Novel sequence-specific primer (SSP) real-time PCR-based genotyping assay for HLA-A, -B, and -C in East African populations.

The layout of the assay is here exemplified for the HLA-A locus, but proceeds similarly for the HLA-B and -C loci. a) After PCR amplification of a genomic region encompassing exons 2 through 3 of HLA-A using locus-specific primers, the amplicon was distributed in 20 separate multiplex SSP real-time PCRs containing sequence-specific primers (colored arrows), variation-insensitive primers (black arrows), and universal fluorescent TaqMan probes (colored stars). Sequence-specific and variation-insensitive primers targeted areas of converse exons. Sequence-specific primers were designed to more efficiently amplify defined targeted alleles (noted next to each reaction). Variation-insensitive primers were used to allow for internal standardization. b) The cross threshold (Ct) values obtained by monitoring amplification with the sequence-specific and internal standardization reagents were then used to assign samples positive or negative reactivities in each reaction (exemplified in the inset by results from reaction HLA-A018). The aggregate reactivity patterns rendered by the array of reactions were used to define the presence or absence of the addressed alleles. See text for details.