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 Genes3

Non-O157 Shiga toxin–producing Escherichia coli (STEC) infections, particularly those caused by the ‘‘big six’’ or ‘‘top six’’ non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of 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 U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx1, stx2, and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes 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 with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P , 0.0001) and eae assay (P , 0.0001), thereby 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. Non-O157 Shiga toxin–producing Escherichia coli (STEC) strains are increasingly reported as causing human infections in the United States. In some European countries, non-O157 STEC cause the majority of the STEC-associated illnesses (15, 17). While E. coli O157:H7 appears to still cause many of the STEC-related outbreaks in the United States, it is estimated that non-O157 STEC strains cause more than twice the number of infections overall compared with E. coli O157:H7 (9, 21). The number of illnesses caused by non-O157 serogroups has been compiled, and the Centers for Disease Control and Prevention (CDC) identified the top six most prevalent serogroups associated with illness (O26, O45, O103, O111, O121, and O145) (9). Correspondingly, multiple Shiga toxin subtypes exist for stx1 (stx1a, stx1c, stx1d) and stx2 (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g) associated with STEC. A recent comprehensive study revealed that 24.3% of ground beef samples in the United States screened positive for stx by PCR (8). In addition, the intimin outer membrane protein, encoded by the eae gene, is another important STEC virulence factor and also has multiple variants and subtypes (9, 17). The intimin gene (eae) was identified in 41.5% of the stx screen–positive beef samples in the United States and remains an important virulence factor when considering the development of detection assays (8). Strains that carry the eae and stx2a variant genes are more frequently associated with severe disease and hemolytic-uremic syndrome; however, STEC strains carrying other stx variants have also been associated with human illness (25). The sequences of the O-antigen gene clusters of the top six non-O157 STEC strains have been determined, and highly variable regions of genes, including the wzx gene (encodes for the O-antigen flippase) have been used to * Author for correspondence. Tel: 215-233-6525; Fax: 215-233-6581; E-mail: [email protected]. { Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 1939 Journal of Food Protection, Vol. 75, No. 11, 2012, Pages 1939–1950 doi:10.4315/0362-028X.JFP-12-087