【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0.964μg/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。更多还原

【Abstract】 Objective: To evaluate the performance of human CYP2 C9 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. The intra-batch precision was evaluated by measuring every genotype sample(including CYP2 C9*3 homozygous wild type, CYP2 C9*3 heterozygous mutant, VKORC1-1639 G>A homozygous mutant and VKORC1-1639 G>A heterozygous mutant) 10 times. The DNA samples were detected by gradient dilution to evaluate the minimum detection limit. Results: The results of real-time PCR were in complete agreement with that of Sanger sequencing, so the accuracy was 100%. The intrabatch of samples with four genotypes was less than 5%. The genotype was detectable when DNA concentration was 0.964 μg/μL. Conclusion: Real-time PCR is a reliable method for the detection of human CYP2 C9 and VKORC1 genotypes. This evaluation model can be applied to the evaluation of other gene polymorphism kits based on fluorescence PCR.更多还原