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

• Published in: PLoS biology Vol. 18; no. 12; p. e3001030
• Main Authors: Reijns, Martin A M, Thompson, Louise, Acosta, Juan Carlos, Black, Holly A, Sanchez-Luque, Francisco J, Diamond, Austin, Parry, David A, Daniels, Alison, O'Shea, Marie, Uggenti, Carolina, Sanchez, Maria C, O'Callaghan, Alan, McNab, Michelle L L, Adamowicz, Martyna, Friman, Elias T, Hurd, Toby, Jarman, Edward J, Chee, Frederic Li Mow, Rainger, Jacqueline K, Walker, Marion, Drake, Camilla, Longman, Dasa, Mordstein, Christine, Warlow, Sophie J, McKay, Stewart, Slater, Louise, Ansari, Morad, Tomlinson, Ian P M, Moore, David, Wilkinson, Nadine, Shepherd, Jill, Templeton, Kate, Johannessen, Ingolfur, Tait-Burkard, Christine, Haas, Jürgen G, Gilbert, Nick, Adams, Ian R, Jackson, Andrew P
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 15.12.2020; Public Library of Science (PLoS)
• Subjects: Biology and life sciences; Coronaviruses; COVID-19; COVID-19 - diagnosis; COVID-19 Testing - economics; COVID-19 Testing - methods; Disease control; Disease transmission; DNA probes; DNA-directed RNA polymerase; Epidemics; Fluorescein; Fluorescent indicators; Genes; Genomes; Humans; Innovations; Medicine and health sciences; Methods and Resources; Microbiological assay; Multiplex Polymerase Chain Reaction - economics; N gene; Nucleocapsids; Open reading frames; Pandemics; Polymerase chain reaction; Prices and rates; Probes; Protocol (computers); Public health; Quality control; Research and Analysis Methods; Respiratory diseases; Reverse Transcriptase Polymerase Chain Reaction - economics; Reverse transcription; Ribonucleic acid; RNA; RNA polymerase; RNA, Viral - analysis; RNA-directed RNA polymerase; SARS-CoV-2 - genetics; SARS-CoV-2 - isolation & purification; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Target detection; Vaccines; Viral diseases; Viruses; United Kingdom; United States > US
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.3001030

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.