大肠杆菌O157-H7论文：快速检测大肠杆菌O157-H7胶体金免疫层析方法的建立

1、大肠杆菌O157:H7论文：快速检测大肠杆菌O157:H7胶体金免疫层析方法的建立【中文摘要】大肠杆菌O157：H7(Escherichia coli O157:H7)是引起出血性肠炎和溶血性尿路综合症的主要致病菌。牛肉、奶制品、蔬菜等食物是其主要传播途径,除此之外,动物和人、人和人的接触也可能导致大肠杆菌O157：H7感染。由于其感染发病快、死亡率高,严重影响了人民的身体健康。因此,快速诊断大肠杆菌O157：H7具有重要的现实意义。本文旨在建立快速、特异性强、适用于大量样品和现场检测的大肠杆菌O157：H7胶体金免疫层析方法。本文采用全菌抗原和细胞碎片抗原分别免疫日本大耳兔,制备了抗大肠杆菌

2、O157：H7多克隆抗体。获得的多克隆抗体效价达106。分别用辛酸-硫酸铵法和磁珠吸附法对多克隆抗体进行纯化,纯化后的抗体通过SDS-PAGE进行蛋白分析,并用斑点杂交对纯化后的抗体进行特异性分析。结果显示,辛酸-硫酸铵法纯化后的抗体仍有较多的杂蛋白,而磁珠吸附法能有效去除这些杂蛋白,得到较纯的抗体。纯化后的多克隆抗体均与其它一些细菌有交叉反应。本文采用胶体金标记商品化的单克隆抗体,通过把多克隆抗体和驴抗鼠抗体(二抗)喷涂于硝酸纤维素膜分别作为检测线(T线)和质控线(C线),研制了大肠杆菌O157：H7胶体金快速检测试纸条,并对其进行了评价。通过实验,确定了试纸条工艺条件如下：标记pH为8.0

3、、蛋白标记量为25g/mL、金标抗体离心转速为4000 r/min、胶体金封闭剂为PEG 20000、T线喷涂浓度为1mg/mL。C线喷涂浓度为1.5mg/mL。用纯培养细菌对试纸条的灵敏度和特异性进行了评价,结果显示试纸条的灵敏度为105个细胞/mL,试纸条除与金黄色葡萄球菌(CMCC26003)和鼠伤寒沙门氏菌(ATCC 13311)有弱阳性反应外,与其它24株常见的细菌无交叉反应。37加速保存实验结果表明,本试纸条常温保质期在15个月以上。在25 g牛肉或莴苣样品中添加10100 CFU的菌,37增菌6h后,试纸条检测为阳性结果。为了进一步提高试纸条的特异性和灵敏度,本文采用提取的大肠杆

4、菌O157：H7鞭毛蛋白免疫Balb/c小鼠,经细胞融合、筛选、克隆化共得到4株能稳定分泌抗大肠杆菌O157：H7单克隆抗体的杂交瘤细胞株,并制备了相应的腹水。配对结果显示,有5对抗体可用于试纸条的制备。【英文摘要】Escherichia coli (E. coli) O157:H7 is an important foodborne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome in human. Human infections with E. coli O157:H7 have usually

5、 been found to follow the consumption of contaminated, improperly prepared beef products, dairy foods, and vegetables. In addition, direct animal-to-person and person-to-person contact can result in E. coli O157:H7 transmission. It has seriously affected the health of human because its fast invasion

6、 and high mortality when infections. Hence, it has important practical significance of developing a rapid method for diagnosis E. coli O157:H7. In this study, a colloid gold immunochromatographic method which is rapid, high specificity and suitable for a great number of samples and on site detection

7、 was established for detection of E. coli O157:H7.Japanese white rabbits were immunized with the whole cell and cell debris of E. coli O157:H7 to prepare polyclonal antibody. The titer of antibody was 106. Caprylic acid ammonium sulphate precipitation and magnetic beads adsorption were used to purif

8、y polyclonal antibodies (pAb). Then SDS-PAGE was used for proteins analysis while Dot blot hybridization was used for analysis the specificity of pAb. The SDS-PAGE results showed that antibodies purified by caprylic acid ammonium sulfate precipitation still had many other proteins; however, the magn

9、etic beads adsorption could effectively remove the other proteins and get pure immunoglobulin. The antiserum after purified reacted with other bacteria.In this study, commercial anti-E. coli O157:H7 monoclonal antibody was labeled with colloidal gold and anti-E. coli O157:H7 polyclonal antibody and

10、donkey anti-mouse IgG were dispensed on the nitrocellulose membrane as the test line and control line respectively. The strips parameters were as follows:The reaction system pH was 8.0, anti-E. coli O157:H7 monoclonal antibody labeling with gold particles was 25g/mL, the gold-antibody conjugate was

11、centrifuged at 4000 rpm/min and blocked with PEG 20000, lmg/mL polyclonal antibody and 1.5 mg/mL donkey anti-mouse IgG were dispensed for the test line and control line respectively. The senitivity and specificity of the strip were determined using pure cultured bacteria. E. coli O157:H7 could be de

