LAMP技术用于志贺菌的检测  

Application of LAMP technology in the detection of Shigella

作　　者：陈传[1] 谢文熙[1] 凌霄[2] 郭震[1] 邵筱[1] 张如胜[3] 魏泉德[3] 

机构地区：[1]广东医学院病原生物学实验室,广东湛江524023 [2]广东医学院第二附属医院 [3]珠海市疾病预防控制中心微生物检验科广东珠海519000

出　　处：《现代预防医学》2014年第5期888-890,893,共4页Modern Preventive Medicine

基　　金：2011年度湛江市科技攻关计划项目(2011C3101005)

摘　　要：目的依据环介导的恒等温扩增技术,建立一种新型的快速、准确、简单并方便检测食物中志贺菌的方法,为全面防治志贺菌导致的食物中毒提供依据。方法本实验针志贺菌侵袭性质粒抗原ipaH基因的6个区域设计6条引物,利用链置换DNA聚合酶,在65℃左右的条件下反应60 min,短时间内快速完成核酸的扩增。反应结束后依据扩增副产物产生的焦磷酸镁沉淀的浊度,或使用SYBR Green I颜色反应,判断ipaH是否为阳性,同时通过,琼脂糖凝胶电泳检测判断扩增产物的大小。结果 13例标准菌中,只有痢疾志贺菌LAMP产物呈混浊,加入SYBR Green I显绿色且琼脂糖凝胶电泳有梯状亮带;痢疾志贺菌稀释106倍(约5 CFU/ml)之后仍然可以被LAMP检出而普通PCR检测不到。结论 LAMP技术可以快速、准确、特异性的检出志贺菌且方法简单、方便、灵敏度高于PCR,适合于室外现场使用。Objective According to the loop-mediated isothermal amplification （LAMP） technology, to establish a fast, accurate, simple detecting system can be used to find Shigella, to provide evidences for control food poisoning due to Shigella. Methods The sequences of six domains of common Shigella was used as target sequences of ipaH to design six special primers; used chain substi- tution DNA polymerase （Bst DNA polymerase） within sixty minutes at constant temperature （about 65 ~C） a nucleic acid amplification reaction can be finished so that a objective fragment can be obtained; ipaH was decided directly according to the turbidity of magne- sium pyrophosphate or SYBR Green I color reaction; meanwhile the size of amplication product was detected by AGE. Results In the 13 strains of bacteria, only Shigella dysenteriae product in LAMP was turbid, after adding SYBR Green I, it was green and with scalariform bright in AGE; with the 106 times dilution of Shigella dysenteriae （approximate 5CFU/ml）, it could be detected by LAMP rather than PCR. Conclusion Fast, accurate, specific LAMP technology for defection of Shigella has characteristics of simple, con- venient, sensitive, compared with PCR, which is suitable for being used outdoor.

关 键 词：环介导恒等温扩增技术 PCR 痢疾志贺菌 检测体系 

分 类 号：R115[医药卫生—公共卫生与预防医学]