建立高分辨熔解曲线分析法检测SF3B1基因突变  

Detection of SF3B1 mutations using high-resolution melting analysis

作　　者：杨静[1] 姚冬明[1] 钱军[1] 王翠竹[1] 杨小飞[1] 林江[2] 李云[1] 陈星星[1] 肖高飞[2] 马吉春[2] 

机构地区：[1]江苏大学附属人民医院血液科,江苏镇江212002 [2]江苏大学附属人民医院中心实验室,江苏镇江212002

出　　处：《江苏大学学报（医学版）》2013年第1期49-52,共4页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81270630);江苏省自然科学基金资助项目(BK2009206);江苏省科技厅临床医学科技专项资金资助项目(BL2012056);江苏省"333工程"资助项目(BRA2011085);2010年镇江市医学重点人才项目;镇江市社会发展资助项目(SH2011056)

摘　　要：目的:建立高分辨熔解曲线分析法(high-resolution melting analysis,HRMA),检测骨髓增生异常综合征(my-elodysplastic syndrome,MDS)患者中SF3B1基因的突变。方法:针对SF3B1基因热点突变位点(密码子E622、H662、K666和K700)设计PCR扩增引物,建立带有温度内标校准品的PCR扩增反应体系,对30例初诊MDS患者的骨髓标本进行PCR扩增,应用HRMA对相应PCR产物进行突变检测,并对HRMA检测结果进行DNA测序验证。结果:HR-MA检测发现2例MDS患者存在异常的熔解曲线,经DNA测序验证1例为K666M杂合子突变,另1例为K700E杂合子突变。所建立的HRMA技术检测SF3B1基因突变的灵敏度为0.05(5%)。结论:成功建立检测SF3B1基因突变的HRMA技术,可快速筛查MDS标本中的SF3B1基因突变。Objective： To establish high-resolution melting analysis（HRMA）assay and detect the SF3B1 mutations in patients with myelodysplastic syndrome（MDS）.Methods： The PCR amplification systems with internal temperature calibrators were established with specific PCR primers against four hotspot mutation sites of SF3B1 gene（E622,H662,K666,and K700）.Thirty MDS samples were amplified and analyzed by using HRMA technique.The results of HRMA were verified by DNA sequencing.Results： Abnormal melting curves were observed by HRMA assay in two MDS samples.DNA sequencing confirmed that these two samples harbored heterozygous K666M mutation and K700E mutation,respectively.The sensitivity of HRMA assay could reach 0.05.Conclusion： The HRMA assay for detecting SF3B1 mutations was established successfully and could be used to screen SF3B1 mutations rapidly in patients with MDS.

关 键 词：高分辨率熔解曲线分析 SF3B1 基因突变 骨髓增生异常综合征 

分 类 号：R393[医药卫生—基础医学]