海水中副溶血弧菌的可视化LAMP快速检测方法  

Rapid detection method of Vibrio parahaemolyticus in seawater based on visual LAMP

作　　者：黄梦诗 杨倩倩[1,2] 张艳[2] 谷阳光[3] 赵俊[2] 张旭志[2] 丁东生[2] 曲克明[2] 

机构地区：[1]上海海洋大学海洋科学学院,上海201306 [2]中国水产科学研究院黄海水产研究所青岛海洋科学与技术国家实验室,青岛266071 [3]中国水产科学研究院南海水产研究所,广州510300

出　　处：《中国渔业质量与标准》2017年第6期58-65,共8页Chinese Fishery Quality and Standards

基　　金：中国水产科学研究院基本科研业务费(2016RC-BR02);黄海水产研究所基本科研业务费(20603022016003);青岛海洋科学与技术国家实验室鳌山科技创新计划项目(2015ASKJ02);山东省重点研发计划项目(2016GSF120008)

摘　　要：副溶血弧菌(Vibrio parahaemolyticus)是沿海地区常见的食源性致病菌之一,评估其在海洋环境中的存在状况对渔业生产与环境监测具有重要意义,但传统方法操作复杂或者设备繁重,往往不适合于现场快速检测。本实验采用环介导等温基因扩增法(loop-mediated isothermal amplification,LAMP),以tlh基因作为其标识基因,建立了海水中该菌的可视化快速检测方法。采用过滤法收集海水中的细菌,以细菌悬液为扩增模板,直接加入LAMP反应液中(免DNA提取与纯化步骤),以羟基萘酚蓝(hydroxynaphthol blue,HNB)为扩增结果指示剂,通过肉眼观察LAMP反应液颜色变化判断阴性、阳性结果。优化条件下,该方法反应约1 h可获得的最低检测限为167 CFU/m L。较之于传统PCR方法,所建立的方法分析周期短(从开始预处理样品到得出检测结果只需~2 h);灵敏度高(~10×PCR)。在验证实验中,应用该方法对青岛近岸海水中副溶血弧菌的检测,所获结果与经典培养方法和PCR方法结果一致。结果表明:该方法使用仪器简单,响应快,操作简易,可为现场快检海水中副溶血弧菌提供技术支持。Vibrio parahaemolyticus （ V. parahaemolyticus） is one of the major foodborne pathogenic bacteria in coastal areas. The evaluation of the current status of this kind of bacteria is extremely significant for both aquaculture and marine environment management. However, the traditional detection methods are unqualified for providing timely detection due to the slow response and expensive equipment. Herein a rapid method was constructed based on a visual loop -mediated isothermal amplification （LAMP） technology to detect V. parahaemolyticus, which has highly conserved genes of tlh. Briefly, bacteria in seawater samples were collected with filtration, the collected bacteria were subsequently used as reaction templates to perform LAMP, without the step of DNA extraction. Positive or negative results were identified finally by the color change of hydroxy naphthol blue with naked eyes. Under the optimized conditions, the detection limit was 167 CFU/mL （ - 1 h）. In comparison with traditional PCR methods, the visual LAMP pro- posed here has the advantage of distinguish efficiency. Including the consumption of sample pretreatment, the whole performance of detection took 2 h. Meanwhile, its sensitivity is about 10 - fold higher than PCR＇ s. The visual LAMP method was also applied to detect V. parahaemolyticus in real seawater samples collected from Qingdao coastal area in this study. The results were in good agreement with those obtained with classical culture method and PCR method. Without the involvement of bulky instrumentation, plus the rapid response and simple operation, this proposed method is favorable to use on - site surveys of target marine microorganisms.

关 键 词：副溶血弧菌 海水 LAMP 免DNA提取 快速检测 

分 类 号：S917.1[农业科学—水产科学]