Erin Crowley, Ben Bastin, Megan Boyle, Jonathan Flannery, M. Joseph Benzinger Jr., Patrick Bird, James Agin, David Goins

Transcription

1 A Comparative Evaluation of the Invisible Sentinel Veriflow Species Method for the Detection of species in Select Foods and Environmental Surfaces November 27, 2013 REVISED December 5, 2013 AOAC Research Institute Independent Laboratory Validation Study Erin Crowley, Ben Bastin, Megan Boyle, Jonathan Flannery, M. Joseph Benzinger Jr., Patrick Bird, James Agin, David Goins Q Laboratories, Inc., 1400 Harrison Ave, Cincinnati, OH A comparative validation study of the Invisible Sentinel Veriflow Species (LS) method for the detection of species was conducted at Q Laboratories, Inc. The validation included an inclusivity, exclusivity, and method comparison evaluation. For the method comparison, the method was compared to the USDA/ FSIS MLG 8.08 (2012) Isolation and Identification of monocytogenes from Red Meat, Poultry and Egg Products, and Environmental Samples for hot dogs, deli turkey and for stainless steel, sealed concrete, plastic and ceramic tile environmental surfaces. Twenty (20) replicates of each food matrix were analyzed at a low inoculum level of CFU/test potion, 5 replicates were analyzed at a high inoculum level of 2-5 CFU/test portion and 5 control replicates were also analyzed at 0 CFU/test portion (un-inoculated). For environmental surfaces twenty (20) replicates were inoculated with ~50 CFU/test area at a level that would produce fractionally positive results, 5 replicate areas were inoculated with ~150 CFU/ test area and 5 control replicates were analyzed at 0 CFU/ test area. Results were analyzed by the Probability of Detection (POD) statistical model. There was no statistically significant difference in the number of positive samples detected by the method and reference methods for all 6 matrices analyzed. For the inclusivity and exclusivity evaluation, all 50 target strains were correctly detected and all 35 exclusivity strains were correctly excluded. The method is a rapid and easy to use alternative method for the detection species in select foods and environmental surfaces. This report presents the analytical results for comparison of Invisible Sentinel Veriflow Species (LS) method to the USDA/ FSIS MLG 8.08 (2012) method: Isolation and Identification of monocytogenes from Red Meat, Poultry and Egg Products, and Environmental Samples. All analyses were conducted at Q Laboratories, Inc. (Cincinnati, OH). All test kits and proprietary media unique to the method were provided by Invisible Sentinel Inc. (Philadelphia, PA). MATERIALS AND METHODS The methodology for this study was followed as outlined in the AOAC Research Institute s Performance Tested Methods Program Protocol: Comparative Evaluation of the Invisible Sentinel Veriflow Species (LS) Method. The study consisted of evaluating a total of 30 samples for each of 2 food matrices and 4 environmental surfaces. Within each sample set, there were 5 un-inoculated samples, 20 low-level inoculated samples and 5 high-level inoculated samples. The target levels of each strain of species used for challenging each food matrix were as follows: colony forming units (CFU) /test portion for the low-level Page 1 of 26

2 inoculation, 2-5 CFU/test portion for the high-level inoculation and 0 CFU/test portion for the un-inoculated control samples. The target levels of each strain of species used for challenging each environmental surface were as follows: ~ 50 CFU/surface areas for the lowlevel inoculation, ~ 150 CFU/surface areas for the high-level inoculation and 0 CFU/surface area for the un-inoculated control samples. The results of the low-level inoculation for both food matrices and environmental surfaces were designed to produce fractionally positive results, those in which ether the reference or candidate method yields 5 to 15 confirmed positive results out of the 20 replicates. Prior to inoculation a background screen of each food matrix was conducted to assure that no natural contamination of the target organism was present in the test matrices. For the method comparison, each matrix was inoculated with commonly associated strains of species as indicated in Table A. Each inoculum was prepared by transferring a pure isolated colony of the specified organism from Trypticase Soy agar with 5% sheep s blood (SBA) into brain heart infusion broth (BHI) and incubating the BHI at 35 ± 2 o C for 24 ± 2 hours. Post incubation, the BHI was diluted and the diluted culture was used to inoculate matrices. For each food matrix, the broth culture inoculum was heat stressed for 10 minutes at 50 o C in a water bath prior to inoculation. The degree of injury of the culture was estimated by plating an aliquot of diluted culture onto Modified Oxford agar (MOX) and Tryptic Soy agar (TSA). The agars were incubated at 35 ± 1 C for 48 ± 2 hours and the colonies were counted. The degree of injury was estimated as nselect ( 1 ) x100 nnonselect where n select = number of colonies on selective agar and n nonselect = number of colonies on nonselective agar. Following inoculation, the bulk lot of test product was mixed thoroughly and was then held at 2-5 C for hours prior to analysis to allow time for the organisms to equilibrate within the sample. Table A: Test Matrices and Inoculating Organisms Matrix Inoculating Organism Matrix Inoculating Organism Deli Turkey Hot Dogs Stainless Steel innocua ATCC monocytogenes ATCC 7644 monocytogenes ATCC 7644 E. faecalis ATCC 10x level Page 2 of 26 Ceramic Tile Sealed Concrete Plastic welshimeri ATCC innocua ATCC ivanovii ATCC For hot dogs, the level of monocytogenes was determined by Most Probable Number (MPN) on the day of analysis by analyzing 5 x 50 g, 20 x 25 g (reference method test portions) and 5 x 10 g inoculated test samples. For the deli turkey, the level of innocua was determined by analyzing 5 x 250 g, 20 x 125 g (reference method test portions) and 5 x 50 g test portions were analyzed. Each test portion was enriched with the appropriate reference method enrichment broth and analyzed by the reference method procedure. The number of positives from the 3 test levels was used to calculate the MPN using the AOAC MPN calculator. ( For environmental surfaces, inoculums were prepared by transferring a pure isolated colony of the specified organism from SBA into BHI and incubating the BHI at 35 ± 2 o C for 24 ± 2 hours. Post incubation, the BHI was diluted and the diluted culture was used to inoculate matrices. For stainless steel and sealed concrete surfaces, 4 x 4 areas were inoculated with

