1 Annual General Meeting 2011 .

Accelerated Detection of Listeria and Salmonella in

Environmental and Food Samples Using PCR Technology Sergiy Olishevskyy1, Melissa Buzinhani1, Morgan Wallace2, Michael Giuffre3 1FoodChek Laboratories Inc., suite 200, 7755 Duplessis, St-Hyacinthe, Quebec, Canada J2R 1S5 2DuPont Nutrition &Health, Experimental Station 400, P.O. Box 8352, Wilmington, Delaware, 19803, USA

3FoodChek Systems, suite 450, 1414 – St. S.W., Calgary, Alberta, Canada, T2R 1J6

Method Protocol Introduction

Results Materials and Methods

References Conclusions

Method Claims Analytes: Listeria spp.

Matrices:

environmental samples

stainless steel

plastic

sealed concrete

food samples

frankfurters

soft Mexican style cheese

bagged fresh spinach

frozen cooked shrimp

cold smoked salmon

Analytes: Salmonella

Matrices:

environmental samples

stainless steel

plastic

food samples

dry pet food

milk chocolate

chocolate liquor

cocoa powder

shell egg

Limit of Detection: one CFU per food sample, or equivalent recovery from 100 cm2

environmental surface.

Step 1 Sample Enrichment

Step 2 Sampling

(with or without dilution)

Step 3 DNA Isolation

Step 4 DNA Amplification

Step 5 Result Analysis

Foodborne infections are among the most widespread public health problems. A wide spectrum of pathogens play a role in foodborne disease. Nontyphoidal

Salmonella enterica serovars and Listeria monocytogenes are classic foodborne pathogens that cause two of the most common foodborne diseases called

salmonellosis and listeriosis respectively. Early and sensitive detection of these foodborne pathogens remains a critical issue in public health policy [1].

The process of isolation and final identification of Listeria and Salmonella with culture and biochemical standard methods are laborious and time-consuming and

can take up to 7–10 days. In recent years, PCR-based methods have been reported as rapid, specific and sensitive alternatives to identify these bacteria from

food and environmental samples [2].

The DuPont™ BAX® System real-time PCR assays were designed to report qualitative results for the detection of Listeria and Salmonella in food and

environmental samples. The processing time is approximately 70 min in the BAX® System Q7 instrument, and the method returns results comparable to the

culture methods but with a significantly faster time to result.

However, to take full advantage of the sensitivity and speed offered by alternative methods of pathogen testing, the food industry requires faster sample

enrichment. This can be achieved using effective selective enrichment media such as Actero™ Listeria Enrichment Media (Actero™ Listeria) and Actero™

Salmonella (Actero™ Salmonella) Enrichment Media developed by FoodChek Systems Inc. for single-step recovery of Listeria [3] and Salmonella [4] from

environmental and food samples.

The aim of the study is to develop and to validate a rapid screening method that significantly minimizes the complexity and length of enrichment for PCR-

based detection of Listeria and Salmonella in environmental and food samples.

In total, 1230 environmental sand food samples were analysed in a performance method comparison study conducted according to the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of

Qualitative and Quantitative Food Microbiological Official Methods of Analysis [5] (Fig. 1).

Detection of Listeria spp. Eight internal and two independent laboratory studies (Fig. 1) were carried out to evaluate the performance of the DuPont™ BAX® System Real-Time PCR Assay for Genus Listeria in

environmental and food samples (Fig. 2) enriched with Actero™ Listeria in comparison with the USDA-FSIS [6] or US FDA [7] reference methods.

Detection of Salmonella. Nine internal and two independent laboratory studies (Fig. 1) were carried out to evaluate the performance of the DuPont™ BAX® System Real-Time PCR Assay for Salmonella in

environmental and food samples (Fig. 3) enriched with Actero™ Salmonella in comparison with the US FDA reference method [8].

