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

摘要

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 TCIDW 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 shuwed that the fluorescent quantitative RT-PCR assay was rapid, specific, sensitive and simple for the detection of PRRSV.%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(R)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.