학술논문
蓝舌病新疆分离株VP7蛋白编码基因序列分析及一步法RT-PCR方法的建立 / Sequence analysis and one-step RT-PCR establishment of VP7 protein coding gene of bluetongue isolated in Xinjiang
Document Type
Academic Journal
Author
Source
新疆农业科学 / Xinjiang Agricultural Sciences. 61(1):253-259
Subject
Language
Chinese
ISSN
1001-4330
Abstract
[目的]分析蓝舌病新疆分离株编码VP7 蛋白S7 基因保守序列,建立蓝舌病新疆分离株一步法RT-PCR方法,为新疆BTV分子流行病学调查及防控提供技术支持.[方法]采用二代测序技术获得新疆分离株S7 基因序列,登陆GenBank对序列进行同源性Blast比对,用软件MEGA 5.0 分析序列差异,根据蓝舌病新疆分离株编码VP7 蛋白S7 基因保守序列,运用软件Oligo6.0 设计引物,对蓝舌病新疆分离株S7 基因进行RT-PCR扩增,并验证该方法的特异性及敏感性.[结果]蓝舌病中国新疆分离株编码VP7 蛋白S7 基因序列与哈尔滨报道BTV分离株S7 基因片段相似度较高为 89.64％,与中国云南报道BTV-29 型分离株、蒙古国BTV分离株S7 基因片段相似度分别为87.49％和87.39％;与德国BTV分离株S7 基因片段相似度为80.70％,其余均无同源性序列.中国新疆分离株S7 基因片段序列与4 条同源序列间存在26 处核苷酸差异,9 处氨基酸差异.[结论]建立的蓝舌病中国新疆分离株S7 基因片段一步法RT-PCR方法具有良好的特异性,仅BTV中国新疆分离株扩增获得目的条带,该方法的敏感性为 4.0×103copies/mL.
[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.
[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.