Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCycler (TM)

Genotyping of human platelet alloantigens (HPA) has become an important pro cedure in the diagnosis and prevention of disorders such as neonatal alloim mune 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 poly morphic site is amplified in the presence of two fluorescently-labelled hyb ridization probes. During the annealing step of the thermal cycling, both p robes bind to their complementary sequences in the amplicon resulting in re sonance energy transfer. thus providing real-time fluorescence monitoring o f PCR. Continuous aquisition of fluorescence data during a melting curve an alysis at the completion of PCR revealed that loss of fluorescence occurred in an allele-specific manner as the detection probe, which was fully compl ementary to the HPA-1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two allel es were readily distinguishable. Using this method, genotyping of 32 sample s was completed within 30 min without the need for any post-PCR sample mani pulation. thereby eliminating the risks of end-product contamination and sa mple tracking errors. The genotypes determined with the LightCycler(TM) wer e identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA-1 on the LightCycler i s a rapid and reliable method that is suitable fur typing both small and la rge numbers of samples.