AOAC-RI ERP Book Micro Jan 19 2017

OMAMAN-35 A : Collaborative Study Manuscript For ERP Use Only January 2017

___________________________________________________________________________ 1 Escherichia coli O157:H7 is a gram-negative, facultative anaerobic, rod-shaped bacterium of the genus 2 Escherichia that is commonly found in the lower intestine of warm-blooded organisms like cattle. Several 3 foodborne outbreaks associated with beef as well as other food products such as produce have been attributed to E. 4 coli O157:H7. Outbreaks are often associated with meat products. The organism has a minimum growth range 5 between 6-7°C and therefore has potential for outgrowth in product when stored at the high end of refrigeration 6 temperatures.The 3M ™ Molecular Detection Assay(MDA) 2 – E. coli O157 (including H7)method uses a 7 combination of bioluminescence and isothermal amplification of nucleic acid sequences to rapidly detect E. coli 8 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. 0 The 3M MDA 2- E. coli O157 (including H7) method allows for the rapid and specific detection of E. coli O157 1 (including H7)in select matrices after as little as 10 to 18 hoursof pre-enrichment using an ISO formulation of 2 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 4 real-time while negative results are displayed after completion of the assay in approximately 60 minutes. 5 Prior to the collaborative study, the 3M MDA 2 – E. coli O157 (including H7) method was validated according to 6 AOAC Guidelines[2] in pre-collaborative evaluation. The objective of the pre-collaborativeevaluationwas to 7 demonstrate that the 3M MDA 2- E. coli O157 (including H7)method could detect E. coli O157 (including H7)in 8 select food matrices as claimed by the manufacturer.For the 3M MDA 2 - E. coli O157 (including H7) pre- 9 collaborative evaluation, four matrices were evaluated: raw ground beef (73% lean) (325g), frozen blueberries (25 0 g), fresh babyspinach (200g), and fresh sprouts (25 g). The frozen blueberries, fresh baby spinach and fresh 1 sprouts were evaluated and compared to U.S. Food and Drug Administration Bacteriological Analytical Manual 2 (FDA BAM) Chapter 4A: Diarrheagenic Escherichia coli [3]. All (100%) E. coli O157 strains were detected with 3 the 3M MDA 2 – E. coli O157 (including H7). None of the (100%) non – E. coli O157 strains were detected by 4 the 3M MDA 2 – E. coli O157 (including H7). 5 The purpose of this collaborative studywas to compare the reproducibility of the 3M MDA 2 - E. coli O157 6 (including H7)method to the United States Department of Agriculture (USDA) Food Safety Inspection Service 7 (FSIS) -Microbiology Laboratory Guidebook (MLG) Chapter 5.09 Detection, Isolation and Identification of 8 Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges [4] for raw ground beef 9 (73% lean). 4 5 In this collaborative study, one matrix,325 g of raw ground beef(73% lean) wasevaluated through a qualitative, 6 unpaired study design due to the different sample enrichments for the 3M MDA 2 – E. coli O157 (including H7) 7 method and the USDA/FSIS MLG Chapter 5.09 reference method.The matrix was obtained from a local retailer 8 and screened for the presenceof E. coli O157:H7by the appropriate reference method prior to analysis . The raw 9 ground beef was artificially contaminated with non-heat stressed cells of E. coli O157:H7, American Type Culture 0 Collection (ATCC) 43895 ® at two inoculation levels: a high inoculation level of approximately 2-5 colony-forming 1 units (CFU)/test portion and a low inoculation level of approximately 0.2-2 CFU/test portion. A set of un- 2 inoculated control test portions (0 CFU/test portion) were also included. 3 Twelve replicate samples from each of the three inoculation levels were analyzed by each method. Two sets of 4 samples (72 total) were sent to each laboratory for analysis by 3M MDA2– E. coli O157 (including H7)or the 5 USDA/FSIS MLG Chapter 5.09 method.(Additionally, collaborators were sent a 60 g test portion and instructed to 6 conduct atotal aerobic plate count (APC) using 3M ™ Petrifilm™Rapid Aerobic Count Plate (AOAC Official 7 Method 2015.13) [5]on the day samples testing was initiated for the purpose of determining the total aerobic 8 microbial load. 9 AOAC Research Institute xpert Review Panel Use Only 0 1 2 3 Collaborative Study Study Design

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