大腸桿菌O157-H7論文：快速檢測大腸桿菌O157-H7膠體金免疫層析方法的建立

1、大腸桿菌O157:H7論文：快速檢測大腸桿菌O157:H7膠體金免疫層析方法的建立【中文摘要】大腸桿菌O157：H7(Escherichia coli O157:H7)是引起出血性腸炎和溶血性尿路綜合癥的主要致病菌。牛肉、奶制品、蔬菜等食物是其主要傳播途徑,除此之外,動(dòng)物和人、人和人的接觸也可能導(dǎo)致大腸桿菌O157：H7感染。由于其感染發(fā)病快、死亡率高,嚴(yán)重影響了人民的身體健康。因此,快速診斷大腸桿菌O157：H7具有重要的現(xiàn)實(shí)意義。本文旨在建立快速、特異性強(qiáng)、適用于大量樣品和現(xiàn)場檢測的大腸桿菌O157：H7膠體金免疫層析方法。本文采用全菌抗原和細(xì)胞碎片抗原分別免疫日本大耳兔,制備了抗大腸桿菌

2、O157：H7多克隆抗體。獲得的多克隆抗體效價(jià)達(dá)106。分別用辛酸-硫酸銨法和磁珠吸附法對多克隆抗體進(jìn)行純化,純化后的抗體通過SDS-PAGE進(jìn)行蛋白分析,并用斑點(diǎn)雜交對純化后的抗體進(jìn)行特異性分析。結(jié)果顯示,辛酸-硫酸銨法純化后的抗體仍有較多的雜蛋白,而磁珠吸附法能有效去除這些雜蛋白,得到較純的抗體。純化后的多克隆抗體均與其它一些細(xì)菌有交叉反應(yīng)。本文采用膠體金標(biāo)記商品化的單克隆抗體,通過把多克隆抗體和驢抗鼠抗體(二抗)噴涂于硝酸纖維素膜分別作為檢測線(T線)和質(zhì)控線(C線),研制了大腸桿菌O157：H7膠體金快速檢測試紙條,并對其進(jìn)行了評價(jià)。通過實(shí)驗(yàn),確定了試紙條工藝條件如下：標(biāo)記pH為8.0

3、、蛋白標(biāo)記量為25g/mL、金標(biāo)抗體離心轉(zhuǎn)速為4000 r/min、膠體金封閉劑為PEG 20000、T線噴涂濃度為1mg/mL。C線噴涂濃度為1.5mg/mL。用純培養(yǎng)細(xì)菌對試紙條的靈敏度和特異性進(jìn)行了評價(jià),結(jié)果顯示試紙條的靈敏度為105個(gè)細(xì)胞/mL,試紙條除與金黃色葡萄球菌(CMCC26003)和鼠傷寒沙門氏菌(ATCC 13311)有弱陽性反應(yīng)外,與其它24株常見的細(xì)菌無交叉反應(yīng)。37加速保存實(shí)驗(yàn)結(jié)果表明,本試紙條常溫保質(zhì)期在15個(gè)月以上。在25 g牛肉或萵苣樣品中添加10100 CFU的菌,37增菌6h后,試紙條檢測為陽性結(jié)果。為了進(jìn)一步提高試紙條的特異性和靈敏度,本文采用提取的大腸桿

4、菌O157：H7鞭毛蛋白免疫Balb/c小鼠,經(jīng)細(xì)胞融合、篩選、克隆化共得到4株能穩(wěn)定分泌抗大腸桿菌O157：H7單克隆抗體的雜交瘤細(xì)胞株,并制備了相應(yīng)的腹水。配對結(jié)果顯示,有5對抗體可用于試紙條的制備?！居⑽恼縀scherichia 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.【關(guān)鍵

