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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION OF PATHOGENS AND THEIR RESPONSES TO ENVIRONMENTAL FACTORS

Location: Molecular Characterization of Foodborne Pathogens

Title: Detection of Escherichia coli O157:H7 in food using a real-time multiplex PCR assay

Authors
item Fratamico, Pina
item Debroy, Chobi - PENNA STATE

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: January 10, 2007
Publication Date: July 8, 2007
Citation: Fratamico, P.M., Debroy, C. 2007. Detection of Escherichia coli O157:H7 in food using a real-time multiplex PCR assay. Abstract. Internatinal Association for Food Protection. P3-69.

Technical Abstract: E. coli O157:H7 is an important food-borne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Sensitive and rapid methods for detection of the pathogen are needed. The purpose of this study was to develop real-time multiplex PCR assays that can be used to detect E. coli O157:H7 in different foods. Samples of apple cider and raw milk (25 ml), and of ground beef and lettuce (25 g) were inoculated with 2 or 20 CFU of E. coli O157:H7 strain 380-94 (possesses the stx1and stx2 genes) in 225 ml of Rapid-Check E. coli O157:H7 Enrichment Broth and incubated at 42 degree C at 100 rpm. One milliliter of the enrichments was removed at 8 and 20 h, and DNA extraction was performed using the PrepMan Ultra reagent. Multiplex PCR assays using TaqMan probes and primers targeting the stx1, stx2, wzyO157 genes in combination with probes and primers targeting either the fliCh7 or the eae genes were performed using Omnimix tablets and the Smart Cycler. The sensitivity of the real-time multiplex PCR assay was 500 CFU/PCR. E. coli O157:H7 was detected in apple cider, raw milk, lettuce, and ground beef samples inoculated with both 2 and 20 CFU/ml or g after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx1, stx2, wzyO157, and eae genes; however, using the assay targeting the stx1, stx2, wzyO157, and fliCh7 gene combination, a positive result was always obtained for the fliCh7 gene using ground beef enrichments. Other primer sets targeting the fliCh7 gene gave similar results; therefore, fliCh7 gene may not be a suitable target for detection of E. coli O157:H7 in ground beef. The real-time multiplex PCR assay targeting the stx1, stx2, eae, and wzyO157 genes is sensitive and specific and can be used for detection of E. coli O157:H7 in apple cider, raw milk, lettuce, and ground beef.

Last Modified: 7/31/2014
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