Rapid Detection Of Food-borne Bacteria Contamination By IMBs-qPCR Method

Foodborne pathogen has been the main reason of foodborne disease meanwhile the hidden danger of food safety. Traditional agar plate cultivation methods are usually used to detect vital bacterial cells contamined in foods. However, this method is a time consuming and laborious process that takes several days, and has limitations in accuracy, specificity and sensitivity. To develop effective detection methods of foodborne pathogen and improving the speed and specificity are of great significant.Immunomagnetic separation(IMS) is a rapid, specific, efficient, and technically simple method that can be used for the separation and enrichment of a target organism directly from complex non-targets matrix. Outer membrane protein(OMP) is specifically owned by gram negative bacteria and usually has strong immunogenicity, closely interact with the immune system and play a significant role in the development of new vaccines. Aim of this study was to explore and develop an IMS-qPCR method based on OMP antibody for efficient isolation of pathogens contamined in food samples.In our experiment, three genes respectively isolated from Salmonella, Yersinia enterocolitica and Campylobacter jejuni were amplified by PCR without signal peptides. Three recombinant proteins(PagC of Salmonella, Ail of Yersinia enterocolitica and CfrA of Campylobacter jejuni) with molecular weight of about 24 kDa, 18 kDa and 75 kDa were expressed in prokaryotic system and purified, which can be used in the preparation of antibodies and immunomagnetic beads.PagC polyclonal antibodies were prepared by injecting recombinant PagC protein to rabbit. The key parameters for preparing the immunomagnetic beads, the coupling rate between antibody and magnetic beads and additive amount of immunomagnetic beads were optimized with different concentrations of Salmonella. Under optimized conditions, the capture efficiency(CE) was greater than 70% against 101 CFU/mL~104 CFU/m L of Salmonella with 0.1 mg immunomagnetic beads. The immunomagnetic beads exhibited high specific binding with Salmonella strains(CE > 70%), and low binding with non-target bacteria(CE < 8%).A qPCR assay targeting pagC gene was designed for specific detection and quantification of Salmonella. The limit of detections was 18 CFU/ml. Detection and quantitative enumeration of Salmonella in pork and milk samples shows good recoveries of 54.34% and 52.07%. In conclusion, the IMBs-qPCR based on PagC is of good efficiency and specifity and the whole process of detection Salmonella is less than 10 hours, which is a promising rapid method to detect Salmonella in emergency.