4 4 5 0 0 8 1
22.6 23.1 26.8 27.0 25.4 28.1 22.8 23.2 25.3 26.7 26.9 28.3
15
0 9 3 7 1 5 5 7 3 4 1 1
22.9 23.2 26.6 27.0 25.3 28.7 23.0 23.4 25.2 27.0 27.0 28.5
16
3 6 3 0 7 0 3 2 4 3 5 1
23.1 23.3 26.6 26.8 25.1 28.4 23.0 23.3 25.3 27.2 27.1 28.6
17
8 2 7 1 9 8 2 6 1 1 6 7
23.4 23.8 26.5 26.9 25.1 28.5 23.0 23.4 25.2 27.3 27.2 28.8
18
7 0 5 8 1 7 6 4 6 0 6 5
23.3 23.6 26.3 26.8 25.4 28.7 22.9 23.1 25.0 26.0 26.6 28.0
19
3 6 1 2 2 4 8 9 8 5 1 9
23.3 23.7 26.5 27.0 25.3 28.4 23.1 23.4 25.2 26.7 27.1 28.6
20
4 0 2 2 1 9 0 3 6 4 6 9
2.41 1.02 2.08 1.07 0.38 1.20 0.75 2.14 0.91 2.61 1.03 0.67
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
22.8 23.9 26.4 27.8 25.0 28.7 23.4 24.7 24.9 26.2 27.2 27.5
1
6 8 9 5 8 7 1 6 6 2 6 4
22.7 23.9 26.4 27.6 24.8 28.8 23.6 24.7 25.0 26.2 27.4 27.7
2
0 2 2 9 5 9 4 5 8 4 5 5
22.8 24.0 26.4 28.1 24.7 28.3 23.6 24.7 24.9 26.4 27.5 28.0
3
1 1 5 0 2 9 2 9 9 2 7 0
22.8 24.1 26.4 28.0 24.6 28.5 23.8 24.6 24.9 26.5 27.7 27.9
4
1 7 0 6 6 0 2 3 2 5 2 6
22.3 23.4 26.6 28.2 25.0 28.1 23.2 24.2 24.7 26.4 27.4 27.7
5
5 2 0 4 4 6 7 7 8 8 5 0
22.7 23.7 26.5 27.9 25.2 28.6 23.3 24.3 25.0 26.4 27.4 27.6
6
3 4 1 5 8 8 1 5 3 8 5 9
23.0 24.2 26.4 28.1 25.1 28.3 23.4 24.7 24.9 26.5 27.6 27.6
7
2 5 0 9 8 6 7 4 0 1 2 9

23.2 24.3 26.4 28.0 24.9 28.4 23.5 24.7 25.1 26.6 27.9 27.5
8
1 7 0 4 1 0 4 4 3 2 0 9
23.2 24.5 26.6 28.1 24.9 28.5 23.4 24.6 25.0 26.0 27.0 27.4
9
5 0 2 0 5 4 5 1 4 9 3 6
23.1 24.3 26.6 27.8 25.1 28.7 23.6 24.8 25.0 26.1 27.3 27.7
10
8 6 0 1 8 6 8 7 6 7 2 7
21.7 23.0 24.9 26.2 24.1 27.4 22.1 23.3 24.4 25.1 26.4 27.5
11
9 3 8 6 7 0 2 6 3 5 9 4
22.0 23.0 25.4 26.7 24.2 27.6 21.9 23.0 24.2 25.4 26.6 27.8
12
2 4 8 9 7 1 2 2 6 6 0 0
21.9 23.1 25.2 26.5 24.2 27.9 22.1 23.3 24.4 25.3 26.6 27.7
13
3 2 4 4 2 0 4 2 7 3 5 1
21.4 22.8 25.2 26.4 24.2 27.7 22.1 23.2 24.4 25.5 26.8 27.7
14
0 3 1 2 3 1 3 3 6 2 1 5
21.3 22.7 25.3 26.5 23.9 27.7 21.6 23.0 24.1 25.5 26.7 27.8
15
1 6 9 4 8 7 6 1 9 6 8 1
21.6 23.0 25.0 26.2 24.2 27.7 21.7 23.1 24.4 25.3 26.5 27.7
16
6 4 5 2 8 6 9 8 4 9 5 2
21.8 23.0 25.2 26.5 24.3 27.7 22.0 23.3 24.5 25.4 26.7 27.6
17
8 1 7 4 3 2 6 6 7 5 4 0
21.8 23.1 25.5 26.6 24.1 27.6 22.0 23.2 24.3 26.6 26.9 27.8
18
8 7 2 6 2 6 5 6 1 5 5 5
22.0 23.0 25.0 26.3 24.1 27.5 21.9 23.2 24.4 25.0 26.1 27.2
19
4 8 6 8 9 5 7 4 3 8 2 5
21.9 23.1 25.3 26.6 24.0 27.6 22.0 23.2 24.2 25.5 26.8 27.7
20
4 8 4 3 5 6 0 5 8 1 0 8
2.79 2.50 2.51 2.90 1.79 1.72 3.54 3.13 1.33 2.12 1.79 0.62
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
22.0 23.3 25.1 26.6 24.3 27.7 22.6 23.7 24.5 25.4 26.7 27.3
1
2 3 3 9 4 2 5 3 8 3 3 6
21.7 22.8 25.1 26.6 24.0 28.1 22.5 23.8 24.3 25.1 26.3 27.1
2
3 8 0 9 2 5 1 7 9 3 9 9
22.1 23.4 25.1 26.7 24.1 28.2 22.5 23.9 24.4 25.2 26.6 27.3
3
1 3 4 8 8 3 7 4 4 0 5 4
21.7 23.0 25.2 26.7 24.1 28.2 22.3 23.5 24.2 25.4 26.7 27.4
4
2 2 4 2 8 1 6 4 9 8 3 3
21.4 22.7 25.2 26.9 23.8 28.3 22.1 23.3 24.1 25.3 26.7 27.3
5
1 3 3 5 5 2 7 3 7 7 1 5
21.7 22.8 26.3 27.1 24.2 28.3 22.2 23.3 24.2 25.3 26.7 27.3
6
5 8 8 0 2 6 1 1 2 0 6 9
21.8 23.0 25.3 27.0 24.1 28.0 22.4 23.6 24.3 25.4 26.8 27.3
7
2 8 2 0 2 9 7 7 7 0 8 8

21.6 23.0 25.3 26.9 24.4 28.1 22.2 23.4 24.0 25.2 26.7 27.4
8
8 8 1 4 9 3 1 8 9 8 2 4
21.8 23.0 25.2 26.7 24.1 27.9 22.2 23.5 24.2 25.1 26.4 27.2
9
8 3 1 6 7 5 7 9 0 6 3 2
22.0 23.2 25.4 26.9 24.3 28.1 22.1 23.3 24.1 25.2 26.4 27.3
10
1 0 3 9 1 0 8 6 2 7 8 0
23.0 23.7 26.2 27.0 25.2 28.6 22.7 23.7 25.2 26.1 27.3 28.7
11
1 6 2 5 9 8 7 5 7 9 0 2
22.9 23.7 26.6 27.3 25.3 28.7 23.2 24.1 25.6 26.6 27.6 28.8
12
3 4 3 9 8 3 8 8 2 7 5 8
22.6 23.5 26.4 27.4 25.1 28.6 23.1 24.0 25.3 26.4 27.1 28.4
13
8 7 4 2 0 3 3 7 4 7 2 6
23.1 23.7 26.5 27.3 25.2 29.1 23.2 24.1 25.2 26.8 27.5 28.9
14
8 7 0 4 4 5 0 0 9 3 9 1
22.4 23.0 26.6 27.5 25.4 28.3 22.5 23.5 25.1 26.4 27.2 28.4
15
5 8 8 8 6 8 2 4 5 5 0 4
22.7 23.5 26.8 27.4 25.6 28.7 22.7 23.6 25.2 26.9 27.4 28.5
16
9 0 0 0 4 5 2 9 5 7 4 0
23.0 23.8 26.7 27.3 25.3 28.5 23.2 23.9 25.4 27.0 27.7 28.7
17
6 1 1 8 1 8 1 4 7 5 0 4
23.2 24.1 26.8 27.6 25.8 28.5 22.9 24.0 25.2 27.2 27.8 28.7
18
1 8 0 0 9 0 9 9 9 8 7 9
23.1 24.0 26.5 27.2 25.2 28.9 22.8 23.8 25.1 26.1 26.8 28.1
19
1 9 9 0 2 9 7 8 9 5 8 7
23.1 24.0 26.1 27.0 25.7 28.6 23.4 24.3 25.6 26.6 27.3 28.8
20
9 4 1 0 0 3 9 0 3 2 6 9
2.81 1.89 2.65 1.10 2.69 1.26 1.82 1.24 2.29 2.88 1.69 2.48
CV
% % % % % % % % % % % %
of the three batches of kits by different operators and within one day range
from 0.55% to 2.52%, the CV of detected Ct values of the three batches by the
same operators and between days range from 0.38% to 3.54%, and both of the
values are less than 5%. Therefore, it can be considered that this kit has good
detection precision under different operators and at different times.
2. Precision test of throat swab samples

The clinical throat swab samples were tested by using three batches of
kits to verify the precision performance of the three batches of kits on clinical
samples. For positive samples of each pathogen, a clinical sample with a
higher concentration was selected and diluted to a low concentration for
precision testing. Each sample was extracted 10 times each time and was
repeatedly tested 10 times and the CV of Ct values were calculated.

Figure1-13 Imprecision test results of positive samples for influenza A virus within
batches (2017001 batch)
Figure1-14 Imprecision test results of positive samples for influenza B virus within

Figure1-15 Imprecision test results of positive samples for respiratory syncytial virus
within batches (2017001 batch)
Figure1-16 Imprecision test results of positive samples for Adenovirus within batches
(2017001 batch)

Figure1-17 Imprecision test results of positive samples for Human rhinovirus within
Figure1-18 Imprecision test results of positive samples for Mycoplasma pneumoniae

Sample Respiratory
Influenza Influenza Human Mycoplasma
No. Syncytial Adenovirus
A virus B virus Rhinovirus pneumoniae
virus
1 29.38 31.57 30.93 29.95 31.48 30.30
2 29.99 31.60 30.58 30.21 30.94 29.30
3 29.15 31.67 30.72 29.85 31.19 29.31
4 29.43 31.62 30.50 29.84 30.78 30.28
5 28.87 31.59 31.32 29.83 31.00 30.54

6 29.05 31.31 31.35 29.41 30.96 29.89
7 29.02 30.73 30.71 29.19 30.92 30.33
8 28.86 30.92 30.86 28.93 30.66 30.44
9 29.10 31.07 30.81 29.16 30.87 30.15
10 28.88 30.95 30.71 29.00 30.48 30.53
CV
1.20% 1.13% 0.92% 1.52% 0.88% 1.53%
value
Sample Respiratory
virus
1 29.83 30.43 30.29 29.51 30.39 29.93
2 30.14 30.49 29.73 29.96 30.35 29.86
3 30.17 30.45 28.95 29.30 29.97 29.24
4 29.81 30.14 30.40 28.94 29.85 30.07
5 29.50 30.24 30.41 29.41 30.04 29.98
6 29.98 30.06 29.86 29.28 30.08 29.99
7 30.33 30.40 29.82 29.07 30.27 28.80
8 29.64 30.01 30.45 29.21 30.12 29.41
9 29.93 30.15 30.48 29.23 30.04 30.31
10 30.13 30.53 30.56 29.24 30.34 30.25
CV
0.85% 0.63% 1.68% 0.94% 0.61% 1.62%
value
Sample Respiratory
virus
1 29.93 31.27 29.85 29.79 31.02 29.63
2 29.75 31.05 29.34 30.10 30.26 29.10
3 29.71 31.21 29.95 29.74 30.78 29.51
4 29.58 30.95 29.64 29.34 30.43 29.11
5 28.81 30.82 29.65 29.65 31.00 29.44
6 28.84 30.26 30.18 29.11 30.81 29.02
7 28.89 30.41 29.52 28.88 30.24 29.20
8 28.98 30.25 29.77 28.81 30.16 29.23
9 28.87 30.68 29.79 29.04 30.15 29.34
10 29.09 30.11 29.66 29.03 30.21 29.37
CV
1.51% 1.38% 0.78% 1.51% 1.17% 0.67%
value

Table 1-14 Ct value of imprecision test among three batches of reagents
Sample Respiratory
virus
1 29.38 31.57 30.93 29.95 31.48 30.30
2 29.99 31.60 30.58 30.21 30.94 29.30
3 29.15 31.67 30.72 29.85 31.19 29.31
4 29.43 31.62 30.50 29.84 30.78 30.28
5 28.87 31.59 31.32 29.83 31.00 30.54
6 29.05 31.31 31.35 29.41 30.96 29.89
7 29.02 30.73 30.71 29.19 30.92 30.33
8 28.86 30.92 30.86 28.93 30.66 30.44
9 29.10 31.07 30.81 29.16 30.87 30.15
10 28.88 30.95 30.71 29.00 30.48 30.53
11 29.83 30.43 30.29 29.51 30.39 29.93
12 30.14 30.49 29.73 29.96 30.35 29.86
13 30.17 30.45 28.95 29.30 29.97 29.24
14 29.81 30.14 30.40 28.94 29.85 30.07
15 29.50 30.24 30.41 29.41 30.04 29.98
16 29.98 30.06 29.86 29.28 30.08 29.99
17 30.33 30.40 29.82 29.07 30.27 28.80
18 29.64 30.01 30.45 29.21 30.12 29.41
19 29.93 30.15 30.48 29.23 30.04 30.31
20 30.13 30.53 30.56 29.24 30.34 30.25
21 29.93 31.27 29.85 29.79 31.02 29.63
22 29.75 31.05 29.34 30.10 30.26 29.10
23 29.71 31.21 29.95 29.74 30.78 29.51
24 29.58 30.95 29.64 29.34 30.43 29.11
25 28.81 30.82 29.65 29.65 31.00 29.44
26 28.84 30.26 30.18 29.11 30.81 29.02
27 28.89 30.41 29.52 28.88 30.24 29.20
28 28.98 30.25 29.77 28.81 30.16 29.23
29 28.87 30.68 29.79 29.04 30.15 29.34
30 29.09 30.11 29.66 29.03 30.21 29.37
CV
1.68% 1.74% 1.94% 1.35% 1.39% 1.73%
value
It can be seen from the above results that, the accuracy of the three
batches of kits to detect clinical samples is 100%, the CV of detected Ct values

2.2.1 Intra-day Imprecision
shown in Figure 1-13 to Figure 1-18 in the intra-batch precision experiment;
the precision detection test results under different operators are shown in
Figure 1-19 to Figure 1-24 (only the amplification curve of the 2017001 batch
of reagents was shown):
Figure 1-19 Intra-day imprecision test results of positive samples for influenza A virus
(2017001 batch)
Figure 1-20 Intra-day imprecision test results of positive samples for influenza B virus
(2017001 batch)

Figure 1-21 Intra-day imprecision test results of positive samples for respiratory syncytial
virus (2017001 batch)
Figure 1-22 Intra-day imprecision test results of positive samples for Adenovirus
(2017001 batch)

Figure 1-23 Intra-day imprecision test results of positive samples for Human rhinovirus
(2017001 batch)
Figure 1-24 Intra-day imprecision test results of positive samples for Mycoplasma
pneumoniae (2017001 batch)
Sample Respiratory
virus
1 29.38 31.57 30.93 29.95 31.48 30.30
2 29.99 31.60 30.58 30.21 30.94 29.30
3 29.15 31.67 30.72 29.85 31.19 29.31
4 29.43 31.62 30.50 29.84 30.78 30.28
5 28.87 31.59 31.32 29.83 31.00 30.54

6 29.05 31.31 31.35 29.41 30.96 29.89
7 29.02 30.73 30.71 29.19 30.92 30.33
8 28.86 30.92 30.86 28.93 30.66 30.44
9 29.10 31.07 30.81 29.16 30.87 30.15
10 28.88 30.95 30.71 29.00 30.48 30.53
11 29.22 30.26 29.87 28.21 30.34 30.33
12 28.86 30.63 30.21 28.44 30.26 29.94
13 29.44 29.98 30.14 28.28 30.08 30.16
14 28.51 30.10 30.00 28.20 29.70 29.75
15 29.21 30.07 30.00 28.41 29.98 29.74
16 29.28 30.18 30.31 28.53 30.12 29.89
17 29.38 30.26 30.34 28.58 30.16 30.09
18 28.78 30.33 30.27 28.55 30.03 29.81
19 29.18 30.01 30.36 28.60 29.95 29.49
20 29.58 30.83 30.97 29.01 30.35 29.94
CV
1.13% 2.00% 1.38% 2.22% 1.59 % 1.24%
value
Sample Respiratory
virus
1 29.83 30.43 30.29 29.51 30.39 29.93
2 30.14 30.49 29.73 29.96 30.35 29.86
3 30.17 30.45 28.95 29.30 29.97 29.24
4 29.81 30.14 30.40 28.94 29.85 30.07
5 29.50 30.24 30.41 29.41 30.04 29.98
6 29.98 30.06 29.86 29.28 30.08 29.99
7 30.33 30.40 29.82 29.07 30.27 28.80
8 29.64 30.01 30.45 29.21 30.12 29.41
9 29.93 30.15 30.48 29.23 30.04 30.31
10 30.13 30.53 30.56 29.24 30.34 30.25
11 29.98 31.42 29.86 29.18 30.77 29.42
12 29.10 31.31 29.55 29.51 30.42 29.24
13 29.68 31.16 30.28 29.13 30.63 28.89
14 28.99 30.32 29.66 29.28 30.51 28.32
15 28.54 31.32 30.11 29.31 30.58 29.38
16 28.25 30.90 30.44 28.83 30.27 28.52
17 28.57 29.53 29.61 28.59 29.79 28.33
18 28.76 30.18 30.11 28.56 30.25 29.26
19 28.46 30.13 29.84 28.54 29.97 29.39
20 28.78 30.07 29.58 28.79 30.19 29.56
CV
2.29% 1.67% 1.40% 1.22% 0.87% 2.06%
value

Sample Respiratory
virus
1 29.93 31.27 29.85 29.79 31.02 29.63
2 29.75 31.05 29.34 30.10 30.26 29.10
3 29.71 31.21 29.95 29.74 30.78 29.51
4 29.58 30.95 29.64 29.34 30.43 29.11
5 28.81 30.82 29.65 29.65 31.00 29.44
6 28.84 30.26 30.18 29.11 30.81 29.02
7 28.89 30.41 29.52 28.88 30.24 29.20
8 28.98 30.25 29.77 28.81 30.16 29.23
9 28.87 30.68 29.79 29.04 30.15 29.34
10 29.09 30.11 29.66 29.03 30.21 29.37
11 28.27 29.57 29.44 28.22 29.57 29.20
12 28.92 27.47 29.38 28.63 28.30 29.54
13 28.95 29.83 29.51 28.24 29.70 29.57
14 28.47 27.91 28.58 27.95 28.92 29.65
15 29.33 29.67 29.80 28.44 29.81 28.85
16 29.11 29.78 29.94 28.23 26.49 29.66
17 29.17 29.69 29.36 28.45 29.88 29.04
18 29.17 29.89 29.64 28.62 28.56 29.59
19 29.07 29.61 29.91 28.38 29.73 29.47
20 29.27 29.90 30.12 28.93 29.68 29.61
CV
1.41% 3.24% 1.18% 2.08% 3.59% 0.84%
value

shown in Figure 1-13 to Figure 1-18; the precision test results performed by
the same operators on alternate days are shown in Figure 1-25 to Figure 1-30
(only the amplification curve of the 2017001 batch of reagents was shown):

Figure 1-25 Inter-day imprecision test results of positive samples for influenza A virus
(2017001 batch)
Figure 1-26 Inter-day imprecision test results of positive samples for influenza B virus
(2017001 batch)

Figure 1-27 Inter-day imprecision test results of positive samples for respiratory syncytial
Figure 1-28 Inter-day imprecision test results of positive samples for Adenovirus
(2017001 batch)

Figure 1-29 Inter-day imprecision test results of positive samples for Human rhinovirus
(2017001 batch)
Figure 1-30 Inter-day imprecision test results of positive samples for Mycoplasma
Sample Respiratory
virus
1 29.38 31.57 30.93 29.95 31.48 30.30
2 29.99 31.60 30.58 30.21 30.94 29.30
3 29.15 31.67 30.72 29.85 31.19 29.31
4 29.43 31.62 30.50 29.84 30.78 30.28
5 28.87 31.59 31.32 29.83 31.00 30.54

6 29.05 31.31 31.35 29.41 30.96 29.89
7 29.02 30.73 30.71 29.19 30.92 30.33
8 28.86 30.92 30.86 28.93 30.66 30.44
9 29.10 31.07 30.81 29.16 30.87 30.15
10 28.88 30.95 30.71 29.00 30.48 30.53
11 30.30 31.26 29.63 29.51 31.00 29.40
12 29.51 30.96 28.87 29.61 30.42 29.24
13 28.99 31.28 29.33 29.34 30.87 28.32
14 29.43 31.05 29.45 29.36 30.40 28.40
15 28.44 31.07 29.88 29.32 31.11 29.17
16 28.57 30.60 29.99 28.99 30.73 28.95
17 28.67 30.63 29.40 28.78 30.40 28.70
18 28.73 30.53 29.37 28.70 30.39 29.32
19 28.80 30.47 29.80 28.50 30.27 29.08
20 28.48 30.28 29.70 28.70 30.05 29.14
CV
1.65% 1.37% 2.43% 1.62% 1.16% 2.41%
value
Sample Respiratory
virus
1 29.83 30.43 30.29 29.51 30.39 29.93
2 30.14 30.49 29.73 29.96 30.35 29.86
3 30.17 30.45 28.95 29.30 29.97 29.24
4 29.81 30.14 30.40 28.94 29.85 30.07
5 29.50 30.24 30.41 29.41 30.04 29.98
6 29.98 30.06 29.86 29.28 30.08 29.99
7 30.33 30.40 29.82 29.07 30.27 28.80
8 29.64 30.01 30.45 29.21 30.12 29.41
9 29.93 30.15 30.48 29.23 30.04 30.31
10 30.13 30.53 30.56 29.24 30.34 30.25
11 29.30 30.63 29.93 28.32 30.44 29.91
12 29.24 30.91 30.12 28.54 30.56 30.07
13 29.63 30.56 30.01 28.29 29.98 29.79
14 28.72 30.22 29.88 28.04 30.47 29.79
15 29.06 30.47 30.02 28.53 30.15 29.65
16 28.98 30.85 30.29 28.73 30.43 28.39
17 29.79 30.94 29.83 28.58 30.61 28.84
18 29.23 30.60 29.67 28.76 30.14 29.22
19 28.75 30.52 30.00 28.68 30.36 29.41
20 29.48 31.02 29.99 29.17 30.78 29.66
CV
1.62% 1.00% 1.24% 1.66% 0.81% 1.74%
value

Sample Respiratory
virus
1 29.93 31.27 29.85 29.79 31.02 29.63
2 29.75 31.05 29.34 30.10 30.26 29.10
3 29.71 31.21 29.95 29.74 30.78 29.51
4 29.58 30.95 29.64 29.34 30.43 29.11
5 28.81 30.82 29.65 29.65 31.00 29.44
6 28.84 30.26 30.18 29.11 30.81 29.02
7 28.89 30.41 29.52 28.88 30.24 29.20
8 28.98 30.25 29.77 28.81 30.16 29.23
9 28.87 30.68 29.79 29.04 30.15 29.34
10 29.09 30.11 29.66 29.03 30.21 29.37
11 29.40 31.08 30.24 29.60 30.87 29.72
12 29.46 31.04 30.02 29.65 30.43 29.84
13 29.59 31.27 30.00 29.57 30.90 30.06
14 29.05 30.96 30.20 29.58 30.24 29.78
15 28.85 30.95 30.39 29.42 30.83 30.27
16 28.62 30.34 30.75 28.93 30.70 29.62
17 28.91 30.53 30.12 28.76 30.19 29.30
18 28.84 30.51 30.32 28.69 30.03 29.56
19 29.18 30.53 30.05 28.61 30.11 30.15
20 29.31 30.60 30.36 28.65 30.10 30.05
CV
1.30% 1.19% 1.15% 1.53% 1.14% 1.26%
value
Conclusion: It can be seen from the above results that, the accuracy of the
three batches of kits to detect clinical samples is 100%, the CV of detected Ct
values of inter-day imprecision test among the three batched of kits by
different operators range from 0.84% to 3.59%; the CV of detected Ct values of
intra-day imprecision test among the three batched of kits by the same
operators range from 0.81% to 2.43%, and both of the values are less than 5%.
Therefore, it can be considered that this kit has good detection precision
under different operators and at different times.
3. Precision test of national reference samples

The precision reference samples CV1 and CV2 in the national reference
samples (batch number 370006-201501) of nucleic acid detection reagents for
influenza A/B virus were tested by using three batches of kits. Three batches
of qualified detection kits in this study were tested on the ABI 7500
instrument to verify the precision performance of the three batches of kits on
the national reference samples. Each sample was extracted 10 times each time
and was repeatedly tested 10 times and the CV of Ct values were calculated.