3 ml of diluted culture and sampled using sampling sponges. For ceramic tile and plastic surfaces, 1 x 1 areas were inoculated with 0.10 ml of diluted culture and sampled using sampling swabs. For the un-inoculated test portions, sterile BHI broth was applied to the test area. In addition to the culture, the stainless steel surfaces were co-inoculated with a competitor organism, Enterococcus faecalis ATCC 29212, at approximately 10x the inoculation level of the culture. Each surface was dried for hours at room temperature (24 ±2 o C). To determine the inoculation level for the environmental surfaces, aliquots of each inoculum were plated onto Tryptic Soy agar (TSA) in triplicate. Sampling sponges and swabs, were pre-moistened with Dey-Engley neutralizing broth (DE) prior to sampling each test area using horizontal and vertical sweeping motions. The sponges and the swabs were held at room temperature for 2 hours prior to analysis. Inclusivity and Exclusivity Evaluation For the inclusivity evaluation, 50 strains of species (24 strains of monocytogenes, 1 strain of grayi sbsp. Murrayi, and 5 strains each grayi, innocua, ivanovii, seeligeri, and welshimeri) were cultured in IS Broth and incubated at 35 o C for 24 hours. The cultures were diluted to 10x the Limit of Detection (LOD = 5 x 10 5 CFU/mL for all species except grayi which has a LOD of 5 x 10 7 CFU/mL) and prepared for analysis with the method. For the exclusivity portion of the validation, 35 closely related non- cultures were enriched in BHI and incubated at temperatures and conditions specific for organism growth. The non-target strains were undiluted and prepared for analysis with the method. If an exclusivity strain produced a presumptive positive result, the strain was re-analyzed following growth in IS broth. All inclusivity and exclusivity strains were randomized and blind-coded prior to analysis. Method Comparison USDA/FSIS MLG 8.08 Reference Method For hot dog samples, 25 g test portions were enriched with 225 ± 5 ml of modified University of Vermont Medium (UVM), homogenized for two minutes, and incubated at 30 ± 2 C for hours. For deli turkey samples, 125 g test portions were enriched with 1125 ± 25 ml of UVM broth, homogenized for 2 minutes and incubated at 30 ± 2 C for hours. For environmental samples, sponges were enriched with 225 ml of UVM and homogenized by hand while swabs were enriched with 10 ml of UVM and mixed by vortex. Sponges and swabs were incubated for hours at 30 ± 2 C. After incubation of all test portions, 0.1 ml of the sample enrichment was transferred to 10 ml of Fraser Broth (FB) containing 0.1 ml of 5% ferric ammonium citrate and incubated at 35 ± 2 C for 26 ± 2 hours. A loopful of the sample enrichment was also streaked to MOX and incubated at 35 ± 2 C for 26 ± 2 hours. After 26 ± 2 hours, FB was examined for any degree of darkening due to esculin hydrolysis. Any FB that displayed darkening was streaked to a MOX plate. If no darkening occurred, FB was re-incubated at 35 ± 2 C for a total of 48 ± 2 hours and re-examined for evidence of darkening. If darkening occurred, the FB was streaked to a MOX plate, if no darkening occurred, samples were considered negative. All FB streaked MOX plates were incubated at 35 C for 26 ± 2 hours. MOX agar plates streaked from the primary enrichment or the FB secondary enrichment were examined after 26 ± 2 hours and if no suspect colonies were present, the MOX agar plate was reincubated for an additional 26 ± 2 hours at 35 C ± 2 C for a total of 48 ± 2 hours. If suspect Page 3 of 26

