血栓性疾病个体化用药指导基因芯片试剂盒的制备和效果评价

Preparation of gene chip detection kit for individualized treatment of thrombotic diseases and evaluation on its effectiveness

目的:探讨血栓性疾病(TD)个体化用药指导基因芯片的制备过程,阐明一种快速、高通量检测氯吡格雷和华法林药物代谢基因的方法。方法:根据氯吡格雷药物代谢基因位点(CYP3A4、CYP2C19*17、CYP2C19*2和CYP2C19*3)和华法林药物代谢基因位点(CYP4F2*3、GGCX、VKORC1-2、VKORC1-1、CYP2C9*2和CYP2C9*3)设计相应的探针和引物,制备TD个体化用药指导的基因芯片检测试剂盒。经过DNA提取、PCR扩增、杂交、洗脱和扫描等步骤对基因芯片的灵敏性、重复性和稳定性进行评价。收集3个地区的150例TD受试者样本并进行药物代谢基因检测,以受试者在临床中接受的用药指导及6个月后患者的疾病恢复情况为依据,评价基因芯片试剂盒的有效率。结果:基因芯片检测2种药物的代谢基因均出现良好的杂交信号;芯片检测PCR扩增产物的最低检测浓度为103 copies·mL-1;对不同批次的3张芯片及同一批次的10张芯片进行平行测定,符合率为100%;芯片具有稳定性,有效期长达6个月;来源于3个地区的150例TD样本验证基因芯片检测试剂盒的有效率在90%以上。结论:成功制备了高通量检测氯吡格雷和华法林药物代谢基因的TD个体化用药指导基因芯片试剂盒,该方法灵敏度高,重复性好,稳定性强,制备效果优良。

Objective:To investigate the preparation process of gene chips for individualized treatment of thrombotic diseases(TD),and to elucidate a rapid and high-throughput method for detecting the drug metabolism genes of clopidogrel and warfarin.Methods:Different kinds of probes and primers were designed according to the clopidogrel drug metabolism gene sites(CYP3A4,CYP2C19*17,CYP2C19*2,and CYP2C19*3)and warfarin drug metabolism gene sites(CYP4F2*3,GGCX,VKORC1-2,VKORC1-1,CYP2C9*2,and CYP2C9*3)to prepare the gene chip detection kit for individualized treatment of TD.The steps of microarray detection were DNA extraction,PCR amplification,hybridization,elution and scanning.The sensitivity,repeatability and stability of the gene chips were evaluated.The samples of 150 subjects with TD from three regions were selected,and the drug metabolism genes were detected.Based on the medical guidances in the clinic and the disease recovery status after 6 months,the effectiveness of gene chip detection kit was determined.Results:The effective hybridization signals were observed in the metabolism genes of two kinds of drugs detected by gene chips;the minimum detection concentration of PCR amplification products was 103 copies·mL-1 detected by gene chip;the coincidence rate of simultaneous determination of three chips in different batches and parallel measurement of 10 chips in the same batch was 100%;the chip was stable and could be stored for at least 6 months;the effective rate of gene chip detection kit in the 150 TD subjects from three regions was more than 90%.Conclusion:The high-throughput gene chip detection kit of individualized treatment of TD to detect the drug metabolism genes of clopidogrel and warfarin are successfully established,and the method has high sensitivity,satisfactory repeatability,strong stability and excellent preparation effect.