Research On IMBs-qPCR Detection Of Food-borne Yersinia Enterocolitica

Yersinia enterocolitica is an important zoonotic pathogen,which causes not only intestinal diseases,but also extra-intestinal syndromes such as arthritis and mesenteric lymphadenitis.It poses a serious threat to human health.At present,the detection method for Y.enterocolitica is the culture method,but the detection steps are very complicated and time-consuming,which has many limitations.The development of a rapid and accurate method for detecting Y.enterocolitica in foods is of great significance in food processing and in ensuring food safety.In this experiment,based on the cloning and expression of pathogen-specific proteins,specific multi-antibody immunomagnetic beads were prepared,and the immunomagnetic bead enrichment and separation technology was combined with quantitative real-time PCR detection technology.A specific,sensitive and rapid detection method for Y.enterocolitica in food was established.The main results are as follows:1.Cloning,expression and purification of Y.enterocolitica specific pathogenic genes:Based on detection of virulence genes in multiple Yersinia strains,and bioinformatic analysis of Y.enterocolitica specific pathogenic outer membrane protein genes ail and ompF,prokaryotic expression vectors were constructed,and recombinant outer membrane proteins Ail and OmpF were expressed and purified at a concentration of 5 mg/mL.2.Preparation and parameter optimization of immunomagnetic beads:Adult male rabbits were immunized with the recombinant outer membrane proteins Ail and OmpF to prepare a polyclonal antibody with a titer of 1:3200.The polyclonal antibody was used to prepare immunomagnetic beads,and the preparation parameters of the immunomagnetic beads were optimized.The results showed that when 25 ?L of the antibody with a concentration of 10 mg/mL and 1 mg of the magnetic beads with a particle size of 1.0-2.0 ?m were shaken at room temperature The coupling efficiency of the prepared immunomagnetic beads was the best when incubating for 6 hours.When using 0.2 mg immunomagnetic beads to capture 101-105 CFU/mL Y.enterocolitica,the capture efficiency was close to 80%.The capture efficiency of other bacteria was less than 10%,which proved that the immunomagnetic beads prepared in this study had good specificity.3.Construction of Y.enterocolitica ompF gene knockout mutant and adsorption and purification of rabbit antisera:The recombinant plasmid pKD46 was resistant to the transformation,and overlapping PCR was used to extend the homologous arm of the transformed fragment to 500 bp,successfully obtained Y.enterocolitica ompF gene knockout mutant according to Red recombination system This strain was used as an absorption strain prepared by OmpF protein-specific polyclonal positive serum to improve the specificity of detection.It can also be used as a negative control strain for this experiment.4.The establishment of immunomagnetic bead capture-fluorescence quantitative PCR(IMBs-qPCR)method to detect Y.enterocolitica:design a specific primer for Y.enterocolitica foxA through online software;use this primer to establish a fluorescent quantitative PCR standard curve,determine the detection limit of 64 CFU/mL;add interference strains such as Salmonella to verify the good specificity of this detection method;use IMB s-qPCR method to detect Y.enterocolitica in artificially contaminated milk and pork,When the concentration of the bacterial solution was 103-104 cfu/mL,the capture rate was greater than 68%.Compared with the culture method,this method has high consistency,and the entire process only takes 12 hours,which greatly shortens the detection time.In summary,this study expressed and purified Y.enterocolitica recombinant outer membrane protein and prepared its specific polyclonal antibody.Non-specific antibodies in serum were removed by adsorption with a knockout mutant strain.The specific capture of Y.enterocolitica in food by using outer membrane protein antibody-conjugated immunomagnetic beads,combined with sensitive and specific fluorescent quantitative PCR technology,to establish an IMBs-qPCR detection method,providing an effective alternative method for rapid detection of Y.enterocolitica in food.