应用甲基化识别定量检测胎儿DNA  

Quantification of fetal DNA by using methylation-based discrimination

作　　者：赵跃宏[1,2] 郭辉 汪杨[1,2] 

机构地区：[1]暨南大学第二临床医学院 [2]深圳市人民医院妇产科,广东深圳518020 [3]深圳市人民医院中心实验室,广东深圳518020

出　　处：《新乡医学院学报》2012年第8期589-591,共3页Journal of Xinxiang Medical University

基　　金：广东省深圳市科技局基金资助项目(编号:2009020169)

摘　　要：目的建立一种应用表观遗传学方法定量检测孕妇血浆中游离胎儿DNA的方法。方法 48例正常孕妇为观察组,10例非孕健康妇女为对照组,抽取外周血10 mL,应用甲基化标志物T-box3基因,经DNA分离、甲基化敏感限制性内切酶消除样本中母源性(非甲基化)DNA。未分解的胎儿DNA在合成寡核苷酸的存在下行竞争性聚合酶链反应扩增,再用基质辅助激光解析电离飞行时间质谱定量检测分析胎儿DNA。结果观察组血浆游离胎儿DNA平均为(148.0±63.5)×103拷贝数.L-1,胎儿DNA占标本中总游离DNA的比例为12%;对照组血浆甲基化DNA平均为(6.0±4.5)×103拷贝数.L-1;2组血标本中甲基化DNA拷贝数差异有统计学意义(P<0.05)。本检测方法的敏感性和特异性分别为99%和100%。结论应用甲基化识别可以对母血中分离出的胎儿DNA进行检测并定量分析,有助于扩大无创伤性产前诊断的临床应用范围。Objective To develop a method of using epigenetic marker to evaluate the cell-free DNA of fetus in mater- nal plasma. Methods Forty-eight pregnant women and ten healthy non-pregnant women were selected as observation group and control group respectively. The peripheral blood samples of 10 mL were drew from objects of the two groups. The assay used marker of T-box 3 （TBX3）, and methylation-sensitive restriction enzymes to eliminate the maternal （unmethylated） fraction of the DNA sample. The undigested fetal DNA fraction was then amplified in the presence of a synthetic oligonucleotide to permit competitive polymerase chain reaction. The free fetal DNA were quantified by MALDI-TOF-MS analysis. Results A mean of （ 148.0 ±63.5） × 10^3 copies · L^-1 of fetal DNA and a mean fetal propotion of 12% in plasma samples was obtained from ob- servation group. A mean of （6.0 ±4.5 ） × 10^3 copies · L-1 of methylated DNA in plasma samples was obtained from control group. The difference of the two groups in copy number of methylated DNA in plasma samples is significant （ P 〈 0.05 ）. The analytical sensitivity and specificity of the method for the assay was 99% and 100% , respectively. Conclusion Methylation- based reaction can detect and quantify the amount of fetal DNA in a sample isolated from maternal plasma. This technology may help explore the clinic application of noninvasive prenatal diagnosis.

关 键 词：甲基化 产前诊断 胎儿DNA 

分 类 号：R394-33[医药卫生—医学遗传学]