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Incubate at 37 ±1°C for 24-30 hours.
1
4.
Invert room temperature (20-25
o
C) lysis tubes to mix. Proceed to next step within 4 hours.
20
2
µLaliquot of each enriched sample are is transferred to separate lysis tubes using a new
3
pipette tip aftereach sample transfer
. Place uncovered samples on a dry bath incubator for 15
4
±2minutes at 100 ± 1
o
C. Following the heat lysis transfer samples to the sterilized lab bench
5
and allow to cool at 18-28 °C for 5-10 minutes.
6
5.
Add 20 µL of each lysed sample and control to separate reagent tubes, and mix by pipetting
7
up and down five times.
Analyze a matrix control tube with the samples for each matrix to
8
verify that no interference with the assay was caused by the matrix. Use sterile Demi Fraser
9
Broth for the Negative Control (NC
).Transfer a 20 µL aliquot to the NC and the Reagent
10
Control (RC) tubes. Add a 20 µL aliquot of a randomly picked sample to the matrix control
11
tube, mixed, and recapped. Using the 3M
™
software, follow prompts to identify samples and
12
controls. Load all samples into the Speed Loader Tray (SLT), place into the Molecular
13
Detection System, and initiate the 3M
™
MDA2
Listeriamonocytogenes
assay. Results are
14
obtained within 75 minutes.
15
16
Interpretation and Test Result Report
17
An algorithm interprets the light output curve resulting from the detection of the nucleic acid
18
amplification. Results are analyzed automatically by the software and are color-coded based on
19
the result. A Positive or Negative result is determined by analysis of a number of unique curve
20
parameters. Presumptive positive results are reported in real-time while Negative and Inspect
21
results will be displayed after the run is completed.
22
23
NOTE:
Even a negative sample will not give a zero reading as the system and 3M Molecular
24
Detection Assay 2 –
Listeriamonocytogenes
amplificationreagents have a “background” relative
25
light unit (RLU).
26
27
In the rare event of any unusual light output, the algorithm labels this as “Inspect.” 3M
28
recommends the user to repeat the assay for any Inspect samples. If the result continues to be
29
Inspect, proceed to confirmation test using your preferred method or as specified by local
30
regulations.
31
32
Confirmation
33
34
Presumptive positive samples
of primary enrichments
were confirmed by following the
35
appropriate reference method confirmation, beginning with transfer from the primary enrichment
36
to secondary enrichment broth (if applicable), followed by subsequent plating and confirmation
37
of isolates using appropriate biochemical and serological methods.
38
39
Validation Study
40
41
Testing was conducted following the procedures outlined in the AOAC Research Institute
42
Performance Tested Methods
SM
Program
validation outline protocol:
Comparative Evaluation of
43
the 3M
™
Molecular Detection Assay 2-Listeria monocytogenes and of the 3M
™
Molecular
44
Detection Assay 2 Listeria
(February, 2015) [8]. The independent study involved a matrix study
45
(9matrices), a lot-to-lot/stability evaluation, and a robustness evaluation. The internal study
46
involved a matrix study (3 matrices) and an inclusivity/exclusivity study.
47
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-30 D/ PTM Validation Report 081501
OMA ERP - June 2016
ERP Use Only