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Buzz about RT-qPCR: An RT-qPCR formulation for SARS-CoV-2 detection using reagents produced at Georgia Institute of Technology.

ABSTRACT: Widespread testing for the presence novel coronavirus SARS-CoV-2 in patients remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. The early testing shortfall in some parts of the US can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can produce the RT-qPCR assay and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. We compare the performance of our in-house kit to a commercial product used for diagnostic testing and describe implementation of environmental testing to monitor surfaces across various campus laboratories for the presence of SARS-CoV-2.

SUBMITTER: Mascuch SJ

PROVIDER: S-EPMC7402063 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR 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 M JM Agarwal Vinayak V Hud Nicholas V NV Glass Jennifer B JB Williams Loren Dean LD Lieberman Raquel L RL

medRxiv : the preprint server for health sciences 20200901

Widespread testing for the presence of the novel coronavirus 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 reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemis ...[more]

PMID: 32766604

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Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has caused a global pandemic beginning in 2020, can be detected by reverse-transcription polymerase chain reaction (RT-PCR). However, owing to the urgent need for a large number of detection kits, the time spent researching and developing these kits has been shortened during the pandemic, and the kits that are being used commercially have not undergone full and independent evaluation. To ensure the accuracy of SARS-CoV-2 test results, performance verification of commercial Real-Time quantitative PCR (RT-qPCR) kits is required.MethodsThe performance of five commercial RT-qPCR diagnostic kits for SARS-CoV-2 used in China was evaluated using a coronavirus disease 2019 (COVID-19) RNA liquid performance verification reference product-manufactured by Guangzhou Bondson (BDS) Biotechnology Co., Ltd.,Guangzhou, China-that uses droplet digital RT-PCR technology combined with fluorescence quantitative PCR. The five kits of Novel Coronavirus 2019-nCoV nucleic acid detection kit (RT-qPCR method) evaluated were Da An (Da An Gene Co., Ltd. of Sun Yat-sen University), Liferiver (Shanghai ZJ Bio-Tech Co., Ltd.), Kinghawk (Beijing Kinghawk Pharmaceutical Co., Ltd.), eDiagnosis (Wuhan Easy Diagnosis Biomedicine Co., Ltd.), and Maccura (Maccura Biotechnology Co., Ltd.). Performance verification criteria included the coincidence rate, limit of detection (LoD), cross-reactivity, precision, and anti-interference. Finally, through the BDS performance verification reference product kit, clinical samples are used to verify its clinical diagnostic efficacy.ResultsThe coincidence rate was 100% for all kits except for Kinghawk, which was 95%. The LoD for Da An, eDiagnosis and Maccura was 250copies/mL, and it was 1000 copies/ml for Liferiver. Kinghawk was not able to detect its advertised LoD of 500 copies/ml. The cross-reactivity test results were all negative. Moreover, all kits had a coefficient of variation less than 5%; however, Liferiver showed the best precision. Da An, Liferiver, and eDiagnosis showed higher sensitivity to the nucleocapsid (N) gene than they did to the open reading frame (ORF) 1ab genes. Anti-interference results for all five kits were positive. The results of clinical diagnostic efficacy were that the specificity of the four kits was 1.000 (0.877-1.000), the sensitivity of Da An was 1.000 (0.850-1.000), Liferiver was 0.964 (0.798-0.998), Maccura was 0.893 (0.706-0.972), and eDiagnosis was 0.857 (0.664-0.953).ConclusionsAll commercial RT-qPCR diagnostic kits for SARS-CoV-2 passed the BDS performance verification, except for Kinghawk (batch No:20200608113) which failed to detect the LoD of 500 copies/mL. Da An and Liferiver have excellent clinical diagnostic specificity and sensitivity. This study can provide guidance for the selection or optimization of RT-qPCR diagnostic test kits for SARS-CoV-2.

Dataset Information

Buzz about RT-qPCR: An RT-qPCR formulation for SARS-CoV-2 detection using reagents produced at Georgia Institute of Technology.

Publications

A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR test kits.

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