SARS-CoV-2Omicron变异株多重微滴式数字PCR定量方法的建立及应用OA北大核心CSTPCD

[Objective]This research is aimed to establish an accurate and efficient multiple droplet digital PCR(ddPCR)quantitative analysis detection method,and to develop a detection system that can simultaneously identify severe acute respiratory syndrome coronavirus(SARS-CoV-2)ORF1ab gene,N gene,E gene,and Omicron variant S gene,so as to improve the diagnostic efficiency of viral infectious diseases and the ability to monitor the risk of transmission.[Method]Based on it,conserved gene sequences were screened,specific primers and probes were designed,reaction systems and amplification procedures were optimized,and the specificity,sensitivity and stability of the method were evaluated.With clinical samples as experimental materials,the established ddPCR method was applied for testing and verification to determine the positive detection rate.[Result]Each pair of primers effectively amplified the target fragment in the multiple ddPCR reaction system.The lower detection limits for SARS-CoV-2 ORF1ab gene,N gene,E gene,and Omicron variant S gene were 0.59,0.68,1.44 and 1.03 copies/μL,respectively.In the nucleic acid tests of 20 clinical samples,a total of 16 positive samples were detected,with a positive rate of 80%(16/20).The coincidence rate was consistent with that of retesting by fluorescence quantitative PCR method.[Conclusion]The multiple ddPCR method established in this study is highly specific and sensitive,and can be used to accurately quantify trace amounts of SARS-CoV-2 in clinical samples.