【作者】 李青；

【导师】 丁世家；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2012， 硕士

【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^-10nM，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^-10⁵CFU 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。更多还原

【Abstract】 Salmonella, as one of the most common pathogens of foodbornedisease worldwide, generally spreads through the consumption ofcontaminated food of animal origin （mainly meat, poultry, eggs and milk）.Pathogenic Salmonella strains cause food poisoning, gastrointestinalinflammation, typhoid fever, and septicemia in both humans and animals. Itconstitutes a major public health burden and represents a significant cost inmany countries. Therefore, sensitive and rapid detection of Salmonella is ofout-most importance to prevent and control the infectious diseases.Electrochemical DNA biosensor has been widely used due to its highsensitivity, simple, fast, good selectivity and low cost. Various methods havebeen used for the detection of Salmonella, including conventional culturemethods, enzyme-linked immunosorbant assay （ELISA） and PCR,but theyall have many disadvantages. In this work, in order to overcome the inherentdisadvantages of traditional methods, a simple strategy for detection ofSalmonella was developed by integrating rapid DNA extraction, specificPCR with an invA gene-based electrochemical DNA sensor. This strategy presented a simple, rapid and sensitive platform for Salmonella detectionand would become a powerful tool for pathogenic microorganisms screeningin clinical diagnostics, food safety and environmental monitoring. Thisdissertation includes the following three parts:Preparation of the invA gene-based electrochemical DNA sensorThe invA gene was used to design specific probes and target forSalmonella by exploring the Gene Bank database. The specificity of primersfor the PCR amplification of invA gene fragment and probes or DNAsensing had been positively verified via the BLAST（Basic Local Alignment Search Tool）. The high sensitivity of the DNAsensor was achieved due to low nonspecific absorption on the electrodesurface, the strong binding of streptavidin-biotin, and efficient signalamplification through catalytic generation of a large number of alkalinephosphatase. The fabrication and hybridization processes were characterizedwith electrochemical impedance spectroscopy, square wave voltammetryand surface plasmon resonance respectively. The linear calibration range fortarget DNA detection was from1pM to10nM with a detection limit of0.5pM and the correlation coefficient of0.9984. DPV responses of the DNAsensor were compared after hybridization with the three differentoligonucleotides, respectively. The reproducibility of the proposed sensorwas investigated by detecting synthetic target DNA at5nM and100pM.The low LOD of the DNA sensor was achieved due to low nonspecific absorption on the electrode surface, the strong binding of streptavidin-biotin,and efficient signal amplification through catalytic generation of a largenumber of alkaline phosphatase. Thus，the electrochemical sensor based onthe highly specific DNA probes for invA gene sequence recognition wassuccessfully developed， which exhibited high sensitivity, satisfactoryselectivity and good reproducibility.PCR amplification of nucleotide sequence within the invA gene ofSalmonellaSalmonella typhimurium strains were cultured under suitableconditions, a simple boiling method was used to extract Salmonella genomeDNA, and PCR was then performed using the genomic DNA extracted fromSalmonella. PCR products were determined by2%agarose gelelectrophoresis and observed under ultraviolet light. The resultsdemonstrated that the amplification of a284bp fragment of invA gene wassuccessfully achieved. PCR was then performed using the genomic DNAextracted from Salmonella with a series of concentrations. PCR product wasdenatured by heating in water bath, and immediately chilled in ice to obtaindenatured ssDNA before detection. The electrochemical DNA sensor wasthen applied to analyze the denatured PCR products. Under optimalconditions, the proposed strategy could quantitatively detect Salmonellafrom10to10⁵CFU mL^-1within3.5h. The sensitivity of the proposedstrategy was further compared with other methods reported previously for the detection of Salmonella including surface plasmon resonance （SPR）,field effect transistor （FET）, fluorescence, magnetoelastic biosensor,capacitive immunosensor, quartz crystal microbalance （QCM）, fiber-opticbiosensor, piezoelectric immunosensor. This proposed strategy possessedthe advantages of excellent sensitivity, rapid detection and low cost, whichwould provide a convenient platform for Salmonella screening inbiomedical diagnostics, food safety, biothreat detection and environmentalmonitoring.更多还原