Temperature‐Resolved Coding for Highly Multiplexed Digital PCR Using MeltArray

ABSTRACT Digital PCR enables sensitive absolute nucleic acid quantification, but its broader application remains constrained by limited multiplexing capacity. Here we report digital MeltArray (dMeltArray), a temperature‐resolved endpoint decoding strategy that transfers MeltArray mediator‐probe chemistry to a static droplet‐imaging digital PCR system. In dMeltArray, target‐specific mediator products generate fluorescent reporter duplexes with programmed melting temperatures. Post‐amplification imaging at discrete temperatures progressively erases lower‐ T m signals while retaining higher‐ T m signals, allowing target‐specific concentrations to be recovered from Poisson‐corrected cumulative counts within each fluorescence channel. This strategy converts melting temperature into a programmable readout dimension requiring no real‐time tracking or full‐curve acquisition, addressing key trade‐offs between multiplexing capacity, workflow simplicity, and cost. dMeltArray can be combined with 5′‐hydrolysis probes, adding an orthogonal persistent‐signal readout in the same optical framework. As proof‐of‐concept applications, we developed a 13‐plex assay for post‐transplantation viruses, an 11‐plex assay for KRAS / BRAF hotspot mutations, and a 14‐plex assay that integrates a 5′‐hydrolysis probe into the 13‐plex panel. These findings establish temperature‐resolved endpoint decoding as a scalable route to multiplex digital PCR and support multidimensional target encoding by combining spectral, thermal and hydrolysis‐probe readouts without requiring full melting‐curve acquisition.