Establishing Reference Genes for Accurate Gene Expression Profiling in Toxigenic Bacillus cereus

<i>Bacillus cereus</i> is a Gram-positive pathogen associated with foodborne illnesses and severe non-gastrointestinal infections. Robust tools for accurate gene transcription analysis are essential for studying toxin gene expression dynamics and deciphering the complex regulatory networks orchestrating the expression of toxin and virulence factors. This study aimed to identify reliable reference genes for normalizing reverse transcription quantitative PCR (RT-qPCR) data in toxigenic <i>B. cereus</i>. An emetic and an enteropathogenic strain were used as model organisms to establish a suitable reference gene set to monitor the dynamics of toxin gene transcription. Ten candidate reference genes were evaluated for their expression stability using geNorm, NormFinder, BestKeeper and the ΔCq method, with the final rankings integrated via RefFinder. Among the tested genes, <i>rho</i>, <i>rpoD</i> and <i>recA</i> were identified as the most stable expressed reference genes across all tested conditions. As shown in this proof-of-principle study, the established reference gene set provides a suitable tool to investigate the influence of extrinsic and intrinsic factors on toxin gene transcription. In conclusion, our newly established reference gene set provides a robust basis for studying toxin gene expression in <i>B. cereus</i> and contributes to a better understanding of its pathogenicity and potential strategies to mitigate its harmful effects.