改进的四引物扩增受阻突变体系PCR方法同时检测叶酸代谢过程相关的四个单核苷酸多态性位点的研究  

A improved tetra-primer amplification refractory mutation system PCR based assay to detect four single nucleotide polymorphisms associated with folate metabolism in a single reaction

作　　者：段素霞 李贵霞[2] 赵梦川 王乐[3] 冯志山[3] 张益[4] 马学军[4] 

机构地区：[1]河北医科大学研究生学院,石家庄050031 [2]河北省儿童医院检验科 [3]河北省儿童医院儿科研究所 [4]中国疾病预防控制中心病毒病预防控制所

出　　处：《中华检验医学杂志》2017年第10期799-804,共6页Chinese Journal of Laboratory Medicine

基　　金：河北省政府资助省级临床医学优秀人才项目;国家重点研发计划（2016YFC1200900）

摘　　要：目的建立改进的四引物扩增受阻突变体系PCR（T－ARMS－PCR）检测方法，实现单管一次性检测叶酸代谢过程相关的4个单核苷酸多态性（SNP）位点：rs1801133，rs1801131，rs1805087和rs1801394。方法方法学建立。于2017年1至4月在河北省儿童医院检验科收集150名体检儿童抗凝全血标本以待验证。利用T－ARMS－PCR原理及多重嵌合引物策略设计rs1801133、rs1801131、rs1805087及rs1801394的特异性内外嵌合引物。通过优化引物浓度和反应条件，经单管PCR反应，结合QIAxcel毛细管电泳分析扩增产物长度，判读150份标本的SNP基因型，同时所有标本经测序法进一步验证。利用SPSS 17.0统计软件卡方检验对150份标本的4个SNP位点分别进行哈迪－温伯格遗传平衡（HWE）检测。结果改进的T－ARMS－PCR结合毛细管电泳分析的方法可在3 h内一次性识别本研究中叶酸代谢过程相关的4个SNP位点的8个不同等位基因，可对150份全血标本的SNP位点准确分型，其分型结果与测序法完全相符。4个SNP位点基因型的分布均符合HWE平衡定律（χ^2rs1801133＝0.69, Prs1801133＝0.40; χ^2rs1801131＝0.21, Prs1801131＝0.64; χ^2rs1805087＝3.32, Prs1805087＝0.07; χ^2rs1801394＝1.91, Prs1801394＝0.17）。结论本研究建立的改进的T－ARMS－PCR结合毛细管电泳分析方法，可准确地同时检测叶酸代谢过程相关的4个SNP位点，为指导人群特异性地补充叶酸提供了一种可能的技术手段。ObjectiveTo develop a tetra-primer amplification refractory mutation system PCR （T-ARMS-PCR） assay for detecting four single nucleotide polymorphisms （SNPs）： rs1801133, rs1801131, rs1805087 and rs1801394 associated with folate metabolism in a single reaction tube.MethodsMethodology was developed. Then, it applied the established method to analyze 150 physical examination children′s anticoagulant blood samples admitted to the Department of Clinical Laboratory, Children′s Hospital of Hebei Province from January 2017 to April 2017. Four sets of chimeric primers consisting of a universal Tag sequence and targeting rs1801133, rs1801131, rs1805087 and rs1801394 fused to the specific sequence were designed according to the T-ARMS-PCR principle and the multiplex chimeric primers strategy. A single tube PCR was then conducted after optimizing the primer concentrations and the reaction conditions. The amplified products were analyzed by QIAxcel capillary electrophoresis, and the corresponding SNP genotypes of 150 samples were identified. Furthermore, all samples were verified by direct sequencing. And the Hardy-Weinberg Equilibrium （ HWE） testing of four SNPs from 150 samples were conducted by SPSS17.0 Chi-square test.ResultsThe improved T-ARMS-PCR combined with capillary electrophoresis can accurately verify eight different alleles of the four SNPs associated with folate metabolism at one time in 3 hours. Four SNPs of 150 whole blood samples were accurately classified and the results were completely consistent with direct sequencing. All the genotype frequencies of these four SNPs were in HWE （χ^2rs1801133=0.69, Prs1801133=0.40; χ^2rs1801131=0.21, Prs1801131=0.64; χ^2rs1805087=3.32, Prs1805087=0.07; χ^2rs1801394=1.91, Prs1801394=0.17）.ConclusionsThe proposed improved T-ARMS-PCR combined with capillary electrophoresis in this study can accurately verify four SNPs associated with folate metabolism in a single reaction tube. This method might be a valuable tool to specifically guide the folate suppl

关 键 词：叶酸 代谢 多态性 单核苷酸 聚合酶链反应 电泳 毛细管 DNA突变分析 DNA引物 

分 类 号：R440[医药卫生—诊断学]