Figure 1-31 The test of national precision reference sample CV1 (2017001-2017003 batch)
Table 1-21 Ct value of precision test of the three batches of kits on the national reference
samples CV1 and CV2
Samples National reference samples

Batch No.
No. CV1 CV2
1 30.94 31.56
2 30.16 30.26
3 30.94 30.78
4 30.06 30.19
5 2017001 31.37 29.07
6 31.80 29.22
7 31.92 29.82
8 31.59 29.40
9 31.75 29.54

10 31.97 29.36
11 29.85 30.77
12 29.89 30.42
13 29.93 30.25
14 29.70 29.86
15 29.90 29.67
2017002
16 29.94 29.58
17 30.47 29.57
18 30.19 29.52
19 31.16 29.60
20 30.06 29.39
21 30.35 29.14
22 29.20 29.04
23 29.82 29.30
24 29.31 29.26
25 28.86 28.87
2017003
26 29.57 28.85
27 29.82 29.40
28 29.55 28.59
29 29.99 29.21
30 30.14 28.62
CV value 2.83% 2.28%
three batches of kits to detect the precision reference samples CV1 and CV2 in
the national reference samples of nucleic acid detection reagents for influenza
A/B virus is 100%, and the CV of detected Ct values of precision between
batches are 2.83% and 2.28%, which are both lower than 5%. It meets the
requirements of national reference sample detection. Therefore, it can be
considered that this kit has good detection precision on national precision
reference samples.

QuantStudioTM 5
Analytical performance evaluation onSLAN-96P automatic

medical PCR analysis system
Precision evaluation
Precision refers to the degree to which multiple parallel test results are
close to each other under the same conditions. The closer the test values, the
higher the precision. Precision reflects the repeatability of the kit operation.

In this experiment, 6 precision reference samples of six respiratory
pathogens nucleic acid diagnostic kits were selected for the imprecision
determination of the kit. Among the 6 reference samples, R1 and R2 are
composed of lentiviral particles containing conserved sequences of influenza
A virus and influenza B virus. The lentivirus of each pathogen is diluted with
normal saline to 5.0 × 104 TU/mL and 5.0 × 103 TU/mL. Each pathogen
lentivirus at the same concentration is mixed in equal volume to R1 and R2.
R3 and R4 are composed of lentiviral particles containing the conserved
sequence of the respiratory syncytial virus. The pathogen lentivirus is diluted
with normal saline to 2.5 × 104 TU/mL and 2.5 × 103 TU/mL to prepare R3 and
R4. R5 and R6 are consisted of cloning plasmids containing adenovirus and
Mycoplasma pneumoniae and lentiviral particles containing the target
conserved sequences of human rhinovirus. The pathogen cloning plasmids
were diluted with normal saline to 5.0 × 104 copise/mL and 5.0 × 103
copise/mL.The lentiviral particles containing the target conserved sequence of
human rhinovirus were diluted with normal saline to 5.0 × 104 TU/mL and 5.0
× 103 TU/mL. The cloning plasmid and lentiviral particles of each pathogen at
the same concentration were mixed in equal volume to R5 and R6. Each
sample was repeatedly tested 10 times, and the detection rate of reference
samples R1, R3 and R5 at medium concentration and reference samples R2,
R4 and R6 at medium concentration were counted and the coefficient of
variance of the detected Ct values of R1-R6 were counted to judge the
precision of this kit.

Figure 2-1 Imprecision test results of negative control within batches (2017001 batch)

Figure 2-2 Imprecision test results of R1 and R2 within batches (2017001 batch)
ect FA FA FA FA
No. M M M M
20.9 23.0 24.9 27.2 24.3 27.9 21.5 23.5 23.3 24.9 27.1 27.6
1
7 9 0 0 5 1 1 6 3 4 0 8
2 20.9 23.0 24.8 27.2 24.2 27.9 21.4 23.5 23.2 24.7 26.9 27.5

4 8 5 0 1 0 7 0 1 0 5 0
21.0 23.0 24.7 26.9 24.2 28.0 21.4 23.4 23.1 24.8 27.1 27.4
3
2 8 2 9 6 5 5 7 7 7 1 3
21.3 23.0 24.8 27.1 24.2 27.7 21.3 23.4 23.1 24.8 26.8 27.3
4
0 0 0 9 6 7 7 8 7 4 7 8
21.0 23.1 24.8 27.2 24.0 27.6 21.4 23.5 23.2 24.9 27.0 27.6
5
9 4 7 8 3 8 4 9 4 4 3 9
21.0 23.1 24.8 27.2 24.1 27.6 21.4 23.5 23.2 24.8 27.0 27.4
6
6 4 2 2 7 4 2 8 4 7 2 1
21.0 23.1 24.9 27.1 24.1 27.6 21.5 23.5 23.2 24.9 26.9 27.4
7
8 8 1 7 9 2 6 9 7 2 8 5
20.9 23.1 24.9 27.1 24.2 27.5 21.5 23.5 23.2 25.1 27.1 27.5
8
7 1 5 8 2 5 0 6 1 1 8 4
20.9 23.1 24.8 27.2 24.1 27.7 21.5 23.5 23.2 24.8 27.0 27.4
9
4 2 9 2 9 3 1 6 2 8 7 7
20.9 23.1 24.8 27.2 24.2 27.7 21.5 23.5 23.2 24.8 27.0 27.5
10
6 4 6 8 4 4 3 6 3 9 8 6
0.52 0.21 0.26 0.30 0.34 0.55 0.27 0.19 0.20 0.41 0.33 0.39
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
20.4 23.1 24.6 27.0 23.7 27.6 21.4 23.4 23.2 26.9 26.7 26.9
1
6 1 8 3 0 2 8 7 0 7 6 7
20.4 23.0 24.6 27.0 23.7 27.5 21.5 23.4 23.2 27.0 26.7 27.0
2
4 4 3 1 0 3 1 1 4 0 3 0
20.5 23.0 24.6 27.0 23.7 27.4 21.4 23.3 23.1 27.0 26.8 27.0
3
1 6 4 0 5 6 9 6 9 2 1 2
20.5 23.0 24.6 27.0 23.5 27.5 20.9 23.3 23.1 26.9 26.7 26.9
4
9 4 1 0 6 5 2 6 3 4 8 4
20.3 22.4 24.6 27.0 23.7 27.5 21.5 23.4 23.2 26.9 26.7 26.9
5
3 7 3 4 0 9 3 7 8 4 6 4
20.7 23.0 24.6 27.0 23.6 27.5 21.0 23.3 23.1 26.9 26.7 26.9
6
9 4 1 3 3 9 1 2 9 3 1 3
20.7 23.0 24.6 26.9 23.5 27.6 21.4 23.3 23.2 26.8 26.6 26.8
7
0 2 2 9 7 4 6 4 0 8 2 8
20.7 23.0 24.6 27.0 23.5 27.6 21.5 23.4 23.2 27.0 26.7 27.0
8
1 4 4 1 7 5 2 5 0 2 6 2
20.4 23.0 24.6 26.9 23.7 27.5 21.4 23.4 23.2 27.0 26.8 27.0
9
3 5 8 9 1 1 7 7 4 3 6 3
20.4 23.0 24.6 27.0 23.6 27.4 21.5 23.4 23.3 26.9 26.8 26.9
10
9 3 6 3 7 8 8 4 0 7 1 7
0.72 0.80 0.10 0.07 0.29 0.24 1.08 0.25 0.21 0.18 0.24 0.18
CV
% % % % % % % % % % % %

ect FA FA FA FA
No. M M M M
21.6 22.8 25.5 26.3 23.6 26.9 21.4 22.7 23.3 25.3 26.2 27.2
1
3 7 7 7 2 6 6 1 2 4 9 0
21.6 22.8 25.6 26.4 23.6 27.1 21.4 22.7 23.2 25.3 26.3 27.2
2
2 9 1 0 2 2 8 2 4 2 0 6
21.6 22.9 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.3 26.3 27.2
3
4 0 6 8 2 8 6 0 6 6 2 5
21.6 22.8 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.5 26.3 27.2
4
1 6 6 7 1 4 6 2 1 3 1 3
21.6 22.8 25.6 26.4 23.6 27.0 21.4 22.7 23.2 25.4 26.3 27.3
5
2 9 7 2 2 4 5 3 9 7 2 2
21.6 22.8 25.5 26.3 23.5 27.0 21.4 22.7 23.3 25.4 26.2 27.1
6
4 5 6 4 9 9 8 2 0 4 8 6
21.6 22.8 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.4 26.3 27.1
7
2 9 7 2 3 0 7 5 9 1 0 9
21.5 22.8 25.5 26.2 23.6 27.0 21.5 22.7 23.2 25.3 26.2 27.2
8
7 6 8 6 3 5 1 5 9 9 5 0
21.6 22.9 25.5 26.3 23.5 27.0 21.4 22.7 23.3 25.4 26.5 27.3
9
5 1 4 1 9 6 7 2 3 9 8 9
21.6 22.8 25.3 26.3 23.6 27.1 21.4 22.7 23.3 25.3 26.2 27.2
10
2 9 9 2 0 4 9 3 5 0 9 4
0.10 0.09 0.28 0.18 0.06 0.23 0.08 0.07 0.18 0.30 0.35 0.25
CV
% % % % % % % % % % % %

Table 2-4 Imprecision test results among three batches of reagents
ect FA FA FA FA
No. M M M M
20.9 23.0 24.9 27.2 24.3 27.9 21.5 23.5 23.3 24.9 27.1 27.6
1
7 9 0 0 5 1 1 6 3 4 0 8
20.9 23.0 24.8 27.2 24.2 27.9 21.4 23.5 23.2 24.7 26.9 27.5
2
4 8 5 0 1 0 7 0 1 0 5 0
21.0 23.0 24.7 26.9 24.2 28.0 21.4 23.4 23.1 24.8 27.1 27.4
3
2 8 2 9 6 5 5 7 7 7 1 3
21.3 23.0 24.8 27.1 24.2 27.7 21.3 23.4 23.1 24.8 26.8 27.3
4
0 0 0 9 6 7 7 8 7 4 7 8
21.0 23.1 24.8 27.2 24.0 27.6 21.4 23.5 23.2 24.9 27.0 27.6
5
9 4 7 8 3 8 4 9 4 4 3 9

21.0 23.1 24.8 27.2 24.1 27.6 21.4 23.5 23.2 24.8 27.0 27.4
6
6 4 2 2 7 4 2 8 4 7 2 1
21.0 23.1 24.9 27.1 24.1 27.6 21.5 23.5 23.2 24.9 26.9 27.4
7
8 8 1 7 9 2 6 9 7 2 8 5
20.9 23.1 24.9 27.1 24.2 27.5 21.5 23.5 23.2 25.1 27.1 27.5
8
7 1 5 8 2 5 0 6 1 1 8 4
20.9 23.1 24.8 27.2 24.1 27.7 21.5 23.5 23.2 24.8 27.0 27.4
9
4 2 9 2 9 3 1 6 2 8 7 7
20.9 23.1 24.8 27.2 24.2 27.7 21.5 23.5 23.2 24.8 27.0 27.5
10
6 4 6 8 4 4 3 6 3 9 8 6
20.4 23.1 24.6 27.0 23.7 27.6 21.4 23.4 23.2 26.9 26.7 26.9
11
6 1 8 3 0 2 8 7 0 7 6 7
20.4 23.0 24.6 27.0 23.7 27.5 21.5 23.4 23.2 27.0 26.7 27.0
12
4 4 3 1 0 3 1 1 4 0 3 0
20.5 23.0 24.6 27.0 23.7 27.4 21.4 23.3 23.1 27.0 26.8 27.0
13
1 6 4 0 5 6 9 6 9 2 1 2
20.5 23.0 24.6 27.0 23.5 27.5 20.9 23.3 23.1 26.9 26.7 26.9
14
9 4 1 0 6 5 2 6 3 4 8 4
20.3 22.4 24.6 27.0 23.7 27.5 21.5 23.4 23.2 26.9 26.7 26.9
15
3 7 3 4 0 9 3 7 8 4 6 4
20.7 23.0 24.6 27.0 23.6 27.5 21.0 23.3 23.1 26.9 26.7 26.9
16
9 4 1 3 3 9 1 2 9 3 1 3
20.7 23.0 24.6 26.9 23.5 27.6 21.4 23.3 23.2 26.8 26.6 26.8
17
0 2 2 9 7 4 6 4 0 8 2 8
20.7 23.0 24.6 27.0 23.5 27.6 21.5 23.4 23.2 27.0 26.7 27.0
18
1 4 4 1 7 5 2 5 0 2 6 2
20.4 23.0 24.6 26.9 23.7 27.5 21.4 23.4 23.2 27.0 26.8 27.0
19
3 5 8 9 1 1 7 7 4 3 6 3
20.4 23.0 24.6 27.0 23.6 27.4 21.5 23.4 23.3 26.9 26.8 26.9
20
9 3 6 3 7 8 8 4 0 7 1 7
21.6 22.8 25.5 26.3 23.6 26.9 21.4 22.7 23.3 25.3 26.2 27.2
21
3 7 7 7 2 6 6 1 2 4 9 0
21.6 22.8 25.6 26.4 23.6 27.1 21.4 22.7 23.2 25.3 26.3 27.2
22
2 9 1 0 2 2 8 2 4 2 0 6
21.6 22.9 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.3 26.3 27.2
23
4 0 6 8 2 8 6 0 6 6 2 5
21.6 22.8 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.5 26.3 27.2
24
1 6 6 7 1 4 6 2 1 3 1 3
21.6 22.8 25.6 26.4 23.6 27.0 21.4 22.7 23.2 25.4 26.3 27.3
25
2 9 7 2 2 4 5 3 9 7 2 2
21.6 22.8 25.5 26.3 23.5 27.0 21.4 22.7 23.3 25.4 26.2 27.1
26
4 5 6 4 9 9 8 2 0 4 8 6
21.6 22.8 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.4 26.3 27.1
27
2 9 7 2 3 0 7 5 9 1 0 9
21.5 22.8 25.5 26.2 23.6 27.0 21.5 22.7 23.2 25.3 26.2 27.2
28
7 6 8 6 3 5 1 5 9 9 5 0
21.6 22.9 25.5 26.3 23.5 27.0 21.4 22.7 23.3 25.4 26.5 27.3
29
5 1 4 1 9 6 7 2 3 9 8 9

21.6 22.8 25.3 26.3 23.6 27.1 21.4 22.7 23.3 25.3 26.2 27.2
30
2 9 9 2 0 4 9 3 5 0 9 4
2.18 0.62 1.61 1.39 1.19 1.10 0.64 1.58 0.24 3.50 1.16 0.87
CV
% % % % % % % % % % % %

1.2.1 Intra-day imprecision
shown in Figure 2-1 to Figure 2-4 in the intra-batch precision experiment; the
precision detection test results under different operators are shown in Figure
2-5 to Figure 2-8 (only the amplification curve of the 2017001 batch of reagents
was shown):
Figure 2-5 Intra-day imprecision test results of negative control (2017001 batch)

ect FA FA FA FA
No. M M M M
20.9 23.0 24.9 27.2 24.3 27.9 21.5 23.5 23.3 24.9 27.1 27.6
1
7 9 0 0 5 1 1 6 3 4 0 8
20.9 23.0 24.8 27.2 24.2 27.9 21.4 23.5 23.2 24.7 26.9 27.5
2
4 8 5 0 1 0 7 0 1 0 5 0
21.0 23.0 24.7 26.9 24.2 28.0 21.4 23.4 23.1 24.8 27.1 27.4
3
2 8 2 9 6 5 5 7 7 7 1 3
21.3 23.0 24.8 27.1 24.2 27.7 21.3 23.4 23.1 24.8 26.8 27.3
4
0 0 0 9 6 7 7 8 7 4 7 8
21.0 23.1 24.8 27.2 24.0 27.6 21.4 23.5 23.2 24.9 27.0 27.6
5
9 4 7 8 3 8 4 9 4 4 3 9
21.0 23.1 24.8 27.2 24.1 27.6 21.4 23.5 23.2 24.8 27.0 27.4
6
6 4 2 2 7 4 2 8 4 7 2 1
21.0 23.1 24.9 27.1 24.1 27.6 21.5 23.5 23.2 24.9 26.9 27.4
7
8 8 1 7 9 2 6 9 7 2 8 5
8 20.9 23.1 24.9 27.1 24.2 27.5 21.5 23.5 23.2 25.1 27.1 27.5

7 1 5 8 2 5 0 6 1 1 8 4
20.9 23.1 24.8 27.2 24.1 27.7 21.5 23.5 23.2 24.8 27.0 27.4
9
4 2 9 2 9 3 1 6 2 8 7 7
20.9 23.1 24.8 27.2 24.2 27.7 21.5 23.5 23.2 24.8 27.0 27.5
10
6 4 6 8 4 4 3 6 3 9 8 6
22.1 22.6 25.9 26.1 23.0 26.4 22.0 22.6 22.7 25.8 25.9 26.5
11
8 9 1 2 9 4 3 0 0 1 8 3
22.1 22.6 25.9 26.1 23.1 26.4 22.0 22.5 22.7 25.7 25.9 26.4
12
5 5 4 0 9 8 3 9 5 1 7 1
22.1 22.6 25.9 26.1 23.1 26.4 22.0 22.5 22.7 25.7 26.0 26.4
13
8 7 0 3 3 6 4 5 9 2 5 6
22.1 22.6 25.9 26.1 23.0 26.4 22.0 22.5 22.7 25.7 26.0 26.5
14
7 8 2 2 5 1 0 8 3 0 7 3
22.2 22.6 25.9 26.1 23.0 26.6 22.0 22.5 22.7 25.6 26.0 26.4
15
0 9 2 2 8 3 2 3 2 9 4 4
22.2 22.6 25.8 26.0 23.0 26.5 22.0 22.5 22.6 25.7 26.0 26.5
16
0 7 3 5 8 3 0 0 9 6 5 7
22.2 22.7 25.8 26.0 23.1 26.5 21.9 22.4 22.6 25.6 26.0 26.5
17
2 1 3 4 1 0 9 9 8 8 1 5
22.2 22.7 25.7 25.9 23.1 26.4 22.0 22.6 22.6 25.6 26.0 26.5
18
1 1 7 9 8 5 2 3 8 9 0 1
22.2 22.6 25.9 26.1 23.1 26.4 22.0 22.5 22.6 25.6 26.0 26.5
19
1 9 1 0 2 8 2 2 3 9 1 5
22.2 22.6 25.8 26.0 23.1 26.4 22.0 22.5 22.7 25.6 26.0 26.5
20
0 9 7 7 0 8 3 2 3 6 0 1
2.77 0.96 2.08 2.15 2.40 2.44 1.29 2.22 1.18 1.68 1.99 1.93
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
20.4 23.1 24.6 27.0 23.7 27.6 21.4 23.4 23.2 26.9 26.7 26.9
1
6 1 8 3 0 2 8 7 0 7 6 7
20.4 23.0 24.6 27.0 23.7 27.5 21.5 23.4 23.2 27.0 26.7 27.0
2
4 4 3 1 0 3 1 1 4 0 3 0
20.5 23.0 24.6 27.0 23.7 27.4 21.4 23.3 23.1 27.0 26.8 27.0
3
1 6 4 0 5 6 9 6 9 2 1 2
20.5 23.0 24.6 27.0 23.5 27.5 20.9 23.3 23.1 26.9 26.7 26.9
4
9 4 1 0 6 5 2 6 3 4 8 4
20.3 22.4 24.6 27.0 23.7 27.5 21.5 23.4 23.2 26.9 26.7 26.9
5
3 7 3 4 0 9 3 7 8 4 6 4
20.7 23.0 24.6 27.0 23.6 27.5 21.0 23.3 23.1 26.9 26.7 26.9
6
9 4 1 3 3 9 1 2 9 3 1 3
20.7 23.0 24.6 26.9 23.5 27.6 21.4 23.3 23.2 26.8 26.6 26.8
7
0 2 2 9 7 4 6 4 0 8 2 8
8 20.7 23.0 24.6 27.0 23.5 27.6 21.5 23.4 23.2 27.0 26.7 27.0

1 4 4 1 7 5 2 5 0 2 6 2
20.4 23.0 24.6 26.9 23.7 27.5 21.4 23.4 23.2 27.0 26.8 27.0
9
3 5 8 9 1 1 7 7 4 3 6 3
20.4 23.0 24.6 27.0 23.6 27.4 21.5 23.4 23.3 26.9 26.8 26.9
10
9 3 6 3 7 8 8 4 0 7 1 7
22.2 22.5 25.8 26.0 24.3 27.7 22.1 22.4 23.9 25.8 26.0 27.6
11
3 8 6 8 6 0 4 1 2 1 7 8
22.2 22.5 25.9 26.0 24.3 27.8 22.1 22.3 23.9 25.8 26.0 27.7
12
4 6 5 5 6 2 0 7 8 0 7 2
22.2 22.5 25.9 26.0 24.3 27.7 22.1 22.3 23.9 25.7 26.0 27.7
13
4 8 8 9 9 3 1 2 6 1 7 7
22.2 22.5 25.9 26.0 24.3 27.6 22.1 22.3 23.9 25.7 26.0 27.7
14
7 7 2 2 6 8 1 3 8 5 9 3
22.3 22.6 25.9 26.0 24.3 27.7 22.1 22.3 23.9 25.7 26.0 27.8
15
4 0 2 8 7 6 6 7 3 4 5 3
22.3 22.6 25.9 26.0 24.3 27.7 22.1 22.3 23.9 25.8 26.0 27.8
16
3 0 0 7 2 9 6 0 4 1 6 3
22.3 22.6 25.9 26.1 24.3 27.7 22.0 22.3 23.9 25.8 26.0 27.8
17
2 0 3 0 2 1 3 3 3 0 6 0
22.3 22.6 25.8 26.1 24.2 27.6 22.1 22.3 23.8 25.7 26.0 27.7
18
2 1 9 0 8 9 0 5 9 6 6 5
22.2 22.5 26.0 26.1 21.7 27.7 22.1 22.3 23.9 25.8 26.0 27.8
19
3 7 0 0 7 1 1 4 8 1 9 7
22.2 22.5 25.9 26.0 24.3 27.7 22.1 22.3 23.9 25.7 26.0 27.6
20
2 7 9 6 8 2 0 7 7 6 2 9
4.17 1.07 2.63 1.81 2.54 0.37 1.84 2.39 1.60 2.33 1.36 1.51
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
21.6 22.8 25.5 26.3 23.6 26.9 21.4 22.7 23.3 25.3 26.2 27.2
1
3 7 7 7 2 6 6 1 2 4 9 0
21.6 22.8 25.6 26.4 23.6 27.1 21.4 22.7 23.2 25.3 26.3 27.2
2
2 9 1 0 2 2 8 2 4 2 0 6
21.6 22.9 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.3 26.3 27.2
3
4 0 6 8 2 8 6 0 6 6 2 5
21.6 22.8 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.5 26.3 27.2
4
1 6 6 7 1 4 6 2 1 3 1 3
21.6 22.8 25.6 26.4 23.6 27.0 21.4 22.7 23.2 25.4 26.3 27.3
5
2 9 7 2 2 4 5 3 9 7 2 2
21.6 22.8 25.5 26.3 23.5 27.0 21.4 22.7 23.3 25.4 26.2 27.1
6
4 5 6 4 9 9 8 2 0 4 8 6
21.6 22.8 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.4 26.3 27.1
7
2 9 7 2 3 0 7 5 9 1 0 9
8 21.5 22.8 25.5 26.2 23.6 27.0 21.5 22.7 23.2 25.3 26.2 27.2

