Dual color microsphere encoded by fluorescent protein and Cy5-labeled anti-Tag ssDNA for multiplex detection of respiratory pathogens.

Respiratory diseases are challenging to diagnose rapidly and accurately due to diverse pathogens, overlapping symptoms, and high infection rates. Here, we developed a suspension array based on magnetic microspheres encoded with blue fluorescent protein (mTagBFP) and Cy5-labeled anti-Tag ssDNA (MB@mTagBFP&anti-Tag-Cy5) for simultaneous detection of five core respiratory pathogens. Multiplex PCR was performed using 5'-Tag-labeled upstream primers and 5'-biotinylated downstream primers, yielding dual-labeled products (Tag and biotin). The resulting complexes (MB@mTagBFP&anti-Tag-Cy5-Tag-dsDNA-phycoerythrin) were analyzed by flow cytometry, achieving a detection limit of 1 copy/μL (3 SD/K). Linear quantification ranged from 101 to 105 copies/μL for respiratory syncytial virus and SARS-CoV-2, and from 101 to 106 copies/μL for influenza A, influenza B, and Mycoplasma pneumoniae. Recovery rates were 89.73-107.25%. Clinical testing of 100 samples yielded 97% sensitivity and 90% specificity, effectively distinguishing single and mixed infections. This integrated method enables rapid, high-throughput, and sensitive detection of respiratory pathogens, offering critical support for early diagnosis, treatment guidance, and epidemiological surveillance, with broad applications in infection control and disease prevention.