CAJ | 학술논문

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 =0.723 3);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-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=0.614 5),PFGE法可分为A～K的11群(相似度为85％,D=0.858 9).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.
목적 연구기우탐침용해곡선분석적다위점용해분형기술(McMLMT)적곽란호균쾌속분형방법.방법 선취하문시CDC우1998-2010년곽란병원감측중종각충수체화수산품등감측양품중분리적곽란호균분리주(ctx-)작위시험균주,공108주.사선곽란호균7개관가기인,침대매개기인설계특이인물화복개다태성위점적4조형광탐침,건립각기인적쌍관쌍색PCR용해곡선분석(PCR-MCA)검측체계,검측108주시험균주,획취28개용점치(Tm치),전화위등위기인편마병학정형별.동시대7개관가기인적다태성구역서렬삼조다위점서렬분형방법(MLST법)진행분형.량충방법적분형수거이BioNumerics연건진행취류분석비교,병여기중부분균주PFGE분형수거비교,평고McMLMT적분변능력화가고성.결과 구건병우화료곽란호균McMLMT분형체계.응용McMLMT분형체계검측곽란호균,108주곽란호균적28개Tm치안관가기인분조병의순서편마후,가분위18형(D =0.723 3);동시삼조MLST방법직접이7개기인적다태성구역서렬진행분형,분형결과여McMLMT-치;기중46주PFGE형별불동적균주,McMLMT법가분위13개용해류형(D=0.614 5),PFGE법가분위A～K적11군(상사도위85％,D=0.858 9).결론 채용McMLMT분형방법가이대비량균주진행쾌속분형,분변력여MLST법상동,여PFGE법재상사도위85％분군적분변력상사.
Objective To develop a high-throughput rapid method for Vibrio (V.)cholerae molecular typing based on Melting Curve-based Multilocus 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 melting-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 allelic genes,standing for the strain's McMLMT 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 BioNumerics software.Results McMLMT 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 allelic 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.