Sergiy Olishevskyy1, Cathy St-Laurent1, Melissa Buzinhani1, Michael Giuffre2, Morgan Wallace3

1FoodChek Laboratories Inc., St-Hyacinthe, Quebec, Canada 2FoodChek Systems Inc., Calgary, Alberta, Canada 3DuPont 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 <1.0×102

Double

Chocolate Ice

Cream

25 g /

375 g MSR0450 1/2b Raw milk 0.7

2.5×103 /

4.0×104

Smoked

Turkey Breast 125 g MSR0439 3b

Raw

turkey 0.3

5.0×104 –

1.0×105

Cured Ham 125 g MSR0452 1/2a Animal

tissues 0.4 <1.0×102

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.

Vanilla1 0.5–2.0 25 10/25 10/25 10/25

Double

Strawberry1 0.3–15.0 50 34/50 34/50 34/50

Pecan and

Caramel1 0.1–6.0 50 28/50 28/50 28/50

Cookie

Dough1 0.1–15.0 50 28/50 28/50 28/50

Double

Chocolate2 0.8–1.0 39 19/39 19/39 19/39

Table 2. Method Performance for 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: 125 g samples; 2375 g samples. Fig. 1. Performance Differences Between Alternative and Reference Methods

ASSAY PRINCIPLE