焦磷酸测序检测NUDT15基因多态性的方法学评价  

Methodological evaluation of pyrosequencing in detecting NUDT15 gene polymorphism

作　　者：张婕妤 初亚男 封利颖 邹秉杰 ZHANG Jie-yu;CHU Ya-nan;FENG Li-ying;ZOU Bing-jie(Department of Clinical Pharmacy,General Hospital of Eastern Theater Command,Nanjing 210002)

机构地区：[1]东部战区总医院临床药学科,南京210002

出　　处：《中南药学》2021年第2期187-190,共4页Central South Pharmacy

基　　金：国家自然科学基金面上项目(No.61871403);江苏省青年医学重点人才(No.QNRC2016889)。

摘　　要：目的建立焦磷酸测序检测NUDT15 R139C基因多态性的方法并对其评价。方法设计引物,考察不同退火温度(55、57、60℃),建立NUDT15多态性分型最佳反应条件。检测不同量的*1*15型DNA样本(10、2、0.4 ng),考察方法的灵敏度;比较焦磷酸测序和Sanger测序分型结果,考察方法的准确度;对*1*1型和*1*15型样本重复检测3次,考察方法的重复性。结果明确扩增引物、57℃退火为焦磷酸测序检测NUDT15基因型的最佳条件,最低可检测0.4 ng基因组DNA;焦磷酸测序法和Sanger测序法的分型结果完全一致,方法准确度100%;批内重复3次检测结果完全一致。结论建立了基于焦磷酸测序的NUDT15 R139C基因多态性分型方法,此方法稳定可靠,可投入临床使用并为个体化使用巯嘌呤类药物提供指导。Objective To establish and evaluate a method to detect NUDT15 R139C gene polymorphism with pyrosequencing.Methods Different primers were designed and different annealing temperature(55,57,and 60℃)were assessed.Sensitivity was evaluated with different amount of*1*15 type DNA samples(10,2,and 0.4 ng).Genotyping results from pyrosequencing were compared with Sanger sequencing to evaluate the accuracy of the method.Samples*1*1 type and*1*15 type were detected for 3 times to evaluate the reproducibility.Results Amplification primers were determined and 57℃annealing temperature was the best selection in detecting NUDT15 genotype with pyrosequencing.The sensitivity of this method was 0.4 ng genomic DNA.The results of pyrosequencing were consistent with those of Sanger sequencing,showing 100%accuracy.The detection results from the 3 times were completely the same.Conclusion The method for NUDT15 genotype determination by pyrosequencing is established,which is stable,reliable,and suitable for guiding individualized thiopurine application.

关 键 词：方法学评价 焦磷酸测序 NUDT15 基因多态性 巯嘌呤类 

分 类 号：R96[医药卫生—药理学]