Development Of CRISPR Nucleic Acid Sensor Based Detection Method For Food Pork Source Components

The adulteration of meat is one of the common food safety problems in China,and it is of great significance to develop a rapid detection method for meat components to ensure food safety.CRISPR technology,as a new molecular diagnostic technology,has broad application prospects in the field of food safety.This study mainly focuses on the nucleic acid detection of pork-derived components.Based on the requirements of high sensitivity and on-site rapid detection,the application of CRISPR/Lb Cas12a technology in the detection of pork-derived components is studied by combining PCR and LAMP technology,and the high sensitivity detection is realized by combining electrochemical sensors with CRISPR/Lb Cas12a technology.The LAMP technology was combined with CRISPR/Lb Cas12a to realize the application of CRISPR/Lb Cas12a detection method which can be interpreted by naked eyes without instruments in the field of meat adulteration detection.The main research results are as follows:（1）PBS method,alkaline lysis method,salt method,CTAB method and two commercial kits were used to extract genomic DNA from pork,and the extraction concentration,purity and extraction time of the six extraction methods were compared.The results showed that the concentration and purity of genomic DNA from pork extracted by alkaline lysis method were close to those of commercial kits,and the time consumption was short,and it could be extracted within 25 min,which could be used for subsequent PCR experiments.The genomic DNA from pork can be detected as low as 500 pg/μL based on PCR.It was also found that the detection results of the processed pork samples were not affected by the processing methods,so the alkaline cracking method was selected as the sample pretreatment method.（2）E.coli BL21（DE3）was used as the expression host,and the recombinant expression of Lb Cas12a protein,the core component of CRISPR/Lb Cas12a system,was carried out.The results showed that the target protein with molecular weight of150.0 k Da was obtained after IPTG induction expression and nickel column purification,and it had cis-and trans-cleavage activity,indicating that the recombinant protein of Lb Cas12a was successfully obtained,which was used for the subsequent detection of pork source components based on CRISPR/Lb Cas12a.（3）An electrochemical nucleic acid detection sensor based on PCR-CRISPR/Lb Cas12a was constructed.MB-hp DNA was used as a probe,and the probe was fixed on the gold electrode based on the gold-sulfur bond self-assembly method.CV and EIS were used to characterize the various steps in the sensor assembly process,and the detection conditions of the sensor were optimized.The results showed that when the working concentrations of MB-hp DNA and Mg²⁺were 0.4μM and 20 m M,respectively,the reaction temperature was 37°C,the reaction time was 60min,and the working concentrations of Lb Cas12a and cr RNA were 200 n M and 100n M,respectively.The linear detection range of the electrochemical nucleic acid detection sensor based on PCR-CRISPR/Lb Cas12a was 25～1000 pg/μL,the linear equation was y=44.49 lgx-40.17,（R²=0.992）,and the minimum detection limit（LOD）was 12 pg/μL.The specific and highly sensitive detection of pork source components could be achieved within 120 min.The established sensor was suitable for the detection of pork samples with various processing methods.Compared with the conventional PCR method,the sensitivity was increased by nearly 40 times.The detection performance of PCR-CRISPR/Lb Cas12a electrochemical nucleic acid sensor in detecting artificial binary meat adulteration model was evaluated.The peak current signal of DPV decreases with the increase of the pork blending ratio（w/w）in beef,and it has a good linear relationship with the logarithmic value of the pork blending ratio.The correlation linear equation is y=21.86 lgx+70.01,（R²=0.981）,the correlation detection linear range is 0.01%～10%,and the minimum detection limit（LOD）is 0.008%.（4）The construction of a single-tube visual detection method for pork source components based on LAMP-CRISPR/Lb Cas12a was studied.Taking the Cytb gene of mt DNA from pork as the detection target gene,the corresponding LAMP primer set was designed,and the detection limit of components from pork was 500 pg/μL,which was equivalent to the sensitivity of conventional PCR detection method.On this basis,in order to realize the synergistic reaction detection of LAMP and CRISPR,the optimal reaction temperatures of LAMP and CRISPR were analyzed.Aiming at the problem that the optimal reaction temperatures of the two systems are different,based on the thermal insulation characteristics of mineral oil,a detection tube for LAMP,CRISPR/Lb Cas12a reaction was designed,and the visual detection of pork source components in a single tube was realized based on LAMP-CRISPR/Lb Cas12a.Under the optimal reaction conditions,from sample pretreatment to detection,the sensitivity of visual detection is 50 pg/μL,and the detection time is 70 min,which is suitable for the detection of pork samples with various processing methods.The detection performance of LAMP-CRISPR/Lb Cas12a visual single-tube method for detecting artificial binary meat adulteration model was evaluated.The detection results show that the fluorescence becomes weaker with the decrease of the adulteration ratio（w/w）of pork in beef.When the adulteration ratio falls below0.05%,the visible fluorescence will no longer be generated.The LAMP-CRISPR/Lb Cas12a visual single tube method can realize the detection of 0.05%adulterated pork in beef（w/w）.