title: Rapid, specific detection of Salmonella Enteritidis in pooled eggs by real-time PCR.
abstract: An assay was developed for the specific detection of Salmonella Enteritidis in eggs with the use of an application of the fluorogenic 5' nuclease assay (TaqMan). In this assay, a segment of the gene sefA specific to Salmonella group D strains such as Salmonella Enteritidis was used. The amplification of the target gene products was monitored in real-time by incorporating a fluorescent dye-labeled gene-specific probe in the PCR reaction. This method correctly detected and distinguished Salmonella Enteritidis from nearly 50 of non-group D Salmonella and other non-Salmonella strains. Detection of the sefA gene was linear for DNA extracted from approximately 10(2) to 10(9) CFU/ml in phosphate-buffered saline and 10(3) to 10(8) CFU/ml in raw egg. In two trials, when applied to detection of Salmonella Enteritidis in homogenized egg pools and compared with conventional culture methods, the newly developed PCR method yielded a 100% correlation with results obtained by a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the culture method. The sensitivity of this assay was approximately less than 1 CFU/600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low concentrations of Salmonella Enteritidis in egg samples.
authors.affiliation: U.S. Food and Drug Administration, CFSAN/OPDFB, 5100 Paint Branch Parkway, College Park, Maryland 20740, USA. kseo@cfsan.fda.gov
authors.forename: K H
authors.initials: KH
authors.lastname: Seo
authors.forename: I E
authors.initials: IE
authors.lastname: Valentin-Bon
authors.forename: R E
authors.initials: RE
authors.lastname: Brackett
authors.forename: P S
authors.initials: PS
authors.lastname: Holt
mesh_terms: D000818:Animals; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis; D004531:Eggs / Q000382:microbiology*; D036781:Fimbriae Proteins; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D005798:Genes, Bacterial; D016133:Polymerase Chain Reaction / Q000379:methods; D012477:Salmonella enteritidis / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial; C080931:sefA protein, Salmonella enteritidis; D036781:Fimbriae Proteins