CRISPR-Cas12a介导的米粉中呕吐型蜡样芽孢杆菌的快速便携式检测研究

呕吐型蜡样芽孢杆菌是存在于高淀粉类米制食品中的典型致病菌。它能分泌耐热、耐酸的呕吐毒素，且不易灭活，具有引发呕吐性中毒甚至致死的风险。目前针对该菌的抗体制备难度大，而现有检测方法存在反应耗时、难以精准定量等技术瓶颈。本项目拟结合CRISPR-Cas12a和血糖仪构建米粉中呕吐型蜡样芽孢杆菌的快速、精准、便携式检测新方法。拟筛选特异性结合呕吐型蜡样芽孢杆菌的适配体作为识别单元，利用三链适配体探针的识别-结构转换特性，激活CRISPR-Cas12a高效切割单链DNA以实现信号放大，利用蔗糖转化酶将呕吐型蜡样芽孢杆菌浓度转换为葡萄糖浓度，通过磁分离及血糖仪对目的菌进行快速、精准、便携式检测，从而实现米粉及其加工过程中呕吐型蜡样芽孢杆菌污染的危害分析及关键点控制。本项目的执行将为防控米制品加工产业链中微生物污染提供重要理论依据，为粮食安全检测提供新的技术平台。

Emetic Bacillus cereus is typical pathogen mainly presents in rice-based food with high starch content. It can secrete heat-resistant and acid-resistant cereulide, which is not easily inactivated and has the risk of vomiting poisoning and even death. Until now, antibody against the target bacteria is difficult to prepare, while the existing detection methods suffer from time-consuming and having difficulty in accurate quantification. This project aims at rapid, accurate and portable detection of emetic Bacillus cereus in rice noodle combining CRISPR-Cas12a and glucose meter. At first, aptamer that specifically binds to emetic Bacillus cereus will be screened as recognition component. The molecular structure characteristic of triple-helix aptamer probe is proposed to activate CRISPR-Cas12a to efficiently cleave single-stranded DNA, thus achieving amplified signal and triggering the concentration variation of invertase in the solution. Then through magnetic separation and utilization of glucose meter, the rapid, accurate and portable quantification detection of emetic Bacillus cereus can be realized. Finally, this strategy is applied to the analysis and critical control of target bacteria pollution in rice noodle and its processing. The implementation of this project will confer theoretical basis for the prevention and control of microbial contamination in the rice-based product processing, and provide a new technical strategy for the food safety test.