Wang Fei, Yang Jun, He Ruyi, Yu Xiao, Chen Shuliang, Liu Yang, Wang Longyu, Li Aitao, Liu Linlin, Zhai Chao, Ma Lixin
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial BiotechnologySchool of Life Sciences, Hubei University, Wuhan, 430062, China.
Hubei Provincial Center for Disease Control and Prevention, Wuhan, 430079, China.
Biosens Bioelectron. 2021 Apr 1;177:112932. doi: 10.1016/j.bios.2020.112932. Epub 2020 Dec 28.
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，我们升级了嗜热栖热菌Argonaute（PfAgo）介导的核酸检测方法，并建立了一个高度灵敏且准确的分子诊断平台，用于大规模筛查新冠病毒感染。简而言之，以从鼻咽拭子或口咽拭子中提取的病毒RNA为模板进行逆转录聚合酶链反应（RT-PCR），以扩增病毒基因组中的保守区域。接下来，将PfAgo、引导DNA和分子信标加入到含有适当缓冲液的PCR产物中，然后在95℃孵育20-30分钟。随后，检测荧光信号。该方法被命名为SARS-CoV-2 PAND。整个过程大约在一小时内完成，与逆转录荧光定量PCR（RT-qPCR）相比，实时荧光定量PCR仪的使用时间从每批>1小时缩短至仅3-5分钟，因此缓解了昂贵的实时PCR仪的短缺问题。此外，由于该平台具有单核苷酸特异性，还被应用于鉴定SARS-CoV-2 D614G突变体。SARS-CoV-2 PAND对临床样本的诊断结果与RT-qPCR检测显示出100%的一致性。