4 colonies were present on the MOX agar plates, these suspect colonies were streaked to Horse Blood Overlay agar (HBO) and incubated at 35 ± 2 o C for 22 ± 4 hours. HBO plates were examined for hemolysis reactions and well isolated colonies were transferred to BHI broth and incubated at 25 C for 24 ± 2 hours. Sample isolates from BHI broth were analyzed for tumbling motility by preparing a wet mount, analyzed by a catalase test and examined for morphology by preparing a gram stain. Additionally, purified HBO isolates were identified using the VITEK GP Biochemical Identification following AOAC OMA Invisible Sentinel Veriflow Species (LS) Method For the method comparison, all food matrices and environmental surfaces were analyzed by the method in an unpaired study design. Additionally, the stainless steel environmental samples were analyzed by the Veriflow monocytogenes (LM) method. For environmental sponges, 200 ml of IS Broth was added to the sampling bag and the sponge was homogenized for 30 seconds. For environmental swabs, 20 ml of IS Broth was added to the swab placed inside a 59 ml (2 oz.) culture bag and homogenized by hand for 30 seconds. For deli turkey test portions, each 125 g sample were enriched with 375 ml of IS Broth and homogenized by stomaching for 2 minutes. For hot dogs test portions, each 25 g sample were enriched with 225 ml of IS Broth and homogenized by stomaching for 2 minutes. All food matrices and environmental samples were incubated at 35 o C for hours. After incubation, 0.5 ml of enrichment was transferred to a 1.5 ml tube containing Buffer A and inverted to mix samples. Sample tubes were boiled in a water bath for 10 minutes and then allowed to cool to room temperature (20-25 o C). From the cooled sample, a 5 µl aliquot was transferred to a thawed PCR reagent tube containing 45 µl of Master Mix. For the evaluation of stainless steel, 5 µl aliquots of each sample were transferred to separate and Veriflow LM PCR reagent tubes for analysis by each assay. All sample PCR reagent tubes were cycled according to the parameters specified in the package insert. The cycling parameters for the amplification procedure are presented in Table B. Following the amplification procedure, 4 drops of Buffer B were added to each sample PCR tube. The entire contents of the PCR tube (200 µl) were transferred to the sample window of a or Veriflow LM assay cassette. The test was allowed to develop for 2 minutes, and then 4 more drops of Buffer B were added to the sample window of the Veriflow assay cassette and allowed to develop for 1 minute. The cassette lever was retracted and the test result was visually interpreted. Regardless of the presumptive results, each sample was confirmed according to procedures outlined in the USDA/FSIS-MLG 8.08 reference method. Final biochemical confirmation was conducted according to VITEK 2 GP Biochemical Identification following AOAC OMA Page 4 of 26

5 Table B: PCR Amplification Parameters for the and LM Methods Stage 1 Description Temperature Time Number of Cycles Incubation 37 C 10 minutes 1 Stage 2 Description Temperature Time Number of Cycles Denaturing 95 C 10 minutes 1 Stage 3 Description Temperature Time Number of Cycles Denaturing 94 C 2 minutes 1 Stage 4 Description Temperature Time Number of Cycles Denaturing 94 C 15 seconds Annealing 55 C 15 seconds 40 Extension 70 C 30 seconds Stage Description Temperature Time Number of Cycles Extension 70 C 1 minute 1 Cooling 4 C 1 minute RESULTS Inclusivity and Exclusivity Results Of the 50 species inclusivity strains analyzed, all 50 were correctly detected as species by the method. Of the 35 exclusivity strains analyzed, 34 strains were correctly excluded by the method. One strain, E. faecium ATCC produced a presumptive positive result and was reanalyzed following the enrichment procedures outlined in the method. The strain was then correctly excluded by the method. Results of the inclusivity and exclusivity evaluation are presented in Tables 1 and 2 of the Appendix. Method Comparison For the method comparison, the probability of detection (POD) was calculated as the number of positive outcomes divided by the total number of trials. The POD was calculated for the candidate presumptive results, POD CP, the candidate confirmatory results, POD CC, the difference in the candidate presumptive and confirmatory results, dpod CP, the candidate method, POD C, the reference method, POD R, and the difference in the confirmed candidate and reference methods, dpod C. A summary of results for the method comparison are presented in Table C. The results of the inoculum heat stress are presented in Table 4 of the Appendix. A summary of the MPN results are presented in Tables 3A-3C of the Appendix. A detailed summary of individual sample Page 5 of 26

