Characterization And Quantification Of Viable Bacillus Cereus In Food Using Flow Cytometry

Bacillus cereusis one of the common high-risk foodborne pathogens,which can cause food contamination from the production,processing and transportation channels and causes1.4%～12%of global food safety incidents according to statistics.Ingesting more than 10~3 CFU/g of viable B.cereus can cause vomiting and diarrhea,and in severe cases,it can be life-threatening.Therefore,rapid quantification of viable B.cereusis the premise and basis of food safety monitoring.The traditional plate counting methods are capable of quantitatively and qualitatively detecting viable B.cereus,but they are time-consuming,laborious,and have a high level of uncertainty;PCR based detection methods have good specificity and are combined with DNA fluorescent dyes to distinguish viable and dead bacteria,but the problems of repeatability and stability of nucleic acid amplificationmake them difficult to achieve direct quantification of viable bacteria;the immunological methods based on the specificity of antigen-antibody binding can specifically label bacteria,but currently effective antibodies to label B.cereus are lacked and these methods therefore cannot distinguish viable and dead bacteria.In conclusion,the existing methods are difficult to meet the requirements of rapidly and accurately quantify viable B.cereus.Therefore,in this study,a FISH probe with complementary 16S r DNA conserved sequence of B.cereus was used to specifically label B.cereus,viability fluorescent dye was used to distinguish viable and dead B.cereuscells,and then counted by flow cytometry,so as to establish a rapid,specific and accurate quantification method for viable B.cereus.In addition,a high precision B.cereus reference material was developed based on this method.The details are as follows:1.A rapid,accurate and quantification method for viable B.cereus was established.The labeling efficiencies of different viable fluorescent probes on B.cereus with different viability states were explored and compared with the plate counting method.The results showed that membrane integrity could accurately characterize the viable state of B.cereus,and PMA was selected to characterize the viable of B.cereus.The labeling efficiencies of antibodies and FISH probe p B394 of B.cereus were compared,and their specificities were verified.These results showed that p B394 could accurately and specifically label B.cereus,and the operation conditions were further optimized to shorten the detection time and improve the detection efficiency.PMA can label all dead bacteria with compromised membrane,while p B394 can specifically label viable B.cereus.When combined with flow cytometric analysis,this method allows for specific and absolute quantification of viable B.cereus.The established PMA-FISH-FCM method can accurately and specifically quantify viable B.cereus within 1.5 h.In addition,this method is capable of distinguishing B.cereus from other Bacillus species such as B.subtilis and B.megaterium.The method shows a good linear relationship with plate counting method(10~2～10~7 cells/g,R~2>0.99).The recovery rate of B.cereus in food matrices is above 96.3%.2.Preparation of B.cereusreference materials.A vacuum freeze-drying method with freeze-drying protective agents was used to prepare B.cereus reference materials.The established PMA-FISH-FCM method and plate counting method were used to determine the value of the B.cereus reference materials,and the uniformity and stability of the reference materials were analyzed.The results showed that the established PMA-FISH-FCM method and plate counting method have the same accuracy in the determination process of B.cereus reference materials.The determination result of B.cereus reference material is 2.49×10~9 CFU/bottle with good uniformity and stability,and can be stably preserved at-20?C for 120 days.It is relatively unstable at 4?C.Therefore,it is recommended to store and transport the B.cereus reference materials at-20?C.This study showedgreat potential to rapidly,accurately,and specifically quantify viable B.cereus cells.It also provides a novel research pattern to rapid and accurate detection pathogenic bacteria in food matrices.The uniformity and stability of the prepared B.cereus reference materialsaresatisfied,which is of great practical significance in the traceability and comparison of laboratory quality control and detection results.It also provides a reference value to the preparation of reference materials for other foodborne pathogenic bacteria.