A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection

Martin A M Reijns, Louise Thompson, Juan Carlos Acosta, Holly A Black, Francisco J Sanchez-Luque, Austin Diamond, David A Parry, Alison Daniels, Marie O'Shea, Carolina Uggenti, Maria C Sanchez, Alan O'Callaghan, Michelle L L McNab, Martyna Adamowicz, Elias T Friman, Toby Hurd, Edward J Jarman, Frederic Li Mow Chee, Jacqueline K Rainger, Marion WalkerCamilla Drake, Dasa Longman, Christine Mordstein, Sophie J Warlow, Stewart McKay, Louise Slater, Morad Ansari, Ian P M Tomlinson, David Moore, Nadine Wilkinson, Jill Shepherd, Kate Templeton, Ingolfur Johannessen, Christine Tait-Burkard, Jürgen G Haas, Nick Gilbert, Ian R Adams, Andrew P Jackson

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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.

Original languageEnglish
Pages (from-to)e3001030
JournalPLoS Biology
Issue number12
Early online date15 Dec 2020
Publication statusE-pub ahead of print - 15 Dec 2020


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