狂犬病病毒微滴式数字PCR检测方法的建立

Development of Droplet Digital PCR Method for Detection of Rabies Virus

为了建立可绝对定量狂犬病病毒(RABV)的微滴式数字PCR(ddPCR)检测方法,本试验根据RABV基因组起始区域设计引物和探针,继而优化引物和探针浓度以及退火温度,并对建立的ddPCR方法的灵敏度、特异性和重复性进行分析。结果显示:ddPCR方法的最佳引物和探针浓度分别为900 nmol/L和250 nmol/L,最佳退火温度为55℃;ddPCR方法的标准曲线相关系数(R~2)为0.999,呈现良好的线性关系;灵敏度高,最低可检测到3.37 copies/μL;特异性良好,与常见犬病原无交叉反应;重复性好,检测结果稳定。临床样品的检测结果显示,本试验建立的狂犬病病毒ddPCR方法与实时荧光定量反转录PCR(RT-PCR)方法的符合率为100%。结果表明,本试验建立的狂犬病病毒微滴式数字PCR方法灵敏度高、特异性强,可用于狂犬病病毒的绝对定量检测。

This study aimed to develop a droplet digital PCR(ddPCR)method for absolute quantitative detection of rabies virus(RABV).Primers and probe were derived from the initial region of the RABV genome,the concentrations of primers and probe and the annealing temperature for the ddPCR method were optimized,and its sensitivity,specificity and repeatability were analyzed.The results showed that the optimal concentrations of primers and probe for the ddPCR method were 900 nmol/L and 250 nmol/L,respectively,with the optimum annealing temperature of 55℃.The correlation coefficient(R 2)of the standard curve of the ddPCR method was 0.999,indicating a clear linear relationship.The ddPCR method exhibited high sensitivity,with a detection limit of 3.37 copies/μL,high specificity,displaying no cross-reaction with common canine pathogens,and excellent repeatability,giving consistent detection results.The coincidence rate of the ddPCR method and the real-time reverse-transcription PCR(real-time RT-PCR)method was 100%for clinical sample detection.Thus,the newly established ddPCR method provides a sensitive,specific,and quantitative means for RABV absolute quantitative detection.