焦磷酸测序检测CYP2C19基因多态性方法的建立.docVIP

焦磷酸测序检测CYP2C19基因多态性方法的建立 　　[摘要] 目的 建立一种基于焦磷酸测序技术的用于CYP2C19基因多态性快速检测的方法。 方法 选择2016年12月～2017年3月于贵州省人民医院行健康体检者50名，提取其基因组DNA，针对CYP2C19*2（681GA）、CYP2C19*3（636GA）、CYP2C19*17（-806CT）3个单核苷酸多态性（SNP）位点分别设计一套PCR扩增引物和焦磷酸测序引物。将PCR扩增产物制备成单链测序模板，用Qiagen PyroMark Q24焦磷酸测序仪进行测序和基因型分析，采用Sanger一代测序技术对焦磷酸测序结果进行验证。 结果 根据出峰的碱基和峰高可清楚判断出3个SNP位点的不同基因型，检测结果与Sanger测序结果完全一致。50名健康体检者中检出CYP2C19超快代谢型1例，快代谢型22例，中代谢性22例，慢代谢型5例。 结论 本研究建立的焦磷酸测序检测CYP2C19基因多态性的方法具有快速、准确、成本低的特点，适用于临床实验室对CYP2C19基因多态性的分型。 　　[关键词] CYP2C19；基因多态性；焦磷酸测序；氯吡格雷 　　[中图分类号] R446.9 [文献标识码] A [文章编号] 1673-7210（2017）10（b）-0011-04 　　Establishment of genotyping methods for polymorphisms of CYP2C19 gene based on pyrosequencing technology 　　YANG Nannan1 XU Qin1 LUO Zhenyuan2 FU Xue2 HUANG Shengwen2▲ 　　1.School of Clinical Laboratory Science， Guizhou Medical University， Guizhou Province， Guiyang 550004， China； 2.Department of Clinical Laboratory， Guizhou Provincial People′s Hospital， Guizhou Province， Guiyang 550002， China 　　[Abstract] Objective To establish a fast detection method for CYP2C19 genotyping based on pyrosequencing technology. Methods Genomic DNA was extracted from 50 blood samples of healthy subjects collected from December 2016 to March 2017 in Guizhou Provincial People′s Hospital， and a set of amplification primers and pyrophosphate sequencing primers were designed according to the single nucleotide polymorphism （SNP） sites of CYP2C19*2 （681GA）， CYP2C19*3 （636GA）， CYP2C19*17 （-806CT）. Single-stranded templates were made from PCR products， then sequencing and genotyping analysis were performed on a Qiagen PyroMark Q24 Sequencer System. The sequencing results were verified via Sanger sequencing. Results Based on the type and peak height of bases of DNA sequence， the genotypes of three SNPs could be identified clearly， which were consistent with Sanger sequencing results. Among the 50 samples of healthy group， 1 case was ultra-rapidmetabolism， 22 cases were rapidmetabolism， 22 cases were medium metabolism and 5 cases we