环介导恒温扩增技术快速检测溶藻弧菌  

ESTABLISHMENT OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF Vibrio alginolyticus

作　　者：丁文超[1] 胡健饶[2] 史雨红[1] 李明云[1] 陈炯[1] 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,宁波315211 [2]杭州师范大学生命与环境科学学院,杭州310036

出　　处：《分子细胞生物学报》2009年第1期70-76,共7页Journal of Molecular Cell Biology

基　　金：长江学者和创新团队发展计划(IRT0734);国家科技支撑计划(2007BAD43B08);浙江省科技厅重点项目(2008C22049);宁波市科技局项目(2007C10081)~~

摘　　要：溶藻弧菌是中国南部水产养殖业中弧菌病的最主要病原菌,其快速检测具有重要意义。根据溶藻弧菌外膜蛋白OmpK基因序列,设计一套引物,通过条件优化,成功建立了针对致病性溶藻弧菌的环介导恒温扩增检测技术(loop-mediated isothermal amplification,LAMP)。应用LAMP技术,在65℃温育1h的条件下扩增溶藻弧菌基因组DNA,琼脂糖凝胶电泳得到特异性梯度条带。该研究建立的LAMP法特异性检出致病性溶藻弧菌,其检测下限比PCR法低一个数量级,相当于n(cell)=38/mL的菌液浓度,灵敏度更高。综合分析表明,LAMP技术是快速、简易、实地诊断溶藻弧菌的理想工具。Vibrio alginolyticus is the main Vibrio pathogen in aquaculture in the south of China. A one step loop-mediated isothermal amplification （LAMP） assay was developed for detection of Vibrio alginolyticus. A set of primers were designed from the OmpK sequence of Vibrio alginolyticus. The assay was optimised to amplify Vibrio alginolyticus DNA by incubation at 65℃ for only 1 h, and required only a simple water bath or heating block to provide a constant temperature of 65℃. LAMP amplification products had a ladder-like appearance when electrophoresed on an agarose gel. The detection limit of the LAMP assay was n（cell）=38/mL which was found to be higher than the commonly used PCR method. The assay was evaluated using clinical samples and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for V ibrio alginolyticus.

关 键 词：溶藻弧菌 OmpK基因 环介导恒温基因扩增检测法 

分 类 号：S941[农业科学—水产养殖]