Construction and optimization of a multiplex PMAxx-qPCR assay for viable Bacillus cereus and development of a detection kit.

Ruirui Hu, Antuo Hu, Zhaoxin Lu, Haibo Zhou, Wanqing Wei, Fengxia Lu, Haizhen Zhao, Xiaomei Bie

Journal of microbiological methods 2023 Apr

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In this study, a PMAxx-qPCR method for the detection and quantification of viable Bacillus cereus (B. cereus) was established based on the cesA gene that is involved in cereulide synthesis, enterotoxin gene bceT and hemolytic enterotoxin gene hblD combined with modified propidium monoazide (PMAxx). The sensitivity detection limit of the method was as follows: the DNA extracted by the kit reached 140 fg/μL, and the bacterial suspension without enrichment reached 2.24 × 101 CFU/mL; 14 nonB. cereus strains of the 17 tested strains all tested as negative, whereas the 2 strains of B. cereus carrying the target virulence gene(s) could be accurately detected. In terms of application, we assembled the constructed PMAxx-qPCR reaction into a detection kit and evaluated its application performance. The results showed that the detection kit has high sensitivity, strong anti-interference capability, and has good application potential. The purpose of this study is to provide a reliable detection method for the prevention and traceability of B. cereus infections. Copyright © 2023. Published by Elsevier B.V.

Citation

Ruirui Hu, Antuo Hu, Zhaoxin Lu, Haibo Zhou, Wanqing Wei, Fengxia Lu, Haizhen Zhao, Xiaomei Bie. Construction and optimization of a multiplex PMAxx-qPCR assay for viable Bacillus cereus and development of a detection kit. Journal of microbiological methods. 2023 Apr;207:106705