Currently, the determination of DNA methylation is still a challenge due to the limited efficiency of enrichment, bisulfite modification, and detection. In this study, a dual-modality loop-mediated isothermal amplification integrated with magnetic bead isolation is proposed for the determination of methylated Septin9 gene in colorectal cancer. Magnetic beads modified with anti-methyl cytosine antibody were prepared for fast enrichment of methylated DNA through specific immunoaffinity (30 min). One-pot real-time fluorescence and colorimetric loop-mediated isothermal amplification were simultaneously developed for detecting methylated Septin9 gene (60 min). The real-time fluorescence generating by SYTO-9 dye (excitation: 470 nm and emission: 525 nm) and pH indicator (neutral red) was used for quantitative and visualized detection of methylated DNA. This method was demonstrated to detect methylated DNA from HCT 116 cells ranging from 2 to 0.02 ng/μL with a limit of detection of 0.02 ± 0.002 ng/μL (RSD: 9.75%). This method also could discriminate methylated Septin9 in 0.1% HCT 116 cells (RSD: 6.60%), suggesting its high specificity and sensitivity. The feasibility of this assay was further evaluated by clinical plasma samples from 20 colorectal cancer patients and 20 healthy controls, which shows the potential application in simple, low cost, quantitative, and visualized detection of methylated nucleic acids.

Graphical abstract

中文翻译：

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双模环介导等温扩增，用于无预处理检测结直肠癌中的 Septin9 甲基化 DNA

目前，由于富集、亚硫酸氢盐修饰和检测的效率有限，DNA甲基化的测定仍然是一个挑战。在这项研究中，提出了一种与磁珠分离相结合的双模态环介导等温扩增，用于测定结直肠癌中甲基化的 Septin9 基因。制备了抗甲基胞嘧啶抗体修饰的磁珠，用于通过特异性免疫亲和（30 分钟）快速富集甲基化 DNA。同时开发了一锅实时荧光和比色环介导的等温扩增，用于检测甲基化的 Septin9 基因（60 分钟）。SYTO-9 染料（激发：470 nm 和发射：525 nm）和 pH 指示剂（中性红）产生的实时荧光用于甲基化 DNA 的定量和可视化检测。该方法被证明可以检测来自 HCT 116 细胞的甲基化 DNA，范围为 2 到 0.02 ng/μL，检测限为 0.02 ± 0.002 ng/μL（RSD：9.75%）。该方法还可以区分 0.1% HCT 116 细胞中的甲基化 Septin9（RSD：6.60%），表明其具有高特异性和灵敏度。通过来自 20 名结直肠癌患者和 20 名健康对照的临床血浆样本进一步评估了该测定的可行性，显示了在简单、低成本、定量和可视化检测甲基化核酸方面的潜在应用。表明其具有较高的特异性和敏感性。通过来自 20 名结直肠癌患者和 20 名健康对照的临床血浆样本进一步评估了该测定的可行性，显示了在简单、低成本、定量和可视化检测甲基化核酸方面的潜在应用。表明其具有较高的特异性和敏感性。通过来自 20 名结直肠癌患者和 20 名健康对照的临床血浆样本进一步评估了该测定的可行性，显示了在简单、低成本、定量和可视化检测甲基化核酸方面的潜在应用。

图形概要