环介导等温扩增技术检测福氏志贺菌的实验研究  

作　　者：彭丁晋[1] 

机构地区：[1]邵阳医学高等专科学校微生物教研室,湖南422000

出　　处：《当代医学》2015年第5期1-3,共3页Contemporary Medicine

基　　金：湖南省教育厅课题(11C1163)

摘　　要：目的 了解并建立福氏志贺菌的快速、特异的环介导等温扩增技术（loop-mediated isothermal amplification,LAMP）检测方法,探讨快速、简便、有效的消化道传播性病原体的临床诊断、饮水检测和环境卫生监测等检测手段。方法 对福氏志贺菌进行细菌培养,然后使用聚合酶链反应（polymerase chain reaction,PCR）和LAMP技术设计4条特异性引物,以及使用克隆等分子生物学方法进行DNA提取,然后对PCR方法与LAMP方法进行对比分析。结果 LAMP扩增具有非常高的特异性,几乎不会产生非特异性扩增;与PCR方法相比,LAMP检测限更低,仅为5个拷贝;LAMP在等温条件下扩增,不会因温度改变而造成时间的损失,在1 h内可将靶序列扩增至109-1010倍,并且不需要模板的热变性。结论 LAMP技术具有特异敏感简单的特点,不需要特殊的仪器,在65℃时1 h即可完成反应,同时检测结果可直接通过肉眼或加入荧光染料即可判断。Objective To study on LAMP detection of Shigella flexner and investigate the fast, simple, effective gastrointestinal borne pathogens of clinical diagnosis, drinking water testing and environmental health monitoring and other detection methods. Methods Shigella flexneri were cultured, and then used the PCR and LAMP technology to design 4 primers, and the use of cloning and other molecular biology methods for DNA extraction, and then the PCR method and LAMP method were compared and analyzed.Results LAMP amplification had very high specificity, there was almost no nonspecific amplification; compared with the PCR method, the detection limit of LAMP was lower, only 5 copies; LAMP amplification under isothermal conditions, no time loss caused by temperature change, within the lh target sequence could be amplified to 109-1010 times. Gonclusion LAMP technology has the characteristics of specific sensitive simple, does not require special equipment, to complete the lh reaction at 65℃, and the detection results can be directly by the naked eye or adding fluorescent dye can be judged.

关 键 词：环介导等温扩增技术 福氏志贺菌 聚合酶链反应 检测 实验研究 

分 类 号：R440[医药卫生—诊断学]