Detection Of Mutton Adulteration Based On Enzyme Linked Immunosorbent Assay

In recent years,the adulteration of meat products in China has become more serious.Mutton has a large proportion in the meat industry and a very high consumer demand.However,unscrupulous traders use other meat species such as ducks,chickens,and pork mixed with mutton,and some even replace mutton.In particular,the adulterated meat products have a higher adulteration rate.At present,the detection technology for raw meat is quite mature.Because the protein by heating will changes,so the identification process of cooked meat products is complicated.The biggest disadvantage of enzyme-linked immunosorbent assay for detecting adulterants in meat is that the protein is denatured during heating and cannot be detected by antibodies.The study found that muscle proteins in animal muscles have a very good thermal stability and specificity in species,such as hot myoglobin,these findings solve the problem of ELISA technology.According to some reports,myoglobin in muscle is a kind of thermostable protein,and mutton myoglobin(MMb)is used as a specific index to detect mutton adulteration by enzyme-linked immunosorbent assay.In this experiment,the amino acid sequence of mutton myoglobin(MMb)was obtained by NCBI(National Center for Biotechnology Information)software,and the amino acid codon of MMb was optimized which was better expressed in E.coli Rosetta(DE3).The entire MMb gene was designed and synthesized via overlapping PCR.The target gene and vector pET-28a were digested with NdeI and XhoI enzymes and then ligated with T4 ligase.Finally,the correct recombinant plasmid MMb-pET-28a was obtained and imported into E.coli Rosetta(DE3),after being expressed under the inducer,the soluble MMb protein was successfully expressed by SDS-PAGE.The MMb was purified by Ni-IDA affinity column and the concentration of MMb fusion protein was determined to be 1 mg/mL.By comparing the MMb with the amino acid sequences of other species,a relatively specific MMb polypeptide segment was obtained,and the peptide was coupled to the carrier protein keyhole limpet hemocyanin(KLH)as a macromolecular antigen in BALB/c.The mice were injected and the spleen cells of the injected mice were taken for cell fusion.After subcloning and screening,the highest titer of 2-1-8cell strains was obtained,and reached 3.2×10~6.The ascitic fluid was prepared from the abdominal cavity of mouse and the titer obtained from Protein G purified was 1:64K.An indirect competitive ELISA for MMb was established.The optimal coating concentration of MMb was 1μg/mL.1%BSA was the optimal blocking solution and was blocked at 37 ~oC for 1 h.The optimal dilution of monoclonal antibody was 1:16000,the optimal dilution of enzyme-conjugated secondary antibody was 1:5000.The fitted linear regression equation was y=-9.5x+102.84,R2=0.9885,5-1000 ng/mL was the best detection range,the minimum detection limit was 1 ng/mL,and the precision mid-plate error was 6.25%,the average error between the plates was 8.15%.The recovery rate was 95.38%-101.78%,and the Monoclonal antibodies of anti-MMb has a good specificity and no cross reaction with ducks,chickens,pigs.The established ELISA detected the mutton content in mixed raw meat and the heated mutton content in mixed cooked meat.