伪狂犬病病毒野毒株与疫苗株的实时荧光定量PCR鉴别方法的建立  

Establishment of a novel real-time PCR for differentiation of field strain from vaccine strain of pseudorabies virus

作　　者：张志[1] 樊雅婷[2] 吴发兴[1] 刘爽[1] 董雅琴[1] 邵卫星[1] 段纲[2] 王树双[1] 李晓成[1] 

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266032 [2]云南农业大学,云南昆明650201

出　　处：《中国兽医科学》2015年第7期680-685,共6页Chinese Veterinary Science

基　　金：科技基础性工作专项(2012FY111000);青岛市民生计划项目(13-1-3-91-nsh)

摘　　要：针对伪狂犬病病毒疫苗株基因组缺失的3.5kb片段,设计3套引物和探针,并用标准的伪狂犬病病毒野毒株和疫苗毒株进行测试,建立了一种新的快速鉴别伪狂犬病病毒野毒株和疫苗株的实时荧光定量PCR方法。该方法的检测极限可达10copies/μL,并与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪细小病毒和猪圆环病毒等不存在交叉反应现象,批内和批间重复性试验的变异系数分别为1.70%±1.07%和2.22%±1.02%,显示该方法具有良好的重复性。在对临床20份样品的检测时,该荧光定量PCR的阳性率为60%,普通PCR的阳性率仅为40%。结果表明,该方法具有特异性高、敏感性强的特点,是一种值得推广的快速诊断技术,可用于猪伪狂犬病的诊断和流行病学调查等。According to the lack of 3.5 kb fragment of the vaccine strain of pseudorabies virus （PRV）, three sets of primers and probes were designed. They were examined using standard field and vaccine strains of PRV, and a novel real-time PCR assay for differentiation of field strain from vaccine strain of PRV was established. The assay could detect 10 eopies/μL of PRV,but was not reactive to classical swine fever virus,porcine reproductive and respiratory syndrome virus,porcine parvovirus, and porcine eircovirus type 2, respectively. Its intra-bateh and inter-batch coefficients of variation were 1. 70% ± 1. 07% and 2.22% ±1.02% ,respectively. The positive rate of was 60% in detecting 20 clinical samples using the novel real-time PCR,while the positive rate was 40% using the conventional PCR. These results suggested that the novel real-time PCR was sensitive, specific and reproducible,and could be used for diagnosis and sur- veillance of pseudorabies.

关 键 词：伪狂犬病病毒 野毒株 疫苗株 实时荧光定量PCR 鉴别 

分 类 号：S852.659.1[农业科学—基础兽医学]