Fast and sensitive multiplexed diagnostic system enabled by real-time solid-phase PCR assay

Multiplexed pathogen detection is pivotal for the simultaneous identification of multiple nucleic acid targets in clinical diagnostics. Fluorescent reporters are widely employed for real-time monitoring of nucleic acid amplification due to their high sensitivity and specificity. However, as the demand for high-throughput multiplexing increases, conventional fluorescence-based real-time PCR approaches become increasingly limited by instrumentation complexity, spectral overlap, and cost. In this study, we present a scalable, real-time, and multiplexed solid-phase PCR (SP-PCR) assay that integrates solid-phase and liquid-phase amplification strategies via spatially resolved probe immobilization on a solid surface array. To realize this method, we designed an integrated fluidic handling platform comprising a dual-chamber PCR system and a novel mechanically actuated valving system to facilitate automated reagent transfer for PCR purification and amplification. After each amplification cycle, the liquid-phase PCR solution is actively separated from the solid-phase-bound probes, thereby eliminating background signal interference and enabling real-time monitoring of SP-PCR signals without the need for complex optical systems. The system demonstrates rapid, real-time detection and quantification of five viral pathogens with a limit of detection as low as 10 copies per reaction within 20 min. This platform offers a promising solution for high-throughput, low-cost, and simple-instrumentation multiplexed pathogen diagnostics.