血浆游离DNA Septin 9基因甲基化检测方法

Detection of free DNA septin 9 gene methylation in plasma

目的:探讨血浆游离DNA Septin 9基因胞嘧啶-磷酸-鸟苷酸(cytosine-phosphoric-guanylic,CpG)位点与结直肠癌的相关性,旨在开发基于血浆的real-time PCR检测体系。方法:采用高通量测序技术对训练样本组织进行甲基化检测,筛选出与结直肠癌临床信息高度一致性的位点,设计基于甲基化敏感性酶切法-real-time PCR检测体系,用于分析血浆与组织的一致性,进一步以100例临床测试样本评估甲基化敏感性酶切法-real-time PCR检测体系的性能。结果:71例训练样本高通量测序筛选出与结直肠癌临床信息高度一致性的位点为CpG 38号位点;基于检测区域筛选的甲基化敏感性酶为BstU1、HhaI、HinP1I;开发的甲基化敏感性酶切法-real-time PCR检测下限为0.5%甲基化程度,Ct值为38.5;临床测试体系的灵敏度为87.27%,特异度为91.49%,阳性预测值为92.31%,阴性预测值为86.00%。结论:Septin 9 CpG 38号位点的甲基化敏感性酶切法-real-time PCR检测体系能高度预测结直肠癌的发生,具有重大的临床应用价值。

Objective:To explore the correlation between cytosine-phosphoric-guanylic(CpG)site of Septin 9 gene and colorectal cancer,and to develop a real-time PCR detection system in plasma in patients with colorectal cancer.Methods:The methylation of training samples was detected by high-throughput sequencing technology,and the sites highly consistent with the clinical information of colorectal cancer were identified.Then the detection system of real-time PCR was designed to analyze the consistency of plasma and tissue based on methylationa sensitive enzyme digestion.Finally,100 clinical trials were conducted to evaluate the performance of the detection system with the methylation sensitive enzyme digestion-real-time PCR.Results:The highly consistent sites,which were selected by high-throughput sequencing from 71 training set samples,was the 38th CpG.Based on the detection region,the screened methylation sensitive enzymes were BstU1,HhaI and HinP1I.The limit for the detection of methylation sensitive enzyme digestion-real-time PCR was 0.5%,and the Ct value was 38.5.The sensitivity was 87.27%,the specificity was 91.49%,the positive predictive value was 92.31%,and the negative predictive value was 86.00%.Conclusion:The 38th CpG site of Septin 9 detected by the detection system of methylation sensitive enzyme digestion-real-time PCR can highly predict the occurrence of colorectal cancer with great clinical application value.