G6PD缺乏症单细胞SNaPshot基因诊断方法的建立  

Establishment of a SNaPshot method for genetic diagnosis of G6PD deficiency in a single cell

作　　者：吴彤华[1,2,3,4] 成金泉 朱元昌 黄菊 马珍珍 尹彪 曾勇 蔡应木[1,5] 

机构地区：[1]汕头大学医学院,汕头515041 [2]深圳中山泌尿外科医院生殖医学中心,深圳518045 [3]深圳中山生殖与遗传研究所,深圳518045 [4]深圳市围着床期生殖免疫重点实验室,深圳518045 [5]汕头大学医学院第一附属医院检验科,汕头515041

出　　处：《生殖医学杂志》2015年第4期304-310,共7页Journal of Reproductive Medicine

基　　金：深圳市科技计划项目(201202200)

摘　　要：目的建立有效可行的葡萄糖-6-磷酸脱氢酶(G6PD)缺乏症单细胞基因诊断技术。方法获取正常人及G6PD基因突变杂合子的单个淋巴细胞,采用多重巢式PCR结合多重SNaPshot技术对G6PD基因突变高发的四个外显子上的12个突变位点进行基因诊断。结果共检测了58个正常和108个杂合子单淋巴细胞,扩增效率为90.97%,等位基因脱扣率为8.08%。结论所建立的多重巢式PCR结合SNaPshot技术可在单细胞水平上快速、准确地检测12个G6PD基因突变位点,可望在临床上实现G6PD缺乏症的植入前遗传学诊断。Objective. To establish an effective and feasible assay for genetic diagnosis of glucose-6-phosphate dehydrogenase（G6PD）based on a single cell. Methods： The single lymphocyte was acquired from normal people and the patients with heterozygote of G6PD gene mutation. Genetic diagnosis was performed based on single cell to detect 12 mutation sites on four high G6PD exons with high mutation rates by a multiplex nested PCR combined with multiplex SNaPshot. Results： Fifty-eight lymphocytes from normal people and 108 lymphocytes from the patients with heterozygote were detected. The amplification efficiency of a single lymphocyte was 90.97% ,and the rate of allele dropout was 8.08%. Conclusions： The established multiplex nested PCR combined with SNaPshot assay can fast and accurately detect 12 mutation sites of G6PD gene based on single cell. It is expected to achieve preimplantation genetic diagnosis of G6PD deficiency in clinic.

关 键 词：葡萄糖-6-磷酸脱氢酶 植入前遗传学诊断 巢式聚合酶链反应 多重SNaPshot技术 等位基因脱扣 

分 类 号：R4[医药卫生—临床医学]