应用多重实时聚合酶链反应结合双标记探针检测人血样中野生型细胞色素 P4502A6 基因型和全基因缺失

Genotyping of wild-type cytochrome P450 2A6 and whole-gene deletion using human blood samples and a multiplex real-time polymerase chain reaction method with dual-labeled probes.

机构信息

Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.

出版信息

Clin Chim Acta. 2015 Feb 20;441:71-4. doi: 10.1016/j.cca.2014.12.018. Epub 2014 Dec 19.

DOI:10.1016/j.cca.2014.12.018

PMID:25532948

Abstract

BACKGROUND

Genetic polymorphisms of human cytochrome P450 2A6 (CYP2A6) are one of the determinants of smoking behavior and/or tobacco-related lung cancer risk in male Japanese smokers. To help identify those at high risk, we developed a multiplex real-time polymerase chain reaction (PCR)-based genotyping method with dual-labeled probes to detect wild-type and whole-gene deletion of CYP2A6 directly from blood samples without DNA isolation.

METHODS

We validated the new real-time PCR method that uses dual-labeled probes by utilizing 116 genomic DNA samples that had been genotyped previously and 33 blood samples.

RESULTS

The new method could discriminate CYP2A6 from highly homologous CYP2A7 and CYP2A13 genes and could also determine CYP2A61 (wild type) and CYP2A64 (whole-gene deletion) alleles in perfect accordance with previous analysis data. Amplification curve profiles were obtained by multiplex real-time PCR assay with CYP2A61 and CYP2A64 primer sets and dual-labeled probes using one-drop blood samples previously genotyped for CYP2A6*1/1, CYP2A61/4, and CYP2A64/*4.

CONCLUSIONS

A real-time multiplex PCR assay for genotyping wild-type CYP2A6 and whole-gene deletion was developed with dual-labeled probes. The new method achieved 100% agreement with data from the conventional PCR method for 116 genomic DNA samples and samples from 33 volunteers, thereby establishing its validity.

摘要

背景

人类细胞色素 P450 2A6（CYP2A6）的遗传多态性是影响日本男性吸烟者吸烟行为和/或与烟草相关的肺癌风险的决定因素之一。为了帮助确定高危人群，我们开发了一种使用双标记探针的多重实时聚合酶链反应（PCR）基因分型方法，可直接从血液样本中检测 CYP2A6 的野生型和全基因缺失，而无需 DNA 分离。

方法

我们利用先前已进行基因分型的 116 个基因组 DNA 样本和 33 个血液样本验证了使用双标记探针的新实时 PCR 方法。

结果

该新方法能够区分高度同源的 CYP2A7 和 CYP2A13 基因，并能与先前的分析数据完全一致地确定 CYP2A61（野生型）和 CYP2A64（全基因缺失）等位基因。使用 CYP2A61 和 CYP2A64 引物组和双标记探针进行多重实时 PCR 分析可获得扩增曲线图谱，该探针先前用于 CYP2A6*1/1、CYP2A61/4 和 CYP2A64/*4 的基因分型。