Light-controlled one-pot RPA-CRISPR/Cas12a biosensor for MRSA detection based on tailed crRNA

IF 3.7 1区化学 Q1 CHEMISTRY, ANALYTICAL

Sensors and Actuators B: Chemical Pub Date : 2025-10-09 DOI:10.1016/j.snb.2025.138925

Zhou Yu , Yan Man , Weilin Li , Cong Shi , Peng Xiao , Qinghai Zhang

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引用次数: 0

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) poses a severe global health threat due to its antibiotic resistance and high mortality rates. Herein, we developed a novel light-controlled one-pot RPA-CRISPR/Cas12a sensing system for rapid, specific, and cost-effective MRSA detection. The sensing system features a "tailed crRNA" containing a photocleavable linker (PC-linker), which blocks target recognition through a self-forming hairpin structure, maintaining the CRISPR/Cas system in an inactive state during RPA. Upon ultraviolet irradiation, the PC-linker is cleaved, restoring crRNA activity and enabling one-pot integration of amplification and detection within a single closed reaction tube. The traditional two-step RPA-CRISPR/Cas12a method demonstrated a linear detection range of 8.9 × 10⁰ – 8.9 × 10⁶ CFU/mL with a limit of detection (LOD) of 6.3 CFU/mL, while the light-controlled one-pot RPA-CRISPR/Cas12a system achieved comparable sensitivity (LOD: 34.7 CFU/mL) and specificity with no cross-reactivity against non-target pathogens. Both systems exhibited excellent recovery rates (87.1–110.2 %) and reproducibility (RSD ≤ 6.22 %) in complex food matrices (lettuce, milk, salmon, sauce beef). This light-controlled approach significantly reduces cross-contamination risks while simplifying workflows, providing a sensitive, reliable, and user-friendly platform for point-of-need MRSA detection in food safety and clinical diagnostics.

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基于尾端crRNA的MRSA检测光控单锅式rna - crispr /Cas12a生物传感器

耐甲氧西林金黄色葡萄球菌（MRSA）因其抗生素耐药性和高死亡率而对全球健康构成严重威胁。在此，我们开发了一种新型的光控单锅RPA-CRISPR/Cas12a传感系统，用于快速，特异性和经济高效的MRSA检测。该传感系统的特点是含有一个“尾状crRNA”，其中包含一个光可切割连接子（pc -link），它通过一个自形成的发夹结构阻止目标识别，使CRISPR/Cas系统在RPA过程中处于非活性状态。在紫外线照射下，pc -连接器被切割，恢复crRNA活性，使扩增和检测在一个封闭的反应管内一锅整合。传统的两步RPA-CRISPR/Cas12a方法的线性检测范围为8.9 × 10⁰- 8.9 × 10⁶CFU/mL，检测限（LOD）为6.3 CFU/mL，而光控单锅RPA-CRISPR/Cas12a系统具有相当的灵敏度（LOD: 34.7 CFU/mL）和特异性，对非目标病原体无交叉反应性。两种体系在复杂食物基质（生菜、牛奶、三文鱼、酱牛肉）中均具有良好的回收率（87.1 - 110.2% %）和重现性（RSD≤6.22 %）。这种光控方法显著降低了交叉污染风险，同时简化了工作流程，为食品安全和临床诊断中的MRSA检测提供了一个敏感、可靠和用户友好的平台。

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来源期刊

Sensors and Actuators B: Chemical 工程技术-电化学

CiteScore

14.60

自引率

11.90%

发文量

1776

审稿时长

3.2 months

期刊介绍： Sensors & Actuators, B: Chemical is an international journal focused on the research and development of chemical transducers. It covers chemical sensors and biosensors, chemical actuators, and analytical microsystems. The journal is interdisciplinary, aiming to publish original works showcasing substantial advancements beyond the current state of the art in these fields, with practical applicability to solving meaningful analytical problems. Review articles are accepted by invitation from an Editor of the journal.