1. IMPROVED REAL-TIME PCR DETECTION OF LISTERIA IN ENVIRONMENTAL
SAMPLES AFTER RAPID SINGLE STEP ENRICHMENT
Sergiy Olishevskyy1, Morgan Wallace2, Michael Giuffre3
1FoodChek Laboratories Inc., Suite 200, 7755 Duplessis, St-Hyacinthe, Quebec, Canada J2R 1S5
2DuPont Nutrition & Health, Experimental Station 400/200 Powder Mill Road, Wilmington, DE, 19803, USA
3FoodChek Systems Inc., Suite 450, 1414-8th Street S.W. Calgary, AB, Canada T2R 1J6
DuPont Nutrition & Health
ESL Building 400, Route 141 & Henry Clay Road,
Wilmington, DE 19880 • Tel: 302.695.5473 •
www.fooddiagnostics.dupont.com
Introduction Materials and Methods
Results and Discussion
Conclusions References
A total of 930 environmental sponge samples were
evaluated in two large-scale, unpaired validation studies in
comparison with the USDA-FSIS and Health Canada
reference methods, including 570 samples analysed by
certified independent laboratories. Detection of Listeria
spp. and/or L. monocytogenes in environmental sponge
samples enriched with Actero™ Listeria using the
DuPont™ BAX® System Real-Time PCR assay was
compared to the USDA-FSIS MLG 8.09 and the Health
Canada MFHPB-30 reference methods (Fig. 3) [3–4].
Bacterial Strains and Environmental Surfaces.
Environmental sponge samples were routinely collected
from two food contact (stainless steel and plastic) and
three non-food contact (sealed concrete, ceramic and
rubber) surfaces artificially contaminated with different
Listeria strains in the presence of high levels of competing
background flora (Table 1 – for the AOAC validation
study; Table 2 – for the Health Canada validation study).
Fig. 3. Performance Evaluation of the BAX®
System RT-PCR Assay to Detect Listeria in
Environmental Samples Enriched with
Actero™ Listeria
Bacterial Strains Selected for Contamination of Environmental Surfaces
Table 1. AOAC Validation Study Table 2. Health Canada Validation Study
Fig. 4. Method Comparison Validation Study
Notes: 1According to the FoodChek Laboratories Inc. Bacterial Strain Collection. 2Bacterial strains used in external laboratory comparison study.
Sample enrichment and RT-PCR detection of Listeria. Enrichment of sponge samples followed by the RT-
PCR detection of Listeria was performed according to Fig. 4.
Presumptive positive result confirmation. BAX®
System results were confirmed from ACTERO™
Listeria enrichments by transferring 0.1 mL enriched
sample to 10 mL of MOPS-BLEB or Fraser broth
followed by incubation at 35°C for 18-28 hours and
streaking onto MOX agar plates. Isolation of typical
Listeria colonies and confirmation by API Listeria
biochemical identification system were carried out as
described in [3–4].
Data analysis. Probability of detection (POD) as well
as χ2 statistical models were used to evaluate the
differences between presumptive and confirmed
results as well as between the alternative and the
reference methods. Results are presented as a
difference (dPOD) between the POD values of the
alternative and the reference methods.
If the 95% CI of a dPOD value does not contain zero, then the difference is statistically significant at the 5% level.
The χ2-analysis results greater than 3.84 were considered indicating a significant difference between two
methods at a 95% confidence level. Main performance parameters for alternative method were calculated
according to [5–6].
The unpaired method comparison studies were performed to evaluate the ability of the BAX RT-PCR technology to detect artificially inoculated Listeria from stainless steel, plastic , sealed concrete , ceramic and rubber environmental
surfaces after only 20 hour s of one-step enrichment with ACTERO™ Listeria medium. The validation studies were conducted according to the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Qualitative and
Quantitative Food Microbiological Official Methods of Analysis and the Health Canada Procedure for the Development and Management of Methods and General Procedures [6–7].
Detection of L. monocytogenes in Environmental Samples Enriched with Actero™ Listeria Using BAX®
System RT-PCR Assay. Based on the POD analysis, no significant difference was noted between the alternative
and reference methods for the detection of L. monocytogenes from enriched plastic (χ2=1.9) and sealed concrete
(χ2=0.4) sponge samples. Stainless steel sponge samples tested using the alternative method showed
significantly better detection (χ2=3.9) in comparison with the reference method (Fig. 5).
Detection of Listeria spp. in Environmental Samples Enriched with Actero™ Listeria Using BAX®
System RT-PCR Assay. Significant differences were observed with stainless steel (χ2=3.9) and sealed concrete
(χ2=5.1) samples tested using BAX® System RT-PCR Genus Listeria assay as compared to the FSIS-USDA
MLG 8.09 method while plastic samples did not show any significant difference (χ2=2.6) (Fig. 6).
Fig. 5. Performance Differences Between the BAX® System RT-PCR
Assay for L. monocytogenes and the USDA-FSIS Method
Method Comparison Study: BAX® System RT-PCR Assay vs the USDA-FSIS MLG 8.09 Method
Fig. 6. Performance Differences Between the BAX® System RT-PCR
assay for Genus Listeria and the USDA-FSIS Method
Detection of Listeria spp. in Environmental Samples Enriched with Actero™ Listeria Using BAX® System
RT-PCR Assay. Based on the POD analysis, no significant difference was noted between the alternative and
reference methods for the detection of Listeria spp. from enriched stainless steel (χ2 = 1.0), plastic (χ2 = 0.5),
sealed concrete (χ2 = 1.0), ceramic (χ2 = 0.4) and rubber (χ2 = 0.1) sponge samples (Fig. 7).