7 6 8 6 3 5 1 5 9 9 5 0
21.6 22.9 25.5 26.3 23.5 27.0 21.4 22.7 23.3 25.4 26.5 27.3
9
5 1 4 1 9 6 7 2 3 9 8 9
21.6 22.8 25.3 26.3 23.6 27.1 21.4 22.7 23.3 25.3 26.2 27.2
10
2 9 9 2 0 4 9 3 5 0 9 4
22.0 23.7 25.7 27.0 23.6 26.9 21.8 23.4 23.4 25.5 27.1 27.0
11
4 1 9 7 2 3 2 7 0 8 8 4
22.0 23.7 25.8 27.1 23.6 26.8 21.8 23.5 23.2 25.5 27.1 26.9
12
4 5 4 8 1 9 8 1 1 7 2 7
22.0 23.7 25.7 27.1 23.6 26.9 21.8 23.5 23.2 25.5 27.1 26.9
13
4 3 8 3 2 7 9 5 2 8 4 8
22.0 23.7 25.6 27.0 23.6 26.9 21.8 23.5 23.1 25.5 27.0 26.7
14
4 4 7 5 4 3 7 0 9 6 6 7
22.0 23.7 25.7 27.0 23.5 26.9 21.8 23.4 23.1 25.2 27.0 26.8
15
1 4 5 8 8 3 3 8 1 6 8 0
22.0 23.7 25.8 27.1 23.5 26.8 21.8 23.5 23.1 25.6 27.1 27.0
16
5 6 5 3 5 1 6 1 5 4 7 3
22.0 23.7 25.8 27.1 23.5 26.8 21.8 23.5 23.1 25.6 27.1 27.0
17
6 8 2 5 6 9 4 6 2 3 3 6
22.0 23.7 25.8 27.1 23.5 27.0 21.8 23.5 23.1 25.6 27.1 26.9
18
6 6 1 1 6 0 7 2 5 2 3 7
22.0 23.7 25.7 27.1 23.5 26.8 21.8 23.5 23.1 25.5 27.0 26.8
19
6 6 8 5 9 6 8 3 5 0 5 8
22.0 23.7 25.8 27.1 23.5 26.9 21.8 23.5 23.1 25.5 27.0 26.9
20
6 8 0 3 6 7 9 3 0 4 7 5
1.00 1.92 0.51 1.48 0.11 0.39 0.93 1.76 0.37 0.46 1.54 0.64
CV
% % % % % % % % % % % %

shown in Figure 2-1 to Figure 2-4; the precision detection test results
performed by the same operators on alternate days are shown in Figure 2-9 to
Figure 2-12 (only the amplification curve of the 2017001 batch of reagents was
shown):
Figure 2-9 Inter-day imprecision test results of negative control (2017001 batch)

ect FA FA FA FA
No. M M M M
20.9 23.0 24.9 27.2 24.3 27.9 21.5 23.5 23.3 24.9 27.1 27.6
1
7 9 0 0 5 1 1 6 3 4 0 8
20.9 23.0 24.8 27.2 24.2 27.9 21.4 23.5 23.2 24.7 26.9 27.5
2
4 8 5 0 1 0 7 0 1 0 5 0
3 21.0 23.0 24.7 26.9 24.2 28.0 21.4 23.4 23.1 24.8 27.1 27.4

2 8 2 9 6 5 5 7 7 7 1 3
21.3 23.0 24.8 27.1 24.2 27.7 21.3 23.4 23.1 24.8 26.8 27.3
4
0 0 0 9 6 7 7 8 7 4 7 8
21.0 23.1 24.8 27.2 24.0 27.6 21.4 23.5 23.2 24.9 27.0 27.6
5
9 4 7 8 3 8 4 9 4 4 3 9
21.0 23.1 24.8 27.2 24.1 27.6 21.4 23.5 23.2 24.8 27.0 27.4
6
6 4 2 2 7 4 2 8 4 7 2 1
21.0 23.1 24.9 27.1 24.1 27.6 21.5 23.5 23.2 24.9 26.9 27.4
7
8 8 1 7 9 2 6 9 7 2 8 5
20.9 23.1 24.9 27.1 24.2 27.5 21.5 23.5 23.2 25.1 27.1 27.5
8
7 1 5 8 2 5 0 6 1 1 8 4
20.9 23.1 24.8 27.2 24.1 27.7 21.5 23.5 23.2 24.8 27.0 27.4
9
4 2 9 2 9 3 1 6 2 8 7 7
20.9 23.1 24.8 27.2 24.2 27.7 21.5 23.5 23.2 24.8 27.0 27.5
10
6 4 6 8 4 4 3 6 3 9 8 6
21.1 23.0 24.5 26.3 24.0 28.1 21.0 22.9 23.2 24.5 26.4 26.8
11
0 8 4 8 2 9 9 7 9 2 4 8
21.1 23.0 24.5 26.4 24.0 27.4 21.0 23.0 23.3 24.5 26.5 26.7
12
4 9 5 2 8 1 8 3 7 1 1 9
21.1 23.1 24.5 26.4 24.0 27.3 21.0 23.0 23.3 24.5 26.4 26.9
13
1 0 6 1 1 5 9 0 8 4 7 4
21.1 23.1 24.5 26.4 24.0 27.4 21.1 22.9 23.4 24.5 26.5 26.7
14
1 0 7 5 5 6 0 9 0 5 0 9
21.1 23.1 24.5 26.4 24.0 27.4 21.0 22.9 23.5 24.4 26.4 26.8
15
0 1 8 7 3 2 6 7 1 8 3 4
21.1 23.1 24.6 26.3 24.0 27.3 21.0 23.0 23.4 24.5 26.4 26.8
16
0 1 6 9 1 8 5 0 1 4 6 8
21.1 23.1 20.0 26.2 24.0 27.3 21.0 23.0 23.4 24.5 26.4 26.8
17
2 1 3 5 0 0 7 3 3 2 4 3
21.1 23.1 24.5 26.3 24.0 27.5 21.1 23.0 23.4 24.5 26.4 26.9
18
3 1 6 8 1 8 0 2 4 4 1 2
21.1 23.1 24.6 26.4 23.7 27.3 21.1 23.0 23.4 24.5 26.4 26.8
19
2 1 0 2 7 9 0 2 0 8 3 6
21.1 23.0 24.5 26.4 24.0 27.3 21.0 23.0 23.4 24.3 26.7 26.9
20
1 8 4 2 0 8 9 3 1 6 1 8
0.41 0.15 4.33 1.54 0.57 0.90 0.97 1.20 0.44 0.86 1.12 1.25
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
20.4 23.1 24.6 27.0 23.7 27.6 21.4 23.4 23.2 26.9 26.7 26.9
1
6 1 8 3 0 2 8 7 0 7 6 7
20.4 23.0 24.6 27.0 23.7 27.5 21.5 23.4 23.2 27.0 26.7 27.0
2
4 4 3 1 0 3 1 1 4 0 3 0
3 20.5 23.0 24.6 27.0 23.7 27.4 21.4 23.3 23.1 27.0 26.8 27.0

1 6 4 0 5 6 9 6 9 2 1 2
20.5 23.0 24.6 27.0 23.5 27.5 20.9 23.3 23.1 26.9 26.7 26.9
4
9 4 1 0 6 5 2 6 3 4 8 4
20.3 22.4 24.6 27.0 23.7 27.5 21.5 23.4 23.2 26.9 26.7 26.9
5
3 7 3 4 0 9 3 7 8 4 6 4
20.7 23.0 24.6 27.0 23.6 27.5 21.0 23.3 23.1 26.9 26.7 26.9
6
9 4 1 3 3 9 1 2 9 3 1 3
20.7 23.0 24.6 26.9 23.5 27.6 21.4 23.3 23.2 26.8 26.6 26.8
7
0 2 2 9 7 4 6 4 0 8 2 8
20.7 23.0 24.6 27.0 23.5 27.6 21.5 23.4 23.2 27.0 26.7 27.0
8
1 4 4 1 7 5 2 5 0 2 6 2
20.4 23.0 24.6 26.9 23.7 27.5 21.4 23.4 23.2 27.0 26.8 27.0
9
3 5 8 9 1 1 7 7 4 3 6 3
20.4 23.0 24.6 27.0 23.6 27.4 21.5 23.4 23.3 26.9 26.8 26.9
10
9 3 6 3 7 8 8 4 0 7 1 7
20.9 22.4 24.6 25.8 22.7 26.3 20.9 22.2 22.1 25.0 26.0 26.3
11
6 8 9 1 4 2 2 6 8 5 2 2
21.0 22.4 24.6 25.8 22.7 26.2 20.9 22.2 22.4 25.0 25.8 26.3
12
2 8 7 7 1 8 3 9 4 9 1 4
21.0 22.4 24.6 25.8 22.7 26.3 20.8 22.2 22.4 25.1 25.8 26.4
13
1 9 4 7 5 6 6 4 0 6 3 2
21.1 22.5 24.6 25.8 22.7 26.3 20.8 22.2 22.4 24.7 25.8 26.1
14
0 2 0 2 7 2 9 6 0 9 5 9
21.2 22.5 24.4 25.8 22.6 26.3 21.0 22.2 22.3 24.9 25.8 26.4
15
0 8 6 5 5 8 2 9 3 6 5 7
21.1 22.5 24.4 25.8 22.5 26.3 20.9 22.2 22.1 25.0 25.8 26.3
16
0 1 6 7 8 5 4 4 3 0 4 7
21.1 22.5 24.4 25.8 22.5 26.4 20.9 22.2 22.2 25.1 25.8 26.4
17
3 2 4 6 5 1 8 1 8 6 6 6
21.1 22.5 24.5 25.8 22.6 26.3 20.6 22.3 22.3 25.1 25.7 26.3
18
1 0 1 3 0 0 6 2 0 8 7 7
20.8 22.4 24.6 25.8 22.7 26.3 20.8 22.3 22.0 25.0 25.8 26.3
19
9 5 8 3 5 4 2 4 1 8 7 3
20.8 22.4 24.6 25.8 22.7 26.3 20.8 22.2 22.3 25.0 25.7 26.3
20
9 7 6 3 7 3 5 8 6 0 8 1
1.36 1.24 0.32 2.27 2.17 2.34 1.48 2.56 2.15 3.81 1.80 1.20
CV
% % % % % % % % % % % %
ect FA FA FA FA
No. M M M M
21.6 22.8 25.5 26.3 23.6 26.9 21.4 22.7 23.3 25.3 26.2 27.2
1
3 7 7 7 2 6 6 1 2 4 9 0
21.6 22.8 25.6 26.4 23.6 27.1 21.4 22.7 23.2 25.3 26.3 27.2
2
2 9 1 0 2 2 8 2 4 2 0 6
3 21.6 22.9 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.3 26.3 27.2

4 0 6 8 2 8 6 0 6 6 2 5
21.6 22.8 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.5 26.3 27.2
4
1 6 6 7 1 4 6 2 1 3 1 3
21.6 22.8 25.6 26.4 23.6 27.0 21.4 22.7 23.2 25.4 26.3 27.3
5
2 9 7 2 2 4 5 3 9 7 2 2
21.6 22.8 25.5 26.3 23.5 27.0 21.4 22.7 23.3 25.4 26.2 27.1
6
4 5 6 4 9 9 8 2 0 4 8 6
21.6 22.8 25.5 26.3 23.6 27.1 21.4 22.7 23.2 25.4 26.3 27.1
7
2 9 7 2 3 0 7 5 9 1 0 9
21.5 22.8 25.5 26.2 23.6 27.0 21.5 22.7 23.2 25.3 26.2 27.2
8
7 6 8 6 3 5 1 5 9 9 5 0
21.6 22.9 25.5 26.3 23.5 27.0 21.4 22.7 23.3 25.4 26.5 27.3
9
5 1 4 1 9 6 7 2 3 9 8 9
21.6 22.8 25.3 26.3 23.6 27.1 21.4 22.7 23.3 25.3 26.2 27.2
10
2 9 9 2 0 4 9 3 5 0 9 4
20.8 22.5 24.5 25.7 23.0 26.4 20.7 22.2 22.5 24.5 25.9 26.5
11
9 7 6 8 0 7 1 8 9 3 2 2
20.9 22.5 24.6 25.9 22.9 26.5 20.6 22.3 22.6 24.5 25.9 26.5
12
1 8 7 0 9 1 7 1 0 2 0 0
20.8 22.5 24.7 25.9 23.0 26.4 20.6 22.2 22.5 24.5 25.9 26.5
13
9 6 6 2 4 6 6 8 7 5 3 5
20.9 22.5 24.6 25.9 23.0 26.4 20.7 22.2 22.6 24.6 25.9 26.6
14
0 6 6 2 2 5 3 9 1 8 7 2
20.9 22.5 24.8 25.9 22.9 26.4 20.7 22.2 22.6 24.5 25.9 26.6
15
2 5 0 3 9 7 4 9 0 6 6 0
20.9 22.5 24.7 25.9 23.0 26.0 20.7 22.3 22.6 24.6 25.9 26.5
16
3 6 8 1 0 7 9 3 2 2 6 4
20.9 22.5 24.7 25.9 22.9 26.4 20.7 22.3 22.5 24.5 25.9 26.5
17
2 6 6 3 9 3 3 0 9 8 1 7
20.9 22.5 24.7 25.9 23.0 26.4 20.6 22.3 22.5 24.5 25.9 26.5
18
3 5 1 2 0 4 8 1 9 8 5 5
20.9 22.6 24.6 25.9 23.0 26.4 20.6 22.2 22.5 24.5 25.9 26.5
19
9 1 9 7 8 7 8 8 4 8 4 9
20.9 22.5 24.7 25.9 22.9 26.4 20.6 22.2 22.5 24.5 26.0 26.5
20
1 8 9 9 9 2 8 9 8 4 0 7
1.70 0.71 1.74 0.87 1.33 1.33 1.87 0.98 1.57 1.72 0.79 1.32
CV
% % % % % % % % % % % %
of the three batches of kits by different operators and within one day range
from 0.11% to 4.17%, the CV of detected Ct values of the three batches by the
same operators and between days range from 0.15% to 4.33%, and both of the
values are less than 5%. Therefore, it can be considered that this kit has good
detection precision under different operators and at different times.
2.Precision test of throat swab samples

The clinical throat swab samples were tested by using three batches of
kits to verify the precision performance of the three batches of kits on clinical
samples. For positive samples of each pathogen, a clinical sample with a
higher concentration was selected and diluted to a low concentration for
precision testing. Each sample was extracted 10 times each time and was
repeatedly tested 10 times and the CV of Ct values were calculated.

Figure 2-13 Imprecision test results of positive samples for influenza A virus within

Figure 2-14 Imprecision test results of positive samples for influenza B virus within
Figure 2-15 Imprecision test results of positive samples for respiratory syncytial virus
Figure 2-16 Imprecision test results of positive samples for Adenovirus within batches
(2017001 batch)

Figure 2-17 Imprecision test results of positive samples for Human rhinovirus within
Figure 2-18 Imprecision test results of positive samples for Mycoplasma pneumoniae
Sample Respiratory
virus
1 29.64 30.20 30.18 29.73 30.47 29.71
2 29.68 30.26 30.21 29.69 30.42 30.21

3 29.61 30.39 30.17 29.81 30.27 30.19
4 29.66 30.42 30.28 29.66 30.25 30.26
5 29.68 30.35 29.72 29.73 29.91 30.30
6 29.69 30.07 30.37 29.76 30.34 30.01
7 29.71 30.37 30.09 29.90 30.48 30.51
8 29.83 30.30 30.17 29.95 30.37 30.28
9 29.65 30.17 29.97 29.83 30.22 30.11
10 29.73 30.33 30.21 29.72 30.32 30.11
CV
0.20% 0.37% 0.60% 0.31% 0.54% 0.70%
value
Sample Respiratory
virus
1 29.24 30.65 30.75 29.38 30.81 30.62
2 29.23 30.43 30.32 29.34 30.42 30.20
3 29.20 30.77 30.74 29.46 30.70 30.37
4 29.25 30.84 30.87 29.12 30.32 30.24
5 29.21 30.42 30.39 29.43 30.45 30.50
6 29.24 30.45 30.93 29.43 30.51 30.51
7 29.34 30.75 30.46 29.60 30.85 31.19
8 29.29 30.48 30.43 29.61 30.41 31.00
9 29.33 30.73 30.79 29.30 30.74 31.18
10 29.38 30.74 30.60 29.38 30.92 30.56
CV
0.21% 0.53% 0.71% 0.48% 0.70% 1.19%
value
Sample Respiratory
virus
1 29.28 30.31 30.85 29.33 30.24 30.50
2 29.43 30.33 30.72 29.41 30.39 30.74
3 29.39 30.11 30.61 29.52 30.34 30.64
4 29.39 30.27 30.83 29.31 30.44 30.76
5 29.37 30.50 30.81 29.36 30.40 30.63
6 29.46 30.24 30.98 29.46 30.38 30.88
7 29.49 30.34 30.81 29.40 30.37 30.83
8 29.27 30.48 30.79 29.46 30.33 30.76
9 29.40 30.21 30.67 29.44 30.46 30.59
10 29.47 30.07 31.04 29.58 30.36 30.83
CV
0.25% 0.46% 0.42% 0.28% 0.20% 0.40%
value

Table 2-14 Ct value of imprecision test among three batches of reagents
Sample Respiratory
virus
1 29.64 30.20 30.18 29.73 30.47 29.71
2 29.68 30.26 30.21 29.69 30.42 30.21
3 29.61 30.39 30.17 29.81 30.27 30.19
4 29.66 30.42 30.28 29.66 30.25 30.26
5 29.68 30.35 29.72 29.73 29.91 30.30
6 29.69 30.07 30.37 29.76 30.34 30.01
7 29.71 30.37 30.09 29.90 30.48 30.51
8 29.83 30.30 30.17 29.95 30.37 30.28
9 29.65 30.17 29.97 29.83 30.22 30.11
10 29.73 30.33 30.21 29.72 30.32 30.11
11 29.24 30.65 30.75 29.38 30.81 30.62
12 29.23 30.43 30.32 29.34 30.42 30.20
13 29.20 30.77 30.74 29.46 30.70 30.37
14 29.25 30.84 30.87 29.12 30.32 30.24
15 29.21 30.42 30.39 29.43 30.45 30.50
16 29.24 30.45 30.93 29.43 30.51 30.51
17 29.34 30.75 30.46 29.60 30.85 31.19
18 29.29 30.48 30.43 29.61 30.41 31.00
19 29.33 30.73 30.79 29.30 30.74 31.18
20 29.38 30.74 30.60 29.38 30.92 30.56
21 29.28 30.31 30.85 29.33 30.24 30.50
22 29.43 30.33 30.72 29.41 30.39 30.74
23 29.39 30.11 30.61 29.52 30.34 30.64
24 29.39 30.27 30.83 29.31 30.44 30.76
25 29.37 30.50 30.81 29.36 30.40 30.63
26 29.46 30.24 30.98 29.46 30.38 30.88
27 29.49 30.34 30.81 29.40 30.37 30.83
28 29.27 30.48 30.79 29.46 30.33 30.76
29 29.40 30.21 30.67 29.44 30.46 30.59
30 29.47 30.07 31.04 29.58 30.36 30.83
CV
0.64% 0.70% 1.10% 0.69% 0.67% 1.13%
value
It can be seen from the above results that, the accuracy of the three
batches of kits to detect clinical samples is 100%, the CV of detected Ct values


2.2.1 Intra-day Imprecision
shown in Figure 2-13 to Figure 2-18 in the intra-batch precision experiment;
the precision detection test results under different operators are shown in
Figure 2-19 to Figure 2-24 (only the amplification curve of the 2017001 batch
of reagents was shown):
Figure 2-19 Intra-day imprecision test results of positive samples for influenza A virus
(2017001 batch)

Figure 2-20 Intra-day imprecision test results of positive samples for influenza B virus
(2017001 batch)
Figure 2-21 Intra-day imprecision test results of positive samples for respiratory
syncytial virus (2017001 batch)
Figure 2-22 Intra-day imprecision test results of positive samples for Adenovirus
(2017001 batch)

Figure 2-23 Intra-day imprecision test results of positive samples for Human rhinovirus
(2017001 batch)
Figure 2-24 Intra-day imprecision test results of positive samples for Mycoplasma
Sample Respiratory
virus
1 29.64 30.20 30.18 29.73 30.47 29.71
2 29.68 30.26 30.21 29.69 30.42 30.21
3 29.61 30.39 30.17 29.81 30.27 30.19
4 29.66 30.42 30.28 29.66 30.25 30.26

5 29.68 30.35 29.72 29.73 29.91 30.30
6 29.69 30.07 30.37 29.76 30.34 30.01
7 29.71 30.37 30.09 29.90 30.48 30.51
8 29.83 30.30 30.17 29.95 30.37 30.28
9 29.65 30.17 29.97 29.83 30.22 30.11
10 29.73 30.33 30.21 29.72 30.32 30.11
11 29.64 30.01 30.47 29.55 29.91 30.50
12 29.70 30.44 30.77 29.64 30.20 30.64
13 29.75 30.40 30.53 29.71 30.52 30.53
14 29.62 30.49 30.61 29.65 30.55 30.57
15 29.65 30.53 30.48 29.65 30.14 30.71
16 29.70 30.55 30.83 29.76 30.28 30.58
17 29.72 30.46 30.63 29.58 30.51 30.63
18 29.64 30.45 30.73 29.51 29.84 30.52
19 29.64 30.33 30.71 29.56 30.31 30.60
20 29.69 30.59 30.38 29.56 30.51 30.56
CV
0.17% 0.37% 0.60% 0.40% 0.69 % 0.86%
value
Sample Respiratory
virus
1 29.24 30.65 30.75 30.75 30.81 30.62
2 29.23 30.43 30.32 30.32 30.42 30.20
3 29.20 30.77 30.74 30.74 30.70 30.37
4 29.25 30.84 30.87 30.87 30.32 30.24
5 29.21 30.42 30.39 30.39 30.45 30.50
6 29.24 30.45 30.93 30.93 30.51 30.51
7 29.34 30.75 30.46 30.46 30.85 31.19
8 29.29 30.48 30.43 30.43 30.41 31.00
9 29.33 30.73 30.79 30.79 30.74 31.18
10 29.38 30.74 30.60 30.60 30.92 30.56
11 29.40 31.22 30.83 29.31 30.85 30.68
12 29.41 30.18 30.66 29.47 30.17 30.62
13 29.26 31.23 30.63 29.37 30.01 30.71
14 29.24 31.19 30.79 29.41 28.52 30.55
15 29.40 31.32 30.47 29.45 30.29 30.76
16 29.41 31.33 30.84 29.68 31.15 30.87
17 29.27 31.18 30.98 29.57 31.20 30.74
18 29.49 31.39 30.68 29.61 31.03 30.57
19 29.53 30.07 30.90 29.56 30.51 30.76
20 29.52 30.25 30.62 29.61 29.69 30.56
CV
0.36% 1.36% 0.63% 2.00% 1.97% 0.85%
value

Sample Respiratory
virus
1 29.28 30.31 30.85 29.33 30.24 30.50
2 29.43 30.33 30.72 29.41 30.39 30.74
3 29.39 30.11 30.61 29.52 30.34 30.64
4 29.39 30.27 30.83 29.31 30.44 30.76
5 29.37 30.50 30.81 29.36 30.40 30.63
6 29.46 30.24 30.98 29.46 30.38 30.88
7 29.49 30.34 30.81 29.40 30.37 30.83
8 29.27 30.48 30.79 29.46 30.33 30.76
9 29.40 30.21 30.67 29.44 30.46 30.59
10 29.47 30.07 31.04 29.58 30.36 30.83
11 29.73 30.35 30.66 29.56 30.35 30.73
12 29.65 30.48 30.99 29.64 30.19 30.92
13 29.72 30.12 30.68 29.67 30.40 30.75
14 29.70 30.25 30.56 29.69 30.38 30.70
15 29.70 30.48 30.69 29.76 30.41 30.39
16 29.61 30.39 30.75 29.62 30.28 30.38
17 29.61 30.52 30.96 29.61 30.34 30.77
18 29.66 30.47 30.91 29.67 30.52 30.69
19 29.78 30.12 30.69 29.55 30.38 30.76
20 29.59 29.34 30.66 29.52 30.35 30.66
CV
0.54% 0.86% 0.47% 0.44% 0.24% 0.47%
value

shown in Figure 2-13 to Figure 2-18; the precision test results performed by
the same operators on alternate days are shown in Figure 2-25 to Figure 2-30
(only the amplification curve of the 2017001 batch of reagents was shown):

