Nauck M S, Gierens H, Nauck M A, März W, Wieland H
Division of Clinical Chemistry, Department of Medicine, University Hospital, Freiburg, Germany.
Br J Haematol. 1999 Jun;105(3):803-10. doi: 10.1046/j.1365-2141.1999.01427.x.
Genotyping of human platelet alloantigens (HPA) has become an important procedure in the diagnosis and prevention of disorders such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and refractoriness to platelet transfusion therapy. We present a single-tube method for HPA-1 genotyping that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. A fragment covering the polymorphic site is amplified in the presence of two fluorescently-labelled hybridization probes. During the annealing step of the thermal cycling, both probes bind to their complementary sequences in the amplicon resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous aquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in an allele-specific manner as the detection probe, which was fully complementary to the HPA-1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two alleles were readily distinguishable. Using this method, genotyping of 32 samples was completed within 30 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The genotypes determined with the LightCyclerTM were identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA-1 on the LightCycler is a rapid and reliable method that is suitable for typing both small and large numbers of samples.
人类血小板同种抗原（HPA）基因分型已成为诊断和预防诸如新生儿同种免疫性血小板减少性紫癜、输血后紫癜以及血小板输血治疗无效等病症的重要手段。我们提出了一种用于HPA-1基因分型的单管方法，该方法将快速循环PCR与用于产物基因分型的等位基因特异性荧光探针熔解曲线相结合。在两种荧光标记的杂交探针存在的情况下，扩增覆盖多态性位点的片段。在热循环的退火步骤中，两种探针均与其在扩增子中的互补序列结合，导致共振能量转移，从而实现PCR的实时荧光监测。在PCR完成后的熔解曲线分析过程中持续采集荧光数据显示，随着与HPA-1b等位基因完全互补的检测探针从模板上熔解，荧光以等位基因特异性方式丧失。通过确定杂交体最大熔解发生时的温度，两种等位基因易于区分。使用该方法，32个样本的基因分型在30分钟内完成，无需任何PCR后样本处理，从而消除了终产物污染和样本追踪错误的风险。与传统PCR和限制性片段长度多态性技术相比，使用LightCyclerTM确定的基因型相同。LightCycler上的HPA-1基因分型是一种快速且可靠的方法，适用于对少量和大量样本进行分型。
