215 / 453
3
forming units (CFU)/test portion and a low inoculation level of approximately 0.2-2 CFU/test
1
portion. A set of un-inoculated control test portions (0 CFU/test portion) were also included.
2
Twelve replicate samples from each of the three inoculation levels were analyzed. Due to a
3
common primary enrichment, 1 set of 325 gram samples (36 total) wassent to each participating
4
technician for analysis by both candidate methods (
mericon
E. coliO157 Screen Plus Pathogen
5
Detection Assay and
mericon
E. coliSTEC O-Type Pathogen Detection Assay) and reference
6
method. The candidate methods were evaluated using both the manual and automated DNA
7
extraction method.Additionally, collaborators were sent a 60 g test portion and instructed to
8
conduct atotal aerobic plate count (APC) using 3M
™
Petrifilm™Rapid Aerobic Count Plate
9
(AOAC Official Method 2015.13) [5] on the day samples were received for the purpose of
10
determining the total aerobic microbial load.
11
12
A detailed collaborative study packet outlining all necessary information related to the study
13
including media preparation, test portion preparation and documentation of results was sent to
14
each collaborating laboratory prior to the initiation of the study.
15
16
Preparation of Inoculum and Test Portions
17
18
The
Escherichia coli
O157:H7ATCC 43895 culture used in this evaluation was propagated onto
19
Tryptic Soy Agar with 5% Sheep Blood (SBA) from a Q Laboratories frozen stock culture stored
20
at -70°C. The organism was incubated for 24 ± 2 hours at 35 ±1°C. Isolated colonies were
21
picked to 10 mL of Brain Heart Infusion (BHI) broth and incubated for 18 ± 0.5 hours at 35
22
±1°C.Appropriate 1:10 dilutions were prepared in Butterfield’s Phosphate Diluent (BPD) based
23
on previously established growth curves for both low and high inoculation levels. Bulk portions
24
of the matrix were inoculated with the diluted liquid inoculum and mixed thoroughly to ensure
25
an even distribution of microorganisms. For the preparation of test portions, 25 g of inoculated
26
test product was mixed with 300 g of un-inoculated test product to prepare 325 g test portions
27
which were packaged in sterile Whirl-Pak
®
bags and shipped to collaborators.
28
To determine the level of
Escherichia coli
O157:H7 in the matrix, a 5-tube most probable
29
number (MPN) was conducted by the coordinating laboratory on the day of the initiation of
30
analysis using the USDA/FSIS-MLG 5.09 reference method. The MPN was determined by
31
analyzing 5 x 650 g test portions,thereference method test portions from the collaborating
32
laboratories and 5 x 160 g test portions. The MPN and 95% confidence intervals were calculated
33
using the LCF MPN Calculator, Version 1.6,
34
( www.lcftld.com/customer/LCFMPNCaclucator.exe ), provided by AOAC Research Institute
35
(RI)[6].
36
37
Test Portion Distribution
38
39
All samples were labeled with a randomized, blind-coded 3-digit number affixed to the sample
40
container. Test portions were shipped on a Thursday via overnight delivery according to the
41
Category B Dangerous Goods shipment regulations set forth by the International Air
42
Transportations Association(IATA).Upon receipt, samples were held by the collaborating
43
laboratory at refrigeration temperature (2-8 °C) until the following Monday when analysis was
44
initiatedafter a total equilibration time of 96 hours.All samples were packed with cold packs to
45
target a temperature of < 7°C during shipment.
46
47
In addition to each of the test portions and a separate APCsample, collaborators received a test
48
portion for each matrix labeled as ‘temperature control’. Participants were instructed to obtain
49
OMAMAN-36 A : Collaborative Study Manuscript
For ERP Use Only
January 2017
AOAC Research Instit te
Expert Review Panel Use Only