175 / 596
1
Results were identical after 24 and 26 hours of sample enrichment. For the low inoculation level
2
of the MDA 2 -
Listeria
assay, there were 15 presumptive positives and 15 confirmed positives
3
following the USDA/FSIS MLG 8.09 reference method confirmation procedure. There were 11
4
observed positives for the reference method. For the high inoculation level, there were 5
5
presumptive positives and 5 confirmed positives following the USDA/FSIS MLG 8.09 reference
6
method confirmation procedure. There were 5 confirmed positives following the reference
7
method. Detailed results of the POD analyses are presented in Tables 9 and 13.
8
9
10
Product Consistency (Lot-to-Lot) and Stability Studies
11
12
Methodology
13
Three lots of MDA 2 -
Listeria
test kits, 1 newly manufactured, 1 at the middle of its expiration,
14
and 1 at or slightly beyond expiration were analyzed to determine the stability of the assay. For
15
the MDA 2 -
Listeria
test kit, one
Listeria monocytogenes
ATCC 7644 isolate was analyzed at a
16
fractional positive level (2-8 positives), and testing 10 replicates. One non-
Listeria
isolate,
17
Enterococcus faecalis
ATCC 29212, was analyzed at the growth level achieved in a non-
18
selective broth, testing 5 replicates.
19
20
Results
21
For the lot-to-lot/stability evaluation of the MDA 2 -
Listeria
assay, there were 6 presumptive
22
positives out of 10 replicates for all three lots at the low inoculation level. For the 5 uninoculated
23
control test portions, there were 0 presumptive positives out of 5 replicates for all three lots. The
24
five control test portions were negative. See Table 16 for lot-to-lot/stability results.
25
26
Robustness Study
27
28
Methodology
29
The robustness evaluated the ability of the method to remain unaffected by minor variations in
30
method parameters that might be expected to occur when the method is performed by an end
31
user. Three different parameters were evaluated, which are presented in Table 18. For each
32
parameter, one
Listeria monocytogenes
ATCC 7644
isolate was analyzed at a fractional positive
33
level (2-8 positives), testing 10 replicates. One non-
Listeria
isolate,
Enterococcus faecalis
ATCC
34
29212, was analyzed at the growth level achieved in a non-selective broth, testing 5 replicates.
35
36
Table B: Robustness Parameters
37
38
Parameter
Low Value
Nominal Value
High Value
Lysis Time
13 minutes
15 minutes
17 minutes
Enrichment Volume
18 µL
20 µL
22 µL
Lysate Volume
18 µL
20 µL
22 µL
39
Table C: Robustness Parameter Variations
40
41
Treatment
Lysis Time
Volume
Volume
# of Replicates
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-29 D/ PTM Validation Report 111501
OMA ERP - June 2016
ERP Use Only