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目的建立单细胞MGB探针荧光PCR检测β地贫的方法以及在β地贫PGD中的应用研究。方法获取β地贫基因携带者单个淋巴细胞,建立稳定的单细胞MGB探针荧光PCR检测技术,并对2例双方均为β地贫基因携带者的夫妇应用MGB探针荧光PCR进行了β地贫的PGD。结果利用单细胞MGB探针荧光PCR检测了β地贫基因携带者的200个单个淋巴细胞的基因型,平均扩增效率为94%,平均等位基因脱扣(ADO)率为2.5%。对两对夫妇进行4个周期PGD,共活检16个胚胎,获得16个卵裂球,其中15个卵裂球扩增成功,扩增效率为93.8%,ADO率为6.2%。14个胚胎经PCR分析后获得明确诊断,移植了4个胚胎,获得1例临床妊娠。孕11周时经腹吸取绒毛,证实为完全正常胚胎,最终分娩了一正常女婴。结论应用单细胞MGB探针荧光PCR技术可对β地贫以及其它单基因遗传病进行植入前遗传学诊断,达到优生目的。
Objective To establish a method for the detection of β-thalassemia by single-cell MGB probe fluorescence PCR and its application in PGD of β-thalassemia. Methods Single lymphocyte of thalassemia carriers was obtained and stable single cell MGB probe was used to detect fluorescence. PCR was performed on two couples of carriers who were carriers of β thalassemia gene by using MGB probe. Poor PGD. Results Single cell MGB probe was used to detect the genotypes of 200 single lymphocytes in carriers of β thalassemia gene. The average amplification efficiency was 94% and the average allele release (ADO) rate was 2.5%. Four cycles of PGD were performed on the two couples. Sixteen embryos were biopsied and 16 blastomeres were obtained. Fifteen blastomeres were successfully amplified, with an amplification efficiency of 93.8% and an ADO rate of 6.2%. Fourteen embryos obtained a definite diagnosis after PCR analysis. Four embryos were transplanted and one case of clinical pregnancy was obtained. At 11 weeks of pregnancy, the abdomen was aspirated and confirmed as a completely normal embryo. Finally, a normal baby girl was delivered. Conclusion The single-cell MGB probe fluorescence PCR technique can be used to pre-implant genetic diagnosis of β-thalassemia and other single-gene genetic diseases and achieve the purpose of eugenics.