6 results are presented in Tables 5-10 of the Appendix. Based upon the results of the study, fractional positive results were obtained for each matrix. Table C: Summary of Results Matrix Deli Turkey Hot Dogs Inoculation Level 24 Hour Enrichment USDA/FSIS MLG 24 Hour Enrichment Presumptive Confirmed Method Presumptive Confirmed Page 6 of 26 USDA/FSIS MLG Method Un-inoculated 0/5 0/5 0/5 0/5 0/5 0/5 Low 8/20 8/20 7/20 13/20 13/20 10/20 High 5/5 5/5 5/5 5/5 5/5 5/5 Matrix Stainless Steel Stainless Steel Inoculation Level 24 Hour Enrichment USDA/FSIS MLG Veriflow LM 24 Hour Enrichment Presumptive Confirmed Method Presumptive Confirmed USDA/FSIS MLG Method Un-inoculated 0/5 0/5 0/5 0/5 0/5 0/5 Low 12/20 12/20 8/20 12/20 12/20 8/20 High 5/5 5/5 5/5 5/5 5/5 5/5 Matrix Sealed Concrete Plastic Inoculation Level 24 Hour Enrichment USDA/FSIS MLG 24 Hour Enrichment Presumptive Confirmed Method Presumptive Confirmed USDA/FSIS MLG Method Un-inoculated 0/5 0/5 0/5 0/5 0/5 0/5 Low 15/20 15/20 14/20 15/20 15/20 13/20 High 5/5 5/5 5/5 5/5 5/5 5/5 Matrix Inoculation Level Ceramic Tile 24 Hour Enrichment Presumptive Confirmed USDA/FSIS MLG Method Un-inoculated 0/5 0/5 0/5 Low 8/20 8/20 11/20 High 5/5 5/5 5/5 Deli Turkey Results For low-level inoculum deli turkey test portions, there were 8 confirmed positives for the method and 7 confirmed positives for the USDA/FSIS-MLG method. All highlevel inoculum test portions were positive and all un-inoculated control samples were negative for both methods. No false positives or negatives were observed. For the low-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 0.40 (0.22, 0.61) was obtained. A dpod CP value of 0.00 (-0.28, 0.28) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R values with 95% confidence intervals of 0.40 (0.22, 0.61) and 0.35 (0.18, 0.57) were obtained. A dpod C value of 0.05 (-0.23, 0.32) was obtained, indicating no significant difference between the method and the reference method confirmed results. For the high-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95%

7 confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. For the un-inoculated control level, a POD CP and POD CC value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. Detailed results of the POD analysis are presented in Tables 11 and 12 of the Appendix. Hot Dogs For low-level inoculum hot dog test portions, there were 13 confirmed positives for the method and 10 confirmed positives for the USDA/FSIS-MLG method. All highlevel inoculum test portions were positive and all un-inoculated control samples were negative for both methods. No false positives or negatives were observed. For the low-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 0.65 (0.43, 0.82) was obtained. A dpod CP value of 0.00 (-0.28, 0.28) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R values with 95% confidence intervals of 0.65 (0.43, 0.82) and 0.50 (0.30, 0.70) were obtained. A dpod C value of 0.15 (-0.15, 0.41) was obtained, indicating no significant difference between the method and the reference method confirmed results. For the high-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. For the un-inoculated control level, a POD CP and POD CC value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. Detailed results of the POD analysis are presented in Tables 11 and 12 of the Appendix. Stainless Steel For low-level inoculum stainless steel test portions, there were 12 confirmed positives for the and Veriflow LM method and 8 confirmed positives for the USDA/FSIS-MLG method. All high-level inoculum test portions were positive and all un-inoculated control samples were negative for both methods. No false positives or negatives were observed. For the low-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 0.60 (0.39, 0.78) was obtained for both the and Veriflow LM methods. A dpod CP value of 0.00 (-0.28, 0.28) was obtained, indicating no significant difference in presumptive and confirmed results for both assays. A POD C (both and Veriflow LM) and POD R values with 95% confidence intervals of 0.60 (0.39, 0.78) and 0.40 (0.22, 0.61) were obtained. A dpod C value of 0.20 (-0.10, 0.46) was obtained, indicating no significant difference between the and Veriflow LM methods and the reference method confirmed results. For the high-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained for both the and Veriflow LM methods. A dpod CP value Page 7 of 26

