Two-Dimensional PCR for Identifying the HLA Alleles Associated with Adverse Drug Reactions

This study aimed to establish a two-dimensional PCR (2D-PCR) methodology capable of simultaneously identifying HLA-A∗31:01, HLA-B∗15:02, HLA-B∗57:01, and HLA-B∗58:01 alleles to prevent adverse drug reactions and guide safe clinical medication use. To achieve this goal, key nucleotide sites were first selected through multiple sequence alignment for the purpose of identifying HLA alleles: c.243T and c.290T for HLA-A∗31:01; c.206C, c.256G, and c.285G for HLA-B∗58:01; c.362T and c.419C for HLA-B∗57:01; and c.1012 + 104T (rs144012689) for HLA-B∗15:02. Using universal tag sequences linked to the 5' end of primers and base-quenched probes, high-throughput identification of multiple target genes was achieved through PCR amplification and melting curve analysis, followed by methodologic optimization and evaluation. Results indicated that the 2D-PCR method has a detection limit of approximately 10¹ copies and achieves high specificity and accuracy. The entire detection process can be completed in approximately 100 minutes, with a cost of less than $1 (USD). Furthermore, 2D-PCR overcomes the limitations of traditional fluorescent channels, providing technical support for the identification of multiple target genes. In conclusion, this study shows that 2D-PCR offers a convenient and rapid approach for human leukocyte antigen allele identification to prevent adverse drug reactions and holds potential for clinical application.