Dataset Information

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

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.

SUBMITTER: Reijns MAM

PROVIDER: S-EPMC7771873 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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

Reijns Martin A M MAM Thompson Louise L Acosta Juan Carlos JC Black Holly A HA Sanchez-Luque Francisco J FJ Diamond Austin A Parry David A DA Daniels Alison A O'Shea Marie M Uggenti Carolina C Sanchez Maria C MC O'Callaghan Alan A McNab Michelle L L MLL Adamowicz Martyna M Friman Elias T ET Hurd Toby T Jarman Edward J EJ Chee Frederic Li Mow FLM Rainger Jacqueline K JK Walker Marion M Drake Camilla C Longman Dasa D Mordstein Christine C Warlow Sophie J SJ McKay Stewart S Slater Louise L Ansari Morad M Tomlinson Ian P M IPM Moore David D Wilkinson Nadine N Shepherd Jill J Templeton Kate K Johannessen Ingolfur I Tait-Burkard Christine C Haas Jürgen G JG Gilbert Nick N Adams Ian R IR Jackson Andrew P AP

PLoS biology 20201215 12

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, ...[more]

PMID: 33320856

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Project description:During the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting variants of concern (VOC) while also providing virus quantitation data. However, detection sensitivities of both RT-qPCR and sequencing can be impacted through losses occurring during sample handling, virus concentration, nucleic acid extraction, and RT-qPCR. Therefore, process limit of detection (PLOD) assessments are required to estimate the gene copies of target molecule to attain specific probability of detection. In this study, we compare the PLOD of four RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab) for detection of SARS-CoV-2 to that of ATOPlex sequencing by seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in greater detection rates than individual assays, suggesting that application of multiple assays is better suited for the trace detection of SARS-CoV-2 from wastewater samples. Furthermore, while ATOPlex offers a promising approach to SARS-CoV-2 wastewater surveillance, this approach appears to be less sensitive compared to RT-qPCR under the experimental conditions of this study, and may require further refinements. Nonetheless, the combination of RT-qPCR and ATOPlex may be a powerful tool to simultaneously detect/quantify SARS-CoV-2 RNA and monitor emerging VOC in wastewater samples.

Dataset Information

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

Publications

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

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