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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Editorial Board Member: Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Schlattmann, Peter / Tate, Jillian R. / Tsongalis, Gregory J.

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Rapid HLA-B27 screening with real-time TaqMan PCR: a clinical validation in the Dutch population

Elianne A. Roelandse-Koop1, a / Bert Buisman1 / Erik J. van Hannen2 / Anneke van der Zee3, b / Wouter Kortlandt1 / Mirjam H.A. Hermans4 / 1, 2, 5 / Rianne van Rhee-Luderer6, c

1Department of Clinical Chemistry, Diakonessenhuis, Utrecht, The Netherlands

2Department of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands

3Laboratory of Medical Microbiology, St. Elisabeth Hospital, Tilburg, The Netherlands

4Molecular Diagnostics, Jeroen Bosch Hospital, ‘s Hertogenbosch, The Netherlands

5Department of Medical Microbiology and Immunology, Diakonessenhuis, Utrecht, The Netherlands

6Unit Molecular Diagnostics, Diakonessenhuis, Utrecht, The Netherlands

aCurrent address: Free University Medical Centre, Amsterdam, The Netherlands.

bCurrent address: Maasstad Hospital, Rotterdam, The Netherlands.

cCurrent address: Centre for Infectious Diseases Friesland, Leeuwarden, The Netherlands and Molecular Diagnostic Centre Friesland, Leeuwarden, The Netherlands.

Corresponding author: Dr. Arend-Jan van Houte, Bosboomstraat 1, 3582 KE Utrecht, The Netherlands Phone: +31-882506697, Fax: +31-882506695

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 49, Issue 12, Pages 1979–1985, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2011.252, September 2011

Publication History

Received:
2010-11-29
Accepted:
2011-08-01
Published Online:
2011-09-06

Abstract

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 β-Globin 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.

Keywords: ankylosing spondylitis; Dutch population; HLA-B27; real-time PCR; TaqMan

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