Construction Of A Novel Biosensing Method Based On Nuclease Cas12a And Its Application To Foodborne Pathogenic Bacteria

The contamination of foodborne pathogenic bacteria such as Salmonella,Staphylococcus aureus and Escherichia coli in the food chain has become an important threat to human health.Salmonella has the problem of high contamination rate and drug resistance,so it is very important to carry out the study of simultaneous detection of virulence genes and drug resistance genes.Nucleic acid detection method is usually used to achieve simultaneous detection of virulence genes and drug resistance genes.CRISPR/Cas system is widely reported as a new generation of molecular diagnostic technology due to its high sensitivity and accuracy,which can be used as a complementary technology for nucleic acid detection.Currently,the detection system of nuclease Cas12a for double-stranded DNA is not fully optimized,and multi-target detection is not widely used,and the application of nanotechnology in the CRISPR detection system is less studied.Therefore,the thesis systematically studied the detection method of Cas12a for foodborne pathogenic bacteria.The detection method for food-borne pathogenic bacteria was constructed by screening Cas12a and preliminary optimization of the detection system.Combined with nucleic acid amplification and fluorescence enhancement effect of gold nanoparticles,the detection sensitivity was improved and the pre-processing steps and detection time were reduced.The main studies are as follows:（1）Study on the kinetics of Cas12a trans enzymatic cleavage and optimization of the detection system.The study compared the trans enzymatic cleavage rates of two species of Cas12a.The maximum reaction rates(V_max)of As Cas12a and Lb Cas12a were 5.9127×10^-9M sec^-1 and 4.2664×10^-9 M sec^-1 respectively.The higher cleavage rate of As Cas12a was selected as the enzyme for the subsequent experiments.The Cas12a detection system was also optimized to achieve sensitive detection of S.aureus and Salmonella.The results showed a detection limit of 10 p M for DNA and 10² CFU/m L for two types bacteria in combination with recombinase polymerase amplification,with good linearity in the range of 10²～10⁸CFU/m L.The spiked recoveries in milk samples ranged from 76.38%～127.15%.（2）Construction of a dual-target detection for Cas12a（Cas12a-Dd）.Based on the optimized Cas12a detection system,the detection sensitivity of the detection has been further improved with a limit of detection of 100 f M for DNA,that was 2 orders of magnitude higher than the previous part,with a linear range of 100 f M～10 n M.Cas12a-Dd was sequentially combined with dual-target polymerase chain reaction and recombinase polymerase amplification,and the clever use of a round cap allowed for the temporary storage of more Cas12a assay solution.The results were achieved by the dual mode readout of microplate reader and UV visualization with the dual detection limit of 1 CFU/m L with a linear range of10⁰-10¹⁰ CFU/m L.Thus,a dual-target detection of Salmonella virulence genes and drug resistance genes was achieved.（3）Construction of the Au-Cas12a fluorescence-enhanced sensing method.To build a more sensitive Cas12a detection method,a fluorescence-enhanced"OFF-enhanced ON"probe was used to the fluorescence-enhanced Cas12a detection system based on the metal-enhanced fluorescence effect and the fluorescence resonance energy transfer effect of gold nanoparticles.The enhanced fluorescence could amplify the signal difference between negative and positive samples.The strongest burst effect was achieved when the ratio of Au NP to Au NC was 1:1.The sensitivity of the Au-Cas12a fluorescence-enhanced sensing method was 10 f M for DNA detection within 40 min and 10 CFU/m L for Salmonella,S.aureus and E.coli,which eliminating the amplification process,simplifying the operation and reducing the detection time by 20%.In conclusion,the thesis focused on the trans cleavage ability of Cas12a and took typical food-borne pathogenic bacteria as the research object.A rapid detection biosensing system for foodborne pathogenic bacteria was constructed and optimized with respect to sensitivity and operability systematically to continuously improve the detection sensitivity.The method eliminated the nucleic acid amplification step and simplified the operation process,which had a great prospect of application in the detection of food-borne pathogenic bacteria.