COVID-19 Real Time PCR Detection Kit (2-gene) (500 tests)
Cat. No.: BMG-QP1568-02
Size: 500 tests
Description
The SARS-CoV-2 Real-Time RT-PCR Assay is designed for specific and qualitative detection of SARS-CoV-2 RNA in specimens such as oropharyngeal swabs, nasopharyngeal swabs or sputum suspected of SARS-CoV-2. The product is intended for use by qualified laboratory
personnel well trained in nucleic acid amplification techniques and in vitro diagnostic procedures. Positive results do not rule out bacterial infection or
co-infection with other viruses. Negative results do not preclude SARS-CoV2 infection and should not be used as the sole basis for patient anagement decisions. Test results must be combined with clinical observations, patient history, and epidemiological information.
Summary and explanation of the test
The novel coronaviruses belong to the β genus. COVID-19 is an acute respiratory infectious disease. People are generally susceptible. Currently,
the patients infected by the novel coronavirus are the main source of infection; asymptomatic infected people can also be an infectious source.
Based on the current epidemiological investigation, the incubation period is 1 to 14 days, mostly 3 to 7 days. The main manifestations include fever,
fatigue and dry cough. Nasal congestion, runny nose, sore throat, myalgia and diarrhea are found in a few cases. The SARS-CoV-2 Real-Time RT-PCR Assay is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in nasopharyngeal or oropharyngeal swabs, sputum.
Principle
The primers and probes are selected from the regions of RNase P, ORF1ab and E gene. Two primer/probe sets are designed for specific detection of SARS-CoV-2. An additional primer/probe set to detect human RNase P gene (as an internal control) is also included. RNA extracted from respiratory specimens is reverse transcribed and amplified in a Real-Time PCR Thermocycler (such as Applied Biosystems 7500 Real-Time PCR Instrument). The fluorescent signal is emitted when the 5’ nuclease active domain of Taq polymerase degrades the probe anneals to target sequence between the forward and the reverse primers. During each cycle, the fluorescence intensity is increased with the additional reporter dye molecules released from their respective probes. The SARS-CoV-2 Real-Time RT-PCR Assay also contains a positive control (Plasmid DNA) and a negative control for testing process monitoring.
Reagents and materials provided
| Item | Component | Quantity | Volume /T |
|
| 100 Tests / Box |
500 Tests / Box |
|||
| 1 | SARS-CoV-2 PCR Mix |
1.5 mL/ tube × 1 | 1.5 mL/ tube × 5 | 14 µL |
| 2 | RT-PCR Enzyme Mix |
110 μL/ tube × 1 | 550 μL/ tube × 1 | 1 µL |
| 3 | SARS-CoV-2 Positive Control |
50 μL/ tube × 1 | 250 μL/ tube × 1 | 5 μL |
| 4 | SARS-CoV-2 Negative Control |
50 μL/ tube × 1 | 250 μL/ tube × 1 | 5 μL |
Storage instructions
Store the kit frozen at -20±5°C and avoid repeated freeze-thaw cycles (less than 5 frozen-thaw times allowed). The product is valid for 12 months, and the expiry date of the product is shown on the label.
Specimen type and preservation
Specimen type: oropharyngeal swabs, nasopharyngeal swabs and sputum.
Specimen preservation: process specimens for viral RNA extraction and nucleic acid detection as soon as possible. Specimens can be stored at 4°C for 24 hours, or frozen at -80°C for longer storage, if not processed immediately. Avoid repeated freeze-thaw cycles during transport and storage of the specimens
Assay PCR reaction setup (at reagent preparation area)
A. Reagent Preparation
To calculate the number (n) of PCR reaction tubes (n= number of specimens to be tested + number of positive controls + number of negative controls), add n x14 μL of SARS-CoV-2 PCR Mix and n x 1 μL of RT-PCR Enzyme Mix to a clean tube, vortex to mix well, centrifuge and aliquot 15 µL of the mix into each PCR reaction tube.
