Molecular Epidemiological Investigation On Epidemic Diarrhea Of ​​Sichuan Pig In

Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is composed of porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) acute diarrheal diseases caused by a pig, its main features are vomiting, diarrhea, dehydration and high piglets mortality. On China’s pig industry in recent years, the disease is particularly serious harm, cause serious economic losses. In this study, RT-PCR technique established for the 2012-2013 epidemic in parts of Sichuan were investigated, while the S gene sequence analysis, master of the prevalence of PED Sichuan, the Sichuan region of the disease prevention and control to provide scientific basis.1. Establishment and Application of RT-PCR detection methodReferring to GenBank reference strains CV777 M gene sequence, the application software design Primers5.0 detection primer pair are expected to amplify specific gene fragments 392bp, and the establishment of a RT-PCR det(?) technology. Optimized PCR reaction conditions, established the optimal reaction system:MgC12 concentration was 1.75 mmol/L, Taq enzyme concentration 0.05U/μL, dNTP concentration of 125 μmol/L, primer concentration was 0.55 pmol/L. Optimal cycle conditions:94 ℃ denaturation for 3 min; 94 ℃ denaturation 30s,54 ℃ annealing gradients do 30s,72 ℃ extension 30 s, repeated for 35 cycles; 72 ℃ supplement for 10 min. Porcine transmissible gastroenteritis virus and porcine rotavirus specificity were used in the control test, while the clinical testing proved that the method worked well, with a high specificity. Experimental results show that the sensitivity of the method can detect the minimum RNA concentration of 138 ng/mL. 75 piglet diarrhea material (small intestine and feces) from 9 regions of Sichuan were detected by RT-PCR method. There were 66 copies of the material for the PEDV positive disease, diarrheal diseases compound PEDV positive rate was 88.00%. The results showed that nine regions were detected in PEDV, concentrated in Sichuan, central, eastern regions. The pigs of all ages could infected, but the death occurred in piglets with ages in less than four weeks old. in addition to the onset of the season outside the winter season, also showed a high incidence of recent summer trend; compared with other diarrheal pathogens (porcine rotavirus, transmissible gastroenteritis virus), diarrhea pathogen Sichuan region, PEDV infection remains dominant.2. Cloning and Analysis of the Sichuan region PEDV partial sequence of the S geneFor S gene primers were designed to amplify the size of 947bp. Select the Sichuan region is detected as a representative sample of nine PEDV positive, the S gene fragment was amplified, and gene sequencing, and 14 S gene sequences in GenBank published abroad PEDV part of comparative analysis, clustering and genetic evolution. The nucleotide and amino acid sequence analysis showed that:the nucleotide sequence as compared with the standard CV777 strains,9 strains were nucleotide point mutation, insertion mutations and deletions; deduced amino acid sequence with the CV777 strain compared 9 PEDV portion S gene insertion or deletion of amino acids are present phenomenon. Phylogenetic analysis showed that the nucleotide:S gene -based phylogenetic tree will be divided into two groups PEDV strains G1 and G2, Gl and G2 which has been divided into two subgroups that G1-1, G1-2 and G2-on 1, G2-2.2012-2013 years 9 gene Sichuan region focused on G2-2 subsets of a large branch, in addition to China early isolates DX (Gansu,2007) alone in the G2-1 branch, and the rest domestic isolates distributed in G1, the G2 group. China in recent years PEDV pandemic strain distribution in which three subgroups, namely G1-1, G2-1, G2-2. G2-2 subgroups, including strains is relatively complex, where both of the previous strains, but also in this study in 2012 in Sichuan Province pandemic strain, as well as earlier foreign isolates CV777, BR1/87. From the S gene sequence homology of view, G2-2 reference strains abroad and within subgroups of the study nine isolates high homology, followed by the reference strains G2-1 subgroup within reference strains and nine isolates lowest homology within G1 group. And nine isolates S gene length compared to the presence of base deletion or insertion phenomenon, leading to their length difference varies. S gene homology analysis results showed that:the Sichuan region 2012-2013 pandemic strain diversity, but most prevalent strains with different strains of the previous epidemic, not only low nucleotide sequence homolpgy, and there a large, number of point mutations and nucleotide base deletion, insertion phenomena. The deduced amino acid sequence compared with standard strain CV777,9 PEDV occurring amino acids are part of the S gene point mutation, insertion or deletion phenomenon, mainly focused on G2-2 sub-branch of a large group, an amino acid insertion or deletion mutations include concentrated in the 39-75aa region, with the standard strain CV777 part of the S protein amino acid sequence homology of 93.7%-96.6% amino acid mutation rate of 3.4%-6.3%, while 9 PEDV strain nucleotide insertion, missing and a large number of amino acid changes caused by mutations in the antigenic sites and molecular characteristics change pending further study.