Ultrasensitive and specific detection of Coxsackievirus A10 using the reverse transcription multiple cross displacement amplification combined with CRISPR/Cas12a system.

Abstract

The escalating global burden of Coxsackievirus A10 (CVA10)-associated hand, foot, and mouth disease (HFMD), coupled with the lack of specific antiviral therapies, has created an urgent need for diagnostic methods that balance sensitivity, speed, and field-deployability, a balance that current molecular tools have yet to achieve. To address this gap, we developed CVA10-RT-MCDA-CRISPR analysis, an integrated platform combining reverse transcription multiple cross displacement amplification (RT-MCDA) with CRISPR-Cas12a-mediated detection. Targeting the conserved VP1 of the CVA10 gene, we designed a set of MCDA primers, along with an engineered CP1 primer and a specific gRNA. The assay achieved a detection limit of 0.28 copies/μL for CVA10 RNA standards and showed no cross-reactivity with non-target pathogens. Performance was validated using 112 clinical specimens, confirming the assay's feasibility in real-world settings. Collectively, these findings establish the CVA10-RT-MCDA-CRISPR assay as a practical solution that bridges the gap between laboratory-grade sensitivity and field-ready simplicity. By integrating isothermal amplification with CRISPR-based detection in a streamlined workflow, this platform not only addresses the specific challenges of CVA10 diagnosis but also exemplifies a versatile diagnostic framework applicable to other emerging pathogens in resource-limited settings.