伪狂犬病病毒野毒株与gE基因缺失疫苗株TaqMan实时荧光定量PCR鉴别方法的建立  

Development of a TaqMan Real-time PCR for Differentiation of Wild-type Strain from gE-deleted Vaccine Strain of Pseudorabies Virus

作　　者：兰德松 顾贵波[3,4] 侯振中[1,2] LAN Desong;GU Guibo;HOU Zhengzhong(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;Key Laboratory of Animal Pathogen Biology,Ministry of Agriculture,Harbin 150030,China;Liaoning Provincial Center for Animal Disease Prevention and Control,Shenyang 110164,China;Liaoning Animal Medical Research Institute,Shenyang 110164,China)

机构地区：[1]东北农业大学动物医学院,哈尔滨150030 [2]农业部动物疫病病原生物学重点实验室,哈尔滨150030 [3]辽宁省动物疫病预防控制中心,沈阳110164 [4]辽宁省动物医学研究院,沈阳110164

出　　处：《中国畜牧兽医》2018年第7期1804-1812,共9页China Animal Husbandry & Veterinary Medicine

基　　金：辽宁省"百千万人才工程"资助项目(2016921040)

摘　　要：为实现对伪狂犬病病毒(pseudorabies virus,PRV)野毒株与gE基因缺失疫苗株的快速、敏感、特异的鉴别诊断,本试验针对PRVgD和gE基因设计了2套特异性引物和TaqMan探针,建立了PRV野毒株与gE基因缺失疫苗株的TaqMan实时荧光定量PCR鉴别方法,对引物和探针浓度、退火温度等进行了优化,对方法进行敏感性、特异性、重复性试验,并进行临床样品检测。结果显示,建立的针对gD、gE基因的TaqMan实时荧光定量PCR方法线性相关系数(R2)分别为0.996和0.980,均呈良好的线性关系;检测限分别为39.4和12.1拷贝/μL;与圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒均无交叉反应;重复性试验结果显示,针对gD基因的批内和批间变异系数分别为1.43%~1.86%、1.10%~2.07%,针对gE基因的批内和批间变异系数分别为0.98%~1.41%、1.12%~1.86%。应用建立的TaqMan实时荧光定量PCR与普通PCR分别对11份临床疑似感染样品进行检测,阳性率分别为36.4%和27.3%。结果表明,该方法敏感性高、特异性强、重复性好,可作为伪狂犬病病毒野毒株与gE基因缺失疫苗株的早期鉴别诊断和定量检测的有效手段。In order to establish a TaqMan Real-time PCR for rapid,sensitive and specific distinguishing the wild strain and gE-deleted vaccine strain of pseudorabies virus（PRV）,two sets of primers and TaqMan probes were designed for gDand gEgenes of PRV,respectively,then a set of 2 novel Real-time PCR were developed for quantitative detection and differentiation of wildtype strain from gE-deleted vaccine strain of PRV.The concentration of primers and probes,the annealing temperature in TaqMan Real-time PCR were optimized,the sensitivity,specificity and reproducibility of the assays were determined,and were applied to the detection of clinical samples.The R2 value of the TaqMan Real-time PCR standard curve were 0.996 and 0.980,respectively,both showed good linear response.The detection limit of the assays were 39.4 and12.1 copies/μL,respectively.The assays were highly specific for PRV,without cross-reaction with other common swine viral pathogens,such as porcine circovirus type 2（PCV2）,classical swine fever virus（CSFV）,porcine reproductive and respiratory syndrome virus（PRRSV）.Intrabatch and inter-batch reproducibility tests showed that the coefficient of variation（CV）were1.43% to 1.86%,1.10% to 2.07%（gD gene）and 0.98% to 1.41%,1.12% to 1.86%（gEgene）,respectively.Applying the TaqMan Real-time PCR and the conventional PCR to detect11 PR suspected clinical samples,they got 36.4%and 27.3%positivity,respectively.All these results indicated that the TaqMan Real-time PCR were both sensitive,specific and reproducible,and could be used as a useful tool for quantitative detection and differentiation of wild-type strain from gE-deleted vaccine strain of PRV.

关 键 词：伪狂犬病病毒(PRV) 野毒株 gE基因缺失疫苗株 TaqMan实时荧光定量PCR 鉴别 

分 类 号：S852.65[农业科学—基础兽医学]