Validation of a Qualitative Detection Method for Influenza A Virus RNA Based on the RT-RPA-CRISPR/Cas13a System.

Seasonal influenza A virus (IAV) frequently causes outbreaks, creating an urgent need for rapid, cost-effective detection. Existing methods have limitations: antigen tests lack sensitivity, RT-qPCR (the gold standard) requires bulky thermal cyclers, isothermal techniques like LAMP suffer from complex primer design and non-specific amplification, and many CRISPR-based diagnostics integrate nanotechnology or microfluidics, increasing cost and complexity without systematic clinical validation. To address these issues, we adapted RT-RPA combined with CRISPR-Cas13a into a simplified qualitative IAV detection platform. The workflow uses isothermal RT-RPA for amplification, Cas13a for specific recognition and trans-cleavage, and a lateral flow strip for visual readout. A rapid nucleic acid release reagent simplifies sample pretreatment. This approach retains the high sensitivity and specificity of nucleic acid testing while eliminating complex equipment. Preliminary performance evaluation showed that the platform produced a clear signal at 10¹ copies/mL of viral genome and exhibited no cross-reactivity with other respiratory viruses. Using RT-qPCR as the gold standard, testing of 78 clinical samples achieved 100% concordance. In summary, this study provides a practical simplification of an established RT-RPA-CRISPR/Cas13a workflow for IAV detection, supported by preliminary clinical validation, and demonstrates its suitability for rapid testing in low-resource settings.