Multiplex quantification of osteomyelitis pathogens based on bead array

Accurate diagnosis of osteomyelitis is hindered by the limitations of conventional culture‑based methods, which lack the capacity for rapid simultaneous identification and quantification of multiple pathogens. Mixed infections involving multiple pathogens often exacerbate clinical outcomes, necessitating rapid and multiplex diagnostic approaches. We developed a multiplex detection assay integrating asymmetric solid-liquid biphasic PCR with a bead array platform, designated ASL-PCR, for the simultaneous identification of five common osteomyelitis‑associated pathogens: Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Streptococcus pneumoniae (S. pneumoniae), Aspergillus fumigatus (A. fumigatus), and Candida albicans (C. albicans). In this method, asymmetric PCR (aPCR) in liquid phase generates single‑stranded amplicons that are captured in real time by primer‑conjugated beads and extended on the bead surface. Biotin‑dCTP is incorporated during amplification, and the resulting products are labeled with streptavidin-phycoerythrin (SA‑PE) and detected by flow cytometry. The assay exhibited a wide dynamic range (2.94 × 10⁶ copies/µL to 7.53 × 10⁸ copies/µL) and high sensitivity, with the limits of detection (LOD) for S. pneumoniae, E. coli, S. aureus, C. albicans, and A. fumigatus being 1.84 × 10⁶ copies/µL, 5.28 × 10⁶ copies/µL, 1.24 × 10⁷ copies/µL, 1.22 × 10⁷ copies/µL, and 1.47 × 10⁷ copies/µL, respectively-sufficient for detecting clinically relevant pathogen loads after amplification. Notably, the assay demonstrated excellent specificity, with cross‑reactivity between any two pathogens below 2.5%. Spike‑and‑recovery experiments in serum yielded recoveries of 88.67% to 108.56%, indicating that the assay maintains high quantification accuracy even in complex biological matrices. Comparison with quantitative real-time PCR (qPCR) demonstrated strong correlation. Collectively, these findings support the proof-of-concept analytical feasibility and preliminary technical performance of the ASL-PCR assay for simultaneous detection of osteomyelitis-associated pathogens.