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

Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University Chelmsford, Chelmsford CM1 1SQ, UK
National Measurement Laboratory, LGC, Queens Rd, Teddington, London TW11 0LY, UK
Author to whom correspondence should be addressed.
Academic Editor: Elzbieta Kierzek
Int. J. Mol. Sci. 2021, 22(16), 8702;
Received: 20 July 2021 / Revised: 31 July 2021 / Accepted: 11 August 2021 / Published: 13 August 2021
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. View Full-Text
Keywords: COVID-19; reverse transcription; qPCR; SARS-CoV-2; molecular diagnosis COVID-19; reverse transcription; qPCR; SARS-CoV-2; molecular diagnosis
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MDPI and ACS Style

Bustin, S.; Kirvell, S.; Huggett, J.F.; Nolan, T. RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm. Int. J. Mol. Sci. 2021, 22, 8702.

AMA Style

Bustin S, Kirvell S, Huggett JF, Nolan T. RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm. International Journal of Molecular Sciences. 2021; 22(16):8702.

Chicago/Turabian Style

Bustin, Stephen, Sara Kirvell, Jim F. Huggett, and Tania Nolan. 2021. "RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm" International Journal of Molecular Sciences 22, no. 16: 8702.

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