猪流行性腹泻病毒强毒株和疫苗株多重RT-PCR鉴别检测方法的建立及应用

Establishment and application of a multiplex RT-PCR assay for differential detection of wild-type and vaccine strains of PEDV

为建立猪流行性腹泻病毒(PEDV)的快速鉴别检测方法,针对PEDV基因序列设计3对特异性引物。第1对引物扩增野毒株(经典株、变异株)和疫苗株ORF3基因198 bp和148 bp片段;第2对引物扩增经典株和疫苗株S基因429 bp片段;第3对引物扩增变异株S基因274 bp片段。经过反应条件的优化,建立了能够同时检测并区分PEDV经典株、变异株及疫苗株的多重RT-PCR检测方法。该方法可以特异地检测PEDV,而与猪的其他重要病原FMDV、CSFV、PRRSV、TGEV、PRo V、PCV2、PRV无交叉反应;对重组质粒标准品的检出下限为2.62×10~2copies/L;重复试验获得了均匀一致的结果。应用该方法检测336份临床病料,PEDV阳性的120份,其中变异株96份、疫苗株21份、变异株和疫苗株混合感染3份。结果表明,本研究所建立的多重RT-PCR检测方法特异性强、敏感性高、重复性好,可用于PEDV经典株、变异株和疫苗株的快速鉴别检测和流行病学调查。

A multiplex RT-PCR assay was established for differential detection of classical, variant and vaccine strains of porcine epidemic diarrhea virus（PEDV） after optimization of the reaction conditions. Three pairs of primers were designed for specifically amplifying wild-type （classical, variant）and vaccine strains PEDV, respectively,of which the first pair of primers was used for amplification of 198 bp and 148 bp ORF3 gene fragments of classical and variant strains, and vaccine strain,the second used for amplification of of 429 bp S gene fragment of classical and vaccine strains,and the third for amplification of 274 bp S gene fragments of variant strain. The assay could specifically amplify PEDY,but not other swine pathogens such as FMDV,CSFV,PRRSV,TGEV,PRoV,PCV2,PRV and so on. The detection limit of PEDV standard recombinant plasmids was 2.62×102 copies/μL. The repeated test acquired uniform results. The assay was used to detect a total of 336 clinical samples and 120 were positive for PEDV,ofwhich 96 were positive for variant strain,21 for vaccine strains,and 3 for variant and vaccine strains. The results indicated that the established multiplex RT-PCR assay was sensitive,specific and repeatable, and could be used as a good tool for differential detection and epidemiological invest- igation of PEDV.