中文摘要：

亚甲基四氢叶酸还原酶基因677C〉T和1298A〉C两个位点的多态性与临床常用抗肿瘤药物甲氨喋呤及氟尿嘧啶的作用密切相关,对这两个位点多态性的检测能指导临床合理用药。为进一步缩短检测时间,降低检测成本,本研究建立了基于全血直接PCR的焦测序检测方法,采用＂HpH Buffer＂直接扩增全血模板,仅需1μL全血样本即可对两个位点进行高效扩增。扩增产物经碱变性法制备单链模板后进行焦磷酸测序,经过条件优化,仅需5μL扩增产物和1μL微球即可完成高灵敏的焦测序反应。为验证方法的准确性,检测了12例临床样本,均能正确检测两个位点的基因多态性。本研究为临床基因多态性检测提供了一种操作简便,耗时短,成本低,准确度高的方法,本方法可用于指导甲氨喋呤和5-氟尿嘧啶的个体化用药。

英文摘要：

The polymorphisms of 677C〉T and 1298A〉C in the methylenetetrahydrofolate reductase gene （MTHFR） play a crucial role in the pharmacogenetics of methotrexate （MTX） and 5-fluorouracil （5-FU）. Detection of the MTHFR gene polymorphisms will give a rational guidance to personalized medicine. In order to further reduce the detection time and costs, we developed a method based on highly sensitive pyrosequencing coupled with PCR using human whole blood as starting material. This method can efficiently detect the two sites by using only 1 μL of human whole blood samples for amplification based on ＂HpH Buffer＂. After optimizing the conditions, single strand DNA denatured by alkali from 5 μL of PCR products and 1 μL of streptavidin-coated beads could be enough for pyrose- quencing. To investigate the accuracy of this method, 12 clinical samples were detected, and all of the MTHFR 677C〉T and 1298A〉C polymorphisms were correctly typed. This study demonstrated an easily-operated, time-saving, inexpensive, and highly accurate method for genotyping clinical samples. The method can be well applied to personalized medicine of MTX and 5-FU.