In the present study, we upgraded Pyrococcus furiosus Argonaute (PfAgo) mediated nucleic acid detection method and established a highly sensitive and accurate molecular diagnosis platform for the large-scale screening of COVID-19 infection. Briefly, RT-PCR was performed with the viral RNA extracted from nasopharyngeal or oropharyngeal swabs as template to amplify conserved regions in the viral genome. Next, PfAgo, guide DNAs and molecular beacons in appropriate buffer were added to the PCR products, followed by incubating at 95 °C for 20–30 min. Subsequently, the fluorescence signal was detected. This method was named as SARS-CoV-2 PAND. The whole procedure is accomplished in approximately an hour with the using time of the Real-time fluorescence quantitative PCR instrument shortened from >1 h to only 3–5 min per batch in comparison with RT-qPCR, hence the shortage of the expensive Real-time PCR instrument is alleviated. Moreover, this platform was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The diagnostic results of clinic samples with SARS-CoV-2 PAND displayed 100% consistence with RT-qPCR test.

在本研究中，我们升级了Pyrococcus furiosusArgonaute（PfAgo）介导的核酸检测方法，为大规模筛查COVID-19感染建立了高灵敏、准确的分子诊断平台。简而言之，以从鼻咽或口咽拭子中提取的病毒RNA为模板进行RT-PCR，以扩增病毒基因组中的保守区域。接下来，将适当缓冲液中的 PfAgo、指导 DNA 和分子信标添加到 PCR 产物中，然后在 95°C 下孵育 20-30 分钟。随后，检测荧光信号。这种方法被命名为 SARS-CoV-2 PAND。整个过程在大约一个小时内完成，与 RT-qPCR 相比，实时荧光定量 PCR 仪的使用时间从 >1 小时缩短到每批次仅 3-5 分钟，从而缓解了昂贵的Real-time PCR仪的短缺问题。此外，由于其单核苷酸特异性，该平台还被用于识别 SARS-CoV-2 D614G 突变体。SARS-CoV-2 PAND 临床样本的诊断结果显示与 RT-qPCR 测试 100% 一致。