CRISPR/Cas12a2-Mediated Ultrasensitive Assay for Rapid Detection of H1N1 Influenza Virus RNA

The H1N1 influenza virus poses a major global public health challenge, highlighting an urgent demand for advanced diagnostic technologies that enable rapid and accurate detection. This study established a novel CRISPR-Cas12a2-based diagnostic platform for highly sensitive and rapid detection of influenza A H1N1 virus. Targeting the conserved M gene, the assay combined optimized crRNA design with the unique dual RNA cleavage and collateral ssDNA degradation activities of SuCas12a2, enabling fluorescence-based detection with a sensitivity of 5 copies/μL. Clinical validation of 205 nasopharyngeal swabs demonstrated 91.22% concordance with real-time qPCR (κ = 0.65) and identified 18 additional positive cases, indicating superior sensitivity. The assay achieved a detection limit of 5 RNA copies/μL within 90 min by combining through a reverse transcription PCR with CRISPR detection. Rigorous specificity testing confirmed 100% discrimination against other respiratory pathogens. These results positioned CRISPR-Cas12a2 as a robust diagnostic alternative, offering advantages in speed and sensitivity. This technology represents a significant advancement in molecular diagnostics, meeting the critical needs for rapid and accurate pathogen detection during outbreaks.