Rapid detection of porcine deltacoronavirus and porcine epidemic diarrhea virus using the duplex recombinase polymerase amplification method.

Li, Gen; Wu, Miaoli; Li, Jinhui; Cai, Weiyou; Xie, Yongsheng; Si, Guangbing; Xiao, Li; Cong, Feng; He, Dongsheng

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) have emerged and spread throughout the porcine industry in many countries and are economically important pathogens causing diarrhea in sows and acute death in newborn piglets. Therefore, a sensitive diagnostic method would be beneficial for the prevention and control of PEDV and PDCoV infection. However, traditional detection methods have a number of drawbacks. This research aimed to establish a rapid detection method of duplex recombinant enzyme-mediated thermostatic amplification (RT-RPA) for PEDV and PDCoV. In this study, eight pairs of primers were designed for each virus according to the conserved domains of both PEDV and PDCoV from the NCBI Genbank, and one pair of primers was selected for each virus following the test results. After optimization of the reaction time, reaction temperature and primer concentration ratio, the duplex RT-RPA assay amplified a 226-bp fragment specifically for PEDV and a 321-bp fragment specifically for PDCoV. Meanwhile, the specificity and sensitivity of the primers and clinical samples were tested to verify the establishment of the RT-RPA method. The sensitivities of the duplex RT-RPA method for PEDV and PDCoV were 1 × 102 copies/μL. The results were consistent with PCR results and showed that a detection method for PEDV and PDCoV duplex RT-RPA was successfully established. In summary, the duplex recombinase polymerase amplification method could offer a promising alternative to the duplex RT-qPCR for detection of PEDV and PDCoV.