液相芯片技术快速检测CYP2C9、CYP2C19、CYP4F2、VKORC1及ABCB1基因多态性

Rapid detection of CYP2C9,CYP2C19,CYP4F2,VKORC1 and ABCBl gene polymorphisms by liquid phase chip technology

目的基于液相芯片技术,建立一种可同时、快速检测细胞色素P4502C9(CytoChrome P4502C9,CYP2C 9)、CYP2C19、CYP4F2、维生素K环氧化物还原酶(vitamin K epoxide reductase,VKORCI)及ATP结合盒亚家族B成员l(ATP-binding cassette subfamily B memberl,ABCB1)等与华法林和氯吡格雷相关药物基因多态性的方法。方法方法学的建立。从Genbank中查找与华法林和氯吡格雷药物相关的8个靶位点附近基因序列,设计特异性引物和探针;通过多重PCR扩增,等位基因特异性引物延伸(allele specific primer extension,ASPE),MagPlex-Tag微球杂交,经液相芯片系统Luminex 200检测荧光信号,确定基因型;优化反应体系并进行方法学评价。收集2017年6月至2018年12月东莞市厚街医院抗血栓治疗患者血液样本,共260例,采用建立的方法检测其8个靶位点,并与测序结果比较。结果本方法检测260例样本结果显示:纯合子荧光强度中位值(median fluorescence intensity,MFI)比率均>0.9或<0.1,杂合子MFI比率均在0.3〜0.6之间;各基因型批内和批间变异系数分别低于6.4%和10.9%;所需DNA最低检测限为0.75ng;260例样本的检测结果与测序结果完全一致。结论本研究采用液相芯片技术,成功建立了快速检测华法林和氯吡格雷相关药物基因型的方法。

Objective T o establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P4502 C 9(CYP2C9),CYP2C19,CYP4F2,Vitamin K epoxide reductase(VKORCJ)and ATP-binding cassette subfamily B m e m b e r 1(ABCBl)gene,which were associated with warfarin and clopidogrel,based on liquid phase chip technology.Methods M e thod establishment.T h e eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank,and the specific primers and probes were designed.Through multiple P C R amplification,followed by allele specific primer extension(ASPE),and MagPlex-Tag microspheres hybridization,the suspension array L uminex 200 system step-by-step,the genotypes were determined by fluorescence signal.T h e reaction system was optimized and its methodological evaluation was performed.260 patients with antithrombotic therapy from D o n g g u a n houjie hospital were recruited in this study form June 2017 to D e c e m b e r 2018.T h e eight genotypes of the 260 patients were detected by the established method,and the results were compared with the sequencing results.Results T h e results of 260 samples showed that allelic median fluorescence intensity(MFI)ratios of homozygotes(mutant/wild-type)were all greater than 0.9 or less than 0.1,and all the allelic M F I ratios of heterozygotes were between 0.3 and 0.6.T h e within run and between run coefficients of variance for allelic M F I ratios were lower than 6.4%and 10.9%,respectively.T h e m i n i m u m D N A template requirements was 0.75ng.T h e genotypes of 260 patients determined by the established method were completely concordant with the sequencing results.Conclusion A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology.