重组酶聚合酶扩增技术快速检测猪肉及其制品中ASFV、PDCoV和SVA

Rapid detection of ASFV,PDCoV,and SVA in pork and its products by recombinant enzyme polymerase amplification

目的建立非洲猪瘟病毒(ASFV)、猪Delta冠状病毒(PDCoV)和A型塞内卡病毒(SVA)多重重组酶聚合酶扩增(RPA)快速检测方法。方法以重组质粒为模板进行RPA检测,并优化温度、时间、引物探针配比等反应条件,建立多重RPA检测方法,应用于实际样本的检测;与实时荧光聚合酶链式反应(qPCR)结果对比,评价所建立方法的准确率。结果本方法可在20 min内实现对3种病毒的同时检测,特异性良好;对ASFV、PDCoV、SVA灵敏度分别为940、770、570 copies/μL;用于实际核酸样本检测,准确率分别为93.33%、100.00%、100.00%。结论本文建立的多重RPA方法快速、便捷,适合猪肉及其制品中ASFV、PDCoV、SVA的现场初筛。

Objective To establish a rapid detection method for multiple recombinant enzyme polymerase amplification(RPA)of African swine fever virus(ASFV),Porcine Delta coronavirus(PDCoV),and Seneca virus type A(SVA).Methods RPA detection was carried out using recombinant plasmid as a template,and reaction conditions such as temperature,time,and primer probe ratio were optimized.Multiple RPA detection methods were established,which were applied to the detection of actual samples,and the accuracy of the established method was evaluated by comparing with the results of real-time fluorescence polymerase chain reaction(qPCR).Results Simultaneous detection of the three viruses can be achieved within 20 min with good specificity.Sensitivity to ASFV,PDCoV,and SVA was 940,770,and 570 copies/μL,respectively.The accuracy rates for actual sample detection were 93.33%,100.00%,and 100.00%,respectively.Conclusion The established multiple RPA method is fast and convenient,and it is suitable for on-site preliminary screening of ASFV,PDCoV,and SVA in pork and pork products.