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 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.
Kuzio
Sylvain
S
Laboratoire d'Analyses Médicales Ruffié and Associés, Bordeaux, France.
Sylvain
Kuzio
K
Hanguehard
Aurélie
A
Aurélie
Hanguehard
H
Morelle
Marc
M
Marc
Morelle
M
Ronsin
Christophe
C
Christophe
Ronsin
R
D017931:DNA Primers; D005434:Flow Cytometry; D020022:Genetic Predisposition to Disease; D005838:Genotype; D015796:HLA-B27 Antigen / Q000032:analysis* / Q000235:genetics; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012307:Risk Factors; D012680:Sensitivity and Specificity; D013167:Spondylitis, Ankylosing / Q000175:diagnosis; D019914:Taq Polymerase
D017931:DNA Primers; D015796:HLA-B27 Antigen; D019914:Taq Polymerase