基于限制性核酸内切酶放大技术的沙门氏菌检测生物传感器

发布时间：2018-09-03 11:16

【摘要】：本文详细介绍了食源性致病菌的危害、常见种类及研究现状。核酸适配体的功能及其特点和应用。限制性核酸内切酶放大技术的特点及其在生物传感器检测中的应用。并对核酸适配体生物传感器做了相关改进,用于检测食源性致病菌。1、我们通过适配体的特异性识别和吸附功能设计了简便、省时、一步检测鼠伤寒沙门氏菌的荧光生物传感器。该方案主要基于链置换放大-聚合酶延伸对目标物进行灵敏检测。实验巧妙设计了一个拱形探针(由aptamer和primer构成)和一个发夹探针(HP)。发夹的3’端和5’端分别修饰了荧光基团和猝灭基团。只有当目标物沙门氏鼠伤寒菌存在的条件下,才能够破坏拱形探针,释放primer链。被释放的primer链打开发夹探针,使猝灭基团和荧光基团远离,产生荧光。同时在聚合酶的延伸作用下,发生连置换反应,使primer链能够重复利用,达到信号放大的目的。因为信号放大和荧光强度检测的本质上的高灵敏性,600 cfu m L-1的致病菌可以在2 h内得到检测,这样比之前报道的检测方法更加的敏感和有效。因此它为发展链置换放大效应基于超灵荧光强度来检测致病菌的发展和食品安全分析提供了良好的应用平台。2、在这项工作中,基于目标物诱导的指数放大反应,我们发明了一种简单、迅速、等温、和超灵敏的均匀比色适配体传感器来对致病菌进行探测。这种适配体-引物探针包括结合鼠伤寒沙门氏菌的适配体和一段引物序列,这种发夹探针包括一个引物单元,这个适配体-引物探针被用于识别目标物并且通过基于目标物而激发指数放大反应(EXPAR)。由于EXPAR耦合DNA酶的放大策略,在目标物致病菌出现的时候,将会在溶液中形成大量大G-四联体低聚物,然后在钾离子和血红素的辅助下,又折叠成为G-四联体/血红素复合物,这种复合物对于过氧化氢有很强的催化活性,并且产生很强的紫外吸收。这种新颖的EXPAR耦合模仿过氧化酶催化放大技术通过比色法来探测致病菌。在最佳条件下,这种生物传感器对于致病菌的检测是非常灵敏的,在4 h之内它的检测限度是80 cfu m L-1.同时,我们的生物传感器对于目标物致病菌的检测有很高的专一性,同时,还有检测快速、廉价、操作简单、不需要进行标记和加入不稳定试剂的优点。因此,这种基于EXPAR耦合模拟DNA酶的比色方法将会成为检测致病菌的有效和实用的平台,其在食品安全分析和环境监测上都有很大的用处。
[Abstract]:In this paper, the harm, common species and research status of foodborne pathogenic bacteria are introduced in detail. Functions, characteristics and applications of aptamers of nucleic acids. The characteristics of restriction endonuclease amplification and its application in biosensor detection. We also improved the aptamer biosensor for the detection of foodborne pathogenic bacteria. We designed a simple and time-saving fluorescent biosensor for the detection of Salmonella typhimurium through the specific recognition and adsorption function of the aptamer. This scheme is mainly based on chain replacement amplification-polymerase extension for sensitive detection of the target. An arched probe (composed of aptamer and primer) and a hairpin probe, (HP)., have been ingeniously designed. The 3 'and 5' ends of the hairpin were modified with fluorescence group and quenching group, respectively. Only in the presence of Salmonella typhimurium could the arched probe be destroyed and the primer chain released. The released primer chain opens the hairpin probe, leaving the quenching group and the fluorescence group away to produce fluorescence. At the same time, under the extension of polymerase, the primer chain can be reused to achieve the purpose of signal amplification. Because of the intrinsic sensitivity of signal amplification and fluorescence intensity detection, the pathogens of 600 cfu mL ~ (-1) can be detected within 2 h, which is more sensitive and effective than the previously reported detection method. Therefore, it provides a good application platform for the development of chain replacement amplification based on superfluorescence intensity to detect pathogenic bacteria and food safety analysis. We have developed a simple, rapid, isothermal, and hypersensitive uniform colorimetric aptamer sensor to detect pathogens. The aptamer primer probe consists of an aptamer bound to Salmonella typhimurium and a primer sequence, and the hairpin probe includes a primer unit. This aptamer primer probe is used to identify the target and to excite the exponential amplification reaction based on the target (EXPAR). Due to the amplification strategy of EXPAR coupled with DNA enzyme, a large number of large G- tetraad oligomers will be formed in the solution when the target pathogen appears, and then folded into G- quadruplex / heme complex with the help of potassium ion and heme. The complex has strong catalytic activity for hydrogen peroxide and strong UV absorption. The novel EXPAR coupled imitating peroxidase catalytic amplification technique was used to detect pathogenic bacteria by colorimetry. Under the optimum conditions, the biosensor is very sensitive to the detection of pathogenic bacteria, and the detection limit is 80 cfu ml -1 within 4 h. At the same time, our biosensors have the advantages of high specificity for the detection of target pathogens, rapid detection, low cost, simple operation, and no need to label and add unstable reagents. Therefore, this colorimetric method based on EXPAR coupled simulation of DNA enzyme will be an effective and practical platform for the detection of pathogenic bacteria, and it will be very useful in food safety analysis and environmental monitoring.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TS207.4;TP212.3

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