基于CRISPR-Cas12a/-Cas13a的食源性致病菌检测新方法的研究

食源性致病菌污染是我国和全球最广泛的食品安全问题之一。CRISPR-Cas是细菌等进化出的一种获得性免疫系统。CRISPR-Cas12a/-Cas13a的作用对象为核酸，具有突出特征，包括可编程性、序列特异性以及底物引发的“附属切割”。本项目充分利用CRISPR-Cas12a/-Cas13a的生物学特性，将其引入致病菌检测领域，建立新型检测平台，解决现有方法存在的一些不足。项目计划探索CRISPR-Cas12a/-Cas13a与传统核酸扩增、分析方法的兼容性；利用其“下拉”能力建立高通量检测方法；利用其附属切割活性建立POCT检测。最后对标目前常用方法，检验所构建方法在真实食品检测中的表现。建立的平台方法能满足快速、高灵敏、高特异性、高通量化、现场化的检测需要。本项目拓展了CRISPR-Cas这一尖端技术的外延，占领了食源性致病菌检测的一个新制高点，为新方法的构建提供理论和物质基础。

Foodborne bacterial contamination is one of the most widespread food safety problems in China and the world. CRISPR-Cas is a kind of acquired immunity. The target of CRISPR-Cas12a/-Cas13a is nucleic acid, and this system has striking characteristics, including programmability, sequence specificity and substrate induced “collateral cleavage”. The proposed project intends to take full advantages of the biological characteristics of CRISPR-cas12a/-cas13a system by introducing it into the field of foodborne bacterial detection and establish a series of novel detection methods to solved unmet problems. This project plans to explore the compatibility of CRISPR-Cas12a/-Cas13a with the traditional nucleic acid amplification and analytical methods; explore the use of its “pull-down” ability to establish high-throughput detection methods; explore the use of its collateral cleavage to establish POCT (Point-Of-Care Testing). At last, the proposed methods will be challenged with are real food detection in comparison with some currently established methods. Our proposed methods have the characteristics of rapidity, high sensitivity, high specificity, high-throughput and on-site for detection. This project will expand the reach of CRISPR-Cas system, a cutting-edge technology, seize a new commanding point in the filed of foodborne bacterial detection, and provides both theoretical as well as material basis for the development of novel foodborne bacterial detection.