【摘要】 根据TaqMan复合荧光探针设计原则,应用Primer 5.0和Oligo 6.0软件设计检测猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)和猪圆环病毒2型(PCV2)的特异性引物和探针,优化反应条件,建立了检测PRRSV、CSFV和PCV2的单项和多重Real-time PCR方法。结果表明,建立的方法具有高度特异性,与其他病原检测无明显交叉反应;对阳性质粒和病毒的最低检测量分别为<101个拷贝和<1TCID50/反应,检测灵敏度比常规PCR检测高100倍。通过对20份临床样本进行检测,多重Real-time PCR检测结果和单项Real-time PCR检测结果一致。建立的多重Real-time PCR方法可用于同时快速检测PRRSV、CSF和PCV2的混合感染,具有灵敏、特异、重复性好并能对样品进行定量检测等优点。更多还原

【Abstract】 According to the reported nucleotide sequences of PRRSV,CSFV and PCV2,three pairs of specific probes were designed.By optimizing the primers and probes’ concentration,a multiplex real-time quantitative PCR assay for detection of these viruses was established.The specificity and sensitivity of the multiplex real-time quantitative PCR assay were analyzed for multiple viruses.The results showed that the specificity was high and no non-specific amplification was achieved from other pathogens.Both multiplex and singleplex assays were consistently able to detect<101 copies of plasmid templates or <1 TCID50 virus/reaction.A total of 20 clinical samples were comparatively detected using the multiplex and singleplex assays.The data showed that the results of multiplex assay were in accordance with that of singleplex assay.Conclusively,a multiplex real-time PCR assay was developed,which is rapid,sensitive,specific and accurate to discriminate the PRRSV,CSFV and PCV2 infection in a single test tube.更多还原