32株大肠埃希菌核酸检测鉴定分析

Detection and identification of 32 Escherichia coli by nuclear acid amplification

目的:对本实验室保存多年的32株大肠埃希菌进行核酸检测鉴定,同时验证所用引物的特异性及改用改良方法的可行性。方法:应用环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术,参照最新LAMP改良方法,对32株大肠埃希菌及其它9株非大肠埃希菌实验对照株分别进行大肠埃希菌malB、不耐热性肠毒素I(heat labile Ienterotoxin,LTI)和耐热性肠毒素I(heat stable I enterotoxin,STI)基因检测。结果:32株大肠埃希菌均扩增出大肠埃希菌malB基因,其中3株均扩增出LTI和STI基因,14株只扩增出LTI基因,1株只扩增出STI基因。整个检测过程仅需1.5 h,可通过肉眼目测绿色钙锰复合物是否生成判断结果。结论:32株大肠埃希菌从基因水平均得到鉴定;试验再次证实参考文献中设计的引物其特异性好;改用改良LAMP方法目测结果直观可行,可免去电泳、拍照两步,具有更快速、简便、经济等特点,极适合基层实验室人员应用于对可疑大肠埃希菌的鉴定检测。

Objective：To validate the specificity of the primers and the feasibility of the improved loop-mediated isothermal amplification（LAMP） method,32 Escherichia coli strains stored in our lab were detected and identified by this method.Methods：According to the technique of LAMP,refering the last improved LAMP method Escherichia coli malB,LTI（heat labile I enterotoxin） and STI（heat stable I enterotoxin） were detected in 32 Escherichia coli strains and 9 non-Escherichia coli laboratory-control strains were.Results：The malB gene were amplified in all 32 Escherichia coli strains,the heat labile I enterotoxin（LTI）gene and the heat stable I enterotoxin（STI）gene were both amplified in 3 Escherichia coli strains.The heat labile I enterotoxin（LTI）gene were amplified in 14 strains while the heat stable I enterotoxin（STI）gene were amplified in 1 strain.The whole detection process only cost 1.5 h,and judging through whether the green Ca-Mn combined substance forming or not,the result can be observed directly by naked eyes.or agarose.Conclusion：32 Escherichia coli strains are identified in the genetic level.The tests confirms that the primers described in the references are specific again,and the improved method is feasible while the steps of electrophoresis and photograph could be cancelled.The method is rapid,simple,and economical,and it is very proper for basic laboratories to detect suspicious Escherichia coli.