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  

Rapid Detection of Vibrio vulnificus by Loop-mediated Isothermal Amplification Combined with Lateral Flow Dipstick Assay

作　　者：王耀焕 王瑞娜[1] 周前进[1] 陈炯[1] 

机构地区：[1]宁波大学生物化学与分子生物学实验室,宁波315211

出　　处：《生物技术通报》2014年第6期81-87,共7页Biotechnology Bulletin

基　　金：国家高技术研究发展计划("863"计划)(2012AA020101;2012AA092001);宁波大学学科项目(xk11341)

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。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 for V. 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.

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 

分 类 号：Q93[生物学—微生物学]