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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION OF PATHOGENS AND THEIR RESPONSES TO ENVIRONMENTAL FACTORS Title: Multiplex real-time PCR assay for the specific detection and quantification of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in ground pork samples

Authors
item Kawasaki, Susumu -
item Fratamico, Pina
item Horikoshi, Naoko -
item Okada, Yukio -
item Takeshita, Kazuko -
item Sameshima, Takashi -
item Kawamoto, Shinichi -

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 4, 2009
Publication Date: May 31, 2010
Citation: Kawasaki, S., Fratamico, P.M., Horikoshi, N., Okada, Y., Takeshita, K., Sameshima, T., Kawamoto, S. 2010. Multiplex real-time PCR assay for the specific detection and quantification of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in ground pork samples. Foodborne Pathogens and Disease. 7(5):549-554.

Interpretive Summary: Salmonella, Listeria monocytogenes, and Escherichia coli O157:H7 are food borne-pathogens capable of causing serious gastrointestinal illness. These three pathogens can be found in similar types of food, including meat and produce. Rapid detection of these pathogens in food is critical to prevent contaminated food from reaching the consumer. A method known as real-time multiplex polymerase chain reaction (PCR) was developed, which involves simultaneous amplification of unique DNA sequences of each pathogen and detection of the PCR products via fluorescent signals. This technique allowed detection of Salmonella, L. monocytogenes, and E. coli O157:H7 simultaneously within 24 hours when each was present in ground pork at a level of one bacterium per 25 grams of pork. As a result, this multiplex real-time PCR assay will be valuable as a screening method for foods contaminated with these pathogens, and will also be useful for identifying the sources of food-borne outbreaks. .

Technical Abstract: Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 are food borne-pathogens capable of causing serious gastrointestinal illness. We previously described simultaneous detection of these pathogens by multiplex PCR in 44 types of spiked food samples, including meat, produce, fish, and dairy products targeting genes specific for each pathogen.. Based on the previous work, a multiplex real-time PCR assay using fluorescent probes was developed to detect and accurately quantify Salmonella spp. L. monocytogenes, and E. coli O157:H7 in ground pork samples. The detection sensitivity for this method was 2.0 x 10E2 CFU/ml for each pathogen, and the quantification range was 10E2 to 10E2 CFU/ml with a high correlation coefficient (R squared greater than 0.99) and high PCR efficiency (99.2 to 84.2 percent). When this protocol was used for the detection of each of the pathogens in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 24 h. As a result, this multiplex real-time PCR assay will be valuable as a screening method for foods contaminated with these pathogens, and will also be useful for identifying the sources of food-borne outbreaks.

Last Modified: 10/30/2014
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