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 × 10