Communication: One-Step HLA-B*59:01 Genotyping by Real-Time Duplex Allele-Specific PCR and Melting Curve Analysis.

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.