Figure 2-25 Inter-day imprecision test results of positive samples for influenza A virus
(2017001 batch)
Figure 2-26 Inter-day imprecision test results of positive samples for influenza B virus
(2017001 batch)

Figure 2-27 Inter-day imprecision test results of positive samples for respiratory syncytial
Figure 2-28 Inter-day imprecision test results of positive samples for Adenovirus
(2017001 batch)

Figure 2-29 Inter-day imprecision test results of positive samples for Human rhinovirus
(2017001 batch)
Figure 2-30 Inter-day imprecision test results of positive samples for Mycoplasma
Sample Respiratory
virus
1 29.64 30.20 30.18 29.73 30.47 29.71
2 29.68 30.26 30.21 29.69 30.42 30.21
3 29.61 30.39 30.17 29.81 30.27 30.19
4 29.66 30.42 30.28 29.66 30.25 30.26

5 29.68 30.35 29.72 29.73 29.91 30.30
6 29.69 30.07 30.37 29.76 30.34 30.01
7 29.71 30.37 30.09 29.90 30.48 30.51
8 29.83 30.30 30.17 29.95 30.37 30.28
9 29.65 30.17 29.97 29.83 30.22 30.11
10 29.73 30.33 30.21 29.72 30.32 30.11
11 29.33 30.38 30.68 29.45 30.45 30.61
12 29.24 30.38 30.74 29.38 30.55 30.79
13 29.40 30.46 30.82 29.51 30.38 30.72
14 29.27 30.62 30.82 29.48 30.36 30.58
15 29.47 30.21 30.57 29.55 30.35 30.70
16 29.38 30.33 30.52 29.54 30.53 30.83
17 29.39 30.47 30.83 29.56 30.56 30.67
18 29.53 30.31 30.69 29.67 30.49 30.69
19 29.39 30.17 30.80 29.40 30.25 30.62
20 29.42 30.52 30.74 29.48 30.47 30.75
CV
0.59% 0.44% 1.10% 0.56% 0.49% 1.02%
value
Sample Respiratory
virus
1 29.24 30.65 30.75 29.38 30.81 30.62
2 29.23 30.43 30.32 29.34 30.42 30.20
3 29.20 30.77 30.74 29.46 30.70 30.37
4 29.25 30.84 30.87 29.12 30.32 30.24
5 29.21 30.42 30.39 29.43 30.45 30.50
6 29.24 30.45 30.93 29.43 30.51 30.51
7 29.34 30.75 30.46 29.60 30.85 31.19
8 29.29 30.48 30.43 29.61 30.41 31.00
9 29.33 30.73 30.79 29.30 30.74 31.18
10 29.38 30.74 30.60 29.38 30.92 30.56
11 29.27 30.40 30.51 29.19 30.47 30.41
12 29.26 30.60 30.65 29.18 30.42 30.51
13 29.22 30.51 30.59 29.17 30.37 30.35
14 29.22 30.60 30.44 29.22 30.51 30.38
15 29.24 30.48 30.42 29.18 30.39 30.37
16 29.23 30.40 30.65 29.16 30.30 30.52
17 29.25 30.64 30.63 29.22 30.26 30.63
18 29.21 30.38 30.47 29.21 30.24 30.49
19 29.22 30.39 30.54 29.18 30.38 30.41
20 29.18 30.27 30.68 29.16 30.49 30.59
CV
0.17% 0.53% 0.55% 0.51% 0.66% 0.90%
value

Sample Respiratory
virus
1 29.28 30.31 30.85 29.33 30.24 30.50
2 29.43 30.33 30.72 29.41 30.39 30.74
3 29.39 30.11 30.61 29.52 30.34 30.64
4 29.39 30.27 30.83 29.31 30.44 30.76
5 29.37 30.50 30.81 29.36 30.40 30.63
6 29.46 30.24 30.98 29.46 30.38 30.88
7 29.49 30.34 30.81 29.40 30.37 30.83
8 29.27 30.48 30.79 29.46 30.33 30.76
9 29.40 30.21 30.67 29.44 30.46 30.59
10 29.47 30.07 31.04 29.58 30.36 30.83
11 29.69 30.56 30.80 29.76 30.36 30.76
12 29.68 30.31 30.53 29.75 30.30 30.57
13 29.66 30.47 30.69 29.78 30.37 30.81
14 29.71 30.70 30.49 29.66 30.25 30.61
15 29.74 30.38 30.48 29.79 30.25 30.49
16 29.76 30.38 30.54 29.94 30.21 30.57
17 29.71 30.41 30.85 29.83 30.15 30.57
18 29.85 30.43 30.77 29.97 30.22 30.59
19 29.78 30.28 30.80 29.79 30.18 30.73
20 29.76 30.29 30.73 29.79 30.61 30.66
CV
0.63% 0.49% 0.49% 0.72% 0.36% 0.38%
value
three batches of kits to detect clinical samples is 100%, the CV of detected Ct
values of inter-day imprecision test among the three batched of kits by
different operators range from 0.17% to 2.00%; the CV of detected Ct values of
intra-day imprecision test among the three batched of kits by the same
operators range from 0.17% to 1.10%, and both of the values are less than 5%.
Therefore, it can be considered that this kit has good detection precision
under different operators and at different times.
3. Precision test of national reference samples

The precision reference samples CV1 and CV2 in the national reference
samples (batch number 370006-201501) of nucleic acid detection reagents for
influenza A/B virus were tested by using three batches of kits. Three batches
of qualified detection kits in this study were tested on the SLAN-96P PCR
instrument to verify the precision performance of the three batches of kits on
the national reference samples. Each sample was extracted 10 times each time
and was repeatedly tested 10 times and the CV of Ct values were calculated.

Table 2-21 Ct value of precision test of the three batches of kits on the national reference
samples CV1 and CV2
Samples National reference samples

Batch No.
No. CV1 CV2
1 29.54 29.35
2 29.72 29.41
3 29.51 29.36
2017001
4 29.69 29.42
5 29.66 29.35
6 29.56 29.40

7 29.68 29.43
8 29.62 29.42
9 29.50 29.39
10 29.86 29.26
11 29.85 29.55
12 29.77 29.57
13 29.61 29.37
14 29.69 29.38
15 29.72 29.41
2017002
16 29.67 29.47
17 29.73 29.42
18 29.85 29.40
19 29.82 29.43
20 29.68 29.44
21 29.57 29.35
22 29.67 29.54
23 29.73 29.40
24 29.70 29.37
25 29.68 29.46
2017003
26 29.79 29.43
27 29.57 29.39
28 29.68 29.45
29 29.70 29.53
30 29.65 29.47
CV value 0.32% 0.23%
three batches of kits to detect the precision reference samples CV1 and CV2 in
the national reference samples of nucleic acid detection reagents for influenza
A/B virus is 100%, and the CV of detected Ct values of precision between
batches are 0.32% and 0.23%, which are both lower than 5%. It meets the
requirements of national reference sample detection. Therefore, it can be
considered that this kit has good detection precision on national precision
reference samples via SLAN-96P PCR instrument.

Analytical Sensitivity

described below.
Minimum detection limit (Analytical sensitivity)

1. Determination of the minimum detection limit
The pathogen culture medium with known pathogen concentration was
diluted to determine the minimum detection limit of this kit. The
concentration of influenza A virus and influenza B virus was calibrated by
using 50% tissue culture infectious dose (TCID50). The concentration of
respiratory syncytial virus, adenovirus, human rhinovirus, and Mycoplasma
pneumonia was calibrated by using the nucleic acid concentration of target
fragment (copies/mL). Each pathogen was diluted with normal saline, and
each gradient of diluted pathogen medium had 3 copies, each of which was
tested 20 times with three batches of kits; In total, each gradient of diluted
pathogen medium was repeatedly tested 60 times. The pathogen
concentration and dilution information are shown in Table 1-1 and Table 1-2.
The diluted strain was used as the test sample and was tested according to the
manual of this kit. The test results are shown in Table 1-3 to Table 1-10
(detection results of the three batches of kits) and in Figure 1-1 to Figure 1-24
(only the test results of the 2017001 batch of reagents was shown).
Table 1-1 Pathogen information and dilution gradient for testing (Influenza A, Influenza
B)
Diluted detection
Pathogen No. Type
concentration(TCID50/mL)
StrainInf A-A A/H1N1 4.0 2.0 1.5 1.0
StrainInf A-B A/H3 4.0 2.0 1.5 1.0
StrainInf A-C A/H5 4.0 2.0 1.5 1.0
StrainInf B-A B/Victoria 4.0 2.0 1.5 1.0
StrainInf B-B B/Victoria 4.0 2.0 1.5 1.0
StrainInf B-C B/Yamagata 4.0 2.0 1.5 1.0
Table 1-2 Pathogen information and dilution gradient for testing
Research data on analytical sensitivity 1

Diluted detection
Pathogen No. Type
concentration(copies/mL)
StrainRSV-A A type 1000 500 400 300
StrainRSV-B B type 1000 500 400 300
StrainRSV-C B type 1000 500 400 300
StrainADV-A 3 type 1000 500 400 300
StrainADV-B 7 type 1000 500 400 300
StrainADV-C 4 type 1000 500 400 300
StrainADV-D 1 type 1000 500 400 300
StrainADV-E 2 type 1000 500 400 300
StrainADV-F 5 type 1000 500 400 300
StrainADV-G 14 type 1000 500 400 300
StrainADV-H 21 type 1000 500 400 300
StrainADV-I 55 type 1000 500 400 300
StrainHRV-A A type 1000 500 400 300
StrainHRV-B B type 1000 500 400 300
StrainHRV-C C type 1000 500 400 300
StrainMP-A 1 type 1000 500 400 300
StrainMP-B 1 type 1000 500 400 300
StrainMP-C 2 type 1000 500 400 300
Figure 1-1 Test results of Strain Inf A-A at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 1-2 Test results of Strain Inf A-B at a concentration of 2.0 TCID50/mL (2017001
batch)

Figure 1-3 Test results of Strain Inf A-C at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 1-4 Test results of Strain Inf B-A at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 1-5 Test results of Strain Inf B-B at a concentration of 2.0 TCID50/mL (2017001
batch)

Figure 1-6 Test results of Strain Inf B-C at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 1-7 Test results of Strain RSV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 1-8 Test results of Strain RSV-B at a concentration of 500 copies/mL (2017001
batch)

Figure 1-9 Test results of Strain RSV-C at a concentration of 500 copies/mL (2017001
batch)
Figure 1-10 Test results of Strain ADV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 1-11 Test results of Strain ADV-B at a concentration of 500 copies/mL (2017001
batch)

Figure 1-12 Test results of Strain ADV-C at a concentration of 500 copies/mL (2017001
Figure 1-13 Test results of Strain ADV-D at a concentration of 500 copies/mL (2017001
batch)
Figure 1-14 Test results of Strain ADV-E at a concentration of 500 copies/mL (2017001
batch)

Figure 1-15 Test results of Strain ADV-F at a concentration of 500 copies/mL (2017001
batch)
Figure 1-16 Test results of Strain ADV-G at a concentration of 500 copies/mL (2017001
batch)

Figure 1-17 Test results of Strain ADV-H at a concentration of 500 copies/mL (2017001
batch)
Figure 1-18 Test results of Strain ADV-I at a concentration of 500 copies/mL (2017001
batch)
Figure 1-19 Test results of Strain HRV-A at a concentration of 500 copies/mL (2017001

batch)
Figure 1-20 Test results of Strain HRV-B at a concentration of 500 copies/mL (2017001
batch)
Figure 1-21 Test results of Strain HRV-C at a concentration of 500 copies/mL (2017001
batch)
Figure 1-22 Test results of Strain MP-A at a concentration of 500 copies/mL (2017001
batch)

Figure 1-23 Test results of Strain MP-B at a concentration of 500 copies/mL (2017001
batch)
Figure 1-24 Test results of Strain MP-C at a concentration of 500 copies/mL (2017001
batch)
Table 1-3 The detection rate of the minimum detection limit determination test for
influenza A virus
Pathogen No. Strain Inf A-A Strain Inf A-B Strain Inf A-C
Detection rate
Concentration Positive Positive Positive Positive Positive Positive
(TCID50/mL) rate percentage rate percentage rate percentage
4.0 60/60 100% 60/60 100% 60/60 100%
2.0 60/60 100% 60/60 100% 60/60 100%
1.5 48/60 80% 50/60 83.3% 48/60 80%
1.0 42/60 70% 36/60 60% 39/60 65%
influenza B virus
Pathogen No. Strain Inf B-A Strain Inf B-B Strain Inf B-C
Detection rate
4.0 60/60 100% 60/60 100% 60/60 100%
2.0 60/60 100% 60/60 100% 60/60 100%

1.5 48/60 80% 48/60 80% 48/60 80%
1.0 35/60 58.3% 36/60 60% 35/60 58.3%
respiratory syncytial virus
Pathogen No. Strain RSV-A Strain RSV-B Strain RSV-C
Detection rate
(copies/mL) rate percentage rate percentage rate percentage
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 60/60 100% 60/60 100%
400 46/60 76% 47/60 78.3% 44/60 73.3%
300 26/60 43% 33/60 55% 36/60 60%
Adenovirus
Pathogen No. Strain ADV-A Strain ADV-B Strain ADV-C
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 60/60 100% 60/60 100%
400 48/60 80% 49/60 81.7% 48/60 80%
300 37/60 61.7% 26/60 43.3% 38/60 63.3%
Adenovirus
Pathogen No. Strain ADV-D Strain ADV-E Strain ADV-F
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 60/60 100% 60/60 100%
400 60/60 100% 60/60 100% 60/60 100%
300 55/60 91.67% 46/60 76.67% 58/60 96.67%
Adenovirus
Pathogen No. Strain ADV-G Strain ADV-H Strain ADV-I
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 60/60 100% 60/60 100%
400 60/60 100% 60/60 100% 60/60 100%
300 56/60 93.33% 58/60 96.67% 58/60 96.67

Pathogen No. Strain MP-A Strain MP-B Strain MP-C
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 59/60 98.3% 60/60 100%
400 47/60 78.3% 47/60 78.3% 44/60 73.3%
300 32/60 53.3% 37/60 61.7% 33/60 55%
Human rhinovirus
Pathogen No. Strain HRV-A Strain HRV-B Strain HRV-C
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 59/60 98.3% 60/60 100%
400 46/60 76.7% 48/60 80% 48/60 80%
300 36/60 60% 35/60 58.3% 35/60 58.3%
Conclusion: The sample with Ct ≤ 40 was judged as positive, and

pathogen concentration with the positive detection rate ≥ 95% was regarded
as the minimum detection limit. According to the above-described test results
on ABI7500 instrument: when the concentration of test sample of influenza A
virus is 4.0 TCID50/mL, all the positive detection rate are 100%; when the
concentration is 2.0 TCID50/mL, all the positive detection rate are higher than
95%; when the concentration is 1.5 TCID50/mL, the positive detection rate of
three strains of influenza A virus are lower than 95%, when the concentration
is 1.0 TCID50/mL, all the positive detection rate are lower than 70%. Therefore,
in order to ensure that the positive detection rate of all samples is ≥ 95%, the
minimum detection limit of this kit for influenza A virus is set to 2.0
TCID50/mL. Similarly, according to the test results of each pathogen on
ABI7500 instrument, the minimum detection limit of this kit for influenza B
virus is set to 2.0 TCID50/mL; the minimum detection limit of this kit for
respiratory syncytial virus, Adenovirus, Human rhinovirus and Mycoplasma
pneumoniae are all set to 500 copies/mL, respectively.
2. Verification of minimum detection limit

2.1 Verification of clinical samples
respiratory syncytial virus, Adenovirus, Mycoplasma pneumoniae and
Human rhinovirus with known pathogen concentration were diluted to verify
the minimum detection limit of this kit. The concentration of influenza A
virus and influenza B virus was calibrated by using 50% tissue culture
infectious dose (TCID50). The concentration of respiratory syncytial virus,
adenovirus, human rhinovirus, and Mycoplasma pneumonia was calibrated

by using the nucleic acid concentration of target fragment (copies/mL). The
samples of each pathogen were diluted with normal saline to the minimum
detection limit of this kit, and the diluted samples were tested by 3 batches of
kits 20 times, respectively.
The concentration of throat swab samples of each pathogen and sample
information are shown in Table 1-11 and Table 1-12. The diluted samples were
used as the test sample and were tested on ABI7500 instrument according to
the manual of this kit. The test results are shown in Table 1-13 to Table 1-18
Table 1-11 Sample information for minimum detection limit verification (Influenza A,
Influenza B)
Concentration
Sample No. Type Source
(TCID50/mL)
Sichuan provincial disease
A/Qingyang/1272/201
Sample Inf A-A prevention and control 2100
4(H3)
center
A/Zhejiang/
Yiwu disease prevention and
Sample Inf A-B DTID-ZJU03/2009(H1 6500
control center
N1)
Loudi disease prevention
Sample Inf A-C A/H5 4400
and control center
Sample Inf B-A B/Victoria prevention and control 6400
center
Sample Inf B-B B/Victoria prevention and control 5500
center
Sample Inf B-C B/Yamagata prevention and control 7100
center
Table 1-12 Sample information for minimum detection limit verification

Concentration
(copies/mL)
Human provincial
Sample RSV-A A type 8.71E+05
children’s hospital
Children’s hospital of
Sample RSV-B B type Nanjing medial 6.73E+04
university
Human provincial
Sample RSV-C B type 7.93E+04
Human provincial
Sample ADV-A 3 type 1.34E+05
Human provincial
Sample ADV-B 7 type 5.74E+05
Sample ADV-C 4 type Human provincial 6.78E+05

Human provincial
Sample HRV-A A type 2.33E+04
Human provincial
Sample HRV-B B type 5.27E+05
Human provincial
Sample HRV-C C type 4.85E+04
Human provincial
Sample MP-A 1 type 2.27E+05
Human provincial
Sample MP-B 1 type 1.24E+05
Sample MP-C 2 type Nanjing medial 6.15E+05
university
Figure 1-25 Test results of sample Inf A-A at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 1-26 Test results of sample Inf A-B at a concentration of 2.0 TCID50/mL (2017001
batch)

Figure 1-27 Test results of sample Inf A-C at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 1-28 Test results of sample Inf B-A at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 1-29 Test results of sample Inf B-B at a concentration of 2.0 TCID50/mL (2017001
batch)

Figure 1-30 Test results of sample Inf B-C at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 1-31 Test results of sample RSV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 1-32 Test results of sample RSV-B at a concentration of 500 copies/mL (2017001
batch)

Figure 1-33 Test results of sample RSV-C at a concentration of 500 copies/mL (2017001
batch)
Figure 1-34 Test results of sample ADV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 1-35 Test results of sample ADV-B at a concentration of 500 copies/mL (2017001
batch)

Figure 1-36 Test results of sample ADV-C at a concentration of 500 copies/mL (2017001
batch)
Figure 1-37 Test results of sample HRV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 1-38 Test results of sample HRV-B at a concentration of 500 copies/mL (2017001
batch)

Figure 1-39 Test results of sample HRV-C at a concentration of 500 copies/mL (2017001
batch)
Figure 1-40 Test results of sample MP-A at a concentration of 500 copies/mL (2017001
batch)
Figure 1-41 Test results of sample MP-B at a concentration of 500 copies/mL (2017001
batch)

Figure 1-42 Test results of sample MP-C at a concentration of 500 copies/mL (2017001
batch)
Table 1-13 Statistical table of positive rate of influenza A virus
Sample Inf A-A Sample Inf A-B Sample Inf A-C

Batch No.
2.0TCID50/mL 2.0TCID50/mL 2.0TCID50/mL
1 32.50 30.97 32.79
2 32.06 33.70 29.29
3 31.92 33.01 32.36
4 31.66 37.25 30.59
5 32.09 33.09 36.02
6 32.11 33.00 32.16
7 32.46 33.66 32.22
8 32.39 35.65 32.43
9 35.50 33.11 34.14
10 32.18 34.30 34.78
2017001
11 32.00 33.40 32.84
12 32.38 32.82 32.31
13 33.42 32.13 32.49
14 32.78 33.01 31.10
15 32.79 33.04 31.42
16 32.20 36.34 31.78
17 31.25 32.80 31.67
18 32.49 30.47 31.56
19 32.57 33.28 30.46
20 34.33 32.85 31.56
21 33.41 34.56 31.55
22 34.41 32.58 31.10
23 34.46 32.80 31.32
24 34.33 31.72 31.88
2017002 25 33.96 33.07 31.75
26 34.05 33.27 35.02
27 34.63 33.09 31.18
28 34.29 33.12 32.09
29 34.46 32.16 31.89

30 33.44 33.24 32.35
31 33.87 33.68 31.39
32 33.85 33.99 33.37
33 34.37 34.22 32.67
34 35.15 32.26 31.46
35 34.54 33.24 31.43
36 34.47 32.38 32.03
37 33.36 33.81 32.54
38 33.34 32.78 32.64
39 34.64 32.27 31.68
40 35.68 33.23 32.73
41 33.37 34.20 32.00
42 33.02 31.57 31.84
43 34.02 31.29 33.97
44 33.76 31.89 33.17
45 33.99 31.76 36.89
46 33.45 32.30 32.59
47 32.65 31.61 32.46
48 34.23 31.35 32.60
49 33.89 31.98 33.06
50 34.53 32.40 32.08
2017003
51 33.10 32.52 33.78
52 33.43 31.43 35.39
53 33.98 31.17 33.67
54 33.63 31.11 32.19
55 34.15 32.02 31.85
56 34.05 31.43 32.30
57 33.49 32.90 32.89
58 33.03 31.55 30.48
59 32.96 31.33 33.03
60 34.10 34.73 33.84
Number of detected
60 60 60
positive samples
Positive rate 100% 100% 100%
Table 1-14 Statistical table of positive rate of influenza B virus
Sample Inf B-A Sample Inf B-B Sample Inf B-C

Batch No.
1 34.53 34.44 34.07
2 33.97 35.95 33.36
3 33.85 34.06 33.26
4 34.35 35.55 34.14
2017001
5 35.12 35.18 35.07
6 34.14 36.24 35.45
7 34.01 35.37 35.34
8 34.25 34.84 33.79

9 34.88 34.98 34.01
10 34.71 35.51 34.35
11 34.32 35.23 35.17
12 34.77 35.85 35.00
13 35.37 34.87 33.83
14 34.83 36.00 34.53
15 35.16 36.03 34.78
16 35.09 34.06 34.86
17 35.85 35.43 34.95
18 35.04 35.25 33.07
19 34.33 35.95 34.18
20 34.30 35.88 32.35
21 34.05 35.52 32.29
22 34.09 33.62 34.24
23 34.87 35.75 32.70
24 34.27 34.05 32.95
25 35.43 34.15 33.49
26 34.90 35.00 33.72
27 35.06 35.16 33.30
28 34.84 34.91 33.34
29 35.21 34.50 33.55
30 35.02 35.21 34.47
2017002
31 34.93 35.87 33.09
32 34.90 34.54 33.99
33 34.31 35.17 33.56
34 34.05 34.61 33.67
35 34.49 35.37 33.35
36 35.15 34.56 33.53
37 35.76 34.58 33.75
38 34.87 35.16 33.79
39 31.31 34.62 32.52
40 34.74 33.91 34.46
41 35.23 33.45 34.94
42 34.50 34.06 33.69
43 36.03 34.21 33.67
44 35.22 34.42 34.02
45 35.62 34.87 34.55
46 35.23 34.27 34.83
47 35.57 34.08 33.92
2017003 48 35.44 34.72 34.15
49 34.93 35.49 34.79
50 35.26 34.41 33.35
51 35.58 36.39 34.70
52 35.30 34.48 36.51
53 35.24 34.83 34.32
54 34.96 34.32 35.04
55 34.87 35.46 34.10

