A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection
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Published:2020-12-15
Issue:12
Volume:18
Page:e3001030
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ISSN:1545-7885
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Container-title:PLOS Biology
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language:en
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Short-container-title:PLoS Biol
Author:
Reijns Martin A. M.ORCID, Thompson Louise, Acosta Juan Carlos, Black Holly A.ORCID, Sanchez-Luque Francisco J.ORCID, Diamond AustinORCID, Parry David A.ORCID, Daniels AlisonORCID, O'Shea Marie, Uggenti CarolinaORCID, Sanchez Maria C.ORCID, O'Callaghan AlanORCID, McNab Michelle L. L.ORCID, Adamowicz MartynaORCID, Friman Elias T.ORCID, Hurd Toby, Jarman Edward J.ORCID, Chee Frederic Li MowORCID, Rainger Jacqueline K., Walker Marion, Drake Camilla, Longman DasaORCID, Mordstein ChristineORCID, Warlow Sophie J., McKay Stewart, Slater Louise, Ansari Morad, Tomlinson Ian P. M.ORCID, Moore DavidORCID, Wilkinson NadineORCID, Shepherd Jill, Templeton KateORCID, Johannessen Ingolfur, Tait-Burkard Christine, Haas Jürgen G.ORCID, Gilbert NickORCID, Adams Ian R.ORCID, Jackson Andrew P.ORCID
Abstract
With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.
Funder
Biotechnology and Biological Sciences Research Council Medical Research Council
Publisher
Public Library of Science (PLoS)
Subject
General Agricultural and Biological Sciences,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Neuroscience
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