荧光定量环介导等温扩增法快速检测食品中沙门氏菌

Rapid detection of Salmonella in food by fluorescent quantitative loop-mediated isothermal amplification

中文关键词:  invA基因  荧光定量环介导等温扩增  沙门氏菌  即时检测

英文关键词:invA gene  fluorescence quantitative loop-mediated isothermal amplification  Salmonella  point-of-care detection

中文摘要:

      目的 建立一种快速简易检测食源性沙门氏菌的实时荧光定量环介导等温扩增(quantitative loop-mediated isothermal amplification, qLAMP)方法。方法 依据沙门菌属invA基因序列设计引物, 并结合短时间增菌构建沙门氏菌快速检测qLAMP法, 使用人工污染样品进行验证。结果 建立的qLAMP法最佳反应时间为40 min, 最佳反应温度是65 ℃, 最佳Mg2+浓度为6 mmol/L, Bst 2.0 WarmStart聚合酶的浓度为0.40 U/μL, 反应特异性良好, 纯培养实验表明方法检出限为100 CFU/mL。将沙门氏菌人工添加至鸡胸肉、果蔬沙拉、山泉水中, 经过7 h缓冲蛋白胨水有效增菌后所建立的qLAMP法在食品基质中的检出限达到5 CFU/25 g。结论 该方法准确、简单易行、实验成本低, 可用于食品中沙门氏菌的快速检测。通过WarmStart Colorimetric LAMP试剂盒无需昂贵仪器即可快速检测, 为沙门氏菌现场即时检测提供技术支持。

英文摘要:

      Objective To establish a rapid and simple quantitative loop-mediated isothermal amplification (qLAMP) method for detecting food-borne Salmonella. Methods The primers were designed based on the invA gene sequence of Salmonella, and the qLAMP method for rapid detection of Salmonella was constructed by combining with short-time enrichment, and verified by using artificially contaminated samples. Results The optimal reaction time, reaction temperature, Mg2+ concentration and Bst 2.0 WarmStart DNA polymerase concentration were 40 min, 65 ℃, 6 mmol/L and 0.40 U/μL, respectively, by using the established qLAMP method, the reaction specificity was good, and the pure culture experiment showed that the detection limit of the method was 100 CFU/mL. Salmonella was manually added into chicken breast, fruit and vegetable salad, and mountain spring water, and the limit of detection of the established qLAMP method in the food matrix reached 5 CFU/25 g after 7 h buffer peptone water for effective enrichment. Conclusion This method is accurate, simple and low in experimental cost, and can be used for rapid detection of Salmonella in food. The WarmStart Colorimetric LAMP kit can be used for rapid detection without expensive instruments, which provides technical support for the scene of point-of-care detection of Salmonella.