8 of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results for both assays. A POD C (both and Veriflow LM) and POD R value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the and Veriflow LM methods and the reference method confirmed results. For the un-inoculated control level, a POD CP and POD CC value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained for both the and Veriflow LM methods. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results for both assays. A POD C (both and Veriflow LM) and POD R value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the and Veriflow LM methods and the reference method confirmed results. Detailed results of the POD analysis are presented in Tables 13 and 14 of the Appendix. Sealed Concrete For low-level inoculum sealed concrete test portions, there were 15 confirmed positives for the method and 14 confirmed positives for the USDA/FSIS-MLG method. All uninoculated control samples were negative and all high inoculum samples were positive for both methods. No false positives or false negatives were observed. For the low-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 0.75 (0.53, 0.89) was obtained. A dpod CP value of 0.00 (-0.26, 0.26) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R values with 95% confidence intervals of 0.75 (0.53, 0.89) and 0.70 (0.48, 0.85) were obtained. A dpod C value of 0.05 (-0.22, 0.31) was obtained, indicating no significant difference between the method and the reference method confirmed results. For the high-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. For the un-inoculated control level, a POD CP and POD CC value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. Detailed results of the POD analysis are presented in Tables 13 and 14 of the Appendix. Ceramic Tile For low-level inoculum ceramic tile test portions, there were 8 presumptive positives and 8 confirmed positives for the method and 11 confirmed positives for the USDA/FSIS- MLG method. All high-level inoculum test portions were positive and all un-inoculated control samples were negative for both methods. No false positives or negatives were observed. For the low-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 0.40 (0.22, 0.61) was obtained. A dpod CP value of 0.00 (-0.28, 0.28) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R values with 95% confidence intervals of 0.40 (0.22, 0.61) and 0.55 (0.34, 0.74) were obtained. A dpod C value with 95% confidence intervals of (-0.41, 0.15) was obtained, indicating no significant difference between the method and the reference method confirmed results. Page 8 of 26

9 For the high-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. For the un-inoculated control level, a POD CP and POD CC value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. Detailed results of the POD analysis are presented in Tables 13 and 14 of the Appendix. Plastic For low-level inoculum plastic test portions, there were 15 presumptive positives and 15confirmed positives for the method and 13 confirmed positives for the USDA/FSIS-MLG method. All high-level inoculum test portions were positive and all uninoculated control samples were negative for both methods. No false positives or negatives were observed. For the low-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 0.75 (0.53, 0.89) was obtained. A dpod CP value of 0.00 (-0.26, 0.26) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R values with 95% confidence intervals of 0.75 (0.53, 0.89) and 0.65 (0.43, 0.82) were obtained. A dpod C value of 0.10 (-0.18, 0.36) was obtained, indicating no significant difference between the method and the reference method confirmed results. For the high-level inoculum, a POD CP and POD CC value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95% confidence intervals of 1.00 (0.57, 1.00) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. For the un-inoculated control level, a POD CP and POD CC value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod CP value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference in presumptive and confirmed results. A POD C and POD R value with 95% confidence intervals of 0.00 (0.00, 0.43) was obtained. A dpod C value of 0.00 (-0.43, 0.43) was obtained, indicating no significant difference between the method and the reference method confirmed results. Detailed results of the POD analysis are presented in Tables 13 and 14 of the Appendix. DISCUSSION The results of this study demonstrated the reliability of the method when compared to the traditional reference methods for the detection of species on environmental surfaces and in select food matrices. The method offers a significant savings in time compared to the USDA/ FSIS MLG 8.08 reference method by producing accurate presumptive results in approximately 27 hours compared to 72 to 96 hours for the reference methods. The method protocol is easy to follow and simplifies the process for the rapid detection of species. The cassette produced distinct positive lines allowing fast, accurate, and easy interpretation of the cassette. The evaluation of the stainless Page 9 of 26

10 1 2 steel environmental samples indicated that the and Veriflow LM methods provided accurate detection of the target analyte in the presence of a competitor organism. Page 10 of 26

