Strategic Diagnostics Inc Technical Bulletin Listeria #3
Application of RapidChek for the Detection of Listeria species in RTE Foods and Environmental Samples
Version 1.004-04-04
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Strategic Diagnostics Inc Technical Bulletin Listeria #3
Introduction:
Listeria species are ubiquitous in nature found in environments such as plants, soil, animals, water, dust and silage. Since Listeria species are frequently found in slaughter animals and thus in raw meat and poultry, the organism can be continuously introduced into the processing environment. It has been well documented the ability of this organism to cross contaminate food contact surfaces, equipment, floors, drains etc and to grow under adverse condition including extremes of heat, salt and temperature. The organism has also been found to thrive in damp environments potentially forming biofilms in the processing environment making it difficult to eliminate during cleaning and sanitizing processes.
Among the six species of Listeria commonly found in foods, L. monocytogenes and L. ivanovii are the pathogenic species, capable of causing human illness, with the former species being most commonly linked to foodborne outbreaks. The symptoms of listeriosis vary, ranging from mild flu like conditions to more severe infections of the blood and brain such as septicemia and meningitis, encephalitis. The infectious dose remains unclear but is thought to be high, and host susceptilibility plays a major role in illness with immunocompromised individuals being the most predisposed populations.
In order to prevent occurrence of listeriosis, government regulations stipulate the total absence of Listeria species in food, as presence of the organism, is indicative of insufficient hygiene practices in the handling of raw materials and finished products. Routine testing of high risk products and other ready to eat foods that may support the growth of Listeria is therefore important , for both the prevention of illness and to avoid costly recalls within the food industry. Conventional enrichment and isolation procedure for Listeria in foods tend to be slow and laborious, requiring 3-5 days for presumptive identification.
Strategic Diagnostics Inc. has developed an effective one step enrichment system for the selective growth, isolation and detection of Listeria species in RTE foods and environmental samples. The system consists of proprietary enrichment media which facilitates the maximal recovery of both sub-lethally injured and healthy cells in a sample matrix, while selectively inhibiting closely related competitive flora, within a 40 h time period. No additions or amendments are needed and the broth also allows the option of autoclaving the media or adding the dehydrated media directly to pre-warmed sterile water just prior to use. After the enrichment process, 400ul of the sample is removed and boiled in a test tube for 5 minutes. A test strip is added directly to the test tube and a result is achieved after 10 minutes. During both development and application of the RapidChekListeria system, the assay was subjected to vigorous reactivity tests utilizing typed and field isolates from a variety of sources including clinical, environmental and food.
Aim:
To evaluate the use of the RapidChek system for the enrichment and detection of Listeriaspecies in a variety of processed foods and environmental samples.
Experimental Protocol:
Deli turkey, hotdogs, pepperoni, smoked fish, cooked shrimp, whole milk, ice cream, potato salad, painted concrete and stainless steel were analyzed by the lateral flow strip method to determine the agreement of the kit performance relative to the corresponding reference methods for each food and surface matrix examined. Replicate samples at two inoculum levels (zero and low) were examined for each matrix. External studies were carried out at SillikersCorporateResearchCenter, to validate the procedure for roast beef, ricotta cheese and rubber surfaces.
Various species and isolates of Listeria were grown in 10 ml TSB broth at 37C for 18 h. Two thousand grams of each food type including deli turkey, hot dogs and pepperoni were inoculated with a heat stressed (50oC for 10 min) low inoculum (1-10 cfu/25g) based on microbial plate count results and homogenized by shaking and mixing manually in a large stomacher bag for 5 minutes. Samples were held at 4oC for 48 h. A 3 tube 4 level MPN (most probable number) was set up using the USDA enrichment procedure, to determine cell levels in the sample after the stress period. In short 100, 10, 1, and 0.1g of inoculated materials were stomached in UVM media, incubated at 30+/- 2C for 22h , transferred to Fraser broth (FB) and incubated for an additional 26 h at 35 +/- 2C. Samples were struck to MOX agar plates and confirmed. The 100, 10 and 1g samples were subsequently used to determine the MPN.
Inoculated meat was divided into two sets of twenty 25 g samples. One set was stomached with 225 ml of the RapidChek Test Media for 1 minute and incubated for 40 h at 30+/- 2C. Samples were heat treated and tested on the lateral flow strip.
A second set of twenty 25 g samples of inoculated materials were enriched in 225 ml of UVM, which was incubated at 30C +/- 2C for 22 h. Samples were then transferred to FB for an additional 26h incubation at 35+/- 2C according to the USDA/FSIS reference protocol for Listeria monocytogenes, Chapter 8. All samples were confirmed using the standard cultural and biochemical tests recommended by USDA/FSIS.
