4种猪腹泻病毒恒温隔绝式荧光RT-PCR检测方法的建立及应用

Establishment and application of an insulated isothermal RT-PCR method for four porcine diarrhea viruses

为鉴别猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PoRV)和猪德尔塔冠状病毒(PDCoV)这4种猪主要腹泻病毒,本研究分别针对PEDV M基因、TGEV M基因、PoRV NSP5基因、PDCoV M基因设计特异性引物和探针,经过方阵法优化恒温隔绝式荧光RT-PCR(iiRT-PCR)反应条件,建立了检测这4种猪腹泻病毒iiRT-PCR方法。用建立的iiRT-PCR方法分别检测PEDV、TGEV、PoRV、PDCoV、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、日本脑炎病毒(JEV),并对iiRT-PCR产物进行琼脂糖凝胶电泳,结果显示该方法特异性强,对PEDV、TGEV、PoRV、PDCoV的检测下限分别为10^(2)、10^(2)、10^(3)、10^(3)TCID_(50)/mL,并具有良好的组内和组间重复性;而荧光定量PCR方法的检测下限分别为10^(1)、10^(2)、10^(2)、10^(2)TCID_(50)/mL,敏感性略高于iiRT-PCR。2种方法对22份临床样品的检测结果符合率为100%,PEDV、TGEV、PoRV、PDCoV阳性率分别为40.91%、9.09%、45.45%、9.09%,其中PEDV与PoRV混合感染率为18.18%,PDCoV与PoRV混合感染率为4.55%。本研究建立的iiRT-PCR为临床现场检测4种猪腹泻病毒提供了一种简便快速的方法。

In order to identify and diagnose four major porcine diarrhea viruses,including porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine rotavirus(PoRV),and porcine delta coronavirus(PDCoV),specific primers and probes were designed based on PEDV M gene,TGEV M gene,PoRV NSP5 gene,and PDCoV M gene,respectively.The iiRT-PCR reaction conditions were optimized using the square array method to establish PEDV,TGEV,PoRV,and PDCoV insulated isothermal RT-PCR(iiRT-PCR)methods.The established iiRT-PCR had high specificity as shown by the results of detecting PEDV,TGEV,PoRV,PDCoV,CSFV,PRRSV,and JEV,and by the results of agarose gel electrophoresis of corresponding iiRT-PCR products.The minimum detection limit of the established iiRT-PCR method for PEDV,TGEV,PoRV,and PDCoV were 10^(2),10^(2),10^(3),and 10^(3)TCID50/m L,respectively,with good intra-group and inter-group repeatability.The minimum detection limits of the fluorescent quantitative PCR methods were 10^(1),10^(2),10^(2),and 10^(2)TCID_(50)/m L,respectively,and the sensitivity was slightly higher than that of iiRT-PCR.The coincidence rate of the two detection methods for 22 clinical samples was 100%.The positive rates of PEDV,TGEV,PoRV and PDCoV were 40.91%,9.09%,45.45%and 9.09%,respectively.The co-infection rate of PEDV and PoRV was 18.18%,and the co-infection rate of PDCoV and PoRV was 4.55%.The method of iiRT-PCR for four porcine diarrhea viruses established in this study provided a reference method for the field detection of PEDV,TGEV,PoRV and PDCoV in clinical practice.