Construction Of Novel Biosensing Of Cas12a Based On Combined CrRNA And Inferior PAM And Its Application In Pathogenic Bacteria Detection

Contamination of foodborne pathogenic bacteria in the food chain has posed a major threat to human health.Among them,the high lethality and drug resistance of Salmonella has been a hot issue in public health,and accurate and rapid Salmonella detection and drug resistance diagnosis has become an urgent need.The new generation of molecular diagnostic technology based on CRISPR has been widely reported for its advantages of high sensitivity and fast diagnostic speed.However,the application of CRISPR-Cas12 a for foodborne pathogenic bacteria detection is relatively rare,and the following problems still exist in the application,which make it difficult to achieve immediate detection(Point of Care Testing(POCT)): the whole process can be operated in as many as fifteen steps,which is prone to the risks of sample contamination,RNA degradation and non-specific amplification;nucleic acid amplification and CRISPR trans-cleavage cannot be performed simultaneously In this paper,we systematically investigated the effects of the nucleic acid amplification and CRISPR trans-cleavage on the viability of the pathogenic bacteria,especially the drug-resistant genome,which has low reproducibility.Based on this,this thesis systematically optimized the Cas12 a assay system and combined crRNA,Protospacer Adjacent Motif(PAM)sequences and hairpin probes to improve the detection sensitivity,shorten the detection time and reduce the equipment and environmental dependence.The main studies are as follows:(1)Optimization of combined crRNA-based Cas12 a lyophilization system and assay kinetics analysis: Immobilized CRISPR-Cas12 a system was constructed,and the lyophilization process exposes Cas protein hydrogen bonds and polar groups,reducing enzyme activity.By adding non-reducing disaccharide(sucrose)or polysaccharide(prulululanose)as stabilizer and mannitol as excipient,the enzyme activity was increased by 4.7 times after lyophilization(calculated by Michaelis-Menten equation);electrolytes and polymers were removed before lyophilization,and the enzyme activity was increased by 2.5 times after lyophilization,and the lyophilization system shortened the operation process from fifteen steps to four steps;optimization of Cas12 a The results showed that the best combination of crRNA Q1-Q4 increased the maximum reaction rate from 1.46sec-1 to 6.12 sec-1 and the Kcat/Km value from 106 to 107,and achieved amplification-free detection.The limit of detection(LOD)of the combined crRNA was 43 CFU/m L,which was 27-fold more sensitive compared to the LOD of 1.1×103 for the independent crRNA.(2)Construction and application of CRISPR time-resolved fluorescence flowmetric chromatography papers based on combinatorial crRNA: Based on the lyophilized CRISPRCas12 system and combinatorial crRNA,a non-amplified CRISPR-Cas12 a time-resolved fluorescence immunochromatographic assay(AFC-TRFIA)was developed to target the quinolone resistance gene of Salmonella(qnr S),reducing the reliance on instrumentation for POCT detection.Efficient immobilization of the capture probe within 10 min was facilitated by amine modification of the capture probe,which increased the steric structure and neutralized the negatively charged phosphate backbone with metal ions.The AFC-TRFIA method based on co-crRNA showed good linearity in the range of 4.9×102-1.6×106 CFU/m L,with R2=0.9981,and the calculated LOD was84 CFU/m L.for different concentrations of genomic DNA(8.2×101-8.2×107 amol/L),the LOD were 2.7× 102 a M,R2=0.9981,and the detection line range was 8.2×102-2.6×106a M.The spiked recoveries for the detection of drug-resistant Salmonella in samples of skim milk powder,raw cow milk,spinach and eggs ranged from 88.46% to 119.91%,with CV less than 8.42%.(3)One-step ultra-sensitive detection method based on inferior pre-interval proximity sequences and hairpin probes: To further improve the detection sensitivity based on previous experiments,inferior pre-interval proximity sequences such as VTTV and TTVT were used to design inferior crRNA,which slowed down the viability of CRISPR trans cleavage at the prereaction stage and allowed isothermal amplification to proceed preferentially.In the one-step reaction in the standard crRNA group,it takes 8-10 min to recognize amplicons,while in the inferior crRNA group,a large number of amplicons are observed in only 2 min,and the maximum reaction rate score is increased from 2.16 sec-1 to 8.12 sec-1 to achieve simultaneous amplification and cleavage,shorten the reaction time and reduce the operation steps.The introduction of a hairpin probe reduces background fluorescence and improves detection sensitivity.The inferior crRNAbased method greatly expands the abundance of available crRNA sequences,making the number of available PAMs theoretically 7 times higher than standard TTTV PAMs(3 sets of 7 inferior crRNA).And the detection limit of 100 CFU/m L could be reached within 30 min with R2 of 0.9901 and0.98797,respectively,and linear range of 100～108 CFU/m L.The spiked recoveries of 79.68% to117.91% with CV less than 7.78% were applied to skim milk powder,raw cow milk,spinach and egg,which fully demonstrated the ultra-sensitive DPAM-LPP The sensitivity and stability of the assay were well demonstrated.In summary,this thesis focused on the trans cleavage ability of Cas12 a,and took drug-resistant Salmonella as the main research object,and firstly constructed a rapid detection sensing system of CRISPR-Cas12 a,and then carried out systematic optimization on sensitivity and operability,and gradually proposed to achieve simultaneous nucleic acid amplification and trans cleavage,simplify the operation process,shorten the reaction time while achieving specific and stable ultra-sensitive It has a promising application in POCT of foodborne pathogenic bacteria and their drug-resistant bacteria.