Monday, July 23, 2012
Exhibit Hall (Rhode Island Convention Center)
Venugopal Sathyamoorthy, U.S. Food and Drug Administration-CFSAN, Laurel, MD
Atin Datta, U.S. Food and Drug Administration-CFSAN, Laurel, MD
Chloe Lee, U.S. Food and Drug Administration-CFSAN, Laurel , MD
Yiping He, U.S. Department of Agriculture-ARS-ERRC, Wyndmoor, PA
Jennifer Sadowski, U.S. Food and Drug Administration, Laurel, MD
Ben Tall, U.S. Food and Drug Administration-CFSAN, Laurel, MD
Barbara McCardell, U.S. Food and Drug Administration-CFSAN, Laurel, MD
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 diseases such as gastroenteritis, listeriosis, hemolytic uremic syndrome, etc. Availability of a rapid, accurate and sensitive method to detect these pathogens will immensely help FDA to carry out its regulatory mission in a time-sensitive fashion. In this context, there is a need for a rapid and simultaneous detection assay for
Salmonella spp,
Escherichia coli O157:H7 and
Listeria monocytogenes in foods
. PCR-based methods for
E. coli,
Salmonella and
Listeria monocytogeneshave been reported. However, a universal selective enrichment medium for simultaneous enrichment and detection of these pathogens from various foods pose challenges.
Purpose: To develop a multiplex real-time PCR method for simultaneous detection of Salmonella spp., E. coli O157 and Listeria monocytogenesin soft cheese.
Methods: Soft cheese (Brie) in a previously described selective medium was spiked with ~330 CFU/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 grown overnight. The culture was then centrifuged; the pellet was resuspended in 0.25 ml of buffer, 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 taqMan probes targeting invA (Salmonella), rfbE (E. coli O157) and hlyA (L. monocytogenes).
Results: After enrichment, all three gene targets of the pathogens have been detected in the cheese spiked with ~330 CFU/g. The ratio of individual concentration of the four antibiotics used had an effect on the sensitivity of the detection of each pathogen.
Significance: The data suggest 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. monocytogenes in soft cheese and may be extended to other commodities.
