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目的探讨应用多重巢式聚合酶链反应(PCR)技术在β地中海贫血(简称β地贫)植入前遗传学诊断(PGD)中的应用。方法获取β地贫基因携带者单个淋巴细胞,建立了稳定的单细胞多重巢式PCR检测技术,可同时检测β珠蛋白基因及与β珠蛋白基因紧密连锁的HumTHO1基因,并对4例已出生的重型β地贫患儿及双方均为β地贫基因携带者的夫妇应用多重巢式PCR进行了β地贫的PGD。结果利用单细胞多重巢式PCR,可以同时检测中国人常见的16种β地贫突变类型,单个淋巴细胞平均扩增效率为91·3%,平均等位基因脱扣(ADO)率为17·0%。对4对夫妇进行4个周期PGD,共活检33个胚胎,获得33个卵裂球,其中30个卵裂球扩增成功,扩增效率为90·9%,ADO率为13·3%。26个胚胎经PCR分析后获得明确诊断,移植了8个胚胎,获得1例临床妊娠。孕17周时经脐带血穿刺,证实为完全正常胚胎,现已出生1名正常女婴。结论应用单细胞多重巢式PCR技术可对β地贫进行植入前遗传学诊断,达到优生目的。
Objective To investigate the application of multiplex nested polymerase chain reaction (PCR) in the preimplantation genetic diagnosis (β-thalassemia) of β thalassemia. Methods The single lymphocyte of β-thalassemia carriers was obtained, and a stable multi-nested single-cell PCR assay was established to detect both β-globin gene and HumTHO1 gene closely linked to β-globin gene. Of children with severe β-thalassemia and both carriers of β-thalassemia carriers had multiple nested PCR for β-thalassexmatic PGD. Results Sixteen kinds of β-thalassemia mutations were detected by single-cell multiplex nested PCR. The average amplification efficiency of single lymphocyte was 91.3%, and the average allele transfer rate was 17 · 0%. Four pairs of PGDs were performed on 4 couples and 33 embryos were biopsied. 33 blastomeres were obtained. 30 blastomeres were successfully amplified, with an amplification efficiency of 90.9% and an ADO rate of 13.3%. Twenty-six embryos were confirmed by PCR analysis, 8 embryos were transplanted and 1 clinical pregnancy was obtained. 17 weeks pregnant by cord blood puncture, confirmed as completely normal embryo, has been born a normal baby girl. Conclusion Single-cell multi-nested PCR can be used to diagnose β-thalassemia before implantation, achieving the goal of eugenics.