Annals of clinical and laboratory science · 2025
Communication: One-Step HLA-B*59:01 Genotyping by Real-Time Duplex Allele-Specific PCR and Melting Curve Analysis.
СтатьяНазвание и аннотация: Перевод в очереди
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Huh Ji Young, Park Geon
Аннотация
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.
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.