Abstract In addition to continued testing for E. coli O157:H7, new regulations in the United States require industry to begin monitoring for six non-O157 Shiga toxin-producing E. coli (STEC) serogroups, referred to as the “top-six” non-O157 STEC (O26, O45, O103, O111, O121, and O145), as these have been most frequently associated with outbreaks and cases of food- borne illnesses in the U.S. These top six serogroups were recently declared as adulterants in beef by the USDA Food Safety and Inspection Service (FSIS), and testing for these serogroups in beef will begin in June, The purpose of this study was to conduct evaluations of three separate multiplex Scorpion probe-based PCR assays in a tableted format for the detection of the top-six STEC serogroups. The three multiplex assay configurations were as follows: Assay 1 – O26, O111, O121, and internal positive control (IPC); Assay 2 – O45, O103, O145, IPC; Assay 3 – eae, stx1/2, IPC. Studies evaluating the sensitivity of each of the real-time assays were conducted using titrations of cell lysates. Cell lysates were also used to conduct inclusivity and exclusivity studies for each assay configuration. Sensitivity of each assay with cell titers in tryptic soy broth for each of the various PCR targets was ≤ 1.0 X 10 4 CFU/mL. Each assay was 100% inclusive for the strains tested (n = per assay). Moreover, no cross- reactivity with closely related strains in any of the assays was observed. These results demonstrate the feasibility of deploying a panel of three novel real-time PCR assay configurations for the detection and monitoring of STEC O groups, as well as the virulence genes, eae, stx 1, and stx 2. The approach demonstrated could easily be expanded to include additional multiplex assays should regulations continue to expand into other O groups or virulence gene markers. Finally, the performance of the detection method based on use of the Scorpion probe-based multiplex PCR assays was compared to the FSIS Microbiology Laboratory Guidebook (MLG 5B.01) method for detection of the top-six STEC in beef. The performance of the multiplex Scorpion probe-based PCR assays (BAX® System) was similar to that of the USDA FSIS method for detecting non-O157 STEC in ground beef and beef trim. Development and Evaluation of Three Scorpion Probe-Based Multiplex Real- time PCR Assays for the Detection of Shiga Toxin-Producing Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 Pina M. Fratamico 1, Jamie L. Wasilenko 2, Daniel R. DeMarco 3, Stephen Varkey 3, Mark Jensen 3, Kyle Rhoden 3, and George Tice 3 1 USDA, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania, U.S.A.; 2 USDA, Agricultural Research Service, Richard Russell Research Center, Athens, Georgia, U.S.A. 3 DuPont Qualicon E400, PO Box 80400, Wilmington, Delaware, U.S.A. Materials and Methods Strain Selection and Preparation The strains used in this study were obtained from a variety of sources, including the DuPont Qualicon culture collection, Michigan State University (Lansing, MI), USDA-ARS-ERRC (Wyndmoor, PA), USDA-Agricultural Marketing Service (AMS; Manassas, VA), and USDA-ARS-NPA MARC (Clay Center, NE). Each strain was grown in BHI at 37ºC overnight (about h) for testing with the BAX® System method as described below. For inclusivity and exclusivity testing, strains were tested at the USDA-ARS-ERRC and at DuPont Qualicon.Sensitivity Each overnight culture was diluted in TSB, and then 20 µL of the dilutions were transferred to a cluster tube with 200 µL prepared BAX® System lysis reagent (lysis buffer + protease). Lysis and protease inactivation were performed by heating samples at 37ºC for 20 min and 95ºC for 10 min, and then cooling at 4ºC for 5 min. PCR tablets were hydrated with 30 µL lysate and processed with the appropriate PCR assay. Positive/negative calls for the real-time PCR assay were made by visual observation of amplification curves. Inclusivity and Exclusivity For inclusivity testing, overnight cultures were diluted 1:10 in BHI, and then diluted to ~10 5 CFU/mL in TSB before transferring to tubes with prepared BAX® System lysis reagent. For exclusivity testing, overnight cultures were diluted 1:10 in BHI before transferring. Lysis, protease inactivation, and sample processing were performed for all inclusivity and exclusivity samples as described above. Positive/negative calls for the real time PCR assay were made by visual observation of amplification curves. Sample inoculation and preparation STEC top-six serogroup strains and E. coli O157:H7 were selected for these studies. An isolated colony of each strain was suspended in 8 mL of brain heart infusion (BHI) broth, incubated at 37˚C for h, and diluted in saline solution for inoculating samples. Ground beef and beef trim were divided into 325-g test portions, and 1 mL of the diluted test strain was added to each sample. Uninoculated samples served as negative controls. Samples were held at 4˚C for 2-3 days to stress the inoculum before enrichment. Sample enrichment Ground beef samples were homogenized with 1.5 L pre-warmed (42˚C) TSB with 2 mg/L novobiocin and incubated at 42˚C for 18 h before testing with the BAX® System. Trim samples were