木薯细菌性萎蔫病菌dnaA基因质粒标准样品的构建

Construction of dnaA gene standard sample of Xanthomonas axonopodis pv.manihotis

中文关键词:  木薯细菌性萎蔫病菌  dnaA基因  重组质粒  标准样品

英文关键词:Xanthomonas axonopodis pv.manihotis  dnaA gene  recombinant plasmid  standard sample

中文摘要:

      目的 构建木薯细菌性萎蔫病菌dnaA基因质粒标准样品。方法 以木薯细菌性萎蔫病菌染色体复制起始因子dnaA特异基因序列为靶标设计引物, 通过 PCR 扩增目的片段, 连接至pMD 18-T 载体, 转化大肠杆菌DH5α, 构建木薯细菌性萎蔫病菌dnaA基因片段的重组质粒。采用PicoGreen DNA分子荧光定量方法对该质粒分子进行定值, 对均匀性、稳定性及标准值进行研究, 制备木薯细菌性萎蔫病菌dnaA基因质粒标准样品。结果 结果表明, 木薯细菌性萎蔫病菌质粒标准样品定值为4.99 μg/mL, 可在?18~?20 ℃低温条件下长时间保存, 在夏季高温期间一周内也可实现传输。结论 该标准样品的研制解决了木薯细菌性萎蔫病菌的溯源参照物问题, 为快速检测木薯细菌性萎蔫病菌奠定了基础。

英文摘要:

      Objective To construct dnaA gene standard sample of Xanthomonas axonopodis pv.manihotis. Method One pair of primers were designed with chromosome replication initiation factor dnaA gene sequence as the target to detect Xanthomonas axonopodis pv.manihotis. The target segment was amplified by PCR and cloned into pMD 18-T vector, then transformed into Escherichia coli DH5α and constructed the recombinant plasmid. Then the value of recombinant plasmid was defined by PicoGreen DNA fluorescence quantitative method. Homogeneity and stability were studied, and the recombinant plasmid including dnaA gene standard sample of Xanthomonas axonopodis pv.manihotis was prepared. Results The standard sample was determined for 4.99 μg/mL. It could be stored at ?18~?20 ℃ for a long time, and also could be transmitted within 1 week at high temperature condition in summer. Conclusion Preparation of the standard sample provides a solution of tracing to the source of the reference, which provides a foundation for the rapid detection of Xanthomonas axonopodis pv.manihotis.