基于荧光量子点调控的食源性致病菌高通量荧光定量PCR检测技术的构建

Foodborn pathogens are primary factors threatening food safety and human health. It’s very necessary to develop a foodborne pathogen detection method that is rapid, economical and high throughput. In this project, a hot start technique mediated by QDs combined with FQ PCR is used to develop a novel foodborne pathogen detecion method which is characterized by high sensitivity, high selectivity, high throughput and low cost. The effects of QDs composites, particle sizes, surface states and concentrations on PCR efficiency and selectivity are researched systematically. A high sensitive, high selective, high throughput and rapid FQ PCR can be constructed by the optimization of a PCR system based on QDs regulated. The hot start mechanism of QDs triggered is investigated by AFM, FCS, CE, enzyme activity assay, and etc. Listeria monocyto, Staphylococcus aureus and Escherichia coli O157∶H7 are chosen as experimental models. A high throughput FQ PCR based on QDs is applied to the detection of foodborne pathogens by the optimization of extraction of genome DNA, the design, synthesis and screening of specific primers and probes. The results of this project will have great theoretical significance and application value to ensure food safety and human health.

食源性致病菌是危害食品安全和人类健康的重要因素。目前缺乏快速、经济、高通量食源性致病菌量检测方法。本项目旨在将量子点（QDs）动态调控的类似“热启动”技术和荧光定量PCR（FQ PCR）相结合，建立高灵敏、高选择性、高通量、低成本的食源性致病菌的检测新方法。系统研究量子点的组成、粒径、表面状态、浓度等对PCR扩增效率和选择性的影响。优化量子点调控PCR扩增条件， 建立高灵敏、高选择性、高通量、快速FQ PCR新方法。通过原子力显微镜，荧光关联谱，毛细管电泳，酶活性测定等方法研究量子点与PCR成分之间的相互作用，揭示量子点的调控机理。以单增李斯特氏菌、金黄色葡萄球菌和肠出血性大肠杆菌为模型，优化致病菌基因组DNA的提取方法，设计、合成、筛选特异性扩增引物和探针，将建立的高通量 FQ PCR技术应用于食源性致病菌的检测。本研究对于保障食品安全及人类健康具有重要的理论意义和应用价值。

高特异性、高灵敏度、高准确性仍是PCR急需解决的关键性问题，目前大量商品化的含有热启动酶的 PCR 反应预混液已应用于 PCR，但其价格昂贵，不适合大量实际样本的常规检测。本项目首先将基于荧光量子点热启动的纳米酶技术应用于荧光定量PCR，系统地对不同的聚合酶、不同DNA来源（人类和质粒）等进行研究，构建高灵敏度、高选择性和高通量荧光定量PCR系统；另外还将基于荧光量子点热启动的纳米酶技术应用于多轮PCR扩增中，分别对不同GC含量、不同片段长度及不同模板来源（人类、海洋微生物及酒窖微生物）进行研究，发现基于量子点热启动的纳米酶优化多轮PCR扩增，使得多轮PCR的扩增到9轮，可广泛应用于低含量模板的扩增；同时本项目还通过紫外、红外以及荧光相关光谱技术（FCS)等对基于量子点热启动的纳米酶的机理进行探讨；最后通过对PCR扩增序列的测序分析，发现基于量子点热启动纳米酶技术没有对PCR的保真性产生影响。