Enhanced sensitivity and scalability in liquid biopsy through integrated high-volume cfDNA extraction and droplet digital PCR mutation detection

Introduction: Detecting rare tumor-derived variants in plasma requires both efficient recovery of cell-free DNA (cfDNA) and highly quantitative mutation analysis. Conventional extraction methods are limited by low input capacity and potential bias toward longer fragments, which can compromise sensitivity for low-frequency alleles. Methods: Plasma cfDNA was extracted using the nRichDX Revolution cfDNA Max20 Kit, a high-volume magnetic-bead-based system, and then compared with the Qiagen QIAamp Circulating Nucleic Acid Kit. Pooled human plasma (1-15 mL, n = 3) and contrived samples spiked with 1% EGFR E746_A750delELREA (COSM6223) and KRAS G12C (COSM516) mutant gBlocks were processed per manufacturer's protocols. cfDNA concentration was measured by Qubit fluorometry, and mutation fractional abundance was quantified using the Bio-Rad QX600™ Droplet Digital™ PCR System (ddPCR™). Linearity, recovery efficiency, and reproducibility were evaluated across input volumes. Results: The nRichDX Revolution cfDNA Max20 Kit demonstrated linear cfDNA recovery across 1-15 mL plasma inputs (R² = 0.98) and maintains scalability up to 50 mL without reconcentration or parallel extractions. When coupled with the Bio-Rad ddPCR System, mutation analysis consistently detected 1% variant allele frequencies, yielding 15-30% higher measured fractional abundance compared to the Qiagen QIAamp Circulating Nucleic Acid Kit, which uses carrier RNA.The nRichDX workflow preserved short cfDNA fragments critical for mutation detection, improving signal-to-background ratios and enhancing analytical sensitivity. Replicate variability remained below 10%, confirming robust precision across input volumes. Conclusions: Integration of the nRichDX Revolution cfDNA Max20 Kit with the Bio-Rad QX600 ddPCR System enhances analytical sensitivity, recovery accuracy, and scalability for cfDNA-based liquid biopsy applications. By maintaining cfDNA integrity and eliminating carrier RNA interference, this workflow enables the reliable quantification of low-frequency variants, supporting clinical research in early detection, therapy monitoring, and minimal residual disease assessment.