Taqman-MGB法检测CYP3A5基因rs776746位点单核苷酸多态性及该位点对他克莫司代谢的影响

doi:10.3969/j.issn.1673-8640.2015.08.014

检验医学 ›› 2015, Vol. 30 ›› Issue (8): 830-834.DOI: 10.3969/j.issn.1673-8640.2015.08.014

• 技术研究与评论·论著 • 上一篇下一篇

Taqman-MGB法检测CYP3A5基因rs776746位点单核苷酸多态性及该位点对他克莫司代谢的影响

金丽兰¹, 蔡刚¹, 林琳¹, 王祥慧²

1. 上海交通大学医学院附属瑞金医院检验科,上海 200025
2.上海交通大学医学院附属瑞金医院泌尿外科、肾脏移植中心,上海 200025

收稿日期:2014-11-04 出版日期:2015-08-30 发布日期:2015-08-28
作者简介:null
作者简介:金丽兰,女,1992年生,学士,技师,主要从事临床检验工作。

通讯作者:王祥慧,联系电话:021-64370045。

Taqman-MGB method for determining CYP3A5 rs776746 site SNP and the influence of this site on the metabolism of tacrolimus

JIN Lilan¹, CAI Gang¹, LIN Lin¹, WANG Xianghui²

1. Department of Clinical Laboratory, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
2. Center for Renal Transplantation, Department of Urinary Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China

Received:2014-11-04 Online:2015-08-30 Published:2015-08-28

摘要/Abstract

摘要：

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*1/*1型18例(10.59%)、CYP3A5*1/*3型65例(38.24%)、CYP3A5*3/*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*3基因型的存在会减弱患者对他克莫司的代谢能力。

关键词: Taqman-MGB实时荧光聚合酶链反应, 方法学评价, 细胞色素酶P450, CYP3A5, 单核苷酸多态性

Abstract:

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.

Key words: Taqman-MGB real-time fluorescence polymerase chain reaction, Methodology evaluation, Cytochrome P450, CYP3A5, Single nucleotide polymorphism

中图分类号:

Q503

金丽兰, 蔡刚, 林琳, 王祥慧. Taqman-MGB法检测CYP3A5基因rs776746位点单核苷酸多态性及该位点对他克莫司代谢的影响[J]. 检验医学, 2015, 30(8): 830-834.

JIN Lilan, CAI Gang, LIN Lin, WANG Xianghui. Taqman-MGB method for determining CYP3A5 rs776746 site SNP and the influence of this site on the metabolism of tacrolimus[J]. Laboratory Medicine, 2015, 30(8): 830-834.

图/表 6

参考文献 11

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引物	序列
Taqman-MGB法引物(F)	5'-TGAAGGGTAATGTGGTCCAAACAG-3'(Fwd:12 094~12 117 bp)
Taqman-MGB法引物(R)	5'-CGAATGCTCTACTGTCATTTCTAACCA-3'(Fwd:12 025~12 051 bp)
探针(CYP3A5*1)	5'-VIC-TTTGTCTTTCAATATCTC-NFQMGB-3'(Fwd:12 072~12 089 bp)
探针(CYP3A5*3)	5'-FAM-TTTGTCTTTCAGTATCTC-NFQMGB-3'(Fwd:12 072~12 089 bp)
测序一轮PCR引物(含M13Tail)(F)	5'-TGTAAACGACGGCCAGTCTGATTAAACTTCACTAGCCCGATT-3' (Fwd: 12 276~12 300 bp)
测序一轮PCR引物(含M13Tail)(R)	5'-CAGGAAACAGCTATGACCCCCATACAGGCAACATGACTTAG-3' (Fwd: 11 799~11 821 bp)
测序反应PCR引物	5'-CTGATTAAACTTCACTAGCCCGATT-3' (Fwd:12 276~12 300 bp)

引物	序列
Taqman-MGB法引物(F)	5'-TGAAGGGTAATGTGGTCCAAACAG-3'(Fwd:12 094~12 117 bp)
Taqman-MGB法引物(R)	5'-CGAATGCTCTACTGTCATTTCTAACCA-3'(Fwd:12 025~12 051 bp)
探针(CYP3A5*1)	5'-VIC-TTTGTCTTTCAATATCTC-NFQMGB-3'(Fwd:12 072~12 089 bp)
探针(CYP3A5*3)	5'-FAM-TTTGTCTTTCAGTATCTC-NFQMGB-3'(Fwd:12 072~12 089 bp)
测序一轮PCR引物(含M13Tail)(F)	5'-TGTAAACGACGGCCAGTCTGATTAAACTTCACTAGCCCGATT-3' (Fwd: 12 276~12 300 bp)
测序一轮PCR引物(含M13Tail)(R)	5'-CAGGAAACAGCTATGACCCCCATACAGGCAACATGACTTAG-3' (Fwd: 11 799~11 821 bp)
测序反应PCR引物	5'-CTGATTAAACTTCACTAGCCCGATT-3' (Fwd:12 276~12 300 bp)

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Taqman-MGB法检测CYP3A5基因rs776746位点单核苷酸多态性及该位点对他克莫司代谢的影响

Taqman-MGB method for determining CYP3A5 rs776746 site SNP and the influence of this site on the metabolism of tacrolimus

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