Establishment And Preliminary Application Of Multiple And SYBR Green I Real Time PCR Detection For Porcine Diarrhea Virus

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus(TGEV), and porcine rotavirus(PoRV) infection are common viral diarrhea, and often mix infection. To identify and differentiate rapidly the causing pathogens of clinical diseases, a multiple RT-PCR was developed. The polymerase chain reaction was optimized to simultaneously detect three pathogens, including porcine epidemic diarrhea virus(PEDV), porcine transmissible gastroenteritis virus(TGEV) and porcine rotavirus(PoRV). Three sets of specific primers were designed according to the sequences of PEDV, TGEV and PoRV at the GenBank. Using three pairs of specific primers, a RT-PCR was established to amplify the conservative regions of the three viruses, respectively. The RT-PCR system would amplify a682bp fragment for PEDV, a480bp for TGEV and a297bp for PoRV simultaneously or separately in the samples, depending on its infection status. But no specific band was amplified from other two viruses. As little as3.3x10copies/μL of PEDV,3.2×103copies/μL of TGEV and2.8×103copies/μL of PoRV. RNA could be detected using gel electrophoresis. Using the RT-PCR,20clinical samples were detected. The data showed that the multiple RT-PCR method was100%coincident with the single RT-PCR, and it can be used for the detection and differential diagnosis of three viruses.Three pairs of nucleotide primers were designed against the sequences in ORF3of PEDV, S of TGEV and VP7of PoRV, yielding three different amplification with size of103bp,115bp and153bp for PEDV, TGEV and PoRV respectively. The recombinant plas’mids pMD-ORF3, pMD-S and pMD-VP7were constructed respectively. The positive recombinant plasmid were identified by PCR and sequenced. The quantitative SYBR green Ⅰ real-time PCR system was developed for detecting and differentiating of the three viruses. A linear relationship was obtained by using the recombinant plasmids. The reaction system and amplification protocol were optimized with primers and parameter of cycle. The results showed good linearity with the coefficient correlation (R2)0.992902,0.987219and0.999045for PEDV, TGEV and PoRV, and the efficiency0.93,0.99,1.15for the three viruses, respectively. The sensitivity limit were less than33copies/μL,32copies/μL and28copies/μL for PEDV, TGEV and PoRV. The inter-and intra-coefficients of variation (CV)were less than5%. A total of twenty fecal specimens from piglets with acute gastroenteritis were collected from Heilongjiang and Neimeng etc. They were tested for PEDV, TGEV and PoRV by real-time PCR and compared with conventional RT-PCR. The positive rate of PEDV, TGEV and PoRV was60%,45%and20%by RT-PCR, while the positive rate was70%,50%and35%by SYBR green I real-time PCR.The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting PEDV, TGEV and PoRV in fecal samples. The developed assay represents a potential tool for laboratory diagnostics and quantitive assay for three viruses.