目的：建立检测CYP3A5基因rs776746位点单核苷酸多态性（ SNP）的Taqman-MGB实时荧光聚合酶链反应（PCR），并分析该位点对他克莫司药物代谢的影响。方法针对CYP3A5基因rs776746位点设计相应引物及双标记探针，建立并评价检测该位点SNP的Taqman-MGB实时荧光PCR；收集170例服用他克莫司患者的全血DNA样本，用建立的Taqman-MGB实时荧光PCR检测CYP3A5基因型，并分析不同CYP3A5基因型与他克莫司血药浓度剂量比的相关性。结果建立的Taqman-MGB基因分析法能够对CYP3A5 rs776746位点基因型进行高效区分，可检测最低含量为0．01 ng水平的DNA样本，并且具有较好的重复性和特异性，其检测结果与测序结果完全一致。170例患者中 CYP3A5倡1／倡1型18例（10．59％）、CYP3A5倡1／倡3型65例（38．24％）、CYP3A5倡3／倡3型87例（51．18％）；其中104例异体肾移植患者3种基因型的血药浓度剂量比分别为（52．47±35．96）、（77．79±33．80）、（134．80±79．35）（ng／mL）／（mg· kg－1· d－1），各组之间差异有统计学意义（P＜0．05）。结论建立了一种敏感性高、特异性好、重复性佳、适用于临床快速检测CYP3A5 rs776746 SNP位点的Taqman-MGB方法。初步分析显示CYP3A5倡3基因型的存在会减弱患者对他克莫司的代谢能力。%Objective To establish Taqman-MGB real-time fluorescence polymerase chain reaction ( PCR ) for determining CYP3A5 rs776746 site single nucleotide polymorphism ( SNP), and to analyze the influence on the metabolism of tacrolimus.Methods The primers and double-labeled probes were designed according to the genotype of CYP3A5 rs776746 site, and the Taqman-MGB real-time fluorescence PCR for SNP was established and evaluated.A total of 170 whole blood DNA samples from patients who took tacrolimus were collected, and CYP3A5 genotype was determined by Taqman-MGB real-time fluorescence PCR.The relationship between CYP3A5 genotype and tacrolimus concentration to dose ratio was analyzed.Results The established Taqman-MGB method can discriminate CYP3A5 rs776746 site efficiently with good reproducibility and specificity and had a detection limit of 0.01 ng for DNA.The results of Taqman-MGB method were consistent with those of sequencing.Among the 170 samples, 18 samples belonged to CYP3A5*1/*1(10.59%), 65 samples were CYP3A5*1/*3(38.24%) and 87 samples were CYP3A5*3/*3 (51.18%).Among the samples collected, 104 samples belonged to renal allograft recipients, and the mean tacrolimus concentrations to dose ratios for 3 genotypes were (52.47 ±35.96), (77.79 ±33.80) and (134.80 ±79.35) (ng/mL)/(mg· kg-1· d-1), and there was statistical significance (P<0.05).Conclusions The Taqman-MGB method for determining CYP3A5 rs776746 site SNP is established, which has high sensitivity, specificity and reproducibility and is a rapid method for clinical practice.Through primary analysis, the existence of CYP3A5*3 genotype do weaken the ability of metabolizing tacrolimus.
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