TECHNICAL REPORT. Faster Recovery and Detection of Listeria using. and Real-Time PCR Assays for Listeria. Prepared by

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1 TECHNICAL REPORT Faster Recovery and Detection of Listeria using Elite Listeria Enrichment Media and Real-Time PCR Assays for Listeria Prepared by Sergiy Olishevskyy, PhD Vice President, Research and Development FoodChek Laboratories Inc. 7755, av Duplessis Saint-Hyacinthe, Quebec J2R 1S5 Canada Phone: This method s performance was reviewed by AOAC RESEARCH INSTITUTE and was found to perform to the manufacturer s specifications. This method s performance was reviewed by AFNOR and was found to perform to the manufacturer s specifications in accordance with the NF Validation general rules and NF102 certification rules. Method has been approved for publication in the HEALTH CANADA Compendium of Analytical Methods.

2 Elite Listeria Enrichment Media competitive advantages. Single-step enrichment Fastest time-to-results Accurate Reduce sample handling Use less enrichment media* Improve workflow Higher throughput Release food products sooner * Depending on matrix 2

3 Technical Report Table of Contents Introduction 4 Performance Advantages of Actero Elite Listeria Enrichment Media 4 In vitro Culture Studies 4 Matrix Studies 5 Conclusion 5 Growth of Uninjured and Heat-Injured L. monocytogenes in Different Culture Media Used for the Enrichment of Foods 6 Growth Kinetics Data 6 Growth of Competing Bacteria in Different Culture Media Used for the Enrichment of Foods 7 Growth of Fastidious Species of Listeria in Different Culture Media Used for the Enrichment of Food Samples 8 Co-culture of Injured L. monocytogenes with Competing Bacteria in Different Media Used for the Enrichment of Foods 9 Table 1. Performance Parameters for the Detection of Listeria Using Real-Time PCR Assays for Listeria in Environmental and Food Samples Enriched with Actero Elite Listeria Enrichment Media (AOAC/Health Canada Method Comparison Validation Studies, ) 10 Single-Step Enrichment of Environmental and Food Samples with Actero Elite Listeria Enrichment Media 11 Actero Elite Enrichment Media product list. 12 Appendix 13 Actero Elite Enrichment Media compatible with any diagnostic system. 14 Actero Elite Enrichment Media and Real-Time PCR Assays for Listeria and Salmonella validated matrices. 15 3

4 Introduction Developed and launched by FoodChek Systems Inc., Actero Elite Listeria Enrichment Media (Actero Elite Media), a new culture medium that combines a nutritive base with selective agents optimized to efficiently target competing flora without affecting the growth of sublethally injured Listeria being used for the enrichment of food and environmental samples. Performance Advantages of Actero Elite Listeria Enrichment Media Recovery of Injured Target Bacterium Selective Growth Single-Step Enrichment In vitro culture studies as well as matrix trials demonstrate an excellent capacity of the Actero Elite Media to resuscitate sublethally injured Listeria due to different stresses related to the food production chain, including desiccation, heating, cold and osmotic shocks, oxidation, etc. The incorporation of various antimicrobial agents as well as Listeria growth promoting factors allows for the selective growth of the target microorganism in Actero Elite Media even in the presence of abundant competing flora. One of the strongest characteristics of the Actero Elite Media is its user-friendliness allowing for a single step enrichment methodology, cutting in the time-to-result detection and cost expenditures. Robust ph Equilibration Efficient buffering system enables an optimal ph control during enrichment of various environmental /food samples. Optimized Culture Temperatures Validated for Various Foods and Environmental Samples Compatible with Different Testing Platforms Robust analytical studies performed in situ demonstrate consistently high performance of the Actero Elite Media at 29 to 35 C of incubation. Better or equal to the reference methods performance of the Actero Elite Media is successfully proven in multiple method comparison validation studies using various environmental and food samples. Application of the Actero Elite Media is validated and/or verified for different platforms of rapid detection of Listeria, including antibody and DNA/RNA-based detection technologies. In vitro Culture Studies Actero Elite Media shows one of the shortest lag phase as well as one of the highest growth rate for L. monocytogenes growing at 35 C for 24 hours. Actero Elite Media demonstrates an excellent capacity in controlling the growth of competing bacteria such as E. faecalis and P.saeruginosa. Actero Elite Media has the strongest capacity to promote the growth of fastidious species of Listeria such as L. ivanovii and L.sgrayi as compared to the other enrichment media tested. In a co-culture model of sublethally heat-injured L. monocytogenes and a high number of the competing bacteria, Actero Elite Media advantages are the selective recovery and growth of the target bacterium. 4

