应用复合多聚酶链反应方法快速鉴别伪狂犬病弱毒疫苗与野毒

Rapid Differentiation of Gene Deleted Vaccine from Wildtype Isolates of Pseudorabies virus by Combined Polymerase Chain Reaction

本研究根据伪狂犬病病毒（ＰｒＶ）共有ｇＢ和疫苗株缺失的ｇＥ基因序列分别设计并合成１对通用（ＰＢ１／ＰＢ２）和１对鉴别引物（ＰＥ１／ＰＥ２），以在我国广泛使用的疫苗株Ｂａｒｔｈａ－Ｋ６１及从国内外收集的野毒株Ｓ、ＳＵ、Ｆ、Ｌ、Ｙ、Ｍｉｎ－Ａ、Ｓｈｏｐｅ、Ｓ（川）、ＳＬ１、１０＃、ＥＡ（鄂Ａ）ＤＮＡ为模板在同一反应管中同时扩增ｇＢ和ｇＥ基因序列建立了复合多聚酶链反应（ＰＣＲ）方法。ＰＣＲ产物经２．０％琼脂糖凝胶电泳显示，从所有１１株野毒ＤＮＡ中都扩增出３７２ｂｐ和５２６ｂｐ两个片段，而Ｂａｒｔｈａ－ｋ６１只扩增出３７２ｐｂ一个片段。酶切分析证明ＰＣＲ产物是特异性的。ｇＢ基因是ＰｒＶ主要保护性抗原基因，是病毒复制必需的，强弱毒都有此基因；Ｂａｒｔｈａ－Ｋ６１株是ｇＥ基因缺失的弱毒。所以，本实验建立的方法通过１次ＰＣＲ即可有效鉴别疫苗毒与野毒。这一方法将在今后根除伪狂犬病计划中发挥重要作用。

In order to differentiate genedeleted vaccine from wildtype viruses,two pairs of primers PB/PB2 and PE1/PE2,were designed based on sequences of Pseudorabies virus (prV) gB and gE gene. A combined PCR with four primers was established and two fragments were amplified with size of 372bp and 526bp from eleven wildtype isloates,S,SU,F,Y,L,MinA,Shope,S(),SL1,10# and EA,but only one fragment with size of 372bp from vaccine strain BarthaK61.PrV gB is a major protective antigen and is essential for virus replication,thus,both vaccine strain and wildtype virus have the gene;gE only presents in wildtype virus and is absent in vaccine strain BarthaK61.Therefore,thecombined PCR would be useful in differentiation of PrV infection from vaccination in future eradication program.