Dual-Strategy Optimization of Reference Systems Enhances Reliability in Tumor DNA Methylation Detection: Primer-Probe Innovation for Low-Concentration Clinical Specimens

DNA methylation biomarkers are crucial for early cancer detection, yet quantitative methylation-specific PCR (qMSP) faces significant challenges with low-concentration clinical samples due to suboptimal reference systems, leading to inadequate sample classification and wasted specimens. To address this, we developed optimized reference primer-probe sets through a dual-phase strategy: first, optimizing published bisulfite-converted ACTB (bisACTB) primers and designing novel CpG-free bisACTB primers, validated via SYBR Green qPCR using bisHela templates; second, coupling top-performing primers with probes for rigorous TaqMan qPCR evaluation across diverse biospecimens (cell lines, gender-matched tissues, plasma/serum). Results identified two optimized bisACTB_103 primer-probe sets exhibiting significantly enhanced sensitivity and specificity in probe-based qPCR, alongside novel high-performing candidates including the CpG-free bisProAB1_F2+R2+P1 set. Comparative analysis revealed bisACTB_103_F2+R2+P2 and bisProAB1_F2+R2+P1 as the top performers overall, demonstrating significantly improved amplification efficiency (p<0.01) across all sample types, with pronounced gains in low-concentration plasma (ΔCt = -1.5, 2.8-fold sensitivity increase). This study establishes two robust reference systems that enhance detection pass rates for challenging low-input samples and provide standardized ΔCt-based quality metrics for reliable qMSP assay validation in clinical diagnostics applications.