5 Matrix Studies Actero Elite Media has a strong ability to provide an ideal growing environment for sublethally injured Listeria in foods and environmental samples to allow for accelerated, reliable and faster detection. Using Real-Time PCR Assays for Listeria, Actero Elite Media significantly reduces the time-to-results to as short as 20 hours, enabling a rapid single-step enrichment to successfully reach the detection limit of the method. Actero Elite Media is granted AOAC-RI Certification and approved for the detection of Listeria spp. and Listeria monocytogenes in food and environmental samples, respectively, using Real-Time PCR Assays for Listeria. In addition, Health Canada Certification is granted for the detection of Listeria spp. in environmental samples. All approved methods have exceptional specificity and speed, helping to reduce costly false positives and inventory holds. Conclusion Actero Elite Listeria Enrichment Media is a proven solution to achieve reliable results in a short time that now sets new standards for rapid detection of Listeria in the food industry. 5

6 Growth of Uninjured and Heat-Injured L. monocytogenes in Different Culture Media Used for the Enrichment of Foods 0.6 Uninjured Listeria 0.6 Heat-Injured Listeria O D n m O D n m Tim e (H ours ) Tim e (H ours ) A c te ro T M L is te ria A 2 4 c te L is ro T M te ria L is E te n ria ric 2U 4 V ML is te ria E n ric h m e n t B ro th (O x o id ) Culture Conditions Legend UL EV B M O x o id Initial Inoculum: 20 CFU/well A Actero c ro Elite Listeria Medium A te ro T M L ria L is te ria BEL B E B O x(o o id x o id ) Medium Volume: 200 μl 2 BLEB 4 Medium A N S R B L is is te te ria ria E n ric h m e n t B ro L Eth B (O ric (Nx eo oid g) e n ) Temperature: 35 C U UVM V M 1 A N S R (N e o g e n ) Time: 22 hours L E B O x o id id B L E B (O x o id ) (O id A N S R (N e o g e n ) Growth Kinetics Data (N Bacterium Kinetics Parameter Actero TM Elite Listeria Enrichment Media Primary Enrichment Media Single-Step Enrichment Media* BLEB UVM Medium A Medium B Uninjured Listeria LPD 11.0 ± ± 0.2* 15.2 ± 0.3* 13.9 ± 0.1* ND GR ± ± 0.005* ± 0.002* ± Heat-injured Listeria LPD 12.2 ± ± 0.1* ND ND GR ± ± 0.005* ND Notes: LDP Lag Phase Duration; GR Growth Rate ND Not Determined: Growth kinetics parameters could not be defined because all growth stages were not observed during the incubation period *Commercially available media allowing single-step enrichment of Listeria *P<0.05 as compared to the corresponding values obtained for Actero Elite Listeria Enrichment Media T im 1 6 e (H 1 o 8 u rs ) (H H o u rs rs )) 6 (H o u rs )

7 Growth of Competing Bacteria in Different Culture Media Used for the Enrichment of Foods 1.5 Pseudomonas aeruginosa 1.0 O D n m O D n m Enterococcus faecalis Culture Conditions Initial Inoculum: 2x10 5 CFU/well Medium Volume: 200 μl Temperature: 35 C A c te ro T M L is te ria Time: 22 hours A 2 4 c te L is ro T M te ria L is E te n ria ric 2U 4 V ML is te ria E n ric h m e n t Legend UL EV B M O x o id A Actero c ro Elite T Listeria Medium A te ro T M M L ria L is te ria BEL B E B O x(o o id x o id ) 2 BLEB 4 AMedium N S R B L is is te te ria ria E n ric ric h m e n t B Lro E th B (O (N x e o o id g ) e n ) U UVM V M 1 A N S R (N e o g e n ) L E B O x o id id B L E B (O x o id ) (O id A N S R (N (N e o g e n ) T im 1 6 e (H 1 o 8 u rs ) T im ime (H (Ho u rs rs )) 7

