LAMP实时浊度法快速检测肠出血性大肠杆菌O157的研究

Rapid Detection of Enterohemorrhagic Escherichia coli O157 by LAMP Real-time Turbidity Method

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。

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.