环介导恒温扩增技术快速检测鼠疫耶尔森菌  

Loop-mediated isothermal amplification for quick detection of Yersinia pestis

作　　者：冯娜[1,2] 周亚洲[2] 范艳晓 汪琼[2] 毕玉晶[2] 韩延平[2] 杨瑞馥[2] 周育森[1,2] 王效义[1,2] 

机构地区：[1]安徽医科大学,合肥230032 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学》2015年第11期868-872,共5页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31430006,81171529);国家重点基础研究发展计划课题(2014CB744405)

摘　　要：目的建立简单、快速环介导恒温扩增技术(LAMP)检测鼠疫耶尔森菌的方法。方法基于鼠疫菌染色体上特异的核酸序列3a设计引物;应用浊度仪和目视法对反应结果进行检测;通过对鼠疫菌和其他近源菌同时进行检测评价方法的特异性;通过对不同稀释浓度的鼠疫菌DNA模板进行LAMP和PCR检测以确定方法的灵敏度。结果用建立的LAMP法对与鼠疫菌近源30种其他菌株进行扩增,结果均为阴性,该方法具有很高的特异性。LAMP法检测鼠疫菌DNA的灵敏度可达到20 pg,比常规PCR法高10倍。检测反应在25 min以内完成。结论该方法具有快速、灵敏、特异、操作简单的特点,有望发展成为现场快速检测鼠疫菌的有效手段。Objective To establish a simple and quick loop-mediated isothermal amplification （LAMP）method for detection of Yersinia pestis.Methods LAMP Primers were designed based on the specific sequence 3a in Y.pestis chromosome.LAMP reaction results were detected using turbidity meter or visual method.The specificity of the constructed method was evaluated by detecting Y.pestis and its closely-related bacteria.The different dilution DNA template was detected with LAMP and PCR to evaluate the sensitivity of the method.Results Thirty strains of bacteria closely related to Y.pestis were detected by the constructed LAMP,and all the results were negative,indicating that the method had a very high specificity.The detection sensitivity of this LAMP assay was 20 pg of DNA per reaction,which was ten-fold that of the regular PCR.The detection reaction was completed in 25 min.Conclusion This LAMP method is quick,sensitive, specific and simple,which is expecked to become an effective method for rapid detection of Y.pestis on the scene.

关 键 词：鼠疫耶尔森菌 环介导恒温扩增技术 检测 

分 类 号：R378.61[医药卫生—病原生物学]