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Escherichia coli
O157:H7 is a gram-negative, facultative anaerobic, rod-shaped bacterium of the genus
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Escherichia
that is commonly found in the lower intestine of warm-blooded organisms like cattle. Several
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foodborne outbreaks associated with beef as well as other food products such as produce have been attributed to
E.
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coli
O157:H7. Outbreaks are often associated with meat products. The organism has a minimum growth range
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between 6-7°C and therefore has potential for outgrowth in product when stored at the high end of refrigeration
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temperatures.The 3M
™
Molecular Detection Assay(MDA) 2 –
E. coli
O157 (including H7)method uses a
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combination of bioluminescence and isothermal amplification of nucleic acid sequences to rapidly detect
E. coli
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O157 (including H7)in select food matrices.The isothermal amplification is a molecular reaction conducted at a
9
constant temperature, eliminating the need for temperature cycling and decreasing the time to results.
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The 3M MDA 2-
E. coli
O157 (including H7) method allows for the rapid and specific detection of
E. coli
O157
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(including H7)in select matrices after as little as 10 to 18 hoursof pre-enrichment using an ISO formulation of
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buffered peptone water (ISO BPW)[1].After enrichment, samples are evaluated using the 3M MDA 2 -
E. coli
3
O157 (including H7)on the 3M
™
Molecular Detection System (MDS). Presumptive positive results are reported in
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real-time while negative results are displayed after completion of the assay in approximately 60 minutes.
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Prior to the collaborative study, the 3M MDA 2 –
E. coli
O157 (including H7) method was validated according to
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AOAC Guidelines[2] in pre-collaborative evaluation. The objective of the pre-collaborativeevaluationwas to
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demonstrate that the 3M MDA 2-
E. coli
O157 (including H7)method could detect
E. coli
O157 (including H7)in
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select food matrices as claimed by the manufacturer.For the 3M MDA 2
- E. coli
O157 (including H7) pre-
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collaborative evaluation, four matrices were evaluated: raw ground beef (73% lean) (325g), frozen blueberries (25
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g), fresh babyspinach (200g), and fresh sprouts (25 g). The frozen blueberries, fresh baby spinach and fresh
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sprouts were evaluated and compared to U.S. Food and Drug Administration Bacteriological Analytical Manual
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(FDA BAM) Chapter 4A:
Diarrheagenic Escherichia coli
[3]. All (100%)
E. coli
O157 strains were detected with
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the 3M MDA 2
– E. coli
O157 (including H7). None of the (100%) non –
E. coli
O157 strains were detected by
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the 3M MDA 2 –
E. coli
O157 (including H7).
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The purpose of this collaborative studywas to compare the reproducibility of the 3M MDA 2 -
E. coli
O157
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(including H7)method to the United States Department of Agriculture (USDA) Food Safety Inspection Service
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(FSIS) -Microbiology Laboratory Guidebook (MLG) Chapter 5.09 Detection, Isolation and Identification of
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Escherichia coli
O157:H7 from Meat Products and Carcass and Environmental Sponges [4] for raw ground beef
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(73% lean).
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Collaborative Study
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Study Design
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In this collaborative study, one matrix,325 g of raw ground beef(73% lean) wasevaluated through a qualitative,
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unpaired study design due to the different sample enrichments for the 3M MDA 2 –
E. coli
O157 (including H7)
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method and the USDA/FSIS MLG Chapter 5.09 reference method.The matrix was obtained from a local retailer
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and screened for the presenceof
E. coli
O157:H7by the appropriate reference method prior to analysis
.
The raw
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ground beef was artificially contaminated with non-heat stressed cells of
E. coli
O157:H7, American Type Culture
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Collection (ATCC) 43895
®
at two inoculation levels: a high inoculation level of approximately 2-5 colony-forming
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units (CFU)/test portion and a low inoculation level of approximately 0.2-2 CFU/test portion. A set of un-
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inoculated control test portions (0 CFU/test portion) were also included.
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Twelve replicate samples from each of the three inoculation levels were analyzed by each method. Two sets of
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samples (72 total) were sent to each laboratory for analysis by 3M MDA2–
E. coli
O157 (including H7)or the
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USDA/FSIS MLG Chapter 5.09 method.(Additionally, collaborators were sent a 60 g test portion and instructed to
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conduct atotal aerobic plate count (APC) using 3M
™
Petrifilm™Rapid Aerobic Count Plate (AOAC Official
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Method 2015.13) [5]on the day samples testing was initiated for the purpose of determining the total aerobic
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microbial load.
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OMAMAN-35 A : Collaborative Study Manuscript
For ERP Use Only
January 2017
AOAC Research Institute
xpert Review Panel Use Only