Establishment And Primary Application Of Multi RT-PCR For Porcine Transmissible Gastroenteritis And Porcine Epidemic Diarrhoea

Transmissible gastroenteritis (TGE) and porcine epidemic diarrhea (PED) are two kinds of highly contagious diseases caused by two different members of the Coronaviridae, which characterized in piglets primarily with vomiting and watery diarrhoea and dehydration. At present,main diagnostic methods to TGE and PED are virus neutralization(VN),immunofluorescence(IF),immune electron microscopy(IEM),ELISA and RT-PCR. With the development of molecular biology, the diagnostic methods of ELISA and RT-PCR are becoming more important than before for their sensitivity and specialization.In this research,TGEV and PEDV were cultured at PK15 cell respectively at first.Then the virus liquid of TGEV and PEDV was stored at -20℃ when the 70% cell presented CPE.TCID50 of TGEV and PEDV was detected by the method of Reed-Muench.The result was as follows: TGEV was 0.63 TCID50,PEDV was 1 .6 TCID50 .The homology of the sequences reported in Genbank of TGEV and PEDV was respectively analyzed and compared each other. According to the results of comparison, the conservative domain of S gene for TGEV and PEDV were selected for PCR amplification.In order to prevent the formation of conformational dimers between 4 primers,two pairs of primers were designed with Primer 5.0,which were under the conditions of G+C(40-60%),18-25bp in length.The PCR products of TGEV and PEDV had specific fragment amplified of 426bp and 584bp respectively. The optimum reaction conditions of TGEV or PEDV single RT-PCR were determined through the determination of primers,MgCl2 and annealing temperature.0.63 TCID50 virus can be detected by the established TGEV RT-PCR,and that was 1.6 TCID50 for PEDV RT-PCR.Furthermore,TGEV and PEDV multi RT-PCR was established after reaction conditions were optimized.The optimal reaction system was carried out as follows:PEDV primers for reaction system was 0.4nmol/L,TGEV primers was 0.6umol/L, MgCl2 was 2 mmol/L,dNTP was 100nmol/L,optimal annealing temperature was 56℃.Cycle conditions: 94℃ 1min-56℃ 50s- 72 ℃ lmin,35 cycles.The last extension was for 10min at 72℃.The multi RT-PCR for TGEV and PEDV established in this research can detect two specific fragments with designed length under the condition. 6.3 TCID50 virus for TGEV, 15.8 TCID50 virus for PEDV can be detected,and other control viruses for HC V,PRV and PPV were negative.The sensitivity of multi RT-PCR decreased anorder of magnitude compared with the TGEV or PEDV single RT-PCR established in this research.50 clinical samples were detected by the multi RT-PCR.The result was as follows:8 positive for TGEV,3 positive for PEDV.Then TGEV accounted for 16%,and PEDV accounted for 6% in the whole positive samples.There was one clinical sample infected with the two viruses. It indicated that the sensitivity and specificity of the multi RT-PCR was good through the primary application.The results of the research supplies a reference method to diagnose the two diseases.