11 Appendix Page 11 of 26

12 Table 1: Inclusivity Summary of Results Organism/ Reference Number/ Serotype grayi 1 ATCC grayi 1 ATCC grayi 1 ATCC grayi 1 ATCC grayi 1 ATCC innocua 2 NCTC AB innocua 1 ATCC A innocua 1 ATCC B innocua 1 ATCC B innocua 1 ATCC BAA-680 6A ivanovii 1 ATCC ivanovii 1 ATCC ivanovii 1 Source Feces, animal (Chinchilla feces) Denmark Standing corn stalks and leaves Standing corn stalks and leaves Standing corn stalks and leaves, USA Standing corn stalks and leaves, USA Veriflow LS Unknown Cow brain Feces, pregnant woman Cabbage Dairy product (cheese) Mexico Sheep, Bulgaria Goat, Belgium ATCC Food, France ivanovii 1 ATCC BAA-139 Washing water, Switzerland ivanovii 1 ATCC BAA Sheep fetus seeligeri 1 ATCC /2B Soil, Germany seeligeri 1 ATCC Intestinal content of Clethrionomys glareolus, Bratislava, Slovakia Organism/ Reference Number/ Serotype seeligeri 1 ATCC A seeligeri 2 NCTC seeligeri 1 ATCC B welshimeri 2 NCTC welshimeri 1 ATCC B welshimeri 3 ATCC welshimeri 1 ATCC B welshimeri 1 ATCC /2B grayi sbsp Murrayi 2 NCTC monocytogenes 4 CWD B monocytogenes 4 CWD B monocytogenes 4 CWD B monocytogenes 4 CWD B monocytogenes 4 CWD /2B monocytogenes 4 CWD B monocytogenes 4 CWD /2B monocytogenes 4 CWD /2B 1. ATCC American Type Culture Collection, 2. NCTC HPA Culture Collection; 3. Culture obtained from Invisible Sentinel Culture Collection 4. University of Vermont 5.National collection of Industrial Marine and Food Bacteria Source Veriflow LS Unknown Unknown Feces, pregnant woman Plant Unknown Unknown Soil Feces, healthy man Unknown Lausanna 1987 LA Outbreak 1985 Not Available Halifax 1983 Not Available Not Available Not Available LA Organism/ Reference Number/ Serotype monocytogenes 5 NCIMB# B monocytogenes 2 NCTC# monocytogenes 1 BAA-751 1/2B monocytogenes monocytogenes monocytogenes A monocytogenes B monocytogenes C monocytogenes D monocytogenes E monocytogenes B monocytogenes /2A monocytogenes /2A monocytogenes /2B monocytogenes A monocytogenes 1 ATCC /2C Source Not Available Human feces Not Available Poultry, England Spinal Fluid of Man, Scotland Ruminant Brain Human Isolate Chicken, England Sheep, USA Chicken, England Spinal Fluid of Child Rabbit, England Not Available Cheese Cheese Human Isolate Page 12 of 26

13 Table 2: Exclusivity Summary of Results Organism/ Reference Number/ Source Alcaligenes faecalis 1 ATCC 8750 Bacillus coagulans ATCC 7050 Campylobacter lari ATCC BAA-1060 Campylobacter jejuni ATCC Candida albicans ATCC Carnobacterium maltaromaticum ATCC Citrobacter freundi 8090 Edwardsiella tarda Enterobacter aerogenes Enterobacter cloacae Enterococcus faecalis Dairy Products Herring gull cloacal swab, Larus argintatus Bovine Feces - Nail Infection - Vacuum Packed Beef Feces, Human - Sputum - Urine Organism/ Reference Number/ Escherichia coli ATCC Escherichia coli 2 BEI NR-4356 Escherichia coli 2 BEI NR-12 Hafnia alvei Klebsiella pneumoniae Kocuria rhizophila 9341 Lactobacillus acidophilus 314 Lactobacillus kefiri Lactobacillus lactis 4797 Lactobacillus plantarum ATCC 8014 Morganella morganii Enterococcus faecium Not Available /- 4 Proteus mirabilis Urine ATCC American Type Culture Collection; 2. Culture obtained from Invisible Sentinel Culture Collection; 3. STS- Culture obtained from the University of Pennsylvania Culture Collection; 4. Sample produced a presumptive positive result from non-selective enrichment and a negative result from the selective enrichment used by the assay Source Veriflow LS Clinical Isolate - Soil - Kefir - Human - Organism/ Reference Number/ Source Proteus vulgaris 6380 Pseudomonas aeruginosa Salmonella enterica subsp. enterica serovar Abaetuba Salmonella enterica subsp. enterica serovar Dublin 3 STS 27 Salmonella enterica subsp. enterica serovar SaintPaul 9712 Shigella sonnei Staphylococcus aureus Staphylococcus epidermidis Staphylococcus haemolyticus Staphylococcus hominis Streptococcus pneumoniae 6302 Clinical Isolate - Clinical Isolate - Creek Water, Zaiman, Argentina Dublin - Human Skin - Human Skin - - Page 13 of 26

