一步法TaqMan~探针实时荧光定量RT-PCR快速检测PRRSV的建立及初步应用  

Development of one-step real-time fluorescent quantitative RT-PCR assay for detection of porcine reproductory syndrome virus strains

作　　者：路斌[1,2] 袁秀芳[1] 徐丽华[1] 李军星[1] 郭勇[1] 王一成[1] 

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021 [2]浙江省海盐县畜牧兽医局,浙江嘉兴730070

出　　处：《浙江农业学报》2012年第3期396-403,共8页Acta Agriculturae Zhejiangensis

基　　金：浙江省重大科技专项农业项目(2008C12049)

摘　　要：根据猪繁殖与呼吸综合征病毒(PRRSV)的序列,设计了1对特异性引物和1条TaqMan探针,建立了一步法检测PRRSV的荧光定量RT-PCR方法。结果表明,建立的方法具有较高的特异性,与PCV,JEV,SFV,PRV,PPV无交叉反应;可以检测到10个拷贝数的模板RNA或1个TCID50的PRRSV,比常规RT-PCR方法的灵敏度高10倍;对同一样品进行4次重复试验,变异系数(CV)<1%,具有较好的重复性。应用建立的荧光定量RT-PCR和常规RT-PCR对72份PRRS疑似病例肺组织进行平行检测,结果荧光定量RT-PCR检出40份阳性,高于常规RT-PCR方法(28份阳性)。建立的荧光定量RT-PCR方法特异性好、灵敏度高,而且快速简便,可用于PRRSV的实验室快速诊断和流行病学调查。To identify porcine reproductive and syndrome virus（PRRSV） rapidly using real-time RT-PCR technique,primers and TaqMan probe were designed based on the PRRSV sequence.The fluorescent quantitative RT-PCR assay for detection of PRRSV was established.Its specificity,sensitivity and stability were examined.The assay was also proven to be specific based on the results that no amplification was found by detecting RNA/DNA from PCV,JEV,SFV,PRV and PPV.10 copies of template RNA or one TCID50 of PRRSV could be detected.The coefficient variation（CV%） of intra/inter-assay for same DNA sample was less than 1%.72 samples were examined by the fluorescent quantitative RT-PCR and the conventional RT-PCR respectively.40 were detected to be infected with PRRSV by the real-time PCR,but only 28 were confirmed to be infected with PRRSV by conventional PCR.These results showed that the fluorescent quantitative RT-PCR assay was rapid,specific,sensitive and simple for the detection of PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒 实时荧光定量RT-PCR 一步法 

分 类 号：S858.28[农业科学—临床兽医学]