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<p>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&#xa0;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&#xa0;min, achieving a detection limit of as low as 10<sup>3</sup> 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.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 发表日期：

    2026年5月23日

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  • Yang Li,
  • Jiufa Zhang,
  • Yijie Wang,
  • Xiaohe Huang,
  • Xinyue Zhang,
  • Cuiping Ma,
  • Chao Shi

摘要

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.

Graphical Abstract