3M Molecular Detection Assay E. coli O157 (including H7) Performance Summary

3M Food Safety Technical Bulletin Number: TB.171058.02 Effective Date: Feb 15, 2012 Supersedes: TB.171058.01 Technology Platform: Pathogens Originating Location: St. Paul, MN 3M Molecular Detection Assay E. coli O157 (including H7) Performance Summary Testing for E. coli O157:H7 is a critical component of food safety programs as infection by this pathogen can result in significant adverse health conditions and economical losses. Faster and more accurate testing methods are required versus traditional microbiology methods which are cumbersome and take more than four days to get results. The 3M Molecular Detection Assay E. coli O157 (including H7) was developed for the rapid and specific detection of this adulterant in food samples after enrichment in 3M Buffered Peptone Water ISO (BPW ISO). Performance of the 3M method was evaluated for selectivity, with a range of food matrices, non-food samples and compared to the reference methods. The limit of detection of the method was also determined. Method Selectivity Inclusivity and Exclusivity Testing Inclusivity is defined as the ability of a method to detect the target analyte from a wide range of strains. Exclusivity is defined as the lack of interference from a relevant range of non-target strains. Pure bacterial cultures were derived from purchased lyophilized preparations or from frozen stock cultures or identified by biochemical methods. a. False negative rate Inclusivity Fifty-two (52) E. coli O157 isolates (Appendix 1) were tested. The E. coli O157 strains were cultured overnight then diluted to a level of less than 10 5 CFU/ml (Colony Forming Unit/mL) prior to testing. A false-negative rate of 0 % (100% inclusivity) was determined. b. False positive rate Exclusivity Fifty (50) non- E. coli O157 isolates (Appendix 2) were tested. The non- E. coli O157 strains were cultured overnight to reach a minimum level of 100X the limit of detection. A false-positive rate of 0 % (100% exclusivity) was determined. Food Studies A food study was conducted to assess the performance of the method (including enrichment broth and assay) in the presence of possible interferences from sample matrix and other organisms. Study Design Several serotypes of E. coli O157 were used to artificially contaminate food samples commonly tested and/or reported to be challenging due to their composition, i.e. high fat, high calcium, high native microflora, etc. Thirty three (33) different food matrices were evaluated as follow: either spiked at a level of 5-7 CFU E. coli O157 per 25 g sample size or enriched as blanks (Appendix 3). All enrichments were performed at a 1:10 dilution in pre-warmed 3M BPW ISO and were incubated for 8 hours (raw meat) or 18 hours (all other foods) at 41.5 C. All artificially contaminated samples were tested using both the 3M Molecular Detection Assay E. coli O157 and the 3M Molecular Detection Matrix Control, while enriched blanks were tested using the 3M Molecular Detection Assay Detection Assay E. coli O157 only. All enrichments were also streaked onto BBL CHROMagar E. coli O157plates, used as reference point. TB. 171058.02 Page 1 of 9

