Abstract

Although RAS mutations are concentrated in specific hotspots, their clinical detection remains reliant on next-generation sequencing (NGS), which is hindered by high costs, long turnaround times, and substantial sample requirements. Herein, we report suppression probe enrichment (SPE), a thermodynamically driven low-frequency-mutation enrichment integrated with multiplex TaqMan probes for ultrasensitive detection of RAS mutations. The SPE assay covers 286 mutation types across the three major RAS genes, which is 86.8% of the clinically relevant mutations in the Cancer Genome Atlas database. It achieves a 20-fold sensitivity improvement over conventional methods, with a limit of detection as low as 0.05% variant allele frequency. Compared to NGS, SPE reduces detection time by approximately 7-fold, lowers costs by 3 orders of magnitude, and requires only 1 mL of plasma. We applied SPE to 57 tissue and 83 blood CRC samples, achieving 93.8% positive concordance and 100% true negative rate relative to NGS. For 30 paired tissue-plasma samples, the overall concordance was 93.33% with a positive concordance of 86.67%. These findings highlight SPE as a cost-effective, rapid, and highly sensitive approach for clinical RAS genotyping in CRC and for guiding targeted therapy.