1. Pathogen Information
SARS-CoV-2 virus, also commonly known as 2019 Novel Coronavirus (COVID-19), is a single-stranded, positive sense RNA virus which is causing an outbreak of serious respiratory problems worldwide. This RNA virus is capable of person-to-person transmission, and spreading to more than 20 countries within 1 month. It is genetically highly related to SARS and MERS coronaviruses. So far, seven coronavirus species including COVID-19 are known to infect human. Four viruses, including 229E, OC43, NL63 and HKU1, typically cause mild cold. However, people infected with the other three, including SARS, MERS and COVID-19, may develop acute and severe respiratory diseases, fever, cough and even death.
2. Test Description
This kit uses One-Step Real-Time PCR and TaqMan-MGB fluorescent probe technology. Primers and probes are designed in the highly conserved regions of the S and N gene sequences of SARS-CoV-2, and human RNase P gene as internal control. This kit has been validated on samples extracted from sputum, nasopharyngeal and throat swabs. Three fluorescent reporters are incorporated into this test: FAM measures the SARS-CoV-2 S gene; VIC measures the SARS-CoV-2 N gene; CY5 measures human RNase P gene as internal control.
3. Storage Conditions
The kit reagents should be stored at -20±10℃ in the dark in a NON-frost-free freezer. When properly stored, the reagents are stable for 12 months when sealed and 2 months after initial use. Avoid and reduce repeated freezing and thawing. The reagents should be discarded after 5 times of freezing and thawing. If reagents will be used intermittently, it is suggested to keep the reagents frozen in aliquots.
4. Compatible Instrument
This kit is developed for Applied Biosesystems™ Real time PCR system 7500/7500fast (ABI 7500 or 7500fast) ，Bio-Rad CFX96. And It can be easily adopted for any quantitative PCR instrument with FAM, VIC and CY5-compatible channels.
5 .Procedures
We recommend each following step shall be carried out in separate areas to minimize cross contamination and ensure test performance. When handling the positive control, please take precautions to avoid contamination of the test samples. Failure to take proper precautions when handling the positive control could result in a false positive result.
5.1 Reagent preparation:
ž Remove the kit from the refrigerator and thaw thoroughly at ambient temperature. Vortex and centrifuge the reagent tubes briefly.
ž Prepare your PCR reaction based on the following pipetting scheme:
|
Items |
Volume/rxn |
|
Solution I |
3μL |
|
Solution II |
12μL |
|
RNA Template* |
5μL |
*RNA template: replace with 5 μL Negative for Neg Ctrl and 5 μL Positive for Pos Ctrl ，respectively.
ž Vortex and centrifuge the PCR plate/tube briefly.
5.2 Detection:
ž Open the instrument and wait for the self-test to finish.
ž Place the prepared PCR plate in the sample bay of the instrument and record the well positions.
ž Thermal cycling settings
|
Phase |
Description |
No. of Cycles |
Temperature |
Duration |
|
1 |
cDNA synthesis |
1 |
50 |
30min |
|
2 |
Initial Denaturation |
1 |
95 |
3min |
|
3 |
Amplification |
40 |
95 |
15s |
|
*60 |
30s |
*Data acquisition at annealing phase
ž FAM, VIC and CY5 reporters are selected for fluorescence detection with NFQ-MGB. For ABI 7500, select ROX for Passive Reference. For other instruments, please refer to the manufacturer’s instructions.
