49Establishment Of Detective Fq-Pcr For Porcine Epidemic Diarrhea Virus And Genomic Sequencing And Phylogenetic Analysis For Fore Strains

Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, is an enveloped, single-stranded RNA virus, causing severe entero-pathogenic diarrhea in swine and incurring heavy economic losses in China. Porcine epidemic diarrhea has re-emerged with devastating impact in central China since October2010. The outbreak of PED mutates. In the immune farms, PED erupted again illustrate that the pandemic strain may have mutated, leading to the decline of immune effects. Phylogenetic analysis will be conducive to the development of more protective vaccines.Firstly, fluorescence quantitative real-time PCR (FQ-PCR) for PEDV was established. The primer and probe were designed and synthesized according to the M gene sequence, then a TaqMan fluorescence quantitative real-time PCR was developed by optimizing reaction conditions and parameters. The method had the advantages of specificity, sensitivity and repidity. In addition, it had a dynamic range of detection between101-108copies/ul. The correlation coefficient of the real-time PCR is greater than0.99, and the amplification efficiency is between90%-110%.Secondly, in this study, Twenty-two pairs of primers were designed and synthesized to amplify the complete genome sequences of AH2012, JS-HZ2012, JS2008and SX2012, based on the genome of PEDVCV777(GenBank No. AF353511). Sequence data were assembled and analyzed using Lasergene software (DNAstar Inc., USA). Multiple sequence alignments were performed using Clustal W. Phylogenetic trees was constructed using the neighbor-joining method with MEGA5. The reliability of the neighbor-joining tree was estimated by bootstrap analysis with1000replicates. The result indicates that the pandemic strains in China after2010are mutant strains and are genetically quite distant from the previous strain CV777.