Establishment Of Differential Methods Of Pedv,tgev And PoRV By SYBR Green I Real?time PCR Assay

Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV) and Porcine rotavirus (PoRV) are major etiological agens of diarrhea and death in piglets. The clinical signs of these diseases are similar,such as watery diarrhea,dehydration, and it is difficult to differentiate of those three disease. Several laboratory diagnosis in pigs are made by virus Isolation, IFA, and ELISA etc., but these methods have some disadvantages of costing long time , low sensitivity and complex. With the development of molecular biology, some reserchers established polymerase chain reaction (PCR) to detect these diseases, which reduced the detection time and improved the detection sensitivity, but these methods can only achieve qualitative detection for nucleic acid and it is hard to achieve accurate quantitative. Therefore, the fast and accurate dfferential detection is required for etiologic diagnosis.Three pairs of oligonucletide primers were designed against the sequences in ORF3 of PEDV, S of TGEV and VP7 of PoRV,yielding three different amplicon with size of 101bp , 109bp and 150bp for PEDV, TGEV and PoRV respectively. The recombinant plasmids pMD-ORF3, pMD-S and pMD-VP7 were constructed respectively. The postive recombinant plasmid were identified by PCR and sequenced. The quantitative SYBR GreenⅠ-based PCR system was developed for the detection and differentiation 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 standard curves constructed using the mean threshold cycle and concentration of virus which ranging from 8.5×108-8.5×102copies/μL,6.8×108-6.8×102copies/μL and 1.3×108-1.3×102copies/μL, respectively. The results showed good linearity with the coefficient correlation (R2) 0.99810,0.99836 and 0.99938 for PEDV ,TGEV and PoRV, and the efficiency 0.93,1.11,0.95 for the three viruses ,respectively. The sensitivity limit were less than 85copies/μL,68copies/μL and 13copies/μL for PEDV, TGEV and PoRV,which was 100-fold higher than the conventional RT-PCR. The inter- and intra- coefficients of variation (CV) were less than 5%.A total of forty-two fecal specimens from piglets with acute gastroenteritis were collected from Henan, Beijing, Guangdong 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 was 66.7%,54.8% and 64.3% by RT-PCR, while the positive rate was 73.8%,66.7%and 78.6%by real-time PCR. The cell culture of three viruses were collected from 4h to 72h after infecting every four-hour ,then the samples were tested by real-time PCR. It showed the highest titers of PEDV was 104copies/μL from 36h to 52h; TGEV reached to 105copies/μL from 44h; PoRV was was 104 copies/μL from 28h to 44h..A duplex real-time PCR that can detect PEDV and TGEV was developed. The specifity of the method was tested using the cDNA of PoRV, PRRSV ,CSFV. The detection limit of the reaction was less than 212 copies/μL of PEDV and 340 copies/μL of TGEV,respectively. The coefficients of variation (CV) of intra-assay were 0.32% and 0.15% for PEDV and TGEV while the coefficients of variation (CV) of inter-assays were 0.32% and 0.35% for PEDV and TGEV.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 and cell culture .The developed assay represents a potential tool for laboratory diagnostics and quantitive assy for three viruses. The duplex real-time PCR for PEDV and TGEV is a simple assay and may be usefull for rapid ,sensitive,and cost-effective etiological diagnostic tool for acute viral gastroenteritis in piglets. In conclusion this assay is impotant for the study of virus proliferation in cells and attenuated vaccine quality control.The assay may prove useful for the study of the diagnostic kits and pathogenic mechanisms of the three viruses.