一种通用型肠道病毒微滴式数字PCR检测方法的建立及应用

Establishment and application of universal droplet digital polymerase chain reaction method for quantification of enterovirus

目的建立一种肠道病毒通用型微滴式数字PCR(ddPCR)的定量检测方法,以实现肠道病毒的量化检测。方法利用肠道病毒标准品对ddPCR反应中的探针浓度、退火温度进行优化,并确定ddPCR本研究确定ddPCR的最佳探针浓度为0.4μmol/L,退火温度为51.0℃,核酸检测范围为3.02～3.59×106copies/μL,检出限为3.02 copies/μL。ddPCR结论本研究建立基于ddPCR肠道通用型的定量方法,可有效地对临床样本中不同血清型的肠道病毒临床样本进行拷贝数定量分析,为临床研究病毒载量的测定提供一种技术。

Objective To establish a quantitative detection method of droplet digital polymerase chain reaction(ddPCR)for identification and quantification of enterovirus.Methods The probe concentration and annealing temperature in ddPCR were optimized using enterovirus standards,and the detection range of ddPCR was determined.Viral load assays were performed on 28 clinical samples using optimized reaction conditions.Results In this study,the optimal probe concentration for ddPCR was 0.4μmol/L,the annealing temperature was 51℃,the nucleic acid detection range was 3.02-3.59×10^6 copies/μL,and the detection limit was 3.02 copies/μL.The linear correlation coefficient of the ddPCR method was 0.993 8,showing a good linear relationship.Conclusion This study established a quantification method based on ddPCR for general enterovirus,which can effectively quantify enterovirus load in clinical samples and provide a technique for the determination of viral load in clinical research.