Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: DETECTION AND TYPING OF FOOD-BORNE PATHOGENS

Location: Molecular Characterization of Foodborne Pathogens

Title: 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

Authors
item Sathyamoorthy, Venugopal -
item Datta, Atin -
item Trach, Larisa -
item HE, YIPING
item Tall, Ben -
item Mccardell, Barbara -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 29, 2013
Publication Date: N/A

Technical Abstract: Introduction: Foodborne diseases affect about 48 million people per year in US. 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 from the circulation, thereby reducing the incidence of food borne 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/25g of Salmonella, E.coli O157:H7 and L. monocytogenes, stomached, and incubated for 2 h 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 in control samples, except that the IAC was positive. 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.

Last Modified: 8/27/2014
Footer Content Back to Top of Page