Establishment of Real-time Recombinase Polymerase Amplification for Detection of Porcine Epidemic Diarrhoea Virus

To develop a precise and rapid diagnosis method for detecting porcine epidemic diarrhoea virus (PEDV), a series of recombinase polymerase amplification (RPA) primers and exo-probes were established based on the highly conserved M gene of PEDV. Then a Real-time RPA assay was developed to detect PEDV using pUC57 plasmid carrying M gene fragment of PEDV as template, and the membrane or nucleotide capsid proteins from TGEV, PRRSV, PCV2 and CSFV were utilized as control. Then the sensitivity and specificity of this Real-time RPA assay was evaluated. The results showed that the Real-time reaction could detect PEDV specifically at 39℃ within 20 min with the detection limit of 10 copies/μL of plasmid DNA, and there was no cross-reaction with other control viral pathogens. Besides, the established Real-time PRA method could successfully detecte the PEDV M gene in the plasma and plasma protein power. The Real-time established in this study was simple, rapid and sensitive, which could be a novel and reliable method for diagnosing and control of PED.