부산대학교 도서관

Human platelet antigen (HPA) genotyping is a valuable tool for typing platelets to assist in the management of alloimmunized thrombocytopenic patients. We describe, for the first time, 5′ nuclease assays (NA) to genotype HPA-5 and -15, and improved 5′-NA to genotype HPA-1, -2 and -3, by utilizing minor groove binder (MGB) and non-fluorescent quencher (NFQ) technology. Superior probe specificity and fluorescent performance is attained through MGB-NFQ probe modifications compared with previous 5′-NA designs.Primers and dye-labelled MGB-NFQ probes were designed and synthesized to detect the single nucleotide polymorphism responsible for each HPA-1, -2, -3, -5 and -15. One-hundred blood samples were tested for the combinations of HPA genotypes 1, 2, 3 and 5 by our traditional sequence-specific primer–polymerase chain reaction (SSP–PCR) method, and 41 blood samples were tested for HPA-15 by SSP–PCR at an external laboratory. These results were then compared with those obtained by using the new 5′-NA.There was complete concordance of results for all samples tested by SSP–PCR and 5′-NA. The 5′-NA offers distinct advantages over non-fluorescent genotyping methods. DNA amplification and allele discrimination occurs in a single closed tube for each antigen with no post-PCR manipulation required. This minimizes the risk of cross-contamination and mislabelling of samples, as well as making the assay less time-consuming to perform. In comparison with other fluorescent assays, the 5′-NA has the highest sample throughput, resulting from the use of a 96-well platform, identical cycling conditions for all assays and the potential for automation.Genotyping for HPA-1, -2, -3, -5, and -15 by the 5′-NA is suitable for routine analysis. The latest 5′-NA design, using MGB probe technology, ensures superior detection of all alleles and is the most versatile fluorescent assay, ideal for both urgent clinical samples and large-scale screening programs. [ABSTRACT FROM AUTHOR]