| Transfusion transmitted virus was also known as Torque teno virus(TTV). Torque teno sus virus(TTSuV) is a member of Anelloviridae, genus Iotatorquevirus and Kappatorquevirus which was confirmed in pigs by American scholars in 1999, and in the following years, infections were worldwide. At present, there are no evidences to confirm that TTSu V could directly lead to swine disease, but it may take synergy pathogenic reation with some certain diseases. Considering its extensive epidemic and potential menace, the diagnosis methods to detect TTSu V2 were established in this study, including indirect ELISA and LAMP. 1. Analysis of open reading frame 1 gene of TTSu V2According to the published sequences of TTSu V2 in GenBank, a pair of primers were designed to amplify the full length of open reading frame 1(ORF1) gene of TTSu V2 by PCR method. Two fragments amplified in this study. Whose nucleotide with TTV2Hn93(JQ664305.1)were 97.2% and 97.7% respectively. The evolutional relationship of ORF1 genome of TTSu V2 were analyzed by MEGA 6.0 and DNA star software. The phylogenetic tree analysis showed that TTSu V2 had four subbranches and the homology of each branches were between 53.6% and 82.2%. The China isolates distributed in four branches which meant four subbranches of TTSu V2 were endemic in China. 2. Prokaryotic expression of truncated gene of ORF1 of TTSu V2Based on the analysis of ORF1 gene of TTSu V2, the truncated gene was amplified by PCR and cloned into the expression vector pcoldⅠto construct the recombinant expression plasmid pcold-TTSuV2-ORF1. Pcold-TTSu V2-ORF1 was transformed into BL21(DE3) cells, and the fusion protein with his-tag was expressed after induced with a concentration of 0.5 m M IPTG for 24 h.The expressed protein was approximated 34 KDa and existed in soluble form.Western blot analysis indicated that the purified protein had good reactogenicity, it could provide a antigen foundation for ELISA. 3. The establishment of indirect ELISA method for TTSuV2 detectionTo establish an indirect ELISA method for detecting TTSu V2, The purified peotein was used as coating antigen, and the dilution and reacation time of serum and HRP labeled rabbit anti pig IgG, the type and reacation time of blocking liquid were optimized respectively. The optimized reaction conditions as follows: the best blocking liquid was 3% neonatal bovine serum, blocking 2 h. The optimal dilution of serum and HRP labeled rabbit anti pig IgG and were 1:400 and 1:4000, which being incubated 45 min and 1 h. Determined by statistical analysis of critical value was 0.182.The specificity and repeatability of this assay was proved in specific and repeated tests. 84 serum samples from three farms were tested and the positive rate were 72%(13/18)、57.5%(19/33)、36.3%(12/33)respectively. The ELISA assay establishment in this study has the capability to detect TTSu V2 antibody clinically. 4. The establishment of LAMP method for TTSu V2 detectionTo establish an LAMP method for detecting TTSu V2, the untranslated region gene and front-end gene of ORF1 was picked for the LAMP assay as the target sequence, and a pair of primers were designed by using biological software website. The results showed optimized reacation conditions of LAMP for detecting TTSuV2 were in a constant temperature at 64℃ for 90 min. The method was able to detect TTSuV2 specificity,which could achieve a detection limit of 100 copies/μL DNA molecules, no cross-reation with other swine diseases. The LAMP assay established in this study which has a characteristic of rapid, sensitivity, specificity is expected to a major mean for detecting TTSu V2. |
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