Asian-type DEL (RHD, c.1227G>A) is relatively prevalent in East Asians and is essential to detect in serological rhesus (Rh) D-negative individuals. Despite the recognized importance of identifying Asian-type DEL, traditional detection methods such as adsorption-elution and Sanger sequencing are complex and time-consuming. A reliable method is required for the rapid and precise detection of Asian-type DEL. A novel multiplex real-time PCR (mRT-PCR) assay using allele-specific TaqMan hydrolysis probes was developed to target the Asian-type DEL variant. Analytical specificity and sensitivity were evaluated using RHD or RHCE synthetic DNAs with 1227G or 1227A. The assay was tested on 315 clinical samples, and the results were compared with Sanger sequencing to assess diagnostic performance. Analytical specificity evaluations confirmed that the assay selectively amplified RHD, 1227A, or 1227G without cross-reactivity to RHCE. Additionally, clinical sample tests showed the assay maintained high specificity and sensitivity even at high nucleic acid concentrations. The Asian-type DEL mRT-PCR assay demonstrated 100% sensitivity and specificity, with complete concordance across all samples compared to reference methods. Compared to Sanger sequencing, the mRT-PCR assay had a shorter analysis time. The assay developed in this study offers a fast, reliable, and accurate approach for detecting Asian-type DEL. This method significantly improves efficiency over conventional techniques and provides a valuable tool for managing transfusion safety and RhD alloimmunization risks in RhD-negative populations.

Gout is a metabolic disorder caused by chronic hyperuricemia, which leads to monosodium urate crystal deposition and subsequent inflammatory arthritis. Genetic factors, particularly those regulating urate transport, play a major role in susceptibility to hyperuricemia and gout. Among these, the functional ABCG2 variants Q126X (rs72552713) and Q141K (rs2231142) are well-established genetic determinants of impaired urate excretion. In this study, we developed a hydrolysis probe-based multiplex real-time polymerase chain reaction (PCR) assay for allele discrimination and genotyping of the ABCG2 Q126X and Q141K variants in separate reactions. This assay demonstrated high analytical specificity without cross-reactivity in both synthetic and clinical samples and maintained reliable genotype detection across clinically relevant DNA concentrations. In method comparison using 1653 clinical samples, the multiplex real-time PCR assay showed complete concordance with Sanger sequencing (Cohen’s κ = 1.00). Combined genotype analysis (Q126X/Q141K) demonstrated a gene dose-dependent association with serum uric acid levels and hyperuricemia risk, consistent with the estimated ABCG2 transport function. These findings indicate that the developed assay provides a reliable and practical molecular tool for rapid ABCG2 genotyping and genetic risk assessment of hyperuricemia and gout.