发现甲基化标记,开发血浆中用于结直肠癌早期检测的有义-反义和双 MGB 探针 PCR 分析法

Yanteng Zhao, Zhijie Wang, Qiuning Yu, Xin Liu, Xue Liu, Shuling Dong, Xianping Lv, Tiao Zhang, Dihan Zhou, Qiankun Yang
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摘要

背景:与粪便检测相比,利用血浆无细胞 DNA(cfDNA)甲基化筛查结直肠癌(CRC)更具挑战性,因为 cfDNA 的丰度较低。因此,开发基于适当标记物的信号放大检测方法对于提高灵敏度至关重要:方法:利用公共数据库中现有的17个450K微阵列数据集(包括组织、健康白细胞(WBC)和血浆中的cfDNA数据)来鉴定常见于CRC和腺瘤的差异甲基化CpGs(DMCs)。候选 DMCs 的甲基化状态是通过与 CRC 和正常组织进行 Sanger 测序确认的。然后开发了一种有义-反义和双 MGB 探针(SADMP)检测方法。随后,使用 SADMP 方法在 712 份血浆样本中验证了这些生物标记物:结果:共有 2237 个 DMCs 在癌症组与正常组和腺瘤组与正常组之间出现重叠。其中,有 75 个 DMCs 在 30 个其他非 CRC 癌症中发生了低甲基化。经过 LASSO 回归,这一数字减少到 8 个。其中,NTMT1 和 MAP3K14-AS1 这两种标记物在经过 WBC 验证和引物/探针设计评估后被确定为有希望的候选标记物。SADMP 技术已证明能将检测信号放大到原始水平的两倍左右。总体而言,双靶标 SADMP 检测对 CRC(I 期:75.0%)的灵敏度为 84.8%,对晚期腺瘤(AA)的灵敏度为 32.0%,对对照组的特异性为 91.5%:结论:在基于血浆的检测中,双目标检测法在检测 CRC 和 AA 方面表现出很高的性能,这表明它可以作为一种很有前途的非侵入性 CRC 检测工具。
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Discovering methylation markers and development of a sense-antisense and dual-MGB probe PCR assay in plasma for colorectal cancer early detection
Background: Screening for colorectal cancer (CRC) using plasma cell-free DNA (cfDNA) methylation is more challenging than stool testing due to the low abundance of cfDNA. Therefore, the development of signal amplification assays based on appropriate markers is essential to increase sensitivity. Methods: A total of 17 existing 450K microarray datasets including tissue, healthy white blood cell (WBC) and plasma cfDNA data from public databases were used to identify differentially methylated CpGs (DMCs) common to CRC and adenoma. The methylation status of candidate DMCs was confirmed by Sanger sequencing with CRC and normal tissues. A sense-antisense and dual MGB probe (SADMP) assay was then developed. Subsequently, the biomarkers were validated in 712 plasma samples using the SADMP method. Results: A total of 2237 DMCs showed overlap between the cancer vs. normal and adenoma vs. normal groups. Of these, 75 were hypomethylated in 30 other non-CRC cancers. After LASSO regression, this number was reduced to eight. Two of these, NTMT1 and MAP3K14-AS1, were identified as promising candidate markers following WBC validation and primer/probe design evaluation. The SADMP technology demonstrated the ability to amplify the detection signal to approximately twice the original level. Overall, the dual-target SADMP assay demonstrated a sensitivity of 84.8% for CRC (stage I: 75.0%), a sensitivity of 32.0% for advanced adenomas (AA), and a specificity of 91.5% in controls. Conclusions: The dual-target assay demonstrated high performance for CRC and AA detection in plasma-based tests, suggesting that it may serve as a promising noninvasive tool for CRC detection.
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