Rapid and accurate quantification of viable Bacillus cereus in foods with a Propidium monoazide (PMA) - Fluorescence in situ hybridization (FISH) - Flow cytometry (FCM) method

Fluorescence-labeled antibodies and viability indicators are routinely employed in conjunction with flow cytometry (FCM) to rapidly quantify viable target bacteria, ensuring food safety. However, the specific detection of Bacillus cereus, a prominent bacterium causing food poisoning, proves challenging because of its close phylogenetic relationship with other Bacillus species. Moreover, the accuracy of viability indicators in identifying viable B. cereus has been underexplored. This study established a rapid and specific method for quantifying viable B. cereus. The membrane-permeable dye propidium monoazide was identified as an effective means to differentiate between viable and non-viable B. cereus cells. Subsequently, fluorescence in situ hybridization was introduced to specifically identify viable B. cereus cells. Furthermore, FCM was utilized for the rapid and accurate quantification of labeled viable B. cereus cells. Following optimization, the method demonstrated notable specificity and robust anti-interference ability, with B. cereus recovery rates in milk powder and chicken breast after pretreatment ranging from 96.3% to 97.7%. Viable B. cereus cells were accurately quantified within 1.5 h, with a linear range of 102 to 107 cells/g. In conclusion, this developed method enables the rapid and specific quantification of viable B. cereus, offering a novel approach to the detection of specific foodborne pathogens.