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1
Internal Study
2
3
Inclusivity and Exclusivity
4
5
Methodology
6
Fifty
frozen
Listeriamonocytogenes
strainsusp
ensions were thawed and
sub-cultured
in brain heart
7
infusion (BHI) broth overnight at 37°C ± 1°C. The cultures were diluted in peptone
salt
8
solution
in order to inoculate between 10 to 100 cells per 225 mL of Demi Fraser. The
9
enrichment broths were incubated for 24 hours at 37°C ± 1°C, and the3M MDA2 -
10
Listeriamonocytogenes
method wasthen performed.
11
Thirty frozen non-
Listeriamonocytogenes
strain suspensions were thawed and sub-cultured
12
grown
in BHI broth overnight at 37°C ± 1°C. The cultures were diluted in buffered peptone water
13
(BPW) or de Man, Rogosa, and Sharpe (MRS) in order to inoculate 10
5
cells/mL.The broths
14
were then incubated for 24 hours at the appropriate incubation temperature in order to have
15
culture to test with the 3M MDA2 –
Listeriamonocytogenes
method.
16
17
Results
18
All 50
Listeriamonocytogenes
strains were detected by the 3M MDA2 -
19
Listeriamonocytogenes
method. None of the 30 non-
Listeriamonocytogenes
strains were detected.
20
See Tables 2 and 3 for study details and results.
21
22
23
Matrix Study
24
25
The matrix study consisted of evaluating a total of 30 un-paired sample replicates for 11
26
matrices, along with a raw chicken leg pieces evaluating 20 sample replicates using two separate
27
lots. Within each sample set, there were 5 uninoculated samples (0 CFU/test portion), 20 low
28
level inoculated samples (0.2-2 CFU/test portion), and 5 high level inoculated samples (2-5
29
CFU/test portion), except for raw chicken leg pieces that were naturally contaminated with the
30
target analyte. The inoculum was prepared by transferring a single
Listeriamonocytogenes
colony
31
from Trypticase soy agar with 5% sheep blood (SBA) into BHI broth and incubating the culture
32
at 35 ± 2
o
C for 24 ± 2 hours. Table 4 presents the sample preparation guidelines for the matrix.
33
All matrices were screened for the presence of the target organism following the appropriate
34
reference method. Additionally, an aerobic plate count (APC) was conducted following the
35
FDA/BAM Chapter 3 reference [9] method to determine the level of background flora in each
36
test matrix prior to inoculation.
37
Prior to inoculation of beef hot dogs, deli turkey, and queso fresco the broth culture inoculum
38
was heat stressed for 10 ± 1 minute at 50 ± 1°C in a water bath. The degree of injury of the
39
culture was estimated by plating an aliquot of diluted culture onto modified Oxford agar (MOX)
40
and Tryptic Soy agar (TSA). The agars were incubated at 35 ± 1°C for 24 ± 2 hours and the
41
colonies were counted. The degree of injury was estimated as:
42
43
44
45
100 )
1(
x
n
n
nonselect
select
−
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-30 D/ PTM Validation Report 081501
OMA ERP - June 2016
ERP Use Only