PCR-HRM技术在华法林最佳用药剂量关联基因单核苷酸多态性检测中的应用  

A Rapid PCR-HRM Genotyping Technique for the Single Nucleotide Polymorphisms of Genes that Influence Response to Warfarin Dose

作　　者：王芳芳[1] 尤崇革[2] 李光迪[1] 潘云燕[1] 郜莉娜[1] 李菲菲[2] 

机构地区：[1]兰州大学第二医院检验科,兰州730030 [2]兰州大学第二医院中心实验室,兰州730030

出　　处：《现代检验医学杂志》2011年第2期32-34,共3页Journal of Modern Laboratory Medicine

摘　　要：目的 最新研究显示华法林最佳用药剂量关联基因CYP2C9,CYP4F2和VKORC1可指导临床个体化用药,该成果的临床应用有赖于临床快速分子诊断方法的建立,为此该论文建立PCR-HRM（High-resolution melting）技术检测华法林最佳用药剂量关联基因单核苷酸多态性（SNP）并进行应用评价.方法 采用国际最前沿的基因突变筛查技术-HRM和新型荧光染料Eva Green对华法林最佳用药剂量关联基因CYP2C9＊2,CYP2C9＊3,CYP4F2 V433M和VKORC1 1173C/T四个SNP位点先进行染料法荧光PCR,接着进行PCR产物的高分辨熔解,依据熔解峰的特点进行SNP分型即PCR-HRM技术建立,最后通过检测临床181例标本进行临床应用评价.结果 在181例服用华法林的患者中检测到CYP2C9＊2杂合子2例,CYP2C9＊3杂合子15例,CYP4F2 V433M杂合子74例,纯合子14例,VKORC1 1173C/T杂合子33例.随机选择各类基因型PCR产物共24例进行测序,验证结果与检测结果一致.结论 PCR-HRM技术检测华法林最佳用药剂量关联基因SNP是一种灵敏、简便、快捷的低成本检测方法,可用于临床常规化分子诊断.Objective The latest studies showed that the Warfarin opitimal dosage associated genes of CYP2C9,CYP4F2, VKORC1 could guide clinical individualized medicine. It was essential to develop a rapid molecular diagnosis method for the clinical application of the achlevement,so this paper reported and evaluated the PCR-HRM technique detecting the single nucleotide polymorphisms （SNPs） of the genes in clinical application. Methods The four SNPs of CYP2C9 ＊ 2,CYP2C9 3,CYP4F2 V433M and VKORC1 1173C/T were detected using fluorescent PCR assay with the international cutting-edge gene mutations screening technology -HRM and new dye Eva Green. HRM was performed immediately after finished the amplification of target fragments,then judged the genotypes based on the characteristics of the ampilcons＇melting peaks. Using this PCR-HRM technnique, 181 cases of clinical specimens were detected and the application was evaluated. Results Heterozygous mutants of CYP2C9 ＊ 2,15 heterozygous mutants of CYP2C9 ＊ 3,14 homozygote and 74 heterozygous mutants of CYP4F2 V433M and 33 heterozygous mutants of VKORC1 1173C/T were found in the 181 cases of patients taking warfarin, which were confirmed by sequencing. Conclusion PCR-HRM technology is a sensitive,simple,fast,low-cost method detecting the SNPs of the genes that influence response to Warfarin dose,which can be used for clinical practice of molecular diagnostics.

关 键 词：华法林 EvaGreen 高分辨熔解技术 单核苷酸多态性 

分 类 号：Q503[生物学—生物化学] Q781