【作者】 余玲玲； 王慧燕； 余坚； 郑晓群； 田可港； 吕建新； 冯晶晶； 郑美琴；

【Author】 YU Ling-ling l,TIAN Ke-gang 2,FENG Jing-jing 3,WANG Hui-yan l,ZHENG Mei-qin 4,YU Jian l,LV Jian-xin 2,ZHENG Xiao-qun l.(1 The Second Affiliated Hospital of Wenzhou Medical College,Wenzhou,Zhejiang,325027,China;2 Wenzhou Medical College, Wenzhou,Zhejiang,325035,China;3 The Hospital of Binjiang,Hangzhou,Zhejiang,311100,China;4 Ophtha lmologic Affiliated Hospital of Wenzhou Medical College,Wenzhou,Zhejiang,325027,China)

【机构】 温州医学院附属第二医院； 温州医学院； 杭州市滨江医院； 温州医学院附属眼视光医院；

【摘要】 目的建立高分辨率熔解曲线分析(High-resolution melting,HRM)技术检测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地贫最常见基因突变:-28(A>G)与IVS-2-654(C>T)为研究位点,以TA克隆技术构建其质粒DNA作为HRM体系的模板与基因分型对照,通过体系优化及一系列方法学评价,建立HRM技术检测β地贫基因突变的方法。同时用所建方法对107例临床疑似β地贫患者DNA样本进行检测,并与直接测序结果进行比较分析。结果所建方法 PCR扩增中未出现污染、没有引物二聚体及非特异性片段的扩增;各不同基因型质粒DNA的Tm值、Ct值的CV均较小,分别为0.03%～2.14%、0.06%～1.78%;可以对浓度范围为1pg～100ng的DNA模板进行基因分型,最低可检出相当于103拷贝的基因组DNA模板;并可以检测低至10%的突变的存在。107例临床疑似β地贫患者DNA样本经该法分析后,共检出48例IVS-2-654(C>T)杂合突变型基因与9例-28(A>G)杂合突变型基因,未发现两位点的纯合突变型基因;其结果与测序所得基因型结果完全一致。结论成功建立的HRM方法是一种操作简便、特异性强、敏感度高、结果准确并能实现快速分析的突变检测技术,不仅在β地贫的临床诊断与产前基因诊断中值得推广使用,还有望成为β地贫的大规模人群筛查不可多得的实验室工具,同时也为β地贫其它突变位点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。更多还原

【Abstract】 Objective To establish a method for detection of gene mutations by high-resolution melting Analysis(HRM) inβ-thalassemia.And to evaluate its clinical application value.Methods According to the distribution ofβ-thalassemia gene mutations in Wenzhou,we selected two common sites:-28 (A>G) and IVS-2-654(C>T) as our experimental subjects.Then,we established a diagnostic assay for detection ofβ-thalassemia gene mutations based on HRM by using the plasmids DNA which we have constructed by TA clone technology as PCR templates and reference gene types.The reaction conditions of experiments and the evaluation of methodology were optimized.At last,107 specimens of patients who seem to have theβ-thalassemia gene mutations were detected by HRM that we have established.The results of HRM analysis were confirmed by direct DNA sequencing.Results The results showed that the specificity of HRM is so high that nonspecific reactions would taken place.From the repeatability analysis, we find that Coefficieni of variation(CV) of different DNA types Tm or Ct is very small.the range varies from 0.03%to 2.14%and 0.06%to 1.78%,respectively.The range for DNA templates concentration that can be identified by HRM varies from lpg to lOOng.We also find that the minimum concentration of DNA templates and the lowest ratio of mutation that can be distinguished by HRM is about 103 copy and 10%,respectively.And 107 specimens of patients who seem to have theβ-thalassemia gene mutations were detected by HRM that we have established.There are 48 IVS-2-654(C>T) heterozygous and 9 -28(A>G) hete rozygous were discovered.The results of HRM were all in line with that of direct DNA sequencing.Conclusion The approach of rapid identification ofβ-thalassemia gene mutations based on HRM is successfully established.It is convenient with good sensitivity and high specificity.The result of it is not only reliable but also stable.And the rapid detection ofβ-thalassemia gene mutations comes true by using it.We believe that it can be used in clinical diagnosis and prenatal diagnosis ofβ-thalassemia.And it can also be a share of technical platforms in the detections of other gene mutations ofβ-thalassemia.更多还原