膜反向斑点杂交法检测广东人G6PD基因6种常见突变  

Detection of Six Common Mutations of Cantonese G6PD Gene by RDB Assay

作　　者：郑卫东[1,2] 张太松[3] 陈冬[1,2] 葛艳芬[1,2] 林婷[1,2] 胡斌[3] 

机构地区：[1]广东省人民医院 [2]广东省医学科学院病理医学部检验科 [3]中山大学达安基因诊断中心,广东广州510080

出　　处：《中山大学学报（医学科学版）》2011年第4期463-466,共4页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省医学科学研究基金(A2009040)

摘　　要：【目的】探讨膜反向斑点杂交法在广东人G6PD基因6种常见突变同步检测中的应用。【方法】用高铁血红蛋白还原试验和G6PD/6-PGD酶活性比值法进行G6PD缺乏症筛查。应用多重PCR方法扩增G6PD基因目的片段。用Premier Primer6.0软件设计和优化针对广东人常见6种G6PD基因突变型的特异性寡核甘酸探针,用乙基-3-二甲氨基丙碳二亚胺(EDC)法处理尼龙膜并固定探针,制成G6PD基因突变检测用膜条。多重PCR扩增产物与固定在膜条上寡核苷酸探针进行杂交,通过显色反应判读结果。以G6PD基因测序为金标准,对膜反向斑点杂交法进行评价。【结果】26例标本中膜反向斑点杂交法检测结果:95A→G突变型5例,1024C→T突变型3例,1376G→T突变型8例,1388G→A突变型7例,392G→T突变型1例,未发现1311C→T突变型,有两例检测为阴性。膜反向斑点杂交法未能测出的2例标本经基因测序证实存在392G→T突变,其余24例膜反向斑点法与基因测序法的结果完全一致,符合率为92.3%(24/26)。【结论】在优化探针设计和杂交条件前提下,膜反向斑点杂交法可用于广东人G6PD基因6种常见突变型的同步检测。[Objective] To explore the application of reverse dot blotting（RDB） assay in the simultaneous detection of the most common six Cantonese G6PD gene mutations.[Methods] Glucose 6 phosphate dehydrogenase（G6PD） deficiency was screened using the methemoglobin reduction test and G6PD/6-PGD ratio method.Multiplex PCR was used to amplify the objective fragments of G6PD gene.The specific oligonucleotide probes were designed and optimized using the Premier Primer 6.0 program.Nylon membrane was treated by（ethyl-3-（dimethylaminopropyl） carbodiimide,EDC） and then was dotted with the specific hybridization probes.The genotypes were judged by colorimetric reaction after the RDB.Moreover,the RDB assay was evaluated by using the sequencing result as a gold standard.[Results] Among 26 severe G6PD deficient patients,five individuals was found carrying 95 A→G mutation,three with 1024 C→T,eight with 1376 G→T,seven with 1388G→A,one with 392 G→T.Moreover,two G392T homozygote identified by DNA sequencing could not be correctly detected by the RDB assay.[Conclusions] The RDB assay could be used to simultaneously detect the most common six Cantonese G6PD gene mutations after optimizing probe design and hybridization condition.

关 键 词：葡萄糖-6-磷酸脱氢酶 基因突变 反向斑点杂交法(RDB) 

分 类 号：R555[医药卫生—血液循环系统疾病]