Septin9基因甲基化检测方法的建立及在结直肠癌血浆标本检测中的应用探讨  

Establishment of methylation detection method for Septin9 gene and its application in plasma specimen detection in colorectal cancer

作　　者：束新华[1] 潘志文[1] 包叶江[1] 金元[1] 叶柳青[1] 李德川[2] 谢玲女[2] 徐笑红[1] 

机构地区：[1]浙江省肿瘤医院检验科,杭州310022 [2]浙江省肿瘤医院大肠外科,杭州310022

出　　处：《国际检验医学杂志》2018年第1期17-20,24,共5页International Journal of Laboratory Medicine

基　　金：浙江省公益技术应用研究计划资助项目(2015C33187)

摘　　要：目的以荧光聚合酶链反应(PCR)技术为基础,构建早期筛查结直肠癌的血液Septin9甲基化检测体系。方法在美国国立生物技术信息中心(NCBI)数据库查找Septin9甲基化的CpG岛位点,设计相应的PCR引物。提取结直肠癌组织和癌旁组织的DNA,PCR扩增并测序确认Septin9基因启动子区高甲基化位点。针对高甲基化位点,设计荧光PCR和Taqman探针检测技术构建血浆标本甲基化检测体系,并对该检测体系的准确度、特异度、重复性及最低检出量进行评估分析。在57例结直肠癌患者和30例健康人血浆标本中进行Septin9甲基化检测,验证该检测体系的可靠性。结果组织标本中经测序确认Septin9基因高甲基化位点。以该位点为靶点设计荧光PCR检测体系,经评估,该检测体系的准确度为100%,特异度和重复性均为100%,最低检出量可达0.1ng/μL。在57例结直肠癌患者血浆标本中,Septin9甲基化位点检测的阳性率为71.92%(41/57),结直肠癌患者病理分期Ⅰ和Ⅱ期阳性率达64.2%;而健康人群血浆中Septin9基因均无甲基化。结论以Septin9基因甲基化位点为靶标的血浆荧光PCR检测体系具有灵敏度、特异度及准确度高的特点,是结直肠癌早期筛查的可靠检测技术,具有良好的临床应用前景。Objective To establish the blood Septin9 methylation detection system for early screening of colorectal cancer based on fluorescence PCR technology. Methods The PCR primer of Septin9 was designed by searching the CpG island site of Septin9 methylation in the NCBI database. The high methylation site of Septin9 gene promoter region was confirmed by PCR amplification and sequencing after extracting DNA from colorectal cancer and para-carcinoma tissues. The fluorescence PCR and TaqMan probe detection technique was designed by aiming at high methylation site for constructing the plasma sample methylation detection sys-tem. Then its accuracy ？ specificity ？ repeatability and minimum detectable amount were performed the assess-ment and analysis. The SETP9 methylation detection was performed in plasma samples from 57 cases of color-ectal cancer and 30 healthy persons. Results The high methylation site of Septin9 gene in tissue samples was confirmed by sequencing. This site served as the target for designing fluorescence PCR detection system. After assessment ？ the accuracy ？ specificity and repeatability of this detection system were 100 % ？ the lowest detection amount reached 0. 1 ng/jLtL. Among the plasma samples in 57 cases of colorectal cancer, the positive rate of Septin9 methylation site detection was 71. 92% （41/57） and the positive rate of in the patients with pathologi-cal stage I and H of colorectal cancer reached 64. 2%. But Septin9 gene had no methylation in plasma of healthy population. Conclusion The plasma fluorescence PCR detection system with Septin9 gene methylation as the target has the characteristics of high sensitivity,high specificity and high accuracy,which is the reliable detection technique for the early screening of colorectal cancer and has good clinical application prospect.

关 键 词：Septin9甲基化 结直肠癌 血浆 早期诊断 

分 类 号：R735.3[医药卫生—肿瘤]