Establishment and application of a multiplex RT-PCR to differentiate wild type and vaccine strains of porcine epidemic diarrhea virus

Due to outbreaks of porcine epidemic diarrhea (PED) and the wide use of attenuated live vaccine, both wild-type and vaccine strains (CV777) are believed to circulate in Chinese pig farms. Thus, identification of different PEDV strains is of epidemiological importance. In this study, a multiplex RT-PCR method was established based on the sequence features of spike (S) gene and ORF3 gene of PEDV5. The method could identify PEDV variant strains, classical wild-type strains and classical vaccine strains. The limit of detection of the RT-PCR was 1.51 x 10(4) copies/uL for plasmids and 1 x 10(1.7) TCID50/100 u L for PEDV, respectively. There were no cross-detections among three different PEDVs and no false detections among six swine pathogens. This assay was used to test 940 samples from China of which 303 samples were PEDV positive, and 289, 5, 10 were positive for variant, classical wild, classical vaccine, respectively. One sample was positive for both variant and classical vaccine PEDV. The variant PEDV5 could be detected in samples from 13 provinces, while classical PEDVs were detected from nine provinces, supporting the prevalence of variant PEDV in China. In summary, this multiplex RT-PCR was a useful tool for the clinical detection and epidemiological survey of PEDV.