摘要
目的建立一种适合基层检验部门及小型实验室使用的快速检测O139群霍乱弧菌的方法。方法应用环介导等温扩增技术（100p-mediated isothermal amplification，LAMP），针对O139霍乱弧菌wbfR基因设计4条引物（2条内引物、2条外引物）；优化LAMP反应条件和反应体系，对反应体系中的引物、dNTP、Mg^2＋／Mn^2＋离子及Calcium等浓度进行优化；并对13株种系背景明确的霍乱弧菌不同实验对照株、30株0139群霍乱弧菌地方分离株、10株01群霍乱弧菌地方分离株、32株其他肠道菌进行检测，验证该方法的特异性；通过肉眼目测或电泳检测比较结果。结果所有0139群霍乱弧菌经LAMP检测均呈绿色并电泳有阶梯状条带为阳性，01群霍乱弧菌及其他肠道菌均检测呈橙色并电泳无相应条带为阴性；该体系最低检测限为63CFU／反应；检测结果在白光下通过肉眼即可判断；从菌株核酸的提取至检测完成仅需1．5h左右。结论本研究建立的LAMP方法能够快速、灵敏、特异地检测O139群霍乱弧菌，无需昂贵的仪器，简单方便，非常适合基层检验部门或小型实验室以及流行病学人员于应急车上或现场监测等使用，值得推广。
Objective To develop a loop-mediated isothermal amplification method for rapid diagnosing Vibrio cholerae O139 in inspection department and small-scale laboratory. Methods Four primers （2 inner primer and 2 outer primer） for the LAMP test were designed by targeting the wbfR gene of Vibrio cholerae O139, and the reaction condition and reaction system of LAMP were optimized. Thirty Vibrio cholerae O139, 13 Vibrio cholerae reference strains, 10 O1 biotype Vibrio cholerae and 32 other enterobaeterias were analyzed and the LAMP results were determined by visual inspection or electrophoretic analysis . Results M1 of the Vibrio cholerae O139＇s amplification products were observed as green by visual inspection and had a ladder-like pattern on the gel, but O1 biotype Vibrio cholerae and other enterobacteria＇s products were displayed as orange by visual examination and had no ladder-like pattern on the gel. In addition, the reaction time of the LAMP method was only 1.5 h and the detection limit of this assay was 63 CFU/reaetion. Conclusion LAMP assay targeting the wbfR gene of Vibrio cholerae O139 is rapid, specific, and sensitive for the detection of Vibrio cholerae O139. This method not only reduced the dependence of complicated equipment but also had a potential for wider use in inspection department, small-scale laboratory, emergency motor vehicles and field survey.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2009年第6期564-567,共4页
Chinese Journal of Microbiology and Immunology
基金
断江省医药卫生科学研究基金资助项目（2008A029）
