遗传 ›› 2011, Vol. 33 ›› Issue (11): 1283-1290.doi: 10.3724/SP.J.1005.2011.01283

• 技术与方法 • 上一篇    

单管PCR-Pyrosequencing快速检测华法林代谢酶基因多态性方法的建立

施宏, 虞闰六, 马金飞, 任绪义   

  1. 杭州迪安医学检验中心, 杭州 310030
  • 收稿日期:2011-03-16 修回日期:2011-06-26 出版日期:2011-11-20 发布日期:2011-11-25
  • 通讯作者: 任绪义 E-mail:renxy@dagene.net

Development of a single-tube PCR-pyrosequencing method for simultaneous and rapid detection of the genetic polymorphism of warfarin metabolizing enzymes

SHI Hong, YU Run-Liu, MA Jin-Fei, REN Xu-Yi   

  1. Research and Development Centre, Hangzhou D. A. Medical Laboratory, Hangzhou 310030, China
  • Received:2011-03-16 Revised:2011-06-26 Online:2011-11-20 Published:2011-11-25
  • Contact: REN Xu-Yi E-mail:renxy@dagene.net

摘要: 文章旨在建立一种单管、快速及高通量的华法林药物代谢酶相关基因多态性的检测方法。通过抽取人外周血DNA, 应用带有生物素标记的扩增引物, 经PCR扩增并制备焦磷酸测序单链模板, 于PyroMark ID焦磷酸测序仪上进行焦磷酸测序, 以Sanger测序法测序结果为对照, 观察分析的准确性。结果显示, 华法林药物代谢酶的3个相关基因多态性(CYP2C9*2、CYP2C9*3、VKORC1(-1693))于单管中可被同时检测, 一次可获得96份DNA的华法林药物代谢相关多态性位点检测结果。经与Sanger测序方法比较, 符合率为100%。结果表明本方法可准确、高通量、快速检测华法林药物代谢酶相关基因多态性, 与单管检测一个位点的焦磷酸测序方法相比, 能有效降低检测成本, 节省检测时间。该方法在个性化医疗上有较大的推广应用价值, 也可以将该平台运用于其他疾病相关基因多态性检测。

关键词: CYP2C9, VKORC1, 华法林, 焦磷酸测序

Abstract: The purpose of this article is to develop a new high throughput method for detecting genetic polymorphism of warfarin metabolism-related genes rapidly in a single tube. Genomic DNA from human peripheral blood was extracted, and amplified with biotinylated primer to obtain single-stranded templates for pyrosequencing. Then, the single-stranded tem-plates were subjected to Pyrosequencing analysis using PyroMark ID instrument. Simultaneously, Sanger sequencing was also applied to sequence the products as a control to check the reliability of the pyrosequencing result.. The results dis-played that three variants of the warfarin metabolism-related genetic polymorphism (CYP2C9*2, CYP2C9*3, and VKORC1(-1693)) could be simultaneously detected using three different sequencing primers in a single-tube (one test), and 96 tests could be carried out each time. Repeat test and reliability test indicated that the agreement between the pyrosequencing and the Sanger sequencing methods was 100%. . All of these demonstrated that pyrosequencing could ac-curately and rapidly detect the genetic polymorphism of the warfarin drug metabolism-related genes with high throughput. Comparing with simplex pyrosequencing, the method established in the present study was much more economical and timesaving. It has a great value in personalized medical treatment and could be extended to the other genetic diseases.

Key words: CYP2C9, VKORC1, warfarin, pyrosequencing