15、詞】大腸桿菌O157:H7 多克隆抗體 膠體金 免疫層析 單克隆抗【英文關(guān)鍵詞】Escherichia coli O157:H7 polyclonal antibody colloidal gold immunochromatography monoclonal antibody【目錄】快速檢測大腸桿菌O157:H7膠體金免疫層析方法的建立摘要3-4ABSTRACT4-5縮略語表6-11第一章 引言11-201.1 E. coli O157:H7的臨床表現(xiàn)和感染11-121.2 E. coli O157:H7的特異性抗原和抗體制備研究進(jìn)展12-141.2.1 E. coli O157:H7的O

16、抗原121.2.2 E. coli O157:H7的H抗原12-131.2.3 其它特異性抗原13-141.3 E. coli O157:H7檢測方法的研究進(jìn)展14-171.3.1 常規(guī)檢測方法14-151.3.2 基于PCR的分子生物學(xué)方法15-161.3.3 免疫學(xué)檢測方法16-171.3.4 新興技術(shù)171.4 膠體金快速檢測技術(shù)的概述17-191.4.1 膠體金的性質(zhì)181.4.2 膠體金制備方法181.4.3 膠體金標(biāo)記抗體18-191.4.4 免疫膠體技術(shù)在微生物檢測中的應(yīng)用191.5 展望19-20第二章 抗大腸桿菌O157:H7多克隆抗體的制備20-312.1 前言202.2

17、材料與方法20-252.2.1 菌株、免疫動(dòng)物202.2.2 主要試劑和儀器設(shè)備202.2.3 相關(guān)試劑和培養(yǎng)基配置20-212.2.4 E. coli O157:H7抗原制備21-222.2.5 其它菌株包被原的制備222.2.6 動(dòng)物免疫22-232.2.7 間接ELISA232.2.8 E. coli O157:H7多克隆抗體效價(jià)監(jiān)測232.2.9 抗體純化23-242.2.10 多克隆抗體特異性評價(jià)24-252.3 結(jié)果與討論25-312.3.1 E. coli O157:H7全菌抗原和細(xì)胞碎片抗原25-262.3.2 多克隆抗體免疫應(yīng)答曲線26-272.3.3 純化多抗電泳分析27-

18、282.3.4 多克隆抗體特異性28-292.3.5 討論29-31第三章 大腸桿菌O157:H7膠體金快速檢測試紙條的制備31-423.1 前言313.2 材料與方法31-353.2.1 菌株313.2.2 主要試劑和儀器設(shè)備313.2.3 相關(guān)試劑與培養(yǎng)基配制31-323.2.4 多克隆抗體和單克隆抗體配對32-333.2.5 膠體金制備333.2.6 標(biāo)記pH值的確定333.2.7 蛋白標(biāo)記量的確定33-343.2.8 標(biāo)記膠體金離心參數(shù)343.2.9 封閉劑的確定343.2.10 檢測線包被濃度的確定34-353.2.11 質(zhì)控線包被濃度的確定353.3 結(jié)果與討論35-423.3.1

19、 多克隆抗體和單克隆抗體配對35-363.3.2 制備的膠體金分析36-373.3.3 標(biāo)記pH值的確定37-383.3.4 蛋白標(biāo)記量的確定383.3.5 標(biāo)記膠體金離心參數(shù)38-393.3.6 封閉劑的確定393.3.7 檢測線包被濃度的確定39-403.3.8 質(zhì)控線包被濃度的確定40-413.3.9 討論41-42第四章 大腸桿菌O157:H7膠體金快速檢測試紙條的評價(jià)42-524.1 前言424.2 材料與方法42-454.2.1 菌株424.2.2 主要試劑和儀器設(shè)備424.2.3 相關(guān)試劑與培養(yǎng)基配制42-434.2.4 試紙條的制備434.2.5 細(xì)菌培養(yǎng)與處理434.2.6 陰性樣品測試434.2.7 試紙條特異性43-444.2.8 試紙條靈敏度444.2.9 食品樣品檢測444.2.10 試紙條保質(zhì)期44-454.3 結(jié)果與討論45-524.3.1 試紙條陰性樣品測試454.3.2 試紙條特異性45-474.3.3 試紙條靈敏度分析47-484.3.4 試紙條保質(zhì)期48-494