猪流行性腹泻病毒猪传染性胃肠炎病毒猪Delta冠状病毒和猪轮状病毒多重RT-PCR检测方法的建立

Establishment of a multiplex RT-PCR for simultaneous detection of PEDV,TGEV,PDCoV and PRoV

针对猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪Delta冠状病毒(PDCoV)和猪轮状病毒(PRoV)基因序列,分别设计特异性引物,扩增PEDV N基因(750 bp)、TGEV M基因(544 bp)、PDCoV N基因(183 bp)和PRoV VP6基因(329 bp)。经过对引物浓度、退火温度等反应条件的优化及特异性、敏感性、重复性试验,成功建立了快速鉴别检测PEDV、TGEV、PDCoV、PRoV的多重RT-PCR方法。该方法仅对PEDV、TGEV、PDCoV、PRoV检测为阳性,对口蹄疫病毒(FMDV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)和猪细小病毒(PPV)等主要病毒的扩增均为阴性。PEDV、TGEV、PDCoV、PRoV重组质粒标准品的检出下限分别为1.57×10~2、1.57×10~3、1.57×10~2、1.57×10~2copies/L。相同条件下的重复性试验获得均匀一致的结果。应用所建立的方法检测2017—2018年采集自广西各地的270份腹泻病料,结果显示,PEDV、TGEV、PDCoV、PRoV的阳性率分别为15.56%、11.48%、10.74%、1.85%,并且存在混合感染现象。上述结果表明,所建立的多重RT-PCR方法可用于PEDV、TGEV、PDCoV、PRoV的快速鉴别检测及流行病学调查。

To establish a rapid method for simultaneous detection of porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine deltacoronavirus(PDCo V) and porcine rotavirus(PRo V),four pairs of specific primers were designed according to gene sequences for amplifying PEDV N gene(750 bp),TGEV M gene(544 bp),PDCo V N gene(183 bp) and PRo V VP6 gene(329 bp),respectively.After optimizing the primer concentration,annealing temperature and other reaction conditions,a specific,sensitive and reproducible multiplex RT-PCR method was established. The assay could specifically amplify the target fragments from PEDV,TGEV,PDCoV and PRoV,but could not amplify the target fragments from FMDV,PRRSV,CSFV,PRV,PCV2 and PPV.The lowest detection limits of PEDV,TGEV,PDCo V and PRo V were 1.57×10^2,1.57×10^3,1.57×10^2,1.57×10^2 copies/L,respectively.The repeatability test underthe same reaction conditions resulted in uniform and consistent results.The established assay was used to detect 270 clinical diarrhea samples collected from Guangxi Province during 2017-2018,and the positive rates of PEDV,TGEV,PDCo V and PRo V were 15.56%,11.48%,10.74% and 1.85%,respectively.There existed co-infections among four viruses.The results indicated that the multiplex RT-PCR for detection of PEDV,TGEV,PDCo V and PRo V was successfully established and could be used for differential detection and epidemiological investigation of these pathogens.