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

Establishment and Application of Multiplex Droplet Digital PCR for SARS-CoV-2 Omicron Variant

【目的】为建立精准、高效的多重微滴式数字PCR(droplet digital PCR,ddPCR)定量分析检测方法,开发可同时鉴别新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)ORF1ab基因、N基因、E基因以及Omicron变异株S基因的检测体系,提高病毒性传染疾病的诊断效率及传播风险监测能力。【方法】通过筛选保守基因序列并设计特异性引物与探针,优化反应体系和扩增程序,对该方法的特异性、灵敏度及稳定性进行评价。以临床样本为实验材料,利用建立的ddPCR方法进行检测和验证,确定阳性检出率。【结果】多重ddPCR反应体系中,各对引物探针对目的片段均能有效扩增,SARS-CoV-2 ORF1ab基因、N基因、E基因以及Omicron变异株S基因灵敏度检测下限分别为0.59、0.68、1.44、1.03 copies/μL。在20份临床样本的核酸检测中,共检出16份阳性样本,阳性率达80%(16/20),经荧光定量PCR方法复测符合率一致。【结论】研究建立的多重ddPCR方法特异性强、灵敏度高,可实现对临床样本中微量新冠病毒的精确定量检测。

【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.