Rapid Differential Diagnosis of Influenza A and B Viruses With RT‐RPA Centrifugal Microfluidic Chip

Influenza A H1N1 and Influenza B are two of the most prevalent airborne viruses, capable of triggering seasonal epidemics that affect millions of individuals globally. Rapid on-site identification of these two viruses is crucial to accurate clinical diagnosis and prompt control of public health risks. This study presents a novel one-step reverse transcription recombinase polymerase amplification (RT-RPA) method integrated with a centrifugal microfluidic chip for rapid differential diagnosis of Influenza A H1N1 and Influenza B viruses. Addressing limitations of conventional techniques, the platform merges reverse transcription and amplification into a single step, reducing aerosol contamination risk and enabling on-site detection. Specific primers and probes targeting H1N1-HA, H1N1-NA, IVB-HA, and IVB-NA genes were designed, achieving detection limits of 10² copies/mL for H1N1-HA and 10¹ copies/mL for other targets within 25 min. The system demonstrated 100% specificity against common respiratory viruses and no cross-reactivity. Validation with 26 aerosol samples collected from public areas using bioaerosol sampler identified two H1N1-positive cases, showing 96.15%-100% consistency with off-chip qRT-RPA. The chip required only 6.25 µL of sample, exhibited high reproducibility (CV <5%). This microfluidic-RT-RPA system offers a practical solution for point-of-care influenza subtyping, enhancing outbreak control and clinical decision-making in resource-limited settings.