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P1-56 A Robust Multiplex Real-time PCR Method for Simultaneous Detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes in Fresh Fruits and Vegetables

Monday, August 4, 2014
Exhibit Hall D (Indiana Convention Center)
Venugopal Sathyamoorthy, U.S. Food and Drug Administration, Laurel, MD
Larisa Trach, U.S. Food and Drug Administration, Laurel, MD
Yiping He, U.S. Department of Agriculture-ARS-ERRC, Wyndmoor, PA
Ben Tall, U.S. Food and Drug Administration, Laurel, MD
Hannah Chase, U.S. Food and Drug Administration, Laurel, MD
Seongeun Hwang, U.S. Food and Drug Administration, Laurel, MD
Boram Lee, U.S. Food and Drug Administration, Laurel, MD
Barbara McCardell, U.S. Food and Drug Administration, Laurel, MD
Atin Datta, U.S. Food and Drug Administration, Laurel, MD
Introduction: On average, about 48 million people per year in the US are affected by foodborne diseases. A major portion of these 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 from the marketplace, and thereby reducing the incidence of foodborne diseases. At present, there is no good method available for the simultaneous detection of these organisms in foods regulated by FDA.

Purpose: This project aims to optimize, and adapt a multiplex real-time PCR method, originally developed for meats at the USDA, to simultaneously detect the presence of Salmonella spp., E. coli O157 and L. monocytogenes in salads, sprouts, cantaloupes, strawberries, mangoes and peanut butter.

Methods: Food samples were spiked with 5-25 CFU/25g of Salmonella, E. coli O157:H7 and L. monocytogenes stomached and incubated for 2 hours at 37°C in Buffered Listeria Enrichment Broth, followed by the addition of nalidixic acid, fosfomycin, cycloheximide and acriflavine and grown overnight. The enriched culture was centrifuged and the pellet was used for the extraction of genomic DNA (Qiagen). The DNAs were used to carry out the multiplex real-time PCR with primers and TaqMan probes specifically targeting invA (Salmonella), rfbE (E. coli O157), hlyA (L. monocytogenes), and an internal amplification control.

Results: All three gene targets of the tested pathogens were detected in the spiked salads, sprouts, cantaloupes, strawberries, mangoes, and peanut butter, with a sensitivity of 5-25 CFU/25g. The gene targets were not detectable in the non-spiked controls. Extraction of template DNA from peanut butter was time consuming because of its viscosity.

Significance: This method can be applied to detecting pathogens from the food matrices involved in outbreaks, thus allowing the FDA to take swift action and prevent the spread of an outbreak.

Back to: Poster Session - Applied Laboratory Methods, Produce, Low-water Activity, Beverages and Water
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