Loop-mediated isothermal amplification for detection of HLA-B*58:01 allele

Abstract

Strong association of human leukocyte antigen (HLA)-B*58:01 allele with allopurinol-induced hypersensitivity was found worldwide, especially in the Han Chinese populations. This study aims to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for rapid detection of HLA-B*58:01. Two sets of LAMP primers targeting exons 2 and 3 of HLA-B*58:01 allele were designed and their annealing temperatures were optimized accordingly. The heating devices for LAMP assay were tested. The analytical sensitivities of the two sets of LAMP primers were determined by 1:10 serial dilution of a positive control with homozygous HLA-B*58:01 allele from 100 ng down to 1 fg. The analytical specificities of the LAMP primers were evaluated by 30 selected University of California, Los Angeles (UCLA) DNA Exchange Program samples with known HLA-B loci typings previously typed by sequencing. Both sets of LAMP primers targeting exons 2 and 3 amplified optimally at 67°C. Thermal cycler is essential in achieving a more precise and specific LAMP result. The sensitivity of the exon 2 LAMP primer set was found to be 1 pg, whereas it was 10 ng for the exon 3 primer set in a 60-min amplification. The LAMP primers were highly specific because LAMP results were perfectly concordant to the sequencing results. The HLA-B*58:01 LAMP assay has compatible sensitivity and specificity to routine genotyping assays, and it is potentially an alternative screening test for the detection of HLA-B*58:01 and ultimately allopurinol-induced hypersensitivity.