Intharuangrung N.Sirikul C.Nimsamer P.Wongsurawat T.Saejia A.Anukul N.Mahidol University2025-10-222025-10-222025-01-01Frontiers in Genetics Vol.16 (2025)https://repository.li.mahidol.ac.th/handle/123456789/112720This study reports an optimized in-house 2-step PCR-SSP assay for rapid, cost-effective detection of HLA-B*57:01 and HLA-B*58:01 in routine pharmacogenomics laboratory. This assay employs allele-specific primers positioned within exon 2–3 boundaries, validated in silico against common HLA-B alleles. Using 30 clinical DNA samples, our PCR workflow (<1 h) showed 100% concordance at 2-field resolution with Oxford Nanopore sequencing performed using ligation-based sequencing kit with PCR barcoding. Cohen’s kappa was 1.00 with 95% CI. The turnaround time and reagent cost per sample were reduced to 1 h of hands-on PCR time and USD 7 per sample, respectively. These do not include DNA extraction or gel electrophoresis analysis. This 2-step PCR-SSP offers a robust alternative for pharmacogenomic screening in resource-limited settings for detecting the HLA-B*57:01 and HLA-B*58:01.Biochemistry, Genetics and Molecular BiologyMedicineArticleSCOPUS10.3389/fgene.2025.16499902-s2.0-10501868312116648021