Sansure Biotech

Six Respiratory Pathogens Nucleic Acid Diagnostic Kit sore throat, fever, cough and other general respiratory tract symptoms. A small number of patients will continue to suffer from high
(PCR-Fluorescence Probing) fever, severe cough, rapid disease progression, respiratory failure, multi-organ functional disorder and other critical diseases in a
short period of time.
【 Reference Number 】 Currently, the primary laboratory diagnostic methods of respiratory tract pathogen include virus isolation and identification, RT-PCR,
S3066E colloidal gold method and etc.
【 Package Specification 】 【 Test Principle 】
24 tests/kit By applying multiplex real-time fluorescence quantitative PCR detection technique, on the fluorescence quantitative PCR
【 Intended Use 】 instrument, this diagnostic kit uses specific primers and fluorescence probes which are designed to target a conserved sequence of
The Six Respiratory Pathogens Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) is intended for the simultaneous qualitative pathogen nucleic acid, accompanied with other ingredients of PCR Mix, to achieve quick detection of respiratory pathogens in the
detection of influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, human rhinovirus and mycoplasma sample through fluorescent signal changes.
pneumonia in human nasopharyngeal swabs(NPS). The detection results can be used for the auxiliary diagnosis of the patients with The PCR detection system uses internal control (positive) to monitor the specimen extraction process and the PCR amplification
respiratory tract pathogen infection and providing the molecular diagnostic basis for the respiratory tract pathogen infection. This kit process by detecting whether the housekeeping genes mRNA that encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
can distinguish the above pathogens, but for the same pathogen, can not distinguish the specific subtypes. in the sample is normal to avoid a false negative result.
The detection results of this diagnostic kit is only for clinical reference, and can not be used as the only criterion of clinical diagnosis. 【 Components of the Diagnostic Kit 】
No. Reagent Name Specification & Qty. Main Ingredients
For in vitro diagnostic use only. For professional use only. 1 Six RP-PCR Mix A 1044 μL/tube × 1 tube Primer, probe, dNTPs , PCR buffer, DEPC water, MgCl2
【 Summary 】 2 Six RP-PCR Mix B 1044 μL/tube × 1 tube Primer, probe, dNTPs , PCR buffer, DEPC water, MgCl2
Respiratory infections are classified into the upper respiratory tract infections and lower respiratory tract infections, refer to the 3 Six RP-Enzyme Mix 72 μL/tube × 1 tube DNA polymerase, mMLV enzyme
pathogen infecting the nose, throat, trachea, bronchi or lungs, principally cause tissue and organs disease of outside of respiratory Six RP-Positive cloning plasmids (7.0 x 103copies/mL) and Lentivirus particles(7.0×103TU/mL)
tract, characterized by fever, sore throat, cough, headache and other symptoms. The respiratory tract pathogen has the 4 1.0 mL/tube × 1 tube
Control containing each pathogen gene sequences diluted with normal saline
characteristics of strong infectivity, rapid spread, short incubation period and acute onset, etc. which seriously harm human health. Six RP-Negative
Influenza Virus is a kind of RNA virus in the Orthomyxoviridae family to lead to human and animal influenza, it causes 5 1.0 mL/tube × 1 tube Normal saline
Control
acute upper respiratory tract infection, spreads rapidly through the air, has periodic pandemics around the world. Human influenza
Six RP-Internal Lentivirus particles(2.0×103TU/mL) containing each pathogen gene
virus are influenza pathogen, classified into three types, namely A , B and C, influenza A is the most harmful, influenza B and 6 50 μL/tube × 1 tube
Control sequences diluted with normal saline
influenza C have weak pathogenicity, not easy to mutate. Influenza A Virus (Inf. A) has many subtypes, so far, there are 18 subtypes
Note:
of HA and 11 subtypes of NA. Influenza B Virus (Inf. B) can be divided into two phylogenetic lineages: Yamagata family and Victoria
1. Self-prepared reagent: Multi-type Sample DNA/RNA Extraction-Purification Kit (Magnetic beads method) (Reference Number :
family.
S1006E) manufactured by Sansure Biotech Inc..
Respiratory Syncytial Virus (RSV) is a member of the RNA viruses in the Paramyxoviridae family. The clinical 2. Do not mix or exchange components from different kit lots.
research shows that the respiratory syncytial virus is the most common pathogen that causes virus pneumonia, which is more 3. All biological samples in the diagnostic kit have been inactivated.
common in infants under the age of three, mainly with the symptoms of hyperpyrexia, nasitis, pharyngitis and laryngitis, and even 4. Materials required but not provided: 1.5 mL DNase-free and RNase-free centrifuge tubes, 0.2 mL PCR reaction tubes, pipette
bronchiolitis and pneumonia. A very few sick infants will have the complications of tympanitis, pleuritis and myocarditis, etc. When tips (10 μL, 200 μL and 1000 μL tips with filters are preferred), desktop centrifuge, desktop vortex mixer, magnetic-bead separator,
the older children and adults get infected, the main symptom is upper respiratory tract infection. various models of pipette guns.

