猪流行性乙型脑炎病毒TaqMan荧光定量PCR方法的建立  

Development of TaqMan real-time PCR assay for detection of Japanese encephalitis virus

作　　者：霍红[1] 李业南[1] 薛遥 郭立平[1] 王晓磊[1] 步志高[1] 华荣虹[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部兽医公共卫生重点开放实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2014年第7期547-550,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：公益性行业(农业)科研专项(201203082)

摘　　要：为建立猪流行性乙型脑炎病毒(JEV)的定量检测方法,本实验根据JEV的E基因保守序列设计一对特异性引物及探针,并以E基因重组质粒作为标准品,建立检测JEV的TaqMan荧光定量PCR方法。结果显示,该方法的灵敏度可达到10个拷贝,是常规PCR灵敏度的100倍。该方法对其它相关猪病病毒的检测显示均为阴性。重复性试验表明该方法的组间和组内的变异系数均小于2%。通过检测未免疫猪和免疫减毒活疫苗猪在感染JEV后的病毒血症样品,将该方法与JEV传统病毒分离方法进行比较,结果表明该方法与乳鼠脑内接种方法灵敏度相同,比细胞分离方法灵敏度高。该方法可以作为临床检测JEV及评价疫苗保护效力的检测方法。In this study, a TaqMan real-time PCR was developed for detection of Japanese encephalitis virus （JEV） with a pair of primers and probe targeting the E gene of the virus. The test results showed that the assay was specific for JEV detection and no crossing-reaction with other related pocine virus, such as the pseudorabies virus, encephalomyocarditis virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus and porcine parvovirus, with a detection limit of 10 copies/μL, which was 100 times more sensitive than the conventional PCR. The repeatability tests showed that the inter- and intra-variation were both less than 2%. The real-time PCR assay had the same sensitivity with that of intracranial inoculation assay in suckling mice, and more sensitive than the BHK-21 cell isolation and identification method. The established assay could be potentially used for detection of JEV in clinical samples and evaluation of the vaccine against JEV infection.

关 键 词：流行性乙型脑炎病毒 TaqMan荧光定量PCR 检测 

分 类 号：S852.65[农业科学—基础兽医学]