LAMP实时浊度法快速检测肠出血性大肠杆菌O157的研究.pdfVIP

现代食品科技 Modern Food Science and Technology 2014, Vol.30, No.7 268 LAMP 实时浊度法快速检测肠出血性大肠杆菌 O157 的研究 李碧霞 1,2，游淑珠 2，邝筱珊 2，冯家望 2，王小玉 2，唐食明 2，成晓维 2，许喜林 1 （1.华南理工大学轻工与食品学院，广东广州 510640）（2.珠海出入境检验检疫局技术中心，广东珠海 519015） 摘要：采用环介导等温扩增（Loop Mediated Isothermal Amplification，LAMP）技术，利用实时浊度仪实时检测 LAMP反应过程 中所产生的焦磷酸镁白色沉淀，实现对扩增反应全过程的监控，建立食品中肠出血性大肠杆菌（Enterohemorrhagic Escherichia coli， EHEC）O157的 LAMP实时浊度法快速检测方法。针对 EHEC O157抗原基因 rfbE的序列设计 4条特异性 LAMP 引物。通过实时浊 度仪在恒温 63 ℃下 1小时完成检测，对方法的特异性、灵敏度、稳定性进行了评价，并进行了食品样品添加试验以评价其应用于实 际样品的效果。结果显示，经优化后，该方法的最低检出限为 2.8×103 CFU/mL，与 PCR法灵敏度比较高 100 倍。样品添加试验能检 出的最低添加菌量为 2.8×102 CFU/25 g（或 mL）。LAMP实时浊度法具有快速、灵敏、特异性高、操作简便的优势，为食源致病菌快 速筛查提供了一种良好的检测模式。 关键词：环介导等温扩增技术；实时浊度仪；大肠杆菌O157；检测 文章篇号：1673-9078(2014)7-268-272 Rapid Detection of Enterohemorrhagic Escherichia coli O157 by LAMP Real-time Turbidity Method LI Bi-xia1,2, YOU Shu-zhu2, KUANG Xiao-shan2, FENG Jia-wang2, WANG Xiao-yu2, TANG Shi-ming2, CHENG Xiao-wei2, XU Xi-lin1 (1.School of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China) (2.The Inspection Technical Center of Zhuhai Entry-Exit Inspection and Quarantine Bureau, Zhuhai 519015, China) Abstract: Real-time monitoring of magnesium pyrophosphate produced from the loop-mediated isothermal amplification (LAMP) reaction was achieved by the turbidimeter. Four specific primers were designed according to the sequences of antigen gene rfbE of EHEC O157. Detection was completed at 63 ℃ on the real-time turbidimeter in 1 h. The specificity, sensitivity, stability of the method was evaluated. Results showed that after optimized, the lowest detection limit was 2.8×103 CFU/mL, and 100 times more sensitive than the PCR, and the minimal adding quantity of 2.8×100 CFU/25 g (or mL) could be detected. LAMP real-time turbidity method is rapid, sensitive, high specificity and easy to operate, which is a good pattern for fast screening of foodborne pathogens. Key words: loop-mediated isothermal amplificati