实时PCR和cycling probe技术检测母血浆游离胎儿DNA筛选重型β地中海贫血胎儿

Cell-free fetal DNA detection in maternal plasma using real-time PCR and cycling probe technology for prenatal screening β-thalassaemia major

目的通过检测孕妇外周血中的游离胎儿DNA来筛选重型β-地中海贫血胎儿。方法选择行产前基因诊断的夫妇6对,孕妇孕周23-26周。血液学检查:胎儿的父亲均为β-地中海贫血17M/N型,孕妇本人为携带除17M/N型之外的另一β-地中海贫血突变类型。针对CD17(A→T)无义突变,设计β-珠蛋白肽链上该等位基因的一对特异性引物和通过cycling probe法分别设计检测正常基因序列和基因突变位点的两条荧光探针,分别用FAM和HEX荧光标记。结合RT-PCR技术检测孕妇外周血中游离胎儿DNA,诊断胎儿是否遗传了其父亲的β地中海贫血17M/N碱基突变位点。同时与脐血血液学检查所诊断的胎儿地贫基因型对照。结果提取的6例孕妇血浆DNA模板中有3例同时显示FAM和HEX荧光信号值阳性结果,即这3例孕妇的胎儿遗传了父亲β-珠蛋白肽链上CD17位点的突变碱基(A→T)。另外3例孕妇血浆DNA模板的FAM信号值阳性,HEX信号值阴性,即所孕胎儿没有遗传父亲的CD17位点的突变碱基。结论利用RT-PCR和cycling probe技术检测孕妇外周血中的游离胎儿DNA可用来筛选患重型地中海贫血的胎儿。

Objective To analyze cell-free fetal DNA in maternal plasma for prenatal screening of β-thalassaemia major. Methods Six couples undergoing prenatal diagnosis of β-thalassaemia （gestational age range 23-26 weeks） were enrolled in this study. The husbands were all carriers of the CD17 （A→T） mutation, and the wives carreid another β-thalassaemia mutation. The allele-specific primers and two fluorescent cycling probes were synthesized for the detection of the CD17（A---＊ T） mutation, using FAM and HEX fluorescence labeling, respectively. The cell-free fetal DNA in the maternal plasma was detected using real-time PCR, and the fetal genotype was confirmed by cord blood conventional prenatal diagnosis. Results In the 6 pregnancies, FAM and HEX fluorescent signals were detected in 3 maternal plasma samples; in the other 3 samples, only FAM fluorescent signals were detected, suggesting the absence of paternally derived CD 17 （A→T） mutation. Conclusion Examination of cell-free fetal DNA in maternal plasma using real-time PCR and cycling probe technology can be effective means for prenatal screening of β-thalassaemia major.