CYP2C19基因多态性检测方法的建立

目的: 建立检测CYP2C19基因多态性的PCR-荧光探针法,为预测CYP2C19代谢药物的疗效提供新的检测试剂盒。方法: 应用Primer Express 3.0软件针对人类CYP2C19基因CYP2C19*2、CYP2C19*3和CYP2C19*17多态性位点设计引物和探针。以经测序确认为杂合突变型、纯合突变型或野生型的人类全血基因组DNA(genomic DNA, gDNA)样本为模板,应用荧光PCR法筛选合适的引物和探针,建立和优化CYP2C19基因多态性检测荧光PCR法。然后用特定浓度且基因型已知的人类全血gDNA样本作为质控品评价该方法的准确性、灵敏度和特异性。最后用100例临床样本验证该方法的临床适用性,并与直接测序结果进行比较,计算Kappa值。结果: 建立的CYP2C19基因多态性检测PCR-荧光探针法,能准确地检测出CYP2C19*2、*3和*17多态性位点杂合突变型和纯合突变型;灵敏度为每个反应体系约1 ng;以基因型已知为野生型的全血gDNA为模板进行检测的结果显示均为野生型。临床样本的检测结果显示与测序结果的一致性较好,Kappa值为1.00。结论: 建立的CYP2C19基因多态性检测方法具有很好的准确性、灵敏度和特异性,且具有很好的临床适用性,可以用于CYP2C19代谢的药物疗效的预测。

Objective: To establish fluorescence-PCR probe method for CYP2C19 gene polymorphism detection, and provide a new detection kit to predict the efficacy of drug metabolism through CYP2C19. Methods: CYP2C19 gene sequence (CYP2C19*2, CYP2C19*3 and CYP2C19*17) was selected as target.The primers and probes were designed with Primer Express 3.0 software. First,heterozygous mutant and homozygous mutant and wild-type samples were confirmed by sequencing using the human whole blood genomic DNA (gDNA) as templates. The right combination of primers and probes were selected. Then, the fluorescence PCR method for CYP2C19 gene polymorphism detection was established and optimized with the accuracy, sensitivity and specificity of fluorescence PCR method with gDNA (the specific concentration and genotype known) as quality control. Finally, the clinical applicability was verified with 100 cases of clinical samples. The results were compared with the direct sequencing, and the Kappa value was calculated. Results: Fluorescence PCR method for CYP2C19 gene polymorphism detection can accurately detect the different polymorphic variants of CYP2C19 gene, including heterozygous mutant and homozygous mutant. The sensitivity of fluorescence PCR detection can reach to 1 ng·reaction^-1.The results of clinical samples show good consistency with the results of sequencing, and the Kappa value is 1.00. Conclusion: Fluorescence PCR method for CYP2C19 gene polymorphism detection proved to be an effective method with better accuracy, sensitivity, specificity and clinical applicability, and can be applied to predict the efficacy of drugs metabolism through CYP2C19.