1. © 3M 2012. 3M is a trademark of 3M.
These studies demonstrated the specificity of the 3M Salmonella and E. coli O157 (including H7)
methods and their compatibility with a variety of foods, environmental and carcass samples.
CONCLUSIONS
1. University of Minnesota Department of Animal Science
2. University of Calgary Salmonella Genetic Stock Center
3. Pennsylvania State University Department of Veterinary and Biomedical Sciences
4. The authors thank Molly Rother, Amber Turner and Olivia Trittipo for their dedicated
laboratory work.
ACKNOWLEDGEMENTS
P2-142: Performance of the 3M™
Molecular Detection System for the Detection of Salmonella and Escherichia coli O157
3M Food Safety, 3M Center, St. Paul, MN, United States: John M. David; Micki L. Rosauer; Cynthia D. Zook
Testing for Salmonella and E. coli O157:H7 is a critical component of food safety programs
as contamination by these pathogens can result in significant adverse health conditions
and economic loss. The 3M™
Molecular Detection Assay Salmonella and the 3M™
Molecular
Detection Assay E. coli O157 (including H7) were developed for the rapid and specific
detection of these contaminants in samples after enrichment. The assays use isothermal
amplification of nucleic acid sequences with high specificity, efficiency and speed, and
bioluminescence to detect the amplification. Presumptive positive results are reported in
real-time while negative results are displayed after the assay is completed.
ABSTRACT
INCLUSIVES
Fifty-two (52) E. coli O157 isolates were tested. The E. coli O157 strains were cultured
overnight then diluted to a level of less than 105
CFU/mL (Colony Forming Unit/mL)
prior to testing. A false-negative rate of 0% (100% inclusivity) was determined.
EXCLUSIVES
Fifty (50) non-E. coli O157 isolates were tested. The non-E. coli O157 strains
were cultured overnight to reach a minimum level of 100X the limit of detection.
A false-positive rate of 0% (100% exclusivity) was determined.
E. coli O157: STUDIES RESULTS
Beef carcasses were tested at different process steps to evaluate the performance of the
method in the presence of possible interference from matrix and/or competing microbiota.
Sixty (60) carcass samples were collected by sampling the right and left side of each
animal, at similar anatomical locations, and then paired into one bag. Swabbing was
performed at the following process steps per USDA guidelines, summarized below.
Sampling Material Carcass Sample Steps
3M™
Cattle/Swine Kit, 25mL BPW
• Prior to hide removal (100cm2
)
• Pre-evisceration (8000cm2
)
• Post-wash (8000cm2
)
Suspensions of E. coli O157 were used to artificially contaminate one of the split samples
at a level of 10 CFU and one duplicate was enriched blank. All enrichment were performed
at 41.5°C for 18 hours then tested using the 3M Molecular Detection Assay E. coli O157
and the 3M Molecular Detection Matrix Control. All enrichments were also streaked onto
chromogenic E. coli O157 agar plates, used as reference point. A chi-square (X2
) test was
used to compare the results for significant differences.
E. coli O157 RESULTS
The method was shown to be compatible with a variety of relevant food matrices. All results
were as expected: negative (blank samples), positive (artificially contaminated at a level
of 5–7 CFU) and valid (matrix control). None of the matrices tested demonstrated inhibition
(total or partial) of the assay.
Target Sample n Accuracy Specificity Sensitivity X2
E. coli O157
Foods 66 100% 100% 100% —
Carcass 60 95% 100% 92% 1.33
No significant differences were observed between the 3M method results and the
chromogenic agar results as indicated by a X2
value of less than 3.84. The method was
shown to be compatible with carcass swabs samples, including fecal containing samples.
The 3M Molecular Detection Matrix Control yielded valid results for all samples tested.
CARCASS STUDY
Studies were conducted to assess the performance of the method (including
enrichment broth and assay) in the presence of possible interferences from sample
matrix and other organisms.
STUDY DESIGN
Several serotypes of E. coli O157 were used to artificially contaminate food samples
commonly tested and/or reported to be challenging due to their composition, i.e. high
fat, high calcium, high native microflora, etc.
