Unit Molecular Diagnostics

Utrecht, Netherlands

Unit Molecular Diagnostics

Utrecht, Netherlands
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Roelandse-Koop E.A.,Diakonessenhuis | Roelandse-Koop E.A.,University of Amsterdam | Buisman B.,Diakonessenhuis | Van Hannen E.J.,St Antonius Hospital | And 8 more authors.
Clinical Chemistry and Laboratory Medicine | Year: 2011

Background: Human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. The B27 allele is present in 90% of patients with this disease, whereas it is present in only 9% of Caucasians. Molecular detection of HLA-B27 is traditionally based on allele specific amplification of exon 2 (Olerup method) or exon 3 (Dominguez method) by PCR, followed by gel analysis. Methods: We developed a real-time TaqMan PCR based on the Dominguez method with a βGlobin PCR as internal control. Results: A total of 544 clinical samples were used to compare the real-time TaqMan PCR with the traditional Dominguez PCR, the traditional Olerup PCR and a commercial Olerup based HLA-B27 detection kit (Olerup SSPTM HLA-B27, GenoVision). While 542 samples gave concordant results, two samples showed discrepancies and were further analyzed. One sample that showed a discrepancy was negative with the traditional Olerup method and positive with the three other procedures. Sequencing analysis showed the presence of HLA-B*2712 in this sample. The other sample, positive with both Olerup based PCRs and negative with both Dominguez based methods, turned out to be positive for HLA-B*2707 by sequence analysis. Conclusions: With a correct result for 543 out of 544 samples (99.8%), we consider our real-time HLA-B27 PCR is a reliable method to detect HLA-B27 in the Dutch population, with reduced hands-on time and contamination risk compared to traditional PCR methods. © 2011 by Walter de Gruyter.

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