基于invA基因的电化学DNA传感器的构建及用于沙门氏菌快速高灵敏的检测

发布时间：2018-02-06 04:48

本文关键词： 电化学生物传感器 DNA检测 PCR 沙门氏菌 invA基因　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：沙门氏菌，是世界范围内最常见的食源性致病菌之一，主要通过污染的动物性食品（主要有肉类、家禽、蛋类和牛奶）传播。致病性沙门菌主要引起人类和动物的食物中毒、胃肠炎、伤寒和败血症等疾病，沙门氏菌不仅是公共卫生健康的重大问题，还给部分国家带来严重的经济负担。因此，为有效地预防和控制疾病发生，建立一种快速、简便、灵敏的检测沙门氏菌的方法是非常必要的。电化学DNA传感器以其灵敏度高，简单快速，选择性好，成本低廉的显著优点而成为一种应用广泛的检测手段。传统检测沙门氏菌的方法主要包括细菌培养，酶联免疫分析(ELISA)和特异性聚合酶链式扩增反应（PCR），但是均存一定的缺点，为克服沙门氏菌传统检测方法的局限性，本研究通过整合快速提取基因组DNA， PCR和构建基于侵袭蛋白A（invA）基因的电化学DNA传感器，发展了一种简单的检测沙门氏菌的策略，该策略能够实现简单、快速、灵敏的检测沙门氏菌，为临床诊断、食品安全和环境监测等领域中致病性微生物的检测提供了有力的工具。本研究主要包括以下三个部分：基于invA基因的DNA电化学传感器的构建通过GeneBank数据库，根据致病性沙门菌特异性invA基因设计靶序列和DNA探针，引物和探针特异性均用局部序列比对基本检索工具（BLAST）比对证实其特异性。通过减少非特异性吸附引起的背景信号，结合链霉亲和素-生物素耦合系统和酶催化底物的作用放大电化学信号来提高传感器的灵敏度。对电极表面的组装和杂交过程进行电化学阻抗（EIS）、方波伏安法（SWV)和表面等离子共振（SPR）表征，该传感器对靶序列响应的线性范围为1pM~(-1)0nM，相关系数为0.9984，检出限（LOD）达0.5pM。传感器的高灵敏度主要是由于电极表面较低的非特异性吸附，生物素链霉亲和素的结合能力及碱性磷酸酶强大的催化能力。通过检测三种不同序列的核苷酸的电化学信号考察传感器的特异性；同时考察两个不同浓度5nM和100pM的靶序列的重现性，变异系数均小于5%。本部分成功构建了针对invA基因的灵敏度高、选择性和重现性好的电化学DNA传感器。电化学传感器检测沙门氏菌培养鼠伤寒沙门氏菌，用水煮破细胞法快速提取细菌基因组DNA，用特异性引物扩增invA基因，作2%琼脂糖凝胶电泳，紫外凝胶电泳成像仪验证PCR产物，，结果表明，成功扩增沙门氏菌invA基因靶序列，片段大小为284bp。提取不同浓度细菌的DNA模板，PCR扩增后，将PCR产物加热后冰浴变性获得单链DNA，在最优的实验条件下，电化学DNA传感器对沙门菌的响应范围为10~(-1)0~5CFU mL~(-1)，灵敏度远远高于其它检测沙门氏菌的传感器包括SPR传感器，荧光传感器，磁电传感器，电容免疫传感器，石英晶体微量天平，光纤传感器及压电免疫传感器。整个检测过程仅需要3.5h，本策略具有灵敏度高，操作方便快速，成本低的优点，为医学疾病诊断，环境和食品卫生中微生物的筛选和检测提供了便利的平台。
[Abstract]:Salmonella is one of the most common foodborne pathogens in the world, mainly through animal food contamination (mainly meat, poultry, eggs and milk). The spread of Pathogenic Salmonella gastroenteritis is mainly caused by human and animal food poisoning, disease and sepsis, typhoid fever, Salmonella is not only a major problem public health, bring serious economic burden to some countries. Therefore, to effectively prevent and control the disease, to establish a rapid, simple and sensitive method for detection of Salmonella is necessary. The electrochemical DNA sensor with its high sensitivity, good selectivity, simple and fast, has the advantages of low cost as a detection method used widely. The traditional method for detection of Salmonella including bacterial culture, enzyme-linked immunosorbent assay (ELISA) and specific polymerase chain reaction amplification type (PCR), but There are some disadvantages, to overcome the limitations of traditional detection methods of Salmonella, this study through the integration of rapid extraction of genomic DNA and PCR based on invasion protein A (invA) DNA electrochemical sensor gene, developed a simple Salmonella detection strategy, this strategy can achieve a simple, fast, detection of Salmonella sensitive, for clinical diagnosis, provide a powerful tool for the detection of pathogenic microorganisms in the field of food safety and environmental monitoring etc.. This study mainly includes the following three parts:
Construction of DNA electrochemical sensor based on invA gene
Through the GeneBank database, according to the pathogenicity of Salmonella specific invA gene target sequence and DNA probes, specific primers and probes were used for basic local alignment search tool (BLAST) than in confirming its specificity. The background signal caused by non-specific adsorption, combined with streptavidin biotin and enzyme coupled system the effect of electrochemical catalyzed signal amplification to improve the sensitivity of the sensor. The electrochemical impedance of the electrode assembly and hybrid process (EIS), square wave voltammetry (SWV) and surface plasmon resonance (SPR) characterization, the sensor of the linear range of the response to the target sequence 1pM~ (-1) 0nM, correlation coefficient 0.9984, the limit of detection (LOD) with high sensitivity of 0.5pM. sensor is mainly due to nonspecific adsorption on the electrode surface is low, the binding ability and catalytic alkaline phosphatase strong affinity to biotin streptavidin Force. Through the electrochemical detection of three different signal sequences of nucleotide specificity of the sensor; the target sequence was also investigated in two different concentrations of 5nM and 100pM of the reproducibility, the coefficient of variation was less than 5%. this section was successfully constructed for invA gene with high sensitivity, selectivity and reproducibility of electrochemical DNA sensor.
Electrochemical sensor for the detection of Salmonella
Cultivation of Salmonella typhimurium, rapid extraction of bacterial genomic DNA boiled broken cell method, invA gene was amplified with specific primers, 2% agarose gel electrophoresis, UV Gel Electrophoresis imaging verification of PCR products, the results show that the successful amplification of Salmonella invA gene target sequence, fragment size of 284bp. extracted by different concentration of bacteria DNA template, PCR amplification, the PCR products after heating the ice bath for degeneration of single stranded DNA, under the optimal conditions, the electrochemical response range of Salmonella DNA sensor 10~ (-1) 0~5CFU mL~ (-1), the sensitivity of the sensor is far higher than other Salmonella SPR sensor, fluorescence sensor, magnetic the sensor, capacitive immunosensor, quartz crystal microbalance, fiber sensor and piezoelectric immunosensor. The whole detection process only needs 3.5H, this method has high sensitivity, easy operation, low cost It provides a convenient platform for the screening and detection of microbes in medical disease diagnosis, environment and food hygiene.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378;TP212.2

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