荧光短片段多重PCR在脊髓性肌萎缩症SMN1基因突变检测中的应用  

Application of quantitative fluorescence multiplex polymerase chain reaction in detection of SMN1 gene mutations in spinal muscular atrophy

作　　者：杨慧 李桂河 谢颖璇 陈海珠 李世举[3] 何瑾[2] YANG Hui;LI Guihe;XIE Yingxuan;CHEN Haizhu;LI Shiju;HE Jin(Department of Neurology,the First Affiliated Hospital,Fuzhou 350005,China)

机构地区：[1]福建中医药大学附属第二人民医院检验科,福州350003 [2]福建医科大学附属第一医院神经内科 [3]福建中医药大学附属第二人民医院神经内科

出　　处：《中国神经精神疾病杂志》2022年第9期519-524,共6页Chinese Journal of Nervous and Mental Diseases

基　　金：福建医科大学启航基金项目(编号:2019QH2027);福建医科大学附属第一医院脑血管及神经系统疑难病症诊治能力提升工程项目(编号:PT-YNBZN2018)。

摘　　要：目的设计特异性扩增引物,建立荧光短片段多重PCR技术检测SMN1基因拷贝数变异的方法,验证方法的检测性能并初步应用于基因拷贝数检测。方法选取107例来自神经系统疾病生物样本库的人外周血DNA样品(包括SMN1基因0拷贝30例、1拷贝47例、2拷贝30例),设计5’端带有FAM荧光基团的SMN1基因7号外显子引物以及三对内参基因引物,利用荧光短片段多重PCR技术,使用基因分析仪片段分析多重PCR的产物,并与相应的MLPA检测结果进行比较。结果特异性扩增SMN1基因7号外显子及内参基因的引物可实现目的片段的扩增,通过计算可得到0拷贝、1拷贝、2拷贝的样本结果。对107例DNA样本的SMN1基因拷贝数荧光短片段多重PCR的检测结果进行分析,实验结果均与MLPA检测结果相一致。结论使用荧光标记多重PCR反应检测SMN1拷贝数可重复性好,精确度高,与MLPA得到的结果高度一致,具有便捷、准确、成本低的优势,可满足临床高准确性的检测要求,可用于SMA新生儿携带者的大规模筛查。Objective To establish the technique of quantitative fluorescence multiplex polymerase chain reaction(QFM-PCR)to detect SMN1 copy number variation by specific primers,and evaluate its clinical application in SMN1 copy number detection.Methods A total of 107 human peripheral blood DNA samples(including 30 samples with 0 copy of SMN1 gene,47 samples with 1 copy of SMN1 gene,and 30 samples with 2 copies of SMN1 gene)were tested by QFM-PCR and multiplex ligation-dependent probe amplification(MLPA).The primers labelled with FAM fluorescence at 5'end for QFM-PCR were designed specifically targeted on exon 7 of SMN1 gene,and three reference genes.The amplicons of QFM-PCR were analyzed using the Gene Analyzer fragment and compared with MLPA results.Results The specific primers of SMN1 exon 7 and the reference genes could achieve the targeted fragments amplification,and the results of samples with 0 copy,1 copy and 2 copies were obtained.The results of SMN1 gene copy number detected by QFM-PCR in 107 samples were consistent with that of MLPA.Conclusions QFM-PCR has the advantages of high repeatability and accuracy for the detection of SMN1 copy number,which is consistent with that of MLPA.And the application of QFM-PCR in SMN1 copy number detection has higher clinical values and better prospects for the large-scale screening in neonatal SMA carriers.

关 键 词：荧光短片段多重PCR 脊髓性肌萎缩症 SMN1基因拷贝数检测 多重连接依赖性探针扩增技术 运动神经元生存基因1 运动神经元生存基因2 神经遗传病 携带者筛查 

分 类 号：R741[医药卫生—神经病学与精神病学]