Establishment and Application of Multiplex Two-step RT-PCR for Detection of Swine Main RNA Virus Pathogens

To develop a multiplex RT-PCR test for detection of swine main RNA virus pathogens,three sets of primers were designed based on the conserved domains of E2 gene of CSFV,Nsp2 gene of PRRSV and E gene of JEV.The conditions of the PCR test were optimized,such as Mg2+ and the concentration of the primers,and annealing and extension step were merged into one step as well.The multiplex two-step PCR test amplified product with size of 576,482 and 375 bp specific for PRRSV,CSFV and JEV respectively.To evaluate the multiplex RT-PCR assay,50 clinical samples were detected.The data showed that the multiple PCR method being 93% coincidence with the single PCRs for the presence of CSFV,PRRSV and JEV.The optimized multiplex RT-PCR is both sensitive,specific and rapid,providing a new method for the clinical diagnosis and epidemiological research of the three diseases.