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_T) 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.

中文翻译：

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数字 PCR 和定量 PCR 与各种 SARS-CoV-2 引物-探针组的比较。

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，这是涉及病毒 RNA 扩增的金标准测试。然而，RT-qPCR 检测在灵敏度和量化方面存在局限性。在这项研究中，我们测试了 qPCR 和液滴数字 PCR (ddPCR) 以检测少量病毒 RNA。RT-PCR 的病毒 RNA循环阈值 (C _T ) 根据引物和探针组的序列在体外显着变化转录本 (IVT) RNA 或病毒 RNA 作为模板，而 ddPCR 的病毒 RNA 拷贝数可使用 IVT RNA、培养的病毒 RNA 和临床样本中的 RNA 进行有效量化。此外，通过这两种方法对临床样本进行了检测，ddPCR 的灵敏度被确定为等于或高于 RT-qPCR。然而，ddPCR 检测更适用于确定参考材料的拷贝数。这些发现表明，使用 ddPCR 定义的参考材料进行的 qPCR 测定可用作病毒 RNA 检测的高度灵敏且兼容的诊断方法。