Dual-Pathway Lateral Flow Assay for Rapid and Sensitive SARS-CoV-2 RNA Detection via CRISPR/Cas13a-Mediated SERS

Reverse transcription-polymerase chain reaction (RT-PCR) has been the gold standard for SARS-CoV-2 detection during the COVID-19 pandemic. However, its requirement for RNA-to-DNA conversion, reliance on centralized laboratory infrastructure, and lengthy turnaround times have limited its application in point-of-care (POC) settings. CRISPR/Cas13a-mediated lateral flow assays (LFAs) have emerged as promising alternatives for direct RNA analysis, yet their two-step workflows introduce procedural complexity and reduce sensitivity. To overcome these limitations, we developed a dual-pathway LFA strip based on surface-enhanced Raman scattering (SERS), which integrates CRISPR/Cas13a-mediated RNA cleavage and SERS detection into a single, portable platform. The device utilizes five vertically stacked paper layers with distinct geometries, enabling sequential CRISPR reaction and SERS quantification through two independent pathways. When tested with SARS-CoV-2 ORF1ab RNA targets, the system exhibited an 80-fold increase in sensitivity and a 10 min reduction in assay time compared to conventional fluorescence assays. Clinical validation using 18 samples (13 positives and 5 negatives) demonstrated high diagnostic accuracy, fully consistent with RT-PCR results. By unifying CRISPR-based RNA recognition and SERS signal amplification in a user-friendly format, this dual-pathway LFA strip offers a rapid, ultrasensitive, and practical diagnostic tool for infectious diseases in POC settings.