Abstract 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.

中文翻译：

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蜡样芽孢杆菌属物种分化的实时 PCR 检测评价

摘要 为了将 Bacillus weihenstephanensis 和 B.pseudomycoides 与蜡状芽孢杆菌组的其他成员区分开来，2011 年提出了实时 PCR，它应用了引物-探针组合 BCFomp2/BCRomp2 与 MotB-FAM-2 和BpmF/BpmR2 分别与 Bpm-FAM-1。然而，随后的一项研究揭示了基于实时 PCR 结果的分类关系与基于多位点序列分型的系统发育分析之间的矛盾。因此，我们通过将实时 PCR 结果与进一步标记物进行比较来评估建议的引物-探针组合的特异性，以识别相应物种。这些标记包括菌落形态、生长温度、16S rRNA 和 cspA 基因序列、panC 进化枝和 ANI 物种。观察到的 PCR 特异性仅为 42%，用于鉴定 B. weihenstephanensis 和 56% 用于检测 B.pseudomycoides 表明建议的引物-探针-组合不是特异性的。BCFomp2/BCRomp2 与 MotB-FAM-2 组合缺乏对 B.weihenstephanensis 的排他性，而 BpmF/BpmR2 与 Bpm-FAM-1 组合缺乏对 B.pseudomycoides 的包容性。