Park Min Young, Lee Kang-Hee, Yang Yong-Jin, Seo Ji Young, Lee Jong Kwon, Jang Ja-Hyun, Sohn Yong-Hak, Han Kyou-Sup, Cho Duck
Department of Molecular Diagnostics, Seegene Medical Foundation, Seoul, Republic of Korea; Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, Republic of Korea.
Department of Molecular Diagnostics, Seegene Medical Foundation, Seoul, Republic of Korea.
J Mol Diagn. 2025 Sep;27(9):841-849. doi: 10.1016/j.jmoldx.2025.05.010. Epub 2025 Jul 15.
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.
亚洲型DEL（RHD，c.1227G>A）在东亚人群中相对普遍，对于血清学检测为恒河猴（Rh）D阴性的个体来说，检测该类型至关重要。尽管识别亚洲型DEL的重要性已得到认可，但传统的检测方法，如吸附洗脱法和桑格测序法，既复杂又耗时。因此需要一种可靠的方法来快速、精确地检测亚洲型DEL。我们开发了一种使用等位基因特异性TaqMan水解探针的新型多重实时PCR检测方法，以靶向亚洲型DEL变异体。使用含有1227G或1227A的RHD或RHCE合成DNA评估分析特异性和灵敏度。该检测方法在315份临床样本上进行了测试，并将结果与桑格测序法进行比较，以评估诊断性能。分析特异性评估证实，该检测方法能选择性地扩增RHD、1227A或1227G，而不会与RHCE发生交叉反应。此外，临床样本检测表明，即使在高核酸浓度下，该检测方法仍保持高特异性和灵敏度。亚洲型DEL多重实时PCR检测方法的灵敏度和特异性均为100%，与参考方法相比，所有样本的结果完全一致。与桑格测序法相比，多重实时PCR检测方法的分析时间更短。本研究中开发的检测方法为检测亚洲型DEL提供了一种快速、可靠且准确的方法。该方法显著提高了检测效率，优于传统技术，为管理RhD阴性人群的输血安全和RhD同种免疫风险提供了一个有价值的工具。
