Enzyme-Linked Immunoassay For The Determination Of Cronobacter Spp. In Food

An indirect-enzyme-linked immunosorbent assay (ELISA) for rapid detection of Cronobacter spp. using polyclonal antibody has been developed. The titer of antibody against the antigen was1:243,000. The dilution of antibody was0.0267μg/mL; the confining liquid was1%skimmed milk in PBS,30min; antibody for detecting should be incubated for1h; HRP-labeled goat anti-rabbit should be incubated for30min.The lowest detectable antigen protein was104-105CFU/mL in pure culture. The twelve other bacteria (Escherichia coli, Citrobacter freundii, Enterobacter cloacae, Proteus mirabilis, Sh.flexneri, Enterobacter agglomerans, Salmonella enteritidis, Bacillus cereus, Methanosarcina, Streptococcus agalactiae, Streptococcus dysgalactiae) were all no cross-reactivity. The whole test procedure could be completed within6h. Pure milk, whole milk powder, whey protein powder free of Cronobacter spp. were inoculated with Cronobacter sakazakii (ATCC29544) at concentration of10-1,1, and10CFU/g.10CFU Cronobacter spp. and1CFU Cronobacter spp. could be detected by14h and0.1CFU Cronobacter spp. could be detected by17h in pure milk and whole milk powder. For whey protein,10CFU Cronobacter spp. and1CFU Cronobacter spp. could be detected by17h and0.1CFU Cronobacter spp. could be detected by20h. All dairy products can be detected after20h of growth in mLST-Vm. Reproducibility was good.In comparison with current culture-dependent detection methods, the ELISA is a rapid, sensitive, and specific method for detection of Cronobacter spp. in dairy products. This method may be used as rapid screening procedure for food monitoring during outbreak situation of Cronobacter spp..