基于核酸靶标免扩增CRISPR/Cas体系的分子速测技术研究进展  

Research Avances in Rapid Detection Technology Based on Nucleic Acid Amplification-free CRISPR/Cas System

作　　者：李柚 张岩 雒嘉炜 徐丹红 马娟 高梦祥[1] 刘华[2] 曾海娟 王金斌 LI You;XU Danhong;ZHANG Yan;LUO Jiawei;MA Juan;GAO Mengxiang;LIU Hua;ZENG Haijuan;WANG Jinbin(College of Life Science,Yangtze University,Jingzhou 434022,China;Biotechnology Research Institute,Shanghai Academy of Agricultural Sciences,Key Laboratory for Safety Assessment(Environment)of Agricultural Genetically Modified Organisms,Ministry of Agriculture and Rural Affairs,Shanghai 201106,China)

机构地区：[1]长江大学生命科学学院,荆州434022 [2]上海市农业科学院生物技术研究所,农业农村部农业转基因生物安全评价(环境)重点实验室,上海201106

出　　处：《中国生物工程杂志》2024年第6期104-115,共12页China Biotechnology

基　　金：上海市科技计划(21N31900800);上海市青年科技英才扬帆计划(20YF1443000);上海市农业科学院卓越团队计划(B-16)资助项目。

摘　　要：快速准确的分子诊断在食品安全、疾病诊断、环境监测等生命科学领域发挥重要作用。成簇规则间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)及相关蛋白(CRISPR-associated protein,Cas)组成的CRISPR/Cas系统因具有高灵敏度、高特异性、便捷性及快速等优势,已成为精确检测核酸靶标的重要分子诊断工具。基于CRISPR/Cas系统的检测通过预扩增步骤提高靶标浓度,但也随之带来气溶胶污染、操作复杂和检测周期长等问题。因此需要积极开发更多免扩增策略,在最大限度保证灵敏度的同时,一步式实现对靶标的检测。综述了免扩增CRISPR系统的重要性,重点阐述了反应影响因素及相关策略对体系的优化作用,以及数字芯片法、表面增强拉曼光谱法和电化学金属电极法对信号放大效果的前沿进展,并对CRISPR/Cas系统的即时检测应用进行展望,以期为免扩增CRISPR/Cas检测系统的发展提供理论指导。Rapid and accurate molecular diagnostics play an important role in food safety,disease diagnosis,environmental monitoring and other life science fields.The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)system has become an effective molecular diagnostic tool due to its advantages such as sensitivity,specificity,convenience and speed.However,most current detection methods based on CRISPR/Cas still rely on the pre-amplification of nucleic acid,which can produce non-specific amplicons,prolong the detection time,and increase the risk of carryover contamination.Therefore,more amplification-free strategies have been developed to maximize the sensitivity while achieving target detection in a single step.In this review,the importance of amplification-free CRISPR systems,influencing factors and related strategies to optimize the system are summarized,followed by the three main strategies to amplify the signal of target amplification-free CRISPR/Cas-based technology,including the digital CRISPR detection platforms,surface-enhanced Raman spectroscopy,and electrochemical(EC)/ECL biosensors.Finally,the future of the CRISPR/Cas system is envisioned to provide theoretical guidance for the application and development of amplification-free CRISPR/Cas detection systems.

关 键 词：成簇规则间隔短回文重复序列(CRISPR) 免扩增检测 体系优化 信号放大 

分 类 号：Q78[生物学—分子生物学]