Development Of IMBs-qPCR Method For Detection Of Food-borne Salmonella

Salmonella is a Gram-negative bacilli bacterium belonging to the Enterobacteriaceae family,which can cause zoonotic diseases and parasitizes in the intestines of animals and humans.It is the most common pathogen causing food poisoning.Most countries currently adopt traditional culture methods for the detection of foodborne diseases.However,due to the complexity and length of the method,it is difficult to achieve rapid and large-scale food detection.Therefore,the establishment of rapid,efficient,accurate and quantitative Salmonella detection technology to improve the detection rate and accuracy is of great significance for global public health safety and emergency treatment of food-borne diseases.1.Analysis and expression of Salmonella-specific pathogenic antigens and antibody preparation:Establish criteria for selecting Salmonella detection genes,requiring that the selected virulence genes are chromosome-encoded and species-specific,and the genes can express outer membrane proteins with multiple epitopes.Combined with bioinformatics analysis,the outer membrane protein genes pagN and pagC of Salmonella were amplified by PCR.High-purity recombinant proteins were obtained by purification of inclusion bodies.The expression levels of the two recombinant proteins both accounted for more than 60%of the bacterial cell expressionand the protein concentrations were 1 mg/mL and 600 ?g/mL.Immunized adult male rabbits to obtain antibody serum,purified and obtain specific polyclonal antibodies with ELISA titers of 1:3200.2.Preparation and parameter determination of immunomagnetic beads:Based on the magnetic response of the prepared PagN immunomagnetic beads to enrich and isolate Salmonella in the sample,and explore the optimal parameters of the Salmonella capture experiment by immunomagnetic beads.The study found that when 30 ?L of PagN polyclonal antibody(0.4 mg)was combined with 1 mg of carboxylated magnetic beads(1-2 ?m)at 37? for 4 h,the efficiency of coupling magnetic beads was the best.The magnetic beads can capture more than 80%of Salmonella in the order of 102-105 CFU/mL,and a clear image of the immunomagnetic beads that successfully captured Salmonella was observed under the scanning electron microscope.3.Construction of Salmonella pagC Gene deletion mutant by Red Recombinant Gene Editing System:pagC deleted strains were used to absorb non-PagC protein-specific Salmonella antibodies by reverse adsorption in order to increase the specificity of magnetic beads coupled with antibodies;As a negative control for virulence gene deletion,the pagC gene deletion mutant strain is used for quality control of the established IMBs-qPCR detection methodIt also provides resources support for the study of Salmonella pagC to carry out the regulation and research on the function of virulence gene.4.Establishment of a rapid detection method for foodborne Salmonella:Immune magnetic beads separation and enrichment and fluorescence quantitative PCR technology(IMBs-qPCR)are combined and integrated to detect Salmonella.Designed primers for Salmonella-specific virulence genes pagC and invA for fluorescent quantitative detection.The results showed that the invA gene has better specificity,sensitivity,and amplification efficiency as a detection primer.The number of colonies captured by immunomagnetic beads can be accurately quantified by establishing a fluorescence quantitative standard curve and evaluating its stability can accurately quantify the number of colonies captured by immunomagnetic beads,and the minimum detection limit is 13 CFU/mL.At the same time,the detection of Salmonella in artificially contaminated foods such as milk and pork is detected by immunomagnetic beads.In the range of 102-105 CFU/mL,the capture rate of Salmonella by immunomagnetic beads is above 70%,which can effectively solve the problems of low detection limit,lengthy time,and poor specificity in the current technology.In summary,this study prepared recombinant protein and antibody of Salmonella membrane protein virulence gene,and the immunomagnetic beads prepared by specific virulence protein antibody can effectively capture Salmonella in food.In combination with specific and sensitive qPCR technology,the Salmonella IMB-qPCR detection technology has enriched the rapid detection of food-borne pathogenic Salmonella and laid the foundation for the further development and application of the kit.