Gene promoter hypermethylation is a vital step in tumorigenesis. This paper set out to explore the use of polymerase chain reaction - surface-enhanced Raman spectroscopy (PCR-SERS) for the detection of gene methylation levels, with a focus on cancer diagnosis.

Methods: PCR with methylation independent primers were used on DNA samples to amplify target genes regardless of their methylation states. SERS was used on the obtained PCR products to generate spectra that contained peak changes belonging to CG and AT base pairs. Multiple linear regression (MLR) was then used to deconvolute the SERS spectra so that the CG/AT ratios of the sample could be obtained. These MLR results were used to calculate methylation levels of the target genes. For protocol verification, three sets of seven reference DNA solutions with known methylation levels (0%, 1%, 5%, 25%, 50%, 75%, and 100%) were analysed. Clinically, blood plasma samples were taken from 48 non-small-cell lung cancer (NSCLC) patients and 51 healthy controls. The methylation levels of the genes p16, MGMT, and RASSF1 were determined for each patient using this method.

Results: Verification experiment on the mixtures with known methylation levels resulted in an error of less than 6% from the actual levels. When applied to our clinical samples, the frequency of methylation in at least one of the three target genes among the NSCLC patients was 87.5%, but this percentage decreased to 11.8% for the control group. The methylation levels of p16 were found to be significantly higher in NSCLC patients with more pack-years smoked (p=0.04), later cancer stages (p=0.03), and cancer types of squamous cell and large cell versus adenocarcinoma (p=0.03). Prediction accuracy of 88% was achieved from classification and regression trees (CART) based on methylation levels and states, respectively.

Conclusion: This research showed that the PCR-SERS protocol could quantitatively measure the methylation levels of genes in plasma. The methylation levels of the genes p16, MGMT, and RASSF1 were higher in NSCLC patients than in controls.

基因启动子高甲基化是肿瘤发生的重要步骤。本文着手探索利用聚合酶链反应-表面增强拉曼光谱（PCR-SERS）检测基因甲基化水平，重点是癌症诊断。

方法：使用不依赖于甲基化的引物对 DNA 样本进行 PCR 扩增目标基因，无论其甲基化状态如何。对获得的 PCR 产物使用 SERS，生成包含属于 CG 和 AT 碱基对的峰值变化的光谱。然后使用多元线性回归 (MLR) 对 SERS 光谱进行解卷积，从而获得样品的 CG/AT 比率。这些 MLR 结果用于计算目标基因的甲基化水平。为了进行方案验证，分析了三组七种已知甲基化水平（0%、1%、5%、25%、50%、75% 和 100%）的参考 DNA 溶液。临床上，血浆样本取自 48 名非小细胞肺癌 (NSCLC) 患者和 51 名健康对照者。使用该方法测定了每位患者的基因 p16、MGMT 和 RASSF1 的甲基化水平。

结果：对已知甲基化水平的混合物进行验证实验，结果与实际水平的误差小于6%。当应用于我们的临床样本时，NSCLC患者中三个靶基因中至少一个的甲基化频率为87.5%，但对照组的这一百分比下降至11.8%。研究发现，吸烟年数较多（p=0.04）、癌症分期较晚（p=0.03）以及鳞状细胞和大细胞癌症类型与腺癌相比（p=0.03）的 NSCLC 患者中 p16 的甲基化水平显着更高）。分别基于甲基化水平和状态的分类和回归树 (CART) 的预测准确率达到 88%。

结论：本研究表明PCR-SERS方案可以定量测定血浆中基因的甲基化水平。NSCLC 患者中基因 p16、MGMT 和 RASSF1 的甲基化水平高于对照组。