Establishment and Application of a Multiplex PCR NGS Method for the Genotyping of HLA-Class I and HPA

被引:0
作者
He, Yanmin [1 ]
Wang, Fang [1 ]
Wu, Zhipan [1 ]
Zhang, Wei [1 ]
Zhu, Faming [1 ]
机构
[1] Blood Ctr Zhejiang Prov, Hangzhou, Peoples R China
关键词
HLA; human platelet alloantigens; multiplex PCR; next-generation sequencing; platelet donor bank; platelet transfusion refractoriness; POLYMERASE-CHAIN-REACTION; REAL-TIME PCR; PLATELET TRANSFUSION; POLYMORPHISMS; ANTIGENS; ALLOANTIBODIES; MUTATIONS; AND-15; TO-21W; SSP;
D O I
10.1111/tan.15716
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Selecting compatible HLA-Class I and/or HPA platelets based on genotyping could alleviate immune platelet transfusion refractoriness (PTR). A fast and reliable method of HLA-Class I and HPA genotyping is necessary to construct a platelet donor bank with known HLA-Class I and HPA genotypes. Ten pairs of specific primers for HLA-A, HLA-B, HLA-C, HPA-1 through HPA-6w, HPA-15 and HPA-21w were designed. The appropriate fragments were optimised for amplification in a single multiplex reaction. After a cleanup step using paramagnetic beads, the amplicon library was prepared and sequenced. To validate the accuracy of the developed method, commercial NGS kits for the genotyping of HLA-A, HLA-B and HLA-C and the TaqMan real-time PCR method in-house for the genotyping of HPA-1 through HPA-6w, HPA-15 and HPA-21w were used to detect all the specimens in parallel. A total of 386 specimens were detected and the results of genotyping HLA-A, HLA-B, HLA-C and HPA-1 through HPA-6w, HPA-15 and HPA-21w were obtained simultaneously, which is 100% consistent between the two methods. Four new HLA alleles, HLA-A*11:451, HLA-A*30:01:26, HLA-B*39:201 and HLA-B*40:538, were also reconfirmed. Two novel SNVs, c.2671C > T and c.2681T > G, in the coding region of ITGA2B were detected, all of which are heterozygous in individuals. A novel NGS method based on multiplex PCR was established to detect HLA-Class I and HPA simultaneously, which is high-throughput, rapid and accurate and could be applied to build a platelet donor bank.
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页数:10
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