DiAGSureTM nCoV-19 Detection Assay (Multiplex, TaqMan based)

Catalogue No.: DG6001-OSTM-50R; DG6001-OSTM-100R; DG6001-OSTM-200R

Kit Components:
Colour Coding 50 tests 100 tests 200 tests
(Caps) Contents Description DG6001-OSTM-50R DG6001-OSTM-100R DG6001-OSTM-200R
Amber WRTaqMan Master Mix Amplification Mix 700 µL 1.4 mL 2 X 1.4 mL
Amber Primer-Probe Mix Amplification Reagent 100 µL 200 µL 400 µL
Red GRTScript Enzyme cDNA Synthesis Reagent 50 µL 100 µL 200 µL
Green Internal Control (IC) Internal Control 275 µL 550 µL 1.1 mL
Yellow Positive control (PC) Positive Control 50 µL 100 µL 200 µL
White Negative control (NC) Negative control 1 mL 1 mL 1 mL
Note: A vial of RNA dilution buffer has been provided, in case the eluted RNA is concentrated and necessitates further dilution. Viral RNA extraction kit;
Disposable gloves; RNase and DNase free PCR tube are not provided with this kit.

Intended Use:
avoided. It is advised to start amplification with at least 100 ng
The DiAGSureTM nCoV-19 Detection Assay (Multiplex, TaqMan based) is
RNA. Note: Freshly extracted RNA should be used. Otherwise,
an in vitro diagnostic (IVD) real-time reverse transcriptase polymerase
RNA should be stored in alcohol at -80°C. Since RNA is a labile
chain reaction (qRT-PCR) test involving TaqMan chemistry intended for
biomolecule, improper handling and storage can lead to significant
the qualitative detection of nucleic acid from severe acute respiratory
loss of titer and may affect the kit’s sensitivity.
syndrome-related coronavirus 2 (SARS-CoV-2) in human
nasopharyngeal swab, oropharyngeal swab, anterior nasal swab,
B. Preparation of amplification reagent:
midturbinate and sputum specimens from individuals with signs and Take the reagents out from the -20˚C freezer and thaw them on
symptoms of infection who are suspected of COVID-19 by their health ice. After thawing, briefly vortex the reagents followed by a short
care provider. This three-plex PCR includes dual targets E-gene and spin. The RT and PCR mixes should be kept on ice.
ORF1ab gene, and a separately configured internal control in a single-
tube reaction. C. One-step RT-qPCR protocol:
Per sample, set up a single reaction according to the following
table: PCR reaction mix composition without Internal Control
Estimated operating time:
(When IC is added during RNA Extraction)
~1.5 hrs Note: If internal control (IC) is required to be added at the time of
RNA extraction, add 5 µL of internal control (IC) per isolation to the
Kit Stability: lysis buffer along with other components of kit used for lysis (as per kit
instructions) and vortex for 5 seconds prior to usage. The final RNA
 Kit is shipped on dry ice and should be stored immediately upon elution volume should be 50 µL.
receipt at –20°C in a constant-temperature freezer. Please refer
to product label for final expiry date. Components Volume per reaction
 This product can be used for 30 days after opening the vials. WRTaqMan Master Mix 12.5 µL
 This product can be used for maximum 7 repeats of freezing Primer-Probe Mix 2 µL
and thawing.
GRTScript Enzyme 1 µL
Positive control (PC)/ Extracted RNA/
Specimen handling & Storage: Negative Control (NC)
10 µL
Total Volume 25.5 µL
After sample collection, perform the test on the same day. Otherwise,
store it in the following condition: 2 to 8˚C for no more than 24 hours. PCR reaction mix composition with Internal Control (When IC is
Store below -20 ˚C for no more than 3 days. Can be stored for a long not added during RNA extraction)
time below -70 ˚C. Repeated freezing-thawing should be avoided. Components Volume per reaction
Transportation: The foam box is sealed with ice for transportation. WRTaqMan Master Mix 12.5 µL
Internal Control (IC) 0.5 µL
Operation procedure: Primer-Probe Mix 2 µL
GRTScript Enzyme 1 µL
A. Sample solution preparation：
Positive control (PC)/ Extracted RNA/ 10 µL
Clinical samples are extracted according to the corresponding
Negative Control (NC)
requirements and steps with the viral RNA extraction kit, and the Total Volume 26 µL
extracted RNA could be directly used for detection. If
samples are not immediately tested after extraction, they can also Note: Set the reaction volume as 25 µL (the final volume is 25.5 µL and
be stored at -70°C, and repeated freezing and thawing should be 26 µL but selecting 25 µL does not affect the sensitivity)

