c)
Homogenize thoroughly by
blending,
stomaching
,
or hand mixing for 2 ±0.2 minutes.
1
Incubate at 37 ±1°C according to Table 1.
2
3
Environmental samples
4
Sample collection devices can be a sponge hydrated with a neutralizing solution to inactivate the
5
effects of the sanitizers. 3M recommends the use of a biocide-free cellulose sponge. Neutralizing
6
solution can be Dey-Engley (D/E) Neutralizing Broth or Letheen Broth. It is recommended to
7
sanitize the area after sampling.
8
9
The recommended size of the sampling area for verifying the presence or absence of the
10
pathogen on the surface is at least 100 cm
2
(10 cm x 10 cm or 4”x4”). When sampling with a
11
sponge, cover the entire area going in two directions (left to right then up and down) or collect
12
environmental samples following your current sampling protocol or according to the FDA BAM,
13
USDA FSIS MLG or ISO 18593 [7] guidelines.
14
15
1.
Allow the Demi-Fraser Broth enrichment medium (includes FAC) to equilibrate to ambient
16
laboratory temperature.
17
2.
Aseptically combine the enrichment medium and sample according to Table 1.
18
3.
Homogenize thoroughly by
blending,vortexing or
stomaching
, or hand mixing
for 2 ±0.2
19
minutes. Incubate at 37 ±1°C for 24-30 hours.
20
4.
Invert room temperature (20-25
o
C) lysis tubes to mix. Proceed to next step within 4 hours.
21
20 µL aliquot of each enriched sample are is transferred to separate lysis tubes using a new
22
pipette tip after each sample transfer. Place uncovered samples on a dry double block heater r
23
for 15 ± 1 minutes at 100 ± 1
o
C. Following the heat lysis transfer samples into the chill
24
block insert and placed onto a sterilized lab bench and allow to cool at 18-28 °C for 5-10
25
minutes.
26
27
5.
Add 20 µL of each lysed sample and control to separate reagent tubes, and mix by pipetting
28
up and down five times. Analyze a matrix control tube with the samples for each matrix to
29
verify that no interference with the assay was caused by the matrix. Use sterile Demi Fraser
30
Broth with FAC for the Negative Control (NC).
31
6.
Transfer a 20 µL aliquot to the NC and the Reagent Control (RC) tubes. Add a 20 µL aliquot
32
of a randomly picked sample to the matrix control tube, mixed, and recapped. Using the 3M
™
33
software, follow prompts to identify samples and controls. Load all samples into the Speed
34
Loader Tray (SLT), place into the Molecular Detection System, and initiate the 3M
™
MDA 2
35
-
Listeria
assay. Results are obtained within 75 minutes.
36
37
Interpretation and Test Result Report
38
An algorithm interprets the light output curve resulting from the detection of the nucleic acid
39
amplification. Results are analyzed automatically by the software and are color-coded based on
40
the result. A Positive or Negative result is determined by analysis of a number of unique curve
41
parameters. Presumptive positive results are reported in real-time while Negative and Inspect
42
results will be displayed after the run is completed.
43
44
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