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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doaj-b2051db7df854c9cb1dedfc7760515522021-08-26T13:52:24ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-08-01228702870210.3390/ijms22168702RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay ParadigmStephen Bustin0Sara Kirvell1Jim F. Huggett2Tania Nolan3Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University Chelmsford, Chelmsford CM1 1SQ, UKMedical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University Chelmsford, Chelmsford CM1 1SQ, UKNational Measurement Laboratory, LGC, Queens Rd, Teddington, London TW11 0LY, UKMedical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University Chelmsford, Chelmsford CM1 1SQ, UKThe 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.https://www.mdpi.com/1422-0067/22/16/8702COVID-19reverse transcriptionqPCRSARS-CoV-2molecular diagnosis |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Stephen Bustin Sara Kirvell Jim F. Huggett Tania Nolan |
spellingShingle |
Stephen Bustin Sara Kirvell Jim F. Huggett Tania Nolan RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm International Journal of Molecular Sciences COVID-19 reverse transcription qPCR SARS-CoV-2 molecular diagnosis |
author_facet |
Stephen Bustin Sara Kirvell Jim F. Huggett Tania Nolan |
author_sort |
Stephen Bustin |
title |
RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm |
title_short |
RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm |
title_full |
RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm |
title_fullStr |
RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm |
title_full_unstemmed |
RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm |
title_sort |
rt-qpcr diagnostics: the “drosten” sars-cov-2 assay paradigm |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1661-6596 1422-0067 |
publishDate |
2021-08-01 |
description |
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. |
topic |
COVID-19 reverse transcription qPCR SARS-CoV-2 molecular diagnosis |
url |
https://www.mdpi.com/1422-0067/22/16/8702 |
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