水产品中创伤弧菌DNA环介导恒温扩增快速检测方法的建立及初步应用  

Establishment of Rapid Detection Method for Vibrio vulnificus in Aquatic Food by Loop-Mediated Isothermal Amplification

作　　者：徐义刚 李苏龙 杨君宏 李丹丹[3] 姜艳春 谢晓峰 崔丽春[4] 

机构地区：[1]黑龙江出入境检验检疫局检验检疫技术中心,哈尔滨150001 [2]哈尔滨市粮食研究所,哈尔滨150076 [3]海南出入境检验检疫局检验检疫技术中心,海口570125 [4]东北林业大学,哈尔滨150040

出　　处：《中国生物工程杂志》2010年第6期96-102,共7页China Biotechnology

基　　金：国家质检总局科技计划项目(2008IK162);黑龙江省博士后基金(LBH-Z09)资助项目

摘　　要：创伤弧菌是一种重要的食源性致病菌,主要存在于河口和海洋环境中,严重危害水产养殖业的发展和人类健康。建立快速、准确、易操作的检测方法对防控创伤弧菌的传染,保障水产养殖业发展和增强食品安全意义重大。基于创伤弧菌vvHA基因,利用一种新型的核酸扩增技术-环介导恒温扩增（loop-mediated isothermal amplification,LAMP）,建立了创伤弧菌LAMP快速检测方法。对11种共46株细菌进行扩增,仅创伤弧菌为LAMP阳性结果,说明LAMP方法具有高度特异性。灵敏度试验结果表明,对创伤弧菌纯培养菌的检测灵敏度为15CFU/ml,对污染食品中创伤弧菌的检测灵敏度为24CFU/g。此法40~60min内即可完成检测,检验检疫实践证明：LAMP方法操作简便、特异性强、灵敏度高且成本低廉,具有良好的应用前景。Vibrio vulnificus is an important foodborne pathogen,which mainly exists in estuaries and the marine environment and seriously harms aquaculture development and human health.Therefore,the development of a technique that can identify the pathogen quickly,accurately and protect aquaculture development and enhance the food safety is of great importance.Here a rapid method for detecting Vibrio vulnificus with loop-mediated isothermal amplification（LAMP）using the vvHA gene of Vibrio vulnificus as target gene were established.Forty-six bacterial strains were selected to test specificity of the LAMP method and only Vibrio vulnificus strain was LAMP positive result,and indicating the high specificity of the LAMP method.The LAMP method could be completed in 40~60min and showed a good sensitivity of 15 CFU/ml for cultured Vibrio vulnificus and of 20 CFU/g for its contaminated food.Finally,25 samples in detected 278 practical samples of shrimps,crabs,oysters and artificial contamination ones were positive results using the LAMP method,which accorded with the detection results according to SN/T 1870-2007.The LAMP assay is a sensitive,rapid simple tool for the detection of Vibrio vulnificus.

关 键 词：创伤弧菌 食源性致病菌 环介导恒温扩增 快速检测 

分 类 号：Q819[生物学—生物工程]