旨在建立一种可快速同时检测猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)、猪A群轮状病毒(porcine rotavirus type A,PoRVA)和猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)三重荧光定量PCR方法.针对PEDV-N、PoRVA-VP6和PDCoV-M基因设计了引物和探针,条件优化后进行性能评估,并与商品化试剂盒检测对比.结果显示:本研究建立的三重RT-qPCR方法具有良好特异性,对PRRSV、PCV2和PRV等阳性核酸不发生扩增;具有较高的敏感性,PEDV、PoRVA和PDCoV的最低检测限均达1 copies·μL-1;重复性良好,组内和组间变异系数均小于1％;样本适应性广,检测不同样本类型时,变异系数均小于1％.与商品化试剂盒对比,本研究建立的方法PEDV和PoRVA的检测符合率为92.5％和97.5％,并对PDCoV的检测范围更广,对其变异毒株仍有较好的检测效果.本研究建立的检测方法具有特异性好、敏感性高、稳定性强和样本适应性广等优势,为临床腹泻样本提供了一种好的检测方法.
Establishment and Preliminary Application of PEDV,PoRVA and PDCoV TaqMan Triple RT-qPCR Assay
This paper aims to establish a triple fluorescence quantitative PCR method for the simultaneous detection of porcine epidemic diarrhea virus(PEDV),porcine rotavirus type A(PoRVA)and porcine deltacoronavirus(PDCoV).Primers and probes were designed for PEDV-N gene,PoRVA-VP6 gene and PDCoV-M gene,and their performance was evaluated after the conditions were optimized,and compared with commercial kits.The results showed that the triple RT-qPCR method established in this study had good specificity and did not amplify positive nucleic acids such as PRRSV,PCV2 and PRV.The detection limits of PEDV,PoRVA and PDCoV were all 1 copies·μL-1.The coefficient of variation within and between groups was less than 1％.The sample has wide adaptability,and the coefficient of variation is less than 1％when different sample types are detected.Compared with commercial kits,the detection coincidence rates of PEDV and PoRVA were 92.5％and 97.5％,and the detection range of PDCoV was wider,and the detection effect of PDCoV variant strains was still good.The detection method established in this study has the advantages of good specificity,high sensitivity,strong stability and wide sample adaptability,which provides a good detection method for clinical diarrhea samples.
porcine epidemic diarrhea virusporcine rotavirus type Aporcine deltacoronavirustriple RT-qPCR
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