A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection
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...
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| Published in | PLoS biology Vol. 18; no. 12; p. e3001030 |
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| Main Authors | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
United States
Public Library of Science
15.12.2020
Public Library of Science (PLoS) |
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| 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. |
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| AbstractList | 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. Introduction The COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) [1], originated in Wuhan (China) in December 2019 and rapidly spread across the globe, resulting in substantial mortality [2,3] and widespread economic damage. [...]a vaccine becomes available, public health strategies centered on reducing the rate of transmission are crucial to mitigating the epidemic, for which effective and affordable testing strategies to enable widespread population surveillance are essential. The N gene was central to the United States of America Centers for Disease Control and Prevention (CDC) in vitro diagnostics emergency protocol, with 3 different primer/probe sets (N1, N2, and N3) used against different portions of this viral gene [9]. [...]see S1 Data. 2019-nCoV, Novel Coronavirus 2019; Cq, cycle quantification; E, envelope; N, nucleocapsid; ORF, open reading frame; qRT-PCR, quantitative reverse transcription PCR; RdRp, RNA-dependent RNA polymerase; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SD, standard deviation. https://doi.org/10.1371/journal.pbio.3001030.g001 In early versions of these protocols, all probes were labelled with fluorescein amidite (FAM), and separate reactions were therefore needed to detect each target. In the final version of our assay, we use previously described primers and probes against the well-established SARS-CoV-2 E and N gene (N1 and N2) targets, as well as a human cellular “sample quality control” and a viral spike-in “extraction control” (Fig 1B and 1C): human RPP30 and Phocine Herpes Virus 1 (PhHV-1, hereafter referred to as PhHV). 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. Better and cheaper SARS-CoV-2 qRT-PCR tests are needed, but it is known that human and viral nucleic acid quantities in swab samples correlate, showing the importance of a human quality control probe. This study describes multiplex assays that perform equally well to commercial tests, but at ~10% of the cost. 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. Introduction The COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) [1], originated in Wuhan (China) in December 2019 and rapidly spread across the globe, resulting in substantial mortality [2,3] and widespread economic damage. [...]a vaccine becomes available, public health strategies centered on reducing the rate of transmission are crucial to mitigating the epidemic, for which effective and affordable testing strategies to enable widespread population surveillance are essential. The N gene was central to the United States of America Centers for Disease Control and Prevention (CDC) in vitro diagnostics emergency protocol, with 3 different primer/probe sets (N1, N2, and N3) used against different portions of this viral gene [9]. [...]see S1 Data. 2019-nCoV, Novel Coronavirus 2019; Cq, cycle quantification; E, envelope; N, nucleocapsid; ORF, open reading frame; qRT-PCR, quantitative reverse transcription PCR; RdRp, RNA-dependent RNA polymerase; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SD, standard deviation. https://doi.org/10.1371/journal.pbio.3001030.g001 In early versions of these protocols, all probes were labelled with fluorescein amidite (FAM), and separate reactions were therefore needed to detect each target. In the final version of our assay, we use previously described primers and probes against the well-established SARS-CoV-2 E and N gene (N1 and N2) targets, as well as a human cellular “sample quality control” and a viral spike-in “extraction control” (Fig 1B and 1C): human RPP30 and Phocine Herpes Virus 1 (PhHV-1, hereafter referred to as PhHV). |
| Audience | Academic |
| Author | Jarman, Edward J Tomlinson, Ian P M Wilkinson, Nadine Longman, Dasa Gilbert, Nick McNab, Michelle L L Friman, Elias T Slater, Louise Sanchez-Luque, Francisco J Reijns, Martin A M Diamond, Austin Ansari, Morad Johannessen, Ingolfur Jackson, Andrew P Rainger, Jacqueline K O'Shea, Marie Warlow, Sophie J Tait-Burkard, Christine Sanchez, Maria C Moore, David Uggenti, Carolina Adamowicz, Martyna Parry, David A Drake, Camilla Daniels, Alison Templeton, Kate Hurd, Toby Haas, Jürgen G O'Callaghan, Alan Shepherd, Jill Acosta, Juan Carlos Thompson, Louise Walker, Marion Mordstein, Christine McKay, Stewart Adams, Ian R Black, Holly A Chee, Frederic Li Mow |
| AuthorAffiliation | 7 The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Edinburgh, United Kingdom 2 The South East of Scotland Clinical Genetic Service, Western General Hospital, NHS Lothian, Edinburgh, United Kingdom 4 Centre for Genomic & Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, United Kingdom 3 Cancer Research UK Edinburgh Centre, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, United Kingdom 1 MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, United Kingdom 6 Division of Infection Medicine, Edinburgh Medical School, The University of Edinburgh, Edinburgh, United Kingdom University of Wisconsin-Madison, UNITED STATES 5 Centre Pfizer-University of Granada-Andalusian Government for Genomics and Oncological Research (Genyo), Granada, Spain 8 The Milner Centre for Evolution, Department of Bio |
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| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/33320856$$D View this record in MEDLINE/PubMed |
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| DOI | 10.1371/journal.pbio.3001030 |
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| DocumentTitleAlternate | A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection |
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| Title | A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection |
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