Universal primer mediated multiplex asymmetric PCR coupled with hybridization-based electrochemical biosensing for rapid, simultaneous and label-free detection of multiple pathogens.

Multiplex PCR has emerged as a powerful tool for simultaneously detecting multiple pathogens that cause similar clinical symptoms within a single reaction. However, its reliance on fluorescently labeled probes and sophisticated optical instrumentation limits its applicability in resource-limited or grassroots settings. To address this limitation, we propose a rapid, label-free biosensing platform that enables multiplex pathogen detection with minimal equipment by coupling multiplex asymmetric PCR with hybridization-based electrochemical sensing. A universal primer mediated PCR system, termed ARMS-HANDS PCR, which combines the amplification refractory mutation system (ARMS) with the homo-Tag-assisted non-dimer system (HANDS), was implemented to suppress primer dimer formation and efficiently generate abundant single-stranded amplicons (ss-amplicons) specific to distinct respiratory pathogens. Unlabeled hairpin probes were employed to rapidly bridge the ss-amplicons and thiol-modified capture probes immobilized on microelectrode array chips, forming ternary hybridization complexes within 90 s that enhance both selectivity and the electrochemical signal of methylene blue. The platform demonstrated accurate detection of four bacterial pathogens in artificial nasal mucus samples within a single reaction in less than 50 min, achieving a detection limit of as low as 103 CFU/mL for target pathogens while effectively discriminating between closely related strains. This approach presents a promising platform for simultaneous detection of multiplex pathogens and is expected to facilitate rapid diagnosis of respiratory infections in clinical and point-of-care settings.