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目的 检测中国人基因组中HIV-1感染相关的特异性协同受体(CCR5)的基因多态性和比较提取人基因组DNA方法的优缺点。方法 我们分别用常规法和柱层析法从1219名中国人外周血PBMC中提取基因组DNA样品,对纯化后的DNA样品质量进行定性和定量分析;并对其基因组DNA中CCR5基因进行了PCR、Southern杂交和直接DNA测序等分析。结果 在中国人基因组中,检测的CCR5基因多态性是wt/wt 1 210例、wt/△32 3例,未检测到CCR5△32/△32纯合子突变,CCR5等位基因突变率为0.125％。常规法提取的基因组DNA所需的外周静脉血多(5ml);DNA的量较少且不稳定;操作步骤复杂。相反,QIAamp Boold Kit柱层析法提取DNA仅需要外周血200μl,可稳定地获得一定量的DNA,操作简便。结论 首次报道了中国人CCR5等位基因突变率是0.125％,提示我国绝大多数人群对嗜巨细胞HIV-1病毒的遗传易感性较高;同时证实试剂盒法提取的DNA更适合于CCR5基因多态性分析。
Objective To detect the gene polymorphism of CCR5 related to HIV-1 infection in Chinese human genome and to compare the advantages and disadvantages of the methods of extracting human genomic DNA. Methods Genomic DNA samples were extracted from 1219 Chinese PBMCs by routine and column chromatography methods. The quality of the purified DNA samples was qualitatively and quantitatively analyzed. The CCR5 gene in genomic DNA was subjected to PCR, Southern hybridization and direct DNA sequencing analysis. Results In the Chinese human genome, CCR5 gene polymorphism was detected in 1210 cases of wt / wt and wt / △ 32, no CCR5 △ 32 / △ 32 homozygous mutation was detected, and the rate of CCR5 allele mutation was 0.125 %. The amount of peripheral venous blood (5 ml) required for routine genomic DNA extraction is small and unstable; the procedure is complicated. In contrast, QIAamp Boold Kit column chromatography requires only 200 μL of peripheral blood to obtain a stable amount of DNA, making it easy to perform. Conclusions The mutation rate of CCR5 allele in Chinese population was 0.125% for the first time, which indicated that the vast majority of Chinese population had a high genetic susceptibility to HIV-1 virus. At the same time, it was confirmed that the DNA extracted by kit method was more suitable for CCR5 gene Polymorphism analysis.