Center for Food Safety and Applied Nutrition (CFSAN)
Microbial Methods and Subtyping Branch
Background: Ph.D.in Microbiology, 2004. University of Chile
Development and validation of Real time PCR for the detection of Salmonella enterica Enteritidis in eggs.
Proposed Regulatory Research Project for the FDA Commissioner's Fellow:
Among non-typhoidal salmonellosis, S. Enteritidis (SE) has emerged as a major egg-associated pathogen. SE transmission to humans has been linked mainly to consumption of contaminated foods containing undercooked eggs. Fresh shell-eggs can be contaminated easily with SE through cracks in the shell by contact with chicken feces or by trans-ovarian infection. Consequently, the increase of consumption of shell eggs and egg products per capita in the United States to approximately 249 eggs per year (American Egg Board, 2008) may have contributed, in part, to increases in foodborne outbreaks including a large multistate outbreak of SE outbreak associated with eggs in the US in 2010. Traditional culture methods for SE detection from shell eggs and liquid whole eggs consist of a series of steps including non-selective pre-enrichment, selective enrichment, and selective/differential plating, and finally biochemical and serological confirmation. The traditional microbiological method for SE isolation from liquid eggs is described in detail in Chapter MLG 4.05 "Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products" by the United States Department of Agriculture (USDA) (http://www.fsis.usda.gov/PDF/MLG_4_05.pdf). This method is labor intensive and takes about two weeks to complete the analysis. Consequently, a need exists for the development and validation of faster screening and detection methods for this pathogen in eggs. The use of PCR or real time PCR (qPCR) for specific pathogen detection in foods has increased in recent years. They are fast and reliable tools for the testing of contaminated foods and had help in preventing outbreaks. In recent years, numerous methods based on Salmonella DNA detection (e.g. invA gene) either by conventional or real-time PCR have been developed. qPCR is faster, is more sensitive than conventional PCR, and provides real-time data avoiding the use of gels. In particular, the invA gene represents a good candidate for Salmonella detection as it is present in all pathogenic serovars described to date. The product of this gene is essential for the organism’s ability to invade mammalian cells and subsequently cause disease. In the case of SE specifically, several PCR and isothermal methodologies has also been developed targeting different genes. Although isothermal amplification techniques have some advantages over qPCR, such as increased detection limit and lower cost, it still has the disadvantage that only 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. In the present study we will develop a fast and accurate qPCR assay for the specific detection of SE in eggs. The proposed method is intended as an initial screening of 24 h pre-enrichments for the presence of Salmonella in eggs. In turn, this method will dramatically decrease the time and effort required during standard microbiological testing, since only positive pre-enrichment samples will be processed.
Ph.D. in Food Science or Microbiology with a background in microbial culture and isolation, basic training in molecular biology techniques such as DNA extraction and PCR.
Selected Recent Publications:
Gonzalez-Escalona N., Brown E. W., and Zhang G. (2012). Development of a multiplex TaqMan real-time PCR (qPCR) assay targeting invA and ttrRSBCA locus for accurately detection of Salmonella in produce and eggs. Food Research International, 48: 202-8.
Zhang G., Brown E. W., and Gonzalez-Escalona N. (2011) Comparison of quantitative real-time PCR, quantitative reverse transcriptase real-time PCR, loop-mediated isothermal amplification and conventional microbiological method by the U.S. Food and Drug Administration for the detection of Salmonella spp. in produce. Appl. Environ. Microbiol., 77, (18), 6495–6501.
González-Escalona, N.; Eric W. Brown; and Guodong Zhang; (2011) Multiplex TaqMan real-time PCR (qPCR) assay targeting invA and prot6E genes for fast and accurate detection of Salmonella Enteritidis. In: Salmonella / Book 3. Edited by: Yashwant Kumar. Intech. ISBN 979-953-307-691-0.
González-Escalona N., Hammack TS, Russell M, Jacobson AP, De Jesús AJ, Brown EW, Lampel KA (2009). Detection of live cells of Salmonella spp. in produce by a TaqMan® based quantitative RT-qPCR targeting invA mRNA. Appl. Environ. Microbiol., 75 (11):3714-20.