猪流行性腹泻病毒和猪德尔塔冠状病毒双重TaqMan荧光RT-PCR检测方法的建立与应用OA

The aim of this study was to develop a one-step duplex real-time RT-PCR assay to simultaneously detect and differentiate porcine epidemic diarrhea virus(PEDV)and porcine deltacoronavirus(PDCoV)so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of pig diarrhea.The specific primers and probes were designed for targeting to the conserved regions of PDCoV M gene and PEDV N genes.This duplex TaqMan real-time RT-PCR assay was developed and optimized for simultaneous detection of PEDV and PDCoV.The results showed this method had high specificity as it specifically detected PEDV and PDCoV without cross-reaction with TGEV,PRoV,PRV and other major porcine viruses.The lowest detection limit was 10 TCID50/mL for PEDV and PDCoV,indicating this assay was highly sensitive.In addition,236 clinical diarrhea samples collected between January 2021 and December 2022 were tested using this method and the positive rates were 45.76%for PEDV and 14.14%for PDCoV,in which the mixed infection rate was 5.08%.The results indicated that the established duplex qRT-PCR method provided a rapid,specific,sensitive and efficient technique for differential detection and epidemiological investigation of PEDV and PDCoV.