Communication: One-Step HLA-B59:01* Genotyping by Real-Time Duplex Allele-Specific PCR and Melting Curve Analysis.

IF 1 4区医学 Q4 MEDICAL LABORATORY TECHNOLOGY

Annals of clinical and laboratory science Pub Date : 2025-07-01

Ji Young Huh, Geon Park

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引用次数: 0

Abstract

Objective: HLA-B*59:01 is associated with severe cutaneous adverse reactions (SCARs) caused by carbonic anhydrase inhibitors, highlighting the need for rapid and reliable genotyping methods. Our previous conventional PCR-based HLA-B*59:01 genotyping required post-PCR electrophoresis, increasing labor and hands-on time.

Methods: We developed a real-time duplex allele-specific PCR with melting curve analysis for HLA-B*59:01 genotyping. Using KOD SYBR qPCR Mix, we differentiated an HLA-B*59:01-specific amplicon (838 bp) and a GNAQ internal control amplicon (912 bp), yielding distinct melting peaks at about 90.3°C and 94.5°C, respectively. Validation was performed on 50 HLA-B*59:01-positive and 100 negative samples with sequence-based typing (SBT) as the reference.

Results: The method demonstrated complete concordance with SBT, achieving 100% sensitivity and specificity. The closed-tube approach eliminated the need for electrophoresis, reducing hands-on time.

Conclusions: This melting curve analysis provides a reliable and labor-efficient alternative for HLA-B*59:01 screening, facilitating clinical implementation for SCAR risk mitigation.

本刊更多论文

通讯：一步HLA-B*59:01基因分型采用实时双工等位基因特异性PCR和熔化曲线分析。

目的：HLA-B*59:01与碳酸酐酶抑制剂引起的严重皮肤不良反应（scar）相关，强调对快速可靠的基因分型方法的需求。我们以前传统的基于pcr的HLA-B*59:01基因分型需要pcr后电泳，增加了劳动和动手时间。方法：建立实时双工等位基因特异性PCR，熔点曲线分析HLA-B*59:01基因分型。使用KOD SYBR qPCR Mix，我们分化出HLA-B*59:01特异性扩增子（838 bp）和GNAQ内控扩增子（912 bp），分别在约90.3°C和94.5°C产生不同的熔化峰。以序列分型（SBT）为参照，对50份HLA-B*59:01阳性样本和100份阴性样本进行验证。结果：该方法与SBT完全吻合，灵敏度和特异度均达到100%。封闭管方法消除了电泳的需要，减少了操作时间。结论：这种熔化曲线分析为HLA-B*59:01筛查提供了可靠且劳动效率高的替代方法，促进了SCAR风险缓解的临床实施。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Annals of clinical and laboratory science 医学-医学实验技术

CiteScore

1.60

自引率

0.00%

发文量

112

审稿时长

6-12 weeks

期刊介绍： The Annals of Clinical & Laboratory Science welcomes manuscripts that report research in clinical science, including pathology, clinical chemistry, biotechnology, molecular biology, cytogenetics, microbiology, immunology, hematology, transfusion medicine, organ and tissue transplantation, therapeutics, toxicology, and clinical informatics.