非洲猪瘟病毒TaqMan探针实时荧光定量PCR检测方法的建立

为建立一种快速、准确、特异的非洲猪瘟病毒（African Swine Fever Virus，ASFV）定量检测方法，根据TaqMan探针荧光定量分析原理，对ASFV核酸进行了实时荧光定量PCR分析．借助计算机软件辅助，对ASFV基因序列及检测引物和探针进行了优化筛选，利用pET-ASFVP72质粒作为参比模板对PCR反应的Mg^2-、引物、探针浓度等参数进行了优化，其最佳浓度分别为4．5mmol／L，400nmol／L和500nmol／L．同时，对灵敏度、特异性、定量线性关系、精确度等进行了评估，最低检出量为10^2个拷贝数的质粒，特异性为100％，CT（Cycle Threshold）值的变异系数CV小于5％．对送检的15份（是否感染ASFV不确定）猪肉样品进行检测，结果全为阴性．试验表明，所建立的实时荧光定量PCR检测方法能够快速、准确、特异、灵敏地对ASFV核酸进行定量分析，从而为非洲猪瘟的检测提供了一个新的、可靠的方法．

Real-Time Fluorescence Quantitative PCR for the Detection of African Swine Fever Virus

In order to develop an assay for rapid,specific,quantitive detection of African swine fever virus,the primers and quantitive TagMan probes were designed and applied to detect ASFV,based on the principle of TagMan probes quantitative assay.With the help of computer softwares,the optimum sequence of ASFV gene nucleotide,primers and probes were used.The plasmid pET-ASFVP72 containing the whole gene of ASFVP72 was used as a control template,the concentration of Mg2+,primers and probes were optimized,which were 4.5 mmol/L,400 nmol/L,500 nmol/L,respectively.At the same time the sensitivity,specificity,reproducibility and quantitation range of the method were also determined,the detection limit was 102 copies plasmids,the specificity was 100%,the CV of CT value was less than 5%.15 histological materials were detected to evaluate the effect of the method.All of the results of histological sample detection were negative.It could be concluded that this method established for quantitation of ASFV was highly sensitive,rapid and easy to handle and showed a very good reproducibility,it provided a newer,more reliable diagnose for the prevention of ASFV.