AS-PCR法检测CLU基因SNP位点方法的建立

Establishment of AS-PCR method for detecting SNP locus of CLU gene

目的 基于等位基因特异性PCR(allele-specific PCR,AS-PCR)原理,建立一种QPCR检测CLU基因与阿尔兹海默病(AD)相关性SNP位点基因型的筛选方法。方法根据CLU基因上的rs11136000,rs2279590和rs9331888 3个SNP位点设计特异性的野生型、突变型引物,使用实时荧光定量PCR(QPCR)对AD患者外周血样本进行SNP基因分型,以DNA基因测序法结果作为参考标准。结果 10例样本的QPCR定量分型结果显示,CLU基因rs11136000位点检测到CC基因型7例,CT基因型3例,未发现TT基因型;CLU基因rs2279590位点检测到CC基因型7例,CT基因型3例,未发现TT基因型;CLU基因rs9331888位点检测到CC基因型3例,CT基因型4例,TT基因型3例。检测结果与基因测序法结论完全一致。结论 基于AS-PCR技术的QPCR检测法可以高效、简洁的对CLU基因上的SNP位点进行基因分型,并为后续大量AD临床样本的SNP筛查提供了一种新的检验方法。

Objective Based on the principle of allele-specific PCR(AS-PCR),a screening method for detecting the genotype of CLU gene and Alzheimer’s disease(AD)-associated SNP locus was established by QPCR.Methods According to rs11136000,rs2279590 and rs9331888 three SNPs on the CLU gene,specific wild-type and mutant primers were designed,and SNP genotyping was performed on peripheral blood samples of AD patients by real-time fluorescence quantitative PCR(QPCR).The results of the sequencing method were used as reference standards.Results Quantitative typing results of QPCR of 10 samples showed that CC genotype was detected in 7 cases of CLU gene rs11136000,CT genotype was detected in 3 cases,and TT genotype was not found.CC genotype was detected in 7 cases of CLU gene rs2279590,CT genotype in 3 cases,and TT genotype was not found.CC genotype,CT genotype and TT genotype were detected in 3 patients with CLU gene rs9331888.The test results are completely consistent with the results of the gene sequencing method.Conclusion Based on AS-PCR,The QPCR method can efficiently and concisely genotype SNP locus on CLU gene,and provide a new test method for SNP screening of a large number of AD clinical samples.