14 Table 3A: MPN Summary Table for Deli Turkey Inoculum Level Deli Turkey 1 innocua ATCC Low-Level Inoculum: (0.2-2 CFU/Test Portion) A B C D E 250g Reference Samples 7/20 50g MPN/Test portion 0.22 Low Conf. Limit MPN/Test Portion High Conf. Limit MPN/Test Portion High-Level Inoculum: (2-5 CFU/Test Portion) A B C D E 250g 5 Reference Samples 5/5 50g - - MPN/Test portion 0.98 Low Conf. Limit MPN/Test Portion 0.46 High Conf. Limit MPN/Test Portion MPN was calculated for Deli Turkey using five 250g, five 50g and the 20 reference method samples and the AOAC LCF MPN Calculator Page 14 of 26

15 Table 3B: MPN Summary Table for Hot Dogs Inoculum Level Hot Dogs 2 monocytogenes ATCC 7644 Low-Level Inoculum: (0.2-2 CFU/Test Portion) A B C D E 50g Reference Samples 10/20 10g - - MPN/Test portion 0.36 Low Conf. Limit MPN/Test Portion High Conf. Limit MPN/Test Portion High-Level Inoculum A B C D E 50g Reference Samples 5/5 10g - - MPN/Test portion 1.19 Low Conf. Limit MPN/Test Portion 0.62 High Conf. Limit MPN/Test Portion MPN was calculated for Hot Dogs using five 50g, five 10g and the 20 reference method samples and the AOAC LCF MPN Calculator Table 3C: Inoculum Summary Table for Environmental Surfaces Matrix Stainless Steel Stainless Steel Sealed Concrete Ceramic Tile Plastic Inoculating Organisms monocytogenes ATCC #7644 Enterococcus faecalis ATCC #29212 innocua ATCC #33090 welshimeri ATCC #43548 ivanovii ATCC #19119 Low-Inoculum Level CFU/Test Area* High-Inoculum Level CFU/Test Area* *Test area was 4 x 4 for Stainless Steel and Sealed Concrete and 1 x 1 for Ceramic Tile and Plastic Page 15 of 26

16 Table 4: Heat Stress Results Matrix Agar CFU Percent Injury Deli Turkey ( innocua ATCC 33090) TSA 1 MOX 2 9.1x10 8 CFU 2.9x10 8 CFU 68.1% Hot Dogs ( monocytogenes ATCC 7644) TSA 1 MOX 2 8.9x10 8 CFU 2.3x10 8 CFU 74.2% 1 TSA: Trypticase soy agar 2 MOX: Modified Oxford Agar Page 16 of 26

17 Table 5: Results of the method vs. the USDA/FSIS-MLG Method for Deli Turkey innocua ATCC #33090 Low-Level 0.22 MPN/Test Portion Sample # Presumptive Confirmed USDA/FSIS-MLG Total 8/20 8/20 7/20 Un-inoculated Total 0/5 0/5 0/5 High-Level 0.98 MPN/Test Portion Total 5/5 5/5 5/5 Page 17 of 26

18 Table 6: Results of the method vs. the USDA/FSIS-MLG Method for Hot Dogs monocytogenes ATCC #7644 Low-Level 0.64 MPN/Test Portion Sample # Presumptive Confirmed USDA/FSIS-MLG Total 13/20 13/20 10/20 Un-inoculated Total 0/5 0/5 0/5 High-Level 1.19 MPN/Test Portion Total 5/5 5/5 5/5 Page 18 of 26

19 Table 7: Results of the and Veriflow LM method vs. the USDA/FSIS-MLG Method for Stainless Steel monocytogenes ATCC #7644 Low-Level 21 CFU/4 x 4 Sample # Presumptive Veriflow LM Presumptive and LM Confirmed USDA/FSIS-MLG Total 12/20 12/20 12/20 8/20 Un-inoculated 0 CFU/4 x Total 0/5 0/5 0/5 0/5 High-Level 110 CFU/4 x Total 5/5 5/5 5/5 5/5 Page 19 of 26

20 Table 8: Results of the method vs. the USDA/FSIS-MLG Method for Sealed Concrete Sample # Presumptive innocua ATCC #33090 Low-Level 60 CFU/ 4 x 4 Confirmed USDA/FSIS-MLG Total 15/20 15/20 14/20 Un-inoculated 0 CFU/ 4 x Total 0/5 0/5 0/5 High-Level 170 CFU/ 4 x Total 5/5 5/5 5/5 Page 20 of 26

21 Table 9: Results of the method vs. the USDA/FSIS-MLG Method for Ceramic Tile welshimeri ATCC #43548 Low-Level 36 CFU/ 1 x 1 Sample # Presumptive Confirmed USDA/FSIS-MLG Total 8/20 8/20 11/20 Un-inoculated 0 CFU/ 1 x Total 0/5 0/5 0/5 High-Level 75 CFU/ 1 x Total 5/5 5/5 5/5 Page 21 of 26