56 36.09 34.78 35.58
57 35.18 34.38 34.54
58 35.50 35.04 34.43
59 36.20 33.35 35.30
60 35.72 33.29 32.95
Number of detected
60 60 60
positive samples
Table 1-15 Statistical table of positive rate of respiratory syncytial virus

Sample RSV-A Sample RSV-B Sample RSV-C
Batch No.
500copies/mL 500copies/mL 500copies/mL
1 33.43 34.66 34.28
2 35.25 35.15 34.15
3 35.24 34.05 33.40
4 34.23 35.38 35.22
5 34.23 35.17 36.07
6 34.48 35.29 35.45
7 34.52 33.83 36.20
8 34.48 33.83 35.08
9 36.01 36.46 33.38
10 34.70 34.38 34.90
2017001
11 33.37 34.28 34.66
12 36.19 34.97 34.44
13 34.30 34.25 35.73
14 35.03 35.65 35.16
15 34.54 34.60 35.42
16 35.05 36.09 34.68
17 33.59 35.44 34.64
18 35.25 34.51 35.63
19 34.15 33.86 35.25
20 34.85 36.11 35.16
21 34.89 33.43 33.33
22 32.88 33.32 35.00
23 35.74 34.52 34.55
24 34.52 35.29 33.39
25 33.07 35.42 33.47
26 35.58 34.28 35.45
27 34.02 34.18 34.25
2017002 28 33.43 33.97 33.42
29 No Ct 33.87 32.73
30 32.85 33.84 33.38
31 34.79 34.83 No Ct
32 33.98 35.17 35.12
33 33.67 34.46 35.65
34 35.16 34.57 33.07
35 34.85 35.08 32.08

36 35.19 35.56 33.65
37 33.56 33.80 33.29
38 33.56 35.51 34.52
39 34.22 34.93 34.45
40 34.42 34.50 33.30
41 35.22 33.69 34.74
42 34.27 33.58 35.27
43 32.92 32.94 34.18
44 34.89 34.79 35.50
45 33.33 35.54 35.28
46 35.57 34.77 35.45
47 33.95 35.66 34.02
48 34.51 34.54 34.01
49 32.82 33.02 36.60
50 33.21 34.40 34.53
2017003
51 33.99 34.19 34.32
52 34.55 34.01 35.11
53 33.13 34.08 34.39
54 32.73 35.13 35.84
55 34.19 34.68 34.74
56 34.74 34.89 36.34
57 33.65 35.78 35.58
58 34.36 34.08 34.65
59 33.34 35.15 34.02
60 33.59 34.77 36.30
Number of detected
59 60 59
positive samples
Positive rate 98.3% 100% 98.3%
Table 1-16 Statistical table of positive rate of Adenovirus
Sample ADV-A Sample ADV-B Sample ADV-C

Batch No.
1 31.84 33.35 31.85
2 31.32 33.07 31.68
3 30.96 31.74 32.02
4 32.20 34.68 32.02
5 32.53 32.88 33.02
6 32.57 32.41 31.58
7 31.86 31.78 32.04
2017001
8 32.58 33.56 30.60
9 31.89 34.09 32.16
10 32.43 34.51 31.68
11 32.92 34.32 33.35
12 31.76 33.28 32.58
13 32.44 33.37 32.00
14 33.59 33.03 32.27

15 32.27 34.46 33.42
16 34.06 32.97 32.92
17 32.38 33.56 31.91
18 31.17 32.50 32.28
19 31.09 35.36 32.43
20 32.54 33.42 33.23
21 33.82 32.41 33.26
22 33.80 33.38 28.78
23 33.52 33.40 32.52
24 33.89 32.88 31.34
25 34.12 33.66 37.41
26 34.28 33.27 32.35
27 34.55 32.53 32.58
28 33.70 32.99 32.67
29 34.36 33.39 34.44
30 34.09 32.75 36.30
2017002
31 34.15 34.17 33.08
32 34.46 33.58 32.72
33 34.45 33.40 31.52
34 34.20 33.51 32.41
35 34.45 33.00 32.04
36 34.04 32.50 34.76
37 34.68 32.29 31.58
38 34.24 31.94 27.70
39 34.67 33.40 31.13
40 33.82 33.74 30.28
41 33.98 31.34 33.50
42 33.34 33.17 32.81
43 33.32 30.06 33.18
44 33.56 35.13 32.90
45 33.43 31.30 32.80
46 33.66 36.32 34.41
47 34.03 32.00 35.34
48 34.12 32.49 32.90
49 33.27 32.79 31.58
50 33.37 33.24 33.20
2017003
51 33.65 35.07 32.59
52 33.72 33.25 34.26
53 34.42 31.55 32.80
54 34.08 32.81 32.69
55 33.55 31.89 35.86
56 34.07 31.77 34.71
57 34.40 32.67 30.16
58 34.24 32.05 32.67
59 36.32 32.90 32.73
60 34.20 31.06 33.61
Number of detected 60 60 60

positive samples
Table 1-17 Statistical table of positive rate of Mycoplasma pneumoniae
Sample MP-A Sample MP-B Sample MP-C

Batch No.
1 33.52 33.85 35.31
2 34.17 33.75 35.39
3 36.88 34.96 34.21
4 35.00 35.71 35.56
5 33.97 35.82 34.94
6 36.73 34.71 35.16
7 35.00 34.54 35.55
8 35.25 34.32 33.50
9 35.39 34.20 35.03
10 33.75 35.30 34.23
2017001
11 35.52 34.77 35.52
12 33.46 34.82 35.13
13 33.55 35.06 34.93
14 34.73 34.34 34.27
15 33.18 35.93 34.68
16 34.73 34.24 35.78
17 34.25 35.20 37.15
18 33.86 35.26 36.06
19 34.06 34.94 35.89
20 34.82 34.87 34.50
21 34.44 34.22 32.56
22 32.81 34.30 No Ct
23 33.71 35.41 34.17
24 35.85 33.93 34.27
25 33.59 34.30 33.44
26 33.22 34.91 33.47
27 33.74 36.56 33.27
28 34.20 35.93 33.51
29 34.05 34.20 33.06
30 32.56 34.17 33.62
2017002
31 34.55 35.33 34.26
32 33.65 34.01 33.38
33 34.24 34.05 34.33
34 32.47 34.24 33.57
35 34.29 34.71 34.63
36 32.86 35.08 33.35
37 32.49 33.30 33.12
38 33.34 34.35 34.03
39 34.11 34.03 34.10
40 33.08 33.96 33.03

41 33.10 34.39 35.16
42 No Ct 34.55 35.23
43 34.72 34.85 35.33
44 33.11 35.69 34.75
45 34.38 34.20 35.38
46 34.76 35.03 33.32
47 34.13 34.67 34.11
48 33.95 35.28 35.32
49 36.19 35.15 34.71
50 33.68 37.01 35.01
2017003
51 33.90 36.30 34.89
52 34.19 33.93 34.45
53 34.05 34.59 35.41
54 33.47 35.54 35.72
55 35.50 34.28 36.76
56 34.19 34.43 35.82
57 34.89 35.30 33.89
58 34.06 33.64 35.61
59 34.46 34.20 34.30
60 34.74 34.17 34.42
Number of detected
59 60 59
positive samples
Positive rate 98.3% 100% 98.3%
Table 1-18 Statistical table of positive rate of Human rhinovirus
Sample HRV-A Sample HRV-B Sample HRV-C

Batch No.
1 33.94 35.02 33.23
2 34.69 34.52 34.86
3 34.04 34.62 33.19
4 35.28 34.79 33.55
5 34.70 35.37 34.67
6 35.13 35.16 33.45
7 34.64 36.17 34.41
8 35.06 34.86 33.49
9 34.94 35.74 34.10
2017001 10 34.67 35.28 34.51
11 34.71 35.77 33.30
12 35.10 34.98 34.53
13 33.86 35.20 33.98
14 34.70 34.75 34.32
15 34.93 35.31 33.46
16 34.27 34.76 34.01
17 34.68 35.33 34.77
18 34.57 35.29 34.40
19 34.56 36.37 33.27

20 35.27 35.39 32.79
21 34.17 34.05 33.25
22 35.58 34.68 33.32
23 33.74 34.78 32.85
24 35.27 34.97 34.09
25 34.90 35.38 33.63
26 36.03 34.81 34.55
27 35.10 34.47 34.08
28 34.48 35.23 34.77
29 34.65 36.11 33.24
30 35.24 34.84 35.67
2017002
31 34.95 37.05 33.92
32 35.51 35.02 34.70
33 34.50 35.31 33.89
34 35.61 34.70 33.32
35 35.67 36.00 34.23
36 34.35 35.24 34.35
37 35.13 34.89 34.43
38 34.98 35.54 34.47
39 35.35 34.00 32.41
40 35.57 33.89 33.59
41 34.11 33.51 34.06
42 35.08 33.41 33.46
43 34.59 33.39 33.24
44 36.41 35.21 34.21
45 34.86 35.58 35.27
46 35.43 33.69 35.77
47 35.04 33.97 35.60
48 36.25 34.13 33.99
49 34.94 34.55 33.92
50 35.93 35.30 34.52
2017003
51 35.36 35.12 35.45
52 35.85 34.62 35.12
53 34.40 34.68 34.06
54 35.28 34.36 34.79
55 36.69 35.01 34.96
56 35.38 34.67 34.92
57 35.98 33.22 35.22
58 35.40 34.23 32.91
59 36.66 32.55 33.81
60 36.45 33.00 32.31
Number of detected
60 60 60
positive samples
Conclusion: At the minimum detection limit of 2.0 TCID50/mL, three

batches of qualified kits were used to test the above three throat swab samples

for influenza A virus 20 times, respectively and the detected positive rate are
all higher than 95%. Therefore, 2.0 TCID50/mL can be used as the minimum
detection limit of this kit for influenza A virus; At the minimum detection
limit of 2.0 TCID50/mL, three batches of qualified kits were used to test the
above three throat swab samples for influenza B virus 20 times, respectively
and the detected positive rate are all higher than 95%. Therefore, 2.0
TCID50/mL can be used as the minimum detection limit of this kit for
influenza B virus; At the minimum detection limit 500 copies/mL, three
for respiratory syncytial virus 20 times, respectively and the detected positive
rate are all higher than 95%. Therefore, 500 copies/mL can be used as the
minimum detection limit of this kit for respiratory syncytial virus; At the
minimum detection limit 500 copies/mL, three batches of qualified kits were
used to test the above three throat swab samples for Adenovirus 20 times,
respectively and the detected positive rate are all higher than 95%. Therefore,
500 copies/mL can be used as the minimum detection limit of this kit for
Adenovirus; At the minimum detection limit 500 copies/mL, three batches of
qualified kits were used to test the above three throat swab samples for
Human rhinovirus 20 times, respectively and the detected positive rate are all
higher than 95%. Therefore, 500 copies/mL can be used as the minimum
detection limit of this kit for Human rhinovirus; At the minimum detection
limit 500 copies/mL, three batches of qualified kits were used to test the above
three throat swab samples for Mycoplasma pneumoniae 20 times, respectively
and the detected positive rate are all higher than 95%. Therefore, 500
copies/mL can be used as the minimum detection limit of this kit for
Mycoplasma pneumoniae.
2.2 Verification of national reference samples

The minimum detection limit reference samples S1-S5 in the national
reference samples of nucleic acid detection kits for influenza A/B virus
(Batches 370006-201501) were tested. S1-S5 were diluted to 2.0 TCID50/mL and
were tested using three batches of qualified kits on ABI7500 instrument, so as
to verify the sensitivity performance of the three batches of kits on national
reference samples. Each sample was extracted 20 times each time and was
repeatedly tested 20 times. The test results were then counted.
Test results are shown in Table 1-19 (Results of the three batches of kits)
and Figure 1-43 to Figure 1-47 (Only the results of 2017001 batch are shown).

Figure 1-43 Test result of the national reference sample S1 at a concentration of 2.0
TCID50/mL (2017001 batch)


Table 1-19 The detected Ct value of the national sensitivity reference samples
Influenza B virus Influenza A virus
Batch No.
S1 S2 S3 S4 S5
1 34.54 34.44 34.00 33.18 32.43
2 34.02 34.16 33.52 32.80 31.84
3 34.01 34.31 33.34 32.51 32.36
4 34.56 34.23 33.47 32.80 29.47
5 34.25 33.92 33.13 32.69 32.80
6 34.21 33.76 32.94 32.43 31.93
7 34.19 34.40 33.15 33.90 31.34
8 34.65 33.80 33.18 32.67 32.61
9 34.26 34.85 34.92 32.12 32.55
10 34.11 33.83 34.84 33.27 32.57
2017001
11 34.28 34.59 34.78 32.52 32.77
12 34.32 33.57 34.97 31.75 32.04
13 34.49 35.21 33.33 32.95 32.63
14 33.94 34.61 34.12 32.69 32.21
15 34.34 35.04 33.76 32.51 31.07
16 34.44 34.45 34.15 32.52 32.29
17 34.20 33.57 33.94 32.48 32.62
18 34.45 36.08 33.61 32.67 32.78
19 33.60 35.55 34.12 32.86 32.91
20 33.98 34.93 33.67 32.36 33.05
21 33.96 35.25 31.93 33.05 31.42
22 33.96 34.92 31.67 31.86 31.47
23 33.59 34.48 31.74 32.17 31.51
2017002
24 33.67 34.07 31.72 32.28 31.49
25 34.00 34.54 31.62 32.11 32.64
26 33.41 33.84 31.47 31.81 32.17

27 33.75 34.83 31.50 32.16 31.95
28 33.78 33.88 32.08 32.13 32.96
29 34.11 34.24 35.86 31.08 32.29
30 33.37 34.62 34.22 32.31 31.66
31 33.40 34.60 34.97 31.75 32.12
32 33.89 34.95 33.99 31.23 31.82
33 33.57 35.00 32.99 32.26 31.18
34 33.62 34.51 33.38 31.71 31.54
35 34.00 34.72 33.07 31.95 31.56
36 33.79 34.63 33.10 31.31 31.18
37 33.40 34.53 33.20 32.11 31.77
38 34.56 35.87 33.07 31.96 31.48
39 34.09 35.34 33.44 32.24 31.77
40 33.73 34.82 33.33 32.14 31.36
41 35.99 35.15 32.27 30.68 31.90
42 35.70 35.69 31.88 31.36 31.79
43 36.08 34.77 31.35 30.14 31.92
44 35.86 35.03 31.12 31.74 31.95
45 35.75 34.85 31.89 31.75 32.04
46 35.54 35.29 32.11 31.28 31.89
47 34.55 35.11 31.86 31.90 31.92
48 35.24 35.03 32.03 31.99 31.91
49 34.85 34.53 32.83 31.31 30.08
50 35.24 34.78 32.96 31.75 31.62
2017003
51 34.66 34.38 32.94 31.74 31.92
52 34.46 34.34 32.19 31.67 29.83
53 34.65 34.16 32.46 31.64 31.91
54 34.50 34.20 32.34 31.58 31.92
55 35.10 34.68 33.06 31.58 32.27
56 35.12 34.35 32.30 31.57 31.85
57 35.19 34.63 33.36 31.41 31.79
58 34.90 35.05 32.94 31.30 31.82
59 34.91 34.74 32.44 31.22 31.53
60 35.00 34.64 32.95 31.48 31.72
Number of detected
60 60 60 60 60
positive samples
Positive rate 100% 100% 100% 100% 100%

batches of qualified kits were used to test the above diluted national reference
samples of nucleic acid detection reagent for influenza A/B virus 20 times,
respectively and the detected positive rate are all 100%; Meanwhile, all the
sensitivity reference samples met the requirements of national reference
sample detection at the concentration of 2.0 TCID50/mL. Therefore, 2.0
influenza A/B virus, respectively.

QuantStudioTM 5
Analytical performance evaluation on SLAN-96P automatic

Minimum detection limit (Analytical sensitivity)

1.Determination of the minimum detection limit
The pathogen culture medium with known pathogen concentration was
diluted to determine the minimum detection limit of this kit. The
concentration of influenza A virus and influenza B virus was calibrated by
using 50% tissue culture infectious dose (TCID50). The concentration of
respiratory syncytial virus, adenovirus, human rhinovirus, and Mycoplasma
pneumonia was calibrated by using the nucleic acid concentration of target
fragment (copies/mL). Each pathogen was diluted with normal saline, and
each gradient of diluted pathogen medium had 3 copies, each of which was
tested 20 times with three batches of kits; In total, each gradient of diluted
pathogen medium was repeatedly tested 60 times. The pathogen
concentration and dilution information are shown in Table 2-1 and Table 2-2.
The diluted strain was used as the test sample and was tested according to the
manual of this kit. The test results are shown in Table 2-3 to Table 2-10
Table 2-1 Pathogen information and dilution gradient for testing (Influenza A, Influenza
Diluted detection
Pathogen No. Type
concentration(TCID50/mL)
StrainInf A-A A/H1N1 4.0 2.0 1.5 1.0
StrainInf A-B A/H3 4.0 2.0 1.5 1.0
StrainInf A-C A/H5 4.0 2.0 1.5 1.0
StrainInf B-A B/Victoria 4.0 2.0 1.5 1.0
StrainInf B-B B/Victoria 4.0 2.0 1.5 1.0
StrainInf B-C B/Yamagata 4.0 2.0 1.5 1.0
B)
Table 2-2 Pathogen information and dilution gradient for testing

Diluted detection
Pathogen No. Type
concentration(copies/mL)
StrainRSV-A A type 1000 500 400 300
StrainRSV-B B type 1000 500 400 300
StrainRSV-C B type 1000 500 400 300

StrainADV-A 3 type 1000 500 400 300
StrainADV-B 7 type 1000 500 400 300
StrainADV-C 4 type 1000 500 400 300
StrainADV-D 1 type 1000 500 400 300
StrainADV-E 2 type 1000 500 400 300
StrainADV-F 5 type 1000 500 400 300
StrainADV-G 14 type 1000 500 400 300
StrainADV-H 21 type 1000 500 400 300
StrainADV-I 55 type 1000 500 400 300
StrainHRV-A A type 1000 500 400 300
StrainHRV-B B type 1000 500 400 300
StrainHRV-C C type 1000 500 400 300
StrainMP-A 1 type 1000 500 400 300
StrainMP-B 1 type 1000 500 400 300
StrainMP-C 2 type 1000 500 400 300
Figure 2-1 Test results of Strain Inf A-A at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 2-2 Test results of Strain Inf A-B at a concentration of 2.0 TCID50/mL (2017001
batch)

Figure 2-3 Test results of Strain Inf A-C at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 2-4 Test results of strain Inf B-A at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 2-5 Test results of strain Inf B-B at a concentration of 2.0 TCID50/mL (2017001
batch)

Figure 2-6 Test results of Strain Inf B-C at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 2-7 Test results of Strain RSV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 2-8 Test results of Strain RSV-B at a concentration of 500 copies/mL (2017001
batch)

Figure 2-9 Test results of Strain RSV-C at a concentration of 500 copies/mL (2017001
batch)
Figure 2-10 Test results of Strain ADV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 2-11 Test results of Strain ADV-B at a concentration of 500 copies/mL (2017001
batch)

Figure 2-12 Test results of Strain ADV-C at a concentration of 500 copies/mL (2017001
Figure 2-13 Test results of Strain ADV-D at a concentration of 500 copies/mL (2017001
batch)
Figure 2-14 Test results of Strain ADV-E at a concentration of 500 copies/mL (2017001
batch)

Figure 2-15 Test results of Strain ADV-F at a concentration of 500 copies/mL (2017001
batch)
Figure 2-16 Test results of Strain ADV-G at a concentration of 500 copies/mL (2017001
batch)
Figure 2-17 Test results of Strain ADV-H at a concentration of 500 copies/mL (2017001
batch)

Figure 2-18 Test results of Strain ADV-I at a concentration of 500 copies/mL (2017001
batch)
Figure 2-19 Test results of Strain HRV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 2-20 Test results of Strain HRV-B at a concentration of 500 copies/mL (2017001
batch)

Figure 2-21 Test results of Strain HRV-C at a concentration of 500 copies/mL (2017001
batch)
Figure 2-22 Test results of Strain MP-A at a concentration of 500 copies/mL (2017001
batch)
Figure 2-23 Test results of Strain MP-B at a concentration of 500 copies/mL (2017001
batch)

Figure 2-24 Test results of Strain MP-C at a concentration of 500 copies/mL (2017001
batch)
influenza A virus
Pathogen No. Strain Inf A-A Strain Inf A-B Strain Inf A-C
Detection rate
4.0 60/60 100% 60/60 100% 60/60 100%
2.0 60/60 100% 60/60 100% 60/60 100%
1.5 47/60 78.3% 49/60 81.7% 46/60 76.7%
1.0 41/60 68.3% 42/60 70% 35/60 58.3%
influenza B virus
Pathogen No. Strain Inf B-A Strain Inf B-B Strain Inf B-C
Detection rate
4.0 60/60 100% 60/60 100% 60/60 100%
2.0 60/60 100% 59/60 98.3% 60/60 100%
1.5 48/60 80% 48/60 80% 48/60 80%
1.0 42/60 70% 19/60 31.7% 43/60 71.7%
respiratory syncytial virus
Pathogen No. Strain RSV-A Strain RSV-B Strain RSV-C
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 58/60 96.7% 60/60 100%
400 46/60 76.7% 49/60 81.7% 48/60 80%
300 24/60 40% 30/60 50% 28/60 46.6%

Adenovirus
Pathogen No. Strain ADV-A Strain ADV-B Strain ADV-C
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 60/60 100% 60/60 100%
400 49/60 81.7% 47/60 78.3% 43/60 71.7%
300 41/60 68.3% 40/60 66.7% 37/60 61.7%
Adenovirus
Pathogen No. Strain ADV-D Strain ADV-E Strain ADV-F
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 60/60 100% 60/60 100%
400 60/60 100% 60/60 100% 60/60 100%
300 55/60 91.67% 42/60 70.00% 59/60 98.33%
Adenovirus
Pathogen No. Strain ADV-G Strain ADV-H Strain ADV-I
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 60/60 100% 60/60 100%
400 60/60 100% 60/60 100% 60/60 100%
300 60/60 100% 60/60 100% 60/60 100%
Pathogen No. Strain MP-A Strain MP-B Strain MP-C
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 60/60 100% 60/60 100%
400 48/60 80% 48/60 80% 49/60 81.7%
300 37/60 61.7% 36/60 60% 37/60 61.7
Human rhinovirus

Pathogen No. Strain HRV-A Strain HRV-B Strain HRV-C
Detection rate
1000 60/60 100% 60/60 100% 60/60 100%
500 60/60 100% 60/60 100% 59/60 98.3%
400 48/60 80% 47/60 78.3% 45/60 75%
300 35/60 58.3% 35/60 58.3 38/60 63.3%
Conclusion: The sample with Ct ≤ 40 was judged as positive, and

pathogen concentration with the positive detection rate ≥ 95% was regarded
as the minimum detection limit. According to the above-described test results
on SLAN-96P instrument: when the concentration of test sample for influenza
A virus is 4.0 TCID50/mL, all the positive detection rate are 100%; when the
concentration is 2.0 TCID50/mL, all the positive detection rate are higher than
95%; when the concentration is 1.5 TCID50/mL, the positive detection rate of
three strains of influenza A virus are lower than 95%, when the concentration
is 1.0 TCID50/mL, all the positive detection rate are lower than 70%. Therefore,
in order to ensure that the positive detection rate of all samples is ≥ 95%, the
minimum detection limit of this kit for influenza A virus is set to 2.0
TCID50/mL. Similarly, according to the test results of each pathogen on
SLAN-96P instrument, the minimum detection limit of this kit for influenza B
virus is set to 2.0 TCID50/mL; the minimum detection limit of this kit for
respiratory syncytial virus, Adenovirus, Human rhinovirus and Mycoplasma
pneumoniae are all set to 500 copies/mL, respectively.
2.Verification of minimum detection limit

