PEDV、TGEV和PRoV多重RT-PCR检测方法的建立及应用

Establishment and Application of a Multiplex RT-PCR Assay for Detection of PEDV,TGEV and PRoV

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 VP6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 ×103 , 1.41 × 102 and 1.41× 103 copies/μL,respectively. The repeated reaction under the same conditions obtained uniform results. The assay was used to detect a total number of 190 clinical samples,of which 42 （22.11%） 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.