单细胞五重巢式PCR检测DMD基因

目的:探索建立单细胞多重巢式PCR检测杜氏肌营养不良症(DMD)基因外显子17、19、44、45、48的技术,及应用于植入前遗传学诊断(PGD)的前景。方法;获取正常男性单个淋巴细胞和无DMD家族史的单个胚胎细胞,行DMD基因外显子17、19、44、45、48的五重巢式PCR,分析扩增成功率、假阳性率和假阴性率。结果:20个单个淋巴细胞5个外显子扩增成功率为97％(97/100)、假阳性率为4％(1/25)、假阴性率为0％(0/25),20个单个胚胎细胞5个外显子扩增成功率为80％(80/100)、假阳性率为0％(0/25)。结论:本文建立的单细胞DMD基因外显子17、19、44、45、48的五重巢式PCR技术具有较高扩增成功率和特异性,应用于DMD的PGD具有现实的可能性。

Single Cell 5-plex Nest PCR for DMD Detection.

Objective: To establish a technique of single cell 5-plex nest PCR for Ducheme muscular dystrophy (DMD) detection and evaluate the possibility of using for preimplantation genetic diagnosis (PGD). Methods:Single lymphocyte in normal males and single blastomere from couple with no family history of DMD were obtained. Single cell 5-plex nest PCR for exons 17,19,44,45 and 48 of DMD gene was established. The success rates of amplification and specialities were analyzed. Results:The success rate of amplification in total 100 exons from 20 single lymphocytes is 97% (97/100) and the false positive rate is 4% (1/25) and the false negative rate is 0%(0/25). The success rate of amplification from the 20 blastomeres is 80% (80/100) and the false positive rate is 0% (0/28). Conclusion: The technique of single cell 5-plex nest PCR for exons 17,19,44,45 and 48 of DMD gene established in this program is highly successful and specific,it is practically possible to use this method for PGD of DMD.