Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs
Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs
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DOI：
10.3390/ijms21124396
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发表时间：
2020-06-01
影响因子：
5.6
通讯作者：
Widera, Marek
中科院分区：
文献类型：
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作者：
Toptan, Tuna;Hoehl, Sebastian;Widera, Marek
新型冠状病毒SARS-CoV-2是急性呼吸道疾病COVID-19的病原体，因其迅速传播而成为全球关注的焦点。与此同时，对检测的需求增加导致试剂和用品短缺，并损害了许多国家诊断实验室的性能。世界卫生组织（WHO）和疾病控制与预防中心（CDC）都推荐使用多个引物和探针对的多步骤RT-PCR分析，这可能会使测试结果的解释复杂化，特别是对于边缘病例。在本研究中，我们描述了一种用于检测SARS-CoV-2 RNA的替代RT-PCR方法，该方法可用于诊断中基于探针的临床分离物检测，也可用于研究实验室中使用低成本的SYBR绿色方法。为了进行评估，我们使用了确诊的SARS-CoV-2感染患者的样本，并进行了RT-PCR检测，同时连续稀释RNA标准品，以确定检测限。我们确定了一个m基因结合引物和探针对，非常适合用于诊断和研究目的的SARS-CoV-2 RNA的定量检测。
The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.