环介导等温扩增技术快速检测沙门菌  

Rapid Detection of Salmonella spp. by Loop-Mediated Isothermal Amplification Method

作　　者：朱胜梅[1] 吴佳佳[1] 徐驰[1] 屈炯[1] 成炜[1] 陈福生[1] 

机构地区：[1]华中农业大学食品科技学院,湖北武汉430070

出　　处：《现代食品科技》2008年第7期725-730,共6页Modern Food Science and Technology

摘　　要：环介导等温扩增(1oop-mediated isothermal amplification,LAMP)是一种在等温条件下高特异、高效、快速地扩增靶序列的DNA扩增新技术。以沙门菌(Salmonellaspp.)为研究对象,根据其特异性的invA基因,设计了一套特异性引物对该基因进行了LAMP,同时优化了其反应条件,建立了沙门菌的LAMP快速检测技术。结果表明,LAMP的最佳反应条件为外引物浓度5pmol/L、内引物浓度40pmol/L,Mg2+浓度6mmol/L,dNTP浓度0.8mmol/L,甜菜碱浓度0.8mmol/L,BstDNA聚合酶8U,反应温度63℃,反应时间1h。在此条件下,LAMP检测沙门菌DNA的敏感度达10fg/反应,且与其他常见的细菌无交叉反应。其对牛奶样品的检出量为102cfu/mL,适合于食品中污染沙门菌的快速检测。Loop-mediated isothermal amplification （LAMP） is a novel DNA amplification technology which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions. According to the specific invA gene of Salmonella spp., a set of specific primers were designed to amplify the special DNA sequences by LAMP. Moreover, the reaction conditions of LAMP were optimized. The results showed that the optimal concentrations of inner primers, exogenous primers, Mg^2＋, dNTP, betaine, Bst DNA polymerasc, reaction temperature and reaction time were 40 pmol/L, 5 pmol/L, 6 mmol/L, 0.8 mmol/L, 0.8 mmol/L, 8U, 63 ℃ and lh, respectively. Under those conditions, the detection limit for Salmonella spp. DNA template was 10fg/tube and no cross reaction with other usual bacteria was found. For Salmonella spp. in milk products, the detection limit of the method was 10^2cfu/mL. So this method was suitable for rapid detection of Salmonella spp. in contaminated foods.

关 键 词：环介导等温DNA扩增 LAMP 沙门菌 快速检测 

分 类 号：TS207.4[轻工技术与工程—食品科学]