四重荧光qRT-PCR检测猪腹泻相关病毒方法的建立

ESTABLISHMENT OF A QUADRUPLEX REAL-TIME RT-PCR FOR DETECTING PEDV, TGEV, PDCOV AND PTV

为建立同时检测猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪delta冠状病毒(Porcinedeltacoronavirus,PDCoV)和猪捷申病毒(Porcine teschovirus,PTV)的四重荧光qRT-PCR检测方法,本研究以PEDV的N基因、TGEV的S基因、PDCoV的M基因和PTV基因组5'非编码基因为检测对象,建立同时检测4种病毒的荧光RT-PCR方法。结果显示,该方法可以特异扩增4种病毒的基因,不能扩增猪呼吸道冠状病毒、伪狂犬病毒、猪瘟病毒、口蹄疫病毒、猪细小病毒、猪繁殖与呼吸综合征病毒和猪圆环病毒2型的基因;PEDV、TGEV、PDCoV和PTV的最低核酸检出量分别为141、68、8和11拷贝数;对100份送检样品进行检测,其中PEDV、TGEV、PDCoV和PTV感染检出率分别为10%、6%、2%和55%。结果表明本研究建立的检测方法具有良好的特异性和敏感性,而且操作快速简便,可用于猪腹泻疫病的临床诊断和流行病学调查。

In this study,a quadruplex real-time RT-PCR was developed using primers specific for Porcine epidemic diarrhea virus(PEDV)N gene,Transmissible gastroenteritis virus(TGEV)S gene,Porcine deltacronavirus(PDCoV)M gene and Porcine teschovirus(PTV)5'-UTR gene.This method specifically amplified fragments of the above 4 viral genes but not Porcine respiratory coronavirus,Pseudorabies virus,Classical swine fever virus,Foot–and-mouth disease virus,Porcine parvovirus,Porcine reproductive and respiratory syndrome virus and Porcine circovirus type 2.Sensitivity testing showed that the detection limits of PEDV,TGEV,PDCoV and PTV were 141 copies,68 copies,8 copies and 11 copies,respectively.Additionally,100 fecal samples from healthy pigs were tested using this method and the positive rates of PEDV,TGEV,PDCoV and PTV were 10%,6%,2%and 55%,respectively.The availability of the quadruplex real-time RT-PCR method would be beneficial to diagnostics and epidemiologic study for diarrhea cases.