1828 Gao et al.: Journal of aoaC InternatIonal Vol. 101, no. 6, 2018 Background: Ready-to-eat (RTE) meats, fruits, and vegetables contaminated by Shiga toxin producing Escherichia coli (STEC) raise serious concerns because they are often consumed directly without further processing. Objective: To evaluate a multiplex PCR for the detection of STEC across food categories. Methods: Samples (25 g) from seven RTE meat and nine fruit and vegetable matrices were inoculated with each of seven STEC (O157:H7, O26, O121, O145, O45, O103, O111) strains targeting 10 CFU/25 g, enriched in 225 mL of modifed tryptone soya broth (mTSB), and tested by a multiplex real-time PCR for stx and eae genes, following U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5B, which was originally validated for meat products and environmental sponge. Results: The mTSB was successful at enriching for STEC in RTE meat, fruit, and vegetable matrices, except for sprouts; however, mEHEC resulted in successful enrichment of target organisms in mung bean sprouts. Suppression of eae results by stx in PCR was observed in six fruit and vegetable matrices. Conversely, suppression of stx gene by eae was not observed. PCR solely targeting eae is recommended if a fruit or vegetable sample tested positive for stx and negative for eae. Despite the signifcant effect from food matrix, strain, and experimental batch, the cycle threshold of PCR was <30 in inoculated samples, and mostly 30–42 and up in uninoculated samples. Conclusions: The multiplex PCR can be adopted for detection of all seven regulated STEC in RTE meat, fruit and vegetable matrices after validation with cut off value selected and justifed based on real samples. C ontamination from Shiga toxin producing Escherichia coli (STEC) is a signifcant and highly relevant concern in food safety. The development of methods for the detection of STEC has been the topic of signifcant interest in Food BioLoGicaL conTaMinanTS Received January 9, 2018. Accepted by AH March 9, 2018. This research is funded by Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) via University of Guelph–OMAFRA Partnership. Corresponding author’s e-mail: agao@uoguelph.ca Evaluation of a Multiplex PCR for Detection of the Top Seven Shiga Toxin-Producing Escherichia coli Serogroups in Ready-to-Eat Meats, Fruits, and Vegetables Anli GAo, Jennifer fischer-Jenssen, colin cooper, honGhonG li, JipinG li, shu chen, and perry MArtos University of Guelph, Laboratory Services Division, 95 Stone Rd W, Guelph, ON N1H8J7 Canada DOI: https://doi.org/10.5740/jaoacint.18-0010 recent decades, especially as STEC serotype, O157:H7, has become recognized as a food-borne pathogen (1). Additionally, as non-O157 strains are increasingly detected as important food-borne pathogens in outbreaks, six other STEC serogroups (O26, O45, O103, O111, O121, and O145) are now being regulated by the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) in raw beef products (2). A number of methods have been developed and evaluated by research groups, such as for the detection of STEC O104 in sprouts (3); O157:H7 and non-O157 in leafy green vegetables, cilantro, sprouts and milk (4, 5); O157 in various food samples (6); O157:H7 in alfalfa sprouts (7); non-O157 in pure culture and food (8), and the USDA Microbiology Laboratory Guidebook (MLG) 5B method for raw meat (9). However, despite these efforts, information on the evaluation of methods for the detection of these seven STEC serogroups in different food categories remains limited (10). To our knowledge, methods for the detection of both O157 and non-O157 STEC across broad food categories have not been reported. Fruits, vegetables, and ready-to-eat (RTE) meats contaminated by STEC raise a more serious concern to public health, unlike raw beef, which is cooked prior to consumption. The objective of this study was to develop a screening method for the detection of these STEC serogroups in fruits, vegetables, and RTE meats prior to bacterial culture. This work presents an adopted multiplex real-time PCR screening method that can be used to detect all seven regulated STEC serogroups across different food categories. Materials and Methods Experimental Design Seven RTE meats (cooked chicken, cooked deli, cooked ham, cooked sausage, dry cured ham, salami, and sausage) and nine fruit and vegetable (apple cider, broccoli, carrot, lettuce, raspberry, spinach, mung bean sprouts, strawberry, and tomato) samples were inoculated with each of the seven STEC strains (Table 1) in a factorial design. The experiments were carried out in eight batches. Each batch consisted of one blank control sample and two inoculated replicate samples from each of the 16 food matrices, with only one STEC strain employed for artifcial inoculation. Seven batches were conducted to cover each of the seven STEC strains. An additional batch of mung bean sprout samples was inoculated with the seven STEC strains individually and enriched in a proprietary media mEHEC (BioControl) when it was observed that modifed tryptone soya broth (mTSB; Oxoid) did not provide successful enrichment of