基于胞内过氧化氢酶作为酶标的免标记等离子体ELISA检测食源性致病菌活菌的研究

Foodborne illness has become a worldwide issue of public health, the most prominent of which microbial hazards. Research on easy to use, sensitive, rapid and automated detection technology for pathogenic bacteria is of great practical significance to suppress outbreaks of foodborne illness and strengthen the food safety. Combining the feature that most pathogenic bacteria are catalase-positive and the high sensitivity of the plasmonic color development based on the enzyme-guided growth of metallic nanoparticles, this project proposes a label-free plasmonic ELISA for the detection of living Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes using their intracellular catalases as enzyme label. After verifying the feasibility of plasmonic color development for both direct measurement and microplate-based immunoassay of the living bacteria in the pre-experiment, we intend to focus on enhancing the capture efficiency and the resulting sensitivity of the proposed assay, by taking the magnetic beads (MBs) as the solid phase and comparing the different bioconjugate methods for the better preparation of the immunological MBs. Then, a soft-driven repetitive immersion mixing and MBs transfer based system will be employed to automate the label-free plasmonic ELISA. Finally, the proposed assay will be further evaluated through collaborative detection of the same practical sample inspected simultaneously by the Hangzhou CDC, who uses the official analysis method of VIDAS® system or culture method. Hopefully, the project may provide new ideas and help pave the way for the R&D of Chinese detection technology and instrument for pathogenic bacteria with independent intellectual property.

食源性疾病已成为当今世界性的公共卫生热点，其中以微生物性危害最为突出。研究简便、灵敏、快速的致病菌自动化检测技术对于遏制食源性疾病的爆发、加强食品安全保障有着重大的现实意义。针对大多数致病菌为过氧化氢酶阳性菌的特点，结合酶致纳米粒子生长的等离子体显色反应的高灵敏性，本项目提出以活菌胞内的过氧化氢酶作为酶标的免标记等离子体ELISA检测鼠伤寒沙门氏菌、大肠杆菌O157:H7和单核细胞增生李斯特菌。在预实验验证了等离子体显色反应用于直接测活菌和酶标板上免疫分析活菌都可行的基础上，拟采用磁珠作为固相，比较抗体不同偶联方式所制免疫磁珠的性能，重点提升活菌的捕获率和检测的灵敏度。再通过往复蘸入式混合的磁珠转运系统提升新方法的自动化程度。最后，同步检测杭州市疾控中心抽检的实际样品，并与其官方标准方法比较结果，力求为我国研制具有自主知识产权的致病菌检测技术和仪器提供新思路。

食源性疾病已成为当今世界性的公共卫生热点，其中以微生物性危害最为突出。研究简便、灵敏、快速的致病菌检测技术对于遏制食源性疾病的爆发、加强食品安全保障有着重大的现实意义。针对大多数致病菌为过氧化氢酶阳性菌的特点，结合酶致纳米粒子生长的等离子体显色反应的高灵敏性，本项目提出以活菌胞内的过氧化氢酶作为酶标的免标记等离子体ELISA检测鼠伤寒沙门氏菌、大肠杆菌O157:H7和单核细胞增生李斯特菌。重点进行了以下研究：(1)等离子体显色条件优化，确保酶活测试的稳定；(2)探索以sEH蛋白为模型分析物免疫分析；(3)过氧化氢酶偶联物的制备及等离子体显色用于致病菌的检测。该研究可为降低食品安全风险、促进食源性疾病预防提供新型的快速检测技术和仪器研发思路。