论文总字数：18909字

摘 要

大肠杆菌广泛存在于自然界中，能够导致腹泻、出血性结肠炎等病症，且潜伏期短、致病性强、发病迅速，起因大多与误食被该菌污染的食物有关，目前检测大肠杆菌的方法主要有以下几类：传统检测方法、免疫学方法、分子生物学方法等，但这些方法基本具有操作技术要求高、设备昂贵、耗时长等不足，因此建立一种快速检测食品中大肠杆菌含量的手段具有重大意义。

本实验基于非特异性磁珠分离技术，非靶向快速分离富集大肠杆菌O157:H7，并进一步优化裸磁珠富集大肠杆菌O157:H7的条件。首先控制孵育时间与孵育温度不变，改变裸磁珠的浓度与双极电极恒定电压，测得加入量为1 mg，恒定电压为5.5 V时，磁珠吸附率最大；再根据所得结果，控制裸磁珠浓度、双极电极恒定电压及孵育温度不变，改变孵育时间，测得孵育15分钟时，富集效率最高；最后控制磁珠加入量为1 mg、恒定电压5.5 V，在梯度温度条件下孵育15分钟，测得温度为37℃时，吸附率最高。在最佳条件下，对大肠杆菌浓度进行定量检测，将所得数据制成图表，得到标准曲线，其相关系数R² = 0.996，检测范围达到10-10⁸ CFU/mL，表明该曲线具有很高的拟合度与准确性。

本实验基于微生物的导电性，有效降低食品复杂基质对浓度测定的影响，顺利进行双极电极传感界面上的电荷传输，再结合双极电极-电致化学发光技术，将检测所得电信号转化为更敏感可测的电致化学发光信号，数值可读，从而实现快速、灵敏、定量检测食品中的大肠杆菌。

关键词：大肠杆菌O157:H7 裸磁珠 双极电极

Rapid quantitative detection of Escherichia coli O157:H7 based on bare magnetic bead and bipolar electrode

Abstract

Escherichia coli O157:H7 (E. coli O157:H7) can cause diarrhea, hemorrhagic colitis symptoms, and the incubation period is short, strong pathogenic, quick, the cause of most related to eating food contaminated with the bacteria. The detection of E. coli O157:H7 method mainly has the following categories: the traditional detection method, immunology, molecular biology, etc. These methods has high operation technical requirements, basic equipment is expensive, time consuming, so rapid detection of E. coli O157:H7 in the food is of great significance.

Based on the non-specific magnetic bead separation technology, this experiment rapidly isolated and enriched foodborne pathogens without targeting, and further optimized the conditions for the enrichment of E. coli O157:H7 by bare magnetic beads. Firstly, the incubation time and incubation temperature were controlled to change the concentration of the bare magnetic bead and the constant voltage of the bipolar electrode. The adsorption rate of the magnetic bead was the maximum when the addition amount was 1 mg and the constant voltage was 5.5 V. According to the results, the concentration of bare magnetic beads, the constant voltage of bipolar electrode and the incubation temperature were controlled, and the incubation time was changed. Finally, the added amount of magnetic beads was controlled to be 1 mg with a constant voltage of 5.5 V, and they were incubated for 15 min under the condition of gradient temperature. When the measured temperature was 37℃, E. coli O157:H7 was the most suitable for growth and reproduction, and the adsorption rate was the highest.

This experiment based on the conductivity of microbial, effectively reduce the food complex matrix on measurement of concentration, smoothly bipolar electrode sensor interface on the charge transfer, coupled with a bipolar electrode-electrically induced chemiluminescence technology, will detect the electrical signal is transformed into more sensitive electrically induced chemical luminescence signal, measurable value can be read, so as to realize rapid, sensitive and quantitative detection of e. coli in food.

Under the best conditions, the concentration of E. coli O157:H7 was quantitatively detected and the obtained data were plotted to obtain the standard curve. The correlation coefficient R² = 0.996 and the detection range reached 10-10⁸ CFU/mL, indicating that the curve has a high degree of fit and accuracy.

Key words: E. coli O157:H7; bare magnetic bead; bipolar electrode

目录

摘 要 I

Abstract II

第一章 绪论 1

1.1食源性疾病现状 1

1.2大肠杆菌 1

1.3大肠杆菌的检测方法及其现状 2

1.3.1传统方法 2

1.3.2免疫学方法 2

1.3.3分子生物学方法 3

1.4裸磁珠的分离富集研究 3

1.5研究意义与研究方法 4

1.5.1研究意义 4

1.5.2研究方法 5

第二章 基于裸磁珠富集及检测大肠杆菌的研究 6

2.1材料与仪器 6

2.1.1仪器与材料 6

2.1.2试剂与溶液 6

2.1.3培养基及其配制体系 6

2.2实验方法 7

2.2.1接种培养大肠杆菌 7

2.2.2不同磁珠浓度对裸磁珠吸附率影响实验 7

2.2.3不同电化学电压对裸磁珠吸附率影响实验 7

2.2.4线性关系的测定 8

2.2.5不同孵育时间对裸磁珠吸附率影响实验 8

2.2.6不同孵育温度对裸磁珠吸附率影响实验 8

第三章 结果与讨论 9

3.1磁珠浓度对裸磁珠吸附率影响实验 9

3.2不同电化学电压对裸磁珠吸附率影响实验 9

3.3线性关系的测定 10

3.4不同孵育时间对裸磁珠吸附率影响实验 11

3.5不同孵育温度对裸磁珠吸附率影响实验 11

第四章 结论与展望 13

4.1结论 13

4.2展望 13

参考文献 15

致谢 17

1. 绪论

1.1食源性疾病现状

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