HLA-B27 genotyping by Fluorescent Resonance Emission Transfer (FRET) probes in real-time PCR

Abstract

Abstract Several polymerase chain reaction (PCR)-based human leukocyte antigen (HLA) genotyping methods are in use, but none is fully satisfactory. The introduction of real-time PCR (rt-PCR) with fluorescence resonance energy transfer (FRET) probes provides a powerful tool to overcome the drawbacks of current methods such as the long processing time and the requirement for post-PCR manual procedures. Here we present evidence that the FRET-fluorotyping principle may resolve HLA-B27 variants, providing a higher resolution in less time than the techniques currently in use. The protocol uses between one and three consecutive amplification reactions depending on the resolution required. The first reaction, aimed at detecting HLA-B27-positive samples, uses β-globin coamplification as control. The second reaction, aimed at resolving most frequent B27 alleles, uses two hybridization probes whose melting temperatures curves allow the classification of HLA-B27 alleles into eight groups. By adding a third reaction, even the rarest alleles associated and not associated to ankylosing spondylitis (AS) may be discriminated. The technique was blindly tested on 60 samples from individuals previously typed and confirmed by standard PCR sequence-specific oligoprobes-PCR sequence and PCR-based typing PCR-SBT (30 B27+, 30 non-B27). There was a complete concordance rate, thus confirming the potential of this new technique for clinical HLA-B27 typing and for HLA typing in general.