8 Growth of Fastidious Species of Listeria in Different Culture Media Used for the Enrichment of Food Samples Listeria grayi O D n m O D n m Listeria ivanovii Culture Conditions Initial Inoculum: 200 CFU/well Medium Volume: 200 μl Temperature: 35 C A c te ro T M L is te ria Time: 22 hours A 2 4 c te L is ro T M te ria L is E te n ria ric 2U 4 V ML is te ria E n ric Legend UL EV B M O x o id A Actero c ro Elite T Listeria Medium A te ro T M M L ria L is te ria BEL B E B O x(o o id x o id ) 2 BLEB 4 AMedium N S R B L is is te te ria ria E n ric h m e n t B Lro Eth B (O ric (Nx eo oid g) e n ) U UVM V M 1 A N S R (N e o g e n ) L E B O x o id id B L E B (O x o id ) (O id A N S R (N (N e o g e n ) T im 1 6 e (H 1 o 8 u rs ) T im ime (H (Ho u rs rs )) 8

9 L o g C F U /m L Co-culture of Injured L. monocytogenes with Competing Bacteria in Different Media Used for the Enrichment of Foods Culture Conditions Listeria Starting Inoculum: 10 3 CFU/mL E. faecalis Starting Inoculum: 10 5 CFU/mL Medium Volume: 10 ml Temperature: 35 C Time: 22 hours Legend Actero Elite Listeria 0 1 :1 0 0 L is te ria + E. fa e c a lis 1:100 Listeria + E. faecalis 1 :1 0 0 L is te ria + E. fa e c a lis 1 :1 0 0 L is te ria + E. fa e c a lis UVM Single-Step Enrichment Broth 9

10 Table 1. Performance Parameters for the Detection of Listeria Using Real-Time PCR Assays for Listeria in Environmental and Food Samples Enriched with Actero Elite Listeria Enrichment Media (AOAC/Health Canada Method Comparison Validation Studies, ) Method Target Matrix Total Tested P N FP FN Relative Sensitivity % Relative Specificity % FP Rate % FN Rate % Test Efficacy % Environmental L. monocytogenes Food TOTAL Real-Time PCR Assays for Listeria Listeria spp. Environmental Food TOTAL Environmental Samples Food Samples TOTAL

11 Single-Step Enrichment of Environmental and Food Samples with Actero Elite Listeria Enrichment Media Time (hours) Fish/Raw Environmental Samples Cheese/Soft Mexican Style Turkey/ Smoked Breast Ham/Cured Ice Cream Concrete/ Sealed Milk/ Pasteurized Frankfurters Plastic Salmon/Cold Smoked Stainless Steel Spinach/Bagged Fresh Shrimp/Frozen Cooked 11

12 Listeria United States Canada Europe Elite Listeria Enrichment Media product list. Our patented Actero Elite Listeria Enrichment Media is a selective bacterial culture medium specifically optimized for the recovery of Listeria spp. from environmental and food samples in a single-step enrichment. We continue to strive to be the food industry s single, trusted provider for pathogen and other microorganism testing supplies and equipment. Actero Elite Listeria Enrichment Media is compatible with a RT-PCR Assay for Listeria and represents the result of true food safety innovation. Microorganism FoodChek Cat. # Product Name Format Description FCM-011 ACTERO ELITE LISTERIA ENRICHMENT MEDIA 500G Specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples. 500G dehydrated media (yields 9.29L rehydrated media). FCM-022 ACTERO ELITE LISTERIA ENRICHMENT MEDIA 2KG A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples. Four (4) 2KG of dehydrated Actero Elite Listeria Enrichment Media. FCM-023 ACTERO ELITE LISTERIA ENRICHMENT MEDIA 10KG A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples. 10KG of dehydrated Actero Elite Listeria Enrichment Media. FCM-045 FCM-046 ACTERO ELITE LISTERIA ENRICHMENT MEDIABOX 5L 10L A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples in liquid format. A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples in liquid format. FCM-202 ACTERO ELITE LISTERIA ENRICHMENT MEDIA 25KG A selective medium, specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental samples in dehydrated format. FCM-203 FCM-204 ACTERO ELITE LISTERIA ENRICHMENT MEDIABAG 3X3L 2X5L A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental sample. A box of 3 x 3L liquid Actero Elite Listeria Enrichment Media. A selective medium specifically optimized for single-step recovery and enrichment of Listeria spp. from food and environmental sample. A box of 2 x 5L liquid Actero Elite Listeria Enrichment Media. 12

