Sequence analysis and one-step RT-PCR establishment of VP7 protein coding gene of bluetongue isolated in Xinjiang
[Objective]According to conserved sequence of VP7 protein coding S7 gene of bluetongue i-solated in Xinjiang,a one-step RT-PCR method was established,the results provides technical support for molecular epidemiological investigation and prevention of BTV in Xinjiang.[Methods]The S7 gene sequence which was obtained by second-generation sequencing,were compared the homology by Blast in GenBank,and analyzed the difference by Mega 5.0.Primers were designed according to conserved sequence of S7 gene enco-ding VP7 protein of bluetongue isolated in Xinjiang by Oligo 6.0.One-step RT-PCR method was estab-lished,then specificity and sensitivity was verified.[Results]Sequence comparison results showed that S7 gene of bluetongue isolated in Xinjiang has highest homology with BTV isolated fragment reported by Harbin,it was 89.64％.The sequence similarity between S7 gene of bluetongue isolated in Xinjiang and other fragment including BTV-29 reported by Yunnan,BTV isolated from Mongolia and German were87.49％,87.39％and 80.70％respectively.Excpt those squences,there was no more homologous sequences.The results showed that there were 26 nucleotide variation and 9 amino acid variation between the S7 gene fragment sequence of Xin-jiang isolate and 4 homologous sequences.[Conclusion]One-step RT-PCR was able to specifically detect S7 gene of bluetongue isolated in Xinjiang,simultaneously with the detection limit of 4.0×103copies/mL.But no genomic RNA or DNA were amplified from BVDV and IBRV.
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