Eu-TRFM免疫层析试纸条检测大肠杆菌O157：H7方法的建立

Establishment of a Eu-TRFM Immunochromatographic Test Strip for Detection of Escherichia coli O157:H7

摘要:

目的 建立一种快速、高效且灵敏的大肠杆菌（Escherichia coli, E. coli） O157：H7检测方法。方法 运用1-乙基-3-（3-二甲基氨基丙基）碳酰二亚胺（EDC）介导法，以时间分辨荧光微球为标记物与抗体进行偶联，使用荧光读取仪定量分析检测大肠杆菌O157：H7。通过对缓冲液的标记pH、时间分辨荧光微球探针添加量、T线抗体质量浓度以及样本与标记抗体的孵育时间进行优化来提高检测灵敏度。结果 在最优条件下，大肠杆菌O157：H7的检测灵敏度可达3.2×10³ CFU/mL，比传统胶体金免疫层析方法提高了10~1 000倍，并且与其他8株常见的食源性致病菌不存在交叉反应。另外，该方法在人工污染的饮用水、牛奶和牛肉实际样品检测中均表现出良好的检测性能。结论 该Eu-TRFM免疫层析试纸条适用于大肠杆菌O157：H7的检测。

Abstract:

Objective The aim of this study is to establish a rapid, efficient, and sensitive method for the detection of Escherichia coli (E. coli) O157:H7.Method The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-mediated method was used to conjugate antibodies with time-resolved fluorescent microspheres (markers), and a fluorescence reader was used for quantitative analysis and detection of E. coli O157:H7. The detection sensitivity was improved by optimizing the pH of buffer for labeling, the amount of time-resolved fluorescent microspheres, the mass concentration of T-line antibodies, and the incubation time of samples and labeled antibodies.Result Under the optimal conditions, the detection sensitivity for E. coli O157:H7 reached 3.2×10³ CFU/mL, which was 10~1 000 times higher than that of colloidal gold immunochromatography, and there was no cross-reaction with other 8 common food-borne pathogens. In addition, the established test strip demonstrated good detection performance for the real samples of artificially contaminated drinking water, milk, and beef.Conclusion The EU-TRFM immunochromatographic test strip is suitable for the detection of E. coli O157:H7.