荧光探针熔解曲线分析法快速检测SMN1基因纯合性缺失  

Probe-based fluorescence melting curve analysis assay for rapid determining of the homozygous deletion of SMN1

作　　者：孙曼娜[1] 娄季武[1] 赵颖[1] 付有晴 刘彦慧[1] 李亮 SUN Man - na;LOU Ji - wu;ZHAO Ying;FU You - qing;LIU Yan - hui;LI Liang(Prenatal Diagnosis Center,Dongguan Maternal and Children Hospital,Dongguan 523000,Guangdong,China)

机构地区：[1]东莞市妇幼保健院产前诊断中心,广东东莞523000 [2]南方医科大学基础医学院医学遗传学教研室,广东广州510515

出　　处：《广东医学》2019年第11期1546-1550,共5页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:81101328);东莞市社会科技发展一般项目(编号:201750715007181);南方医科大学创新驱动推进项目(编号:CX2015N011)

摘　　要：目的建立一种快速检测SMN1基因纯合缺失的荧光探针熔解曲线分析方法。方法收集正常成人的外周血标本94份,经MLPA检测过的已知SMN1和SMN2基因拷贝数的样本238例。采用荧光探针熔解曲线分析技术,分别对SMN1基因c.840C>T和c.835-45G>A进行基因分型,根据基因分型结果判断是否存在SMN1基因exon7纯合缺失。对SMN1基因和SMN2基因拷贝数比值为2∶2、2∶0和0∶2基因型样本进行10倍梯度稀释,评估检测体系的敏感性。选取SMN1基因和SMN2基因拷贝数比值为2∶2、2∶0和0∶2基因型样本各8例,不同时间检测3次,用于评估检测体系的重复性。结果该方法可以方便地检测SMN1基因exon7纯合缺失,灵敏度至少为0.05ng基因组DNA。应用此方法检测94例正常成人样本,未发现SMN1基因exon7纯合缺失;对238例已知基因型样本进行检测,结果显示准确率为100%。结论荧光探针熔解曲线分析方法可以快速准确检测SMN1基因exon7纯合缺失,操作简单实用,适合大规模人群筛查和产前分子诊断。Objective To develop a probe - based fluorescence melting curve analysis assay for homozygous dele- tion of SMN1 exon7. Methods Peripheral blood samples from 94 healthy individuals were collected,and 238 samples with genotypes identified by MLPA were collected.The probe based fluorescence melting curve analysis was developed to determine the genotypes of c.840C > T and c.835 - 45G > A;homozygous deletion of SMN1 exon7 was identified by the genotypes of the two nucleotides.The sensitivity of the assay was evaluated using 10 - fold serial dilutions on SMN1 and SMN2 gene copy number ratios of 2∶ 2,2∶ 0 and 0∶ 2 genotype samples;and reproducibility was evaluated using each 8 samples with 2∶ 2,2∶ 0 and 0∶ 2 genotypes.Results Homozygous deletion of SMN1 exon7 could be easily determined from different shapes of melting curves,and the minimum sensitivity was 0.05 ng gDNA.No homozygous deletion of SMN1 exon7 was identified in 94 healthy individuals,and 100% diagnostic accuracy rates was observed in 238 samples with identified genotypes.Conclusion The probe - based fluorescence melting curve analysis assay is a rapid and relia- ble method for large - scale population screening and prenatal diagnosis of SMA patients caused by homozygous deletion of SMN1 exon7.

关 键 词：脊髓性肌肉萎缩症 基因缺失 生存运动神经元基因 熔解曲线分析 

分 类 号：R394.3[医药卫生—医学遗传学] R596.2[医药卫生—基础医学]