title: In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences.
abstract: Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.
authors.affiliation: National Veterinary Institute; Department of Bacteriology; Uppsala, Sweden; Department of Biomedical Sciences and Veterinary Public Health; Swedish University of Agricultural Sciences (SLU); Uppsala, Sweden.
authors.forename: Joakim
authors.initials: J
authors.lastname: Ågren
authors.affiliation: National Institute for Public Health and the Environment; Centre for Infectious Disease Control; Laboratory for Zoonoses and Environmental Microbiology; Bilthoven, the Netherlands.
authors.forename: Raditijo A
authors.initials: RA
authors.lastname: Hamidjaja
authors.affiliation: National Food Institute; Technical University of Denmark; Søborg, Denmark.
authors.forename: Trine
authors.initials: T
authors.lastname: Hansen
authors.affiliation: Central Veterinary Institute of Wageningen University and Research Centre; Lelystad, the Netherlands.
authors.forename: Robin
authors.initials: R
authors.lastname: Ruuls
authors.affiliation: University Paris-Est Anses; Animal Health Laboratory; Maisons-Alfort, France.
authors.forename: Simon
authors.initials: S
authors.lastname: Thierry
authors.affiliation: National Food Institute; Technical University of Denmark; Søborg, Denmark.
authors.forename: Håkan
authors.initials: H
authors.lastname: Vigre
authors.affiliation: National Institute for Public Health and the Environment; Centre for Infectious Disease Control; Laboratory for Zoonoses and Environmental Microbiology; Bilthoven, the Netherlands.
authors.forename: Ingmar
authors.initials: I
authors.lastname: Janse
authors.affiliation: National Veterinary Institute; Department of Bacteriology; Uppsala, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Sundström
authors.affiliation: National Veterinary Institute; Department of Bacteriology; Uppsala, Sweden.
authors.forename: Bo
authors.initials: B
authors.lastname: Segerman
authors.affiliation: Central Veterinary Institute of Wageningen University and Research Centre; Lelystad, the Netherlands.
authors.forename: Miriam
authors.initials: M
authors.lastname: Koene
authors.affiliation: National Food Institute; Technical University of Denmark; Søborg, Denmark.
authors.forename: Charlotta
authors.initials: C
authors.lastname: Löfström
authors.affiliation: National Institute for Public Health and the Environment; Centre for Infectious Disease Control; Laboratory for Zoonoses and Environmental Microbiology; Bilthoven, the Netherlands.
authors.forename: Bart
authors.initials: B
authors.lastname: Van Rotterdam
authors.affiliation: University Paris-Est Anses; Animal Health Laboratory; Maisons-Alfort, France.
authors.forename: Sylviane
authors.initials: S
authors.lastname: Derzelle
mesh_terms: D000818:Animals; D000881:Anthrax / Q000175:diagnosis*; D001408:Bacillus anthracis / Q000235:genetics* / Q000302:isolation & purification*; D001409:Bacillus cereus / Q000235:genetics; D001413:Bacillus thuringiensis / Q000235:genetics; D002876:Chromosomes, Bacterial; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
keywords: Bacillus anthracis; chromosomal marker; detection; diagnostic sensitivity; in silico analysis; inter-laboratory trial; qPCR; specificity