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Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

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Irenge, Leonid M. [UCL] Durant, Jean-François [UCL] Tomaso, Herbert [Bundeswehr Institute of Microbiology, Munich, Germany] Pilo, Paola [University of Bern] Olsen, Jaran S. [Norwegian Defence Research Establishment (FFI), Norway] Mahillon, Jacques [UCL] Gala, Jean-Luc [UCL]

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Document type Article de périodique (Journal article) – Article de recherche – Journal Article, Research Support, Non-U.S. Gov't, Validation Studies
Access type Accès restreint
Publication date 2010
Language Anglais
Journal information "Applied Microbiology and Biotechnology" - Vol. 88, no. 5, p. 1179-1192 (2010)
Peer reviewed yes
Publisher Springer (New York)
issn 0175-7598
e-issn 1432-0614
Publication status Publié
Affiliations UCL - Cliniques universitaires Saint-Luc
UCL - SSS/IREC - Institut de recherche expérimentale et clinique
UCL - SST/ELI/ELIM - Applied Microbiology
MESH Subject Virulence - genetics ; Sequence Analysis, DNA ; Sequence Alignment ; Sensitivity and Specificity ; Ribose - analogs & derivatives - genetics ; Polymorphism, Single Nucleotide ; Polymerase Chain Reaction - methods ; Plasmids ; Phylogeny ; Phosphotransferases - genetics ; Nucleotides - genetics ; Genes, Bacterial ; DNA, Bacterial - analysis - genetics ; Base Sequence ; Bacteriological Techniques ; Bacterial Typing Techniques ; Bacillus anthracis - classification - genetics - isolation & purification - pathogenicity ; Bacillus - genetics ; Adenylosuccinate Synthase - genetics
Keywords Bacillus anthracis ; LNA probe ; PCR ; Environmental samples
Links
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Bibliographic reference Irenge, Leonid M. ; Durant, Jean-François ; Tomaso, Herbert ; Pilo, Paola ; Olsen, Jaran S. ; et. al. Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples. In: Applied Microbiology and Biotechnology, Vol. 88, no. 5, p. 1179-1192 (2010)
Permanent URL http://hdl.handle.net/2078.1/34999