PfAgo-based detection of SARS-CoV-2

• DOI: 10.1016/j.bios.2020.112932
• Publication Date: 2020-12-28T17:16:34Z
• Authors: Fei Wang; Jun Yang; Ruyi He; Xiao Yu; Shuliang Chen; Yang Liu; Longyu Wang; Aitao Li; Linlin Liu; Chao Zhai; Lixin Ma
• Keywords: 0301 basic medicine; SARS-CoV-2; Archaeal Proteins; Biomedical Engineering; Biophysics; COVID-19; General Medicine; Biosensing Techniques; Article; Recombinant Proteins; 3. Good health; Pyrococcus furiosus; 03 medical and health sciences; Limit of Detection; COVID-19 Nucleic Acid Testing; Nasopharynx; Argonaute Proteins; Electrochemistry; Humans; Point Mutation; RNA, Viral; Biotechnology

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.