3种猪繁殖障碍性病毒Real-time PCR快速检测方法的建立

Development of a multiplex Real-time fluorescent quantitative PCR assay for simultaneous detection of PRV,PPV and PCV2

【目的】建立可同时检测猪伪狂犬病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒Ⅱ型(PCV2)的多重实时荧光定量PCR方法。【方法】根据GenBank数据库中PRV、PPV和PCV2的核苷酸序列,设计3对特异性引物和探针,以10倍系列稀释的阳性质粒为模板,优化反应条件,建立检测PRV、PPV和PCV2的多重Real-time PCR方法,并对其敏感性、重复性和特异性进行检验;分别采用单项和多重Real-time PCR方法,对临床收集的42份疑似病料进行检测,比较2种方法的符合率。【结果】特异性和灵敏度试验表明,建立的多重Real-time PCR检测方法具有高度特异性,与其他病原无明显交叉反应;检测灵敏度高,可检出1.0×101拷贝/μL的阳性质粒或1TCID50/mL的病毒样品。用多重Real-time PCR对42份临床疑似病料进行检测,其检测结果与单重Real-time PCR结果完全一致,表明多重Real-time PCR方法是可行的。【结论】建立了可同时检测PRV、PPV和PCV2的多重Real-time PCR方法,该法具有快速、灵敏、特异和重复性好等优点。

【Objective】 The study developed a multiplex real-time fluorescent quantitative PCR which can simultaneously detect and discriminate porcine pseudorabies virus（PRV）,porcine parvovirus（PPV） and porcine circovirus type 2（PCV2）.【Method】 According to the nucleotide sequences of PRV,PPV and PCV2 from GenBank,3 pairs of specific primers and probes were designed.The positive plasmid diluted by 10 times was used as template to establish a multiplex Real-time PCR assay by optimizing the reaction conditions.Sensitivity,reproducibility and specificity assays were determined.42 clinical suspected disease materials were detected by the established multiplex Real-time PCR assay and compared with the result of singleplex assay.【Result】 The results of specificity and sensitivity assays showed that the specificity of the established multiplex Real-time PCR assay was high,and there was no cross reaction with other pathogeneses.The detection sensitivity of the multiplex assay was high with a detection limit of 1.0×101 copies/μL of plasmid or 1 TCID50/mL virus templates.42 clinical samples detected by the multiplex assay were consistent with the results of singleplex assay.【Conclusion】 A multiplex Real-time PCR assay was developed for simultaneously detecting and discriminating PRV,PPV and PCV2 in a single test tube.The established method is rapid,sensitive,specific and accurate.