General protocol. Each type of sample was split into three sets. The first set of 40 samples was inoculated in bulk with the appropriate target strain at a low concentration in order to achieve fractional (between 25 and

75%) positive results. The second set of 10 samples was similarly inoculated using a high level of target strain that generally did not exceed 10 times the low level of inoculation. Finally, the third portion of 10 samples

remained uninoculated. Half of the samples were analysed using the BAX® System method and then confirmed by transferring to the secondary enrichment according to the appropriate reference method protocol. The

other half of samples were analyzed using the appropriate reference method. Food samples. All the foods sample types were purchased at local grocery stores and screened for the presence of the target bacteria.

Target strains were heat stressed in order to achieve a 50–80% injury of the inocula prior to the artificial contamination of the frankfurter,

frozen cooked shrimp, milk chocolate and chocolate liquor samples. Lyophilized Salmonella strains were used to inoculate dry pet food

and cocoa powder. Soft Mexican style cheese and shell egg samples were inoculated with unstressed target strains. The inoculated

and uninoculated samples were stabilized at 4°C for 48–72 h (soft Mexican style cheese fresh bagged spinach, frankfurters, cold

smoked salmon, shell egg), at room temperature (dry pet food, milk chocolate, chocolate liquor, cocoa powder) or at -20°C (frozen

cooked shrimp) for at least two weeks prior to testing. For each food type, the concentration of the inoculum was confirmed using the

MPN procedure. The samples were enriched with Actero™ Listeria or Actero™ Salmonella at 35°C for 16-26 hours (See Table 1, and

Fig. 4 and Fig. 5 for specific enrichment times for each target for each matrix).

Environmental samples. Environmental surfaces (stainless steel, plastic and sealed concrete) of 100 cm2 were inoculated with low

concentration of pure target bacterium cultures or with mixed cultures of the target and non-target bacteria (approx. ratio 1:10) prepared

using 10% nonfat dry milk. The high level of inoculation did not exceed 10 times the low level of inoculation. For each type of

environmental matrix, 40 surfaces of 100 cm2 were inoculated with 250 µL of the low level mixed culture by spreading many drops over

the entire surface. A first set of 10 surfaces was similarly inoculated using the high level mixed culture. Another set of 10 surfaces was

then inoculated with 250 µL of 10% nonfat dry milk as negative control. The surfaces were dried for 18 to 20 h at room temperature (22

2°C) inside a closed safety cabinet. Each plate was swabbed using non-bactericidal sampling sponge. The sponges were placed in

sterile sample bags and kept at room temperature for at least 2 h before enrichment with Actero™ Listeria or Actero™ Salmonella at

35°C for 14-20 hours (Table 1).

1.Hanson et al. 2012. Estimating global mortality from potentially foodborne diseases: an analysis using vital registration data. Popul Health

Metr 2012, 10: 5.

2.Lopes-Campos G., Martinez-Suarez J.V., Aguado-Urda M. 2012. Detection, identification, and analysis of foodborne pathogens. In:

Microarray detection and characterization of foodborne pathogens. SpringerBriefs in Food, Health, and Nutrition. 126 p.

3.Claveau et al. 2014. Detection of Listeria spp. using Actero Listeria Enrichment Media. J AOAC Int, 97 (4): 1127-1136.

4.Tremblay et al. Actero™ Salmonella/STEC Enrichment Media Promotes the Recovery of Salmonella spp. and STEC strains. Inside

Laboratory Management. 2015. In Press..

5.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.

6.USDA-FSIS. 2012. Isolation and identification of Listeria monocytogenes from red meat, poultry, egg, and environmental samples. In:

Microbiology Laboratory Guidebook, Chapter 8.09.

7.Hitchins A.D., Jinneman K. 2014. Chapter 10. Detection and enumeration of Listeria monocytogenes in foods. In: FDA Bacteriological

Analytical Manual.

8.Andrews W.H., Jacobson A., Hammack T. 2014. Chapter 5. Salmonella. In: FDA Bacteriological Analytical Manual.