Thirty three (33) different food matrices were evaluated as follow: either spiked
at a level of 5–7 CFU E. coli O157 per 25g sample size or enriched as blanks. All
enrichments were performed at a 1:10 dilution in pre-warmed 3M BPW-ISO and were
incubated for 8 hours (raw meat) or 18 hours (all other foods) at 41.5°C. All artificially
contaminated samples were tested using both the 3M Molecular Detection Assay
E. coli O157 and the 3M™
Molecular Detection Matrix Control, while enriched blanks
were tested using the 3M Molecular Detection Assay E. coli O157 only. All enrichments
were also streaked onto chromogenic E. coli O157 plates, used as reference point.
E. coli O157: FOOD CARCASS SAMPLE STUDIES
SIMPLIFIED FOR PRODUCTIVITY
INCLUSIVES
One hundred and four (104) Salmonella isolates were tested. The Salmonella strains were
cultured overnight in 3M™
BPW-ISO, then diluted to a level of less than 105
CFU/mL (Colony
Forming Unit/mL) prior to testing.
EXCLUSIVES
Fifty (50) non-Salmonella isolates were tested. The non-Salmonella strains were cultured to
reach a minimum level of 100X the limit of detection prior to testing.
Performance Cultures Result
Inclusivity
104 strains including following Salmonella subspecies:
enterica, salamae, arizona, diarizonae, houtenae, bongori and indica
99%
Exclusivity 50 strains including Citrobacter, Enterobacter, E. coli, Proteus, Shigella, Yersinia, etc. 100%
Salmonella: STUDIES RESULTS
A variety of environmental samples and poultry rinses were
tested to evaluate the performance of the method in the presence
of possible interference from matrix and/or competing microbiota.
One hundred and forty two (142) different environmental
samples, including poultry drag swabs, were collected in
duplicate from various farms. Surfaces tested included concrete
floors and ceiling, wooden roosts, metal feeders, and stainless
steel equipment and pipes.
Eighty-four (84) bird rinses were provided by multiple poultry
processing plants. Sample collection devices, hydrating
solutions and enrichment volumes are summarized out right.
Technology Comparisons
Sample Sampling Device Hydrating Solution 3M BPW-ISO Enrichment Volume
Poultry carcass rinse None BPW 30mL
Surfaces 3M™
Dry-Sponge 10mL D/E Neutralizing Broth 50mL
Poultry fecal drag swab 3M™
Dry-Sponge with String 10mL BPW 50mL
Salmonella Studies Results
Target Sample n Accuracy Specificity Sensitivity X2
Salmonella
Foods 67 99.3% 100% 98.5 0.00
Bird Rinse 84 97.6% 100% 93.9% 1.33
Environmental 142 99% 100% 95% 0.00
Salmonella: ENVIRONMENTAL POULTRY RINSE STUDIES
Studies were conducted to assess the performance of the method in the presence of possible
interferences from sample matrix and other organisms. Strains of Salmonella Typhimurium,
S. Enteritidis and S. Newport were used to artificially contaminate food samples commonly
reported in outbreaks and/or reported to be challenging due to their composition, i.e. high fat,
high calcium, high native microflora, etc.
Sixty-seven (67) different food matrices were evaluated as follows: either spiked at a level of
7–13 CFU Salmonella per sample size or as enriched blanks. All enrichments were performed
at a 1:10 dilution in pre-warmed 3M™
BPW-ISO and were incubated for 18 hours at 37°C. All
artificially contaminated samples were tested using both the 3M Molecular Detection Assay
Salmonella and the 3M Molecular Detection Matrix Control, while enriched blanks were tested
using the 3M Molecular Detection Assay Salmonella only. All enrichments were also streaked
onto chromogenic Salmonella and/or XLD agar plates, as a reference point.
