Development of a Reverse Transcription Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Visual and Highly...

Zika virus (ZIKV), a single-stranded positive-sense RNA virus of the Flaviviridae family (Flavivirus genus), causes acute febrile illness and severe congenital anomalies. Serological cross-reactivity with Dengue virus (DENV) and Chikungunya virus (CHIKV) complicates diagnosis, underscoring the urgency of developing specific point-of-care tests for early detection, outbreak mitigation, and reduced misdiagnosis risks. This study established and optimized an RPA-CRISPR/Cas12a assay for the rapid, visual, and highly specific detection of ZIKV. Primers and crRNAs targeting the highly conserved capsid (C) gene were designed, and the assay was systematically evaluated for its specificity, sensitivity, reproducibility, and clinical applicability. The RPA-CRISPR/Cas12a assay enabled naked-eye detection under UV light within 35 min. It demonstrated single-copy sensitivity (1 copy/μL), no cross-reactivity with DENV 1-4, CHIKV, or Japanese encephalitis virus (JEV), and 100% concordance with RT-qPCR in clinical validation. Repeatability tests showed low variability (coefficient of variation (C.V.) < 15%), confirming robust reproducibility. This instrument-free platform integrates rapid visual detection, single-copy sensitivity, high specificity, and field-deployable features, making it particularly suitable for point-of-care testing (POCT) in resource-limited settings. The developed assay provides critical support for early outbreak containment and prenatal screening in ZIKV-endemic regions.