Highly Sensitive and Specific Lateral Flow Detection for DNA Methylation Based on GIaI-Mediated Specific-Terminal-Mediated Polymerase Chain Reaction

Sensitive and specific detection of DNA methylation is crucial for the early diagnosis of various human diseases, particularly cancers. However, conventional methylation detection methods often face challenges in balancing both sensitivity and specificity. In this study, we present a novel approach that integrates the high specificity of methylation-dependent restriction endonuclease (GlaI) digestion with the amplification efficiency of specific terminal-mediated polymerase chain reaction (STEM-PCR). This combination enables selective amplification of methylated DNA, which is then detected through lateral flow detection (LFD), providing a simple, visual readout. As a proof of concept, a STEM-PCR-LFD assay was applied to detect methylated <i>Septin 9</i>, a biomarker for colorectal cancer. The assay demonstrated a sensitivity of approximately 0.1% (10 copies of methylated template per reaction), with no cross-reactivity observed when 10,000 copies of unmethylated DNA were included as background. Furthermore, the assay was validated with ten formalin-fixed paraffin-embedded (FFPE) tissue samples, achieving 100% consistency with standard real-time STEM-PCR. This method offers a highly sensitive, specific, and accessible platform for DNA methylation detection, with potential for early disease diagnosis.