Establishment Of Nested PCR Detection Method Of Porcine Torque Teno Virus Type 1(TTV1) And 2(TTV2) And Cloning And Analysis Of Complete Genome

According to the different genotypes and TTV1 (GU570198.1) and TTV2 (AY823991.1) of the sequence published in GeneBank, the two pairs of primers were designed to detect TTV1 and TTV2 by nested PCR method. Several blood samples of pigs were collected to extract viral DNA and amplify TTV1 and TTV2 specific target genes by nested PCR. The suspected fragments were recycled, cloned and sequenced to determine the TTV1 and TTV2 virus. Then the positive TTV DNA was used as a template to optimize nested PCR reaction conditions. The methods were verified through repetition, sensitivity and specificity of tests. Finally, the optimized PCR detection methods of TTV 1 and TTV2 were used to detect the 127 serum samples collected from Sichuan. The results show that the nested PCR detection methods established were favourable in repeatability, sensitivity and specificity. So the methods were suitable for large-scale clinical testing.According to the published genomic sequence of TTV1 and TTV2 (GeneBank accession number AB076001.K GU456386.1),the pairs of specific PCR/hested-PCR primers were designed to amplify the full length of viral genome from blood serum samples of porcine infected TTV1 and TTV2 in Sichuan province. The fragment amplified was cloned,sequenced and spliced.Then, the homology and evolutional relationship of TTV1/TTV2 complete genome and reference strain sequences were analyzed by Blasts DNAStar and Vector NTI advance 10 software. The result of homology analysis showed that the complete genome of TTV1 were 2852bp and 2917bp in length,as well as complete genome of TTV2 were 2802 bp and 2798bp in length. TTV1-SCK TTV1-SC2's genome of Sichuan isolated strains shared 81%-95%and 82%-94%homology with reference sequences respectively,and TTV2-SC1,TTV2-SC2's genome of Sichuan isolated strains shared 88%-99% and 80%-96% homology with reference sequences respectively.The phylogenetic tree analysis showed that the closest genetic relationship of TTV1-SC1 and TTV1-SC2 isolated strains were TTV1Bj10 strain (HM633251.1) and TTVSH0822/2008 strain (GU450331.1),as well as the closest genetic relationship of TTV2-SC1 and TTV-SC2 isolated strains and PTTV2c-VA strain (GU456386.1).According to analysis antigenicity, hydrophobicity and secondary structure of TTV-ORF1 protein, we speculated the epitopes of TTV1-SC1-ORF1 concentrated in the three areas of 155aa-173aa,409aa-423aa and 470aa-497aa and the epitopes of TTV1-SC2-ORF1 concentrated during the three areas of 46aa-62aa,169aa-175aa and 321aa-337aa. The two strains of TTV2 were highly homologous of primary structure, and the differences of amino acid did not affect the antigenicity of the proteins.We hypothesized that the epitopes of TTV2-SC1-ORF1 and TTV2-SC2-ORF1 concentrated in the four areas of 268aa-279aa, 379aa-390aa,505aa-51 laaand517aa-533aa. The further experiment were needed to prove the correctness of antigenic sites we predicted.The study would play a biological foundation to understand the morphology structure and genie function of porcine torque tero virus.