Fully integrated centrifugal microfluidic platform for rapid HLA-B∗58:01 allele identification using duplex RPA assay

Abstract

We developed a fully integrated, portable centrifugal microfluidic platform to detect HLA-B∗58:01 allele, a strong genetic risk factor for severe hypersensitivity reactions to allopurinol. This allele is located within the human leukocyte antigen (HLA) locus, which is characterized by its polymorphism and high GC content. The platform is capable of performing on chip lysis, duplex recombinase polymerase amplification (RPA) and real-time fluorescence detection of genomic DNA directly from buccal swab samples. A localized contact heating module was customized to enable fast and efficient sample lysis using a thermophilic proteinase, and a build-in internal control was incorporated to minimize diagnostic errors. All reagents are pre-stored in a disposable microfluidic chip and the swab samples can be added directly without any pre-treatment, achieving a fully automated, walk-away test. As a verification, our platform could simultaneously detect the HLA-B∗58:01 allele and the β-globin gene as an internal control, with a limit of detection (LOD) of 30 pg/μL and 3 pg/μL, respectively. Validation using buccal swabs from 12 volunteers demonstrated 100 % concordance with the gold-standard sequencing-based typing (SBT) methods. The platform demonstrated high specificity, reproducibility, and reliability. Compared to SBT, our platform significantly reduces the time-to-result (50 min vs. up to 16 h) while minimizing extensive manual labor. Its fully automated integration of sample lysis and genomic DNA analysis provides a new direction for pharmacogenetic screening, enabling personalized medicine in resource-limited settings.