Adenoviruses(Adv) belongs to Adenoviridae, with seven species (A to G) based on different immunology, biology and 【 Storage and Stability 】

biochemical characterization, approximately 52 serotypes. Different serotype has different organ compatibility and will cause 1. The diagnostic kit should be stored in a sealed pouch at -20±5°C and protected from light. The shelf life of the kit is 12 months.

corresponding clinical manifestation, which can infect respiratory tract, gastrointestinal tract, urethral canal and bladder, eye, liver Please see the date of manufacture and expiry date on the outer package.

and etc. 2. The reagents keep valid and stable within the expiry date if not used. As long as the container of the reagent is opened, the

Human Rhinovirus (HRV) belong to Enteroviruses of the picornavirus family, is a set of small molecular single strand plus RNA freeze/thaw cycles should not exceed three.

virus, is an important pathogenic factor in respiratory tract virus infection, mainly cause the common cold in adults, in addition to the 3. The reagents keep valid and stable before the expiry date on the outer package when transporting for 7 days in a sealed foam

upper respiratory tract infection, also can cause diseases such as bronchitis and bronchopneumonia, acute asthma and other box containing coolant with the temperature lower than 20°C.

diseases in infants and children. 【 Compatible Instrument 】

Mycoplasma Pneumonia (MP) is a common pathogens of community-acquired pneumonia, can cause mycoplasma pneumonia The diagnostic kit is applicable to ABI7500 and SLAN-96P PCR instrument.

after MP infection, mainly pathological changes of mycoplasma pneumonia are interstitial pneumonia, sometimes complicated with 【 Specimen Requirements 】

bronchial pneumonia, and mainly transmitted via droplets, with the highest incidence in adolescent, incubation time for M. 1. Applicable specimen type : Nasopharyngeal swabs.

pneumoniae infection is approximately 2 to 3 weeks. The clinical symptoms are relatively mild, most patients only have headache, 2. Collection of specimen :

