猪伪狂犬病毒基因分型PCR检测方法的建立  

Establishment of PCR methods for genotyping porcine pseudorabies virus

作　　者：胡铭哲 董伟仁[1] 颜焰[1] 顾金燕 周继勇[1] HU Ming-zhe;DONG Wei-ren;YAN Yan;GU Jin-yan;ZHOU Ji-yong(Zhejiang University Center for Veterinary Sciences,Key Laboratory of Animal Virology of Ministry of Agriculture,Zhejiang University,Hangzhou,310058)

机构地区：[1]浙江大学动物医学中心农业农村部动物病毒学重点实验室,浙江杭州310058

出　　处：《中国兽医科学》2021年第8期983-992,共10页Chinese Veterinary Science

基　　金：浙江省科技重点研发计划项目(2018C02028,2020C02011)。

摘　　要：为建立一种基因Ⅰ型和Ⅱ型猪伪狂犬病毒(PRV)鉴定方法,对PRV全基因组序列进行生物信息学分析,在Ⅱ型PRV gC基因上鉴定到一段不同于Ⅰ型PRV的47 bp长的多位点稳定差异基序。该方法能特异性区分基因Ⅰ型和Ⅱ型PRV,检测猪瘟病毒、猪德尔塔冠状病毒、猪流行性腹泻病毒、猪蓝耳病病毒、猪圆环病毒2型及3型时呈阴性。两种分型方法针对Ⅰ、Ⅱ型PRV的最低检测限均为1×10^(4)copies/L。44份猪组织病料的单重及双重PCR检测均显示,5份样品为Ⅰ型PRV,1份样品为Ⅱ型PRV。结果表明,本研究建立的单重和双重PRV分型检测方法均能准确鉴定PRV基因型,双重PCR分型方法更便捷,本研究为PRV流行病学调查及持续性感染猪的清群净化提供有效技术手段。To establish a method for identifying genotype Ⅰ and Ⅱ of porcine pseudorabies virus(PRV),the bioinformatics analysis of the PRV full genome sequence were performed,and a conservative difference motif present in 47 bp nucleotide sequence was found in the gC gene of PRV Ⅱ which is different from PRVⅠ.Genotype specific primers were designed and screened based on the conserved difference,and the simplex and duplex PCR systems were established to identify PRV Ⅰ and PRV Ⅱ.Both systems can specifically identify genotype Ⅰ and Ⅱ of PRV,and had no cross reaction with CSFV,PDCo V,PRRSV,PEDV,PCV2 and PCV3.The detection limits of two systems were both 1×10^(4) copies/L.Clinical detection results showed that 5 samples were PRVⅠtype and 1 samples was PRV Ⅱ out of 44 samples by simplex and duplex PCR systems.These results showed that both the simplex and the duplex PCR systems established in this study could accurately identify the genotypes of PRV,while the latter was more convenient and effective to simultaneously detect PRV genotypes,which could provide efficient methods for the epidemiology of PRV and the eradication of swine with persistent infection.

关 键 词：猪伪狂犬病毒 基因分型 双重PCR 

分 类 号：S852.6591[农业科学—基础兽医学]