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目的:采用三重PCR技术检测缺失型杜氏肌营养不良(Duchenne muscular dystrophy,DMD)基因部分外显子和性别鉴定位点amelogenin基因,探讨该技术对DMD家系中致病基因携带者进行植入前遗传学诊断(preimplantation genetic diagnosis,PGD)的可行性。方法:显微操作取正常男性、正常女性和DMD基因8号、47号外显子缺失型DMD患者单个淋巴细胞及无DMD家族史夫妻行体外授精-胚胎移植(in vitro fertilization pre-embryo transfer,IVF-ET)术后废弃的单个卵裂球细胞,采用三重PCR技术扩增DMD基因8号、47号外显子和amelogenin基因。结果:64枚正常人单个淋巴细胞的DMD基因8号、47号外显子和amelogenin基因扩增阳性率分别为93.8%,93.8%和95.3%,假阳性率为2.8%;48枚8号外显子缺失型患者单个淋巴细胞的DMD基因47号外显子和amelogenin基因扩增阳性率均为95.8%,假阳性率为3.3%;48枚47号外显子缺失型患者单个淋巴细胞的DMD基因8号外显子和amelogenin基因扩增阳性率均为95.8%,假阳性率为0;40枚单卵裂球细胞DMD基因8号、47号外显子和amelogenin基因扩增阳性率分别为82.5%,80.0%和77.5%,假阳性率为0。结论:本研究所建立的单细胞三重PCR技术具有较高的扩增阳性率和特异性,有望在缺失型DMD家系中进行PGD的临床应用。
OBJECTIVE: To detect the amelogenin gene in exon and sex determination sites of the degenerative Duchenne muscular dystrophy (DMD) gene by triplex PCR, and to explore the effect of this technique on the preimplantation of the carriers of DMD pedigree Feasibility of preimplantation genetic diagnosis (PGD). Methods: The normal male and normal female and DMD gene exon 8 and exon 47 deletion type DMD patients with single lymphocytes and without DMD family history husband and wife in vitro fertilization - embryo transfer (IVF -ET), a single blastomere was used to amplify DMD gene exon 8 and exon 47 and amelogenin gene by triplex PCR. Results: The positive rate of DMD gene exon 8, exon 47 and amelogenin of single lymphocyte in 64 normal individuals were 93.8%, 93.8% and 95.3%, respectively, and the false positive rate was 2.8%. 48 exon 8 The positive rate of DMD gene exon 47 and amelogenin gene deletion in single lymphocyte of deletion type patients were 95.8% and the false positive rate was 3.3%. The 48 DMD gene exon 47 deletions in DMD gene No. 8 The positive rate of amelogenin gene amplification and amelogenin gene amplification were 95.8% and the false positive rate was 0. The positive rates of DMD gene exon 8, exon 47 and amelogenin gene amplification in 40 single blastomere were 82.5% and 80.0%, respectively 77.5%, false positive rate of 0. Conclusion: The single-cell triplex PCR technique established in this study has a high positive rate and specificity of amplification and is expected to be used in the clinical application of PGD in the missing DMD pedigrees.