Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA
Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA
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DOI：
10.1371/journal.pone.0236564
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发表时间：
2020-07-24
期刊：
影响因子：
3.7
通讯作者：
Tang, Patrick
中科院分区：
文献类型：
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作者：
Hasan, Mohammad Rubayet;Mirza, Faheem;Tang, Patrick
为了避免RNA提取试剂的有限可用性，我们旨在开发一种直接RT-qPCR检测鼻咽拭子中SARS-CoV-2的方案，而无需RNA提取。通过几种商业和实验室开发的方法对在通用病毒转运（UVT）培养基中收集的SARS-CoV-2和其他冠状病毒阳性的鼻咽标本进行预处理，并使用不同的RT-qPCR主混合物在不提取RNA的情况下进行RT-qPCR检测。将结果与涉及RNA提取的标准方法的结果进行比较。标本在65 ℃下孵育沿着10分钟，同时使用TaqPathT 1-Step RT-qPCR Master Mix，可提供比许多其他测试条件更高的SARS-CoV-2 RNA检测分析灵敏度。优化的直接RT-qPCR方法证明了6.6x10（3）拷贝/ml的检测限和高重现性（变异系数= 1.2%）。在132例SARS-CoV-2鼻咽标本中，与标准方法相比，优化方法的敏感性、特异性和准确性分别为95%、99%和98.5%。此外，通过两种方法获得的RT-qPCR C-T值呈正相关（Pearson相关系数r = 0.6971，p = 0.0013）。直接法的PCR抑制率为8%，而标准法为9%。我们通过直接RT-qPCR检测SARS-CoV-2 RNA的简单方法可以帮助实验室在试剂短缺的情况下继续检测病毒，或者在资源有限的情况下扩大其检测能力。
To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65 degrees C for 10 minutes along with the use of TaqPathT 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x10(3) copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR C-T values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, p = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.