7. Real-time RT-PCR for the routine diagnosis of COVID19
There is currently only one test routinely used to diagnose active SARS-CoV-2 infection. This is RT-PCR (“real-time reverse transcriptase polymerase chain reaction”), which specifically detects the presence of viral nucleic acid in clinical samples. The test takes a few hours to complete.
PCR is a widely used technique in molecular biology that is able to target a specific region of a genome and make billions of copies of it. In the context of diagnostics, this means that PCR can be used to detect even very low numbers of viral genomes in clinical material. However, PCR only works on DNA, not RNA. Since SARS-CoV2 has an RNA genome, it must be first converted into DNA before it can be added to a PCR test. This is achieved using an enzyme called reverse transcriptase (RT). The DNA produced by reverse transcriptase is commonly known as cDNA (short for complimentary DNA) and is the right sort of “template” for PCR. Viral RNA is first extracted from swabs of the nose or throat or samples of sputum using commercial RNA extraction kits. The extracted RNA is added to a “one step” reaction mix that combines the RT and the PCR stages of the assay. The PCR targets a small fragment of a gene called RdRP. This PCr is not actually SARS-Cov2 specific, but will detect all “Sabecoviruses”, including SARS-CoV1 and many very similar viruses that have been detected in bats around the world. To distinguish SARS-CoV2 from these other viruses, a probe specific to SARS-CoV-2 is included in the reaction mix that produces a fluorescent signal when it specifically binds to the amplified RdRP fragment. The amount of fluorescence produced is directly proportional to the number of copies of the SARS-Co-V-2 RdRP gene fragment, hence the viral load can be measured.
Evaluation of the SARS-CoV-2 real-time RT-PCR assay has indicated that its limit of detection is about 4 viral RNA molecules – i.e. it is exquisitely sensitive. Furthermore, evaluation has failed to detect non-specificity – the assay only detects SARS-Cov2 RNA and not RNA from any other coronavirus (or anything else). Laboratories routinely performing the SARS-CoV-2 RT-PCR assays employ stringent control measures to ensure that assays work correctly (i.e. positive and negative controls) and that cross-contamination of RNA between samples does not occur.