细胞外游离DNA (cfDNA) 的碎片化和低浓度增加了对cfDNA甲基化检测技术的挑战。传统的亚硫酸盐转化方法由于操作繁琐和加剧cfDNA降解而不适合cfDNA甲基化分析。在此，我们提出了一种温度程序酶反应的cfDNA甲基化分析单管方法。使用内切酶轻微识别DNA甲基化以避免cfDNA降解，并且两阶段扩增反应显著提高了GC富集序列的检测灵敏度。以vimentin为目标，检测灵敏度为10个甲基化DNA的拷贝数。同时，该方法可以准确地从1000倍的未甲基化DNA背景中量化目标序列的甲基化水平。此外，成功地检测到20个临床血浆样本中的甲基化vimentin基因。结果表明，健康志愿者和结直肠癌患者vimentin基因的甲基化水平存在显著差异。这些结果使我们相信，该方法作为亚硫酸盐转化方法的补充，在DNA甲基化分析方面具有巨大的应用潜力。版权所有©2023 Elsevier B.V.。保留所有权利。

The fragmentation and low concentration of cell-free DNA (cfDNA) pose higher challenges for the cfDNA methylation detection technologies. Conventional bisulfite conversion-based methods are inadequate for cfDNA methylation analysis due to cumbersome operation and exacerbating cfDNA degradation. Herein, we proposed temperature-programmed enzymatic reactions for cfDNA methylation analysis in a single tube. Endonuclease was used to mildly recognize DNA methylation to avoid the degradation of cfDNA. And two stages of amplification reactions significantly improved the detection sensitivity for GC-rich sequence. With vimentin as the target, the detection sensitivity was 10 copies of methylated DNA. Meanwhile, the proposed method can accurately quantify the methylation level of target sequence from 1000-fold of unmethylated DNA background. Further, the methylated vimentin gene in 20 clinical plasma samples was successfully detected. The results shown significant differences in methylation levels of the vimentin gene between healthy volunteers and colorectal cancer patients. These results lead us to believe that the proposed method has great application potential for DNA methylation analysis as a complement to bisulfite conversion-based methods.Copyright © 2023 Elsevier B.V. All rights reserved.