目的 首次培养建立HPA永生化细胞系,并首次研制HPA基因分型国家参考盘.方法 筛选、招募、采集志愿者的新鲜外周血样本,经 E B病毒转化,培养建立HPA永生化细胞系.细胞扩大培养后提取HPA细胞系基因组gDNA,采用Qubit进行浓度检测、Nanodrop进行纯度质检,琼脂糖凝胶电泳分析基因组的完整性,将质检合格的gDNA浓度稀释至30 ng/μL,20 μL分装,组盘制备为HPA国家参考品盘,并进行复溶稳定性(-20℃和25℃之间反复冻融3次)和均匀性验证.结果 成功建立了27 株永生化HPA细胞系,提取的gDNA完整且纯度和均匀性良好,27例DNA样本冻融前后浓度无明显改变,相对偏差在±10.0%以内.结论 成功建立了HPA基因分型国家参考盘,为多种HPA基因分型检测试剂盒的质量评价提供了实物标准.
The first development of the HPA genotyping national reference panel
Objective To establish HPA immortalized cell lines and to develop a national refer-ence panel for HPA genotyping for the first time.Methods Fresh peripheral blood samples were obtained from volunteers,screened,and recruited.These samples were then transformed by EB virus to establish HPA immortalized cell lines.Following expansion of the cell culture,genomic DNA from the HPA cell line was ex-tracted.The concentration was detected using Qubit,purity was inspected using Nanodrop,and the integrity of the genome was analyzed using agarose gel electrophoresis.The qualified gDNA was diluted to 30 ng/μL,with 20 μL packaged separately.These samples were prepared as the HPA national reference panel,and under-went redissolved stability testing(repeated freezing and thawing at-20℃and 25℃three times)as well as uni-formity verification.Results 27 immortalized HPA cell lines were successfully established,and the extracted gDNA was complete,with good purity and uniformity.There was no significant change in the concentration of the 27 DNA samples before and after freeze-thaw,with a relative deviation of±10.0%.Conclusion A na-tional reference panel for HPA genotyping has been successfully established,offering physical standards for evaluating the quality of various HPA genotyping detection kits.
National reference panelHPAImmortalized cell linesQuality evaluationIn vitro diagnostic reagents
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