Restriction Endonuclease-Assisted Recombinase Polymerase Amplification: Application towards the Detection of ctDNA

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.