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United States Department of Agriculture

Agricultural Research Service

Research Project: Genomic and Proteomic Analysis of Foodborne Pathogens

Location: Molecular Characterization of Foodborne Pathogens

Title: Evaluation of a multiplex real-time PCR method for detecting Shiga toxin-producing Escherichia coli in beef and comparison to the FSIS microbiology laboratory guidebook method

Authors
item FRATAMICO, PINA
item Wasilenko, Jamie
item Garman, Bradley -
item Demarco, Daniel -
item Varkey, Stephen -
item Jensen, Mark -
item Rhoden, Kyle -
item Tice, George -

Research conducted cooperatively with:
item

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 23, 2013
Publication Date: October 13, 2013
Repository URL: http://handle.nal.usda.gov/10113/58409
Citation: Fratamico, P.M., Wasilenko, J.L., Garman, B., Demarco, D., Varkey, S., Jensen, M., Rhoden, K., Tice, G. 2013. Evaluation of a multiplex real-time PCR method for detecting Shiga toxin-producing Escherichia coli in beef and comparison to the FSIS microbiology laboratory guidebook method. Journal of Food Protection. 77:180-188.

Interpretive Summary: Food-borne pathogenic bacteria known as non-O157 Shiga toxin-producing Escherichia coli (STEC) belonging to six specific types (also known as serogroups), namely serogroups O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. STEC O157:H7 was classified as an adulterant in beef in 1994 by the Food Safety and Inspection Service (FSIS), and the additional six STEC serogroups were declared as adulterants in 2011. Cattle and other ruminants are reservoirs for these pathogens, thus food of bovine origin may be a vehicle for infection with STEC. Rapid and sensitive methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to ensure that food contaminated with STEC does not reach the consumer. The purpose of this study was to evaluate the performance of polymerase chain reaction-based methods known the BAX System real-time STEC suite and the BAX System real-time PCR assay for E. coli O157:H7 and to compare these methods to the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the pathogens in ground beef and beef trim samples. Generally, results of the BAX System assays were similar to those of the MLG methods for detecting non-O157 STEC and O157:H7; however, the BAX assays were more rapid and easier to perform. This study demonstrates the feasibility of deploying the BAX assays for the detection and monitoring for the six non-O157 STEC serogroups and O157:H7 in beef, and they are very useful for food industry for the detection of STEC. The BAX system approach for non-O157 STEC detection could easily be expanded to include additional assays should regulations continue to expand into other serogroups of public health concern.

Technical Abstract: The “top-six” non-O157 STEC (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of food-borne illnesses have been declared as adulterants in beef by the USDA Food Safety and Inspection Service (FSIS), and regulatory testing for these serogroups in beef began in June, 2012. The purpose of this study was to evaluate the DuPont™ BAX® System method for detecting these top-six STEC and E. coli O157:H7. For STEC, the BAX® System real-time STEC suite was evaluated, including a Screening assay for the stx and eae virulence gene markers and two panel assays to identify the target serogroups (Panel 1 detects O26, O111, O121; Panel 2 detects O45, O103, O145). For O157:H7, the BAX® System real-time PCR assay for E. coli O157:H7 was used. Sensitivity of each assay for the different PCR targets was =1.23 x 103 CFU/mL. Each assay was 100% inclusive for the strains tested (n = 20-50 per assay), and no cross-reactivity with closely related strains in any of the assays was observed. The performance of the BAX® System method was compared to the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the top-six STEC and O157:H7 in ground beef and beef trim. Generally, results of the BAX® System method were similar to those of the MLG methods for detecting non-O157 STEC and O157:H7. Reducing or eliminating novobiocin in mTSB may improve the ability to detect STEC O111 strains, since one O111-inoculated beef trim sample returned a negative result when enriched in mTSB with 8 mg/L novobiocin but was positive when enriched in mTSB without novobiocin. This study demonstrates the feasibility of deploying a panel of real-time PCR assay configurations for the detection and monitoring for the top six STEC and O157:H7 in beef. The approach could easily be expanded to include additional multiplex assays should regulations continue to expand into other O groups or virulence gene markers.

Last Modified: 8/27/2014
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