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目的对简单序列重复区间聚合酶链反应(ISSR-PCR)进行改进和优化,以建立一种改进型霍乱弧菌分子分型和遗传溯源技术。方法通过设计、筛选优良扩增引物和优化反应体系对ISSR-PCR进行技术改进,并选择75株霍乱弧菌作为方法学的分型测试样品进行实验验证。结果经筛选以引物ISSR_GA5分型效果最佳。对ISSR-PCR实验条件进行优化,确定最佳反应体系:Mg2+浓度为2.0mmol/L,dNTPs浓度为0.3mmol/L、引物浓度为1.2μmol/L。采用改进型ISSR-PCR技术能正确区分霍乱弧菌菌株中的产毒株(流行株)和非产毒株(非流行株)、O1群/O139群和非O1/非O139群以及本地株和外地株。结论建立的改进型ISSR-PCR技术分型效率高,为我国卫生检疫和疾控部门开展霍乱等重要传染病的防控和疫情溯源提供了新的、简便实用的技术方法。
Objective To improve and optimize Simple Sequence Repeat Polymerase Chain Reaction (ISSR-PCR) to establish an improved molecular typing and genetic tracing of Vibrio cholerae. Methods The technique of ISSR-PCR was optimized by designing and screening excellent amplification primers and optimizing the reaction system. 75 strains of V. cholerae were selected as the methodological typing test samples for experimental verification. The results of screening by the primer ISSR_GA5 best typing. Optimize the experimental conditions of ISSR-PCR to determine the best reaction system: Mg2 + concentration of 2.0mmol / L, dNTPs concentration of 0.3mmol / L, primer concentration of 1.2μmol / L. The improved ISSR-PCR technique can correctly distinguish the virulent and non-virulent strains (non-epidemic strains), O1 / O139 and non-O1 / non-O139 and local strains in V. cholerae strains and Foreign strains. Conclusion The improved ISSR-PCR technique has high typing efficiency, which provides a new, simple and practical technical method for the prevention, control and epidemic tracking of important infectious diseases such as cholera in China’s health quarantine and disease control departments.