®
 D. Real-time PCR Instrument set up:
Set up the PCR tubes/strips/plate in the real time machine and label the
sample slots accordingly. Select the sample type (Unknown/PC/NTC)
and select the acquisition channel for each slot as follows:
the primers did not cross-react with pathogens with similar symptoms.
The primers were tested on Influenza A virus (H1N1, H3N2 and H5N1),
Influenza B virus, Influenza C virus, Respiratory Synctitial virus, Dengue
virus, Streptococcus pneumoniae and Mycoplasma pneumoniae and no
non-specific amplification was obtained.

Gene Channel Source Detection Detection limit:

wavelength (nm) wavelength (nm) The absolute sensitivities were obtained using synthetic RNA as a
ORF1ab gene FAM (Green) 470 510 template for detection. The Limit of Detection (LoD) was found to be 5
E-gene HEX/TET/VIC 530 555 RNA copies/reaction for both E and ORF1ab genes under in vitro
(Yellow)
IC Cy5 (Red) 625 660
conditions.

Safety information:
E. PCR Cycling conditions: The DiAGSureTM nCoV-19 Detection Assay (Multiplex, TaqMan based)
No. of is for laboratory use only. Use proper safety measures while handling
Step Temperature (°C) Time
cycles clinical samples, like wearing mask, gloves, lab-coat, etc.
RT reaction 50 15 min 1
Hold stage 95 5 min 1 Technical assistance:
95 10 sec
PCR Stage 45 Satisfaction of the customers is our utmost priority. For any kind of
60 40 sec*
*Acquisition step (Green/Yellow/Red Channel) technical assistance, always feel free to reach out to us at
tech.support@gccbiotech.co.in.
F. Instrument compatibility
The DiAGSureTM nCoV-19 Detection Assay (Multiplex, TaqMan based) Ct cut-off for each fluorescent channel:
is compatible with a broad range of real-time PCR platforms.
Performance of the kit has been verified on the following systems: Target Ct Value Interpretation
Applied Biosystems® 7500/7500 Fast Real-Time PCR System, ORF1ab gene SARS-CoV-2 ORF1ab gene
Ct≤40
Quanstudio 3, Bio-Rad CFX96, Qiagen Rotor Gene Q, Roche (FAM) positive
Lighcycler® 480. E gene (TET) Ct≤40 Coronavirus E gene positive
IC (Cy5) Ct≤28 Internal control positive
G. Performance Evaluation:
The positive control (PC) should give Ct values around 20 (3) for ORF1ab
and E genes.
Specificity:
The target sequences detected in this kit are the conserved regions of
the novel coronavirus (SARS-CoV-2) E and ORF1ab genes.
The two primer sets used in the assay failed to give any amplification
from human genomic DNA extracted from blood and sputum eliminating
the possibility of non-specific annealing with human DNA. Furthermore,

Interpretation of the results:

Assessment of clinical specimen test results should be performed after the positive and negative controls have been examined and determined to be valid. If the
controls are not valid, the patient results cannot be interpreted.
Refer to the table below for the validity and the interpretation of each specimen result according to the results of each channel.

Target ORF1ab gene E-gene IC

Interpretation Results
Fluorophore* FAM/ Green TET/Yellow Cy5/Red
2019-nCoV All Target Results are valid. Result for SARS-CoV-2
Case-1 Positive Positive/Negative Positive/Negative Positive RNA is Detected.
Sample is repeated once. If the repeated result
remains “PRESUMPTIVE POSITIVE”;
2019-nCoV additional confirmatory testing may be conducted if
Case-2 Negative Positive Positive/Negative Presumptive it is necessary to differentiate
Positive between SARS-CoV-2 and other SARS-like viruses
for epidemiological purposes or
clinical management.
2019-nCoV
Case-3 Negative Negative Positive Report results to sender
Negative
Sample should be repeated once from extraction. If
a second failure occurs, it is reported to sender as
Case-4 Negative Negative Negative Invalid
invalid and recommend recollection if patient is still
clinically indicated.

* If the target gene signal (Green and Yellow Channel) is strong, the CY5 (IC) may be negative.