霍乱弧菌分子分型和溯源技术ISSR-PCR的改进  

Technical improvement of ISSR-PCR to genotype and genetically trace Vibrio cholerae

作　　者：莫秋华[1] 谭华[1] 王琪[1] 安胜利[2] 冯子力[1] 伍碧梅[1] 汪海波[1] 林继灿[1] 杨泽[1] 

机构地区：[1]珠海出入境检验检疫局国际旅行卫生保健中心,广东珠海519020 [2]南方医科大学公共卫生与热带医学学院生物统计学系

出　　处：《中国病原生物学杂志》2014年第9期795-799,共5页Journal of Pathogen Biology

基　　金：国家质检总局科技计划项目(No.2009IK206)

摘　　要：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 modify and optimize an inter-simple sequence repeat-polymerase chain reaction（ISSR-PCR）technique ein order to establish a modified method to molecularly type and genetically trace Vibrio cholerae. MethodsFifteen primers were designed and screened while various PCR parameters,including the concentration of Mg^2＋,dNTPs,and primers,were optimized step by step.Seventy-five strains of V.cholerae were then selected as samples for molecular typing. Results Screening indicated that the primer ISSR_GA5was the most effective at typing V.cholerae.An optimized PCR system had 2.0mmol/L of 2＋,0.3mmol/L of dNTPs and 1.2μmol/L of primer.This optimized ISSR-PCR assay generated quality polymorphic fingerprints with a single round of PCR and it accurately distinguished toxigenic and non-toxigenic strains,O1/O139 and non-O1/non-O139 serogroup strains,and local and foreign strains. ConclusionThe modified ISSR-PCR assay has improved ability to type samples and may provide a novel,simple,and practical technique for control and tracing of infectious diseases such as cholera at border health and quarantine stations and disease control departments.

关 键 词：霍乱弧菌 简单序列重复区间聚合酶链反应 分子分型 遗传溯源 

分 类 号：R378.3[医药卫生—病原生物学]