2.1Verification of clinical samples
respiratory syncytial virus, Adenovirus, Mycoplasma pneumoniae and
Human rhinovirus with known pathogen concentration were diluted to verify
the minimum detection limit of this kit. The concentration of influenza A
virus and influenza B virus was calibrated by using 50% tissue culture
infectious dose (TCID50). The concentration of respiratory syncytial virus,
adenovirus, human rhinovirus, and Mycoplasma pneumonia was calibrated
by using the nucleic acid concentration of target fragment (copies/mL). The
samples of each pathogen were diluted with normal saline to the minimum
detection limit of this kit, and the diluted samples were tested by 3 batches of
kits 20 times, respectively. The concentration of throat swab samples of each
pathogen and sample information are shown in Table 2-11 and Table 2-12. The
diluted samples were used as the test sample and were tested on SLAN-96P
instrument according to the manual of this kit. The test results are shown in
Table 2-13 to Table 2-18 (detection results of the three batches of kits) and in
Figure 2-25 to Figure 2-42 (only the test results of the 2017001 batch of
reagents was shown).
Table 2-11 Sample information for minimum detection limit verification (Influenza A,
Influenza B)

Sample No. Type Source Concentration
(TCID50/mL)
Sichuan provincial
A/Qingyang/1272/2014(
Sample Inf A-A disease prevention and 2100
H3)
control center
A/Zhejiang/
Yiwu disease prevention
Sample Inf A-B DTID-ZJU03/2009(H1N1 6500
and control center
)
Loudi disease
Sample Inf A-C A/H5 prevention and control 4400
center
Sichuan provincial
Sample Inf B-A B/Victoria disease prevention and 6400
control center
Sichuan provincial
Sample Inf B-B B/Victoria disease prevention and 5500
control center
Sichuan provincial
Sample Inf B-C B/Yamagata disease prevention and 7100
control center
Table 2-12 Sample information for minimum detection limit verification

Concentration
(copies/mL)
Human provincial
Sample RSV-A A type 8.71E+05
Sample RSV-B B type Nanjing medial 6.73E+04
university
Human provincial
Sample RSV-C B type 7.93E+04
Human provincial
Sample ADV-A 3 type 1.34E+05
Human provincial
Sample ADV-B 7 type 5.74E+05
Human provincial
Sample ADV-C 4 type 6.78E+05
Human provincial
Sample HRV-A A type 2.33E+04
Human provincial
Sample HRV-B B type 5.27E+05
Human provincial
Sample HRV-C C type 4.85E+04
Human provincial
Sample MP-A 1 type 2.27E+05
Human provincial
Sample MP-B 1 type 1.24E+05

Sample MP-C 2 type Nanjing medial 6.15E+05
university
Figure 2-25 Test results of sample Inf A-A at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 2-26 Test results of sample Inf A-B at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 2-27 Test results of sample Inf A-C at a concentration of 2.0 TCID50/mL (2017001
batch)

Figure 2-28 Test results of sample Inf B-A at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 2-29 Test results of sample Inf B-B at a concentration of 2.0 TCID50/mL (2017001
batch)
Figure 2-30 Test results of sample Inf B-C at a concentration of 2.0 TCID50/mL (2017001
batch)

Figure 2-31 Test results of sample RSV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 2-32 Test results of sample RSV-B at a concentration of 500 copies/mL (2017001
batch)
Figure 2-33 Test results of sample RSV-C at a concentration of 500 copies/mL (2017001
batch)

Figure 2-34 Test results of sample ADV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 2-35 Test results of sample ADV-B at a concentration of 500 copies/mL (2017001
batch)
Figure 2-36 Test results of sample ADV-C at a concentration of 500 copies/mL (2017001
batch)

Figure 2-37 Test results of sample HRV-A at a concentration of 500 copies/mL (2017001
batch)
Figure 2-38 Test results of sample HRV-B at a concentration of 500 copies/mL (2017001
batch)
Figure 2-39 Test results of sample HRV-C at a concentration of 500 copies/mL (2017001
batch)

Figure 2-40 Test results of sample MP-A at a concentration of 500 copies/mL (2017001
batch)
Figure 2-41 Test results of sample MP-B at a concentration of 500 copies/mL (2017001
batch)
Figure 2-42 Test results of sample MP-C at a concentration of 500 copies/mL (2017001
batch)
Table 2-13 Statistical table of positive rate of influenza A virus

Sample Inf A-A Sample Inf A-B Sample Inf A-C
Batch No.
1 33.24 33.32 34.07
2017001 2 33.27 34.76 33.64
3 34.42 34.79 33.61

4 33.95 34.90 34.67
5 34.40 33.21 34.29
6 33.45 33.33 34.29
7 36.22 33.41 33.56
8 33.93 32.93 34.26
9 35.87 34.25 33.08
10 33.92 34.64 33.42
11 33.19 34.99 34.30
12 34.21 34.41 34.92
13 35.01 33.89 34.00
14 33.68 34.20 34.89
15 33.75 33.64 35.62
16 35.00 37.04 34.40
17 33.06 34.96 33.55
18 34.46 34.68 34.35
19 33.75 33.90 33.83
20 34.36 37.66 36.24
21 33.49 33.75 34.02
22 34.45 34.35 33.93
23 33.69 34.22 34.37
24 34.27 35.01 34.94
25 33.67 34.01 34.01
26 37.72 34.25 33.33
27 33.40 35.64 33.63
28 37.95 33.55 34.28
29 33.07 33.75 34.39
30 34.49 34.72 34.49
2017002
31 34.51 33.93 34.53
32 34.64 34.54 33.81
33 32.99 33.92 36.70
34 33.09 33.97 34.18
35 33.17 33.64 36.31
36 32.69 38.82 35.01
37 34.00 33.32 36.09
38 34.37 34.76 34.60
39 34.70 34.79 34.92
40 34.15 34.90 34.70
41 33.72 34.45 33.73
42 35.19 36.09 33.84
43 34.43 36.27 33.83
44 35.12 36.72 33.13
45 33.63 33.84 35.75
2017003
46 33.77 35.14 33.46
47 35.53 35.74 35.45
48 34.06 34.70 34.24
49 34.18 34.07 35.29
50 34.84 33.64 33.86

51 34.66 33.61 34.19
52 35.49 34.67 33.95
53 34.45 34.29 35.01
54 34.70 34.29 33.74
55 36.55 33.56 37.90
56 33.97 34.26 34.51
57 34.20 33.08 33.91
58 35.20 33.42 35.55
59 34.36 34.30 35.54
60 35.02 34.92 36.07
Number of detected
60 60 60
positive samples
Table 2-14 Statistical table of positive rate of influenza B virus

Sample Inf B-A Sample Inf B-B Sample Inf B-C
Batch No.
1 34.79 35.52 35.11
2 34.52 34.94 34.36
3 34.31 34.45 33.85
4 33.49 35.46 33.72
5 34.33 35.61 35.50
6 34.11 34.90 34.97
7 33.43 36.09 34.99
8 34.82 35.38 34.33
9 35.04 35.64 34.91
10 34.13 35.38 35.07
2017001
11 34.53 35.15 35.44
12 35.15 34.35 34.61
13 34.35 35.19 34.57
14 34.89 34.93 34.60
15 34.50 34.28 35.13
16 35.15 35.69 34.33
17 35.06 35.91 35.42
18 34.15 35.00 35.13
19 34.33 35.38 35.27
20 34.56 36.02 34.50
21 35.11 36.06 33.27
22 34.84 35.13 35.46
23 34.63 35.52 35.78
2017002
24 33.81 36.14 34.61
25 34.65 35.35 35.26
26 34.41 35.91 34.93

27 33.74 35.51 34.47
28 35.14 36.16 35.98
29 35.36 36.05 35.86
30 34.46 35.14 35.07
31 34.85 35.33 34.46
32 35.47 35.55 34.31
33 34.68 35.34 36.31
34 35.22 35.68 35.71
35 34.83 35.09 35.79
36 35.48 34.83 34.93
37 35.38 34.69 35.62
38 34.47 34.78 35.80
39 34.65 35.76 36.17
40 34.89 35.36 35.25
41 35.66 35.06 34.33
42 34.76 35.76 34.95
43 34.94 35.35 34.42
44 35.17 34.39 33.70
45 34.98 34.70 34.04
46 35.30 35.56 34.77
47 34.65 34.61 33.95
48 34.46 34.71 34.05
49 34.32 34.75 33.99
50 34.38 34.69 33.98
2017003
51 35.38 35.09 34.37
52 34.96 35.28 34.55
53 34.74 36.25 35.34
54 35.63 35.48 34.70
55 35.19 35.78 34.99
56 35.52 34.60 33.94
57 35.40 36.29 34.35
58 35.92 34.90 34.52
59 37.22 35.80 34.01
60 34.71 34.76 33.86
Number of detected
60 60 60
positive samples
Table 2-15 Statistical table of positive rate of respiratory syncytial virus
Batch No. Sample RSV-A Sample RSV-B Sample RSV-C

1 35.62 34.64 34.70
2 34.21 34.71 34.41
3 34.55 34.53 34.37
4 34.01 34.39 34.09
5 33.82 34.24 34.31
6 34.07 34.82 33.02
7 34.66 35.47 34.39
8 No Ct 34.35 34.20
9 34.01 35.33 33.12
10 35.25 34.40 37.19
2017001
11 34.31 34.18 33.38
12 33.87 34.64 34.71
13 34.48 .35.54 34.00
14 34.10 34.25 33.72
15 34.02 34.01 34.73
16 35.20 34.45 33.16
17 34.46 34.70 33.56
18 34.11 34.02 34.88
19 34.23 35.61 34.99
20 33.97 33.85 33.12
21 34.07 33.66 34.17
22 34.13 33.77 33.25
23 34.12 32.82 34.68
24 33.98 33.05 33.63
25 33.83 34.29 36.13
26 33.92 33.59 33.42
27 34.79 33.36 34.06
28 33.90 33.55 34.88
29 34.21 33.74 33.63
30 34.00 33.45 32.60
2017002
31 33.40 34.24 34.19
32 33.64 32.31 34.85
33 34.76 34.05 35.38
34 33.83 33.81 33.68
35 33.60 33.56 34.45
36 33.88 No Ct 34.36
37 34.08 33.69 33.94
38 33.66 33.64 33.59
39 34.75 34.05 35.11
40 33.09 34.10 33.43

41 34.38 33.80 33.73
42 34.29 32.82 32.90
43 34.91 32.98 34.37
44 35.10 32.98 33.38
45 34.25 32.92 33.75
46 35.12 37.13 33.57
47 34.81 33.27 33.57
48 34.25 33.86 33.33
49 34.76 33.18 33.53
50 35.80 33.23 32.34
2017003
51 38.78 35.08 33.60
52 34.43 34.13 33.52
53 34.42 33.28 32.45
54 34.45 34.87 35.87
55 34.40 33.80 32.66
56 34.02 34.27 33.76
57 34.62 34.03 33.29
58 34.21 34.01 33.03
59 34.55 33.75 33.96
60 34.01 33.95 32.43
Number of detected
59 59 60
positive samples
Positive rate 98.3% 98.3% 100%
Table 2-16 Statistical table of positive rate of Adenovirus

Sample Sample
Sample ADV-B
Batch No. ADV-A ADV-C
1 33.51 36.73 33.93
2 34.08 34.56 36.18
3 33.98 33.81 34.71
4 34.74 33.73 34.46
5 33.77 35.48 34.37
6 34.00 35.01 35.79
7 35.19 35.26 35.17
2017001
8 33.32 36.23 34.53
9 33.49 33.38 33.18
10 34.45 34.15 33.18
11 33.69 33.62 34.60
12 34.27 34.84 36.23
13 33.67 34.57 34.40
14 33.72 33.94 34.83

15 33.40 34.61 34.91
16 37.95 33.96 34.63
17 33.07 34.71 34.36
18 34.49 34.77 34.99
19 34.51 33.77 33.63
20 34.64 33.80 35.74
21 34.68 34.25 34.70
22 34.42 34.64 36.34
23 33.67 34.99 36.63
24 37.26 34.41 37.02
25 34.41 33.89 34.05
26 33.20 34.20 35.37
27 33.51 33.64 35.97
28 34.07 37.04 34.92
29 36.24 34.96 34.28
30 34.30 34.68 33.85
2017002
31 33.56 33.90 33.81
32 33.47 37.66 34.91
33 35.17 34.66 34.52
34 34.71 33.44 34.52
35 34.99 33.75 33.76
36 35.92 34.43 34.50
37 33.14 36.73 33.28
38 33.90 34.56 33.62
39 33.37 33.81 34.55
40 34.57 33.73 34.92
41 33.76 34.40 33.60
42 35.25 33.55 33.16
43 35.31 34.35 33.15
44 35.37 33.83 34.15
45 33.60 35.24 33.77
46 33.75 35.00 33.77
47 33.82 35.19 33.10
2017003 48 33.32 No Ct 33.75
49 34.72 33.93 32.65
50 35.13 36.18 32.97
51 35.52 34.71 33.76
52 34.88 34.46 34.41
53 34.33 34.37 33.54
54 34.67 35.79 34.39
55 34.09 35.17 35.02

56 37.85 34.53 33.91
57 35.46 33.18 33.09
58 35.17 33.18 33.80
59 34.33 34.60 33.37
60 38.67 36.23 34.74
Number of detected
60 59 60
positive samples
Positive rate 100% 98.3% 100%
Table 2-17 Statistical table of positive rate of Mycoplasma pneumoniae

Sample MP-A Sample MP-B Sample MP-C
Batch No.
1 34.80 34.76 34.16
2 33.99 34.36 34.07
3 36.69 34.49 33.63
4 33.53 34.21 33.32
5 33.98 36.98 34.73
6 34.29 34.91 33.17
7 34.24 34.32 33.69
8 34.47 36.76 34.42
9 36.81 35.34 33.61
10 34.13 34.18 33.81
2017001
11 36.56 37.19 34.30
12 34.16 33.87 34.73
13 33.79 34.45 34.43
14 33.44 34.56 34.10
15 34.76 34.50 33.84
16 34.16 34.77 33.61
17 34.26 37.32 33.92
18 34.01 34.40 33.81
19 34.20 37.03 34.50
20 34.87 34.43 33.39
21 34.04 34.01 33.88
22 33.97 34.67 34.09
23 34.49 36.86 34.59
24 34.56 33.74 35.08
25 33.68 33.75 34.74
2017002
26 34.69 33.79 34.39
27 34.21 33.46 34.13
28 34.02 33.45 33.87
29 .4.41 34.54 34.19
30 35.29 33.57 34.10

31 37.79 33.85 34.83
32 34.10 33.45 33.67
33 34.10 33.39 33.21
34 34.13 33.50 33.77
35 33.77 33.94 34.04
36 33.75 No Ct 34.54
37 35.14 33.40 33.98
38 33.90 34.36 34.57
39 34.21 32.86 34.18
40 33.75 33.30 33.63
41 34.01 32.82 33.54
42 35.25 36.55 34.19
43 34.31 33.05 36.71
44 33.87 34.28 32.76
45 34.48 33.68 33.41
46 34.10 33.41 33.33
47 34.02 34.38 32.86
48 35.20 32.83 32.61
49 34.46 33.24 33.80
50 34.11 34.47 32.50
2017003
51 34.23 34.59 33.15
52 33.97 32.82 33.69
53 35.57 33.89 32.91
54 34.62 34.30 33.13
55 34.08 33.70 33.56
56 36.34 33.37 33.90
57 34.80 32.71 33.64
58 33.99 33.95 33.38
59 36.69 33.04 33.11
60 33.53 35.24 32.96
Number of detected
60 59 60
positive samples
Positive rate 100% 98.3% 100%
Table 2-18 Statistical table of positive rate of Human rhinovirus

Sample
Sample HRV-B Sample HRV-C
Batch No. HRV-A
1 34.79 35.91 34.46
2 34.52 35.01 34.83
2017001
3 34.31 35.19 34.45
4 33.49 35.42 35.42

5 34.33 35.23 34.33
6 34.11 35.55 34.42
7 33.43 34.92 34.97
8 34.82 34.72 35.22
9 35.04 34.58 34.10
10 34.13 34.63 34.07
11 34.53 35.63 35.04
12 35.15 35.22 34.78
13 34.35 35.00 34.39
14 34.89 35.88 33.10
15 34.50 35.46 35.42
16 35.15 35.78 34.51
17 35.06 35.65 34.47
18 34.15 36.18 34.49
19 34.33 37.49 33.80
20 34.56 34.97 34.73
21 34.04 35.78 36.29
22 34.11 36.32 35.85
23 35.09 37.63 35.97
24 34.68 35.11 35.09
25 34.45 36.25 35.22
26 35.35 36.54 35.46
27 34.89 36.11 35.36
28 35.24 36.21 35.72
29 35.12 36.05 35.10
30 35.63 34.68 35.52
2017002
31 36.94 33.62 35.05
32 34.43 35.80 36.20
33 35.59 35.52 34.93
34 35.88 35.85 35.03
35 35.46 35.13 35.66
36 35.55 36.65 35.92
37 35.38 34.70 34.67
38 34.01 35.86 34.64
39 32.96 35.32 35.77
40 35.14 35.23 35.42
41 35.66 34.14 32.29
42 34.29 34.59 34.38
2017003 43 33.24 34.83 34.70
44 35.42 35.18 33.64
45 35.12 32.97 34.28

46 35.45 35.12 34.00
47 34.74 35.45 33.57
48 36.21 34.31 34.90
49 34.31 34.95 34.70
50 35.46 34.64 33.94
51 34.91 34.20 33.50
52 34.84 35.65 33.38
53 34.67 35.50 35.02
54 34.94 34.73 34.58
55 35.07 34.17 34.58
56 34.87 34.03 34.00
57 35.21 35.93 34.52
58 33.66 35.35 34.68
59 34.45 35.39 35.03
60 34.42 34.63 34.27
Number of detected
60 60 60
positive samples

for influenza A virus 20 times, respectively and the detected positive rate are
all higher than 95%. Therefore, 2.0 TCID50/mL can be used as the minimum
detection limit of this kit for influenza A virus; At the minimum detection
limit of 2.0 TCID50/mL, three batches of qualified kits were used to test the
above three throat swab samples for influenza B virus 20 times, respectively
and the detected positive rate are all higher than 95%. Therefore, 2.0
influenza B virus; At the minimum detection limit 500 copies/mL, three
for respiratory syncytial virus 20 times, respectively and the detected positive
rate are all higher than 95%. Therefore, 500 copies/mL can be used as the
minimum detection limit of this kit for respiratory syncytial virus; At the
minimum detection limit 500 copies/mL, three batches of qualified kits were
used to test the above three throat swab samples for Adenovirus 20 times,
respectively and the detected positive rate are all higher than 95%. Therefore,
500 copies/mL can be used as the minimum detection limit of this kit for
Adenovirus; At the minimum detection limit 500 copies/mL, three batches of
qualified kits were used to test the above three throat swab samples for
Human rhinovirus 20 times, respectively and the detected positive rate are all
higher than 95%. Therefore, 500 copies/mL can be used as the minimum
detection limit of this kit for Human rhinovirus; At the minimum detection
limit 500 copies/mL, three batches of qualified kits were used to test the above
three throat swab samples for Mycoplasma pneumoniae 20 times, respectively
and the detected positive rate are all higher than 95%. Therefore, 500
copies/mL can be used as the minimum detection limit of this kit for
Mycoplasma pneumoniae.

The minimum detection limit reference samples S1-S5 in the national
reference samples of nucleic acid detection kits for influenza A/B virus
(Batches 370006-201501) were tested. S1-S5 were diluted to 2.0 TCID50/mL and
were tested using three batches of qualified kits on SLAN-96P instrument, so
as to verify the sensitivity performance of the three batches of kits on national
reference samples. Each sample was extracted 20 times each time and was
repeatedly tested 20 times. The test results were then counted.
Test results are shown in Table 2-19 (Results of the three batches of kits)
and Figure 2-43 to Figure 2-47 (Only the results of 2017001 batch are shown).

Table 2-19 The detected Ct value of the national sensitivity reference samples

Influenza B virus Influenza A virus
Batch No.
S1 S2 S3 S4 S5
1 32.33 32.62 33.18 32.75 32.96
2 32.45 32.76 33.12 32.75 33.07
3 33.06 33.23 33.11 33.29 32.28
4 32.87 32.46 32.92 33.07 32.91
5 33.18 33.10 33.07 32.83 33.03
6 32.68 33.13 30.15 33.24 32.94
7 32.65 32.66 32.62 33.02 32.83
8 32.67 32.19 33.20 33.29 33.23
9 32.49 32.98 33.20 32.94 32.88
10 32.55 33.62 32.72 33.20 33.06
2017001
11 32.68 32.74 33.29 32.89 32.79
12 32.87 32.88 32.65 32.84 32.79
13 32.42 32.48 33.13 33.18 32.98
14 32.49 33.35 33.24 32.90 32.65
15 32.44 32.29 32.84 32.87 33.17
16 32.17 32.46 32.91 32.95 32.84
17 32.72 33.31 32.73 33.22 33.16
18 32.40 32.35 32.83 32.79 33.03
19 32.71 32.59 32.63 32.91 32.66
20 33.05 32.82 33.07 33.25 32.42
21 32.24 33.92 31.66 31.90 31.68
22 32.05 34.11 31.77 31.98 31.35
23 32.47 33.66 31.81 31.99 31.19
24 31.84 33.77 31.71 32.01 31.22
25 32.21 34.05 31.64 31.98 23.75
26 27.63 34.51 31.41 32.17 31.40
27 32.15 33.60 31.78 31.87 31.69
28 32.04 33.43 31.77 31.92 31.93
29 32.12 34.06 32.02 32.23 31.54
2017002
30 32.06 33.33 31.77 32.02 31.54
31 32.41 33.80 31.85 32.23 31.06
32 32.15 33.82 32.35 32.05 30.84
33 32.35 33.89 31.93 32.23 31.56
34 32.19 33.69 31.97 31.96 28.91
35 32.28 34.19 32.08 31.94 32.34
36 32.18 34.07 32.08 32.18 31.16
37 32.06 33.85 31.99 31.99 31.77
38 32.19 34.31 32.09 32.15 31.52

39 31.78 33.74 31.98 31.90 31.73
40 32.45 33.46 31.71 31.82 31.55
41 30.32 33.40 29.77 31.51 31.84
42 30.35 33.62 31.74 31.81 31.95
43 30.44 33.76 31.88 31.86 31.88
44 30.28 33.40 31.70 31.50 31.95
45 33.93 33.35 31.66 31.95 32.05
46 33.71 33.24 31.75 31.85 31.80
47 34.07 33.66 31.68 32.03 32.02
48 33.85 33.76 31.92 31.73 31.79
49 34.26 33.62 31.75 31.85 32.09
50 34.18 33.86 31.72 31.59 31.99
2017003
51 34.17 33.94 31.71 31.65 31.58
52 33.48 33.86 31.74 31.82 31.92
53 34.22 33.68 31.71 31.71 31.92
54 33.61 33.97 31.70 31.55 31.76
55 33.82 33.66 31.61 31.71 31.73
56 33.68 33.53 31.85 31.63 31.87
57 33.75 33.39 31.71 31.67 31.76
58 33.59 33.96 31.46 31.58 31.78
59 34.13 33.49 31.56 31.58 31.72
60 33.97 33.71 31.75 31.84 31.90
Number of
detected positive 60 60 60 60 60
samples
Positive rate 100% 100% 100% 100% 100%

batches of qualified kits were used to test the above diluted national reference
samples of nucleic acid detection reagent for influenza A/B virus 20 times,
respectively and the detected positive rate are all 100%; Meanwhile, all the
sensitivity reference samples met the requirements of national reference
sample detection at the concentration of 2.0 TCID50/mL. Therefore, 2.0
influenza A/B virus, respectively.

