一步法人类CYP2C19基因分型检测在临床中的应用及其意义

Clinical application and significance of one-step humanCYP2C19 genotype detection

摘要:

目的 探索一步实时荧光PCR检测CYP2C19基因分型的方法。方法采用人类CYP2C19基因分型检测试剂盒(PCR-荧光探针法)对全血或人基因组进行一步法实时荧光PCR检测,同时采用焦磷酸测序和试剂盒进行对比。结果一步法检测结果准确性为100%。重复性检测显示,该法对各个基因型Ct值进行变异系数(CV值)的统计学分析,CV值均小于5%,对已知基因型的浓度的检测范围为0.2～125 ng,对杂合突变型基因组DNA(1 ng)和血液样本(十倍稀释)进行7次检测,变异系数(CV值)均小于5%,对特异性样品、EDTA抗凝样本、枸橼酸钠抗凝样本、乳糜血样本、血红素污染样本的基因型都有较好的检测效果。结论一步法检测方法准确性高,检测范围宽,重复性、特异性和灵敏度均能达到人基因组DNA样本的检测的水平,满足临床对人CYP2C19基因分型检测的目的。

Abstract:

Objective To verify the analysis capabilities of one-step real-time PCR method in the directly detect CYP2C19 gene classification.MethodsUsing one-step real-time fluorescent PCR detection with human CYP2C19 genotyping kit (Coyotebio) to detect whole blood or genome,and compared with the pyrosequencing method and other qPCR kit.ResultsAccuracy of one-step real-time PCR is 100%.The coefficient of variation (CV) of repeatability for all genotypesare was less than 5%.To detect known genes of different concentration,each sample can be detected correctly.Genomic DNA of hybrid mutation type (1 ng) and blood samples (10 times dilution),7 times repeating detection,the coefficient of variation (CV) of sensitivity are less than 5%.For EDTA anticoagulant samples,the samples of sodium citrate anticoagulation,chyle blood samples,heme pollution samples,each sample genotypes can be correctly detected.ConclusionOne-step detection method is of high accuracy and wide detection range,repeatability,sensitivity and specificity all can reach the level of genome DNA test samples.So one-step detection method could meet the clinical CYP2C19 gene classification testing purpose.