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目的 建立五重巢式PCR技术同步检测单细胞DMD基因和性别 ,探讨该技术在杜氏肌营养不良症的植入前诊断 (DMD -PGD)的可行性。方法 获取正常男性、女性单个淋巴细胞和无DMD家族史的单个胚胎细胞 ,用五重巢式PCR技术检测DMD基因外显子 17、19、44、48和SRY基因。结果 在正常男性单个淋巴细胞扩增成功率为 96 7% (145 /15 0 ) ,假阳性率为 4% (1/2 5 ) ,假阴性率为 0 (0 /2 5 )。在正常女性单个淋巴细胞扩增成功率为 98% (147/15 0 ) ,假阳性率为 0 (0 /2 5 ) ,假阴性率为 0 (0 /2 5 )。 15个胚胎细胞扩增成功率为 10 0 % (6 6 /75 ) ,假阳性率为 0 (0 /2 5 )。结论 本文建立的单细胞DMD基因外显子 17、19、44、48和SRY基因五重巢式PCR技术具有较高的敏感性和特异性 ,有望应用于DMD -PGD
Objective To establish a five-fold nested-PCR technique for simultaneous detection of single-cell DMD gene and sex and to explore the feasibility of this technique in pre-implantation diagnosis of Duchenne muscular dystrophy (DMD-PGD). Methods Single embryo cells from normal male and female lymphocytes and without DMD family history were obtained. Exon 17, 19, 44, 48 and SRY genes of DMD gene were detected by five-fold nested PCR. Results The success rate of single lymphocyte expansion in normal male was 96 7% (145/158), the false positive rate was 4% (1/2 5), and the false negative rate was 0 (0/25). In normal women, the success rate of single lymphocyte expansion was 98% (147/15 0), the false positive rate was 0 (0/25) and the false negative rate was 0 (0/25). The success rate of 15 embryo cell expansion was 10 0% (6 6/75), the false positive rate was 0 (0/25). Conclusion The single-nested DMD gene exon 17, 19, 44, 48 and SRY gene five-nested PCR have high sensitivity and specificity and are expected to be used in DMD-PGD