Evaluation of a real-time PCR assay for the differentiation of Bacillus cereus group species

For the differentiation of the species Bacillus weihenstephanensis and B. pseudomycoides from other members of the B. cereus group a real-time PCR was suggested in 2011, which applies the primer-probe-combinations BCFomp2/BCRomp2 with MotB-FAM-2 and BpmF/BpmR2 with Bpm-FAM-1, respectively. However, a subsequent study revealed contradictions between taxonomic relationships based on real-time PCR results and phylogenetic analyses based on multi-locus sequence typing. Hence, we evaluated the specificity of the suggested primer-probe-combinations by comparing real-time PCR results with further markers for the identification of the respective species. These markers comprised colony morphology, growth temperatures, 16S rRNA- and cspA gene sequences, panC clade and ANI species. Observed PCR specificities of only 42% for the identification of B. weihenstephanensis and 56% for the detection of B. pseudomycoides indicate that the suggested primer-probe-combinations are not specific. The BCFomp2/BCRomp2 with MotB-FAM-2 combination lacks exclusivity for B. weihenstephanensis, whereas the BpmF/BpmR2 with Bpm-FAM-1 combination lacks inclusivity for B. pseudomycoides. •Taxonomic classification based on a real-time PCR was evaluated.•panC typing and ANI determination were used to support species classification.•Primer-probe BCFomp2/BCRomp2-MotB-FAM-2 lacks exclusivity for B. weihenstephanensis.•Primer-probe BpmF/BpmR2-Bpm-FAM-1 lacks inclusivity for B. pseudomycoides.