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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 method for detecting chimeric mutation of SCN1A gene by TaqMan real-time PCR and investigate the genetic background of infants with severe myoclonic epilepsy (SMEI). Methods The peripheral blood whole genome DNA of one case of SMEI children and their parents was extracted. The mutations of SCN1A gene were amplified by PCR and sequenced directly. The mutant plasmid, the wild-type plasmid, the TaqMan-MGB probe and the primer were then synthesized according to the c.302G> A mutation carried by the child. Mutants and wild-type plasmids were also studied to further analyze the specificity and sensitivity of the method. Finally, TaqMan real-time PCR was used to find out if their parents were chimeric SCN1A mutants. Results The established TaqMan real-time PCR showed no nonspecific amplification and high sensitivity, and a mutation of 1.6% could be detected in the wild-type background with 107 copies / ml. Direct sequencing found that there was SCN1A c.302G> A heterozygous mutation in his children, his father found a small number of suspicious mutations in the background, but the mother did not find. Using TaqMan real-time PCR confirmed that the father of children with c.302G> A chimeric mutation. Conclusion TaqMan real-time quantitative PCR can effectively improve the detection rate of chimeric mutation of SCN1A gene, which is helpful to prenatal and postnatal care and genetic counseling.