The Establishment Of Differential Detection Method To Three Different Porcine Diarrheal Pathogene And Gene Sequence Analysis To COE And ORF3 Of PEDV

Porcine Epidemic Diarrhea(PED)and Transmissible Gastroenteritis(TGE) are respectively cause by porcine epidemic diarrhea virus(PEDV) and Transmissible Gastroenteritis Coronavirus(TGEV) which are acute and highly contagious disease of intestinal canal. Porcine of all ages are susceptible, Especially for age within one week. The Porcine delta Coronavirus(PDCoV)was found as an newly discovered swine diarrhea pathogens when PEDV outbreak in America. Since 2013, the PDCoV arise when PEDV and TGEV were widespread exist. These three diseases have similar clinical Symptoms, vomiting and watery diarrhea, through which are difficult to identify.In order to evaluate if the PDCOV would promote the prevalence of PEDV and TGEV, We established a triplex PCR method to quickly and accurately differetiate pathogen of TGEV PEDV and PDCOV, which is the first time in domestic. In order to improve the accuracy and sensitivity of detection of PEDV,reducing the occurrence of false positive in the process of experiment, We established RT-nested PCR to detect low content of PEDV. Meanwhile,in order to discuss the recessive infection of PEDV in central China.We make analysis to COE gene and ORF3 gene of 20 strains prevalent PEDV.According to the conservative sequence N gene of Coronavirus,we designed three pairs of specific primers. Then we optimized different system and reaction condition. We Determine the PEDV, TGEV concentration is 0.24?M, PDCOV concentration 0.32?M,10×PCR buffer solution is 1m M, the DNA Taq polymerase final concentration of 2.5 U, the d NTP final concentration is 0.2 mmol/L, annealing temperature is 56 ?. There are no target fragment be found when we use above method to detect PRRSV?CSFV?PRV?PPV. The highest sensitivity of Multiple RT-PCR is 33.4 pg of PEDV,322.3 pg of PDCOV,28.56 pg of TGEV.It turned out that the method have good sensitivity and specificity,which would provide a good way to differential diagnosis quickly and accurately in pathogen of diarrhea. We detected 149 clinical samples through triplex RT-PCR and RT-PCR, PEDV positive rate is 46.3%,PDCOV positive rate is 1.4%, no TGEV and mixed infection be found,which have the same result with RT-PCR. It means PEDV is highly prevalent in this area.Because of the low content template of PEDV, it is difficult to get satisfactory results with RT-PCR,A RT-nested PCR was established to detect PEDV. After the specificity and sensitivity test,we found that none of PCR products were amplified from TGEV, PRRSV,GARV and CSFV. The minimum concentration of template is 284pg/ul for RT-PCR,while the RT-nested PCR is 2.84pg/ul. The result show that the RT-nested PCR have higher sensitivity, so it reduce the occurrence of false positive in the process of experiment.ORF3 gene is a virulence genes of PEDV,Which determine the virulence. Compared and analyzed 20 strains of PEDV ORF3 gene sequences we found that the nucleotide homology is between 95.5% to 100%, amino acid homology is between 95.5% to 100%.Comparing different domestic PEDV strain, we know that the nucleotide and amino acid of PEDV have similar mutations. The evolutionary tree of PEDV analysis shows that 20 PEDV pandemic strain located on the G2 branch,It is distant relative with classic strain CV777 of PEDV and close relative with other domestic strain. The analysis of S gene epitope of PEDV show that the nucleotide homology is between 95.8% to 100%,amino acid homology is between 95.1% to 100%.Nucleotide and amino acid have 11 or 5 common mutations respectively. CEO gene genetic analysis show that JXTJ and JXBS strain located on the G1 branch, other 18 strains on G2,they belong to the same branch with DR13 strain, distant away relative with the classic strain CV777,it means the COE gene mutation changes its antigenicity,which explained why the PEDV is still exist in pigs after they have injected PEDV vaccination.Therefore the analysis of COE gene and ORF3 gene make us better to know the Epidemic strains in genetic variation so that we can make diagnosis and prevent this disease accurately and rapidly...