An improved PCR-CTPP assay for the detection of ADH1B Arg48His polymorphism
Shupeng Jiang1, Yongqing Tong1, Rui Zhao1
1Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, China.
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.
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