What is a reverse-transcription polymerase chain reaction (RT-PCR) assay test for coronavirus disease 2019 (COVID-19)?

Updated: Apr 02, 2021
  • Author: James J Dunn, PhD, D(ABMM), MT(ASCP); more...
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With regard to RT-PCR assays, given that all coronaviruses have an RNA genome, it is necessary to synthesize complementary DNA (cDNA) from the RNA genome through reverse transcription, followed by PCR amplification of the cDNA with specific primers for the SARS-CoV-2 genes of interest. While all NAATs that utilize RT-PCR detect SARS-CoV-2 in this way, there are many variations that can be applied for the actual detection of the amplified genes. Most common is the use of a real-time RT-PCR, which employs fluorescence to detect the amount of amplified DNA in real time. A frequently utilized example of this is TaqMan hydrolysis. [34]  In real-time RT-PCR, the amount of gene target present in the sample typically determines the number of PCR cycles (known as the cycle threshold [Ct] value) needed before SARS-CoV-2 is detected.

All current SARS-CoV-2 RT-PCR assays with EUA approval are labeled only for the qualitative detection of gene targets specific to the virus and are not approved for quantitative measurement of the amount of virus present in the sample. While the Ct value can be reduced by increasing the amount of gene target in the sample, it can be influenced by many other factors as well, including the quality of the specimen collection technique, sample type (eg, NP sample vs saliva), gene target, and assay. There are no current recommendations for the use of Ct values in patient management, and more research on viral load kinetics is needed.

An alternative to fluorescent detection includes sequencing of the viral genome. While this has had limited applications in the detection of SARS-CoV-2, it can play an important role in determining whether a patient has become re-infected, as it may be possible to compare the sequence of the original isolate with that of a second one. [35]

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