title: Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples.
abstract: 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.
authors.affiliation: Defence Laboratories Department, Belgian Armed Forces, Brussels, Belgium.
authors.forename: Léonid M
authors.initials: LM
authors.lastname: Irenge
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Durant
authors.forename: Herbert
authors.initials: H
authors.lastname: Tomaso
authors.forename: Paola
authors.initials: P
authors.lastname: Pilo
authors.forename: Jaran S
authors.initials: JS
authors.lastname: Olsen
authors.forename: Vincent
authors.initials: V
authors.lastname: Ramisse
authors.forename: Jacques
authors.initials: J
authors.lastname: Mahillon
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Gala
mesh_terms: D000265:Adenylosuccinate Synthase / Q000235:genetics; D001407:Bacillus / Q000235:genetics; D001408:Bacillus anthracis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D015373:Bacterial Typing Techniques; D001431:Bacteriological Techniques; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D005798:Genes, Bacterial; D009711:Nucleotides / Q000235:genetics; D010770:Phosphotransferases / Q000235:genetics; D010802:Phylogeny; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012266:Ribose / Q000031:analogs & derivatives / Q000235:genetics; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014774:Virulence / Q000235:genetics
chemical_list: C434798:2'-O-4'-C-methylene-ribofuranosyl nucleotide; D004269:DNA, Bacterial; D009711:Nucleotides; D012266:Ribose; D010770:Phosphotransferases; D000265:Adenylosuccinate Synthase