Rapid and Visual Detection of Coxsackievirus A6 Using Reverse Transcription Multiple Cross Displacement Amplification Combined with Nanoparticle-Based Biosensor Platform

Coxsackievirus A6 (CVA6) is the most common causative pathogen of hand, foot, and mouth disease (HFMD) in children under 5 years of age and has caused multiple outbreaks in recent years. Currently, no effective vaccines or antiviral treatments are available. The present study introduces a detection assay, termed CVA6-RT-MCDA-LFB, which combines reverse transcription multiple cross displacement amplification (RT-MCDA) with a nanoparticle-based lateral flow biosensor (LFB). Ten specific primers targeting the VP1 gene region were designed for CVA6-RT-MCDA. The assay demonstrated an analytical sensitivity of 33 copies per reaction, with 100% specificity and no cross-reactivity against non-CVA6 strains. Clinical performance was evaluated using 70 anal swab samples and 42 throat swab samples, demonstrating 100% concordance between CVA6-RT-MCDA-LFB and commercial quantitative real-time PCR(qRT-PCR). The entire detection process could be completed within 1 h, including sample preprocessing (15 min), isothermal amplification (40 min), and result confirmation (1-2 min). This rapid turnaround, combined with simplicity and high accuracy, makes CVA6-RT-MCDA-LFB a promising Point-of-Care Testing (POCT) for CVA6, particularly in resource-limited settings.