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Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/COVID-19) RT-PCR Kit contains pairs of specific primers and Taqman probes for the conserved regions of the SARS-CoV-2 (2019 Novel Coronavirus) N gene and ORF1ab gene. The SARS-CoV-2 nucleic acid in the specimens is quantitatively analyzed using one-step real-time PCR detection technology after RNA extraction.
Research Articles on SARS-CoV-2/COVID-19 RT-PCR Kit
||Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/COVID-19)
||Store all components at -20 °C in the dark. Avoid repeated freeze/thaw cycles.
||No interference was observed with: Azithromycin, Ceftriaxone, Histamine hydrochloride, Interferon alpha, Levofloxacin, Lopinavir, Meropenem, Oseltamivir, Oxymetazoline, Paramivir, Phenylephrine, Ribavirin, Ritonavir, Sodium chloride, Tobramycin, Zanamivir.
No cross-reactivity was observed with: influenza A H1N1, H3N2, H5N1 and H7N9; influenza B Yamagata and Victoria; respiratory syncytial virus types A and B; parainfluenza types I, II and III; Adenovirus types 1, 2, 3, 4, 5, 7 and 55; Enteroviruses A, B, C and D; Epstein-Barr virus; Measles virus; Human cytomegalovirus; Rotavirus; Norovirus; Mumps virus; Varicella-zoster virus; Mycoplasma pneumoniae; Chlamydia pneumoniae; Legion bacteria; Pertussis; Haemophilus influenzae; Staphylococcus aureus; Streptococcus pneumoniae; Streptococcus pyogenes; Klebsiella pneumoniae; Mycobacterium tuberculosis; human genomic DNA.
| Component || Quantity and Volume || Contents|
| Reaction Solution|| 2 × 850 µl ||5X RT-PCR Buffer, specific primers and probes|
|Enzyme Solution || 2 × 150 µl || Reverse Transcriptase, DNA Polymerase, dNTP, Mg2+|
|Negative Control || 1 × 400 µl || Pseudovirus containing internal standard fragments|
| Positive Control || 1 × 400 µl || Pseudovirus containing target fragments and internal standard fragments|
|Material Required But Not Provided
- RT-PCR Tubes
- RT-PCR Instrument
- Nucleic acid extraction and purification reagents
|Directions for use
- Bring all components to room temperature. Centrifuge at low speed for 10 seconds.
- Create a reaction mixture using the following reagents. It is recommended to multiply each of these volumes by the number of reactions being carried out. Mix the reagents well.
(For example, when carrying out 10 reactions, this would use 170 μl of Reaction Solution and 30 μl of Enzyme Solution, for a total volume of 200 μl.)
- Add the mixture to a sterile centrifuge tube and mix thoroughly. Centrifuge at 2000 RPM for 10 seconds. Aliquot into PCR tubes (20 µl/tube).
- Cover all PCR tubes and label them appropriately. All kit reagents can now be stored back to -20 °C.
PCR (Nucleic acid amplification region)
- Carry out RNA extraction using an RNA extraction kit. It is recommended to also subject the Positive Control and Negative Control to the same RNA extraction method.
- Centrifuge the PCR tubes containing the reagent prepared earlier at low speed for 10 seconds.
- Add 5 μl of corresponding sample to the sample PCR tubes. Add 5 μl of positive and negative control to the respective positive and negative control PCR tubes.
- Centrifuge the PCR tubes at low speed for 10 seconds.
Analysis of Results
- Set up the PCR machine and allow it to warm up. Ensure all PCR tubes are covered tightly before placing them in the machine.
- Program the appropriate PCR cycling protocol on your RT-PCR instrument. A suggested thermal cycle is below.
Note: Acquisition must be performed at the end of the Annealing and Extension stages.
|Step||Temperature (°C)||Time (seconds)||Cycles |
|Annealing and Extension||55||45|
- Select the fluorescent channel (FAM/VIC/Cy5) of the instrument for testing.
- SARS-CoV-2 N gene: FAM
- SARS-CoV ORF1ab gene: VIC/HEX
- Internal standard: Cy5
- Follow the instrument software instructions to generate cycle threshold (Ct) values from the acquired data. Once the Ct values have been calculated from the qPCR data, the samples can be judged as follows:
- If both the FAM and VIC Ct values for a sample is at or below 40, that sample is positive for both the SARS-CoV-2 N gene and ORF1ab gene.
- If both the FAM and VIC Ct values do not appear or if both the FAM and VIC Ct values are above 40, that sample is negative.
- If the VIC Ct is at or below 40, but the FAM Ct does not appear or is above 40, the sample is positive for the SARS-CoV-2 ORF1ab gene, but not the N gene. It is recommended to re-test the sample.
- If the FAM Ct is at or below 40, but the VIC Ct does not appear or is above 40, the sample is positive for the SARS-CoV-2 N gene, but not the ORF1ab gene. It is recommended to re-test the sample.
- The positive control should show Ct values at or below 32 for both FAM and VIC, and no Ct values for Cy5; if not, the test is invalid.
- The negative control should show Ct values at or below 32 for Cy5 only, and no Ct values for both FAM and VIC; if not, the test is invalid.
|FAM (SARS-CoV-2 N gene)||VIC (SARS-CoV-2 ORF1ab gene)||Outcome|
|Positive||Positive||Positive for both SARS-CoV-2 N gene and ORF1ab gene|
|Positive||Negative||Positive for SARS-CoV-2 N gene, re-test sample|
|Negative||Positive||Positive for SARS-CoV-2 ORF1ab gene, re-test sample|
|Negative||Negative||Negative for both SARS-CoV-2 N gene and ORF1ab gene|
||Shipped within 5-10 working days.
||This product is for research use only.