PfAgo-based detection of SARS-CoV-2

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. [Display omitted] •Highly rapid, sensitive and accurate nucleic acid detecting method for the clinical diagnosis of COVID-19 based on Argonaute protein.•Alleviating the shortage of the expensive Real-time PCR instrument by shortening the using time of this instrument to only 3–5 min per batch.•Single input guide was exploited and guide DNA directing the second round of site-direct cleavage of PfAgo reached 91 nt.•An Argonaute-based method suitable for large-scale genotyping of SARS-CoV-2 variants.