Single-Tube Six-Channel Ultrafast PCR with Melting-Curve Discrimination for Seven Respiratory Pathogens

Expanding single-tube multiplexing beyond the number of fluorescence channels on a real-time PCR instrument is a persistent challenge for point-of-care testing (POCT). Here, we break this barrier by leveraging melting temperature (Tm) as an extra information dimension. By employing a probe-cleavage-mediated reporter-duplex strategy, we circumvent the conventional limitation that hydrolyzed TaqMan probes cannot participate in postamplification melting analysis. Two FAM-labeled probes, each containing an engineered 5'-terminal nontarget extension, generate cleavage-dependent reporter amplicons with distinctly different melting temperatures (ΔTm = 10.3 °C), enabling precise differentiation between BV (72.5 °C) and BY (82.8 °C) within a single optical channel. Combined with a lyophilized-bead cartridge that automates nucleic acid release (4 min) and an ultrafast PCR run (16 min), the platform detects eight targets─seven respiratory pathogens (SARS-CoV-2, influenza A H1N1/H3N2/H5N1 clade 2.3.4.4b, influenza B Victoria/Yamagata, and Mycoplasma pneumoniae) plus one internal control (B2M)─using only six optical channels. The assay achieves a limit of detection of 5 copies per reaction (R2 > 0.97, amplification efficiency 96-104%) with no cross-reactivity against 55 nontarget microorganisms, and intra/inter-assay CVs < 5%. The lyophilized beads remain stable for 12 months at 25 °C (ΔCt < 1.5). In 272 clinical throat swabs, the system shows 100% agreement with single-plex qPCR. Mixed BV/BY infections produce two distinct melting peaks, with the low-abundance peak detectable down to a 400:1 ratio under the tested conditions. This six-channel ultrafast PCR system reliably detects eight targets in one tube within 20 min without extra hardware, providing a generic strategy for higher-order multiplexing in point-of-care settings.