基于串联CRISPR-Cas13a的食品包装病原微生物污染免扩增分子检测

Amplification-free molecular detection of pathogenic microbial contamination in food packaging based on tandem CRISPR-Cas13a

中文关键词:  病原微生物  规律间隔成簇短回文重复序列和规律间隔成簇短回文重复序列相关蛋白13a  免扩增检测  食品包装  食品安全

英文关键词:pathogenic microorganisms  clustered regularly interspaced short palindromic repeats-associated protein 13a  amplification-free detection  food packaging  food safety

中文摘要:

      目的 开发一款基于串联规律间隔成簇短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)和CRISPR相关蛋白13a (CRISPR-associated protein 13a, Cas13a)组成的CRISPR-Cas13a快速、灵敏的食品包装病原微生物检测技术。方法 利用CRISPR-Cas13a技术的特异识别和信号放大特性, 同时基于对病原RNA多靶点识别的串联设计, 通过一个病原RNA分子激活多个Cas13a, 反式切割反应体系中的RNA信号报告分子并伴随荧光信号产生, 协同提升信号放大效率, 从而实现对病原体的灵敏检测。结果 该方法对病原微生物及RNA的检出限分别为800 copies/mL和18.7 pmol/L, 能够在较短时间内完成病原RNA检测, 同时能够准确区分不同病原微生物。测试新型冠状病毒(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)假病毒污染的食品包装样品显示, 方法测定的回收率在89.71%~102.56%。结论 本研究提供了一种快速、经济且操作简便的食品包装病原微生物污染检测方法, 具备较好的准确性。对食源途径风险微生物的监控具有潜在应用价值。

英文摘要:

      Objective To develop a rapid and sensitive detection technology for pathogenic microorganisms in food packaging based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 13a (Cas13a) system. Methods The method leveraged the specific recognition and signal amplification characteristics of the CRISPR-Cas13a technology. Multiple guide RNAs were designed to target different sites on pathogenic RNA, enabling a single RNA molecule to activate multiple Cas13a enzymes simultaneously. This cascade triggered trans-cleavage of RNA reporter molecules in the reaction mixture, generating a detectable fluorescent signal. The tandem design synergistically enhanced signal amplification efficiency, thereby improving detection sensitivity. Results The limits of detection of this method for pathogenic and RNA were 800 copies/mL and 18.7 pmol/L, respectively, and the detection could be completed in a short time, accurately distinguishing different pathogens. Testing food packaging samples contaminated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudvirus showed recovery rates ranging from 89.71% to 102.56%. Conclusion This study presents a rapid, cost-effective and easy-to-operate method for detecting pathogenic microbial contamination on food packaging surfaces, indicating high accuracy, offering potential application value for monitoring microbial risks in foodborne transmission pathways.