本文關(guān)鍵詞： 電化學(xué)生物傳感器 DNA檢測 PCR 沙門氏菌 invA基因　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：沙門氏菌，是世界范圍內(nèi)最常見的食源性致病菌之一，主要通過污染的動物性食品（主要有肉類、家禽、蛋類和牛奶）傳播。致病性沙門菌主要引起人類和動物的食物中毒、胃腸炎、傷寒和敗血癥等疾病，沙門氏菌不僅是公共衛(wèi)生健康的重大問題，還給部分國家?guī)韲?yán)重的經(jīng)濟(jì)負(fù)擔(dān)。因此，為有效地預(yù)防和控制疾病發(fā)生，建立一種快速、簡便、靈敏的檢測沙門氏菌的方法是非常必要的。電化學(xué)DNA傳感器以其靈敏度高，簡單快速，選擇性好，成本低廉的顯著優(yōu)點(diǎn)而成為一種應(yīng)用廣泛的檢測手段。傳統(tǒng)檢測沙門氏菌的方法主要包括細(xì)菌培養(yǎng)，酶聯(lián)免疫分析(ELISA)和特異性聚合酶鏈?zhǔn)綌U(kuò)增反應(yīng)（PCR），但是均存一定的缺點(diǎn)，為克服沙門氏菌傳統(tǒng)檢測方法的局限性，本研究通過整合快速提取基因組DNA， PCR和構(gòu)建基于侵襲蛋白A（invA）基因的電化學(xué)DNA傳感器，發(fā)展了一種簡單的檢測沙門氏菌的策略，該策略能夠?qū)崿F(xiàn)簡單、快速、靈敏的檢測沙門氏菌，為臨床診斷、食品安全和環(huán)境監(jiān)測等領(lǐng)域中致病性微生物的檢測提供了有力的工具。本研究主要包括以下三個部分： 基于invA基因的DNA電化學(xué)傳感器的構(gòu)建 通過GeneBank數(shù)據(jù)庫，根據(jù)致病性沙門菌特異性invA基因設(shè)計(jì)靶序列和DNA探針，引物和探針特異性均用局部序列比對基本檢索工具（BLAST）比對證實(shí)其特異性。通過減少非特異性吸附引起的背景信號，結(jié)合鏈霉親和素-生物素耦合系統(tǒng)和酶催化底物的作用放大電化學(xué)信號來提高傳感器的靈敏度。對電極表面的組裝和雜交過程進(jìn)行電化學(xué)阻抗（EIS）、方波伏安法（SWV)和表面等離子共振（SPR）表征，該傳感器對靶序列響應(yīng)的線性范圍為1pM~(-1)0nM，相關(guān)系數(shù)為0.9984，檢出限（LOD）達(dá)0.5pM。傳感器的高靈敏度主要是由于電極表面較低的非特異性吸附，生物素鏈霉親和素的結(jié)合能力及堿性磷酸酶強(qiáng)大的催化能力。通過檢測三種不同序列的核苷酸的電化學(xué)信號考察傳感器的特異性；同時考察兩個不同濃度5nM和100pM的靶序列的重現(xiàn)性，變異系數(shù)均小于5%。本部分成功構(gòu)建了針對invA基因的靈敏度高、選擇性和重現(xiàn)性好的電化學(xué)DNA傳感器。 電化學(xué)傳感器檢測沙門氏菌 培養(yǎng)鼠傷寒沙門氏菌，用水煮破細(xì)胞法快速提取細(xì)菌基因組DNA，用特異性引物擴(kuò)增invA基因，作2%瓊脂糖凝膠電泳，紫外凝膠電泳成像儀驗(yàn)證PCR產(chǎn)物，，結(jié)果表明，成功擴(kuò)增沙門氏菌invA基因靶序列，片段大小為284bp。提取不同濃度細(xì)菌的DNA模板，PCR擴(kuò)增后，將PCR產(chǎn)物加熱后冰浴變性獲得單鏈DNA，在最優(yōu)的實(shí)驗(yàn)條件下，電化學(xué)DNA傳感器對沙門菌的響應(yīng)范圍為10~(-1)0~5CFU mL~(-1)，靈敏度遠(yuǎn)遠(yuǎn)高于其它檢測沙門氏菌的傳感器包括SPR傳感器，熒光傳感器，磁電傳感器，電容免疫傳感器，石英晶體微量天平，光纖傳感器及壓電免疫傳感器。整個檢測過程僅需要3.5h，本策略具有靈敏度高，操作方便快速，成本低的優(yōu)點(diǎn)，為醫(yī)學(xué)疾病診斷，環(huán)境和食品衛(wèi)生中微生物的篩選和檢測提供了便利的平臺。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R378;TP212.2

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