combined with 1.5 L pre-warmed (42˚C) TSB (without novobiocin) and incubated at 42˚C for 18 h before testing with the BAX® System. For the USDA method, all samples were homogenized with 975 mL room- temperature mTSB with 8 mg/L novobiocin, bile salts, and casamino acids, and then incubated at 42˚C for 18 h before testing with both the USDA and BAX® System methods. BAX ® System testing method Twenty microliters of enrichment were added to 200 µL prepared lysis reagent (150 µL protease and 12 mL lysis buffer). Lysis was performed by heating for 20 min at 37˚C and 10 min at 95˚C, then at 4˚C for at least 5 min. Thirty microliters of each lysate were used to hydrate a PCR tablet for the STEC Screening, STEC Panel 1, STEC Panel 2, and real-time E. coli O157:H7 assays. All PCR tubes were loaded into the BAX® System Q7 instrument. All samples were culture confirmed using the USDA confirmation method described below. USDA FSIS testing method DNA extraction was performed according to the FSIS MLG procedure 5B.01 (step 5B.6.1). PCR master mix was prepared as described in the MLG procedure 5B Appendix 1, then 20 µL master mix was mixed with 5 µL extracted DNA in the wells of the plate. The plate was centrifuged briefly, and then loaded into a real-time PCR cycler and processed to screen for stx and eae. Samples that returned positive results for both stx and eae were further processed using three multiplex real-time PCR reactions to identify the six target STEC serogroups, as described in MLG 5B.01. USDA FSIS confirmation method (all samples) Each sample enrichment was filtered using a sterile 40 µm cell strainer, and then 1 mL of the filtered enrichment was added to 20 µL O-specific immunomagnetic capture beads in a microcentrifuge tube. Enrichments were processed according to the protocol described in FSIS MLG procedure 5B.01. Discussion and Conclusions The results of these studies demonstrate that:  BAX® System performance was equivalent to or better than the USDA FSIS method for detecting non-O157 STEC in ground beef and beef trim.  The BAX® System accurately detected and identified the “top six” non-O157 STEC strains.  There was good sensitivity and inclusivity from the assays, as well as full exclusivity. P-058 Results The results for the first round of ground beef testing are summarized in Table1. All samples tested with the BAX® System returned the appropriate positive results for each assay. Three samples tested - those spiked with E. coli O111, O121 and O145 – also returned positive results that identified the presence E. coli O103. These samples were found to be naturally contaminated with an E. coli O103 strain, which caused the samples to return negative results during the pre-screen for virulence factors but positive results for the specific O group during the study. All BAX® System results correlated 100% with the results of the USDA PCR method. The results for the second and third rounds of ground beef testing are summarized in Tables 2 and 3. All samples tested with the BAX® System returned positive results for the Screening assay for stx and eae, and the Panel 1 and Panel 2 assays correctly identified each of the “top six” strains. All BAX® System results correlated 100% with the results of the USDA PCR method. The results for the first round of beef trim testing are summarized in Table 4. All samples tested with the BAX® System returned the appropriate positive results for each assay, and all BAX® System results correlated 100% with the results of the USDA PCR method. E. coli O45 was not tested is this round due to mislabeling of the strain selected for spiking, which caused samples to be spiked with E. coli O103 instead of E. coli O45. Because of this spiking error, these samples were removed from the study results. The results for the second round of beef trim testing are summarized in Table 5. All samples tested with the BAX® System returned positive results for both the Screening assay for stx and eae, and the Panel 1 and Panel 2 assays correctly identified each of the “top six” strains. In this round of testing, beef trim samples spiked with E. coli O111 and enriched with the USDA FSIS method failed to return positive results or culture confirm, while samples enriched with the BAX® System method were correctly detected. It is possible that these discrepant results were caused by the higher concentration of novobiocin in the reference method enrichment medium (8 mg/L compared to no novobiocin), which may have inhibited the growth of the target E. coli strain during enrichment. Table 6 shows the sensitivity of each assay with cell titers in TSB for each of the various PCR targets. These results demonstrate that for all organisms tested, the lower limit of detection was less than or equal to 1.2 X 10 3 CFU/mL. Table 7 shows a summary of the results from inclusivity testing. All of the strains tested were detected by the appropriate panel or screening assay. Table 8 shows a summary of the results from exclusivity testing. Neither of the panel assays nor the screening assay indicated any cross reactivity with the organisms tested. United States Department of Agriculture Agricultural Research Service