Pittayabumrung Ornuma, Leelayuwat Chanvit, Romphruk Amornrat V, Simtong Piyapong
Medical Technology Program, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
Department of Clinical Immunology and Transfusion Sciences, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
Transfus Med Hemother. 2025 Feb 12;52(3):169-177. doi: 10.1159/000544107. eCollection 2025 Jun.
The molecular basis of the gene found in serologically D- phenotype individuals differs with race/ethnicity. Therefore, we aimed to develop a single-tube multiplex PCR-sequence specific primer (multiplex PCR-SSP) to detect variant alleles commonly found in serologically D- phenotype individuals in a Thai population.
In total, 205 blood samples with a serologically D- phenotype were tested using a single-tube multiplex PCR-SSP targeted exons 1, 4, 7, 10, and c.1227G>A in exon 9 in combination with a hybrid , and results were confirmed by direct DNA sequencing.
In a single-tube multiplex PCR-SSP, three patterns of amplified exons were observed: total deletion of the gene, Asian-type DEL, and hybrid. The allele frequencies of , , and hybrid were 83.4%, 12.9%, and 2%, respectively. All of the Asian-type DEL samples present the allele (predicted RhCE phenotype: C/E+).
This study successfully established a simple and reliable molecular diagnostic platform for analyzing variant alleles commonly found in serologically D- phenotype individuals in a Thai population. This technique could enable broader (Asian-type DEL) analyses in high-prevalence areas such as Thailand and other countries in East and Southeast Asia, serving as an example for blood bank routine settings.
血清学D抗原阴性个体中发现的基因的分子基础因种族/民族而异。因此，我们旨在开发一种单管多重聚合酶链反应-序列特异性引物（多重PCR-SSP），以检测泰国人群血清学D抗原阴性个体中常见的变异等位基因。
总共使用针对外显子1、4、7、10以及外显子9中c.1227G>A的单管多重PCR-SSP结合杂交技术对205份血清学D抗原阴性的血样进行检测，并通过直接DNA测序对结果进行确认。
在单管多重PCR-SSP中，观察到三种外显子扩增模式：基因完全缺失、亚洲型DEL以及杂交型。、和杂交型的等位基因频率分别为83.4%、12.9%和2%。所有亚洲型DEL样本均呈现等位基因（预测RhCE表型：C/E+）。
本研究成功建立了一个简单可靠的分子诊断平台，用于分析泰国人群血清学D抗原阴性个体中常见的变异等位基因。该技术能够在泰国以及东亚和东南亚其他国家等高流行地区进行更广泛的（亚洲型DEL）分析，为血库常规检测提供了范例。
