副溶血弧菌tdh基因LAMP检测技术的建立  

Establishment of Loop-Mediated Isothermal Amplification for Detecting Vibrio Parahaemolyticus tdh Gene

作　　者：程晓艳[1,2] 刘庆慧[1] 黄倢[1] 

机构地区：[1]农业部海洋渔业资源可持续利用重点开放实验室中国水产科学院黄海水产研究所,山东青岛266071 [2]中国海洋大学食品科学与工程学院,山东青岛266003

出　　处：《中国食品学报》2012年第8期156-162,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(30871942);农业部对虾行业专项(201103034)

摘　　要：环等温扩增技术(Loop-mediated isothermal amplification,LAMP)是一种在等温条件下高特异性、高效、快速地扩增靶基因的DNA扩增技术。根据副溶血弧菌特异性的毒力基因tdh保守序列,本文设计1套特异性引物对该基因进行环等温扩增,同时对反应条件进行优化,建立携带tdh基因的致病性副溶血弧菌的LAMP快速检测技术。结果表明,LAMP最适反应在60.8℃恒温、45min内完成,与其它常见的细菌无交叉反应。LAMP方法的细菌DNA最低检出限为35.5fg/反应管,灵敏度较PCR方法高10倍。对扇贝样品中分离的菌株的检测表明,LAMP方法对检测致病性副溶血弧菌具有良好的可靠性。Loop-mediated isothermal amplification（LAMP） is a novel DNA amplification technology which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. In this paper, LAMP was applied for rapid detection of Vibrio parahaemolyticus. According to the sequence of the specific tdh gene of Vibrio parahaemolyticus, a set of primers was designed to amplify the specifial DNA sequences by LAMP. Moreover, the reaction conditions were optimized. The results showed the optimum LAMP assay for the rapid detection of Vibrio parahaemolyticus was performed at 60.8 ℃ for 45 min. No crossreaction was found with other common bacteria which showed a good specificity. The LAMP assay had an detection limit of 35.5 fg/tube, which was 10 times lower than that of PCR assay. The LAMP assay could be finished within an hour requiring only a regular laboratory water bath for reaction. The detection of the strains isolated from scallop also proved the LAMP assay reliability.

关 键 词：副溶血弧菌 环介导等温核酸扩增 耐热直接溶血毒素基因(tdh) 快速检测 

分 类 号：TS207.4[轻工技术与工程—食品科学]