Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Rt-Qpcr Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

Version 1 : Received: 20 July 2021 / Approved: 27 July 2021 / Online: 27 July 2021 (13:36:36 CEST)
Version 2 : Received: 31 July 2021 / Approved: 3 August 2021 / Online: 3 August 2021 (15:34:05 CEST)

A peer-reviewed article of this Preprint also exists.

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. 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.

Journal reference: Int. J. Mol. Sci. 2021, 22, 8702
DOI: 10.3390/ijms22168702

Abstract

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 test 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. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target as 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 significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

Keywords

COVID-19; reverse transcription; qPCR; SARS-CoV-2; molecular diagnosis

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