基于CRISPR-SERS超灵敏感知的食源性致病菌多重检测新方法研究

食源性致病菌是引发食源性疾病的主要来源之一。研发灵敏度高、特异性强的多重快速检测方法对保障国家食品安全具有重要作用。本项目拟以SERS芯片为平台，通过引入标记有拉曼活性分子的ssDNA或RNA作为信号报告探针，巧妙建立传感策略，构建CRISPR-SERS传感器。以常见的致病菌目的基因为检测靶标，首先建立CRISPR/Cas12a-SERS和CRISPR/Cas13-SERS单重检测平台，并揭示其超灵敏感知机理。在此基础上，通过引入不同拉曼活性分子标记的信号报告探针，实现多信号输出，建立CRISPR-SERS多重检测平台。探索拉曼增强最大时SERS芯片的制备条件、CRISPR与靶标结合的最佳孵育条件以及探针分子在SERS芯片表面的最佳固定条件，通过不同峰下拉曼信号的变化实现对食源性致病菌的多重快速检测。本项目不仅解决了SERS传感器特异性差的难题，还为食源性致病菌的多重检测提供一种新方法。

Food-borne pathogens are one of the main reasons that causes food-borne diseases. It is important to ensure the country's food safety by developing rapid detection methods for multiple microorganisms with high sensitivity and specificity. This project intends to develop a CRISPR-SERS sensor based on the SERS chip as detection platform. The sensing strategy was smartly established by introducing an ssDNA or RNA labeled with Raman active molecule as signal report probe. Aiming at some target genes of food-borne pathogens, a CRISPR/Cas12a-SERS and CRISPR/Cas13-SERS detection platform for single target was firstly developed and the ultra-sensitive sensing mechanism was explored. On this basis, the multiple detection platform with multiple signal output ability was developed by introducing signal reporter probes labeled with different Raman active molecules. Some experiment conditions, including the preparation parameter of the SERS chip Raman with the highest enhancement factor, the incubation time for the binding of CRISPR and the target, the immobilization conditions of the probe on the SERS chip, were fully investigated. The Raman signal change at different perks can be used to indicate the concentration of target DNA, thus the multiple and rapid detection method for food-borne pathogens. In this project, we can not only solve the problem of poor specificity of SERS sensors, but also provide an alternative method for the multiple detection of foodborne pathogens.