409 / 596
NOTE:
Even a negative sample will not give a zero reading as the system and 3M Molecular
1
Detection Assay 2 -
Listeria
amplification reagents have a “background” relative light unit
2
(RLU) reading.
3
4
In the rare event of any unusual light output, the algorithm labels this as “Inspect.” 3M
5
recommends the user to repeat the assay for any Inspect samples. If the result continues to be
6
Inspect, proceed to confirmation test using your preferred method or as specified by local
7
regulations.
8
9
Confirmation
10
11
Presumptive positive primary enrichment samples were confirmed by following the appropriate
12
reference method confirmation, beginning with transfer from the primary enrichment to
13
secondary enrichment broth (if applicable), followed by subsequent plating and confirmation of
14
isolates using appropriate biochemical and serological methods.
15
16
Validation Study
17
18
Testing was conducted following the procedures outlined in the AOAC Research Institute
19
Performance Tested Methods
SM
Program
validation outline protocol:
Comparative Evaluation of
20
the 3M
™
Molecular Detection Assay 2 - Listeria monocytogenes and of the 3M
™
Molecular
21
Detection Assay 2 - Listeria
(February, 2015) [8]. Three brands of Demi Fraser Broth with FAC
22
were used for this validation study; X, Y, and Z. The independent study involved a matrix study
23
(10 matrixes), a lot-to-lot/stability evaluation, and a robustness evaluation using brand Y. The
24
internal study involved a matrix study (4 matrixes) and an inclusivity/exclusivity study using
25
either brand X or Z.
26
27
Internal Study
28
29
Inclusivity and Exclusivity
30
31
Methodology
32
33
Fifty frozen
Listeria
strain suspensions were thawed and sub-cultured in Brain Heart Infusion
34
(BHI) broth overnight at 37°C ± 1°C. The cultures were diluted in peptone salt solution in order
35
to inoculate between 10 to 100 cells per 225 mL of Demi Fraser with FAC. The enrichment
36
broths were then incubated for 24 hours at 37°C ± 1°C, and the 3M MDA 2 -
Listeria
method
37
was then performed and confirmed according to ISO 11290-1/A1. See Table 2a.
38
39
Five Thirteen
additional
Listeria
strains were tested at 3M (St. Paul, MN). The
five thirteen
40
frozen
Listeria
strain suspensions were thawed
, or lyophilized cultures were revived,
and
then
41
streaked onto Sheep Blood Agar (SBA). The SBA plates were incubated at 37°C ± 1°C
42
overnight. A single isolated colony from the SBA plate was sub-cultured in 10 mL of Demi
43
Fraser Broth with FAC overnight at 37°C ± 1°C. The cultures were diluted using Demi Fraser
44
broth with FAC, and the 3M MDA 2 –
Listeria
method was then performed. See Table 2b.
45
46