基于多位点探针熔解曲线的霍乱弧菌快速分型方法研究  

Development of a rapid molecular typing method for Vibrio cholerae using melting curve-based multilocus melt typing

作　　者：黄仕杰[1] 刘赞赞 温慧欣[1] 李莉[1] 李庆阁[2] 黄建炜[1] 

机构地区：[1]厦门市疾病预防控制中心检验科,361021 [2]厦门大学生命科学学院

出　　处：《中华预防医学杂志》2015年第2期122-127,共6页Chinese Journal of Preventive Medicine

基　　金：福建省科技计划重点项目（2008Y0301）

摘　　要：目的研究基于探针熔解曲线分析的多位点熔解分型技术（McMLMT）的霍乱弧菌快速分型方法。方法选取厦门市CDC于1998-2010年霍乱病原监测中从各种水体和水产品等监测样品中分离的霍乱弧菌分离株（ctx-）作为试验菌株，共108株。筛选霍乱弧菌7个管家基因，针对每个基因设计特异引物和覆盖多态性位点的4条荧光探针，建立各基因的双管双色PCR熔解曲线分析（PCR—MCA）检测体系，检测108株试验菌株，获取28个熔点值（Tm值），转化为等位基因编码并确定型别。同时对7个管家基因的多态性区域序列参照多位点序列分型方法（MLST法）进行分型。两种方法的分型数据以BioNumerics软件进行聚类分析比较，并与其中部分菌株PFGE分型数据比较，评估McMLMT的分辨能力和可靠性。结果构建并优化了霍乱弧菌McMLMT分型体系。应用McMLMT分型体系检测霍乱弧菌，108株霍乱弧菌的28个Tm值按管家基因分组并依顺序编码后，可分为18型（D=0．7233）；同时参照MLST方法直接以7个基因的多态性区域序列进行分型，分型结果与McMLMT一致；其中46株PFGE型别不同的菌株，McMLMT法可分为13个熔解类型（D=0．6145），PFGE法可分为A～K的11群（相似度为85％，D=0．8589）。结论采用McMLMT分型方法可以对批量菌株进行快速分型，分辨力与MLST法相同，与PFGE法在相似度为85％分群的分辨力相似。Objective To develop a high-throughput rapid method for Vibrio（ V. ） cholerae molecular typing based on Melting Curve-based Muhiloeus Melt Typing （McMLMT）. Methods Seven housekeeping genes of V. cholerae were screened out, and for each gene, the specific primers were designed for correspondent genes as well as 4 probes covering polymorphism loci of sequences. After optimizing all parameters, a method of mefting-curve analysis following asymmetric PCR was established with dualfluorescent-reporter in two reaction tubes for each gene. A set of 28 Tm-values was obtained for each strain and then translated into a set of code of allelie genes, standing for the strain＇s MeMLMT type （MT）. Meanwhile, sequences of the 7-locus polymorphism were typed according to the method of MLST. To evaluate the efficiency and reliability of McMLMT, the data were compared with that of sequence-typing and PFGE using BioNumeries software. Results MeMLMT method was established and refined for rapid typing of V. cholerae that a dozen of strains can be finished testing in a 3-hours PCR running using 96-well plates. 108 strains were analyzed and 28-Tm-values could be grouped and encoded according to 7 housekeeping gene to obtain the code set of allelie genes,and classified into 18 types （D = 0. 723 3）. Sequences of the 7 genes＇ polymorphism areas were directly clustered into the same 18 types with reference to MLST method. 46 of the strains, each represented a different PFGE type, could be classified into 13 types （D = 0. 614 5） with McMLMT method and A - K groups at 85% similarity （ D = 0. 858 9 ） with PFGE method. Conclusion McMLMT method is a rapid high-throughput molecular typing method for batches of strains with a resolution equal to MLST method and comparable to PFGE group.

关 键 词：弧菌 霍乱 细菌分型技术 聚类分析 

分 类 号：R378.3[医药卫生—病原生物学] R440[医药卫生—基础医学]