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United States Department of Agriculture

Agricultural Research Service

Research Project: DETECTION AND TYPING OF FOOD-BORNE PATHOGENS

Location: Molecular Characterization of Foodborne Pathogens

Title: A robust multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes in fresh fruits and vegetables

Authors
item Sathyamoorthy, Venugopal -
item Trach, Larisa -
item He, Yiping
item Tall, Ben -
item Chase, Hannah -
item Hwang, Seongeun -
item Lee, Boram -
item Mccardell, Barbara -
item Datta, Atin -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 25, 2014
Publication Date: N/A

Technical Abstract: Introduction: On average, about 48 million people per year in the U.S. are affected by food borne 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 the specific pathogens in contaminated foods so that they can be eliminated from the circulation, and thereby reducing the incidence of food borne 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 evaluate, 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 degrees C in a previously described medium (BLEB), 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 more time consuming because of its viscosity. Significance: The feasibility of applying this method to outbreak food matrices will allow the FDA and other organizations to take swift action and prevent the spread of an outbreak.

Last Modified: 7/22/2014
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