Establishment and application of SYBR Green I fluorescence quantitative RT-PCR method for detection of Porcine epidemic diarrhea virus

ABSTRACT

To develop a real-time fluorescent quantitative PCR method for rapid, accurate, sensitive and quantitative detection of porcine epidemic diarrhea virus (PEDV), according to the highly conserved nucleotide sequence of S gene reported by GenBank, a pair of PEDV S gene specific primers were designed, and a fluorescent quantitative RT-PCR detection method using SYBR Green I as the dye was established. The clinical samples suspected of PEDV infection were tested and compared with the results of ordinary RT-PCR. The results showed that the established standard curve of the SYBR Green I fluorescence quantitative RT-PCR method had a good linear relationship. The linear correlation coefficient R2=1, its amplification efficiency E=2.03, and the melting curve was a sharp single peak. The amplification of transmissible gastroenteritis virus, porcine parvovirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine deltacoronavirus and porcine rotavirus was negative and had strong specificity. The lowest detection concentration of 1 x 101 copies/L was 100 times more sensitive than that of the ordinary RT-PCR method. The coefficient of variation of intra- and inter-assay repeatability test were both less than 2%, with good repeatability and stability. Comparing the test results of 36 clinical samples, the total coincidence rate with ordinary RT-PCR was 88.89%. The results show that the established real-time fluorescent quantitative RT-PCR detection method has strong specificity, good reproducibility, and high sensitivity, which is of great significance for the rapid and quantitative detection of PEDV.