Sergiy Olishevskyy 1 , Cathy St-Laurent 1 , Melissa Buzinhani 1 , Michael Giuffre 2 , Morgan Wallace 3 1 FoodChek Laboratories Inc., St-Hyacinthe, Quebec, Canada 2 FoodChek Systems Inc., Calgary, Alberta, Canada 3 DuPont Nutrition & Health, Wilmington, Delaware, USA INTRODUCTION The ability of L. monocytogenes to proliferate in various foods at refrigeration temperatures and survive even after deep freezing makes the occurrence of this foodborne pathogen in ready-to-eat (RTE) foods of particular concern. It is especially threatening to the deli meat and dairy industries if fast and reliable detection methods are not applied. Since L. monocytogenes in RTE food can be present at low concentration with sub-lethal injury during food processing, an enrichment step is crucial to resuscitate injured cells and allow sufficient growth for detection. The objective of this study was to validate a sensitive and rapid method for L. monocytogenes detection in deli meat and dairy products. MATERIALS AND METHODS CONCLUSIONS REFERENCES Optimization studies resulted in significant reduction in the enrichment phase with incubations of 18-22 hours for dairy products (Table 2) and 24 hours for deli meats. No false positive or false negative results were obtained. The candidate method was equivalent to the reference methods (Fig. 1). High performance and reliability of the candidate method were confirmed using ice cream with different flavors as well as 375 g samples of double chocolate ice cream (Table 3) contaminated with different levels of L. monocytogenes. Deli meat (cold smoked turkey and cured ham) and dairy product (double chocolate ice cream and pasteurized milk) samples were artificially contaminated with sub-lethally heat-stressed L. monocytogenes of different serotypes (Table 1) and stabilized for 48–72 hours at 2–8°C or for 14 days frozen. Samples were enriched in Actero™ Listeria Enrichment Media, then processed with the DuPont™ BAX® System Real-Time PCR Assay for L. monocytogenes. A total of 240 artificially contaminated food samples were examined to evaluate performance of the candidate method in comparison with the appropriate USDA- FSIS or US FDA reference method [1, 2]. Additionally, efficacy of the method was evaluated with four other ice cream flavors (vanilla, strawberry, caramel with pecan and cookie dough) as well as with 375 g samples of double chocolate ice cream using Method Detection Limit methodology. Probability of detection (POD) statistical model was used to evaluate the differences between the alternative and reference methods. Method performance parameters including relative sensitivity, relative specificity, false positive rate, false negative rate, and test efficacy were calculated. Single step enrichment of deli meat and dairy samples with Actero™ Listeria medium allowed to reduce significantly incubation time of the samples. The BAX® System method showed high accuracy and reliability for detection of L. monocytogenes in deli meat and dairy products with performance equivalent to the reference method. 1. Hitchins, A. D. 2013. Chapter 10. Detection and enumeration of Listeria monocytogenes in foods. In: FDA Bacteriological Analytical Manual. 2. USDA-FSIS. 2012. Isolation and Identification of Listeria monocytogenes from red meat, poultry, egg, and environmental samples. In: Microbiology Laboratory Guidebook, Chapter 8.09. FAST DETECTION OF LISTERIA MONOCYTOGENES IN DELI MEAT AND DAIRY PRODUCTS Table 1. Food Matrices and Inoculating Microorganisms Food Matrix Sample Size L. monocytogenes strain Background flora , CFU/sample # Serotype Origin MPN, CFU / sample Pasteurized Milk 25 g MSR0441 4c Beef manure 1.5 <1 .0 × 10 2 Double Chocolate Ice Cream 25 g / 375 g MSR0450 1/2b Raw milk 0.7 2.5 × 10 3 / 4.0 × 10 4 Smoked Turkey Breast 125 g MSR0439 3b Raw turkey 0.3 5.0 × 10 4 – 1 .0 × 10 5 Cured Ham 125 g MSR0452 1/2a Animal tissues 0.4 <1 .0 × 10 2 AOAC VALIDATION PROTOCOL RESULTS AND DISCUSSION Table 3. Detection of L. monocytogenes in Ice Cream Using the BAX® System Assay Ice Cream Flavor MPN, CFU/sample N Presumptive Positives Confirmed Positives (US FDA BAM 10) L. monocytogenes Listeria spp. Vanilla 1 0.5–2.0 25 10/25 10/25 10/25 Double Strawberry 1 0.3–15.0 50 34/50 34/50 34/50 Pecan and Caramel 1 0.1–6.0 50 28/50 28/50 28/50 Cookie Dough 1 0.1–15.0 50 28/50 28/50 28/50 Double Chocolate 2 0.8–1.0 39 19/39 19/39 19/39 Table 2. Method Performance f or Double Chocolate Ice Cream Samples Enriched for 18 hrs with Actero™ Listeria Matrix / medium dilution BAX® System Assay Sample # P N FP FN Relative sensitivity, % Relative specificity, % FP rate, % FN rate, % Test efficacy, % 1:5 L. monocytogenes 46 11 33 0 2 84.6 100.0 0.0 5.7 95.7 Listeria spp. 9 33 0 4 69.2 100.0 0.0 10.8 91.3 1:7 L. monocytogenes 46 22 23 0 1 95.7 100.0 0.0 4.2 97.8 Listeria spp. 22 23 0 1 95.7 100.0 0.0 4.2 97.8 Notes: P – positive, N – negative; FP – false positive; FN – false negative. Notes: 1 25 g samples; 2 375 g samples. Fig. 1. Performance Differences Between Alternative and Reference Methods ASSAY PRINCIPLE