CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19

Abstract Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We ha...

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Autores principales:	Stephen Bustin, Amy Coward, Garry Sadler, Louise Teare, Tania Nolan
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/61cde46ea3594d7aacd4dfe2fba6afbd
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spelling	oai:doaj.org-article:61cde46ea3594d7aacd4dfe2fba6afbd2021-12-02T12:03:14ZCoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-1910.1038/s41598-020-79233-x2045-2322https://doaj.org/article/61cde46ea3594d7aacd4dfe2fba6afbd2020-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79233-xhttps://doaj.org/toc/2045-2322Abstract Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique five plex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test’s reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 min. It was 100% sensitive and 100% specific when tested on 23 RNA samples extracted from COVID-19 positive patients and five COVID-19 negative patients. We also propose using multiple cycle fluorescence detection, rather than real-time PCR to reduce significantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantification cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid-April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype.Stephen BustinAmy CowardGarry SadlerLouise TeareTania NolanNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-13 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Stephen Bustin Amy Coward Garry Sadler Louise Teare Tania Nolan CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19
description	Abstract Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique five plex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test’s reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 min. It was 100% sensitive and 100% specific when tested on 23 RNA samples extracted from COVID-19 positive patients and five COVID-19 negative patients. We also propose using multiple cycle fluorescence detection, rather than real-time PCR to reduce significantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantification cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid-April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype.
format	article
author	Stephen Bustin Amy Coward Garry Sadler Louise Teare Tania Nolan
author_facet	Stephen Bustin Amy Coward Garry Sadler Louise Teare Tania Nolan
author_sort	Stephen Bustin
title	CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19
title_short	CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19
title_full	CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19
title_fullStr	CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19
title_full_unstemmed	CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19
title_sort	cov2-id, a miqe-compliant sub-20-min 5-plex rt-pcr assay targeting sars-cov-2 for the diagnosis of covid-19
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/61cde46ea3594d7aacd4dfe2fba6afbd
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CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19

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