猪肉及制品中HEV、PEDV和PDCoV三重qPCR方法的建立与应用

Establishment and Application of Triple-qPCR for HEV,PEDV and PDCoV in Pork and Its Products

人兽共患病引发的动物源性食品安全事件频发,为从食品原料源头上控制和保障其食用安全,控制猪肉及其制品消费成本,本研究构建了可同时检测猪肉及制品中戊型肝炎病毒(Hepatitis E virus,HEV)、猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)和猪δ冠状病毒(Porcine delta corona virus,PDCoV)三重实时荧光定量PCR(Real-time Quantitative PCR,qPCR)方法。结果表明,三重qPCR方法只能扩增出3种目的病毒的特异性基因片段,特异性好;对HEV、PEDV和PDCoV三种病毒的最低检测限分别为6.02、6.98、6.92 copies/μL;组内和组间变异系数(CV%)在0.10%~3.00%之间,重复性好。将所建立方法应用于248份出口猪肉及制品和282份生猪粪拭子的检测,同时以相应病毒标准检测方法进行平行检测,结果显示猪肉及制品中三种病毒的检出率均为0%,与标准方法检测结果一致。生猪粪拭子中,该方法对PEDV、PDCoV和HEV三种病毒的检出率分别为1.06%、3.19%、0.35%,标准方法检出率分别为1.06%、3.19%、0%。研究表明,建立的三重qPCR检测方法能准确快速地检测猪肉及制品或生猪样品中三种病毒,为保障生鲜猪肉及其制品市场流通和阻断病毒食源性传播提供技术支持。

Animal-derived food safety incidents caused by zoonotic diseases occur frequently.In order to control and ensure the safety of pork consumption from its raw material source and to control the cost of pork and its products consumption,this study constructed a triple real-time fluorescent quantitative PCR(qPCR)method that can simultaneously detect hepatitis E virus(HEV),porcine epidemic diarrhea virus(PEDV),and porcineδcorona virus(PDCoV)in pork and its products.The results showed that the triple qPCR method could only amplify the specific gene fragments of three target viruses,which had high specificity.The minimum detection limits of the three viruses(HEV,PEDV,and PDCoV)were 6.02,6.98 and 6.92 copies/μL respectively.The coefficient of variation(CV%)within and between groups ranged from 0.10%to 3.0%,with good repeatability.The established method was applied to detect 248 samples of pork products and exports and 282 raw pig manure swabs,and parallel detection was also conducted using corresponding virus detection methods for comparison.The detection rates of porcine epidemic diarrhea virus,porcine deltacoronavirus,and hepatitis E virus in pork products and exports were all 0%,which was consistent with the results of the corresponding virus detection methods.The detection rates of the three viruses(PEDV,PDCoV,and HEV)using this method were 1.06%,3.19%and 0.35%,respectively.The detection rates of the standard method for these three viruses were 1.06%,3.19%and 0%,respectively.The study showed that the established triplex qPCR detection method can accurately and rapidly detect three viruses in pork and its products or pig samples,providing technical support for ensuring the market circulation of fresh pork and its products and blocking the transmission of viral foodborne diseases.