ARS | Publication request: Detection of non-O157 STEC in ground beef using the GeneDisc real-time PCR system

Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 15, 2012
Publication Date: December 15, 2012
Citation: Fratamico, P.M., Bagi, L.K. 2012. Detection of non-O157 STEC in ground beef using the GeneDisc real-time PCR system. Frontiers in Cellular and Infection Microbiology. DOI: 10.3389/fcimb.2012.00152.

Interpretive Summary: Food-borne pathogenic bacteria known as Shiga toxin-producing Escherichia coli (STEC) belonging to nine specific types (also known as serogroups), namely serogroups O26, O45, O91, O103, O111, O113, O121, O145, and O157:H7 are responsible for the majority of the STEC infections in the United States and worldwide, representing a major public health concern. Cattle and other ruminants are reservoirs for these pathogens, thus food of bovine origin may be a vehicle for infection with STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop control strategies. This study describes a method for detection of these pathogens in ground beef, which consists of using a kit referred to as the GeneDisc system. This method is based on the use of polymerase chain reaction assays targeting genes that are specific for each of the STEC serogroups, thus allowing rapid detection and identification. The STEC strains were also isolated from the ground beef enrichments using an agar medium known as Rainbow Agar O157. The pathogens were detected when present in low levels in ground beef, and were able to be readily isolated and confirmed. This work provides a rapid and reliable method that can be used by regulatory agencies and the food industry for detection in food and for isolation of STEC serogroups that are important public health threats.

Technical Abstract: Certain non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups have emerged as important public health threats. The development of methods for rapid and reliable detection of this heterogeneous group of pathogens has been challenging. A GeneDisc real-time PCR assay was evaluated for detection of the stx1, stx2, eae, and ehx genes and a gene that identifies the O157 serogroup, followed by a second GeneDisc assay targeting serogroup-specific genes of STEC O26, O45, O91, O103, O111, O113, O121, O145, and O157. The ability to detect the non-O157 STEC serogroups in ground beef samples artificially inoculated at a level of ca. 2-20 CFU/25 g and subjected to enrichment in mTSB or BPW was similar. All samples were detected following enrichment, and results showed amplification of the correct set of genes for each strain. Samples inoculated with STEC serogroups O26, O45, O103, O111, O121, O145, and O157 were subjected to immunomagnetic separation, and isolation was achieved by plating onto Rainbow agar O157. Colonies were confirmed by PCR assays targeting stx1, stx2, eae, and serogroup-specific genes. Thus, this work demonstrated that the GeneDisc assays are rapid, sensitive, and reliable and can be used for screening ground beef and potentially other foods for non-O157 STEC serogroups that are important food-borne pathogens worldwide.