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

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（Salmonella spp.）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5 pmol/L、内引物浓度40 pmol/L，Mg2＋浓度6 mmol/L，dNTP浓度0.8 mmol/L，甜菜碱浓度0.8 mmol/L，Bst DNA聚合酶8 U，反应温度63 ℃，反应时间1 h。在此条件下，LAMP检测沙门菌DNA的敏感度达10 fg/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102 cfu/mL，适合于食品中污染沙门菌的快速检测。

Abstract:

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, Mg2＋, dNTP, betaine, Bst DNA polymerase, reaction temperature and reaction time were 40 pmol/L, 5 pmol/L, 6 mmol/L, 0.8 mmol/L, 0.8 mmol/L, 8 U, 63 ℃ and 1h, respectively. Under those conditions, the detection limit for Salmonella spp. DNA template was 10 fg/tube and no cross reaction with other usual bacteria was found. For Salmonella spp. in milk products, the detection limit of the method was 102 cfu/mL. So this method was suitable for rapid detection of Salmonella spp. in contaminated foods.