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Nasopharyngeal swabs : Use sterile swabs to insert the nostril 3 ~ 6 cm in the direction of the earlobe and the tip of the nose, or on Table 1 Proposed sample Plate Layout (96-well-plate)
the nasal palates of the both nasal passages, stay for 15 seconds, then slowly rotate out. Put the swab into a sterile tube
containing sampling solution, Plug the mouth of the tube tightly, seal and send it for detection. The top part of the swab should be PCR-Mastermix A PCR-Mastermix B
made from artificial material (e.g. polyester or terylene), the rod of the swab should be made from aluminum or plastics. It is not Positive Sample 7 Positive Sample 7
recommended to use cotton head swab with wooden handle. Calcium alginate swab sampling is not recommended. Control Control
3. Storage and delivery of specimens: Negative Sample 8 Negative Sample 8
Specimens collected via the above-mentioned method can be stored at 4°C for detection within 48 hours, or stored below -20°C for Control Control
three months, or stored below -70°C for longer than three months. Multiple freeze/thaw cycles should be avoided. Specimens Sample 1 Sample 9 Sample 1 Sample 9
should be transported in a sealed frozen pitcher with ice or in a sealed foam box with ice. Sample 2 ... Sample 2 ...
【 Test Method 】 Sample 3 Sample 3
1. Preparation of reagent (performed at “reagent preparation region”) Sample 4 Sample 4
1.1 Take out each component from the diagnostic kit and place them at room temperature. Allow the reagents to equilibrate at room
Sample 5 Sample 5
temperature, then vortex each of them respectively for later use.
Sample 6 Sample 6
1.2 Mix Six RP-Internal Control and Six RP-Negative Control in proportion : Add 10μL Six RP-Internal Control into every 200 μL Six
3. PCR Amplification (Refer to user manual of each instrument to adjust the settings.)
RP-Negative Control, then fully mix them to make a Six RP-Negative Control containing internal control, centrifuge it
3.1 Place PCR reaction tubes into the specimen wells of the amplification instrument. Set up the Six RP-Positive Control, Six
instantaneously for later use.
RP-Negative Control and unknown specimens in the corresponding sequence and input sample information.
1.3 According to the quantity of test specimens, Six RP-Positive Control and Six RP-Negative Control, pipette appropriate quantity
3.2 Select PCR test channel:(take ABI7500 as example)
of Six RP-PCR Mix A and Six RP-Enzyme Mix ( Six RP-PCR Mix A 43.5 μL/test + Six RP-Enzyme Mix 1.5 μL/test), mix them
3.2.1 PCR-Mastermix A
thoroughly to make a PCR-Mastermix A, centrifuge it instantaneously for later use. Pipette appropriate quantity of Six RP-PCR Mix
a) Select FAM channel (Reporter: FAM, Quencher: None) to test influenza A virus.
B and Six RP-Enzyme Mix ( Six RP-PCR Mix B 43.5 μL/test + Six RP-Enzyme Mix 1.5 μL/test), mix them thoroughly to make a
b) Select HEX or VIC channel (Reporter: HEX/VIC, Quencher: None) to test influenza B virus.
PCR-Mastermix B, centrifuge it instantaneously for later use.
c) Select CY5 channel (Reporter: CY5, Quencher: None) to test respiratory syncytial virus.
1 specimen 10 specimens 24 specimens
d) Select ROX channel (Reporter: ROX, Quencher: None) to test internal control.
Six RP-PCR Mix A (μL) 43.5 435 1044 e) Set Sample Volume: 50.

Six RP-Enzyme Mix (μL) 1.5 15 36 3.2.2 PCR-Mastermix B

a) Select FAM channel (Reporter: FAM, Quencher: None) to test adenovirus.
Note: The above configuration is just for your reference and to ensure enough volume of the PCR-Mastermix A, more
b) Select HEX or VIC channel (Reporter: HEX/VIC, Quencher: None) to test human rhinovirus.
volume of the actual pipetting may be required.
c) Select CY5 channel (Reporter: CY5, Quencher: None) to test mycoplasma pneumonia.
1 specimen 10 specimens 24 specimens
d) Select ROX channel (Reporter: ROX, Quencher: None) to test internal control.
Six RP-PCR Mix B (μL) 43.5 435 1044 e) Set Sample Volume: 50.

