Validation of allele-specific polymerase chain reaction for DNA typing of HLA-B27.
To find a specific method for HLA-B27 typing for the diagnosis of rheumatic disorders, we extensively tested the single-step B27-specific polymerase chain reaction (PCR) described by Dominguez et al. (Immunogenetics 1992;36:277-82). This method, which relies on specific primer recognition of a sequence in the third exon (unique to the B27-allele), was used for screening of 270 characterized blood samples, 57 of which were B27-positive. The method proved to be both sensitive and specific: It unambiguously identified all B27-positive samples and produced no false-positive results. For approximately 1% of the samples, we had to repeat DNA isolation and PCR to obtain a clear control amplification signal. In contrast to the specificity of the PCR method, parallel-performed flow cytometry gave ambiguous results in 3% of the samples because of antibody cross-reactivity. Flow cytometry and the PCR method described were similar in labor and costs. Therefore, we conclude that the proposed single-step PCR is feasible in a routine laboratory and would improve the reliability of HLA-B27 typing.
Steffens-Nakken
H M
HM
Department of Clinical Chemistry, Hospital Medisch Spectrum Twente, Enschede, The Netherlands.
Zwart
G
G
van den Bergh
F A
FA
D000483:Alleles; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D005091:Exons; D005434:Flow Cytometry; D005914:Globins / Q000235:genetics; D015796:HLA-B27 Antigen / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012216:Rheumatic Diseases / Q000175:diagnosis* / Q000276:immunology
D015796:HLA-B27 Antigen; D005914:Globins; D004247:DNA