Rapid and simultaneous on-site detection of influenza A(H1N1) virus and Mycoplasma pneumoniae nucleic acids using magnetic RPA ratiometric fluorescent lateral flow assay.

Influenza A(H1N1) virus [InfA (H1N1)] and Mycoplasma pneumoniae (MP) are key pediatric respiratory pathogens with overlapping symptoms, necessitating rapid and simultaneous on-site detection of nucleic acids for optimal clinical management. Herein, we developed a finger-actuated and contamination-resistant PEARL chip integrating magnetic bead-based pathogen enrichment and cascade duplex recombinase polymerase amplification (RPA) for a ratiometric fluorescence lateral flow assay (LFA), which allows both visual and quantitative detection of InfA (H1N1) and MP. 3-Aminophenylboronic acid (APBA)-functionalized magnetic beads enabled rapid target enrichment from complex matrices within 3 min, with capture efficiencies up to 97.8%. The ratiometric LFA strips operate based on fluorescence color ratio variation, which arises from the signal overlap of two fluorescent nanomaterials tagged with detection and capture antibodies. With the ratiometric LFA readout, the duplex RPA-facilitated assay yield visual detection limits of 100 copies/mL for InfA (H1N1) and 1000 copies/mL for MP, respectively. Moreover, a custom smartphone-based portable device and its accompanying application are available as an optional approach to enable quantitative analysis within the defined dynamic range. The entire PEARL chip process is completed within 20 min. Across 93 pediatric clinical samples, PEARL demonstrated strong concordance with qPCR, achieving 100% sensitivity for H1N1 and 93.3% sensitivity for MP, with 100% specificity for both pathogens. Overall, the PEARL chip provides a rapid, sensitive, and user-friendly platform for simultaneous detection of pediatric respiratory pathogens, holding great promise for decentralized and point-of-care diagnostic applications.