基于足点域介导的DNA链置换信号放大技术的构建及食源性致病菌高灵敏度检测研究

A novel signal amplification strategy based on toehold-mediated strand displacement will be developed for the ultrasensitive detection of food pathogenic bacteria. In the presence of the target, the catalyst C will be released from the dsDNA. After the substrate S and fuel F being added, the catalyst C will be recycled through the toehold hybridization and branch migration reaction and amount of signal strand SP will be released. Strong signal can be gained by detecting the SP. The new signal amplification strategy, which no need of nuclease, based on toehold hybridization coupling branch migration reaction will be firstly fabricated by detecting the released SP through the recycle and strand displacement. This signal amplification strategy can solve the scientific problems of needing the nuclease in DNA-based signal amplification. It also can enrich and develop the theory of signal amplification. The one-to-one recognition chemiluminescence triple Au nanoparticles (OR-CLTri-AuNPs) probe will be prepared with gold nanoparticles, chemiluminescence reagent, recognition and occupation DNA. The Salmonella typhimurium, choosed as the interested target, will be sensitively and selectively detected with the developed signal amplification strategy combined with the magnetic bead capturing and separation and OR-CL-Tri-AuNPs probe. The feasibility and reliability of the developed method will be evaluated by the determination of Salmonella typhimurium in real samples and microbiological examination controlled experiment and molecular biological detection. This study will provide a new and effective design idea and experimental basis for high sensitive food pathogenic bacteria determination and new analytical method and technology.

本课题拟开展基于足点域介导的DNA链置换信号放大技术的构建及食源性致病菌高灵敏度检测研究。课题拟利用足点域杂交反应和分支迁移技术，以目标物适体互补序列为催化剂C、基质S和燃料F实现催化剂C的循环利用，产生大量信号链SP，建立足点域介导的DNA链置换信号放大新方法；结合功能化磁性微珠的捕获和分离功能，以一对一识别的化学发光三胶体金纳米粒子为探针，构建基于足点域介导的DNA链置换信号放大化学发光分析新技术；以鼠伤寒沙门氏菌为模式分析测定对象构建高灵敏度和高选择性食源性致病菌分析测定新方法。利用微生物检验标准方法以及分子生物学方法对照等对新方法进行评价，并对实际样品中鼠伤寒沙门氏菌进行测定；对建立的分析测定新方法进行拓展。解决现有DNA循环信号放大技术中需要酶参与反应的限制性科学问题，丰富和发展信号放大的理论；为高灵敏度食源性致病菌检测提供有效的设计思路及实验依据。

建立快速、灵敏测定食源性致病菌的分析检测方法对食品安全、临床诊断、环境监测以及生物防御具有重要理论意义和实用价值。本项目按原计划完成了所有的研究内容，建立了鼠伤寒沙门氏菌、金葡萄球菌、副溶血性弧菌、单增李斯特菌、棒曲霉毒素、微囊藻毒素六种食源性致病菌和毒素和一种食品违禁添加品雷托帕明测定新方法；并围绕足点域介导新分析方法的构建开展了相关研究。.首先，利用足点域杂交反应和分支迁移技术，构建了足点域介导的DNA链置换信号放大技术，结合一对一识别化学发光三胶体金纳米粒子标记实现信号放大，建立了鼠伤寒沙门氏菌化学发光测定新方法。检测限为63 cfu/mL，以检测限为标准进行比较，项目所建立的方法比未经足点域介导的DNA链置换、直接以ABEI标记DNA为探针提高154倍和1×103倍。所建立的方法和提出的测定思路为高效、准确、灵敏的鼠伤寒沙门氏菌分析测定提供有效的设计思路及实验依据。利用金属有机凝胶（MOG(Fe)）对氨苄西林-H2O2的化学发光催化现象，构建了以氨苄西林为识别和捕获元素、以MOG(Fe)为自催化剂的高灵敏度化学发光发测定金葡萄球菌的新方法。合成了Cu/Co/Ni三元合金，以其为催化剂，以氨苄西林为识别元素，构建了测定单增李斯特菌的化学发光新方法。利用催化发卡自组装循环放大化学发光技术，建立了副溶血性弧菌检测新方法。发现在催化发卡自组装循环碱基配对中错配碱基的存在，能够降低背景信号、提高信噪比，据此构建了错配发卡自组装循环放大化学发光技术检测鼠伤寒沙门氏菌的新方法。基于位阻效应，构建了Cu/Co双金属纳米片催化化学发光效应检测微囊藻毒素的方法；还将该方法拓展应用于癌细胞的分析测定中：基于Cu/Co双金属纳米片和生物条形码细胞及位阻效应检测人急性淋巴白血病细胞CCRF-CEM。以血糖仪为检测器，建立了棒曲霉毒素测定新方法。该项目的研究结果将为食品中食源性致病菌和毒素含量的测定提供新的分析方法和重要信息，对开发新型分析技术具有重要意义；为足点域介导的链置换信号放大提供理论依据和实验技术。