建立TaqMan实时定量PCR检测SCN1A基因嵌合突变  

Establishment of Taq Man fluorescence quantitative PCR to detect SCN1A mosaic mutation

作　　者：朱喆[1,2] 朱萌华[3] 王震[3] 陈秉宇[3] 郝珂[3] 蒋璐茜[3] 李凯强[3] 吕建新[1,3] 

机构地区：[1]温州医科大学检验医学院和生命科学学院,浙江温州325035 [2]浙江省宁波市第二医院输血科,浙江宁波315010 [3]杭州医学院附属人民医院,浙江省人民医院输血科,浙江杭州310014

出　　处：《中国优生与遗传杂志》2017年第8期7-9,34,共4页Chinese Journal of Birth Health & Heredity

基　　金：浙江省医药卫生科技计划项目(2015KYA028,2016DTB001,2014KYA233,2016KYB012);浙江省公益技术应用研究分析测试项(2015C37001);浙江省中医药项目(2014ZB007,2014ZQ005)

摘　　要：目的建立TaqMan实时定量PCR检测SCN1A基因嵌合突变的方法,调查婴儿严重肌阵挛癫痫(SMEI)家系的遗传背景。方法抽提1例SMEI患儿及其父母的外周血全基因组DNA,对SCN1A基因外显子PCR扩增后直接测序检测突变位点。然后根据患儿所携带的c.302G>A突变,合成突变型质粒、野生型质粒、TaqMan-MGB探针和引物。并以突变型及野生型质粒为研究对象,进一步分析方法的特异性及敏感度。最后用TaqMan实时定量PCR探寻其父母是否为SCN1A基因突变嵌合子。结果建立的TaqMan实时定量PCR未发现非特异性扩增,且敏感度高,在107copies/ml的野生序列背景下能检测出1.6%的突变量。直接测序发现患儿存在SCN1A c.302G>A杂合突变,父亲发现少量可疑突变背景,而母亲未发现。运用TaqMan实时定量PCR证实了患儿父亲含有c.302G>A嵌合突变。结论 TaqMan实时定量PCR能有效提高SCN1A基因嵌合突变的检出率,对优生优育和遗传咨询有较大帮助。Objective：To establish a Taq Man fluorescence quantitative PCR method to analyze the genetic background of a family with severe myoclonic epilepsy in infancy（SMEI）. Methods：Genomic DNA were extracted from the peripheral blood of SEMI child and his parents. SCN1 A gene mutation were detected by direct DNA sequencing. Then according to the mutation site,we designed SCN1 A wide-type plasmid,mutate plasmid,primers and Taq Man-MGB probe. The specificity and sensitivity of Taq Man quantitative PCR were further assessed by amplifying the recombinants containing wide-type and mutate plasmids. Finally,we took this method to search for the parental mosaicisms in the selected family,and compared with direct sequencing. Results：Non-specific amplifications were not found in Taq Man quantitative PCR. As for its sensitivity,we could approximately detect 1.6% mutate rate in the background of 107copies/ml wide-type gene. SCN1 A c.302GA heterozygous mutation were found in the SMEI child,his father showed some background but his mother showed no mutation. We verified the child′s SCN1 A mutation was inherited from his mosaicism father by Taq Man quantitative PCR. Conclusion The Taq Man fluorescence quantitative PCR was a high specific and sensitivity method for rapid detection of SCN1 A mosaic mutation in epilepsy families,and had a great helpful in and genetic counseling.

关 键 词：TaqMan实时定量PCR 癫痫 婴儿严重肌阵挛性癫痫 Ⅰ型电压门控钠离子通道α亚基 嵌合突变 

分 类 号：R440[医药卫生—诊断学] R742.1[医药卫生—临床医学]