同时检测PRRSV美洲型经典株、变异株和TJM-F92疫苗株的多重TaqMan荧光定量RT-PCR方法的建立及应用

Establishment and application of a multiplex TaqMan real-time RT-PCR for differential detection of classical, highly pathogenic and TJM-F92 vaccine strains of North American genotype PRRSV

为建立猪繁殖与呼吸综合征病毒(PRRSV)的快速鉴别检测方法,本研究针对PRRSV美洲型经典株、变异株及弱毒活疫苗TJM-F92株的基因序列差异,设计了2对特异性引物和3条TaqMan探针,经过反应条件的优化,建立了能够同时检测并区分PRRSV美洲型经典株、变异株及TJM-F92疫苗株的多重TaqMan荧光定量RT-PCR方法。结果显示,该方法具有特异性强、敏感性高、重复性好的特点,与猪其他常见病毒无交叉反应;经典株、变异株、TJM-F92疫苗株重组质粒标准品的检出下限均为2.68拷贝/μL,比常规PCR敏感100倍;组内及组间重复试验的变异系数均小于1.5%。此外,该方法对324份疑似PPRSV感染的临床样品进行检测,结果显示PRRSV阳性114份,其中美洲型变异株105份、经典株6份、TJM-F92疫苗株3份,变异株和经典株混合感染4份。本研究建立的多重TaqMan荧光定量RT-PCR方法可以为PRRSV美洲型野毒株、疫苗株的快速鉴别检测及流行病学调查提供有效的技术手段。

In this study, a multiplex TaqMan real-time RT-PCR assay was established for differential detection of the classical （C）, highly pathogenic （HP） and TJM-F92 vaccine strains of North American （NA） genotype porcine reproductive and respiratory syndrome virus （PRRSV）. The assay utilized two pairs of specific primers and three TaqMan probes designed according to the different genomic sequences among C-PRRSV, HP-PRRSV and TJM-F92 strain. The assay was highly specific and sensitive with the detection limits of 2.68 copies/μL for each standard plasmid of C-PRRSV, HP-PRRSV and TJM-F92, but no amplification for FMDV, CSFV, PRV, PPV and PCV2. In addition, the coefficient of variation was less than 1.5% for both intra-assay and inter-assay. Moreover, the established assay was used to detect PRRSV in 324 clinical samples and 114 samples were positive for PRRSV, of which 105 samples were positive for HP-PRRSV, 6 samples for C-PRRSV, 3 samples for TJM-F92 and 4 samples for both HP-PRRSV and C-PRRSV coinfections. The results indicated that this assay could be used as an effective tool for rapid detection and epidemic surveillance of PRRSV.