【佳學(xué)基因檢測】通過定量多重甲基化特異性 PCR (QM-MSP) 測定對 DNA 甲基化進(jìn)行定量

靶向基因檢測2萬多重要性

挖掘在《腫瘤致病基因檢測與轉(zhuǎn)移潛能分析》收錄《Methods Mol Biol》在?2018;1708:473-496發(fā)表了一篇題目為《通過定量多重甲基化特異性 PCR (QM-MSP) 測定對 DNA 甲基化進(jìn)行定量》腫瘤靶向藥物治療基因檢測臨床研究文章。該研究由Mary Jo Fackler,?Saraswati Sukumar?等完成。促進(jìn)了腫瘤的正確治療與個性化用藥的發(fā)展，進(jìn)一步強(qiáng)調(diào)了基因信息檢測與分析的重要性。

腫瘤靶向藥物及正確治療臨床研究內(nèi)容關(guān)鍵詞：

亞硫酸氫鹽,細(xì)胞, DNA甲基化,甲基化特異性 PCR,質(zhì)量管理-MSP,定量,組織。

腫瘤靶向治療基因檢測臨床應(yīng)用結(jié)果

定量多重甲基化特異性 PCR (QM-MSP) 方法敏感地量化 DNA 甲基化的定義特征是兩步 PCR 方法，用于對臨床樣本中多達(dá) 12 個基因的面板進(jìn)行多重分析，且 DNA 量賊少。在先進(jìn)步中，對于多達(dá) 12 個測試的基因，一對基因特異性引物（正向和反向）在一個 PCR 反應(yīng)中同時并以多重方式擴(kuò)增同一基因的甲基化和非甲基化拷貝。這種不依賴甲基化的擴(kuò)增步驟在 36 個 PCR 循環(huán)后產(chǎn)生高達(dá) 109 個拷貝/μL 的擴(kuò)增子。在第二步中，先進(jìn)個反應(yīng)（步驟 1）的擴(kuò)增子使用實(shí)時 PCR 和兩個獨(dú)立的熒光團(tuán)以標(biāo)準(zhǔn)曲線定量，以檢測同一孔中每個基因的甲基化/未甲基化 DNA（例如，6FAM 和 VIC） .在 100,000 個參考基因拷貝中可檢測到一個甲基化拷貝。以連續(xù)規(guī)模報告甲基化。對于基因組，正常 DNA 甲基化的賊高水平（高于該水平的樣本被稱為陽性）是通過使用接受者操作特征 (ROC) 得出的，賊大限度地提高了區(qū)分正常/良性與腫瘤 DNA 的測定特異性和敏感性。 QM-MSP 可應(yīng)用于新鮮或固定導(dǎo)管細(xì)胞、導(dǎo)管液、乳頭液、細(xì)針抽吸物、核心活檢和腫瘤組織切片的臨床樣本。

腫瘤發(fā)生與反復(fù)轉(zhuǎn)移國際數(shù)據(jù)庫描述：

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.