Analytical Specificity

described below.
1 Analytical specificity
Negative reference samples N1 (measles virus), N2 (mumps virus), N3
(rubella virus), N4 (Staphylococcus aureus), N5 (Escherichia coli), N6
(Pseudomonas aeruginosa), N7 (human parainfluenza virus type I), N8
(normal throat swab)and N9 (normal throat swab) required in the product
technical requirements of the six respiratory pathogens nucleic acid
diagnostic kit (PCR-fluorescence probing), and other pathogens that have
homology in their nucleic acid sequence and are likely to cause the same or
similar clinical symptoms, including: cytomegalovirus, coxsackie virus type A,
human metapneumovirus, bacillus pertussis, chlamydia pneumoniae,
haemophilus influenzae, saliva Streptococcus, Streptococcus pneumoniae,
Neisseria meningitidis, Mycobacterium tuberculosis, human parainfluenza
type 2 and type 3 virus, Epstein-Barr virus, Cryptococcus, Aspergillus
fumigatus, Aspergillus flavus, Candida albicans, Legionella pneumophila and
Intestine Daovirus type 71 were used as the test samples. Three qualified kits
were used to test the samples on the same ABI7500 instrument. And the
specificity of the kit was tested by detecting the negative and positive
coincidence rate. Test results are as follows (only the amplification curves of
the 2017001 batch of reagents are shown):
Studies on analytical specificity 1

Figure 1-1 FAM channel amplification curve of samples for analytical specificity (2017001
batch of reagents)
Figure 1-2 HEX channel amplification curve of samples for analytical specificity (2017001
batch of reagents)
Figure 1-3 CY5 channel amplification curve of samples for analytical specificity (2017001
batch of reagents)
Table 1-1 Test results of variable samples by using three batches of reagents
Batch&Result 2017001 batch 2017002 batch 2017003 batch
Sample FAM HEX CY5 FAM HEX CY5 FAM HEX CY5
Positive control 28.43 29.83 29.80 28.47 28.59 30.14 28.50 29.83 29.98
(Mix A, B) 28.68 30.24 30.08 29.03 28.72 30.73 28.77 30.24 29.85
N1 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
Cytomegaloviru No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
s

Coxsackie virus
No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
type A
Human No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
metapneumovir
us
Bacillus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
pertussis
Chlamydia No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
pneumoniae
Haemophilus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
influenzae
Streptococcus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
salivarius
pneumoniae
Neisseria No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
meningitidis
Mycobacterium No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
tuberculosis
Parainfluenza
Type 2
Parainfluenza
Type 3
Epstein-Barr No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
virus
Cryptococcus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
Aspergillus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
fumigatus
flavus
Candida No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
albicans
Legionella No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
pneumophila
Enterovirus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
type 71
Conclusion: It can be seen from Figure 1-1 to Figure 1-3 and Table 1-1 that,
the positive rate of negative reference samples N1-N8 and other pathogens
that have homology in nucleic acid sequence and are likely to cause the same
or similar clinical symptoms detected by using this detection kit developed by
our company is all 0%. Therefore, this kit has good specificity, and no clinical
cross-reaction occurs.

Negative reference samples NC01-NC06 in the national reference

samples (Batch No. 370006-201501) of nucleic acid detection reagents for
influenza A/B virus were tested on ABI7500 instrument by using three
qualified kits to verify the sensitivity performance of the three batches of kits
on national reference samples. The samples were extracted and tested one
time each time. The test results were then counted.
Test results are shown in Table 1-2 (Results of three batches of reagents)
and Figure 1-4 to Figure 1-6 (Only the results of 2017001 batch of reagents are
shown).
Figure 1-4 FAM channel amplification curve of national reference samples NC01-NC06
(2017001 batch of reagents)
Figure 1-5 HEX channel amplification curve of national reference samples NC01-NC06

Figure 1-6 CY5 channel amplification curve of national reference samples NC01-NC06
(Mix A, B) 32.70 32.83 33.06 32.27 32.91 33.72 31.92 32.87 33.59
NC01 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
2. Research on internal and external interfering substances

2.1 Anti-internal and external interfering substances effects
To investigate the impact of possible endogenous substances (such as
purified mucin, heme, urea, sodium chloride, human whole blood, etc.) in the
test sample on the test results, and to examine the effects of treatment drugs
for respiratory pathogens in the sample (such as oxymetazoline hydrochloride,
dexamethasone, zanamivir, cefmenoxime hydrochloride, menthol, etc.) on
PCR, Lentiviral particles containing the target conserved sequence of
rhinovirus, and cloning plasmids containing the target conserved sequence of
adenovirus and Mycoplasma pneumoniae were used as the research objects.
Lentiviral particles and cloning plasmids of each pathogen were diluted with
normal saline to a concentration of 7.0 × 103 TU/mL (and copies/mL), and were
then mixed with equal volume to form the test sample. The test sample was
divided into several parts, and each part was added with the possible internal
and external interfering substances listed in Table 1-3 and was tested
according to the operating method in the manual of this kit. The test sample
without interfering substances was used as the control. The test results of
three batches of kits on ABI7500 instrument are shown in Table 1-4 to Table
1-6 and in Figure 1-7 to Figure 1-12 (Only the test results of 2017001 batch of
reagents are shown).
Table 1-3 The added internal and external interfering substances and its concentration

Final
Sample Sample Inhibitor Final
Inhibitor name concentratio
No. No. name concentration
n
Oxymetazoline
Hydrochloride
Cefmenoxime
Hydrochloride
Purified
F5 Zanamivir 100 μg/mL F13 20 μg/mL
mucin
Absolute
F7 Azithromycin 100μg/mL F15 20%(v/v)
ethanol
Human
whole blood
Figure 1-7 Test curve of the effect of internal and external interfering substances on
influenza A virus (2017001 batch)

influenza B virus (2017001 batch)
respiratory syncytial virus (2017001 batch)
Adenovirus (2017001 batch)
Human rhinovirus (2017001 batch)

Mycoplasma pneumoniae (2017001 batch)
Table 1-4 Ct value of the tested effects of anti-internal and external interfering
substances (2017001 batch)
PCR mix A PCR mix B
Cont 30.7 30.7 31.0 30.8 31.2 31.4 31.4 30.9 31.2 31.1 31.6 31.6
rol 6 9 3 0 3 0 2 8 3 0 2 1
30.7 30.8 30.9 30.9 30.8 31.9 31.0 31.3 31.5 31.2 31.4 31.5
F1
4 5 6 8 5 6 3 8 8 4 9 4
30.8 30.9 31.1 31.0 31.6 32.2 31.4 31.4 31.4 31.4 31.4 31.6
F2
9 3 0 8 2 6 6 5 8 4 5 2
31.0 30.7 31.3 30.6 32.0 31.6 31.7 31.5 31.4 31.2 32.2 31.9
F3
8 2 5 4 5 6 4 0 4 2 3 0
31.2 31.1 31.3 31.1 31.7 31.7 31.7 31.8 31.3 31.2 32.0 31.7
F4
1 8 1 8 5 6 0 2 4 5 0 1
31.1 31.2 31.4 31.0 31.6 31.7 31.6 31.5 31.5 31.3 31.9 31.4
F5
1 6 1 6 0 6 9 9 8 9 4 7
30.7 31.3 31.5 31.3 31.5 31.3 31.8 31.5 31.5 31.3 31.7 31.5
F6
3 0 5 6 2 0 2 4 0 3 9 6
31.1 31.1 31.0 31.0 31.7 30.8 32.0 31.5 31.3 31.2 31.7 32.0
F7
2 8 4 6 1 2 0 8 6 6 3 0
30.8 30.9 30.5 30.7 31.7 31.4 31.3 31.4 31.0 30.6 31.3 31.6
F8
9 1 7 7 6 8 5 9 8 7 3 2
31.4 31.0 31.0 30.8 31.1 31.8 31.4 31.8 31.1 31.0 31.5 31.8
F9
0 1 7 6 7 5 2 3 8 6 1 9
30.9 31.0 31.1 31.2 31.4 31.8 31.4 31.3 31.3 31.1 31.7 31.4
F10
8 1 6 1 0 4 0 6 0 7 0 8
31.2 31.0 31.3 31.4 31.2 31.8 31.3 31.4 31.1 30.9 31.4 31.3
F11
4 3 1 0 7 1 2 9 7 7 0 0
31.1 30.7 31.2 31.1 30.7 31.2 31.8 31.6 31.2 31.1 31.8 31.4
F12
1 0 8 5 5 2 2 5 4 8 8 0
30.9 31.2 30.6 31.4 31.4 31.0 31.6 31.5 31.2 31.1 31.6 31.9
F13
3 9 7 0 3 7 4 3 2 2 4 0

30.9 31.3 31.2 30.8 31.7 31.1 31.2 31.9 31.1 31.0 31.4 31.9
F14
8 4 0 9 7 7 5 0 0 6 9 0
30.9 31.3 31.2 31.0 31.4 31.0 31.3 32.2 31.3 31.2 31.6 32.0
F15
8 1 0 5 7 6 4 6 6 3 0 4
30.9 30.9 30.6 30.6 30.7 31.4 31.2 31.3 30.9 30.5 31.6 31.5
F16
3 1 7 7 9 4 3 2 3 5 7 2
PCR mix A PCR mix B
Cont 31.0 30.6 31.1 31.1 31.0 31.2 31.0 30.9 31.2 30.2 31.1 31.3
rol 4 4 0 9 6 5 3 9 7 6 6 8
30.3 31.0 31.2 31.5 31.2 31.5 31.1 31.0 31.2 31.2 30.9 31.2
F1
5 2 0 7 5 7 5 0 0 1 4 2
30.6 30.7 31.2 31.4 31.5 31.6 31.0 31.2 31.2 31.2 31.2 31.2
F2
5 6 9 3 6 1 8 4 5 9 7 4
30.8 30.5 31.0 31.1 31.7 31.2 31.5 31.4 31.4 31.2 31.8 31.5
F3
7 0 6 4 8 9 1 3 9 8 5 8
30.9 30.9 30.9 31.3 30.4 31.7 31.2 31.4 31.5 31.1 31.4 31.6
F4
1 9 3 5 1 4 5 3 8 5 3 5
30.7 30.6 31.1 31.4 31.6 31.6 31.3 31.2 31.4 31.6 31.3 30.9
F5
0 5 5 2 9 8 9 9 2 1 1 6
30.6 30.7 31.6 31.8 31.5 31.1 31.3 31.4 31.5 31.1 31.5 31.1
F6
2 9 6 8 5 6 0 2 0 7 6 3
30.7 30.9 31.8 31.6 31.9 31.8 31.3 31.3 31.2 31.4 31.9 31.4
F7
6 9 0 8 3 1 9 9 2 1 0 5
30.3 30.4 31.4 31.3 31.2 32.1 31.0 31.2 31.0 31.0 32.0 31.7
F8
5 4 8 6 9 3 5 5 7 6 1 1
31.0 30.6 31.3 31.5 31.2 31.5 31.1 31.1 31.1 31.0 31.7 31.6
F9
2 7 3 6 3 7 5 1 3 7 5 5
31.0 30.9 31.3 31.5 31.9 31.6 31.1 31.2 31.2 31.2 31.5 31.6
F10
8 0 4 0 1 0 7 9 7 1 2 2
30.8 30.9 30.6 31.1 31.4 32.0 31.1 31.4 31.2 31.3 32.0 31.3
F11
6 6 4 8 2 7 3 1 0 2 4 5
31.0 30.6 30.6 31.0 31.4 31.6 31.3 31.3 31.4 31.3 31.8 31.4
F12
3 8 2 1 4 5 3 7 1 7 2 7
30.9 30.9 31.3 31.3 31.1 31.6 31.2 31.6 31.2 31.5 31.4 31.3
F13
9 5 8 4 6 6 3 8 4 6 5 6
30.3 30.5 31.6 32.0 31.6 31.9 31.3 31.1 31.3 31.3 31.2 31.4
F14
0 8 4 2 7 1 5 7 8 0 8 3
30.3 30.7 31.2 32.0 31.7 32.4 31.1 31.2 31.3 31.5 31.1 31.3
F15
8 0 0 1 7 0 4 6 7 9 6 1
30.7 30.6 31.3 31.6 31.4 31.1 30.9 31.1 30.7 31.1 31.6 32.0
F16
0 2 0 8 7 6 9 1 8 2 0 0

PCR mix A PCR mix B
Cont 30.7 30.7 31.0 30.9 31.5 30.9 30.5 30.9 31.2 31.0 31.3 31.1
rol 9 9 7 5 9 0 8 3 7 6 3 6
31.0 31.3 31.1 31.5 31.4 30.7 31.1 31.2 31.1 31.2 31.2
F1 3077
9 6 7 0 3 6 8 7 8 1 4
30.8 31.0 31.2 31.1 31.3 30.7 31.3 31.3 31.4 31.5 31.3 31.4
F2
0 7 0 2 9 8 4 4 2 8 1 0
31.0 31.1 31.0 31.4 31.5 31.6 31.8 31.7 31.2 31.4 31.3 31.3
F3
5 9 0 3 0 1 4 5 9 3 9 4
30.9 31.0 31.6 31.4 31.5 31.3 31.4 31.5 31.6 31.2 31.6 31.1
F4
0 8 3 0 1 8 6 9 2 3 0 7
30.9 31.2 31.5 31.5 31.8 31.5 31.4 31.8 31.6 31.4 31.5 31.3
F5
8 4 1 6 3 9 2 4 2 6 8 8
31.1 31.3 31.4 31.3 31.1 31.6 31.5 31.2 31.4 31.4 31.2 31.2
F6
4 6 3 6 1 8 0 5 6 8 7 5
31.3 31.2 31.3 31.3 31.4 31.4 31.4 31.1 31.3 31.2 31.1 31.4
F7
0 1 4 7 1 8 5 9 3 4 3 2
30.9 30.9 30.9 31.0 31.4 31.2 31.3 31.0 31.2 31.0 31.2 31.4
F8
7 1 9 2 9 6 5 9 3 0 7 8
31.2 31.3 31.0 31.1 31.2 31.5 31.2 31.3 31.3 31.0 31.2 31.3
F9
3 1 1 9 4 8 8 5 3 0 3 0
31.1 31.1 31.5 31.7 31.4 31.3 31.3 31.3 30.6 31.1 31.3 31.2
F10
4 6 1 1 8 9 7 3 1 6 1 0
31.0 31.1 31.2 31.5 31.3 31.3 31.5 31.3 31.2 31.2 31.3 31.3
F11
6 3 8 0 8 8 5 9 4 4 2 4
31.2 31.2 31.4 31.4 31.5 31.6 31.5 31.4 31.3 31.5 31.3 31.5
F12
0 4 4 5 9 5 7 8 0 1 1 7
31.1 31.3 31.9 31.8 31.8 31.5 31.4 31.4 31.3 31.4 31.3 31.7
F13
9 8 0 3 1 3 6 0 6 9 6 0
31.2 31.3 31.5 31.3 31.5 31.1 31.5 31.3 31.2 31.2 31.2 31.6
F14
3 2 9 6 5 6 8 5 2 6 9 1
31.1 31.2 31.8 31.6 31.5 31.6 31.4 31.2 30.0 31.2 31.3 30.7
F15
9 1 1 7 3 5 0 0 7 8 1 2
30.8 31.0 30.6 30.7 30.5 31.5 31.3 31.4 30.9 30.9 31.1 30.9
F16
4 1 3 9 2 0 5 7 2 1 2 8
Conclusion: It can be seen from the test results of the above mentioned
three batched of reagents that, on the ABI7500 instrument, the test results of
weak positive samples containing the above-mentioned internal and external
interfering substances are not significantly different from those of the control
group (weak positive samples that do not contain interfering substances).
Therefore, it can be shown that this kit has a good anti-interference ability
against these internal and external interfering substances.
2.2 Verification of anti-internal and external interfering substances

Positive throat swab samples for influenza A virus, influenza B virus,
rhinovirus at low concentration were used as the test samples and were
divided into several parts. Each part was added with the possible internal and
external interfering substances in PCR listed in Table 1-3 and was tested
according to the operating method in the manual of this kit. Magnetic bead
method was used to extract nucleic acid and the test sample without
interfering substances was used as the control. Test results are shown in Table
1-7 to Table 1-9 and in Figure 1-13 to Figure 1-18 (Only the test results of
2017001 batch of reagents are shown).
Figure 1-13 Verified curve of the effect of internal and external interfering substances on

Table 1-7 Ct value of the verified effects of internal and external interfering

Mycoplasm
Respiratory
Influenza A Influenza B Respiratory a Human
syncytial
virus virus Adenovirus pneumonia rhinovirus
virus
e
1 2 1 2 1 2 1 2 1 2 1 2
Contr
28.04 27.42 30.71 28.95 28.05 27.64 31.88 30.57 31.12 32.09 31.39 30.58
ol
F1 27.02 27.34 30.29 28.98 28.10 27.90 30.63 30.82 31.85 32.08 31.18 30.84
F2 27.27 27.37 29.30 29.11 28.20 28.04 31.05 31.02 31.93 31.71 31.39 30.94
F3 27.28 27.06 29.96 29.38 28.22 27.85 31.32 31.12 31.91 31.71 31.22 31.09
F4 27.44 27.35 29.96 29.19 28.57 27.82 31.45 31.24 31.72 32.58 31.19 31.10
F5 27.32 27.31 30.10 30.02 28.79 28.19 31.01 31.22 31.89 32.11 30.94 31.04
F6 27.38 27.18 30.26 29.29 26.58 27.81 31.24 31.30 31.90 32.09 30.83 31.01
F7 27.81 26.94 30.04 29.37 27.10 28.25 30.91 31.17 31.86 31.73 31.20 31.11
F8 26.78 27.66 31.27 28.47 26.34 26.59 30.60 30.53 31.90 32.40 30.24 30.38
F9 26.71 26.89 28.82 29.22 26.76 26.76 30.76 30.93 31.98 32.06 30.76 30.78
F10 27.54 27.52 29.09 29.26 27.49 26.96 30.94 31.10 32.19 32.36 30.84 30.75
F11 27.33 26.64 28.88 29.21 28.12 27.31 31.27 31.45 32.04 31.45 31.06 30.91
F12 26.34 26.64 29.21 28.16 28.11 27.06 31.31 31.41 32.23 32.28 31.07 31.16
F13 26.53 26.70 28.53 29.08 27.83 27.65 30.86 31.32 32.00 32.21 30.86 31.04
F14 25.83 26.98 28.29 28.22 28.24 26.58 31.30 31.24 32.83 32.16 31.30 31.01
F15 26.96 27.07 29.64 28.97 28.32 26.33 30.93 31.22 32.10 31.81 30.65 30.76
F16 26.82 28.50 29.68 29.67 27.37 26.27 30.42 30.66 32.19 32.38 30.90 30.73
Mycoplasm
Respiratory
syncytial
virus
e
1 2 1 2 1 2 1 2 1 2 1 2
Contr
26.46 26.53 29.70 29.38 27.14 26.89 29.73 29.92 31.50 32.07 30.81 30.11
ol
F1 26.66 27.13 29.61 29.65 27.20 27.45 30.09 30.02 31.53 31.67 30.18 30.15
F2 27.19 27.65 29.67 29.91 27.39 27.33 30.26 30.38 31.80 31.96 30.54 30.53
F3 27.41 27.54 29.31 29.29 27.57 27.60 30.54 30.41 31.57 31.13 30.44 30.33
F4 27.54 27.67 29.59 29.25 27.65 27.72 30.16 31.02 31.85 31.91 30.62 30.30
F5 27.44 27.63 29.51 29.61 27.93 27.65 30.27 30.27 31.57 31.75 29.85 30.63
F6 27.31 27.15 29.16 29.51 27.67 27.50 30.29 30.25 31.37 31.70 30.37 30.23
F7 27.32 27.53 28.98 29.43 27.49 27.65 30.04 30.30 31.60 31.38 30.31 30.69
F8 27.34 27.40 29.44 29.07 27.20 27.03 29.88 30.19 31.79 31.94 30.08 30.11
F9 27.26 27.48 29.73 29.47 27.53 27.23 30.19 30.24 32.35 31.43 30.00 30.37
F10 27.41 27.62 29.61 29.39 27.31 27.30 30.39 30.27 31.35 31.22 30.38 30.57
F11 27.35 27.40 29.30 28.75 27.67 27.34 30.54 30.56 31.66 32.08 30.30 30.56
F12 27.59 27.42 29.40 29.63 27.43 27.59 30.53 30.80 32.12 31.07 30.30 30.64

F13 27.72 27.56 29.71 29.57 27.75 27.83 30.31 30.44 32.04 31.83 30.35 30.36
F14 27.43 27.43 29.48 29.20 27.77 27.33 30.29 30.31 31.73 31.90 30.45 30.66
F15 27.16 27.37 29.67 29.37 27.43 27.46 29.90 30.24 31.35 31.54 30.25 29.97
F16 27.50 27.45 29.46 29.21 26.77 26.98 30.07 30.14 31.82 31.50 30.07 30.01
Mycoplasm
Respiratory
syncytial
virus
e
1 2 1 2 1 2 1 2 1 2 1 2
Contr
26.79 26.78 29.17 29.22 26.90 27.20 29.95 30.11 31.19 30.66 31.10 30.15
ol
F1 26.85 27.08 29.39 29.41 27.16 27.35 30.48 30.31 30.48 30.73 30.41 30.64
F2 27.24 27.23 28.35 29.58 27.27 27.62 30.26 30.47 30.24 30.62 30.46 30.43
F3 27.32 27.24 29.61 29.16 27.52 27.67 30.80 30.80 30.61 30.31 30.77 30.67
F4 27.64 27.22 29.52 29.36 27.67 27.86 30.58 30.78 30.94 30.76 30.36 31.50
F5 27.29 27.36 29.46 29.83 27.91 27.83 30.67 30.60 30.84 30.35 30.51 30.41
F6 27.57 27.17 29.50 29.47 27.47 27.41 30.39 30.67 31.25 31.03 29.66 30.22
F7 26.98 27.35 29.27 30.02 27.51 27.80 30.34 30.53 30.70 30.87 30.43 30.26
F8 27.00 27.14 28.94 29.49 27.65 27.63 30.29 30.10 30.30 30.29 30.24 30.22
F9 27.23 27.17 29.23 29.39 27.12 27.76 30.58 30.63 30.56 30.85 30.14 30.32
F10 27.56 27.10 29.47 29.41 27.53 27.75 30.49 30.72 30.90 30.32 30.13 30.45
F11 27.13 27.30 30.01 29.47 27.66 27.69 30.69 30.84 30.79 30.26 30.53 30.53
F12 27.63 27.48 29.78 29.59 27.81 27.33 30.81 30.89 31.16 30.84 30.39 30.49
F13 27.18 27.57 29.48 29.83 27.82 27.56 30.56 31.00 30.86 30.35 30.29 30.61
F14 27.60 27.41 29.29 29.61 27.68 27.40 30.62 30.55 30.45 30.49 29.73 30.27
F15 27.29 27.24 29.25 29.78 27.48 27.26 30.19 30.46 30.50 30.65 30.25 30.28
F16 27.09 27.06 29.10 29.44 27.60 27.04 30.09 30.37 31.10 29.41 30.06 30.22
three batched of reagents that, on the ABI7500 instrument, the test results of
Therefore, it can be shown that, under the experimental conditions of this kit,
these internal and external substances below the above-mentioned
concentration have no effect on the test results of the kit.

