22 / 199
B
ird
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
1, 2017
83
The method was awarded PTM Certification No. 111501 on
November 3, 2015.
http://news.3m.com/press-release/3ms-next-generation-molecular-detection-assay-listeria-receives-aoac-
ptm-validation.
The purpose of this collaborative study was to compare
the reproducibility of the 3M MDA 2–
Listeria
method to
U.S. Department of Agriculture (USDA), Food Safety and
Inspection Service (FSIS)
Microbiology Laboratory Guidebook
(MLG) Chapter 8.09 “Isolation and identification of
Listeria
monocytogenes
from red meat, poultry and egg products, and
environmental samples” (5) for deli turkey (125 g) and raw
chicken breast fillet (25 g).
Collaborative Study
Study Design
In this collaborative study, two matrixes, deli turkey and raw
chicken breast fillet, were evaluated. The matrixes were obtained
from a local retailer and screened for the presence of
Listeria
by
the USDA/FSIS MLG 8.09 reference method. The raw chicken
breast fillet was artificially contaminated with fresh unstressed
cells of
L. monocytogenes
[American Type Culture Collection
(ATCC) 7644] and the deli turkey was artificially contaminated
with heat-stressed cells (Table 1) of
L. monocytogenes
(ATCC
19115) at two inoculation levels: a high inoculation level of
approximately 2–5 CFU/test portion and a low inoculation level
of approximately 0.2–2 CFU/test portion. A set of uninoculated
control test portions (0 CFU/test portion) was also included.
Twelve replicate samples from each of the three inoculation
levels were analyzed by each method. Two sets of samples
(72 samples total) were sent to each laboratory for analysis by
3M MDA 2–
Listeria
and the USDA/FSIS MLG 8.09 reference
method due to the different sample enrichment procedures for
each method. Additionally, collaborators were sent a 60 g test
portion and instructed to conduct a total aerobic plate count
(APC) using 3M Petrifilm Rapid Aerobic Count Plate (AOAC
Official Method
2015.13
; 6) on the day the samples were received
for the purpose of determining the total aerobic microbial load.
A detailed collaborative study packet outlining all necessary
information related to the study—including media preparation,
test portion preparation, and documentation of results—was
sent to each collaborating laboratory prior to the initiation of
the study. A conference call was then conducted to discuss
the details of the collaborative study packet and answer any
questions from the participating laboratories.
Preparation of Inocula and Test Portions
The
Listeria
cultures used in this evaluation were propagated
onto tryptic soy agar (TSA) with 5% sheep blood agar from
a Q Laboratories frozen stock culture stored at –70°C. Each
organism was incubated for 24 ± 2 h at 35 ± 1°C. Isolated
colonies were picked to brain heart infusion broth, 10 mL,
and incubated for 18 ± 0.5 h at 35 ± 1°C. Raw chicken breast
fillet was inoculated in bulk using the fresh broth culture. Prior
to inoculation of the deli turkey, the culture suspension was
heat-stressed at 55 ± 1°C in a water bath for 15 ± 0.5 min to
obtain 50–80% injury, as determined by plating onto selective
modified Oxford agar (MOX) and nonselective TSAwith yeast.
The degree of injury was estimated as follows:
−
×
1
100
select
nonselect
n
n
where
n
select
= number of colonies on selective agar and
n
nonselect
= number of colonies on nonselective agar.Appropriate dilutions
of each culture were prepared in Butterfield’s phosphate diluent
based on previously established growth curves for both low
and high inoculation levels. Bulk portions of each matrix
were inoculated with the diluted liquid inoculum and mixed
thoroughly to ensure even distribution of microorganisms. The
inoculated raw chicken breast fillet was packaged into separate
30 g test portions in sterile Whirl-Pak bags and shipped to the
collaborators. For the analysis of the deli turkey, 25 g inoculated
test product were mixed with 100 g uninoculated test product to
prepare 125 g test portions that were packaged in sterile Whirl-
Pak bags and shipped to the collaborators.
To determine the level of
Listeria
in the matrixes, a five-
tube most probable number (MPN) was conducted by the
coordinating laboratory on the day of the initiation. To
determine the MPN for the deli turkey test portions, the
coordinating laboratory analyzed 5 × 250 and 5 × 65 g test
portions for each inoculation level. Test portions were enriched
using a 1:10 dilution scheme in University of Vermont medium
(UVM) and analyzed according to the USDA/FSIS MLG 8.09
reference method. Additionally, all test portions submitted by
the collaborating laboratories for low and high levels were
included in the MPN analysis. The MPN and 95% confidence
intervals were calculated using Least Cost Formulations, Ltd
MPN Calculator, Version 1.6, provided by the AOAC Research
Institute (7). For the raw chicken breast fillet, the MPN of the
high and low inoculated levels was determined by analyzing
5 × 50 g test portions, the reference method test portions from
the collaborating laboratories, and 5 × 10 g test portions.
Test Portion Distribution
All samples were labeled with a randomized, blind-coded,
three-digit number affixed to the sample container. Test portions
were shipped on a Thursday via overnight delivery according
to category B dangerous goods shipment regulations set forth
by the International Air Transportations Association. The
two matrixes were shipped consecutively, with collaborating
laboratories performing the analysis on one matrix at a time.
Table 1. Results of heat-stress injury
Matrix
Test organism
a
CFU/MOX
b
CFU/TSA
c
Degree of
injury
d
Deli
turkey
L. monocytogenes
,
ATCC 19115
9.1 × 10
8
2.5 × 10
9
60.8%
Raw
chicken
breast
fillet
L. monocytogenes
,
ATCC 7644
NA
e
NA Raw chicken
product is not
heat-treated, so
it was not
necessary to
injure the cells
a
ATCC=American Type Culture Collection.
b
Selective agar.
c
Nonselective agar.
d
Cultures were heat-stressed for 10 min at 55°C in a recirculating water
bath.
e
NA=Not applicable.