Detection Method Of Bacillus Cereus Based On Nucleic Acid And Application In Food Safety Evaluation

With the improvement of people's living standards and the richness of food types,food safety issues have increasingly attracted people's attention.Bacillus cereus,a natural existence food-borne pathogen that can easily cause food poisoning,primarily causes two type of food-borne disease,emetic and diarrheal syndrome.However,many cases may be unreported or are misdiagnosed in a clinical setting because the associated symptoms are usually similar to Staphylococcus aureus intoxication?Bacillus cereus emetic type?or Clostridium perfringens food poisoning?Bacillus cereus diarrheal type?,respectively.Therefore,the development of rapid and accurate on-site detection of Bacillus cereus is of great significance to related fields such as food safety and disease prevention.Based on the literatures and the development status of domestic and foreign industries,this paper has sorted out the detection methods of Bacillus cereus based on nucleic acid,discussed the advantages and disadvantages,and then established two nucleic acid detection methods with simple operation and fast response.At the same time,according to the requirements in the"WS/T 82-1996 Diagnostic criteria and principles of management of food poisoning by Bacillus cereus",the methods established in this paper have been evaluated for food safety,mainly including the standard compliance,practical analysis and application prospect analysis.The specific research contents and results are as follows:?1?An assembled Pasteur pipette device to integrate nucleic acid extraction,amplifification and detection was developed to detect Bacillus cereus in a sample-to-answer format.Denaturation Bubble-mediated Strand Exchange Amplifification?SEA?was chosen to perform isothermal amplifification.This system only required a plastic Pasteur pipette and a normal fifilter paper without any modifification to incorporate nucleic acids detection.Visual readout was achieved by using a special dye,further simplifying result judgment process.The detection limit for Bacillus cereus is 1.0×10⁴ CFU/mL in pure cultures.The whole diagnostic procedure could be completed within 50 min,dramatically decreasing the detection time.In a word,the assembled Pasteur pipette device,combined with a homemade metal bath,possesses great potential for sample-to-answer application in resource-limited setting.,and provided more reliable technical solutions for the field of food safety monitoring.?2?An ultrasensitive electrochemical DNA?E-DNA?sensing strategy utilizing cooperative proximity hybridization based on a G-quadruplex?G4?probe was developed,which could specifically hybridize with its target DNA in homogenous solution.Such an E-DNA sensing strategy allows for ultrasensitive DNA detection with a detection limit as low as 2.82×10^-15M and a linear response range from1.0×10^-9to 1.0×10^-?15?M.More importantly,this homogenous proximity hybridization strategy could expand to colorimetric assay by incorporating G4 probe with hemin to form DNAzyme,which could effectively catalyze ABTS to generate a visual color change,making it a promising strategy for point-of-care testing.Also it provided the possibility to realize the rapid and sensitive detection of Bacillus cereus in the future.?3?The application of nucleic acid detection method for Bacillus cereus in food safety evaluation was introduced.The detection limit for Bacillus cereus in artifificially contaminated milk is 1.0×10⁵CFU/mL without enrichment and 1.0×10⁰CFU/mL following 12 h enrichment.According to the"WS/T 82-1996 Diagnostic criteria and principles of management of food poisoning by Bacillus cereus",the determination of the number of Bacillus cereus in poisoned food is generally?10⁵CFU per gram of food,so the detection limit of the detection method of Bacillus cereus established in this paper conforms to national standards.In the future,it can be more geographically targeted and very convenient for on-site point-of-care testing and food safety control in remote areas,and has very broad development prospect.