实时荧光环介导等温扩增技术快速检测牛肉中的大肠杆菌O157

Rapid detection of Escherichia coli O157 in beef by real-time fluorescence loop-mediated isothermal amplification

中文关键词:  实时荧光  环介导等温扩增  大肠杆菌O157  rfbE基因  牛肉  快速检测

英文关键词:real-time fluorescence  loop-mediated isothermal amplification  Escherichia coli O157  rfbE  beef  rapid detection

中文摘要:

      目的 建立实时荧光环介导等温扩增技术(real-time fluorescence loop-mediated isothermal amplification, RF-LAMP)快速检测大肠杆菌(Escherichia coli, EHEC)O157的分析方法。方法 针对大肠杆菌O157编码O抗原的rfbE基因设计引物。对该方法进行特异性验证, 同时对大肠杆菌O157:H7纯培养物的灵敏度和人工污染牛肉的检出限进行测定, 对61份牛肉样品进行RF-LAMP检测, 并与GB 4789.36-2016方法进行比较, 评价RF-LAMP方法的敏感性、特异性和准确度。结果 10株大肠杆菌O157呈阳性结果, 21株非大肠杆菌O157呈阴性结果, 该方法特异性良好。纯培养物检测的灵敏度为5.1 CFU/mL, 人工污染的牛肉样品的检出限为5.1 CFU/g。结论 本研究建立的RF-LAMP技术特异性好、灵敏度高、操作简单, 可实时监测扩增反应, 避免了繁琐的电泳过程, 实现了对大肠杆菌O157的快速检测, 对大肠杆菌O157引起的食源性疾病的预防和控制具有重要意义。

英文摘要:

      Objective To establish a method for rapid determination of Escherichia coli (EHEC) O157 by real-time fluorescence loop-mediated isothermal amplification (RF-LAMP). Methods The primers of E. coli O157 were designed based on the rfbE gene encoding the O antigen. The specificity of the method was verified, the sensitivity of pure culture of E. coli O157:H7 and the limit of detection of artificial contaminated beef were determined. A total of 61 beef samples were investigated by RF-LAMP and compared with GB 4789.36-2016 to evaluate the sensitivity, specificity and accuracy of the RF-LAMP method. Results Total 10 E. coli O157 strains were identified as positive, however, 21 non- E. coli O157 strains were negative, which showed this method had great specificity. The sensitivity in pure culture by RF-LAMP was 5.1 CFU/mL. The limit of detection of artificially contaminated beef samples was 5.1 CFU/g. Conclusions The RF-LAMP technology established in this study has good specificity, high sensitivity and simple operation, and can monitor the amplification reaction in real time, avoiding the complicated electrophoresis process. This study realizes the rapid detection of E. coli O157 and has significant importance for the prevention and control of food-borne diseases caused by E. coli O157.