高分辨熔解曲线分析法检测急性髓细胞白血病IDH2基因突变  

High-resolution melting curve analysis for detection of isocitrate dehydrogenase 2mutations in acute myeloid leukemia

作　　者：薛娣[1] 邵睿[1] 林江[2] 肖高飞[2] 钱军[3] 姚冬明[2] 柴海彦[3] 李云[3] 杨静[3] 王翠竹[3] 陈星星[3] 

机构地区：[1]江苏大学附属人民医院病理科,江苏镇江212002 [2]江苏大学附属人民医院实验中心,江苏镇江212002 [3]江苏大学附属人民医院血液科,江苏镇江212002

出　　处：《临床检验杂志》2012年第9期664-667,共4页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省科技厅临床医学科技专项课题(BL2012056);江苏省自然科学基金(BK2009206);江苏省"333工程"科研项目资助计划(BRA2011085);镇江市社会发展项目(SH2010015;SH2011056)

摘　　要：目的建立高分辨熔解曲线分析(HRMA)技术检测急性髓细胞白血病(AML)中可溶性异柠檬酸脱氢酶2(IDH2)基因突变。方法针对IDH2突变位点设计PCR扩增引物,建立带有温度内标校准品的PCR扩增体系,对98例AML患者标本进行PCR扩增,用HRMA技术对PCR产物进行突变分析,并通过DNA测序技术对HRMA结果进行验证。结果对98例AML样本的PCR产物进行HRMA检测,发现5例存在异常熔解曲线,对此5例标本及随机选择的HRMA显示为野生型的35例IDH2标本进行DNA测序验证,结果表明,存在异常熔解曲线的5例标本为4例R140Q突变和1例R172K突变,其余35例经DNA测序证实为野生型IDH2;对不同浓度梯度的R140Q质粒HRMA检测结果表明其灵敏度为2%。结论成功建立HRMA技术检测IDH2突变,其可用于AML标本的临床检测。Objective To establish high-resolution melting curve analysis（HRMA） technique for detecting isocitrate dehydrogenase 2（IDH2） mutations in acute myeloid leukemia（AML）.Methods The primers for IDH2 mutations were designed and PCR system with internal temperature calibrators was established.The blood samples from 98 AML patients were amplified and analyzed by HRMA.Direct DNA sequencing was performed to confirm the results of HRMA.Results For 5 of the 98 AML samples abnormal melting curves were identified in HRMA.The 5 samples with abnormal melting curves and 35 randomly selected samples with wild-type DNA detected by HRMA were checked by sequencing,and R140Q mutation was found in 4 samples and R172K mutation was in 1 sample.The 35 samples were also confirmed as wild-type by DNA sequencing.The sensitivity of two percent was determined by HRMA for plasmid DNA with different concentrations of R140Q mutant.Conclusion The rapid,easy and sensitive HRMA method was established and may be used for high-throughput screening of IDH2 mutations in AML patients.

关 键 词：高分辨熔解曲线分析 可溶性异柠檬酸脱氢酶2 基因突变 急性髓细胞白血病 

分 类 号：R733.71[医药卫生—肿瘤]