猪流行性腹泻病毒疫苗株与野毒株PCR-HRM鉴别检测方法的建立

Development of PCR-HRM method for differentiating vaccine strain from wild strain of porcine epidemic diarrhea virus

基于新型的PCR产物分析技术-高分辨率熔解曲线（HRM），大量分析猪流行性腹泻病毒（PEDV）全基因序列的特点，建立鉴别PEDVcV777疫苗株与野毒株的PCR-HRM分析方法。在ORF1保守区域设计区分引物，同时用普通的RT-PCR方法进行对比。检测19份临床样品中，PCR-HRM方法检测均为阳性且鉴定为野毒株，普通RT-PCR之后凝胶电泳分析检测18份阳性，为野毒株。结果表明，PCR-HRM方法具有特异性好，灵敏度高，PCR扩增之后产物无需开盖分析，极大地避免了污染问题，是一种新型的鉴别检测方法。

Based on the new PCR post-analysis technique--PCR-HRM analysis and analysis of a large number of gene sequence of PEDV,a kind of PCR-HRM analysis was established for differ- entiating CV777 vaccine strain from wild-type strain. A pair of primer targeted the ORF1 conserved regions was designed and compared with conventional RT-PCR methods. A total of 19 clinical samples were positive and identified as wild strains utilizing PCR-HRM method,normal gel e- lectrophoresis analysis of post PCR amplication detected that 18 were positive and identified as wild strains. PCR-HRM is a high and specificity sensitivity method for identifying vaccine strain from wild strains,without the need for opening the lid after PCR amplication which called avoid pollution problems to a large extent.