Six RP-Enzyme Mix (μL) 1.5 15 36 3.3 Set cycle parameters (the time parameter varies according to instruments):
Step Temperature Time Cycle No.
Note: The above configuration is just for your reference and to ensure enough volume of the PCR-Mastermix B, more
1 Reverse transcription 50°C 30 min. 1
volume of the actual pipetting may be required.
2 Pre-denaturation 95°C 1 min. 1
2. Processing and loading of specimens (performed at “specimen processing region”)
Denaturation 95°C 15 sec.
2.1 Processing of specimens 3 45
Annealing, extension and fluorescence detection 60°C 30 sec.*
Add 200 μL of test specimen, Six RP-Negative Control and Six RP-Positive Control respectively into 1.5 mL centrifuge tube. Use
4 Device cooling(Optional) 25°C 10 sec. 1
Multi-type Sample DNA/RNA Extraction-Purification Kit (Magnetic beads method) (Reference Number :S1006E) manufactured by
*Note: Due to ABI 7500’s technical specification, it can not be set at 30 sec., but at 31 sec. or 32 sec.
Sansure Biotech Inc. to extract the nucleic acid as per the product manual.
When the settings are completed, save the settings and carry out the reaction procedure.
2.2 Loading of specimens
4. Result Analysis (Refer to user manual of each instrument to adjust the settings, take ABI7500 as example.)
2.2.1 Add 5 μL of aboved prepared test specimen, Six RP-Positive Control and Six RP-Negative Control respectively into
Results will be saved automatically when reactions are completed. Adjust Start, End and Threshold values of Baseline of the graph
corresponding 0.2mL PCR reaction tube, then add 45 μL PCR-Mastermix A to every tube, cover the tube lid.
according to analysis result (Users can adjust the values according to the actual situation. Start value can be set between 3-15, and
2.2.2 Add 5 μL of aboved prepared test specimen, Six RP-Positive Control and Six RP-Negative Control respectively into
End value between 5-20. Adjust the amplification curve of negative control to be flat or below threshold). Click “Analyze” to
corresponding 0.2mL PCR reaction tube, then add 45 μL PCR-Mastermix B to every tube, cover the tube lid.
implement the analysis and make sure each parameter satisfy the requirements given in “5. Quality Control”. Go to “Plate” window
2.2.3 It is suggested to use the spotting method as shown in Table 1 to detect the sample.
to record Ct value.

