为建立猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)及猪轮状病毒(PRoV)的快速鉴别检测方法,本试验针对 PEDV、TGEV、PRoV 的基因组序列设计3对特异性引物 PEDV-N、TGEV-M 和 PRoV-VP6,分别扩增 PEDV N 基因、TGEV M 基因和 PRoV VP 6基因。经优化反应条件,成功建立了能同时检测并区分PEDV、TGEV、PRoV 的多重 RT-PCR 方法。该方法可特异扩增 PEDV、TGEV、PRoV 相应的基因片段,而与猪瘟病毒(CSFV)、猪口蹄疫病毒(FMDV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV2)均无交叉反应；对 PEDV、TGEV、PRoV 基因重组质粒标准品的检出限分别为1.41×103、1.41×102和1.41×103拷贝/μL；在相同条件下重复试验可获得一致的结果。应用该方法对临床采集的190份腹泻病料进行检测,结果PEDV 阳性42份,阳性率22.11%；TGEV 阳性58份,阳性率30.53%；PRoV 阳性34份,阳性率17.89%,且存在不同病毒混合感染的现象。结果表明,所建立的多重 RT-PCR 方法具有特异性强、敏感性高、重复性好的优点,可用于 PEDV、TGEV 和 PRoV 的临床检测和流行病学调查。%In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV)and porcine rotavirus (PRoV)after optimization of the reaction conditions.Three pairs of primers PEDV-N, TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP 6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV, but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseud-orabies virus (PRV),porcine parvovirus (PPV)and porcine circovirus type 2 (PCV2).The detec-tion limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10 3 ,1.41×10 2 and 1.41 ×10 3 copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 1 90 clinical samples,of which 42 (22.1 1%)samples were positive for PEDV,58 (30.53%)samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.
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