Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC)eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coliO157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed,eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coliO157:H7 when ≥103 CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥104 CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥102 CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥104 CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed,eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.

Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called "non-top-7" have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7-12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle.

A same day protocol for the detection of Escherichia coli O157:H7 by Pathatrix (a novel immuno‐capture method) and Colortrix (a rapid ELISA test) was developed. This study indicated that Pathatrix system, which circulated the pre‐enriched (4.5 h) samples over paramagnet beads with antibodies (0.5 h), increased the concentration of E. coli O157:H7 by 1.2–2.6 log CFU/25 g in 0.5 h from enrichment broth (modified EC broth with novobiocin or buffered peptone water). Colortrix, a rapid ELISA system, was used to detect the pathogens in another 0.25 h. Excellent correlation (100%) between positive Pathatrix/Colortrix (5.25 h system) results and positive Pathatrix/plating method with cefixime tellurite sorbitol‐MacConkey agar, with initial concentration of 0.7 to 2.1 log CFU/25 g was obtained. This new protocol offers a significant benefit to the meat industry allowing compliance with a “test” and “hold” program for E. coli O157: H7 in ground beef.

Two 5’nuclease‐based PCR methods (PCR‐LS‐50B and PCR‐7200) were evaluated to determine their sensitivity for detecting Escherichia coli O157:H7 from pure cultures and in food samples enriched in different media and after different incubation periods. The PCR‐7200 method was able to detect E. coli O157:H7 at ± 102 CFU/mL in pure culture in both mECB and EEB. In spiked meat samples, the PCR‐7200 procedure was capable of detecting the eaeA gene at lower concentrations than the PCR‐LS‐50B procedure, regardless of the meat type or enrichment medium. Escherichia coli O157:H7 spiked at 0.3 CFU/mL was detectable after 9 h in EEB, but it was not detected in mECB within 24 h. An enrichment time of 4 h in mECB was needed to detect E. coli O157:H7 when spiked at higher levels (41 CFU/mL). The detection levels reported in this study are similar with other reported PCR‐based detection techniques for E. coli O157:H7, however, the 5’nuclease‐based assays are less labor intensive and capable of higher sample throughput because of their automated detection and analysis steps.