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建立和应用多重PCRNGS方法,用于HLA-I类和HPA的基因定型

Yanmin He¹, Fang Wang¹, Zhipan Wu¹

¹Blood Center of Zhejiang Province, Hangzhou, China.

|October 29, 2024

概括

一种新的多重PCR方法能够快速准确地同时对HLA-I类和人类血小板抗原 (HPA) 进行基因定型. 这种高通量方法对于建立血小板捐赠银行和预防输血折射性至关重要.

科学领域:

免疫遗传学免疫遗传学
分子生物学分子生物学
输血医学输血医学

背景情况:

免疫血小板输血折射性 (PTR) 是一个重要的临床挑战.
人类白细胞抗原 (HLA) - 一类和人血小板抗原 (HPA) 的基因定型对于兼容的血小板输血至关重要.
需要一种快速,可靠的HLA和HPA基因定型方法,以建立具有良好的特征的血小板供体银行.

研究的目的:

开发和验证一种新的高通量方法,用于同时进行HLA-Class I和HPA基因型定型.
为了能够高效地建立一个具有已知的遗传特征的血小板供体银行.
改善匹配血小板的选择并降低PTR.

主要方法:

针对HLA-A,HLA-B,HLA-C,HPA-1至HPA-6w,HPA-15和HPA-21w的特定原料设计.
优化单个多重PCR反应的片段放大.
使用下一代测序 (NGS) 进行图书馆准备和测序,在对磁珠清理后.

主要成果:

与商业NGS套件和TaqMan实时PCR相比,从386个标本中同时实现HLA-Class I和HPA的基因定型,达到100%的一致性.
再次确认了四个新的HLA基因 (HLA-A*11:451,HLA-A*30:01:26,HLA-B*39:201,HLA-B*40:538) 的发现.
在ITGA2B基因中发现了两种新的单核酸变异 (SNV).

结论:

一种新的基于NGS的多重PCR方法为同时检测HLA-I类和HPA提供了高吞吐量,快速和准确的手段.
这种方法适用于建立全面的血小板供体银行.
开发的方法可以通过促进兼容的血小板选择,显著帮助预防免疫PTR.

关键词:

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