TECHNICAL REPORT

Strategies for PCR based detection of Burkholderia pseudomallei DNA in paraffin wax embedded tissues

R M Hagen¹, Y P Gauthier², L D Sprague³, D R Vidal², G Zysk⁴, E-J Finke¹ and H Neubauer¹

¹ Institut für Mikrobiologie der Bundeswehr, Neuherbergstr. 11, D-80937 München, Germany
² Unité de Microbiologie, Centre de Recherches du Service de Santé des Armées Emile Pardé, La Tronche, France
³ Klinik und Poliklinik für Strahlentherapie und Radiologische Onkologie der TU-München, Klinikum rechts der Isar, Ismaningerstr. 22, D-81675 München, Germany
⁴ Institut für Medizinische Mikrobiologie und Virologie, Universität Düsseldorf, Geb. 22.21, Universitätsstr. 1, D-40225 Düsseldorf, Germany

Correspondence to:
Correspondence to:
Dr H Neubauer, Institut für Mikrobiologie, Sanitätsakademie der Bundeswehr, Neuherbergstr. 11, D-80937 München, Germany;
320027945810\|[ndash ]\|0001{at}t-online.de

Recently, several cases of melioidosis imported to Europe have been reported. The diagnosis of the acute or chronic infection remains challenging. This report describes an optimised protocol for fast and reliable DNA preparation for use in two different polymerase chain reaction (PCR) assays, namely: (1) a seminested PCR assay targeting a genus specific sequence of the ribosomal protein subunit 21 (rpsU) gene and (2) a nested PCR assay targeting the gene encoding the filament forming flagellin (fliC). Various strains of Burkholderia spp, strains of closely related genera, and spleen tissue samples of experimentally infected mice were investigated. The combination of PCR and sequencing of the amplicons resulted in high sensitivity and specificity. These procedures may allow rapid, sensitive, and reliable detection of B pseudomallei DNA in routinely formalin fixed and paraffin wax embedded samples, thus providing a safe diagnostic tool and avoiding the cultivation of a risk group 3 agent. In addition, this method could be useful for retrospective histopathological investigations.

Keywords: Burkholderia pseudomallei; melioidosis; polymerase chain reaction; pathology

Abbreviations: CFU, colony forming units; PCR, polymerase chain reaction

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