Multiplexed, universal probe-based rare variant detection with USE-PCR.

Polymerase chain reaction (PCR) is an essential tool in research and diagnostics but is limited by the number of resolvable targets, reliance on target-specific probes, and assay-specific data interpretation. To overcome these challenges, we introduce Universal Signal Encoding PCR (USE-PCR), a novel approach combining universal hydrolysis probes, amplitude modulation, multispectral encoding, and standardized analysis for robust, scalable target detection. Using 32 synthetic templates, USE-PCR demonstrates a mean target identification accuracy of 92.6% ± 10.7% at high template copy and 97.6% ± 4.4% at low template copy, with linear correlation coefficients of 0.99 across four dPCR platforms and a dynamic range spanning four orders of magnitude. Integrating USE-PCR with RNase H-based detection chemistry enables 32 single nucleotide variants to be called simultaneously with up to 86.5% accuracy in cancer cell lines. Together, these results position USE-PCR as a transformative platform for high throughput, multiplexed analyte detection with applications in research and clinical settings.