Fluorotyping of HLA-DRB by sequence-specific priming and fluorogenic probing.

Abstract

Similar to our recently described HLA-A and -C fluorotyping strategies, the aim of this study was to develop a sequence-specific primed polymerase chain reaction (PCR-SSP)-based fluorotyping method for HLA-DRB. Applying the fluorogenic 5' nuclease assay, it is possible to increase the sample throughput rate by abolishing all labor-intensive post-amplification steps. Additionally, problems related to contamination are eliminated. The method relies on the 5'-3' exonuclease activity of the Taq-DNA Polymerase which cleaves a target-specific and individually labelled fluorogenic probe during successful PCR. Different labelled probes specific for different targets can be applied in a single PCR, allowing independent detection of the specific HLA and the internal control product. The probe used to detect the HLA-DRB specific amplicons was labeled at its 5' end with FAM as the reporter and further 3' with TAMRA as the quencher. The probe hybridized within the 2nd exon to a conserved region which was covered by all primer mixes. In case of amplification, the cleavage of the fluorogenic probe led to an interruption of the TAMRA-mediated quenching effect and generated a significant increase of the FAM fluorescence. The HLA-DRB fluorotyping information was based on the FAM fluorescence released by 24 individual primer mixes. A TET-TAMRA-labelled probe was used to indicate amplification of the internal control sequence in each PCR reaction. So far, 170 PCR typed clinical samples representing all serologically defined HLA-DRB specificities were analyzed using this fluorotyping method. The results were 100% concordant with those obtained by conventional agarose gel detection.