13 APPENDIX 13

14 Elite Enrichment Media compatible with any diagnostic system. Current Listeria spp., Listeria monocytogenes and Salmonella spp. Validations Diagnostic Instrument External Validation License/ Certification # Pathogen Matrix Method Developer AOAC PTM (3) Listeria spp. and Listeria monocytogenes Variety of food and environmental surfaces BAX System RT-PCR (1) AOAC PTM (3) Salmonella spp. Health Canada (5) MFLP-79 Listeria spp. Variety of food and environmental matrices Stainless steel, ceramic, plastic, sealed concrete, and rubber Hygiena, LLC Atlas System (2) AOAC PTM (3) Listeria spp. Stainless steel, PVC and sealed concrete Roka Bioscience, Inc. Agilent AriaMx AFNOR (4) EGS 38/ AOAC PTM (3) , & Listeria spp. and Listeria monocytogenes Listeria spp. and Listeria monocytogenes Variety of food and environmental surfaces Variety of food and environmental surfaces Eurofins GeneScan Bio-Rad CFX96 Touch DeepWell AFNOR (4) EGS 38/ AOAC PTM (3) , & Listeria spp. and Listeria monocytogenes Listeria spp. and Listeria monocytogenes Variety of food and environmental surfaces Variety of food and environmental surfaces Eurofins GeneScan Xpress LM AOAC PTM (3) Listeria monocytogenes Stainless steel, plastic, ceramic tile Crystal Diagnostics MICT Reader Health Canada (5) MFLP-99 Listeria spp. Environmental surfaces AOAC PTM (3) Listeria spp. Environmental surfaces FoodChek Systems Inc. AOAC PTM (3) Salmonella spp. Variety of egg formats and environmental surfaces (1) Formerly an asset of DuPont Nutrition & Health (2) Acquired by IEH Laboratories & Consulting Group (3) This method s performance was reviewed by AOAC RESEARCH INSTITUTE and was found to perform to the manufacturer s specifications. (4) This method s performance was reviewed by AFNOR and was found to perform to the manufacturer s specifications in accordance with the NF Validation general rules and NF102 certification rules. (5) Method has been approved for publication in the HEALTH CANADA Compendium of Analytical Methods. 14

15 Elite Enrichment Media and Real-Time PCR Assays for Listeria and Salmonella validated matrices. ACTERO ELITE LISTERIA ENRICHMENT MEDIA ACTERO ELITE SALMONELLA ENRICHMENT MEDIA AOAC RI-PTM License # AOAC RI-PTM License # (SUITE) AOAC RI-PTM License # AOAC RI-PTM License # (SUITE) Internally Validated Concrete/Sealed Fish/Raw 25 g Stainless Steel Almonds/Raw 375 g Flour/Wheat 25 g Stainless Steel Ham/Cured 125 g Plastic Beef/Ground 25 g Plastic Ice Cream 25 g Cheese/Soft Mexican Style Frankfurters Salmon/Cold Smoked Shrimp/Frozen Cooked Spinach/Fresh Bagged 25 g Milk/Pasteurized 25 g 125 g Turkey/Smoked Breast Chocolate/ Liquor Chocolate/Milk Bar 125 g Cocoa/Powder 25 g 25 g Eggs/Shell 25 g Beef/Ground 375 g 25 g Butter/Peanut 25 g 20 Eggs Chicken/Carcass Rinse Chicken/Ground 30 ml 25 g Pet Food/Dry 25 g Egg/Dried Whole 100 g 25 g Pet Food/Dry 375 g Egg/Liquid Whole* 25 g 100 g Onions/ Dehydrated Pepper/Black Whole 25 g 375 g Parsley/Dried Pepper/Black Whole 25 g 25 g Raisins/Dried 25 g *Pasteurized 15

16 Elite Enrichment Media cutting edge food safety solutions. FOODCHEK SYSTEMS INC. GLOBAL Corporate Head Office: 450, Street SW Calgary, Alberta, T2R 1J6 Canada Terry Macartney Executive Vice President, Sales and Product Development North America Toll Free: x 4 Phone: x 4 tmacartney@foodcheksystems.com UNITED STATES Carl (Chip) Zerr III Vice President, Product Integration Phone: czerr@foodcheksystems.com foodcheksystems.com Contact us today to purchase our products or ask for a free sample.