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Inspect, proceed to confirmation test using your preferred method or as specified by local
1
regulations.
2
3
Confirmation
4
5
Presumptive positive primary enrichment samples were confirmed by following the appropriate
6
reference method confirmation, beginning with transfer from the primary enrichment to
7
secondary enrichment broth (if applicable), followed by subsequent plating and confirmation of
8
isolates using appropriate biochemical and serological methods.
9
10
Validation Study
11
12
Testing was conducted following the procedures outlined in the AOAC Research Institute
13
Performance Tested Methods
SM
Program
validation outline protocol:
Comparative Evaluation of
14
the 3M
™
Molecular Detection Assay 2 - Listeria monocytogenes and of the 3M
™
Molecular
15
Detection Assay 2 - Listeria
(February, 2015) [8]. Three brands of Demi Fraser Broth with FAC
16
were used for this validation study; X, Y, and Z. The independent study involved a matrix study
17
(10 matrixes), a lot-to-lot/stability evaluation, and a robustness evaluation using brand Y. The
18
internal study involved a matrix study (4 matrixes) and an inclusivity/exclusivity study using
19
either brand X or Z.
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21
Internal Study
22
23
Inclusivity and Exclusivity
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25
Methodology
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27
Fifty frozen
Listeria
strain suspensions were thawed and sub-cultured in Brain Heart Infusion
28
(BHI) broth overnight at 37°C ± 1°C. The cultures were diluted in peptone salt solution in order
29
to inoculate between 10 to 100 cells per 225 mL of Demi Fraser with FAC. The enrichment
30
broths were then incubated for 24 hours at 37°C ± 1°C, and the 3M MDA 2 -
Listeria
method
31
was then performed and confirmed according to ISO 11290-1/A1. See Table 2a.
32
33
Five additional
Listeria
strains were tested at 3M (St. Paul, MN). The five frozen
Listeria
strain
34
suspensions were thawed and streaked onto Sheep Blood Agar (SBA). The SBA plates were
35
incubated at 37°C ± 1°C overnight. A single isolated colony from the SBA plate was sub-
36
cultured in 10 mL of Demi Fraser Broth with FAC overnight at 37°C ± 1°C. The cultures were
37
diluted using Demi Fraser broth with FAC, and the 3M MDA 2 –
Listeria
method was then
38
performed. See Table 2b.
39
40
Thirty frozen non-
Listeria
strain suspensions were thawed and sub-cultured in BHI broth
41
overnight at 37°C ± 1°C. The cultures were diluted in Buffered Peptone Water (BPW) or de
42
Man, Rogosa, and Sharpe (MRS) in order to inoculate 10
5
cells/mL. The broths were then
43
incubated for 24 hours at the appropriate incubation temperature in order to have culture to test
44
with the 3M MDA 2 -
Listeria
method. See Table 3.
45
46
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-29 D/ PTM Validation Report 111501
OMA ERP - June 2016
ERP Use Only