Single-reaction KRAS variant profiling via processor-mediated PCR: Enhanced SNV discrimination using the iProbe

Accurate discrimination of single-nucleotide variants (SNVs) remains a major limitation of quantitative PCR (qPCR) and digital PCR (dPCR). Even with optimized hydrolysis probes, subtle thermodynamic differences between alleles often lead to cross-reactivity, reduced sensitivity near the limit of detection, and the need for multiple singleplex reactions. These constraints are particularly problematic for oncology applications requiring detection of low-frequency variants across closely spaced mutation sites. KRAS mutations in codons 12 and 13 represent clinically actionable drivers in multiple solid tumors, influencing prognosis and response to targeted therapies. However, the extreme proximity of these variants makes simultaneous, highly specific detection difficult; most current PCR-based assays require separate reactions or exhibit off-target signal among neighboring alleles. We developed processor-mediated PCR, a detection architecture that decouples target amplification from readout using a universal “processor” oligo and universal fluorescent probes. This chemistry supports high multiplexing, reduced assay cost, and tunable specificity independent of fluorophore channel limitations. In parallel, we engineered a modular multi-domain probe structure, iProbe, that generates signal only upon complete hybridization to a target variant, enabling exceptional SNV discrimination. To demonstrate this platform, we created a single-tube KRAS G12/G13 assay combining processor-mediated PCR with domain-engineered iProbes. In dPCR, the assay simultaneously detected all major KRAS codon 12 and 13 mutants with no detectable cross-reactivity. Sensitivity studies confirmed reliable detection at allele frequencies down to 0.01% (1:10,000 mutant:wild-type) with clean digital separation. Processor-mediated PCR with iprobes provides a powerful solution to longstanding specificity and multiplexing barriers, enabling cost-effective, highly selective detection of clinically relevant SNVs in a single reaction.