Detection Of Emetic Bacillus Cereus Based On DNA Hairpin Self-assembly Signal Amplification

Bacillus cereus is a common foodborne pathogen,which can cause two kinds of food poisoning symptoms,emesis and diarrhea,associated with cereulide and enterotoxins respectively.Compared with enterotoxins,the cereulide is well-tolerated underdifferentstressconditions,andisproducedintheemeticB.cereus-contaminated food.According to reported research,only 10~3 CFU/g of B.cereus in food can cause emetic syndrome,and considering that emetic B.cereus has caused fatalities,a versatile and reliable method for detecting emetic B.cereus is urgently needed.DNA hairpin self-assembly signal amplification is a enzyme-free signal amplification method,it can be driven by Gibbs free energy or position entropy to make the hairpin DNA assemble.And this method is widely used in the field of modern analysis because of its simplicity,selectivity and stability.In this study,in order to shorten the detection time and further improve the sensitivity,we developed DNA hairpin self-assembly signal amplification combined with flow cytometric bead and graphene oxide fluorescent sensor for detection of the emetic B.cereus in milk.The chapters are as follows:In the first chapter,the research progress in application of DNA self assembly strategy was reviewed.In the second chapter,we first established hybridization chain reaction-based flow cytometric bead sensor for the detection of emetic B.cereus in milk.Using biotin-modified probes as a"bridge"between magnetic microspheres and HCR products,the fluorescence intensity of magnetic beads was used as a signal output and measuring the fluorescence signal on the surface of the MBs to detect the emetic B.cereus.Agarose gel electrophoresis and fluorescence confocal microscope were used to verify the feasibility of the established method,moreover,the amount of biotinylated probe,the concentration of FAM-labeled H1/H2,and the HCR reaction time were optimized,and the sensitivity and specificity of this method were also studied.Based on the optimal reaction conditions,the limit of detection of this method in pure culture was 7.6×10~0 CFU/mL,and the LOD in spiked milk was 9.2×10~2 CFU/mL.In the third chapter,we established a new method for the detection of emetic B.cereus based on the catalytic hairpin DNA self-assembly-graphene oxide fluorescent sensor.DNA from emetic B.cereus was amplified by PCR and target ssDNA generated by heat denaturation,and the CHA reaction was initiated by the target ssDNA to generate a duplex to detach the GO surface to recover the fluorescent signal.Based on the optimal reaction conditions,the LOD in pure culture was 6.2×10~1 CFU/mL;the LOD in spiked milk was 5.9×10~2 CFU/mL.