【作者】 杨宇；

【导师】 牛承岗；

【作者基本信息】 湖南大学 ， 环境工程， 2016， 硕士

【摘要】 随着全球工业的发展,环境污染和食品安全问题日趋严重。环境保护和食品安全己成为本世纪影响人类生存和发展的重要问题。肠出血性大肠杆菌(Enterohemorrhagic escherichia coli, EHEC)是人类食源性以及水源性病原体,食用被该菌污染的食物会导致腹泻和肠炎,严重时将导致出血性结肠炎和溶血性尿毒症综合症,甚至死亡。EHEC以大肠杆菌0157：H7血清型为代表菌株,呈暴发流行趋势,且不断发生变异。建立一种快速、准确、简便检测大肠杆菌的方法,对环境监测和临床医学流行病学诊断有现实意义。因此,为了研究大肠杆菌的高灵敏快速检测方法,本文建立了一种基于萘酰亚胺标记的DNA探针和磁性四氧化三铁氧化石墨烯分离的大肠杆菌0157：H7检测方法。实验结果表明,在一定条件下,0157：H7数量的对数值和荧光强度与空白值荧光强度的比值(F/F0)呈线性关系,线性范围150～1.5×106CFU/mL,经过富集后检出限可达100 CFU/mL。该方法灵敏度高,检出限低,耗时较短,操作简单,为致病菌的高灵敏检测提供了新思路。本文将标记了萘酰亚胺的ssDNA(单链DNA)作为捕获探针吸附在磁性氧化石墨烯表面,当目标ssDNA存在时,捕获探针与目标ssDNA部分杂交,利用磁场将目标ssDNA捕获并进行富集,去除原溶液,然后加入释放探针溶液完成杂交,形成完整双链,使标记了荧光的捕获探针从磁性氧化石墨烯的表面释放出来,通过测定溶液荧光强度可以实现大肠杆菌0157：H7的高灵敏检测。本文对合成的目标DNA T和培养的大肠杆菌0157：H7均进行了检测试验,前者用来初步探索实验的可行性并优化实验条件,后者作为本实验体系的应用,对大肠杆菌的检测进行进一步的探索。论文研究内容和取得的研究成果主要体现在以下几个方面：(1)本实验合成了水溶性良好、荧光强度稳定的N-羟乙基-4-N(氨乙基)-1,8-萘酰亚胺(4-N-aminoethyl-N-hydroxyethyl-1,8-naphthalimide, AHA)并将其作为磁性富集荧光法检测大肠杆菌的荧光剂,为本实验测量出良好的荧光信号强度提供保障。(2)使用荧光分光光度计定量测量溶液荧光强度,建立检测大肠杆菌的新体系。以合成目标DNA为待测物,对各项实验条件进行优化,具体包括杂交温度、杂交时间、吸附时间、MGN浓度、释放探针R浓度和富集倍数。在最优条件下,通过检测不同浓度待测物存在时的荧光强度,研究待测物浓度和荧光强度之间的线性关系。(3)培养大肠杆菌0157：H7细菌溶液,通过平板菌落计数法确定其细菌数量,并用试剂盒提取其DNA,用以作为待测物,建立了对大肠杆菌检测的新方法,并检测该方法的灵敏度和实际应用性。更多还原

【Abstract】 Along with the development of the global industrialization process, the environment pollution and the food security problem have become more and more serious. Environmental issues and food security problem have become one of the most important problem which deeply influence human survival and development in this century. Enterohemorrhagic escherichia coli (EHEC) is a kind of human foodborne and waterborne pathogens. When eating the food contaminated with the bacteria, diarrhea and enteritis would attack our health. More seriously, it may lead to a hemorrhagic colitis and hemolytic uremic syndrome, and even go to a death. EHEC is represented by O157:H7, which showed atrend of outbreak and mutate constantly. It is of gteat significance for environment detection and clinical epidemiological diagnosis to establish a rapid detection mothod. In consequence, A novel sensitive assay is investigated for the highly sensitive and rapid detection of Escherichia coli O157:H7. It is based on magnetic Fe3O4/graphene oxide (MGO) and DNA probes which is modified by 4-N-aminoethyl-N-hydroxyethyl-1,8-naphthalimide (AHA). The results suggest that the linear relationship is found between log (CFU/mL of E.coli) and the ratio of fluorescence intensities (F/Fo) from 150 to 1.5×106 CFU/mL. The detection limit is 100 CFU/mL. The highly sensitive, rapid and simple means is demonstrated to be a noble tool for the detection of pathogenic bacteria.In this paper, the mothod is based on magnetic Fe3O4/graphene oxide (MGO) and DNA probes which is modified by 4-N-aminoethyl-N-hydroxyethyl-1,8-naphthalimide (AHA). First, the ssDNA labelled with AHA was employed as a capture probe and adsorbed on the surface of MGO. When the target ssDNA appeared, capture probe hybridized with it in part. Then target ssDNA was fished and enriched via external magnetization to remove the original solution. Afterwards, a solution of release probe was added to the magnetic particles to complete the hybridization which resulted in separating the AHA from MGO. Then, high sensitive detection of E.coli O157:H7 would achieved by measuring the fluorescence intensity. In this paper, the target DNA T synthesized by Shanghai biological engineering corporation, and E. coli O157:H7 liquid bacterial germ were carried on the test. The former was employed to explore the feasibility of the test preliminarily, and the latter acted as the application of this system for the further exploring of detecting E.coli.The main results of this present work were concluded as follows:1. In this test,4-N-aminoethyl-N-hydroxyethyl-1,8-naphthalimide was synthesized. This material show superior water solubility and it is employed as fluorescer agent.2. High sensitive detection of E.coli O157:H7 had achieved by measuring the fluorescence intensity. The target DNA T synthesized by Shanghai biological engineering corporation was employed to explore the feasible conditions of the experiment, including hybridization temperature, hybridization time, absorption time, concentrations of MGO, concentration of release probe R and times of enrichment. Under the optimal conditions, the linear relationship is researched between log(CFU/mL of E.coli) and the ratio of fluorescence intensities (F/Fo).3. Cultivation of E.coli O157:H7 was conducted. The bacterial quantity was determined by plate colony counting method. Meanwhile, the DNA was extracted through gene extraction box which acted as the determinand under test. A novel method for detection of E.coli is successfully established, including studying the sensitivity and practical applications of this method.更多还原