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.

Influence of HLA Alleles on Shedding of Kaposi Sarcoma–Associated Herpesvirus in Saliva in an African Population

“…For sequencing-based typing of HLA-DRB1 (Protrans), 14 HLA-DRB1 group-specific primer mixes as well as 1 positive and 1 negative control were run in parallel for each sample, to separately amplify exon 2 of the HLA-DRB1 allele groups. PCR product detection was done by fluorescence readout using a fluorescence resonance energy transfer probe [40]. Exon 2, which encodes the polymorphic first domain of the HLA-DR b-1 chain, was sequenced forward and reverse from either 1 positive amplification reaction (in case of group homozygosity) or different positive amplification reactions (in case of group heterozygosity).…”

PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities

“…This value corresponds to an amount of amplicons which was generated in a few cycles if a large amount of templates was present initially or after many cycles if the PCR started with few templates. Real-time TNA was reported to match the sensitivity of protocols for post-PCR analysis by DNA-binding dyes (1), radiolabeled primers (6), qualitative nested PCR (25,29) or hybridization techniques (30).…”

Development of a new HLA-DRB real-time PCR typing method

“…Compared with all the rtPCR-based approaches described elsewhere [5][6][7][8][9][10], our typing method has some clear advantages. Postamplification steps are avoided, thus saving work and reducing the risk of contamination; the time required for typing may be shortened to as little as 2.5 hours if the initial steps of reagents and sample loading into reaction trays could be finally automated.…”

“…Previous studies have shown that real-time PCR (rtPCR)-based HLA typing, also called fluorotyping, does not require post-PCR processing and may be fully automated [5][6][7][8][9][10]. In previous reports on the application of rtPCR to HLA typing, it was used as an equivalent to PCR-SSP with a fluorogenic locus-specific probe, but no attempt has been made to use the potential of this technology to discriminate between the alleles of a given locus.…”