实时荧光等温扩增法检测4种常见食源性致病菌
目的 建立实时荧光等温扩增法(loop-mediated isothermal amplification, LAMP)检测金黄色葡萄球菌、沙门氏菌、副溶血性弧菌、单核细胞增生李斯特氏菌4种常见食源性致病菌的分析方法。方法 根据4种致病菌特异性基因合成LAMP引物, 在反应前加入荧光染料, 在63 ℃条件下反应45 min; 反应结果可以根据扩增曲线变化直接判断。并通过在实际样品中添加标准菌株菌液, 测定了其在增菌0、6、24 h的检测灵敏度。结果 金黄色葡萄球菌、沙门氏菌、单核细胞增生李斯特氏菌等基因组DNA的LAMP检测灵敏度为1 pg, 副溶血性弧菌的检测灵敏度为10 pg; 在样品加标实验中, 其灵敏度在6 h分别可达到101、100、100、102 CFU/mL。结论 该方法用于食源性致病菌的快速检测, 具有可实时监控反应过程、反应快速, 灵敏度和特异性高等优点, 适合于基层食品安全监管领域现场快速检测。
Objective To establish a method for the determination of Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus and Listeria monocytogenes by real-time loop-mediated isothermal amplification (LAMP). Methods The LAMP primers were designed according to the sequences of 4 kinds of food-borne pathogens specific genes, respectively. Target DNA was amplified within 45 min at an isothermal temperature of 63 ℃ and the result was directly analyzed by the amplification curve. Meanwhile, the detection sensitivity of the strains at 0, 6 and 24 h was determined by adding standard bacteria strains to the food samples. Results The limit of detection of the LAMP assay was about 1 pg for DNA per reaction with genomic templates of Staphylococcus aureus, Salmonella, Listeria monocytogenes strains, and 10 pg for Vibrio parahaemolyticus. After 6 h enrichment, the limit of detection for artificially contaminated food samples could reach 101, 100, 100 and 102 CFU/mL, respectively. Conclusion This method provides a useful tool for the rapid and sensitive detection of food-borne pathogenic bacteria, and has the advantages of real-time monitoring of reaction process, rapid reaction, high sensitivity and specificity, which is suitable for rapid on-site detection in the field of food safety supervision in the primary layer.
标题：实时荧光等温扩增法检测4种常见食源性致病菌
英文标题：Detection of 4 kinds of food-borne pathogenic bacteria by real-time loop-mediated isothermal amplification method
作者：
杨丽霞 长沙市食品药品检验所
曾思思 长沙市食品药品检验所
钟菲菲 长沙市食品药品检验所
中文关键词:食源性致病菌,实时荧光等温扩增法,快速检测,
英文关键词:food-borne pathogenic bacteria,real-time loop-mediated isothermal amplification method,rapid detection,
发表日期：2019-05-31
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