A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types

Peter R. Wielinga*, Raditijo A. Hamidjaja, Joakim Ågren, Rickard Knutsson, Bo Segerman, Martina Fricker, Monika Ehling-Schulz, Astrid de Groot, Jane Burton, Timothy Brooks, Ingmar Janse, Bart van Rotterdam

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

52 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)S137-S144
JournalInternational Journal of Food Microbiology
Issue numberSUPPL. 1
Publication statusPublished - 1 Mar 2011

Bibliographical note

Funding Information:
This work was supported by the European Union-funded Integrated Project BIOTRACER (contract # 036272 ) under the 6th RTD Framework.


  • Bacillus anthracis
  • Bacillus cereus
  • Bacillus thuringiensis
  • Multiplex PCR
  • Real-time PCR
  • Zoonoses


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