Method Comparison Study: BAX® System RT-PCR Assay vs MFHPB-30 Method
1. Performance of both DuPont™ BAX® System RT-PCR assays to detect Listeria spp.
and L. monocytogenes in environmental samples enriched with Actero™ Listeria is
equivalent or superior to the USDA-FSIS MLG 8.09 and the Health Canada MFHPB-30
reference methods.
2. Only 20 hours of enrichment are sufficient to successfully recover and detect
Listeria spp. and/or L. monocytogenes from environmental samples when the
combination of the Actero™ Listeria and DuPont™ BAX® Real-Time PCR assays is
used.
1.Zwietering MH, et al. 2016 Relevance of microbial finished product testing in food safety management. Food Control 60: 31–43.
2.Crandall PG et al. 2011. Minimizing the risk of Listeria monocytogenes in retail delis by developing employee focused, cost effective training. Agric Food Anal Bacteriol 1(2):
159–173.
3. USDA-FSIS. 2012. Isolation and Identification of Listeria monocytogenes from red meat, poultry, egg, and environmental samples. In: Microbiology Laboratory Guidebook,
Chapter 8.09. http://www.fsis.usda.gov/wps/wcm/connect/1710bee8-76b9-4e6c-92fc-fdc290dbfa92/MLG-8.pdf?MOD=AJPERES
4.Pagotto, F., Heber K., and J. Farber. 2011. MFHPB-30. Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples. In: Volume 2,
Compendium of Analytical Methods.
5.Microbiological Methods Committee Evaluation Division, Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch (HPFB), Sir Frederick G.
Banting Research Centre. March 2011. Annex 4.4: Procedure For The Statistical Evaluation And Calculation Of Performance Parameters Of A New Alternative Qualitative
Method Compared To A Reference Cultural Method. In: Volume 1, Compendium of Analytical Methods.
6.Microbiological Methods Committee Evaluation Division, Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch (HPFB), Sir Frederick G.
Banting Research Centre. April 2008. Procedure for the Development and Management of Food Microbiological Methods. In: Volume 1, Compendium of Analytical Methods.
7.AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces Official Methods of Analysis of
AOAC INTERNATIONAL, 19th Ed., Appendix J, AOAC INTERNATIONAL, Gaithersburg, MD. http://www.eoma.aoac.org/app_j.pdf
Notes: P – positive result.
N – negative result.
FP – false positive result.
FN – false negative result.
Relative sensitivity – (true positives / true positives + false negatives) × 100.
Relative specificity – (true negatives / true negatives + false positives) × 100.
False negative (FN) rate – (false negatives / true positives + false negatives) × 100.
False positive (FP) rate – (false positives / true negatives + false positives) × 100.
Test Efficacy – (true positives +true negatives / true positives+ false positives+ true negatives + false negatives) × 100.
Fig. 7. Performance Differences Between the BAX® System RT-PCR
Assay for Genus Listeria and MFHPB-30 Method
BAX® System RT-PCR Assay Performance Analysis
The performance parameters of relative sensitivity, relative specificity, false positive rate, false negative rate, and
test efficacy were calculated for the alternative method and presented in Table 3.
Table 3. Performance Parameters for Alternative Method
Listeria monocytogenes is an emerging foodborne bacterium responsible for listeriosis that continues to be
problematic for the food industry. Furthermore, one of the particular concerns is a post-processing contamination
of ready-to-eat food, including hot dogs and deli meats that has caused major outbreaks lately. In fact, during
slicing, peeling, packaging, etc., the ready-to-eat products may be re-contaminated by L. monocytogenes that
results in subsequent growth of the pathogen during chilled storage (Fig. 1). The source of re-contamination may
be another piece of contaminated product (cross-contamination), or a biofilm surviving the cleaning and
disinfection procedure on various food contact and non-food contact surfaces (Fig. 2) [1].
Today, assurance of food safety moves more and more from end- or finished product testing to proactive food
safety managing. A finished product testing is no more considered as the most efficient measure to control
L. monocytogenes in ready-to-eat product. Therefore, environmental monitoring is recognized as an important
strategy to control Listeria in food processing facilities. As a part of this program, environmental sample testing is
commonly used to verify L. monocytogenes contamination in all food-contact areas as well as in many non-food
processing areas (Fig. 2) [1]. Moreover, detection of any Listeria species in environmental samples is considered
as an indication of a potential risk to find L. monocytogenes in manufactured food.
Fig. 1. L. monocytogenes Contamination Risks During Deli Meat Processing
Conventional microbiological methods
used for the Listeria detection are
usually very sensitive and remain the
“gold standard” as compared to other
methods, but they are very time-
consuming and are not user friendly.
Although a negative result can be
confirmed in 3–4 days, the time for a
positive result is usually 5–7 days from
sample collection. Currently, PCR-based
methods are rapid and the most
frequently used alternative for
presumptive detection of Listeria in food
and environmental samples. However,
high sensitivity of the PCR-based
methods should be completed using
efficacious enrichment methods to obtain
faster results as food industry need.
The goal of this study was to evaluate a sensitive and rapid method for monitoring L. monocytogenes and
Listeria genus in environmental samples using a rapid, single-stage enrichment in Actero™ Listeria Enrichment
Media (Actero™ Listeria) followed by processing with the BAX® System real-time PCR assays for Listeria and
L. monocytogenes.
Fig. 2. Potential Sources of Recontamination of Post-
Cooking Products by L. monocytogenes