Microbiological Analysis Team, Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea.
National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea.
J Microbiol Biotechnol. 2021 Mar 28;31(3):358-367. doi: 10.4014/jmb.2009.09006.
The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (C) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.
世界卫生组织（WHO）已宣布 2019 年冠状病毒病（COVID-19）为国际卫生紧急事件。目前的诊断测试基于逆转录-定量聚合酶链反应（RT-qPCR）方法，这是一种涉及病毒 RNA 扩增的金标准测试。然而，RT-qPCR 检测在灵敏度和定量方面存在局限性。在这项研究中，我们同时测试了 qPCR 和液滴数字 PCR（ddPCR）以检测低水平的病毒 RNA。使用体外转录（IVT）RNA 或病毒 RNA 作为模板的 RT-PCR 中病毒 RNA 的循环阈值（C）显著根据引物和探针集的序列而变化，而 ddPCR 中病毒 RNA 的拷贝数则有效地用 IVT RNA、培养的病毒 RNA 和临床样本中的 RNA 进行定量。此外，通过两种方法对临床样本进行了检测，并且确定 ddPCR 的灵敏度等于或高于 RT-qPCR。然而，ddPCR 检测更适合于确定参考物质的拷贝数。这些发现表明，ddPCR 定义的参考物质的 qPCR 检测可以用作病毒 RNA 检测的高度敏感和兼容的诊断方法。
