环介导等温扩增联合横向流动试纸条快速检测创伤弧菌检测方法的建立.docxVIP

·技术与方法·生物技术通报BIOTECHNOLOGYBULLETIN2014年第6期环介导等温扩增联合横向流动试纸条快速检测创伤弧菌 检测方法的建立王耀焕王瑞娜周前进陈炯（宁波大学生物化学与分子生物学实验室，宁波 315211）摘 要 ： 环介导等温扩增技术（LAMP）与横向流动试纸条（LFD）检测联合应用，建立了一种新的快速、便捷的创伤弧菌 检测方法。针对创伤弧菌的外膜蛋白 TolC 基因设计 6 条特异性引物和 1 条异硫氰酸荧光素（FITC）标记的探针。生物素标记的 LAMP 扩增产物能够特异性地与 FITC 标记的探针杂交，杂交产物经 LFD 检测。优化后的扩增温度和时间为 63℃反应 35 min，加 上细菌基因组 DNA 提取步骤，完成检测仅需要 80 min。LAMP-LFD 方法可特异性地检出创伤弧菌，对哈维氏弧菌等 9 种水产品常 见病原菌的检测均呈阴性 ；对纯细菌培养物的检测灵敏度为 3.7×102 CFU/mL 或 7.4 CFU/ 反应，是利用外引物建立的常规 PCR 检 测的 100 倍。结果表明，该方法能够准确、快速、灵敏地检出创伤弧菌，可应用于创伤弧菌污染的水产品的检测。关键词 ： 创伤弧菌 外膜蛋白 TolC 基因 环介导等温扩增技术 横向流动试纸条检测Rapid Detection of Vibrio vulnificus by Loop-mediated Isothermal Amplification Combined with Lateral Flow Dipstick AssayWang Yaohuan Wang Ruina Zhou Qianjin Chen Jiong（Laboratory of Biochemistry and Molecular Biology，Ningbo University，Ningbo 315211）Abstract: A novel and rapid loop-mediated isothermal amplification（LAMP）combined with chromatographic lateral flow dipstick（LFD） assay was developed to detect Vibrio vulnificus. A set of six primers and a fluorescein isothiocyanate（FITC）-labeled probe that recognized V. vulnificus outer membrane protein TolC gene were designed. Biotinylated LAMP amplicons were hybridized exclusively with the FITC-labeled probe and detected by LFD assay. The assay was optimized and could detect V. vulnificus by incubation at 63℃ for only 35 min, and the whole detection procedure from extraction of bacterial genomic DNA to the visualization of the amplicons by LFD last 80 min. V. vulnificus could be accurately detected by LAMP-LFD, and no amplification could be observed when another 9 bacterial genomic DNA were used. The sensitivity forV. vulnificus detection in pure culture was 3.7×102 CFU/mL or equivalent to 7.4 CFU per reaction, which is 100 times higher than that of PCR assay. The results indicate that LAMP-LFD is an accurate, rapid and sensitive tool for V. vulnificus detection and can be used for detection of V. vulnificus in contaminated aquatic foods.Key words: Vibrio vulnificus Outer mem