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5. Quality Control Detect 6 enterprise precision references (R1~R6) repeatedly for 10 times, R1 and R2 are positive for influenza A virus and
The test result is treated as valid if all the conditions in the table below are met for the same test. Otherwise the test result is treated influenza B virus and negative for other pathogens. R3 and R4 are positive for respiratory syncytial virus and negative for other
as invalid and needs to be re-tested. pathogens. R5 and R6 are positive for adenovirus, human rhinovirus and mycoplasma pneumonia, and negative for other
Six RP-Negative Control Six RP-Positive Control pathogens. The variation coefficient (CV, %) of the Ct value of the corresponding pathogen detection channel for R1 ~ R6 is not
Ct value Respectively use PCR-Mastermix A and Respectively use PCR-Mastermix A and greater than 5.0%.
PCR-Mastermix B to detect Six RP-Negative Control, PCR-Mastermix B to detect Six RP-Positive Detect 6 enterprise limit of detection references (SQ1 ~ SQ6). When the concentration of SQ1 is not higher than 2.0TCID50/mL,
Six RP-Internal Control is detected positive, there is Control, there is typical S-shape amplification curve the test results are positive for influenza A virus and negative for other pathogens. When the concentration of SQ2 is not higher than
typical S-shape amplification curve and Ct ≤ 40 at at FAM, HEX(or VIC) , CY5 channel and 27≤ Ct ≤ 2.0TCID50/mL, the test results are positive for influenza B virus and negative for other pathogens. When the concentration of SQ3 is
ROX channel, no amplification curve (no Ct) or Ct >40 33, no amplification curve (no Ct) or Ct >40 at ROX not higher than 500.0copies/mL, the test results are positive for respiratory syncytial virus and negative for other pathogens. When
at FAM, HEX(or VIC) , CY5 channel. channel . the concentration of SQ4 is not higher than 500.0copies/mL, the test results are positive for adenovirus and negative for other
【 Reference Range 】 pathogens. When the concentration of SQ5 is not higher than 500.0copies/mL, the test results are positive for mycoplasma
Draw ROC curve based on clinical research data, through the research on reference values, the Ct reference value of target gene is pneumonia and negative for other pathogens. When the concentration of SQ6 is not higher than 500.0copies/mL, the test results
determined to be 40, and the Ct reference value of internal control is determined to be 40. are positive for human rhinovirus and negative for other pathogens.
【 Explanation of Detection Result 】 Use this kit to detect National reference for influenza A/B virus nucleic acid test reagent (batch no. : 370006-201501) : Positive
1. Determination for positive and negative result : conformity rate : The test results of PC01 and PC02 are both positive for influenza B virus and negative for influenza A virus. The
Typical S-shape amplification curve, and the Ct ≤ 40, which is positive. test results of PC03, PC04, PC05 and PC06 are positive for influenza A virus and negative for influenza B virus. The positive
No amplification curve (No Ct) or Ct > 40, which is negative. conformity rate (+/+) is 6/6.
The details are as below: Negative conformity rate : The test results of NC01~NC06 are both negative for influenza virus. The negative conformity rate -/-) is
6/6.
FAM channel HEX/VIC channel CY5 channel ROX channel
Amplification results Precision (CV, %) : Detect precision references CV1 and CV2 respectively and repeatedly for 10 times, the test results of CV1 are
No Ct or No Ct or No Ct or No Ct or
Mix type Ct ≤ 40 Ct ≤ 40 Ct ≤ 40 Ct ≤ 40 positive for influenza B virus and negative for influenza A virus, the test results of CV2 are positive for influenza A virus and negative
Ct > 40 Ct > 40 Ct > 40 Ct > 40
for influenza B virus, and the CV of Ct value is not greater than 5.0%.
Inf. A Inf. A Inf. B Inf. B RSV RSV
PCR-Mastermix A Internal Internal Limit of Detection : The titer of sensitivity reference (S1) is no less than 2.1x101TCID50/L, the test result is positive for influenza B
Positive Negative Positive Negative Positive Negative
Control Control virus and negative for influenza A virus. The titer of S2 is no less than 2.0TCID50/L,the test result is positive for influenza B virus and
PCR-Mastermix B ADV ADV HRV HRV MP MP
Positive Negative negative for influenza A virus. The titer of S3 is no less than 2.5X10-1TCID50/L, the test result is positive for influenza A virus and
Positive Negative Positive Negative Positive Negative
negative for influenza B virus. The titer of S4 is no less than 1.0TCID50/L, the test result is positive for influenza A virus and negative
2. For the positive specimen, there is no requirement for the internal control test results. For the negative specimen (none of the six for influenza B virus. The titer of S5 is no less than 9.8X10-1TCID50/L, the test result is positive for influenza A virus and negative for
types of respiratory pathogens is detected), the internal control test should be positive (Ct ≤ 40). If internal control Ct >40 or no Ct, influenza B virus.
the test result is treated as invalid, an investigation should be performed to find out the reasons, please collect specimen and Interfering substance references tests show that the common drug for respiratory infections (such as oxymetazoline hydrochloride,
retest the specimen. (If repeated tests still produce invalid results, please contact Sansure Biotech.) dexamethasone, cefmenoxime hydrochloride, menthol, zanamivir, ribavirin, azithromycin) under normal dosage will not interfere
3. Determination of gray zone: If the fluorescence signal of the sample increased significantly at FAM or HEX/ VIC or CY5 channel, with this kit. There is no cross reaction with common respiratory pathogens, like measles virus, mumps virus, rubella
but the Ct > 40, then the specimen is in gray zone, need to be retested. If the retest result is still in gray zone, then it is positive. virus, staphylococcus aureus, Escherichia coli, pseudomonas aeruginosa, human parainfluenza virus Type I, cytomegalovirus,
【 Limitations of Detection Method 】 enterovirus, human metapneumovirus, bordetella pertussis, chlamydia pneumonia, haemophilus influenzae, streptococcus
The detection result is related to the quality of specimen collection, delivery, processing and storage. If there are any mistakes, it salivarius, streptococcus pneumoniae, neisseria meningitidis, mycobacterium tuberculosis and etc.
will lead to an incorrect result. Cross-contamination occurring in specimen processing may result in a false positive result. This The limit of detection of this kit:
kit is designed for detection of conservative area for pathogens, the mutations are rare, but it is not excluded that gene mutations
Pathogen Limit of Detection Pathogen Limit of Detection
in the pathogen during the epidemic, which may lead to false negative results.
Influenza A virus 2.0TCID50/mL Adenovirus 500.0copies/mL
【 Product Performance Index 】
Influenza B virus 2.0TCID50/mL Mycoplasma pneumonia 500.0copies/mL
Use this kit to detect the enterprise reference :
Respiratory syncytial virus 500.0copies/mL Human rhinovirus. 500.0copies/mL
Detect 12 enterprise positive references (P1~P12), P1, P2, P3 and P4 are positive for influenza A virus and negative for other
【 Precautions 】
pathogens. P5 and P6 are positive for influenza B virus and negative for other pathogens. P7 is positive for respiratory syncytial
1. The product can only be used for in vitro diagnosis. Please read the product manual carefully before operation.
virus and negative for other pathogens. P8, P9 and P10 are positive for adenovirus and negative for other pathogens. P11 is
2. Please learn and be familiar with the operation procedures and precautions for each instrument before test. Please make sure
positive for mycoplasma pneumonia and negative for other pathogens. P12 is positive for human rhinovirus and negative for other
quality control for each test.
pathogens.
3. Laboratory management shall strictly follow management practices of PCR gene amplification laboratory, laboratory personnel
Detect 9 enterprise negative references (N1 ~ N9), N1 ~ N9 are all negative.

