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Title: RAPID, SPECIFIC DETECTION OF SALMONELLA ENTERITIDIS IN POOLED EGGS BY REAL TIME PCR

Author
item SEO, KUNHO - FDA - COLLEGE PARK, MD
item VALENTIN-BON, I.E. - FDA - COLLEGE PARK, MD
item BRACKETT, R.E. - FDA - COLLEGE PARK, MD
item Holt, Peter

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/2/2003
Publication Date: 5/1/2004
Citation: Seo, K., Valentin-Bon, I., Brackett, R., Holt, P.S. 2004. Rapid, specific detection of salmonella enteritidis in pooled eggs by real time pcr. Journal Of Food Protection. Vol 67, No.5, p864-869.

Interpretive Summary: Industry and regulatory laboratories continue to search for simple and rapid methods to detect human pathogens in food samples. Salmonella Enteritidis (SE) is a bacterial pathogen that can infect intact table eggs and ultimately cause foodborne outbreaks of human gastroenteritis following consumption of these contaminated eggs. A rapid technique is described using the real-time polymerase chain reaction assay for detection of SE in egg contents. The assay detected and distinguished SE from 50 other paratyphoid salmonellae isolates. The concentrations of SE that the assay detected in egg contents was between 100 and one billion cells in buffer-extracted egg contents and between 1000 and 100 million in raw egg. Detection of SE by this method required only 2 days whereas detection of SE by normal culture techniques required 5 days. The rapidity, simplicity, specificity and sensitivity of this assay make it potentially an important tool for use various laboratories to detect SE in food products.

Technical Abstract: An assay was developed for the specific detection of Salmonella Enteritidis (SE) in eggs, using a novel 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 SE was used. The amplification of the target gene products was monitored in real-time by incorporating fluorescent dye-labeled gene-specific probes in the PCR reaction. This method correctly detected and distinguished SE from nearly 50 of non-group D Salmonella and other non-Salmonella strains. Detection of SEF14 gene was linear for DNA extracted from approximately 100 - 1000000000 CFU/ml in PBS and 1000 - 100000000 CFU/ml in raw egg. In two trials, when applied to detection of SE in homogenized egg pools and compared with conventional culture methods, the newly developed PCR method yielded a 100% correlation with results obtained using a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the cultural method. The sensitivity of this assay was approximately less than 1 CFU per 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 SE in egg samples.