The method was shown to be compatible with a variety of relevant food matrices. All results were as expected: negative (blank samples), positive (artificially contaminated at a level of 5-7 CFU) and valid (matrix control). None of the matrices tested demonstrated inhibition of the assay. Carcass Study Beef carcasses were tested at different process steps to evaluate the performance of the method in the presence of possible interference from matrix and/or competing microbiota. Study Design Sixty (60) carcass swab samples were collected by sampling the right and left side of each animal, at similar anatomical locations and paired in one bag. Swabbing was performed at the following process steps per USDA guidelines 1 : pre-hide removal, preevisceration, and post-wash. Sample collection devices and hydrating solution used in this study are summarized below. Sampling Material 3M Cattle/Swine kit, 25 ml BPW Process Steps Prior to hide removal (100cm 2 ) Pre-evisceration (8000 cm 2 ) Post-wash (8000 cm 2 ) Suspensions of E. coli O157:H7were used to artificially contaminate one of the duplicates at a level of 10 CFU and one duplicate was enriched blank. All enrichment were performed at 41.5 C for 18 hours then tested using the 3M Molecular Detection Assay E. coli O157and the 3M Molecular Detection Matrix Control. All enrichments were also streaked onto chromogenic BBL CHROMagar E. coli O157 agar plates, used as reference point. A Chi-square (X 2 ) test was used to compare the results for significant differences. Target n Accuracy % Specificity % Sensitivity % X 2 E. coli O157:H7 60 95 100 92 1.33 Accuracy, specificity and sensitivity of the 3M method were calculated according to the statistical formula in Appendix 4. No significant differences were observed between the 3M method results and the chromogenic agar results as indicated by a X 2 value of less than 3.84.The method was shown to be compatible with carcass swabs samples, including fecal containing samples. The 3M Molecular Detection Matrix Control yielded valid results for all samples tested. Method Comparison A study was performed with contaminated samples to obtain fractionally positive results, those in which at least one of the methods (candidate or reference) yields 5-15 positive results out of 20 replicates examined for the low level of inoculation, for each matrix tested. The 3M Molecular Detection Assay E. coli O157 method was compared to the USDA FSIS MLG Chapter 5.05 2 Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products reference method for raw ground beef and the US FDA-BAM Chapter 4A Diarrheagenic Escherichia coli 3, Section N reference method for sprouts and spinach. Twenty (20) replicates of each matrix were analyzed at a level of 2-5 CFU per test potion. Five (5) control replicates were also analyzed at 0 CFU/test portion (uninoculated). Samples were conditioned to mimic natural contamination. Two time points, 8 hours and 18 hours, were analyzed for raw ground beef and sprouts. TB. 171058.02 Page 2 of 9

Sample Contamination level, MPN per sample size(g) Reference Method Χ 2 8 hours Χ 2 18 hours Raw ground beef 3.75 / 325 USDA MLG 5.05 0 0.76 Sprout 2.33 / 25 FDA-BAM 4A 0.61 1.52 Spinach 3/ 200 FDA-BAM 4A Not Tested 2.01 Results using the 3M Molecular Detection E. coli O157 method at either 8 or 18 h enrichment [matrix dependant] were found to be equal to or better than the reference method results in relevant matrices, as indicated by a Chi Square (X 2 ) value of less than 3.84. Limit of Detection (LOD) The limit of detection of a method is defined as the lowest concentration point where reliable analytical results can be obtained. This may vary with different serotypes and methods. For the 3M Molecular Detection Assay E. coli O157 this has been demonstrated to be 1-5 CFU/ sample size. The LOD of a qualitative assay is often determined following enrichment such that at least 1 CFU in the sample size has sufficiently multiplied and reached a level to be detected. Pathogen detection technologies have varying inherent LOD. For example, LODs for immunoassays are generally 10 5-10 6 CFU/mL whereas PCR LODs can be less than or equal to 10 4 CFU/mL of target pathogen in the enrichment 4, 5. LOD may also vary by food matrix, bacteria in the sample and the comparison method. One way to measure the LOD is to perform an extensive comparison study against a reference method using multiple food matrices and multiple organisms. The LOD will depend heavily on all variables within the study, such as: the food matrix, the bacteria in the sample and the comparison reference method. Another way to measure LOD is to test suspensions of pure target pathogen and of known concentrations, and then test the assay for its ability to detect the target. 3M internal studies have shown that the average LOD of the 3M Molecular Detection Assay E. coli O157 is approximately 1.8 x 10 4 CFU/mL in 3M BPW ISO. References: 1. US Department of Agriculture (USDA) Incident Investigation Team Methodology for Escherichia coli (E. coli) O157:H7 in Beef Slaughter Establishments. On-line http://www.fsis.usda.gov/pdf/iit_methodology_for_ecoli.pdf 2. US Department of Agriculture (USDA) Microbiology Laboratory Guidebook on-line 2011. MLG 5.05 Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products 3. US Food and Drug Administration Bacteriological Analysis Manual on-line 2011. Chapter 4A Diarrheagenic Escherichia coli. 4. van der Z, Huis in t Veld JH. 1997. Rapid and Alternative Screening Methods for Microbiological Analysis. Journal of AOAC International. 80(4): 934-940. 5. Mandal PK, Biswas AK, Choi K, Pal UK. 2011. Methods for Rapid Detection of Foodborne Pathogens: An Overview. American Journal of Food Technology. 6 (2): 81-102. Trademarks: BBL is a trademark of Becton, Dickinson and Company. CHROMagar is a trademark of Dr. A. Rambach. TB. 171058.02 Page 3 of 9

