单细胞二重巢式PCR诊断β地中海贫血

Diagnosing β-Thalassemia by Single Cell Duplex-nested PCR

【目的】探讨单细胞水平诊断β地中海贫血(β-thalassemia)的可靠性,为开展β地中海贫血的胚胎植入前遗传学诊断(preimplantationgeneticdiagnosis,PGD)打下基础。【方法】采用二重巢式PCR并结合扩增特异的突变系统检测技术(amplificationrefractorymutationsystem,ARMS)扩增含有CD41-42突变位点的β地中海贫血患者的单淋巴细胞及正常人单淋巴细胞和单卵裂球的β-珠蛋白基因的5个常见突变位点(CD41-42、IVS-Ⅱ-654、CD17、CD71-72和nt.-28位点),扩增产物经过2%的琼脂糖凝胶电泳检测。【结果】患者的88个单淋巴细胞的扩增成功率为95.5%,等位基因脱扣的发生率为4.8%,诊断准确率为95.2%;正常人的41个单淋巴细胞的扩增成功率均为95.1%,假阳性率为0;正常人的34个单卵裂球细胞的PCR扩增成功率为88.2%;35管细胞洗脱液空白对照组假阳性扩增率为0。【结论】单细胞二重巢式PCR并结合ARMS技术诊断β地中海贫血是稳定的、可靠的,适用于β地中海贫血的PGD。

[Objective]To research for the reliability of diagnosing β-thalassemia on single cell level and to make a basis for preimplantation genetic diagnosis （PGD）. [Methods]Five frequent mutations of β-globin gene at loci CD41- 42, CD17, nt.-28, CD71-72 and IVS-Ⅱ654 were amplified by duplex-nested PCR together with amplification refractory mutation system （ARMS） with single lymphocyte of the patient and the healthy adult as well as single blastomere of the healthy adult. The products of PCR were detected by 2% agarose gel electrophoresis. [Results]The amplification success rate of 88 single lymphocytes for the patient was 95.5%, the incidence of allele dropout was 4. 8%, and the diagnosis accurate rate was 95.2%; the amplification success rate of 41 single lymphocytes for the healthy adult was 95.1% ; the amplification success rate of 34 single blastomeres for the healthy adults was 88.2% ; the pseudopositive amplification rate of 35 tubes of blank with washing cell solution was 0. [Conclusion]It is stable and reliable to diagnose β-thalassemia by duplex-nested PCR together with ARMS, which is suitable for PGD of β- thalassemia, too.