HRM技术检测β-地中海贫血IVS-2-654（C〉T）与．28（A〉G）突变方法的建立及初步应用  

Establishment of a method for detection of IVS.2-654 （ C 〉 T） and -28 （ A 〉 G） gene mutations by high-resolution melting analysis in β-thalassemia and its clinical application

作　　者：余玲玲[1] 田可港[2] 冯晶晶 王慧燕[1] 郑美琴[4] 郑晓群[1] 吕建新[2] 

机构地区：[1]温州医学院附属第二医院检验科,325027 [2]温州医学院检验与生命科学学院 [3]杭州市滨江医院病理科 [4]温州医学院附属眼视光医院检验科

出　　处：《中华检验医学杂志》2012年第8期730-735,共6页Chinese Journal of Laboratory Medicine

基　　金：温州市科技计划资助项目，浙江省医药卫生科技计划资助项目，浙江省公益性技术应用研究计划资助项目

摘　　要：目的建立基于高分辨率熔解曲线（HRM）技术快速筛查β-地中海贫血（简称β-地贫）基因突变的方法，并初步探讨其临床应用价值。方法选取温州地区人群β-地贫常见基因突变位点IVS-2-654（C〉T）与-28（A〉G）作为研究位点，采用TA克隆技术构建其质粒DNA作为模板或基因分型对照，建立基于HRM技术检测β-地贫基因突变的方法，并对该方法的特异性、重复性、灵敏度等指标进行方法学评价。收集2009年11月至2010年10月温州医学院附属第二医院、育英儿童医院临床疑似p-地贫患者117例，抽取外周血标本，提取外周血白细胞DNA，用该方法进行IVS．2-654（C〉T）与-28（A〉G）位点的HRM检测，并将检测结果与双向直接测序做比较。结果建立的HRM技术可对β-地贫IVS-2-654（C〉T）与-28（A〉G）突变位点进行检测，未见非特异性扩增片段；不同基因型HRM检测的批内、批间熔解温度（Tm）值的变异系数（cv）均〈0．1％；该法最低可检出10^3拷贝的DNA模板，并可检测低至10％的突变存在。117份临床疑似β-地贫患者样本经该法检测，45份为IVS·2-654（C〉T）杂合突变型，9份为一28（A〉G）杂合突变型，未发现2个位点的纯合突变型基因；此结果与直接测序所得基因型结果完全一致。结论建立的HRM技术操作简便，特异性强，灵敏度高，可用于β-地贫基因突变筛查，并为β-地贫其他突变位点的检测及基因组单核苷酸多态性分型等提供了一个通用的技术平台。Objective To establish a method for detection of gene mutations in β-thalassemia by high-resolution melting （HRM） and study its preliminary clinical application. Methods Two common mutations [ IVS-2-654 （ C 〉 T ） and -28 （ A 〉 G ） ] of β-thalassemia in Wenzhou city population were selected. The plasmid DNA fragments of these mutations were constructed by TA clone technology as PCR templates or genotyping controls. A method for detection of β-thalassemia gene mutations based on HRM analysis was established and its specificity, sensitivity and repeatability were methodologically evaluated. One hundred and seventeen patients with clinically suspected β-thalassemia from Second Affiliated Hospital and Yu ying Children＇s Hospital of Wenzhou Medical College were enrolled into this study. The genomic DNA was extracted from whole blood cells and detected by HRM method. The results were compared with the direct sequencing data. Results HRM method could detect the mutations [ IVS-2-654 （ C 〉 T） and -28 （ A 〉 G） ] of β-thalassemia and the results did not show any non-specific amplified fragments. All within-run and between-run coefficients of variation for different DNA types＇ Tm were smaller than 0. 1%. And minimum 103 copies of DNA of each assay and 10% mutation could be determined by this method. One hundred and seventeen patients with clinically suspected 13-thalassemia were detected with HRM and all the results were in accordance with direct DNA sequencing. There were 45 IVS-2-654（ C 〉 T）heterozygous mutation and 9-28 （A 〉 G）heterozygous mutation and none homozygous mutation. Conclusion The method of rapid identification of 13-thalassemia gene mutations based on HRM analysis is successfully established, which is a convenient, rapid, specific, sensitive and accurate technique for screening gene mutations in [3-thalassemia as well as a general technical platform to identif~ other gene mutations.

关 键 词：Β-地中海贫血 突变 高分辨率熔解曲线 

分 类 号：R556[医药卫生—血液循环系统疾病]