【作者】 梁磊；

【导师】 张伟；

【作者基本信息】 河北农业大学 ， 农产品加工及贮藏工程， 2011， 硕士

【摘要】 1885年,德国科学家T.Escherich首次发现大肠杆菌O157:H7,它是引起腹泻的重要致病菌。大多数食物中毒都可追溯到牛肉和未消毒的牛奶,而且感剂量小于100个细菌即可引起感染。目前对大肠杆菌O157:H7的检测方法主要有传统的常规培养法、免疫学检测方法和以PCR为代表的分子生物学检测方法。传统方法操作繁琐,检测时间长,而且灵敏度较低;免疫学检测方法特异性和灵敏度不理想;PCR方法敏感、快速,但由于需要昂贵的仪器设备、繁琐的电泳过程,难以在基层普及和推广。采用环介导等温扩增（Loop-mediated isothermal amplification,LAMP）技术,检测食品中的大肠杆菌O157:H7,具有灵敏度高,特异性强,检测方法简便,耗时短,检测成本低等特点,适宜在基层普及和推广。本研究针对大肠杆菌O157:H7（IQCC 10102） rfbE保守序列（Genbank S83460）,运用在线引物设计软件（http://primerexplorer.jp/e/index.html）设计LAMP引物。对BST酶添加量、dNTP浓度、镁离子浓度等扩增条件进行了优化,确定25μL的LAMP反应体系为:内引物（FIP和BIP）各1.6μM,外引物（F3和B3）各0.2μM,环引物（LB）0.8μM,0.6 mM dNTP,3.0 mM MgCl₂,10×Bst DNA聚合酶反应缓冲液,8 U Bst DNA聚合酶大片段,1μL DNA模板和灭菌双蒸水。LAMP反应过程是:64℃水浴锅中反应20 min,然后将其放入80℃水浴锅中,水浴10 min终止反应,通过肉眼观察有无白色焦磷酸镁沉淀,判断是否发生LAMP反应。为了比较LAMP检测大肠杆菌O157:H7灵敏度和人工污染的检出限,以PCR方法作对照。PCR引物是LAMP的两条外引物（F3和B3）。结果表明:LAMP检测大肠杆菌O157:H7的灵敏度为9.8 CFU/mL,人工污染牛肉的检出限为68 CFU/g。PCR检测大肠杆菌O157:H7的灵敏度为980 CFU/mL,人工污染牛肉的检出限为6.8×10³ CFU/g。采用试剂盒法提取DNA,从样品处理到报告结果,LAMP方法约耗时2 h,PCR方法约耗时3 h。利用LAMP方法对19株肠道致病菌进行特异性试验。结果表明:三株大肠杆菌O157为阳性,其他16株菌均为阴性。初步研究了3种模板制备方法对LAMP检测牛肉中大肠杆菌O157:H7的影响。结果表明,LAMP方法对制备模板DNA的要求不高。研究表明:LAMP技术是一种检测程序简单、灵敏度和特异性较高的基因检测技术,在大肠杆菌O157:H7的快速检测方面有一定开发潜力,为快速检测食源性致病菌构建了一个新的技术平台。更多还原

【Abstract】 Escherichia coli O157: H7 was firstly reported by T.Escherich in 1885 and listed as a major food-borne pathogen which can cause diarrhea. Most food-borne intoxication affairs drived from beef and plain milk, and less than 100 individual bacteria can result in intoxication. At present time, the main methods for isolation and identification of E. coli O157: H7 include routine cultivation method, immunologic test and PCR. Traditional method for routine detection of E. coli O157: H7 is complex, time-consuming and lower sensitivity. The immunologic test had low sensitivity. The PCR methods provide powerful tools for rapid, specific, and sensitive detection of food-borne pathogens. However, owing to the expensive systems required and complicate electrophoretic analysis, this application is still not very common in laboratories. A loop-mediated isothermal amplification （LAMP） technology, which can provide a sensitive, specific, simple and cost-effective test for the rapid detection of E. coli O157: H7, was conducted in this assay. It can popularize and promote in common laboratories.This research used the rfbE gene sequence of E. coli O157: H7 （Genbank S83460） as target sequences with the help of Primer Explorer software （http://primerexplorer.jp/e/index.html） for LAMP primers design. The reaction conditions were optimized including the amount of Bst DNA polymerase, dNTPs and Mg2+ concentration etc. The LAMP mixture was made in 25μL of reaction mixture containing a 1.6μM concentration of each inner primer （FIP and BIP）, a 0.2μM concentration of each outer primer （F3 and B3）, 0.6 mM of each dNTP, 3.0 mM MgCl₂, 10×Bst DNA polymerase reaction buffer, 8 U of the Bst DNA polymerase large fragment, 1μL of isolated DNA templates and sterilized double-distilled water. The mixture was incubated at 64℃for 20 min and then heated at 80℃for 10 min to terminate the reaction. The positive reaction can be determined by the observable presence of the white precipitate in the reaction mixture.For an assessment of the sensitivity and detection limit of artificial contamination of LAMP, PCR with the two outer primers （F3 and B3） of LAMP was performed. We extracted DNA using the commercial kit. The detection limit of pure bacterial culture was 9.8 CFU/mL and the detection limit of artificially contaminated chicken sample was 68 CFU/g with LAMP detection for about two hours. In contrast, the detection limit of pure bacterial culture was 980 CFU/mL and the detection limit of artificially contaminated beef sample was 6.8×10³ CFU/g with PCR detection for about three hours. Our results showed this LAMP assay had a high specificity and time-saving for the detection of E. coli O157: H7 by amplifying a fragment of rfbE of three E. coli O157 strains while other sixteen non- E. coli O157 strains.The effects of three methods of DNA extraction from E. coli O157: H7 in beef sample on detection of E. coli O157: H7 were compared. The results showed that LAMP had less requirements for performing a successful detection.The result indicated that LAMP had the potential to replace PCR because of its simplicity, rapidity, specificity and cost-effectiveness. In conclusion, we developed a new and rapid molecular biological method for specific detection of E. coli O157: H7 in food sample as a basis of a technology platform.更多还原