【佳学基因检测】通过定量多重甲基化特异性 PCR (QM-MSP) 测定对 DNA 甲基化进行定量

靶向基因检测2万多重要性

挖掘在《肿瘤致病基因检测与转移潜能分析》收录《Methods Mol Biol》在 2018;1708:473-496发表了一篇题目为《通过定量多重甲基化特异性 PCR (QM-MSP) 测定对 DNA 甲基化进行定量》肿瘤靶向药物治疗基因检测临床研究文章。该研究由Mary Jo Fackler, Saraswati Sukumar 等完成。促进了肿瘤的精准治疗与个性化用药的发展，进一步强调了基因信息检测与分析的重要性。

肿瘤靶向药物及精准治疗临床研究内容关键词：

亚硫酸氢盐,细胞, DNA甲基化,甲基化特异性 PCR,质量管理-MSP,定量,组织。

肿瘤靶向治疗基因检测临床应用结果

定量多重甲基化特异性 PCR (QM-MSP) 方法敏感地量化 DNA 甲基化的定义特征是两步 PCR 方法，用于对临床样本中多达 12 个基因的面板进行多重分析，且 DNA 量最少。在第一步中，对于多达 12 个测试的基因，一对基因特异性引物（正向和反向）在一个 PCR 反应中同时并以多重方式扩增同一基因的甲基化和非甲基化拷贝。这种不依赖甲基化的扩增步骤在 36 个 PCR 循环后产生高达 109 个拷贝/μL 的扩增子。在第二步中，第一个反应（步骤 1）的扩增子使用实时 PCR 和两个独立的荧光团以标准曲线定量，以检测同一孔中每个基因的甲基化/未甲基化 DNA（例如，6FAM 和 VIC） .在 100,000 个参考基因拷贝中可检测到一个甲基化拷贝。以连续规模报告甲基化。对于基因组，正常 DNA 甲基化的最高水平（高于该水平的样本被称为阳性）是通过使用接受者操作特征 (ROC) 得出的，最大限度地提高了区分正常/良性与肿瘤 DNA 的测定特异性和敏感性。 QM-MSP 可应用于新鲜或固定导管细胞、导管液、乳头液、细针抽吸物、核心活检和肿瘤组织切片的临床样本。

肿瘤发生与复发转移国际数据库描述：

The defining feature of the Quantitative Multiplex Methylation-Specific PCR (QM-MSP) method to sensitively quantify DNA methylation is the two-step PCR approach for a multiplexed analysis of a panel of up to 12 genes in clinical samples with minimal quantities of DNA. In the first step, for up to 12 genes tested, one pair of gene-specific primers (forward and reverse) amplifies the methylated and unmethylated copies of the same gene simultaneously and in multiplex, in one PCR reaction. This methylation-independent amplification step produces amplicons of up to 109 copies per μL after 36 cycles of PCR. In the second step, the amplicons of the first reaction (STEP 1) are quantified with a standard curve using real-time PCR and two independent fluorophores to detect methylated/unmethylated DNA of each gene in the same well (e.g., 6FAM and VIC). One methylated copy is detectable in 100,000 reference gene copies. Methylation is reported on a continuous scale. For the gene panel, the highest level of normal DNA methylation above which a sample would be called positive is derived by using Receiver Operating Characteristic (ROC), maximizing assay specificity and sensitivity to distinguish between normal/benign versus tumor DNA. QM-MSP can be applied to clinical samples of fresh or fixed ductal cells, ductal fluid, nipple fluid, fine needle aspirates, core biopsies, and tumor tissue sections.