RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pus...

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Bibliographic Details
Published in:	International journal of molecular sciences 2021-08, Vol.22 (16), p.8702
Main Authors:	Bustin, Stephen, Kirvell, Sara, Huggett, Jim F., Nolan, Tania
Format:	Article
Language:	eng
Subjects:	Annealing Assaying Automation Coronaviruses COVID-19 Diagnostic software DNA-directed RNA polymerase Errors Flaw detection molecular diagnosis Nucleotide sequence Polymerase chain reaction qPCR Quality assurance Reverse transcription RNA polymerase RNA-directed RNA polymerase SARS-CoV-2 Sensitivity analysis Severe acute respiratory syndrome coronavirus 2 Temperature effects Viral diseases
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Summary:	The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first assays targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level, and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail, and our findings reveal some limitations but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Nevertheless, whilst our data show that some errors can be tolerated, it is always prudent to confirm that the primer and probe sequences complement their intended target, since, when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case, it is unlikely that a mismatch will result in poor specificity or a significant number of false-positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.
ISSN:	1422-0067 1661-6596 1422-0067