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P3-36 Simultaneous Detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes in a Variety of Cheeses and Spinach Using a Multiplex Real-time PCR Method

Wednesday, July 31, 2013
Exhibit Hall (Charlotte Convention Center)
Venugopal Sathyamoorthy, U.S. Food and Drug Administration-CFSAN-DVA, Laurel, MD
Atin Datta, U.S. Food and Drug Administration-CFSAN-DVA, Laurel, MD
Larisa Trach, U.S. Food and Drug Administration-CFSAN-DVA, Laurel, MD
Yiping He, U.S. Department of Agriculture-ARS-ERRC, Wyndmoor, PA
Ben Tall, U.S. Food and Drug Administration-CFSAN, Laurel, MD
Barbara McCardell, U.S. Food and Drug Administration-CFSAN-DVA, Laurel, MD
Introduction: Foodborne diseases affect about 48 million people per year in U.S. A major portion of these foodborne illnesses are caused by Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes. Hence, it is important to identify these pathogens in contaminated foods so that they can be eliminated, thereby reducing the incidence of foodborne diseases. At present there is no method available for the simultaneous detection of all three organisms in contaminated foods regulated by FDA.

Purpose: This project aims to evaluate, optimize and adapt a real-time PCR method, originally developed at USDA, to simultaneously detect the presence of Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes in soft-cheese and spinach.

Methods: Five different cheeses and spinach in a previously described selective medium were spiked with 5-25 CFU/25 g of Salmonella, E.coli O157:H7 and L. monocytogenes, stomached and incubated for 2 hours at 37°C followed by the addition of nalidixic acid, fosfomycin, cycloheximide and acriflavine and then grown overnight. The samples were then used for the extraction of genomic DNA using a DNA extraction kit (Qiagen). The DNAs were used to carry out the real-time PCR with primers and TaqMan probes targeting invA (Salmonella), rfbE (E. coli O157) and hlyA (L. monocytogenes) as well as an internal amplification control (IAC).

Results: All three gene targets of the tested pathogens were detected in the spiked cheeses and spinach samples after enrichment, with a sensitivity level of 5-25 CFU/25g. As expected, the gene targets were not detectable except that the IAC was positive in control samples.

Significance: The availability and optimization of this method for the simultaneous detection of Salmonella spp., E. coli O157 and Listeria monocytogenes in different cheeses and spinach will allow the FDA and other organizations to take action and prevent spread of an outbreak.

Back to: Poster Session 3 - Applied Laboratory Methods, Novel Laboratory Methods, Produce, Risk Assessment
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