基于微流控高分辨率熔解曲线的基因分型研究

Study on Genotyping in High-Resolution Melting on Microfluidics

目的建立一种用于人类乙醇脱氢酶2(acetaldehyde dehydrogenase 2, ALDH2)基因单核苷酸多态性位点准确快速分型的方法。方法将微流控芯片、实时荧光聚合酶链式反应(polymerase chain reaction,PCR)与高分辨率熔解曲线技术相结合。采用聚合物材料制作微流控芯片,在确保熔解过程中温度变化的均一性和精确性的同时,可显著改善热能传输,提高热循环速度,缩短分析时间,从而实现对单核苷酸多态性(single nucleotide polymorphism,SNP)位点的快速检测与分析。结果在自主研制的微流控高分辨率熔解曲线分析系统上,以标准质粒为实验材料,测试了该系统的最低检测限可达10 copies/μL。同时,成功地实现了人类ALDH2基因三种基因分型(AA/GG/AG)的检测。结论微流控高分辨率熔解曲线分析系统为ALDH2基因分型提供了一种准确快速的检测方法,对指导临床硝酸甘油的个性化用药具有重要意义。

Objective To find a method that can strain human acetaldehyde dehydrogenase 2( ALDH2) in single nucleotide polymorphism site accurately and rapidly.Methods Microfluidic chips and real-time fluorescence PCR were combined with high-resolution melting curve technology. While ensuring the uniformity and accuracy of the temperature change in the melting process, the heat transfer was significantly improved, the speed of thermal cycle increased, and the analysis time was shortened. Thereby,SNP loci were quickly detected and analyzed.Results Based on an independently developed microfluidic high-resolution melting curve analysis system, using standard plasmid as experimental material, the minimum detection limit of the system was tested to 10 copies/μL. At the same time,three types of human ALDH2 gene genotypes(AA/GG/AG) were successfully detected.Conclusions The microfluidic high-resolution melting curve analysis system provides an accurate and rapid detection method for ALDH2 genotyping, which is of great significance to guide clinical personalized use of nitroglycerin.