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A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

ABSTRACT: Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

SUBMITTER: Mascuch SJ

PROVIDER: S-EPMC7667971 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Mascuch Samantha J SJ Fakhretaha-Aval Sara S Bowman Jessica C JC Ma Minh Thu H MTH Thomas Gwendell G Bommarius Bettina B Ito Chieri C Zhao Liangjun L Newnam Gary P GP Matange Kavita R KR Thapa Hem R HR Barlow Brett B Donegan Rebecca K RK Nguyen Nguyet A NA Saccuzzo Emily G EG Obianyor Chiamaka T CT Karunakaran Suneesh C SC Pollet Pamela P Rothschild-Mancinelli Brooke B Mestre-Fos Santi S Guth-Metzler Rebecca R Bryksin Anton V AV Petrov Anton S AS Hazell Mallory M Ibberson Carolyn B CB Penev Petar I PI Mannino Robert G RG Lam Wilbur A WA Garcia Andrés J AJ Kubanek Julia J Agarwal Vinayak V Hud Nicholas V NV Glass Jennifer B JB Williams Loren Dean LD Lieberman Raquel L RL

The Journal of biological chemistry 20200903 46

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic ...[more]

PMID: 32883809

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Project description:Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID.

Dataset Information

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Publications

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Similar Datasets

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