Validation of allele-specific polymerase chain reaction for DNA typing of HLA-B27

Abstract

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.

B*27 in molecular diagnostics: Impact of new alleles and polymorphism outside exons 2 and 3

“…The presence of HLA‐B27 is used as a diagnostic marker for ankylosing spondylitis and related spondyloarthropathies and has been the subject of many studies, including molecular typing reports (8–19, 37, 38) . Several advantages of molecular typing of HLA‐B*27 compared with serological methods have been mentioned: [1] potentially more accurate typing resulting from the reduced risk of false‐positive results caused by possible cross‐reactivity of antibodies and of false‐negative results as a result of low B27 expression or bacterial infection; [2] no requirement of viable cells and thus no restrictions to shipment and storage of cells, enabling batchwise testing; [3] and cost effectiveness (9, 11–15, 17, 39) .…”

“…The second mix used in our PCR‐SSP contained the B*27 specific 5′primer described previously (12–14, 16) . As already identified at that time B*2707 was not amplified with this primer.…”

“…DNA typing is potentially more accurate and there is no requirement for viable cells. The different molecular typing techniques that have been used for HLA‐B*27 determination are ligation of probes (4) , PCR‐restriction fragment length polymorphism (RFLP) (5–7), polymerase chain reaction sequence‐specific primer (PCR‐SSP) (8–14) , Hybridisation with sequence‐specific oligonucleotides (PCR‐SSO) (13, 15–17) and sequence‐based typing (SBT) (18, 19) . All these methods have to cope with the rapidly increasing allelic diversity that is observed for the HLA genes.…”

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

“…Besides flow cytometry techniques, PCR based methods are widely used in routine clinical HLA‐B*27 testing . Molecular genotyping of HLA‐B*27 is mostly based on sequence‐specific PCR using primers complementary to a sequence in the second or third exon unique to the B*27 ‐allele, as described by Olerup and Dominguez et al, respectively.…”

Correction

“…HLA-B27 typing of AS patients was performed by flow cytometry and typing of healthy controls was carried out by polymerase chain reaction as described previously. 3 The HLA-B27 positive and negative data we acquired for case and control groups merely represented phenotype rather than exact genotype. Therefore the D' value between the HLA-B27 and TNFa loci could not be calculated using the phenotype results of HLA-B27.…”