伪狂犬病病毒微滴数字PCR定量检测方法的建立

摘要

A droplet digital polymerase chain (ddPCR) detection method,based on gB gene of pseudorabies virus (PRV) was developed to improve the detection and quantification of PRV.The concentration of primer,probe concentration and annealing temperature in ddPCR reaction were optimized.The specificity and sensitivity of the established ddPCR method were also determined and applied to the detection of field samples.In results,the optimal primer concentration was 0.9 μmol · L-1,the probe concentration was 0.25 μmol · L-1,and the annealing temperature was 60 ℃ for ddPCR.The R2 value of ddPCR standard curve was 0.998,showed ddPCR had a good linear response.The Limit of Detection of ddPCR was 6.1 copies · μL-1,lower than that of fluorescence quantitative PCR (qPCR).The coefficient of variation was 2.81％,the ddPCR was specific for detecting PRV.Field samples were detected by ddPCR,qPCR and virus isolation.Compared with the virus isolation,the sensitivity of ddPCR method was 87.5％,specificity was 96.2％,and the coincidence rate was 97％.The results indicate that the new ddPCR assay provides a specific,sensitive tool for quantitative detection of PRV.%为了建立伪狂犬病病毒(PRV)微滴数字PCR(droplet digital PCR,ddPCR)的检测方法,以伪狂犬病病毒gB基因为靶基因,建立了可对其准确定量的微滴数字PCR方法.对ddPCR反应中的引物浓度、探针浓度、退火温度进行了优化.对建立的ddPCR方法的特异性和敏感性也进行测定,并且应用于临床样品的检测.结果表明,ddPCR的最佳引物浓度为0.9μmol·L-1,探针浓度为0.25 μmol·L-1,退火温度为60℃.ddPCR方法线性相关系数(R2)为0.998,显示呈良好的线性关系.检测限为6.1 copies·μL-1,比荧光定量PCR (Real Time PCR,qPCR)的检测限低,变异系数为2.81 ％,与其他病毒无交叉反应.通过对临床样品的检测,与病毒分离比较,ddPCR方法的敏感性为87.5％,特异性为96.2％,符合率为97％.结果表明新建立的ddPCR方法,敏感性高、特异性强,可用于伪狂犬病病毒的定量检测.