Ke Lihui, Zhao Hang, Shan Hongbo, Chen Yicheng, Cai Yongsheng, Wang Yang, Wei Bo, Du Minghua
Department of Thoracic Surgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China.
College of Health Science and Environmental Engineering, Shenzhen Technology University, Shenzhen 518118, China.
Micromachines (Basel). 2025 Mar 28;16(4):387. doi: 10.3390/mi16040387.
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 , 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.
DNA甲基化的灵敏且特异检测对于多种人类疾病尤其是癌症的早期诊断至关重要。然而，传统的甲基化检测方法在平衡灵敏度和特异性方面常常面临挑战。在本研究中，我们提出了一种新方法，该方法将依赖甲基化的限制性内切酶（GlaI）消化的高特异性与特异性末端介导的聚合酶链反应（STEM-PCR）的扩增效率相结合。这种组合能够实现甲基化DNA的选择性扩增，然后通过侧向流动检测（LFD）进行检测，提供简单的可视化读数。作为概念验证，一种STEM-PCR-LFD检测方法被应用于检测甲基化的，这是一种结直肠癌的生物标志物。该检测方法显示出约0.1%的灵敏度（每个反应10个甲基化模板拷贝），当包含10000个未甲基化DNA拷贝作为背景时未观察到交叉反应。此外，该检测方法在十个福尔马林固定石蜡包埋（FFPE）组织样本中得到验证，与标准实时STEM-PCR达到100%的一致性。该方法为DNA甲基化检测提供了一个高度灵敏、特异且易于使用的平台，具有早期疾病诊断的潜力。
