| Porcine transmissible gastroenteritis virus(TGEV) was initially identified as the etiological agent of transmissible gastroenteritis in swine, which could lead to large-scale pig farms viral diarrhea disease outbreak and a large number of piglets severe diarrhea,dehydration and eventually death. The highest mortality rate could be up to 100%, adult pig mortality was low, but it would improve feed conversion, increase the cost of feeding, it has brought huge economic losses to the swine industry. The disease was first reported in 1946 in the United States, then takes on a world-wide trends, China's disease reported in the1960 s. In recent years, viral diarrhea posing a serious hazard to our country's swine industry.So the prevalence of the disease as well as variations in research, and design a very fast and accurate detection method have great significance.The intestinal materials of died piglets with clinical diarrhea symptom in a pig farm of South China was collected, inoculated and cultured in PK-15 cells. Identification by RT-PCR, the result verified that this isolated virus is transmissible gastroenteritis virus, and named TGEV CN12 strain. Then continuous cultured in PK-15 cells to 120 passages.Referring to the TGEV sequences in Gen Bank, primers were designed. The complete genome of F5 and F80 were amplified by RT-PCR, after cloned and sequenced, the whole genome c DNA sequence was obtained. Using biological software to process and analyze for the sequencing results. That would provide some reference for the study of the virus in pathogenic, diagnosis prevention and molecular biology.The isolation and identification of TGEV results indicated that PK-15 cell began to for the sequencing results. That would provide some reference for the study of the virus in pathogenic, diagnosis prevention and molecular biology.The isolation and identification of TGEV results indicated that PK-15 cell began to appear CPE after 3 passages. CPE was shorten and intensified with increased passages.The50% tissue culture infective dose(TCID50) of F5?F20?F40?F60?F80?F100?F120 of CN12 strain respectively was 10-4.25/0.1m L?10-5.23/0.1m L?10-5.67/0.1m L?10-5.89/0.1m L?10-6.67/0.1m L ? 10-7.33/0.1m L ? 10-7.26/0.1m L. Sequence analysis revealed that, in the long-term of cell cultured, there were no inserted nucleotides or deletions in the whole genome sequence of CN12 strain. But there were only 6 nucleotide mutations in the whole genome sequence. Compared with 10 reference strains from domestic and foreign, deletion of 140 continuous nucleotides were found in ORF3 a of CN12 strain. Sequencing results showed that TGEV CN12 strain belongs to the field strains infection.Comparative Analysis on gene homology showed that CN12 strain had a high homology with H strain and Miller strain. The phylogenetic tree analysis revealed that there was a close genetic relationship with H strain and Miller strain.In order to establish a fast, accurate, sensitive and simple method for detection of TGEV, we referenced the latest detection technology research progress of China and abroad,designed a set of primer of conserved region of S gene. By continuous optimizing reaction condition, the detection method of a complete set of TGEV SYBR Green?real-time PCR was established. To compare with traditional methods of RT-PCR, this method for detecting TGEV have the advantages of saving time, specific, sensitive, reproducible and quantitative analysis, avoiding the false positive in the work is happended, improving the detection greatly efficiency and providing big convenience for the clinical diagnosis of TGEV. |
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