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.

Development of a Cell Culture Method To Isolate and Enrich Salmonella enterica Serotype Enteritidis from Shell Eggs for Subsequent Detection by Real-Time PCR

“…All reactions were run at a final volume of 25 l using the SmartCycler II apparatus (Cepheid, Sunnyvale, CA). The primers SEF14-F and SEF14-R and probe SEF14-P were previously designed and shown to specifically target a sequence found on the Salmonella serotype Enteritidis gene sefA (37). This fluorogenic probe contains a 5Ј Cy3 fluorophore and a 3Ј black hole quencher.…”

“…A variety of methods have been developed in order to expedite the detection of salmonellae in eggs, including GeneQuence DNA hybridization, PCR analysis, and enzyme-linked immunosorbent assay (3,27,37). However, these methods require lengthy enrichment steps prior to the application of the respective methods.…”

Multiplex TaqMan Real-Time PCR (qPCR) Assay Targeting prot6E and invA Genes for Fast and Accurate Detection of Salmonella Enteritidis

“…Sdf1 is highly specific for SE but is missing in SE phage types (PT) 6A, 9A, 11, 16, 20, and 27 and besides that were only tested on pure cultures (Malorny et al, 2007a). SefA gene is also present in all members of S. enterica serogroup D (Gallinarum, Pollorum, Dublin, Rostock, and Typhi, among others) which might lead to false positives results (Seo et al, 2004;Malorny et al, 2007a) and therefore it is not recommended for specific identification of SE. prot6E is present in the SE 60 kb virulence plasmid, which is present in most SE (>90%) (Chu et al, 1999;Helmuth and Schroeter, 1994;Clavijo et al, 2006) and therefore was our target of choice for SE specific detection by qPCR.…”

“…Among the most common gene targets used for SE detection by qPCR are: 1) Sdf1, a chromosomal fragment (Agron et al, 2001); 2) sefA, encoding for fimbrial antigen SEF14 (Seo et al, 2004); and 3) prot6E, encoding for a unique surface fimbriae (Malorny et al, 2007a;Clavijo et al, 2006). Sdf1 is highly specific for SE but is missing in SE phage types (PT) 6A, 9A, 11, 16, 20, and 27 and besides that were only tested on pure cultures (Malorny et al, 2007a).…”

“…The product of this gene is essential for the organism's ability to invade mammalian cells and subsequently cause disease (Galan and Curtiss, III, 1991;Galan JE et al, 1992). In the case of SE in specific, several PCR and isothermal methodologies has also been developed targeting different genes (Seo et al, 2004;Malorny et al, 2007a;O'Regan et al, 2008;Hadjinicolaou et al, 2009). Although isothermal amplification techniques has some advantages over qPCR, such as increased detection limit and lower cost; still has the disadvantage that a single target can be used at a time and lacks internal control for monitoring possible inhibitors of the reaction that might exist in the food matrix analyzed.…”