Development of a Multiplex Real-Time PCR Assay for the Rapid Detection of the Asian-Type DEL

Asian-type DEL (RHD, c.1227G>A) is relatively prevalent in East Asians and is essential to detect in serologic 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 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 multiplex real-time PCR assay demonstrated 100% sensitivity and specificity, with complete concordance across all samples compared with reference methods. Compared with Sanger sequencing, the multiplex real-time 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.