Establishment and Performance Evaluation of a Multiplexed TET2-APOBEC-Mediated cfDNA Methylation Detection Workflow Using qPCR and dPCR Readouts.

Background/Objectives: Bisulfite-based cell-free DNA (cfDNA) methylation assays enable the detection of clinically valuable epigenetic biomarkers but often cause DNA degradation and inconsistent conversion efficiency, limiting performance in low-input liquid biopsy samples. We aimed to develop and evaluate a fully enzymatic cfDNA methylation workflow that preserves DNA integrity and supports quantitative clinical detection. Methods: The assay integrates TET2-mediated oxidation and APOBEC3A deamination with RNase H2-guided primer design, uracil-DNA glycosylase error suppression, and dual-probe detection compatible with quantitative PCR (qPCR) and digital PCR (dPCR). Performance was assessed using serial dilutions of methylated HT29 DNA, unmethylated controls, and plasma cfDNA from colorectal cancer (CRC) patients and healthy donors. Analytical sensitivity, linearity, and concordance between platforms were evaluated. Results: The 40-marker panel demonstrated higher cumulative methylation scores and more frequent methylation-positive signals in CRC cfDNA compared to controls. dPCR confirmed single-molecule resolution and clear discrimination between methylated and unmethylated templates, with occasional double-positive partitions consistent with mixed allelic methylation. Signal intensity across the dilution series followed a four-parameter logistic model, achieving detection sensitivity below 0.2% methylated DNA. qPCR and dPCR results showed strong correlation across the HT29 dilution series (R2 = 0.80) and high concordance in classifying CRC and healthy samples. Conclusions: This TET2-APOBEC-based enzymatic cfDNA assay enables sensitive, quantitative, sequencing-free methylation detection under gentle conditions, supporting its application in early colorectal cancer screening and routine clinical liquid biopsy workflows.