B. Addition of Viral RNA
Add 5 μL of viral RNA extracts of the specimen or positive/negative control to each PCR tube according to the reaction preparation table below for testing. Then immediately cap the tube tightly, briefly spin the mixture and move it to the amplification area for RT-PCR detection
- PCR Reaction Preparation
| Component | Volume per test |
| SARS-CoV-2 PCR Mix | 14 μL |
| RT-PCR Enzyme Mix | 1 μL |
| SARS-CoV-2 PCR Negative Control / SARS-CoV-2 PCR Positive Control / RNA of specimen to be tested | 5 μL |
| Total Reaction Volume | 20 μL |
C. RT-PCR amplification (at Amplification Area, use Applied Biosystems 7500 Real-Time PCR System as an example)
- 1. Place the complete PCR reaction tubes into the fluorescent quantitative PCR analyzer and label positive control, negative control and testing specimen on the Thermocycler software.
- 2. Enter the PCR reaction volume: 20 μL.
- 3. Select fluorescence detection channels: JOE, FAM and Cy5 channels. Cy5 is the E gene indicator channel, FAM is the ORF1ab indicator channel, and JOE is the RNase P gene indicator channel (internal control).
- 4. Select PCR cycle parameter setting following the instructions below, save the file and start the PCR run.
- 5. Analyze the results according to your thermocycler manufacturer’s instructions. If the positive and negative control samples do not meet their respective requirements, the PCR run must be repeated.
| Cycles | Step | Time | Temperature |
| 1 | Reverse transcription | 15 minutes | 50 ºC |
| 1 | Initial denaturation | 30 seconds | 95 ºC |
| 45 | Denaturation | 30 seconds | 95 ºC |
| Annealing/Extension | 45 seconds | 60 ºC |
Determination of results
1. The results can only be determined when the results of both controls meet their requirements.
2. Criterion for a positive specimen: a specimen can be reported as positive when FAM, Cy5 and JOE channels are all positive.
3. Criterion for a negative specimen: a specimen can be reported as negative when the JOE channel is positive and the FAM and Cy5 channels are negative.
| Channel | Gene | Results Interpretation |
| Cy5 | E | Positive: If both Cy5 and FAM have exponential increase curves with Ct ≤ 37. Negative: If both Cy5 and FAM have a Ct value >40. Inconclusive: Only one channel’s Ct ≤ 37 and the other one is 37< Ct<40. Or both Cy5 and FAM channels’ 37 |
| FAM | ORF1 ab | |
| JOE (VIC/HEX) | RNase P | Ct ≤ 37, It’s a valid result if the Ct ≤ 37, otherwise it’s invalid. |
Compatibility of real-time PCR thermocyclers
| Manufacturer | Model |
| Applied Biosystems | 7500 Real-Time PCR System |
| 7300 plus Real-Time PCR System |
Product performance
Product analysis performance evaluation results:
- 1. The analytical sensitivity of this kit is 200 copies/mL.
- 2. Cross-reaction: no cross-reaction with other pathogens such as seasonal influenza A (H1N1) virus, novelinfluenza A (H1N1-2009) virus, influenza AH3N2, H5N1, H7N9,influenza B Yamagata,influenza B Victoria, RSV A, RSV B, parainfluenza I, parainfluenza II, parainfluenza III, adenovirus types 1, 2, 3, 4, 5, 7 & 55, enterovirus types A, B, C and D, hMPV (human metapneumovirus), EB virus, measles virus, human cytomegalovirus, rotavirus, norovirus, mumps virus, varicella zoster virus, mycoplasma pneumoniae, chlamydia pneumoniae, legionella, bordetella pertussis, haemophilus influenzae, staphylococcus aureus, streptococcus pneumoniae, streptococcus pyogenes, klebsiella pneumoniae, mycobacterium tuberculosis, aspergillus fumigatus, candida albicans, candida glabrata, cryptococcus neoformans, coronavirus (HKU1, OC43, NL63, 229E), SARS-CoV-1, MERS coronavirus.
REFERENCE
- National Health Commission & State Administration of Traditional Chinese Medicine. Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 7). [R]. March 3, 2020
