猪Delta冠状病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪轮状病毒多重PCR检测方法的建立和应用

Establishment and application of the multiplex PCR method for detection of PDCoV, PEDV, TGEV and PRoV

旨在建立快速准确检测与猪腹泻相关的猪Delta冠状病毒(PDCoV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PRoV)多重PCR检测方法。针对PDCoV、PEDV、TGEV、PRoV的基因组序列设计了4对特异性引物,分别扩增PDCoV N基因(447 bp)、PEDV M基因(204 bp)、TGEV M基因(612 bp)、PRoV VP6基因(329 bp),经过对反应条件的优化,成功建立了能同时特异性检测PDCoV、PEDV、TGEV、PRoV的多重RT-PCR,且特异性好。应用该检测方法对上海及周边地区养殖场临床猪腹泻病料进行检测,结果检出PEDV阳性样品16份,其中PEDV和TGEV混合感染1份,PDCoV和PRoV均未检出,与单一RT-PCR检测结果完全吻合。该快速检测方法的成功建立,将有助于提高对PDCoV、PEDV、TGEV、PRoV的病原学调查和疾病监测,为生态养殖提供技术储备。

This study was to establish a rapid and accurate multiplex PCR assay for detection of porcine deltacoronavirus(PDCoV),porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),and porcine rotavirus(PRoV).Four pairs of specific primers were designed for genome sequencing of PDCoV,PEDV,TGEV and PRoV so as to amplify the PDCoV N(447 bp),PEDV M(204 bp),TGEV M(612 bp)and PRoV VP6(329 bp)genes,respectively.Then,the reaction conditions were optimized,and a multiplex RT-PCR capable of simultaneously detecting PDCoV,PEDV,TGEV and PRoV was successfully established without cross-reaction.The method was used to detect 17 pig samples with clinical diarrhea diseases on pig farms in the Shanghai area.The results showed that 16 samples were PEDV positive,of which one sample was positive of PEDV and TGEV mixed infection.However,PDCoV and PRoV were not detected.These results were in good agreement with those of single RT-PCR.The present method was successfully established.It would be applicable to the clinical detection and pathogen investigation of PDCoV,PEDV,TGEV,PRoV,which was a technical support for ecological breeding of live stock.