Salmonella: FOOD STUDIES
E. coli O157: FOODS
Meat
– 75% lean ground beef, raw
– 85% lean ground beef, raw
– 96% lean ground beef, raw
– 93% lean ground beef, raw
– Grass fed ground beef, raw
– 85% lean grass fed ground
beef, raw
– Raw beef trim 1
– Raw beef trim 2
– Raw pork
Dairy
– 1% pasteurized milk
– 4% pasteurized milk
– Lactose free milk
– Raw milk 1
– Raw milk 2
– Raw milk 3
Produce
– Apple juice
– Organic apple juice
– Blended fruits juice
– Raw organic cucumber
– Raw conventional
cucumber
– Raw tomato
– Alfalfa sprouts
– Organic salad blend
(Sprouts, clover, alfalfa,
broccoli sprouts)
– Daikon radish sprouts
– Bagged baby spinach
– Bagged hearts of lettuce
– Organic arugula
Processed Foods
– Refrigerated peanut
butter milk chocolate
chip cookie dough
– Refrigerated chocolate
chip dough
– Refrigerated pie crusts
– Thin crispy crust frozen
pepperoni pizza
– Sausage and
pepperoni pizza
– 5 cheese
frozen lasagna
Salmonella: FOODS
Meat
– Turkey breast, raw, ground
– Turkey, raw, ground
– Chicken, raw, ground
– Chicken, frozen nuggets
– Chicken, crispy breast strips
– Breaded nuggets
(chicken – substitute)
– Beef, raw, ground, 80% lean
– Beef, raw, ground, 85% lean
– Beef, raw, ground, Angus steak
Dairy
– Milk, instant non fat dry
– Milk, fluid, 1% milk fat
– Milk, fluid, 4% milk fat
– Milk, lactose free, 1%
– Buttermilk (dry)
– Ice cream, vanilla
– Ice cream, butter pecan
– Ice cream, toasted almond fudge
– Frozen mozzarella sticks
– Cheese, processed block
– Cheese, processed slices
– Cheese, processed spread
Egg Products
– Liquid egg substitute
– Egg, raw shell
– Egg, pre-cooked, shelled
– Egg white, liquid, 100%
– Egg (dried whole)
– Egg (dried egg white)
Produce
– Breaded jalapenos
with cheddar cheese
– Mushrooms, breaded fried
– Lettuce, hearts, pre-washed
– Arugula, bagged, pre-washed
– Carrot juice
– Apple juice
– Orange juice from concentrate*
– Orange juice, pulp free
Seafood
– Breaded fish sticks, minced
– Frozen beer-batter fillets
– Grilled fish fillet, lemon pepper
– Cooked frozen shrimp
– Shrimp, canned
– Shrimp, raw frozen
Confectionery Products
– Chocolate bar, 72% cacao
– Chocolate, dark chocolate almond
– Chocolate covered peanuts
– Chocolate, milk chocolate
Spices and Condiments
– Paprika
– Black pepper
– Parsley flakes, dried
– Cinnamon*
– Oregano*
Pet Foods
– Dog food, dry
– Dog food, canned
– Dog food
– Soft bacon wrapped sweet potato
intermediate moisture dog treat
Nuts
– Peanut butter
– Hazelnut spread
– Sunflower seed spread
Other Processed Foods
– Whey protein
– Vanilla nutritional drink
– Chocolate nutritional drink
– Milk-based infant formula (2)
– Soy-based infant formula
– Cereal, crispy rice
– Cereal, golden puffs
– Cereal, blueberry
*Dilution required
3M Method Salmonella
Sample Enrichment Incubation
18–24 hr at 37°C (± 1°)
3M Method E. coli O517 (including H7)
Sample Enrichment Incubation
8–24 hr at 41.5°C (± 1°)
3M Method Listeria
Sample Enrichment Incubation
24–28 hr at 37°C (± 1°)
Transfer 20µL enriched
sample to lysis tube.
LS
20µL
Transfer 20µL
lystate to reagent tubes
containing lyophilized pellet.
Transfer closed tubes
to Speed Loader Tray.
Performs amplifications in 75 minutes.
Automated and color coded real-time results.
TTR:
10–30 hours
99-101ºC
Heat 15 minutes
at 100°C (±1°).
0-20ºC
Cool 10 minutes
on chill block.
Let sit 5 minutes.
Place Speed Loader Tray
into instrument. Close the
lid and start run.
One
Protocol
for All
Pathogen
Targets