伪狂犬病病毒TaqMan双重实时荧光定量PCR检测方法的建立与初步应用  

Establishment and Preliminary Application of TaqMan Duplex Real-Time Fluorescence Quantitative PCR Assay for Detection of Porcine Pseudorabies Virus

作　　者：吕玉金[1] 张世军 吴凤笋[1] 赵攀登[1] 刘玲玲[1] 李文刚[1] 饶宝 LYU Yu-jin;ZHANG Shi-jun;WU Feng-sun;ZHAO Pan-deng;LIU Ling-ling;LI Wen-gang;RAO Bao(College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Muyuan Foods Co.,Ltd.,Nanyang 473000,China)

机构地区：[1]河南牧业经济学院动物医药学院,河南郑州450046 [2]河南农业大学动物医学院,河南郑州450002 [3]牧原食品股份有限公司,河南南阳473000

出　　处：《中国兽医杂志》2022年第5期22-28,共7页Chinese Journal of Veterinary Medicine

基　　金：河南省科技攻关(222102110252);河南牧业经济学院博士启动资金(2020HNUAHEDF017);河南牧业经济学院科研创新基金项目(XKYCXJJ2020012)。

摘　　要：为建立快速、特异、灵敏的鉴别伪狂犬病病毒(PRV)野毒株和疫苗株的方法,本试验基于PRV gB和gE基因保守区域序列,设计了2对特异性的引物和探针,探针标记不同发光基团,以区分野毒株和疫苗株。通过对引物的筛选和反应条件的优化,建立了可鉴别PRV野毒株和疫苗株的TaqMan双重实时荧光定量PCR方法,并对该方法的特异性、灵敏性、重复性和干扰性等进行评估。结果显示,该方法能同时特异性地检测出PRV野毒株和疫苗株,而与PPV、PCV2、PRRSV、CFSV、JEV等无交叉反应;该方法灵敏性高,对PRV gE和gB基因最低检测量分别为27.1 copies/μL和2.74 copies/μL;该方法重复性好,变异系数小于1%;用该方法对18份临床样本进行PRV gB和gE基因检测,且与普通PCR方法相比,符合率高达100%。结果表明,本试验建立的TaqMan双重实时荧光定量PCR方法可用于临床样本中PRV野毒株和疫苗株的鉴别检测,为猪伪狂犬病防制和净化提供了一种有效的技术手段。In order to establish a rapid,specific,sensitive method for differentiation of porcine pseudorabies virus(PRV)wildtype strains and vaccine strains,this study designed two pairs of specific primers and two probes from the conserved regions of PRV gB and gE genes,the probes were labeled with different luminophores to distinguish wildtype strains and vaccine strains.By screening the primers and optimizing the reaction conditions,a TaqMan duplex real-time fluorescent quantitative PCR(qPCR)was established,and the specificity,sensitivity,repeatability and interference of the method were evaluated.The results showed that this qPCR specifically detected PRV wild strains and vaccine strains simultaneously,but did not react with PPV,PCV2,PRRSV,CFSV and JEV;this qPCR was highly sensitive,with the minimum detection amounts of PRV gE and gB genes at 27.1 copies/μL and 2.74 copies/μL,respectively,and had the coefficient of variation less than 1%.Preliminary assessment of this qPCR with 18 swine tissue samples revealed a high coincidence rate(up to 100%)with the standard PCR.Thus,this new established TaqMan duplex real-time fluorescent quantitative PCR allows differential detection of PRV wildtype and vaccine strains in clinical samples,and provides a technical means for improved control and prevention of porcine pseudorabies.

关 键 词：伪狂犬病病毒 GE基因 GB基因 实时荧光定量PCR 

分 类 号：S853.65[农业科学—临床兽医学]