利用环介导等温核酸扩增技术(LAMP)快速检测霍乱弧菌

Rapid detection of Vibrio cholerae by Loop-Mediated Isothermal Amplification

目的:建立基于环介导等温核酸扩增技术(LAMP)的霍乱弧菌快速检测方法,为霍乱疫情监测与预防提供及时的应急检测服务和参考结果。方法:通过Genbank检索、下载霍乱弧菌ctxA基因序列,使用BLAST、GENtle、DNAMan等相关核酸序列分析软件比对分析确定目标序列,使用PrimerExplorer v4软件设计霍乱弧菌LAMP引物。以SYBR Green I作为荧光剂在荧光定量PCR仪上实时检测LAMP反应结果,作为本研究的主要研究方式。通过优化反应温度、时间、组成浓度等实验条件对LAMP检测方法进行改进,并与PCR方法进行比较与评价。结果:从分属不同种属的20株实验菌株中分别正确地检出所有5株产霍乱毒素的霍乱弧菌菌株,对菌株纯培养物的检出限达到了5 cfu/μl(10 cfu/reaction)。结论:本研究建立的霍乱弧菌LAMP检测方法是一种灵敏、特异、快速、简便的霍乱弧菌检测方法,适于基层单位推广使用,对霍乱疾病监测与暴发后疫情应急检测具有重要意义。

Objective：The aims of this study were to research Loop-Mediated Isothermal Amplification（LAMP）,a novel technology of nucleic acid amplification,so to establish a rapid,reliable and importantly easy-to-use detection method of V.cholerae,providing references and sevices to the early diagnosis of pathogen and disease survey of Cholera.Methods： The target sequences of the V.cholerae ctxA genes（available from Genbank） were analyzed and selected for LAMP usage by some softwares,BLAST,GENtle,DNAMan and so on.The LAMP primers were designed by special software,PrimerExplorer version 4.The LAMP assay for V.cholerae was developed on the realtime PCR cycler using fluorescent SYBR Green I for detection,improved through a series of optimization tests including temperature,time and component concentration of reaction,and compared with a ordinary PCR assays aiming to familiar target genes,ace,rtxA and ctxA of V.cholerae.Results： The LAMP assay correctly identified all 5 strains of CT-producing V.cholerae with ctxA primers,from 20 tested bacteria strains comprising 8 genera.The detection limits of the assay in pure cultrue were determined to be 5 cfu/μl（10 cfu per reaction） of V.cholerae cells.Conclusion： The LAMP assay is a more rapid,reliable,simple detection method of V.cholerae,worthy of popularization,with important significance to disease survey and emergency detection of Cholera.