HNB-RT-LAMP Coupled with CRISPR/Cas12a for the Simultaneous Detection of Enteroviruses and Enterovirus A71 in a Single Tube

作者：Tianchi Zhuang, Xingtong Chen, Wenwu Zhao, Zihan Zhou, Chang Gao, Yan Cui, Yun Liu, Minghui Ji · 发表于：ACS Omega · 年份：2025 · DOI：10.1021/acsomega.4c09729 · 被引用次数：6 · 研究领域：Viral Infections and Immunology Research、Advanced biosensing and bioanalysis techniques、Cytomegalovirus and herpesvirus research

High Resolution Image Download MS PowerPoint Slide Background: Enterovirus (EV) infections encompass a spectrum of diseases, with severe cases often linked to enterovirus A71 (EV-A71), highlighting its significance in EV screening. Although the CRISPR/Cas12a diagnostic system shows promise in nucleic acid detection, enhancing specificity and sensitivity when combined with reverse transcription-loop-mediated isothermal amplification (RT-LAMP), its limitation lies in targeting a single sequence, thus preventing the simultaneous detection of both EVs and EV-A71. Results: This study presents a novel one-tube strategy by integrating HNB (hydroxynaphthol blue)-RT-LAMP with CRISPR/Cas12a for the simultaneous detection of EVs and EV-A71 in a single tube. The assay initially detects EVs using an HNB colorimetric approach under natural light, followed by the specific detection of EV-A71 through CRISPR/Cas12a under blue light. The limit of detection for EVs was 10 copies/μL, and for EV-A71, it was 1 copy/μL. Clinical sample assays demonstrated that, compared to qPCR, the accuracy of HNB-LAMP-CRISPR detection for EVs and EV-A71 was 95.7 and 100%, respectively. Significance: In summary, this strategy offers a reliable and user-friendly approach for EV screening. Also worth mentioning is that the provided method has beneficial effects on rapid visualized detection.