Appendix 1: E. coli O157 inclusivity test strains Genus, species Serotype ID Escherichia coli O157 TICC 4289, FSD 944 Escherichia coli O157 TICC 2850, FSD 756 Escherichia coli O157 TICC 2860, FSD 759 Escherichia coli O157 TICC 2861, FSD 760 Escherichia coli O157 TICC 2862, FSD 761 Escherichia coli O157 TICC 2863, FSD 762 Escherichia coli O157:H- TICC 3042, FSD 808 Escherichia coli O157:H- TICC 2373, FSD 724 Escherichia coli O157:H- TICC 2374, FSD 725 Escherichia coli O157:H- TICC 2375, FSD 726 Escherichia coli O157:H- TICC 2376, FSD 727 Escherichia coli O157:H- TICC 2377, FSD 728 Escherichia coli O157:H- TICC 2378, FSD 729 Escherichia coli O157:H- TICC 2379, FSD 730 Escherichia coli O157:H- TICC 2384, FSD 735 Escherichia coli O157:H- TICC 2380, FSD 731 Escherichia coli O157:H7 NCTC 12900, ATCC 700728 Escherichia coli O157:H7 TICC 2382, FSD 733 Escherichia coli O157:H7 TICC 2383, FSD 734 Escherichia coli O157:H7 TICC 2385, FSD 736 Escherichia coli O157:H7 ATCC 20644 Escherichia coli O157:H7 ATCC 43894 Escherichia coli O157:H7 ATCC 35150 Escherichia coli O157:H7 RC43R Escherichia coli O157:H7 K20 Escherichia coli O157:H7 ATCC BAA-1882 Escherichia coli O157:H7 ATCC BAA-460 Escherichia coli O157:N PSU 93.0134 Escherichia coli O157:N PSU 93.0135 Escherichia coli O157:N PSU 93.0136 Escherichia coli O157:N PSU 93.0137 Escherichia coli O157:N PSU 90.0040 Escherichia coli O157:NM PSU 90.2730 Escherichia coli O157:H5 PSU 3.1177 Escherichia coli O157:H5 PSU 3.1180 TB. 171058.02 Page 4 of 9

Appendix 1: E. coli O157 inclusivity test strains (cont.) Genus, species Serotype ID Escherichia coli O157:H7 PSU 8.0416 Escherichia coli O157:H7 PSU 8.0566 Escherichia coli O157:H7 PSU 93.0061 Escherichia coli O157:H7 PSU 93.0138 Escherichia coli O157:H7 PSU 7.1485 Escherichia coli O157:H7 PSU 7.1495 Escherichia coli O157:H12 PSU 7.1171 Escherichia coli O157:H12 PSU 8.0370 Escherichia coli O157:H12 PSU 8.2974 Escherichia coli O157:H29 PSU 3.1162 Escherichia coli O157:H29 PSU 3.1163 Escherichia coli O157:H29 PSU 7.1269 Escherichia coli O157:H29 PSU 8.0576 Escherichia coli O157:H42 PSU 6.1659 Escherichia coli O157:NM ATCC 700377 Escherichia coli O157:H7 ATCC 700599 Escherichia coli O157 T2850 / FSD756 Abbreviations: TICC - Tecra International Culture Collection; ATCC - American Type Culture Collection; NTCT- National Collection of Type Cultures; CDC - Center for Disease Control; PSU Penn State University; others - isolates from naturally contaminated samples TB. 171058.02 Page 5 of 9

