title: A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types.
abstract: Bacillus anthracis is closely related to the endospore forming bacteria Bacillus cereus and Bacillus thuringiensis. For accurate detection of the life threatening pathogen B. anthracis, it is essential to distinguish between these three species. Here we present a novel multiplex real-time PCR for simultaneous specific identification of B. anthracis and discrimination of different B. anthracis virulence types. Specific B. anthracis markers were selected by whole genome comparison and different sets of primers and probes with optimal characteristic for multiplex detection of the B. anthracis chromosome, the B. anthracis pXO1 and pXO2 plasmids and an internal control (IC) were designed. The primer sets were evaluated using a panel of B. anthracis strains and exclusivity was tested using genetically closely related B. cereus strains. The robustness of final primer design was evaluated by laboratories in three different countries using five different real-time PCR thermocyclers. Testing of a panel of more than 20 anthrax strains originating from different locations around the globe, including the recent Swedish anthrax outbreak strain, showed that all strains were detected correctly.
authors.affiliation: National Institute for Public Health and the Environment, Centre for infectious Disease Control, Laboratory for Zoonoses and Environmental Microbiology, Antonie van Leeuwenhoeklaan 9, PO Box 1, Bilthoven, The Netherlands. peter.wielinga@rivm.nl
authors.forename: Peter R
authors.initials: PR
authors.lastname: Wielinga
authors.forename: Raditijo A
authors.initials: RA
authors.lastname: Hamidjaja
authors.forename: Joakim
authors.initials: J
authors.lastname: Agren
authors.forename: Rickard
authors.initials: R
authors.lastname: Knutsson
authors.forename: Bo
authors.initials: B
authors.lastname: Segerman
authors.forename: Martina
authors.initials: M
authors.lastname: Fricker
authors.forename: Monika
authors.initials: M
authors.lastname: Ehling-Schulz
authors.forename: Astrid
authors.initials: A
authors.lastname: de Groot
authors.forename: Jane
authors.initials: J
authors.lastname: Burton
authors.forename: Tim
authors.initials: T
authors.lastname: Brooks
authors.forename: Ingmar
authors.initials: I
authors.lastname: Janse
authors.forename: Bart
authors.initials: B
authors.lastname: van Rotterdam
mesh_terms: D001408:Bacillus anthracis / Q000145:classification* / Q000302:isolation & purification / Q000472:pathogenicity; D001409:Bacillus cereus / Q000145:classification / Q000235:genetics; D001413:Bacillus thuringiensis / Q000145:classification / Q000235:genetics; D017931:DNA Primers; D016133:Polymerase Chain Reaction / Q000379:methods*; D014774:Virulence / Q000235:genetics
chemical_list: D017931:DNA Primers