一种用于检测ADH1B基因Arg48His多态性的改良PCR-CTPP检测方法。
An improved PCR-CTPP assay for the detection of ADH1B Arg48His polymorphism.
作者信息
Jiang Shupeng, Tong Yongqing, Zhao Rui, Xiong Ge, Qiao Bin, Li Yan
机构信息
Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, China.
出版信息
J Clin Lab Anal. 2018 Feb;32(2). doi: 10.1002/jcla.22268. Epub 2017 Jun 28.
BACKGROUND
ADH1B Arg48His polymorphism is associated with the development of alcohol-related diseases. In this study, we aimed to explore an improved polymerase chain reaction with confronting two-pair primers (PCR-CTPP) assay for the detection of ADH1B Arg48His polymorphism.
METHODS
A mismatch was introduced at the 3' end of each of the two allele-specific to increase the specificity of the reaction. But beyond that, a new mismatch at-3 positions of outer primers was designed to decrease the efficiency of the aforementioned primers and depresses the amplification of an internal nonspecific DNA control. A total of 180 samples from healthy volunteers Han Chinese were tested to evaluate this new assay.
RESULTS
The protocol of PCR-CTPP was successful for genotyping of ADH1B Arg48His. The results from the improved PCR-CTPP assay were confirmed by Sanger sequencing, and correct genotyping rates were 100%.The genotype frequencies were 49.44% (89 cases) for His/His, 46.67% (84 cases) for Arg/His, and 3.89% (seven cases) for Arg/Arg respectively.
CONCLUSIONS
This improved PCR-CTPP assay is simple, rapid, cost-effective, and reliable, specific for the detection of ADH1B Arg48His polymorphism in most clinical diagnostic laboratories.
背景
乙醇脱氢酶1B（ADH1B）基因的精氨酸48组氨酸（Arg48His）多态性与酒精相关疾病的发生有关。在本研究中，我们旨在探索一种改进的双引物对扩增阻滞突变系统聚合酶链反应（PCR-CTPP）检测方法，用于检测ADH1B基因的Arg48His多态性。
方法
在两条等位基因特异性引物的3'端引入错配以提高反应的特异性。除此之外，在外引物的-3位置设计一个新的错配以降低上述引物的扩增效率，并抑制内部非特异性DNA对照的扩增。共检测了180例汉族健康志愿者的样本，以评估这种新方法。
结果
PCR-CTPP方法成功用于ADH1B基因Arg48His位点的基因分型。改进的PCR-CTPP检测结果经桑格测序验证，正确基因分型率为100%。His/His、Arg/His和Arg/Arg基因型频率分别为49.44%（89例）、46.67%（84例）和3.89%（7例）。
结论
这种改进的PCR-CTPP检测方法简单、快速、经济有效且可靠，适用于大多数临床诊断实验室检测ADH1B基因的Arg48His多态性。
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