Optimization of a rapid dot-blot immunoassay for detection of Salmonella enterica serovar Enteritidis in poultry products and environmental samples

摘要

An immunoassay was developed for the detection of Salmonella serovar Enteritidis in poultry and environmental samples. This assay consisted of a two-step procedure that involved an enrichment step using whole egg homogenate (EH) as the enrichment medium and detection by a monoclonal antibody (MAb)-based dot-blot assay. Egg homogenate enriched Salmonella Enteritidis was heated to 100degreesC for 10 min in the presence of cholic acid, a detergent, to liberate the lipopolysaccharide (LPS) antigen in gelled egg matrix. This was subsequently transferred onto a nitrocellulose membrane for detection with MAb 2F11. Several commercially available media were compared with egg homogenate for their relative ability to resuscitate and propagate Salmonella Enteritidis to detectable levels. Incubation in EH, trypticase soy broth (TSB), and lactose broth (LB) resulted in comparable levels of Salmonella Enteritidis as demonstrated by viable plate counts. Salmonella Enteritidis grown in TSB exhibited the greatest visual intensity showing a positive test when tested by the dot-blot assay. Incubation time necessary to detect one cfu of Salmonella Enteritidis was reduced from 20 to 10 h using TSB as the enrichment broth. Addition of ferrous sulphate or ferrioxamine E or cholic acid in the enrichment broth had negligible negative effects on the growth of Salmonella. Salmonella Enteritidis when incubated with a mixture of naturally contaminated or artificially inoculated competitive micro-organisms in environmental samples at a ratio of 1: 102, was able to reproduce to detectable numbers for the immunoassay. This method was able to detect all phage types (PT 1, 6, 7, 8, 13, 13a, 14b, 21 and 28) with unique ribopatterns. The results demonstrated that Salmonella Enteritidis, when preenriched in a medium containing ferrous sulphate or cholic acid, could be readily detected in the presence of 100-fold higher competition of other microorganisms.