A novel blocker displacement qMSP assay for DNA methylation analysis at single-base resolution.

Accurate quantification of DNA methylation at single-CpG resolution remains a challenge for conventional qMSP, which typically lacks the specificity to discriminate solitary methylated sites. Here, we present a novel blocker displacement qMSP (BD-qMSP) assay that integrates a rationally designed blocker oligonucleotide to suppress unmethylated sequences while enabling robust amplification of single methylated CpGs. Through systematic optimization of blocker design, primer/probe modification, and annealing conditions, BD-qMSP achieved high discrimination efficiency with a detection limit as low as 0.01%. Notably, the assay's performance is not affected by the methylation status of neighboring CpG sites, ensuring reliable quantification of individual targets. In clinical colorectal cancer tissues, BD-qMSP accurately distinguished cancers from adjacent normal samples with 100% diagnostic consistency. This method provides a simple, cost-effective, and scalable platform for precise methylation analysis, ideally suited for single-site biomarker validation and early cancer detection.