22 Table 10: Results of the method vs. the USDA/FSIS-MLG Method for Plastic Sample # Presumptive ivanovii ATCC #19119 Low-Level 140 CFU/ 1 x 1 Confirmed USDA/FSIS-MLG Total 15/20 15/20 13/20 Un-inoculated 0 CFU/ 1 x Total 0/5 0/5 0/5 High-Level 311 CFU/ 1 x Total 5/5 5/5 5/5 Page 22 of 26

23 Table 11: Food Matrices, Presumptive vs. Confirmed POD Results Matrix Deli Turkey Strain innocua MPN a /Test Portion N b Presumptive Confirmed x c d e POD CP 95% CI x POD CC 95% CI dpod CP f 95% CI g , , , (0.11, 0.47) , , , (0.46, 2.07) , , , , , , 0.43 monocytogenes 0.64 Hot Dogs , , , 0.28 (0.36, ) , , , 0.43 (0.62, 2.25) a MPN = Most Probable Number is based on the POD of reference method test portions across labs using the AOAC MPN calculator, with 95% confidence interval b N = Number of test potions c x = Number of positive test portions d POD CP = Candidate method presumptive positive outcomes divided by the total number of trials e POD CC = Candidate method confirmed positive outcomes divided by the total number of trials f dpod CP = Difference between the candidate method presumptive result and candidate method confirmed result POD values g 95% CI = If the confidence interval of a dpod does not contain zero, then the difference is statistically significant at the 5% level Page 23 of 26

24 Table 12: Food Matrices, Candidate vs. Reference POD Results Matrix Deli Turkey Strain innocua MPN a /Test Portion N b Candidate Reference x c d e POD C 95% CI x POD R 95% CI dpod C f 95% CI g , , , (0.11, 0.47) , , , (0.46, 2.07) , , , , , , 0.43 monocytogenes 0.64 Hot Dogs , , , 0.41 (0.36, ) , , , 0.43 (0.62, 2.25) a MPN = Most Probable Number is based on the POD of reference method test portions across labs using the AOAC MPN calculator, with 95% confidence interval b N = Number of test potions c x = Number of positive test portions d POD C = Candidate method confirmed positive outcomes divided by the total number of trials e POD R = Reference method confirmed positive outcomes divided by the total number of trials f dpod C = Difference between the candidate method confirmed results and candidate method confirmed result POD values g 95% CI = If the confidence interval of a dpod does not contain zero, then the difference is statistically significant at the 5% level Page 24 of 26

25 Table 13: or Veriflow LM Environmental Surfaces, Presumptive vs. Confirmed POD Results Matrix Strain CFU/Test Area a N b Presumptive Confirmed x c d e POD CP 95% CI x POD CC 95% CI Stainless Steel () Stainless Steel (Veriflow LM) monocytogenes 7644 monocytogenes 7644 dpod CP f 95% CI g , , , , , , , , , , , , , , , , , , 0.43 Sealed Concrete Ceramic Tile innocua welshimeri , , , , , , , , , , , , , , , , , , 0.43 Plastic ivanovii , , , , , , , , , 0.43 a CFU/Test Area = Results of the CFU/Test area were determined by plating the inoculum for each matrix in triplicate b N = Number of test potions c x = Number of positive test portions d POD CP = Candidate method presumptive positive outcomes divided by the total number of trials e POD CC = Candidate method confirmed positive outcomes divided by the total number of trials f dpod CP = Difference between the candidate method presumptive result and candidate method confirmed result POD values g 95% CI = If the confidence interval of a dpod does not contain zero, then the difference is statistically significant at the 5% level Page 25 of 26

26 Table 14: or Veriflow LM Environmental Surfaces, Candidate vs. Reference POD Results Matrix Strain CFU/Test Area a N b Candidate Reference x c d e POD C 95% CI X POD R 95% CI Stainless Steel (Veriflow LS) monocytogenes 7644 dpod C f 95% CI g , , , , , , , , , 0.43 Stainless Steel (Veriflow LM) Sealed Concrete Ceramic Tile monocytogenes 7644 innocua welshimeri , , , , , , , , , , , , , , , , , , , , , , , , , , , 0.43 Plastic ivanovii , , , , , , , , , 0.43 a CFU/Test Area = Results of the CFU/Test area were determined by plating the inoculum for each matrix in triplicate b N = Number of test potions c x = Number of positive test portions d POD C = Candidate method confirmed positive outcomes divided by the total number of trials e POD R = Reference method confirmed positive outcomes divided by the total number of trials f dpod C = Difference between the candidate method confirmed results and candidate method confirmed result POD values g 95% CI = If the confidence interval of a dpod does not contain zero, then the difference is statistically significant at the 5% level Page 26 of 26