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method confirmation procedure. There were 5 confirmed positives following the reference
1
method. Detailed results of the POD analyses are presented in Tables 9 and 13.
2
3
Plastic (Enviroswab enriched in 10 mL)
4
5
Results were identical after 24 and 26 hours of sample enrichment. For the low inoculation level
6
of the MDA 2 -
Listeria
assay, there were 15 presumptive positives and 15 confirmed positives
7
following the USDA/FSIS MLG 8.09 reference method confirmation procedure. There were 11
8
observed positives for the reference method. For the high inoculation level, there were 5
9
presumptive positives and 5 confirmed positives following the USDA/FSIS MLG 8.09 reference
10
method confirmation procedure. There were 5 confirmed positives following the reference
11
method. Detailed results of the POD analyses are presented in Tables 9 and 13.
12
13
14
Product Consistency (Lot-to-Lot) and Stability Studies
15
16
Methodology
17
Three lots of MDA 2 -
Listeria
test kits, 1 newly manufactured, 1 at the middle of its expiration,
18
and 1 at or slightly beyond expiration were analyzed to determine the stability of the assay. For
19
the MDA 2 -
Listeria
test kit, one
Listeria monocytogenes
ATCC 7644 isolate was analyzed at a
20
fractional positive level (2-8 positives), and testing 10 replicates. One non-
Listeria
isolate,
21
Enterococcus faecalis
ATCC 29212, was analyzed at the growth level achieved in a non-
22
selective broth, testing 5 replicates.
23
24
Results
25
For the lot-to-lot/stability evaluation of the MDA 2 -
Listeria
assay, there were 6 presumptive
26
positives out of 10 replicates for all three lots at the low inoculation level. For the 5 uninoculated
27
control test portions, there were 0 presumptive positives out of 5 replicates for all three lots. The
28
five control test portions were negative. See Table 16 for lot-to-lot/stability results.
29
30
Robustness Study
31
32
Methodology
33
The robustness evaluated the ability of the method to remain unaffected by minor variations in
34
method parameters that might be expected to occur when the method is performed by an end
35
user. Three different parameters were evaluated, which are presented in Table 18. For each
36
parameter, one
Listeria monocytogenes
ATCC 7644
isolate was analyzed at a fractional positive
37
level (2-8 positives), testing 10 replicates. One non-
Listeria
isolate,
Enterococcus faecalis
ATCC
38
29212, was analyzed at the growth level achieved in a non-selective broth, testing 5 replicates.
39
40
Table B: Robustness Parameters
41
42
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