Appendix 2: Non- E. coli O157 exclusivity test strains Genus, species Serotype ID Escherichia coli O103:H2 TICC 2431 Escherichia coli O111:NM TICC 2432 Escherichia coli O91:H- TICC 2480 Escherichia coli O26:H11 TICC 2481 Escherichia coli O91:H21 TICC 2483 Escherichia coli O111:H2 TICC 2484 Escherichia coli O118:H- TICC 2485 Escherichia coli O46:H16 TICC 2486 Escherichia coli O88:H- TICC 2487 Escherichia coli O111:H- TICC 2488 Escherichia coli O:108 TICC 2929 Escherichia coli O:121 TICC 2930 Escherichia coli O:114 TICC 3128 Escherichia coli O:55 TICC 3129 Escherichia coli O:124 TICC 3130 Escherichia coli ATCC 25922 Escherichia hermanii TICC 2490 Escherichia vulneris TICC 4479 Klebsiella oxytoca ATCC 51817 Salmonella enterica Typhimurium ATCC 51812 Citrobacter brakii ATCC29063 Cronobacter sakazakii ATCC 29544 Edwardsiella tarda NCTC 10396 Enterobacter amnigenus ATCC 51816 Enterobacter cloacae ATCC 23355 TB. 171058.02 Page 6 of 9

Appendix 2: Non- E. coli O157 exclusivity test strains (cont.) Genus, species ID Proteus mirabilis ATCC 43071 Proteus vulgaris ATCC 13315 Hafnia alvei ATCC 51815 Serratia ficara TICC 4475 Serratia liquefaciens ATCC 51814 Serratia marcesens ATCC 14041 Shigella sonnei ATCC 25931 Listeria grayi ATCC 19120 Listeria grayi ATCC 25401 Listeria grayi ATCC 25402 Listeria grayi ATCC 25403 Listeria grayi ATCC 700545 Listeria innocua 3M 19B Listeria innocua 3M 21A Listeria innocua ATCC 33090 Listeria innocua ATCC 33091 Listeria innocua ATCC 51742 Listeria ivanovii ATCC 19119 Listeria ivanovii ATCC 49953 Listeria monocytogenes ATCC 19111 Listeria monocytogenes ILSI J1-031 Listeria monocytogenes ILSI J1-049 Listeria monocytogenes ILSI JI-094 Listeria monocytogenes ILSI J1-110 Listeria monocytogenes ILSI J1-158 Abbreviations: ATCC - American Type Culture Collection; NTCT- National Collection of Type Cultures; TICC - Tecra International Culture Collection; ILSI International Life Science Institute; others - isolates from naturally contaminated samples TB. 171058.02 Page 7 of 9

Appendix 3 Food Study List Meat 75% lean ground beef, raw 85% lean ground beef, raw 96% lean ground beef, raw 93% lean ground beef, raw Grass fed ground beef, raw 85% lean grass fed ground beef, raw Raw beef trim 1 Raw beef trim 2 Raw pork Dairy 1% pasteurized milk 4% pasteurized milk Lactose free milk Raw milk 1 Raw milk 2 Raw milk 3 Produce Apple juice Organic apple juice Blended fruits juice Raw organic cucumber Raw conventional cucumber Raw tomato Alfalfa sprouts Organic salad blend (Sprouts, clover, alfalfa, broccoli sprouts) Daikon radish sprouts Bagged baby spinach Bagged hearts of lettuce Organic arugula Processed foods Refrigerated peanut butter milk chocolate chip cookie dough Refrigerated chocolate chip dough Refrigerated pie crusts Thin crispy crust frozen pepperoni pizza Frozen sausage & pepperoni pizza 5 cheese frozen lasagna TB. 171058.02 Page 8 of 9

Appendix 4 Calculation and Interpretation Reponses Chromogenic agar Positive (R+) Chromogenic agar Negative (R-) 3M method Positive (A+) +/+ Positive Agreement (PA) -/+ Positive Deviation (PD) as ( ) 3M method Negative (A-) +/- Negative Deviation (ND) as( ) -/- Negative Agreement (NA) Accuracy (AC), Specificity (SP), and Sensitivity (SE) of the 3M method were calculated as follow: AC = ( ) x 100% SP = x 100% Where N = total number of samples SE = x 100% Food Safety 3M Center Building 275-5W-05 St. Paul, MN 55144-1000 USA 1-800-328-6553 www.3m.com/3mmoleculardetectionsystem 3M is a trademark of 3M. Please recycle. Printed in U.S.A. 3M 2012. All rights reserved. 70-2009-9521-8 TB. 171058.02 Page 9 of 9