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

<b>Background:</b> 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. <b>Results:</b> 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. <b>Significance:</b> 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.