基于随机扩增多态性DNA分析的霍乱弧菌分子分型方法的建立

   莫秋华1，谭华1，安胜利2，杜坚1，林继灿1，刘志明1，王琪1，涂承宁1，叶立青1，杨泽1

    1.珠海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

    摘要：目的 建立基于随机扩增多态性DNA分析（Randomly amplifiedpolymorphic DNA,RAPD）的霍乱弧菌简便快速分子分型方法。方法　选择16株霍乱弧菌代表性菌株，包括O1群、O139群和非O1/非O139群等血清群，通过40条随机引物的RAPD-PCR实验，筛选出理想的随机引物，要求其产生的DNA指纹图谱多态性高，并有足够的分辨力进行分子分型。结果　通过一系列RAPD-PCR实验和统计学聚类分析，从40条随机引物中筛选出2条最符合要求的引物。16株霍乱弧菌的分析结果显示，2条引物对于O1群和O139群产毒株、O1群非产毒株和非O1/非O139群均有很好的聚类分辨率。结论　本研究筛选获得了2条随机引物并建立了基于RAPD的分子分型方法，该方法简便、省时、省力，适用于地方疾控部门和口岸检疫部门的基层实验室开展霍乱监测和流行病学溯源工作。

    关键词：霍乱弧菌;随机引物;随机扩增多态性分析

    中图分类号：R378.3　文献标识码：B

    Establishment of molecular typing method forvibrio cholerae based on randomly amplified polymorphic DNAanalysis

    MO Qiu-hua*, TAN Hua, 'AN Sheng-li, DU Jian,LIN Ji-can, LIU Zhi-ming,

    WANG Qi, TU Cheng-ning, YE Li-qing, YANGZe

    *Zhuhai International Travel Health CareCenter, Zhuhai, Guangdong 519020, China

    Abstract:   Objective  To establish a simple and rapid molecular typing method for vibriocholerae based on randomly amplified polymorphic DNA (RAPD)analysis.   Methods   A total of 16representative strains of V. cholerae, including members of the O1,O139, and non-O1/non O139 serogroups, and a total of 40 randomprimers were used to develop the RAPD-PCR experiments, in order toscreen out the ideal primers by which the obtained DNAfingerprinting patterns must have high polymorphism and sufficientresolving power in molecular typing.  Results   After a series of RAPD-PCR experiments combinedstatistical clustering analysis, two most satisfactory primers wereselected from the 40 random primers. A finer resolution of theclustering of the toxigenic O1/O139, non-toxigenic O1 andnon-O1/non O139 subtypes were obtained by using these two primersin RAPD-PCR for the 16 strains of V. cholerae.  Conclusion    In this study, we screened out twoideal random primers and established the corresponding RAPD-basedmolecular typing method. This method is easy, timesaving andlabor-saving, especially suitable for the basic laboratories of CDCand port quarantine departments to perform surveillance andepidemiological traceability of cholera.

    Key words:   Vibrio cholerae;Random primer; Randomly amplified polymorphic DNA

    《中国国境卫生检疫杂志》