Real-time PCR based HLA-B*27 screening directly in whole blood.

Geiger, Kathrin; Zach, Christina; Leiherer, Andreas; Fraunberger, Peter; Drexel, Heinz; Muendlein, Axel (2020). Real-time PCR based HLA-B*27 screening directly in whole blood. HLA : immune response genetic, 95(3), pp. 189-195. Wiley 10.1111/tan.13767

Text GeigerK_Real-timePCRbasedHLA-B27_HLA_2019.pdf - Published Version Restricted to registered users only Available under License Publisher holds Copyright. Download (1MB)

The linkage between the occurrence of human leucocyte antigen B*27 (HLA-B*27) and ankylosing spondylitis or other related spondyloarthritides is well documented. PCR based methods are widely used for HLA-B*27 screening. To refine HLA-B*27 testing we aimed at establishing a real-time PCR protocol to detect the HLA-B*27 allele directly in blood samples, without DNA extraction. HLA-B*27 analysis was performed by two real-time PCRs using TaqMan primer-probe assays for B*27 specific amplification of exon 2 or exon 3 of the HLA-B gene together with a mutant of Taq polymerase for direct blood PCR. Conditions for direct blood PCR were optimized and the reliability of the direct blood PCR protocol was evaluated by re-genotyping over 200 blood samples from patients who previously underwent routine DNA-based HLA-B*27 testing. Heating blood samples at 95°C for 10 minutes significantly improved PCR performance. Results from real-time PCR based HLA-B*27 testing directly in blood of over 200 patients were in 100% concordance with results obtained by routine DNA-based HLA-B*27 genotyping. In summary, we present a reliable real-time PCR protocol for HLA-B*27 screening directly in whole blood supporting fast clarification of the presence of ankylosing spondylitis or other spondyloarthritides in suspected cases.

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