Abstract

Circulating tumor DNA (ctDNA) represents an invaluable liquid biomarker with significant applications in early cancer screening, therapeutic efficacy evaluation, and recurrence monitoring. However, ctDNA detection faces some challenges: low abundance in plasma cell-free DNA (cfDNA), difficulty in distinguishing mutant from wild-type signals using conventional methods, and loss of short fragments due to length heterogeneity. To address these biological challenges, we developed Restriction Endonuclease-assisted Recombinase Polymerase Amplification (REA-RPA). Its core advantage is the continuous, specific degradation of wild-type sequences during amplification, which reduces false positive risks by minimizing non-specific signals and enhances detection sensitivity via mutant target enrichment. Based on REA-RPA, we developed a one-pot real-time fluorescent strategy with genotyping probes to suppress residual wild-type signals. We also extended REA-RPA to a primerless system, utilizing wild-type digestion products as endogenous primers. This approach overcomes conventional primer design constraints and detects ctDNA fragments of varying lengths. Using the APC c.4012C > T mutation as a proof-of-concept, our results demonstrated that the one-pot real-time fluorescent detection strategy can identify mutations abundance as low as 0.01 %, while the primerless genotype switching strategy is capable of detecting mutations at a 0.1 % abundance. These findings highlight the exceptional sensitivity and accuracy of REA-RPA, and underscore its translational potential in clinical applications such as early cancer detection and treatment monitoring.