关键词: DNA polymerase RNA RT-qPCR SARS-CoV-2 coronavirus formulation infectious disease polymerase chain reaction (PCR) protein purification reverse transcriptase reverse transcription ribonuclease inhibitor virus

Mesh : Biotechnology / methods COVID-19 / diagnosis virology COVID-19 Nucleic Acid Testing / methods Humans Reagent Kits, Diagnostic / economics supply & distribution Real-Time Polymerase Chain Reaction / methods SARS-CoV-2 / genetics isolation & purification Technology Transfer Universities / economics

来  源:   DOI:10.1074/jbc.RA120.015434   PDF(Sci-hub)   PDF(Pubmed)

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.
摘要:
在有效治疗之前,对个体中是否存在新型冠状病毒严重急性呼吸道综合症冠状病毒2(SARS-CoV-2)的广泛测试对于控制COVID-19大流行仍然至关重要。测试中的挑战可以追溯到最初的供应短缺,专业知识,和/或通过定量RT-PCR(RT-qPCR)检测病毒所需的仪器,最健壮的,敏感,和目前可用的特定测定。在这里,我们表明,配备适当专业知识和基础设施的学术生物化学和分子生物学实验室可以复制市售的SARS-CoV-2RT-qPCR测试试剂盒和回填管道短缺。佐治亚理工学院COVID-19测试套件支持小组,由教师组成,工作人员,佐治亚理工学院生物技术学院的学员,合成了多重引物和探针,并配制了由内部生产的酶和蛋白质组成的主混合物。我们的内部套件与用于诊断测试的商业产品相比具有优势。我们还开发了一种环境测试协议,可以轻松监测表面是否存在SARS-CoV-2。我们的蓝图应该很容易被其他机构的研究团队复制,我们的协议可能会被修改和调整,以便在更多资源有限的环境中检测SARS-CoV-2。
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