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

ABSTRACT 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 10 2 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.