猪伪狂犬病病毒野毒SYBR-Green Ⅰ实时荧光定量PCR方法的建立

摘要

We designed a pair of specific primers according to the sequence of gene gI/gE in porcine pseudorabies virus (PRV), and established the SYBR-Green Ⅰ real-time quantitative PCR method for the detection of wild-type PRV.The sensitivity of this method reached 100 copy/μL.This method exhibited good specificity and repeatability, and it could prevent from the cross reactions between PRV and other porcine pathogens such as PCV2, PPV, CSFV and JEV.After piglets were challenged with high-virulent PRV, the virus in pig tissue could be detected more sensitively by fluorescent quantitative real-time PCR than by traditional PCR.This method can be used for the quantitative assay of wild-type PRV in the tissue (such as brain, lung, tonsil and lymph nodes) samples of pig, and it also can be used for the early clinical detection of wild-type PRV and the quantitative analysis of the infectious degree of PRV.%根据猪伪狂犬病病毒(PRV)gI/gE基因序列,设计一对特异性引物,建立了鉴别PRV野毒感染的荧光定量PCR方法.该方法灵敏度达到102拷贝/μL,与猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、猪瘟病毒(CSFV)、乙脑病毒(JEV)不发生交叉反应,具有良好的特异性和重复性;用PRV强毒攻毒仔猪,荧光定量PCR比普通PCR能更灵敏检出组织带毒量.该方法可以用于猪的组织样品,如脑、肺、扁桃体、淋巴结等样品中伪狂犬野毒的定量,可用于临床伪狂犬野毒感染的早期检测和定量分析猪伪狂犬病毒感染程度.