Fig. 1. Comparison Method Study Design Fig. 2. Validation Studies for Detection of Listeria spp. Fig. 3. Validation Studies for Detection of Salmonella

Target Sample Type Sample

Size

Enrichment Medium Volume

BAX® System

Method

Reference

Method L

iste

ria

Environmental samples 100 cm2 90 mL 225 mL

Frankfurters 125 g 750 mL 1125 mL

Fresh bagged spinach, soft

Mexican style cheese, frozen

cooked shrimps, cold smoked

salmon

25 g 150 mL 225 mL

Salm

onella

Environmental samples 100 cm2 90 mL 225 mL

Dry pet food 25 g 225 mL

225 mL 375 g 2625 mL

Milk chocolate, cocoa powder 25 g

175 mL

Chocolate liquor 225 mL

Shell egg 20 eggs 1000 mL 2000 mL

Table 1. Enrichment Medium Volume Recommended for BAX® System Method

Notes: *Fresh bagged spinach, soft Mexican style cheese, frozen cooked shrimps, cold smoked salmon.

**Detection time includes sampling, DNA isolation and processing in the BAX® System Q7 instrument. Notes: **Detection time includes sampling, DNA isolation and processing in the BAX® System Q7 instrument.

The unpaired method comparison studies were performed to

evaluate the ability of the BAX® System method to detect artificially

inoculated Listeria spp. and Salmonella in environmental and food

samples enriched with Actero™ Enrichment Media. The studies

were conducted according to the AOAC INTERNATIONAL Methods

Committee Guidelines for Validation of Qualitative and Quantitative

Food Microbiological Official Methods of Analysis.

Enrichment Parameter Optimization Studies. For each

environmental and food matrix selected for the method comparison

study, the main parameters of single step enrichment with Actero™

Media have been optimized. Optimal medium volume (Table 1) and

incubation time (Fig. 4 and Fig. 5) of samples were determined. As

a result, enrichment of Listeria spp. from environmental and food

samples enriched with Actero™ Listeria was significantly reduced

(up to 20-26 hours of incubation) as compared to the reference methods (approx. 38-48 hours of incubation) (Fig. 4).

Table 2. Method Comparison Study for Detection of Listeria spp.

Table 3. Method Comparison Study for Detection of Salmonella

For recovery of Salmonella from environmental and food samples, 14-28 hours of incubation in Actero™ Salmonella were found efficacious in comparison to 44-52 hours recommended by the reference method (Fig. 5). Significant reduction of the medium volume used for recovery of Listeria and Salmonella has been achieved for almost all environmental and food matrices (Table 1). BAX® System Method vs FSIS-USDA / US FDA Method Comparison Study for Listeria spp. Detection. Based on the POD analysis, no significant difference was noted between the BAX® System method and

the appropriate reference method for the detection of Listeria spp. in plastic environmental sponge samples as well as in fresh bagged spinach, frozen cooked shrimp and cold smoked salmon samples. Stainless

steel, sealed concrete, soft Mexican style cheese and frankfurter samples tested using the BAX® System method showed significantly superior detection of Listeria spp. as compared to the appropriate reference

methods (Table 2). Only one false negative result was found for stainless steel samples. No false positive outcomes were observed for any environmental and food matrices.

1. The results of the method comparison validation studies demonstrate that performance of the DuPont™ BAX® System real-time PCR assays for Listeria spp. and Salmonella are equivalent or superior to the USDA-FSIS or US FDA reference methods for detecting the target organisms in environmental and food samples enriched with Actero™ Media.

2. These alternative methods are reliable, accurate, and faster than the USDA-FSIS and US FDA reference methods for detection of Listeria and Salmonella.

BAX® System Method vs US FDA Method Comparison Study for Salmonella Detection. Based on statistical

analysis using the POD model, no significant difference was observed between the BAX® System method and the

reference method for the detection of Salmonella in dry pet food, milk chocolate, cocoa powder, shell egg, and

stainless steel and plastic surface samples (Table 3). For chocolate liquor, the candidate method showed

significantly superior performance than the reference method. Neither false negative nor false positive outcomes were observed for any food or environmental samples. 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. The χ2-analysis results greater than 3.84 were considered indicating a significant difference between two methods at a 95% confidence level.

Fig. 4. Enrichment Time Optimized for Recovery of Listeria spp. Fig. 5. Enrichment Time Optimized for Recovery of Salmonella

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