12、tected at a minimum of 105 cells/mL. Cross reaction test with 26 bacteria strains showed that only Staphylococcus aureus (CMCC 26003) and Salmonella typhimurium (ATCC 13311) had slight cross reaction, whereas the rest 24 strains had no cross reaction. The preservation test at 37indicated that the st

13、rips could be stored at room temperature for more than 15 months. The strips showed positive result after inoculating E. coli O157:H7 in samples (beef or lettuce) at 10 to 100 CFU/25 g and incubating for 6 h.In order to increase the specificity and sensitivity of the assay, Balb/c mice were immunize

14、d with flagellum proteins isolated from E. coli O157:H7. After fusion and cloning, four hybridoma cells which would stable secret antibodys against E. coli O157:H7 were abtained and prepared ascitic fluid. The matching results showed that 5 pairs of antibodies could be used for strip preparation.【关键

15、词】大肠杆菌O157:H7 多克隆抗体 胶体金 免疫层析 单克隆抗【英文关键词】Escherichia coli O157:H7 polyclonal antibody colloidal gold immunochromatography monoclonal antibody【目录】快速检测大肠杆菌O157:H7胶体金免疫层析方法的建立 摘要 3-4 ABSTRACT 4-5 缩略语表 6-11 第一章 引言 11-20 1.1 E. coli O157:H7的临床表现和感染 11-12 1.2 E. coli O157:H7的特异性抗原和抗体制备研究进展 12-14 1.2.1 E. c

16、oli O157:H7的O抗原 12 1.2.2 E. coli O157:H7的H抗原 12-13 1.2.3 其它特异性抗原 13-14 1.3 E. coli O157:H7检测方法的研究进展 14-17 1.3.1 常规检测方法 14-15 1.3.2 基于PCR的分子生物学方法 15-16 1.3.3 免疫学检测方法 16-17 1.3.4 新兴技术 17 1.4 胶体金快速检测技术的概述 17-19 1.4.1 胶体金的性质 18 1.4.2 胶体金制备方法 18 1.4.3 胶体金标记抗体 18-19 1.4.4 免疫胶体技术在微生物检测中的应用 19 1.5 展望 19-20

17、第二章 抗大肠杆菌O157:H7多克隆抗体的制备 20-31 2.1 前言 20 2.2 材料与方法 20-25 2.2.1 菌株、免疫动物 20 2.2.2 主要试剂和仪器设备 20 2.2.3 相关试剂和培养基配置 20-21 2.2.4 E. coli O157:H7抗原制备 21-22 2.2.5 其它菌株包被原的制备 22 2.2.6 动物免疫 22-23 2.2.7 间接ELISA 23 2.2.8 E. coli O157:H7多克隆抗体效价监测 23 2.2.9 抗体纯化 23-24 2.2.10 多克隆抗体特异性评价 24-25 2.3 结果与讨论 25-31 2.3.1 E

18、. coli O157:H7全菌抗原和细胞碎片抗原 25-26 2.3.2 多克隆抗体免疫应答曲线 26-27 2.3.3 纯化多抗电泳分析 27-28 2.3.4 多克隆抗体特异性 28-29 2.3.5 讨论 29-31 第三章 大肠杆菌O157:H7胶体金快速检测试纸条的制备 31-42 3.1 前言 31 3.2 材料与方法 31-35 3.2.1 菌株 31 3.2.2 主要试剂和仪器设备 31 3.2.3 相关试剂与培养基配制 31-32 3.2.4 多克隆抗体和单克隆抗体配对 32-33 3.2.5 胶体金制备 33 3.2.6 标记pH值的确定 33 3.2.7 蛋白标记量的确

19、定 33-34 3.2.8 标记胶体金离心参数 34 3.2.9 封闭剂的确定 34 3.2.10 检测线包被浓度的确定 34-35 3.2.11 质控线包被浓度的确定 35 3.3 结果与讨论 35-42 3.3.1 多克隆抗体和单克隆抗体配对 35-36 3.3.2 制备的胶体金分析 36-37 3.3.3 标记pH值的确定 37-38 3.3.4 蛋白标记量的确定 38 3.3.5 标记胶体金离心参数 38-39 3.3.6 封闭剂的确定 39 3.3.7 检测线包被浓度的确定 39-40 3.3.8 质控线包被浓度的确定 40-41 3.3.9 讨论 41-42 第四章 大肠杆菌O15

20、7:H7胶体金快速检测试纸条的评价 42-52 4.1 前言 42 4.2 材料与方法 42-45 4.2.1 菌株 42 4.2.2 主要试剂和仪器设备 42 4.2.3 相关试剂与培养基配制 42-43 4.2.4 试纸条的制备 43 4.2.5 细菌培养与处理 43 4.2.6 阴性样品测试 43 4.2.7 试纸条特异性 43-44 4.2.8 试纸条灵敏度 44 4.2.9 食品样品检测 44 4.2.10 试纸条保质期 44-45 4.3 结果与讨论 45-52 4.3.1 试纸条阴性样品测试 45 4.3.2 试纸条特异性 45-47 4.3.3 试纸条灵敏度分析 47-48 4.3.4 试纸条保质期 48-49 4.3.5 食品样品检测 49-50 4.3.6 讨论 50-52 第五章 抗大肠杆菌O157