基于荧光定量 PCR 技术的人类 CYP2C9 和 VKORC1 基因多态性检测方法学评价
| 作者: |
1戎国栋,
1沈丹婷,
1陈丹,
1赵鸿,
1吴蕾,
1张洁心,
1王芳,
1黄珮珺,
1徐婷
1 南京医科大学第一附属医院检验学部，南京 210029 |
| 通讯: |
徐婷
Email: tingxu@njmu.edu.cn |
摘要
目的: 对基于荧光定量PCR技术的人类CYP2C9和VKORC1基因多态性检测试剂盒进行性能评 价。方法: 选取20例已通过Sanger测序确定基因分型的临床样本编盲，按照试剂盒流程进行实时 荧光PCR检测，评价其准确度;对4种基因型样本(CYP2C9*3纯合野生型、CYP2C9*3杂合突变 型、VKORC1-1639G>A纯合突变型和VKORC1-1639G>A杂合突变型)重复检测10次，评价其批内 精密度;DNA样本梯度稀释，评价最低检测限。结果: 实时荧光PCR法与测序结果对比分析， 结果全部一致，准确度100%。不同基因型批内精密度均≤5%，批内精密度可接受。DNA浓度在 0.964 μg/μL仍可检测标本的基因型。结论: 实时荧光PCR技术对人类CYP2C9和VKORC1基因分型检 测在方法学上稳定可靠，此评价模式可适用于基于荧光PCR技术的其他基因多态性试剂盒的评价。
关键词：
实时荧光PCR
CYP2C9
VKORC1
药物代谢酶
基因多态性
Evaluation of human CYP2C9 and VKORC1 gene polymorphisms detection based on uorescent quantitative PCR
CorrespondingAuthor: Xu Ting Email: tingxu@njmu.edu.cn
Abstract
Objective: To evaluate the performance of human CYP2C9 and VKORC1 polymorphism detection kit based on fluorescent quantitative PCR. Methods: The genotypes of 20 samples were detected by both Sanger sequencing and real-time PCR blindly to evaluate the accuracy of this kit. e intra-batch precision was evaluated by measuring every genotype sample (including CYP2C9*3 homozygous wild type, CYP2C9*3 heterozygous mutant, VKORC1-1639G>A homozygous mutant and VKORC1-1639G>A heterozygous mutant) 10 times. e DNA samples were detected by gradient dilution to evaluate the minimum detection limit. Results: e results of real-time PCR were in complete agreement with that of Sanger sequencing, so the accuracy was 100%. e intra- batch of samples with four genotypes was less than 5%. e genotype was detectable when DNA concentration was 0.964 μg/μL. Conclusion: Real-time PCR is a reliable method for the detection of human CYP2C9 and VKORC1 genotypes. is evaluation model can be applied to the evaluation of other gene polymorphism kits based on uorescence PCR.
Keywords：
real-time uorescence PCR
CYP2C9
VKORC1
drug metabolism enzyme
gene polymorphism