Title: Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes Authors
Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 6, 2012
Publication Date: November 1, 2012
Citation: Wasilenko, J.L., Fratamico, P.M., Narang, N., Tillman, G.E., Ladely, S.R., Cray Jr, W.C. 2012. Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes. Journal of Food Protection. 75(11):1939-50. Interpretive Summary: Food-borne bacterial pathogens known as Shiga toxin-producing Escherichia coli (STEC) cause severe bloody diarrhea, and infection can lead to a disease called hemolytic uremic syndrome or death. Due to the significant public health impact of certain STEC that are known as the top six serogroups and the notable prevalence of STEC in cattle, the Food Safety and Inspection Service recently declared these pathogens as adulterants in beef. Methods for detection of these pathogens in beef have been established; however, there was a need for modifications to make the methods more robust and to have the ability to detect the widest range of STEC. Thus, polymerase chain reaction (PCR)-based assays were designed to detect most of the Shiga toxin genes and the genes that encode for all of the subtypes of the intimin protein (known as eae), which are important STEC virulence genes. A new method to extract the DNA from ground beef enrichment samples was also designed, which is more cost effective and improves the sensitivity of the PCR. Finally, the PCR assays were adapted for use on a new detection platform, which provides a more sensitive and high-throughput system. The improved method allows for more sensitive and reliable detection of the top six STEC serogroups in beef, and can be used by the FSIS and the food industry to ensure that beef contaminated with these pathogens does not reach the consumer.
Technical Abstract: Non-O157 Shiga toxin-producing E. coli (STEC) infections, particularly those caused by the “big six”/”top six” non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Due to their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the USDA FSIS detection methods for screening beef samples for non-O157 STEC target the stx1, stx2, and eae virulence genes, and the 16S rRNA gene as the internal control in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting the wzx gene in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx1d, stx2e, and stx2g, are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared to a previously utilized procedure that was based on a commercially available reagent. The extraction method had a significant effect on the cycle threshold values for the assay, therefore increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx1d, stx2e, and stx2g, and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.