20/04/2020
Detection of SARS-CoV-2 using qRT-PCR
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1- Collect specimens (e.g. Nasopharyngeal swab)
2- Preserve specimens in the Viral Transport Medium (VTM)
3- Extract the viral RNA using a Viral RNA Mini Kit.
4- Prepare the qRT-PCR Mix (e.g. Superscript III one step RT-PCR).
5- Set the RT-PCR program and launch the reaction:
• Reverse transcription: 50 °C for 15 min (01 cycle)
• Denaturation: 95 °C for 3 min (01 cycle)
• Amplification: 95 °C for 15 s, 58 °C for 30 s (40-50 cycles)
• Cooling: 40 °C for 30 s and then 4 °C (∞).
qPCR Principle
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In quantitative PCR we usually use a specific fluorescent probe in addition to primers in order to detect the amplification of the target gene. The probe sequence is labeled at the 5’ end with a fluorescent reporter dye (F) and at the 3’ end with a quencher dye (Q). In case the probe does not anneal to a specific sequence, the fluorescent reporter will stay physically close to the quencher which will cause the quenching of the reporter’s fluorescence. However, if the probe anneals to a specific sequence on the target gene, it will be degraded by the 5’-3’ exonuclease activity of Taq DNA polymerase. The fluorescent reporter will be then released from the quencher which will be no longer close enough to absorb the emission from the reporter dye. The fluorescent signal is thus generated and detected by CCD camera in a LightCycler.
The threshold cycle or Ct value is the number of cycles required for the fluorescent signal to cross the fluorescence threshold (the real signal could so be distinguished from background signal). Ct level is inversely proportional to the amount of target RNA in the sample. The lower is the Ct level the bigger is the amount of target RNA in the sample.
Abbreviations
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qRT-PCR: quantitative Reverse Transcription-Polymerase Chain Reaction
cDNA: complementary DNA
F: fluorophore
Q: quencher
Ct: threshold cycle
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References
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1- Filion, M., 2012. Quantitative Real-Time PCR In Applied Microbiology. Norfolk: Caister Academic Press.
2- Corman VM, Landt O, Kaiser M. et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3):pii=2000045.
3- World Health Organization.
