脑胶质瘤<em>IDH2</em>基因突变HRM检测方法的初步建立

中国生物工程杂志

2013, Vol. 33

Issue (12): 57-63

技术与方法

脑胶质瘤IDH2基因突变HRM检测方法的初步建立

王银辉¹, 张海燕¹, 杨传红¹, 张伟², 赖晃文¹, 陈晓东², 王捷²

1. 广州军区广州总医院医学实验科广州 510010;
2. 广州军区广州总医院病理科广州 510010

Preliminary Establishment of High Resolution Melting Analysis for Detecting IDH2 Gene Mutations in Gliomas

WANG Yin-hui¹, ZHANG Hai-yan¹, YANG Chuan-hong¹, ZHANG Wei², LAI Huang-wen¹, CHEN Xiao-dong², WANG Jie²

1. Department of Medical Research, General Hospital of Guangzhou Military Command, Guangzhou 510010, China;
2. Department of Pathology, General Hospital of Guangzhou Military Command, Guangzhou 510010, China

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摘要： 异柠檬酸脱氢酶2（IDH2）基因突变常常发生于WHOⅡ-Ⅲ级的星形细胞瘤，少突胶质细胞瘤，和大部分继发性胶质母细胞瘤中。在临床上，IDH2 突变已经显示对胶质瘤患者有诊断和预后意义。拟通过提取胶质瘤石蜡包埋组织DNA，优化PCR反应条件，初步建立IDH2 基因突变高分辨率熔解曲线（high resolution melting，HRM）检测方法。并与直接测序法对比，观察反应体系的检测灵敏度和稳定性。响应面分析结果证明20μl HRM 反应体系中，引物浓度0.6μmol/L，模板量45ng，Mg²⁺浓度2.75mmol/L，退火温度52.5℃为最佳。HRM检测 IDH2 基因突变的结果和直接测序法一致，但灵敏度更高。表明所建立HRM 方法简单，特异性强，准确可靠，可为IDH2 突变检测的临床应用提供借鉴和依据。

关键词： IDH2突变; 胶质瘤检测; 高分辨率熔解曲线分析

Abstract: Isocitrate dehydrogenase2(IDH2) mutations have recently been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas, as well as secondary glioblastomas. Since the mutations have been associated with better clinical outcome, they have shown diagnostic and prognostic value. It was performed with optimizing conditions to preliminary establish a high resolution melting for the detecting of IDH2 mutations in routine formalin-fixed tissues of brain biopsies. IDH2 mutations in paraffin-embedded tissues of gliomas were detected by HRM and direct sequencing, estimating the stability and sensitivity of reaction system and reaction conditions. The optimal annealing temperature was 52.5 ℃. The optimization of 20 μl-HRM reaction system including 0.6 μmol/L primer, 45 ng DNA template,2.75 mmol/L Mg²⁺, was established by response surface Design. The results were consistent with the direct sequencing but have a higher sensitivity. It showed that HRM was simple, specific, accurate and reliable method. It would provide the reference for the clinical application of detection IDH2 mutations.

Key words: IDH2 mutation Gliomas detection High resolution melting

收稿日期: 2013-09-24 出版日期: 2013-12-25

ZTFLH:

R739

通讯作者: 王捷，E-mail：laputawangyinhui@gmail.com E-mail: laputawangyinhui@gmail.com

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引用本文:

王银辉, 张海燕, 杨传红, 张伟, 赖晃文, 陈晓东, 王捷. 脑胶质瘤IDH2基因突变HRM检测方法的初步建立[J]. 中国生物工程杂志, 2013, 33(12): 57-63.

WANG Yin-hui, ZHANG Hai-yan, YANG Chuan-hong, ZHANG Wei, LAI Huang-wen, CHEN Xiao-dong, WANG Jie. Preliminary Establishment of High Resolution Melting Analysis for Detecting IDH2 Gene Mutations in Gliomas. China Biotechnology, 2013, 33(12): 57-63.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I12/57

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