For smoked fish, cooked shrimp, ice cream, potatoe salad, and milk the FDA reference method was used as a comparison. Listeria inoculum was set up as previously described. Each Inoculated food was divided into two sets of twenty 25 g samples. One set was stomached with 225 ml of the RapidChek Test Media for 1 minute and incubated for 40 h at 30+/- 2C. Samples were heat treated and tested on the lateral flow strip.
A second set of twenty 25 g samples of inoculated cheese were enriched in 225 ml of BLEB, which was incubated at 30C +/- 2C for 4 h. Samples were then supplemented with an addition of selective agents and incubated an additional 44 h at 30 +/- 2C. as described in the FDA reference protocol for Listeria monocytogenes, Chapter 10. All samples were confirmed using the standard cultural and biochemical tests recommended by FDA/BAM.
For environmental swabs of painted concrete and stainless steel, the Listeria inoculum was grown in 10 ml TSB broth at 37C for 18 h. One square inch painted surfaces/stainless steel were inoculated based on microbial plate count with 1x104 cfu/4 inch2 in 10% skim milk and allowed to dry for 24 hours. Swab samples were taken using sterile swabs moisten with D/E broth. The entire 1 inch2 surface was scrubbed with a single swab for approximately 30 seconds. One set of 25 swabs was added to tubes containing 10 ml of the RapidChek Test Media and incubated for 40 h at 30+/- 2C. Samples were heat treated and tested on the lateral flow strip.
A second set of 25 swab samples were enriched in 225 ml of of UVM, which was incubated at 30C +/- 2C for 22 h. Samples were then transferred to FB for an additional 26h incubation at 35+/- 2C according to the USDA/FSIS reference protocol for Listeria monocytogenes, Chapter 8. All samples were confirmed using the standard cultural and biochemical tests recommended by USDA/FSIS.
Procedures carried out at Silliker were similar to those described above for the additional matrices.
Results :
Method Comparision of RapidChek to the cultural reference method for the Detection of Listeria species in Processed Foods
Food / Strain / Total# / MPN/
25g / Rapid
Chek
Positive / Reference method Positive
Hot dogs / L. welshimeri / 5
20 / 0
2.3 / 0
16 / 0
16
Pepperoni / L. mono
3b / 5
20 / 0
1.1 / 0
10 / 0
12
*Roast Beef / L. innocua / 5
20 / 0
0.2 / 0
14 / 0
9
*Ricotta Cheese / L. mono
4b / 5
20 / 0
0.6 / 0
12 / 0
13
Deli
Turkey / L. mono
1/2b / 5
20 / 0
0.4 / 0
14 / 0
13
Ice Cream / L. mono
4b / 5
20 / 0 / 0
20 / 0
16
Milk / L. mono
3a / 5
20 / 0
17 / 0
18 / 0
Smoked
Fish / L. mono 7 / 5
20 / 0
5.0 / 0
14 / 0
16
Cooked
Shrimp / L. mono
3c / 5
20 / 0
0.2 / 0
11 / 0
11
Potato
Salad / L. mono
1/2a / 5
20 / 0
1.1 / 0
16 / 0
18
Method Comparision of RapidChek to the cultural reference method for the Detection of Listeria species in environmental samples
Food / Strain / Total# / MPN/
25g / Rapid
Chek
Positive / Reference method Positive
Concrete / L. mono
4b / 5
20 / 0
1x10^4 / 0
18 / 0
10
Stainless
Steel / L. innocua / 5
20 / 0
1x10^4 / 0
18 / 0
15
*Rubber / L. mono
4b / 5
20 / 0
1x10^3 / 0
9 / 0
3
*Matrices carried out at SillikersCorporateResearchCenter, Chicago, IL.
Discussion and Conclusions:
The use of the lateral flow test as well as the MOX plate with the 40h proprietary enrichment method has demonstrated excellent accuracy, sensitivity and specificity throughout this study The 2 methods have been shown to be capable of detecting very low levels (1cfu/25g) of Listeria spp. in a variety of food and select environmental matrices. There was excellent method agreement between the lateral flow system and the cultural confirmation. Results from select food matrices indicate that levels as low as 0.2cfu/25g can be successfully detected although overall, these studies demonstrates that low levels (1-10cfu/25g) of Listeria can be recovered and detected in a food matrix in a minimum of 40 h. The RapidChek system, however, offers a faster turnaround time than the traditional reference method with significant savings on time and labor.
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Strategic Diagnostics Inc
111 Pencader Drive
Newark, DE 19702
Phone: 800-544-8881
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Version 1.004-04-04
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