One-tube method for complete HPA-1 genotyping by PCR using sequence-specific primers.

Human platelet antigens (HPA) can be targets for antibody responses that cause life-threatening thrombocytopenia following platelet transfusions or pregnancy. As an aid to diagnosis and prevention, serologic and DNA-based methods have been developed to type HPA. Of the DNA-based strategies, those using the polymerase chain reaction (PCR) are very sensitive, but often require processing of amplification products. Sequence-specific primers (SSP) in the PCR eliminate the need for extensive handling of reaction products beyond gel electrophoresis. However, current methods require a separate reaction for each allele being typed. In this report we describe a method to simultaneously and completely genotype both alleles of HPA-1 in a single PCR. In addition, because the absence of an amplification product might also show the failure of a SSP, we introduced a recombinant template that can only be amplified by the SSP, thus ensuring primer performance and the identified genotype.