1. 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