MOLECULAR CHARACTERIZATION OF PATHOGENS AND THEIR RESPONSES TO ENVIRONMENTAL FACTORS
Location: Molecular Characterization of Foodborne Pathogens
Title: Detection of E. coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx1, stx2, wzy, and the fliCh7 or eae Genes
Submitted to: Journal of Food Analytical Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 24, 2010
Publication Date: April 23, 2010
Citation: Fratamico, P.M., Debroy, C. 2010. Detection of E. coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx1, stx2, wzy, and the fliCh7 or eae Genes. Journal of Food Analytical Methods. 3:330-337.
Interpretive Summary: The bacterium known as enterohemorrhagic E. coli O157:H7 is an important cause of food-borne illness. Drinking water, produce, and food of bovine origin, particularly ground beef, have been linked to outbreaks. Therefore, rapid and reliable methods for detection of this bacterium are needed by the food industry and regulatory agencies to prevent food contaminated with E. coli O157:H7 from reaching the consumer. A detection method involving a technique known as real-time multiplex polymerase chain reaction was developed to detect E. coli O157:H7 in ground beef, lettuce, raw milk, and apple cider. The technique involves the amplification of specific segments of genes found specifically in E. coli O157:H7. With this method, E. coli O157:H7 was detected in these foods within 24 h and when the contamination level of the pathogen was less than or equal to 2 bacteria in 25 g or 25 ml of food. Use of this assay can potentially permit the food industry and regulatory agencies to more readily evaluate foods and other types of samples for the presence of E. coli O157:H7, thus enhancing the safety of the food supply.
E. coli O157:H7 is an important food-borne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml), and ground beef and lettuce (25 g) were inoculated with 2 or 20 CFU of E. coli O157:H7 380-94 (possesses the stx1and stx2 genes) in 225 ml of RapidCheck E. coli O157:H7 Enrichment Broth and incubated at 42 degrees 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 per PCR. E. coli O157:H7 was detected in apple cider, raw milk, lettuce, and ground beef samples inoculated with 2 or 20 CFU per milliliter or gram after both 8 and 20 hours 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 target gene combination, a positive result was always obtained for the fliCh7 gene using ground beef enrichments. Therefore, the 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 food.