链球菌适配体制备及应用

发布时间：2018-03-01 17:33

本文关键词： 指数富集配体的系统进化技术寡核苷酸适配子化脓链球菌无乳链球菌磁性纳米材料　出处：《江南大学》2013年硕士论文　论文类型：学位论文

【摘要】：指数富集配体的系统进化技术（Systematic Evolution of Ligands by ExponentialEnrichment，SELEX），是90年代初研制成功的一种新的组合化学技术。适配子（Aptamer）实质是利用SELEX技术，从体外人工合成的随机寡核苷酸（ssDNA）文库中经过数轮反复筛选，得到的与靶分子具有极高特异性和亲和力的一段寡核苷酸序列。目前，已报道的食源性致病菌的检测方法多种多样，但各有其优缺点。本研究通过细胞SELEX技术（Cell-SELEX）筛选，得到了两种致病菌（化脓链球菌和无乳链球菌）的寡核苷酸适配子，并且建立了基于寡核苷酸适配子识别的新型检测技术。本文首先体外合成一个80nt，中间40个随机碱基序列的ssDNA文库，以化脓链球菌为靶标，通过Cell-SELEX技术及反筛SELEX技术（counter SELEX）进行12轮重复筛选，将筛选得到的DNA序列进行克隆、测序。运用DNAMAN软件、RNAstructure软件对其进行一级结构及二级结构分析；通过羧基荧光素（FAM）标记筛选得到的ssDNA适配子，运用流式细胞术考察了适配子与目标化脓链球菌适配子结合的亲和力和特异性，筛选得到两条亲和力高、特异性强的适配子，其解离常数分别为44.25±5.203nmol/L、53.89±7.586nmol/L，与化脓链球菌的结合率为77.05%、72.65%。同时，体外合成另一个相同长度、不同引物的寡核苷酸文库，，以无乳链球菌为靶标，采用与化脓链球菌类似的方法得到了可以与无乳链球菌特异结合的7条寡核苷酸适配子，对其进行FAM荧光标记，通过流式细胞术从中筛选得到两条高亲和力、高特异性适配子，与无乳链球菌的结合率分别为77.33%、80.95%，解离常数为78.27±12.85nmol/L，49.88±6.871nmol/L。随后，本研究以磁珠-适配子复合物为捕获探针，以FAM荧光素标记的适配子为显示探针，成功构建了灵敏、快捷的食源性致病菌荧光检测新方法。在实验优化条件下，化脓链球菌菌落个数在70-7100cfu/mL范围内时，与荧光强度呈良好线性关系，检出限为70cfu/mL，牛奶样品中的加标回收率为83.9%-103.3%；无乳链球菌菌落个数在50-5200cfu/mL范围内时，与荧光强度呈良好线性关系，检出限为50cfu/mL，牛奶样品中的加标回收率为95.2%-102.5%。
[Abstract]:Systematic Evolution of Ligands by ExponentialEnrichmentSelex, a new combinatorial chemical technique developed at the beginning of 90s, is based on the use of SELEX technique. A sequence of oligonucleotides with high specificity and affinity to the target molecule was obtained from the in vitro synthetic random oligonucleotide ssDNA library after several rounds of repeated screening. The reported methods for the detection of foodborne pathogenic bacteria are various, but each has its own advantages and disadvantages. In this study, the oligonucleotide aptamers of two pathogenic bacteria (Streptococcus pyogenes and Streptococcus lactobacillus) were obtained by cell SELEX technique (Cell-SELEX). A new detection technique based on oligonucleotide aptamer recognition was established. In this paper, we first synthesized an 80nt ssDNA library with 40 random base sequences in vitro. Using Streptococcus pyogenes as the target, we carried out 12 rounds of repeated screening by Cell-SELEX and reverse sieve SELEX technique, and cloned the selected DNA sequence. Sequencing. The primary structure and secondary structure of DNAMAN were analyzed by DNAMAN software. The ssDNA adaptor was screened by carboxyl fluorescein (FAM) labeling. The affinity and specificity of aptamer combined with target Streptococcus pyogenes aptamer were investigated by flow cytometry. Two aptamers with high affinity and strong specificity were obtained. Their dissociation constants were 44.25 卤5.203 nmol / L (53.89 卤7.586nmol / L), and the binding rate with Streptococcus pyogenes was 77.05nmol / L (77.05nmol / L). At the same time, another oligonucleotide library of the same length and different primers was synthesized in vitro. Using Streptococcus lactis as the target, seven oligonucleotide adapters were obtained by using a method similar to that of Streptococcus pyogenes. Two high affinity, high specific aptamers were screened by flow cytometry, and the binding rate of them to Streptococcus lactis was 77.33nmol / L. The dissociation constant was 78.27 卤12.85nmol / L (49.88 卤6.871 nmol / L), and the binding rates were 77.33nmol / L and 80.95nmol / L, respectively, and the dissociation constant was 78.27 卤12.85nmol / L ~ + 49.88 卤6.871 nmol / L respectively. Subsequently, a sensitive and fast fluorescent detection method for foodborne pathogenic bacteria was successfully constructed using magnetic bead aptamer complex as capture probe and aptamer labeled with FAM fluorescein as display probe. When the colony number of Streptococcus pyogenes was within the range of 70-7100 cfur / mL, it had a good linear relationship with the fluorescence intensity, the detection limit was 70 cfumL, the recovery rate in milk samples was 83.9 -103.3%, and the number of Streptococcus pyogenes colonies in the range of 50-5200 cfur / mL showed a good linear relationship with the fluorescence intensity. The detection limit was 50cfu-mL, and the recoveries in milk samples were 95.2- 102.5.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R155.5

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