Development And Application Of Real-Time Fluorescent Quantitative Pcr Approach For Pedv Detection

Porcine epidemic diarrhea (PED) is an enteric infection disease caused by Porcine epidemic diarrhea virus (PEDV) and characterized by severe enteritis and diarrhea, leading with a mortality rate of piglets up to90%.The wide prevalence and spreading of the disease brought out huge loss in many nations and regions in the world. In the present study, a real-time PCR assay by TaqMan probe approach for PEDV detection was established and was used for PEDV detection.1. Establishment of Real-time PCR for PEDV detectionA pair of primers was designed according to the PEDV N gene sequence in GenBank. The Nl gene,a fragment of449bp, was amplied by PCR from field samples inoculated with PEDV. The PCR products were cloned into pMD18-T Easy vector and identified,the recombinant plasmids named pMD18-T/N1were obtained. A pair of primers and a TaqMan probe were designed according to the published N gene sequences of PEDV in GenBank.The Real-time PCR assay was carried out by quantitave concentration of serial10fold dilutions of pMD18-T/N1DNA by optimizing circulation parameters. A standard curve was achieved and the result showed that the sensitive of this method was1.0×101copy/μL and the liner relation was excellent.The final values on initiative concentration of1.0×109,1.0×108,1.0×107copy/μL DNA were7.03,9.93and13.55, respectively, the coeffients of variation were0.9%,1.1%and0.6%, all below5%. Furthermore, we found the sensitivity of Real-time PCR was prior to that of PCR by detecting the positive plasmid.2. Application of Real-time Fluorescence Quantitative PCR Technique for Detecting PEDVPEDV was detected by both reverse transcription polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative PCR in total of97samples, in which14were serial passage viruses in vero cells,52samples of artificially infected swine and31clinical cases. The results showed that32of the tested samples produced positive by real-time fluorescence quantitative PCR, while general RT-PCR only showed25positive. The results indicated that real-time fluorescence quantitative PCR method is sensitive than general RT-PCR. High sensitivity and specificity of our method were confirmed and it could be one of the effective methods for PEDV detection.