Rapid screening for HLA-B27 by a TaqMan-PCR assay using sequence-specific primers and a minor groove binder probe, a novel type of TaqMan trade mark probe

HLA-B27 is strongly associated with ankylosing spondylitis (AS). As typing for HLA-B27 is routinely performed by serological methods, false-positive results can be generated. Therefore, several more accurate molecular methods have been developed for HLA-B27 genotyping. We describe a real-time PCR me...

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Bibliographic Details
Published in:Journal of immunological methods 2004-04, Vol.287 (1-2), p.179-186
Main Authors: Kuzio, Sylvain, Sylvain, Kuzio, Hanguehard, Aurélie, Aurélie, Hanguehard, Morelle, Marc, Marc, Morelle, Ronsin, Christophe, Christophe, Ronsin
Format: Article
Language:English
Subjects:
DNA Primers
Flow Cytometry
Genetic Predisposition to Disease
Genotype
HLA-B27 Antigen - analysis
HLA-B27 Antigen - genetics
Humans
Reverse Transcriptase Polymerase Chain Reaction - methods
Risk Factors
Sensitivity and Specificity
Spondylitis, Ankylosing - diagnosis
Taq Polymerase
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Summary:HLA-B27 is strongly associated with ankylosing spondylitis (AS). As typing for HLA-B27 is routinely performed by serological methods, false-positive results can be generated. Therefore, several more accurate molecular methods have been developed for HLA-B27 genotyping. We describe a real-time PCR method for the detection of the HLA-B27 allele using sequence-specific primers (SSP) combined with a fluorogenic MGB probe (minor groove binder probe), a novel type of TaqMan probe. The MGB increases the melting temperature (T(m)) of the probe, allowing the use of shorter probes. Moreover, the use of a non-fluorescent quencher (NFQ) attached to the MGB probe improves the efficiency of fluorescence quenching, thus providing a very low fluorescent background. We tested this method on 150 subjects (41 HLA-B27 positive and 109 HLA-B27 negative) who underwent routine HLA-B27 serological testing by flow cytometry (FC). Serology and our TaqMan assay gave identical results in all cases and no false positive or negative results were observed. A graphical representation of fluorescence and normalized reporter signal (DeltaRn) values demonstrated that HLA-B27 positive and HLA-B27 negative samples formed two tight clusters making it possible to clearly differentiate between HLA-B27 positive and negative samples. This single tube PCR method for the detection of HLA-B27 should be particularly suitable for the routine analysis of large numbers of samples in the laboratory.
ISSN:0022-1759