Establishment and Clinical Application of a Multiplex Reverse Transcription-PCR for Porcine Epidemic Diarrhea Virus, Porcine Transmissible Gastroenteritis Virus and Porcine Group A Rotavirus

A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) was developed for the detection of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine group A rotavirus (GAR). Three pairs of primers were designed to target the M gene, N gene, and VP7 gene of PEDV, TGEV, GAR, respectively, and the multiplex RT-PCR was developed and optimized. The results of the multiplex RT-PCR and routine single RT-PCRs were compared using samples collected in the field. In laboratory testing, the detection limit of the multiplex RT-PCR is ～35 pg RNA of combined TGEV-PEDV-GAR vaccine. In the field trial, 75 fecal specimens collected from pigs with diarrhea, in the central area of China, were simultaneously tested by the multiplex RT-PCR and by routine single RT-PCRs to evaluate the relative sensitivity and specificity of the multiplex RT-PCR. The results indicate that this new assay is equal in quality to the routine RT-PCR assays (sensitivities were 92%, 100%, 100% for PEDV, TGEV, GAR, respectively; specificity was 100% for all three viruses). The multiplex RT-PCR, with high sensitivity and specificity, provides a new and alternative tool for the detection of PEDV, TGEV and GAR.