Based On CRISPR System For The Identification Of Pork Adulteration In Meat Products

Meat has always played an important role in human diets around the world,but with the rapid growth of meat consumption,the phenomenon of meat adulteration emerge in succession,and meat quality control is of paramount importance.Due to its cheap price,similar color and texture,pork is always incorporated into beef,lamb and other high-priced meat products by criminals.These malicious acts not only affect human health,but also cause economic losses and serious violations of consumers’religious beliefs.In recent years,the identification technologies of porcine-derived ingredients are mostly limited to laboratory,expensive instruments and professionals to analyze complex results,which cannot meet the requirements for the accurate and rapid identification of porcine-derived ingredients in meat products.As a new type of gene editing technology,CRISPR system was reformed into a sensitive nucleic acid detection tool because of its trans-cleavage and highly specific sequence recognition ability.At present,CRISPR system has been used for the detection of viruses and bacteria,but there are few reports on the identification of porcine-derived ingredients.The aim of this study was to establish a LAMP-CRISPR/Lb Cas12a detection system for the rapid detection of porcine-derived ingredients in meat products.The main research results of this study are as follows:1)In the CRISPR system,cr RNA-mediated target DNA binding by Cas12a–cr RNA additionally results in trans-cleavage of non-target DNA.The Lb Cas12a protein and cr RNA are both indispensable.In order to obtain the Lb Cas12a protein with cis-and trans-cleavage activities,the p ET30a-Lb Cas12a recombinant expression vector was constructed,and then the Lb Cas12a protein was successfully obtained.By optimizing the optimal reaction temperature is 37℃,the magnesium ion concentration in the CRISPR detection buffer to 80m M,and optimizing the molar concentration ratio of Lb Cas12a protein to cr RNA is 1:2,the Lb Cas12a detection system in this study can detect target DNA at a minimum of 80 p M.2)In order to construct a CRISPR/Lb Cas12a system for the detection of porcine-derived ingredients,firstly,after comparing the efficiency and purity of DNA extracted by several animal tissue DNA kits,Tiangen tissue genomic DNA extraction kit was selected as the DNA extraction kit.At the same time,through sequence alignment,cytb gene in mitochondrial DNA was selected as the target gene.After designing cr RNA and verifying its activity,the whole pork genome was detected by CRISPR/Lb Cas12a.However,it was found that the CRISPR/Lb Cas12a detection system was difficult to achieve the detection of the whole pork genome.It is necessary to increase the concentration of target DNA through in vitro amplification and improve the sensitivity of the CRISPR/Lb Cas12a system.3)In order to increase the concentration of target DNA and improve the sensitivity of the CRISPR/Lb Cas12a system,our study selected LAMP to amplify pork mitochondrial DNA.Primer Explore V5 was used to design LAMP primers.Through the screening of primer sets,C-3,D-1 and D-2 primer sets were selected.After the optimization of magnesium ion concentration,d NTPs concentration,inner and outer primer concentration ratio,Bst DNA polymerase concentration in the LAMP reaction system,the optimal amplification systems of the three groups of primers are as follows:the outer primer concentration of C-3,D-1 and D-2 primer sets is 0.2μM,the inner primer concentration of C-3,D-1 primer sets is 1.6μM,while the inner primer concentration of D-2 primer set is 2.0μM;The concentration of d NTPs is 1m M;the concentrations of Mg²⁺of the C-3,D-1 and D-2 primer sets are 4m M,5m M,4m M,respectively;the final concentrations of Bst DNA polymerase of the C-3,D-1 and D-2 primer sets are 0.4U/μL,0.4U/μL,0.48U/μL,respectively.After evaluating the specificity and sensitivity of the three primer sets,the C-3 primer set can detect 1%adulterated porcine-derived ingredients in beef at least,while D-1 and D-2 can only detect 10%adulterated porcine-derived ingredients.Fortunately,the three sets of primers is specific,and can provide a basis for LAMP-CRISPR detection system.4)By combining LAMP technology with CRISPR/Cas system,a LAMP-CRISPR/Lb Cas12a system for detecting porcine-derived ingredients was established.The samples were amplified by LAMP,and then the LAMP products were added to the CRISPR/Lb Cas12a detection system,and the fluorescent signal could be detected.In order to verify the specificity and sensitivity of the LAMP-CRISPR/Lb Cas12a detection system,the pork adulteration model was simulated by mixing pork with beef.LAMP-CRISPR detection system of C-3 and D-1 can detect 0.05%and 0.1%pork adulterated in beef,respectively.In summary,our study preliminarily established a LAMP-CRISPR/Lb Cas12a system for the detection of porcine-derived ingredients,which can directly observe the detection results with the naked eye,and provide a new way for meat adulteration detection.