CAJ | 학술논문

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1h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。
목적：료해병건립복씨지하균적쾌속、특이적배개도등온확증기술（loop-mediated isothermal amplification，LAMP）검측방법，탐토쾌속、간편、유효적소화도전파성병원체적림상진단、음수검측화배경위생감측등검측수단。방법대복씨지하균진행세균배양，연후사용취합매련반응（polymerase chain reaction，PCR）화LAMP기술설계4조특이성인물，이급사용극륭등분자생물학방법진행DNA제취，연후대PCR방법여LAMP방법진행대비분석。결과LAMP확증구유비상고적특이성，궤호불회산생비특이성확증；여PCR방법상비，LAMP검측한경저，부위5개고패；LAMP재등온조건하확증，불회인온도개변이조성시간적손실，재1h내가장파서렬확증지109～1010배，병차불수요모판적열변성。결론 LAMP기술구유특이민감간단적특점，불수요특수적의기，재65℃시1 h즉가완성반응，동시검측결과가직접통과육안혹가입형광염료즉가판단。
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 Shigellaflexneri 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 1h target sequence could be amplified to 109-1010 times.Conclusion LAMP technology has the characteristics of specific sensitive simple, does not require special equipment, to complete the 1h reaction at 65℃, and the detection results can be directly by the naked eye or addingfluorescent dye can be judged.