Breaking the channel barrier: same-fluorescence, different-Tm probe strategy enables seven-target respiratory virus...

Respiratory pathogens with overlapping symptoms-including SARS-CoV-2, influenza A/B, H5/H7/H10 avian influenza, and RSV-co-circulate and co-infect, demanding rapid multiplex screening at points of care. Conventional multiplex real-time PCR is constrained by hardware channel capacity (≤6), making it difficult to detect seven targets while distinguishing influenza A and B in a single tube. This work addresses the contradiction between high-order multiplexing and limited fluorescence channels without sacrificing speed or sensitivity.

We developed a six-channel ultra-fast PCR platform detecting seven respiratory viruses in a single tube within 20 min, using a same-fluorescence, different-Tm probe strategy. Influenza A and B share the FAM channel yet generate distinct melting peaks (∼12 °C difference, 84.8 °C vs. 72.3 °C), enabling accurate typing via melting curve analysis. Primers, probes, and enzymes are pre-embedded as lyophilized beads in sealed cartridges for fully automated sample-to-result operation. The assay achieved 100% detection at 5 copies/test for all targets, linearity (R² > 0.95) across 5-10⁵ copies/test, 90-110% efficiencies, and no cross-reactivity against 39 non-target pathogens. Cartridges remained stable for 12 months at 25 °C. Validation on 271 clinical specimens showed 100% agreement with sequencing-verified single-plex qPCR.

This work provides the first integration of melting-curve-based same-channel multiplexing with lyophilized cartridge technology on an ultra-fast PCR platform. By overcoming channel limitations while maintaining speed and sensitivity, the system offers a practical, field-deployable screening tool for decentralized respiratory pathogen detection in resource-limited settings.