Rapid confirmation of polymyxin-cloth enzyme immunoassay for group D salmonellae including Salmonella enteritidis in eggs by polymerase chain reaction

Abstract

A rapid, simple and inexpensive combined screening test and confirmation procedure was developed for group D Salmonellae (e.g. Salmonella enteritidis) in eggs. S. enteritidis inoculated into egg samples was pre-enriched in a minimal volume of enrichment broth, which was then assayed by the polymyxin-cloth enzyme immunoassay (polymyxin-CEIA): cholate-extracted lipopolysaccharide antigens were captured on polymyxin-coated polyester cloth and detected by sequential reactions with factor 0:9 rabbit antiserum, anti-rabbit antibody-peroxidase conjugate and peroxidase substrate. Presumptive positive results obtained in the polymyxin-CEIA were confirmed by subjecting pre-enriched samples directly to a polymerase chain reaction (PCR) technique targeting Salmonella-specific invA gene sequences. The polymyxin-CEIA gave positive results in the assay of 18 different S. enteritidis strains inoculated into eggs, all of which were confirmed by the invA PCR. This entire procedure, combining the steps of sample pre-enrichment in a minimal volume of broth, polymyxin-CEIA screening and rapid confirmation by PCR, gave a short turn around time from sample collection to completion of the analysis of within 26 h.