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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 multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes in soft cheese

Authors
item Sathyamoorthy, V -
item Datta, A -
item Lee, C -
item He, Yiping
item Sadowski, J -
item Tall, B -
item Mccardell, B -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 22, 2012
Publication Date: N/A

Technical Abstract: Introduction: Illnesses due to the ingestion of bacterial pathogens in contaminated food cause enormous cost to our nation both medically and economically. Hence, it is important to detect the specific pathogens in contaminated foods that cause various diseases such as gastroenteritis, listeriosis, and hemolytic uremic syndrome etc. Availability of a rapid, accurate, and sensitive method to detect these pathogens will immensely help FDA to carry out the regulatory mission in a time sensitive fashion. In this context, there is a need for rapid and simultaneous detection of Salmonella spp, Escherichia coli O157:H7, and Listeria monocytogenes in foods. PCR based methods for E.coli, Salmonella, and Listeria monocytogenes have been reported. However,use of an universal selective enrichment medium for simultaneous enrichment and detection of these pathogens from various foods poses challenges. Purpose: To develop a multiplex real-time PCR method for simultaneous detection of Salmonella spp., E. coli O157 and Listeria monocytogenes in soft cheese. Methods: Soft cheese (Brie) was spiked with ~3,300 CFU/ 25 g of Salmonella, E.coli O157:H7 and L. monocytogenes, in a previously described selective medium, stomached, and incubated for 2 hours at 37 degrees C followed by the addition of nalidixic acid, fosfomycin, cycloheximide, and acriflavine and grown overnight. The culture was then centrifuged; the pellet was resuspended, boiled for 15 minutes, and sonicated. The sonicated sample was centrifuged and the supernatant was used as a template to carry out the multiplex qPCR with primers and probes targeting invA (Salmonella), rfbE (E.coli O157), and hlyA (L. monocytogenes) Results: All three pathogens could be detected at a level ~3,300 CFU/25 g of cheese after the enrichment or ~33,000 CFU/ml of the inoculum. The ratio of individual concentrations of the four antibiotics used had an effect on the sensitivity of the detection of each pathogen. Significance: The data suggests that all three pathogens can be simultaneously detected in cheese. This method has the potential to be widely used to simultaneously screen for the presence of Salmonella, E. coli O157, and L. monocytogens in soft cheese and may be extended to other commodities.

Last Modified: 12/19/2014
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