QuantStudioTM 5

Analytical performance evaluation on SLAN-96P automatic

1 Analytical specificity
Negative reference samples N1 (measles virus), N2 (mumps virus), N3
(rubella virus), N4 (Staphylococcus aureus), N5 (Escherichia coli), N6
(Pseudomonas aeruginosa), N7 (human parainfluenza virus type I), N8
(normal throat swab)and N9 (normal throat swab) required in the product
technical requirements of the six respiratory pathogens nucleic acid
diagnostic kit (PCR-fluorescence probing), and other pathogens that have
homology in their nucleic acid sequence and are likely to cause the same or
similar clinical symptoms, including: cytomegalovirus, coxsackie virus type A,
human metapneumovirus, bacillus pertussis, chlamydia pneumoniae,
haemophilus influenzae, saliva Streptococcus, Streptococcus pneumoniae,
Neisseria meningitidis, Mycobacterium tuberculosis, human parainfluenza
type 2 and type 3 virus, Epstein-Barr virus, Cryptococcus, Aspergillus
fumigatus, Aspergillus flavus, Candida albicans, Legionella pneumophila and
Intestine Daovirus type 71 were used as the test samples. Three qualified kits
were used to test the samples on the same SLAN-96P instrument. And the
specificity of the kit was tested by detecting the negative and positive
coincidence rate. Test results are as follows (only the amplification curves of
the 2017001 batch of reagents are shown):
Figure 2-1 FAM channel amplification curve of samples for analytical specificity (2017001
batch of reagents)

Figure 2-2 HEX channel amplification curve of samples for analytical specificity (2017001
batch of reagents)
Figure 2-3 CY5 channel amplification curve of samples for analytical specificity (2017001
batch of reagents)
(Mix A, B) 28.37 30.07 30.19 31.08 30.63 31.77 28.02 30.79 30.47
Cytomegaloviru No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
s
Coxsackie virus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
type A
metapneumovir

us
Bacillus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
pertussis
Chlamydia No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
pneumoniae
Haemophilus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
influenzae
salivarius
pneumoniae
Neisseria No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
meningitidis
Mycobacterium No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
tuberculosis
Parainfluenza
Type 2
Parainfluenza
Type 3
Epstein-Barr No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
virus
Cryptococcus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
fumigatus
flavus
Candida No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
albicans
Legionella No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
pneumophila
Enterovirus No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
type 71
Conclusion: It can be seen from Figure 2-1 to Figure 2-3 and Table 2-1 that,
the positive rate of negative reference samples N1-N8 in nucleic acid
detection and other pathogens that have homology in nucleic acid sequence
and are likely to cause the same or similar clinical symptoms detected by
using this detection kit developed by our company is all 0%. Meanwhile, the
internal standard samples are all positive. Therefore, this kit has good
specificity, and no clinical cross-reaction occurs.

Negative reference samples NC01-NC06 in the national reference
samples (Batch No. 370006-201501) of nucleic acid detection reagents for
influenza A/B virus were tested on SLAN-96P instrument by using three
qualified kits to verify the sensitivity performance of the three batches of kits
on national reference samples. The samples were extracted and tested one
time each time. The test results were then counted.

Test results are shown in Table 2-2 (Results of three batches of reagents)
and Figure 2-4 to Figure 2-6 (Only the results of 2017001 batch of reagents are
shown).
Figure 2-4 FAM channel amplification curve of national reference samples NC01-NC06
Figure 2-5 HEX channel amplification curve of national reference samples NC01-NC06

Figure 2-6 CY5 channel amplification curve of national reference samples NC01-NC06
(Mix A, B) 34.64 34.31 34.77 34.50 34.17 34.90 34.91 33.88 35.34
2. Research on internal and external interfering substances

2.1 Anti-internal and external interfering substances effects
To investigate the impact of possible endogenous substances (such as
purified mucin, heme, urea, sodium chloride, human whole blood, etc.) in the
test sample on the test results, and to examine the effects of treatment drugs
for respiratory pathogens in the sample (such as oxymetazoline hydrochloride,
dexamethasone, zanamivir, cefmenoxime hydrochloride, menthol, etc.) on
PCR, Lentiviral particles containing the target conserved sequence of
rhinovirus, and cloning plasmids containing the target conserved sequence of
adenovirus and Mycoplasma pneumoniae were used as the research objects.
Lentiviral particles and cloning plasmids of each pathogen were diluted with
normal saline to a concentration of 7.0 × 103 TU/mL (and copies/mL), and were
then mixed with equal volume to form the test sample. The test sample was
divided into several parts, and each part was added with the possible internal
and external interfering substances listed in Table 2-3 and was tested
according to the operating method in the manual of this kit.
The test sample without interfering substances was used as the control.
The test results of three batches of kits on SALN-96P instrument are shown in
Table 2-4 to Table 2-6 and in Figure 2-7 to Figure 2-12 (Only the test results of

Table 2-3 The added internal and external interfering substances and its concentration
Sample Final Sample Final

Inhibitor name Inhibitor name
No. concentration No. concentration
Oxymetazoline
Hydrochloride
Cefmenoxime
Hydrochloride
F5 Zanamivir 100 μg/mL F13 Purified mucin 20 μg/mL
Oxymetazoline
F7 100 μg/mL F15 SDS 20%(v/v)
Hydrochloride
F8 Dexamethasone 60 μg/mL F16 EDTA 20%(v/v)


Table 2-4 Ct value of the tested effects of anti-internal and external interfering substances
(2017001 batch)
PCR mix A PCR mix B
Cont 29.9 30.2 31.5 31.7 31.3 31.0 29.0 29.0 30.5 30.8 31.6 31.1
rol 6 4 7 1 3 7 4 2 0 1 6 5
30.0 30.2 31.6 31.8 31.2 31.6 28.9 28.8 30.7 30.6 31.4 31.6
F1
6 5 9 0 1 0 6 2 6 8 7 0
30.0 30.0 31.6 31.6 31.3 31.3 28.9 28.8 33.6 30.7 31.4 31.3
F2
9 8 3 0 6 9 5 8 6 9 5 9
30.1 30.0 31.2 31.4 31.1 31.2 28.9 28.7 30.7 30.6 31.8 31.2
F3
5 9 4 9 2 3 1 6 5 2 1 8
30.3 30.2 31.5 31.7 31.4 31.1 28.9 28.7 30.9 30.8 31.3 31.5
F4
3 8 1 0 5 5 0 4 7 2 6 9
30.3 30.1 31.4 31.6 30.8 31.2 28.9 28.8 30.8 30.9 31.5 31.7
F5
8 8 0 7 7 9 7 0 2 7 4 1
30.2 30.1 31.2 31.4 30.8 31.0 28.9 28.9 30.7 30.5 31.1 31.7
F6
9 5 0 7 2 8 1 1 5 5 3 4
30.2 30.1 31.3 31.5 31.0 30.0 29.0 28.0 30.7 30.6 31.1 31.1
F7
0 0 9 2 7 8 2 5 9 9 6 8
30.1 30.1 31.2 31.5 31.4 31.3 28.9 28.9 30.8 30.8 31.4 31.3
F8
0 9 8 1 9 0 8 1 8 1 1 4
30.0 30.3 31.6 31.3 31.2 31.5 28.4 28.6 30.6 30.7 31.3 31.4
F9
3 0 1 0 9 7 5 7 1 8 5 2
30.2 30.1 31.6 31.5 31.2 31.5 29.0 28.8 30.8 30.8 31.5 31.5
F10
7 8 6 8 9 1 0 7 1 3 4 4
30.3 30.2 31.4 31.6 31.3 31.3 28.9 28.9 30.6 30.8 31.5 31.4
F11
4 8 4 3 3 5 3 8 5 1 8 8
30.4 30.4 31.3 31.5 31.7 31.4 28.9 28.5 30.8 30.7 31.5 31.4
F12
0 3 1 5 0 9 9 4 3 5 7 6
30.3 30.4 31.6 31.6 30.8 31.0 28.8 28.7 30.7 30.7 31.4 31.5
F13
1 5 1 8 8 1 0 8 7 6 3 0

30.4 30.3 31.7 31.6 30.9 31.1 28.9 29.0 30.7 30.8 31.6 31.6
F14
9 0 8 6 4 5 6 7 0 6 4 1
30.3 30.3 31.1 31.7 30.8 31.0 28.8 28.9 30.6 30.7 30.9 31.2
F15
1 0 5 2 8 1 4 1 0 4 5 3
30.4 30.1 31.6 31.4 31.2 30.9 28.8 28.8 30.7 30.8 31.5 31.3
F16
4 7 0 6 6 1 9 6 4 7 5 3
PCR mix A PCR mix B
Cont 28.9 29.1 31.0 30.3 30.6 30.9 28.0 28.0 31.0 31.2 31.8 31.6
rol 1 9 6 7 0 5 8 5 4 2 1 6
29.0 29.1 30.9 31.0 30.2 31.2 28.0 28.0 31.1 31.1 32.0 31.7
F1
0 0 7 4 4 1 7 5 7 3 8 1
28.7 28.8 31.1 31.2 30.6 31.2 28.0 28.0 31.2 31.0 32.3 32.6
F2
0 7 4 2 2 1 4 6 8 5 7 2
28.8 28.8 31.0 31.0 30.9 31.2 28.0 28.1 31.1 31.1 32.1 31.9
F3
9 8 6 3 2 4 4 3 8 5 3 7
29.0 28.8 30.9 30.8 31.1 31.3 28.1 28.0 30.9 31.2 32.1 31.8
F4
3 5 9 4 6 2 6 9 6 1 5 5
29.0 28.8 30.9 30.9 30.7 31.3 28.1 28.0 30.9 31.2 31.9 31.9
F5
0 3 5 5 5 1 7 8 5 0 6 9
28.8 29.0 30.8 30.8 30.8 31.4 28.1 28.1 30.9 31.2 32.2 32.3
F6
8 0 4 9 8 3 2 1 6 0 1 5
28.9 28.9 31.0 31.2 30.9 31.5 28.0 28.3 30.9 31.0 32.0 31.9
F7
0 5 5 6 5 1 4 0 6 7 3 7
28.9 28.8 30.8 30.9 31.0 31.1 27.9 28.0 31.0 31.1 32.0 31.6
F8
8 7 3 5 2 3 3 0 0 6 6 4
28.8 28.8 31.0 30.9 31.3 31.2 28.0 27.9 31.1 31.0 32.2 32.2
F9
8 4 0 6 0 3 8 8 0 3 7 0
28.9 28.8 31.0 30.7 31.3 31.3 28.0 27.9 31.1 30.3 32.1 31.9
F10
0 7 5 8 0 9 0 9 6 2 3 1
28.7 28.9 31.0 31.0 31.0 31.0 28.1 28.0 31.1 31.1 32.1 31.9
F11
9 4 6 4 3 6 1 1 3 9 6 6
29.0 28.8 31.0 31.2 31.1 31.0 28.0 28.0 30.9 31.0 32.1 32.3
F12
3 8 9 8 7 5 6 4 2 6 9 7
28.8 28.8 31.0 31.1 30.8 31.0 28.1 28.0 31.0 31.1 31.7 31.8
F13
0 5 1 2 7 9 3 2 2 3 6 5
29.0 28.8 31.0 31.0 30.8 31.3 28.0 28.0 31.1 31.0 31.9 31.7
F14
6 0 7 5 8 7 2 2 7 2 8 5
28.9 28.8 31.1 31.2 30.7 31.6 27.9 28.0 31.0 31.1 32.1 32.1
F15
3 8 0 3 3 5 4 4 3 8 9 8
28.9 28.9 30.8 31.0 31.2 31.1 28.0 28.1 31.2 31.0 32.2 32.1
F16
3 7 9 8 4 4 0 7 0 5 6 4

PCR mix A PCR mix B
Cont 28.6 28.5 31.1 31.0 31.3 31.1 28.1 28.1 30.6 30.3 31.4 31.7
rol 2 5 0 2 0 7 2 0 7 8 3 3
28.5 28.5 30.2 31.1 31.1 31.4 28.1 28.1 30.4 30.4 31.8 31.6
F1
2 4 4 1 2 6 4 7 9 6 0 1
28.5 28.5 31.1 31.3 31.6 31.3 28.0 27.9 30.3 30.5 32.2 31.4
F2
5 8 7 0 2 0 4 4 0 8 5 8
28.6 28.5 31.0 31.1 31.1 31.6 28.0 28.1 30.2 30.4 31.8 31.8
F3
9 9 4 6 7 4 7 0 8 9 8 4
28.6 28.5 29.6 30.9 31.3 31.1 28.0 28.1 30.5 30.5 31.7 31.7
F4
0 8 7 4 9 2 5 8 8 7 2 8
28.6 28.6 31.0 30.9 30.7 31.0 28.2 28.1 30.5 30.5 31.4 31.6
F5
1 7 2 4 0 7 5 3 1 9 1 2
28.6 28.6 31.0 31.1 31.0 31.3 28.1 28.1 30.7 30.6 31.5 31.5
F6
8 8 8 3 5 8 5 5 7 1 4 6
28.7 28.7 30.9 31.0 31.1 31.4 28.1 28.1 30.7 30.4 31.3 31.7
F7
5 3 7 2 8 4 9 6 1 4 0 6
28.8 28.6 31.1 31.1 31.2 31.2 28.0 28.0 30.5 30.6 31.3 31.3
F8
2 4 3 3 9 5 8 9 8 2 4 2
28.4 28.6 31.1 30.9 31.2 31.1 28.1 28.1 30.5 30.5 31.8 31.4
F9
6 8 3 9 9 0 4 4 7 2 6 9
28.5 28.7 31.0 31.1 31.1 31.2 28.1 28.0 30.6 30.6 31.5 31.6
F10
0 9 3 2 4 9 5 7 3 0 8 7
28.4 28.7 31.1 31.0 31.2 31.2 28.1 28.0 30.5 30.6 31.5 31.6
F11
5 7 9 6 9 9 0 6 8 1 6 7
28.7 28.6 31.1 31.0 31.0 31.3 28.1 28.2 30.5 30.5 31.7 32.0
F12
2 9 1 8 4 0 0 5 1 3 0 5
28.7 28.7 30.9 31.0 31.2 31.1 28.1 28.0 30.6 30.8 31.6 31.7
F13
9 9 6 5 0 0 4 8 1 4 3 3
28.7 28.6 31.1 31.2 30.9 31.3 28.1 28.2 30.6 31.2 31.6 31.6
F14
1 6 5 1 7 1 1 7 0 3 6 9
28.8 28.8 31.3 31.2 31.0 31.0 28.0 28.0 30.7 31.0 32.0 31.5
F15
7 3 3 3 8 9 6 9 3 8 3 5
28.6 28.6 31.1 30.9 31.3 30.8 28.0 28.8 30.5 30.4 32.1 31.7
F16
5 3 4 6 1 8 7 7 1 7 1 6
three batched of reagents that, on the SLAN-96P instrument, the test results of
Therefore, it can be shown that this kit has a good anti-interference ability
against these internal and external interfering substances.

2.2 Test of removal ability of PCR inhibitor
Positive throat swab samples for influenza A virus, influenza B virus,
rhinovirus at low concentration were used as the test samples and were
divided into several parts. Each part was added with the possible internal and
external interfering substances in PCR listed in Table 2-3 and was tested
according to the operating method in the manual of this kit. Magnetic bead
method was used to extract nucleic acid and the test sample without
interfering substances was used as the control. Test results are shown in Table
2-7 to Table 2-9 and in Figure 2-13 to Figure 2-18 (Only the test results of


Table 2-7 Ct value of the verified effects of anti-internal and external

interfering substances (2017001 batch)
Mycoplasm
Respiratory
syncytial
virus
e
1 2 1 2 1 2 1 2 1 2 1 2
Contr
28.33 28.42 30.98 31.00 30.08 30.20 29.94 29.90 32.90 32.19 31.18 31.06
ol
F1 28.28 28.39 31.00 30.92 30.44 30.21 29.89 29.90 32.49 32.40 31.00 30.91
F2 28.31 28.22 31.25 30.98 30.12 29.95 29.91 29.95 32.45 32.82 30.72 30.88
F3 28.26 28.25 31.02 31.21 30.20 30.19 29.90 29.90 32.47 32.52 30.81 30.89
F4 28.32 28.30 30.50 30.96 30.22 30.16 29.95 30.05 32.66 32.25 30.71 30.99
F5 28.33 28.23 31.04 31.05 30.30 30.15 29.97 29.96 33.03 32.36 31.02 30.91
F6 28.30 28.29 30.90 31.05 30.38 30.43 29.93 29.94 33.04 32.55 30.92 30.96
F7 28.15 28.26 31.04 30.92 30.36 30.14 29.89 29.96 33.14 32.99 30.84 30.89
F8 28.39 28.41 31.00 31.04 30.13 30.07 29.61 29.81 32.17 32.28 30.88 30.82
F9 28.34 28.35 30.90 31.18 30.13 30.27 29.82 29.95 32.44 32.26 30.72 30.92
F10 28.27 28.35 31.09 30.94 30.23 30.19 29.94 29.96 32.52 32.60 30.94 31.02
F11 28.30 28.35 31.07 31.09 30.35 30.26 29.90 30.00 32.62 32.55 30.82 31.01
F12 28.41 28.49 30.93 30.97 30.33 30.27 29.97 29.85 32.78 32.75 30.88 30.89
F13 28.45 28.49 31.13 30.97 30.22 30.14 30.08 29.94 33.10 32.75 31.03 31.00
F14 28.38 28.37 30.94 30.95 30.39 30.30 30.6 30.03 32.61 33.33 30.89 31.15
F15 28.31 28.50 31.12 30.99 30.16 30.35 30.12 29.84 32.57 32.64 30.86 30.94
F16 28.40 28.41 31.07 31.01 30.36 30.12 30.00 29.85 32.91 31.96 30.88 30.98

Mycoplasm
Respiratory
syncytial
virus
e

1 2 1 2 1 2 1 2 1 2 1 2
Contr
28.13 28.28 31.08 31.11 29.87 30.24 29.96 29.52 31.65 32.24 30.02 30.10
ol
F1 28.14 28.15 31.02 31.13 30.30 30.27 30.11 29.88 31.62 32.42 30.17 30.18
F2 28.26 28.24 30.89 31.15 30.17 30.10 30.10 30.09 31.94 32.64 30.36 30.18
F3 28.59 25.54 30.93 31.03 30.33 30.11 29.99 30.14 31.89 32.04 30.11 30.13
F4 28.40 28.28 31.07 30.89 30.67 30.83 29.91 30.05 32.08 32.23 30.21 30.11
F5 28.34 28.38 30.96 31.10 30.15 30.03 29.98 29.89 32.02 32.16 30.00 29.93
F6 28.18 28.32 30.87 31.07 30.37 30.14 29.94 30.14 32.63 31.61 30.12 30.14
F7 28.16 28.21 31.13 31.41 30.36 30.10 30.05 29.90 31.94 31.93 30.01 29.89
F8 28.25 28.27 31.40 31.07 30.20 30.21 29.93 29.94 31.37 31.55 30.31 30.09
F9 28.21 28.42 31.07 31.00 30.33 30.27 29.97 29.81 31.72 31.69 30.14 30.37
F10 28.59 28.55 31.18 31.10 29.45 30.49 29.96 29.95 32.04 32.43 30.29 30.10
F11 28.42 28.36 31.22 31.04 30.27 30.20 29.93 29.98 31.65 31.66 29.94 30.40
F12 28.50 28.43 31.19 32.11 30.32 30.20 29.89 30.07 31.75 32.70 30.30 30.13
F13 28.45 28.53 31.92 30.70 30.31 30.22 29.89 30.00 31.95 32.04 30.30 28.46
F14 28.33 28.56 31.13 31.07 30.58 30.69 29.93 29.91 31.78 32.08 30.10 30.27
F15 28.31 28.18 31.02 30.98 30.14 30.25 29.91 30.02 31.81 31.59 30.06 30.11
F16 28.14 28.14 31.12 31.23 30.20 30.25 30.07 30.24 32.24 31.61 30.46 30.26

Mycoplasm
Respiratory
syncytial
virus
e
1 2 1 2 1 2 1 2 1 2 1 2
Contr
28.28 28.23 30.88 31.03 30.33 30.52 29.83 29.81 31.55 32.34 30.38 30.02
ol
F1 28.31 28.31 30.91 30.98 30.80 30.67 29.79 29.87 31.89 32.28 30.05 30.23
F2 28.21 28.16 31.05 31.22 30.83 30.86 29.83 29.83 32.46 32.40 30.31 30.09
F3 28.14 28.08 30.90 31.03 30.97 30.58 29.98 29.98 31.66 32.11 30.10 30.08
F4 28.14 28.06 31.25 31.32 30.55 30.52 29.97 29.96 32.70 31.87 30.05 30.18
F5 28.22 28.12 31.16 30.98 30.55 30.16 29.70 29.82 32.04 33.35 30.20 30.14
F6 28.16 28.18 30.92 31.26 30.79 30.67 29.63 29.82 32.08 32.88 30.21 30.12
F7 28.17 28.10 31.01 31.01 30.38 30.62 29.69 29.96 31.59 32.93 30.25 30.06
F8 28.14 28.12 31.18 31.07 30.41 30.35 30.01 29.96 31.61 31.87 30.11 30.00
F9 28.26 29.00 30.94 31.27 30.41 30.72 29.99 30.13 31.83 31.44 29.86 30.48
F10 28.20 28.16 31.03 31.27 30.53 30.49 30.11 29.93 31.82 31.96 30.15 29.98
F11 28.19 28.14 31.38 31.17 30.37 30.46 29.96 30.06 32.52 31.93 30.14 29.63
F12 28.12 28.26 31.32 31.17 30.46 30.12 30.16 29.95 32.12 32.19 30.12 29.94
F13 28.15 28.19 31.22 31.18 30.64 30.59 29.98 29.94 32.00 32.18 30.02 30.39
F14 28.15 28.24 31.17 31.19 31.57 31.23 29.93 29.99 31.65 32.14 30.21 30.16

F15 28.17 28.19 31.30 30.92 30.81 30.50 29.57 29.71 31.94 32.66 29.93 30.13
F16 28.22 28.32 31.09 31.10 30.71 30.58 30.11 29.94 32.66 31.11 30.11 30.19
three batched of reagents that, on the SLAN-96P instrument, the test results of
Therefore, it can be shown that, under the experimental conditions of this kit,
these internal and external substances below the above-mentioned
concentration have no effect on the test results of the kit.

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Sansure Biotech Inc.

Six Respiratory Pathogens Nucleic Acid Diagnostic Kit

Determination of cut-off value

2. Samples used for determining cut-off value

Determination of cut-off value 1

3. Methods of determining cut-off value

4. Specific test data about determining cut-off value

Determination of cut-off value 2

4.1.1.2 Serial dilution, detection sensitivity determination

Figure 2. Amplification results of 0.2~2.0 × 103 TCID50/mL influenza A virus

Determination of cut-off value 3

Figure 3. Amplification results of samples with 2.0 TCID50/mL influenza A virus

Determination of cut-off value 4

Figure 4. Amplification results of samples with 1.5 TCID50/mL influenza A virus

Figure 5. Amplification results of samples with 1.0 TCID50/mL influenza A virus

4.1.2 Influenza B virus

Determination of cut-off value 5

Figure 7. Amplification results of 0.2~2.0 × 103 TCID50/mL influenza B virus

Determination of cut-off value 6

Figure 8. Amplification results of samples with 2.0 TCID50/mL influenza B virus

Determination of cut-off value 7

Figure 9. Amplification results of samples with 1.5 TCID50/mL influenza B virus

4.1.3 Respiratory syncytial virus

Determination of cut-off value 8

Figure 12. Amplification results of 100~2.0E+06 copies/mL RSV

Table 5. Ct value of samples with 100~2.0E+06copies/mL RSV

Determination of cut-off value 9

200 Undetermined 36.38

Figure 13. Amplification results of samples with 600 copies/mL RSV

Determination of cut-off value 10

Figure 14. Amplification results of samples with 500 copies/mL RSV

Figure 15. Amplification results of samples with 400 copies/mL RSV

Determination of cut-off value 11

shown in Figure 16.

4.1.4.2 Serial dilution, detection sensitivity determination

Figure 17. Amplification results of samples with 100~2.0E+06 copies/mL Adenovirus

Determination of cut-off value 12

2.0E+03 33.33 30.41

Figure 18. Amplification results of samples with 600 copies/mL Adenovirus

Determination of cut-off value 13

Figure 19. Amplification results of samples with 500 copies/mL Adenovirus

Figure 20. Amplification results of samples with 400 copies/mL Adenovirus

4.1.5 Human rhinovirus

Determination of cut-off value 14

4.1.5.2 Serial dilution, detection sensitivity determination

Figure 22. Amplification results of samples with 100~2.0E+06 copies/mL Human

Table 9. Ct value of samples with 100~2.0E+06 copies/mL Human Rhinovirus

Determination of cut-off value 15

2.0E+04 30.70 27.78

Determination of cut-off value 16

4.1.6 Mycoplasma pneumoniae

Determination of cut-off value 17

4.1.6.2 Serial dilution, detection sensitivity determination

Figure 27. Amplification results of samples with 100~2.0E+06 copies/mL Mycoplasma

Determination of cut-off value 18

2.0E+04 30.65 27.84

Figure 28. Amplification results of samples with 600 copies/mL Mycoplasma