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Sansure Biotech
must receive professional training, test processes must be performed in separated regions, all consumables should be for single
Sansure Biotech Inc.
use only after sterilization, special instruments and devices should be used for every process, all lab devices used in different
processes and regions should not be cross-used. Add.: No. 680, Lusong Road, Yuelu District, 410205 Changsha, Hunan Province,
4. All specimens for detection should be handled as if infectious. Wear laboratory coats, protective disposable gloves and change the
PEOPLE’S REPUBLIC OF CHINA
gloves often to avoid cross-contamination between samples. Handling of specimens and waste must meet relevant requirements
outlined in local, state and national regulations. Tel.: +86-731-88883176

5. Before use, all reagents must be fully thawed at room temperature and mixed thoroughly. Fax: +86-731-88884876
6. When testing samples that do not contain human endogenous genes, there is no requirement for internal control (ROX channel).
Web: www.sansure.com.cn
【 Bibliography 】
1. Walsh, M. P., et al. 2010. Computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory
disease pathogen. J. Clin.Microbiol. 48:991–993. Obelis S.A
2. J. Mahony, S. Chong, F. Merante, et al.2007.Development of a Respiratory Virus Panel Test for Detection of Twenty Human
Bd. Général Wahis 53, 1030 Brussels, BELGIUM
Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay, JOURNAL OF CLINICAL MICROBIOLOGY, p.
2965–2970. Tel: + (32) 2.732.59.54

Jayme Parker, Nisha Fowler, Mary Louise Walmsley, et al. Analytical Sensitivity Comparison between Singleplex Real-Time PCR Fax: + (32) 2.732.60.03
and a Multiplex PCR Platform for Detecting Respiratory Viruses, PLOS ONE, 2015, 10(11).
E-Mail : mail@obelis.net
【 Symbols 】

Symbols Meanings Symbols Meanings

In Vitro Diagnostic Medical Device Date of Manufacture

Use By Consult Instructions for Use

For Professional Use Only

Temperature Limitation Manufacturer

Lot Number Reference Number

Authorized representative in
Number of Tests
the European Community

This product fulfills the

Any warnings and/or precautions to requirements of the European
take Directive 98/79 EC for in vitro
diagnostic medical devices.

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