170 / 596
OAA agar plates, these suspect colonies were streaked to HBO and incubated at 37 ± 1
o
C for 18-
1
24 hours. HBO plates were examined for hemolysis reactions and well-isolated colonies were
2
transferred to BHI broth and incubated at 25°C for 24 ± 2 hours. Sample isolates from BHI broth
3
were analyzed for tumbling motility by preparing a wet mount, analyzed by a catalase test and
4
examined for morphology by preparing a Gram stain. Additionally, purified HBO isolates were
5
identified using the VITEK
®
GP Biochemical Identification following AOAC OMA 2013.02.
6
7
8
3M
™
Molecular Detection Assay 2 - Listeria
9
10
All 25 g samples were analyzed by the 3M
™
MDA 2 -
Listeria
were enriched with 225 mL of
11
Demi-Fraser Broth with the addition of ferric ammonium citrate; all test portions were then
12
homogenized by stomaching thoroughly for 2 ± 0.2 minutes and incubated at 37 ± 1 °C for 24-28
13
hours. For the 125 g deli turkey test portions, samples were enriched with 1125 mL of Demi-
14
Fraser Broth with the addition of ferric ammonium citrate; test portions were then homogenized
15
by stomaching thoroughly for 2 ± 0.2 minutes and incubated at 37 ± 1 °C for 24 hours. For
16
whole melons, test portions were enriched with approximately 1.5 times the weight of the whole
17
melon in Demi Fraser with ferric ammonium citrate and incubated at 37 ± 1 °C for 26 hours.
18
Stainless steel sponge samples were enriched with 225 mL of Demi-Fraser Broth with the
19
addition of ferric ammonium citrate; samples were homogenized by hand thoroughly for 2 ± 0.2
20
minutes and incubated at 37 ±1 °C for 24 and26 hours. Sealed concrete environmental surface
21
sponge samples were enriched with 100 mL of Demi-Fraser Broth with the addition of ferric
22
ammonium citrate; samples were thoroughly homogenized by hand for 2 ± 0.2 minutes and
23
incubated at 37°C for 24 and 26 hours. Plastic environmental surface swab samples were
24
enriched with 10 mL of Demi-Fraser Broth with the addition of ferric ammonium citrate;
25
samples were homogenized by vortexing for 2 ± 0.2 minutes and incubated at 37 ±1 °C for 24
26
and 26 hours.
27
28
Prior to analysis, lysis tubes were brought to room temperature (20-25
o
C) by placing the tubes on
29
a sanitized bench top for 16-18 hours. Lysis tubes were inverted to mix up to four hours before
30
use. The lysis tubes were then de-capped and the rubber cap was discarded. A 20 µL aliquot of
31
each sample was transferred to separate lysis tubes; a new pipette tip was used after each sample
32
transfer. Uncovered samples were placed in the 3M Molecular Detection Heat Block Insert and
33
heated for15±1 minutes at 100 ± 1
o
C. Following the heat lysis, samples were transferred into the
34
Chill Block Insert and placed on a sanitized laboratory bench and were allowed to cool at 18-
35
28°C for 5-10 minutes.
36
37
A 20 µL aliquot of each lysed sample and control was added to separate reagent tubes, and
38
samples were mixed by pipetting up and down five times. A Matrix Control tube was analyzed
39
with the samples for each matrix to verify that no interference with the assay was caused by the
40
matrix. Also lysed was a sterile Demi Fraser Broth with FAC tube for the kit Negative Control
41
(NC). A 20 µL aliquot was transferred to the NC and the Reagent Control (RC) tubes. A 20 µL
42
aliquot of a randomly picked sample was added to the Matrix Control tube, mixed, and recapped.
43
Using the 3M
™
software, prompts were followed to identify samples and controls. All samples
44
were loaded into the Speed Loader Tray, placed into the Molecular Detection System, and the
45
3M
™
MDA 2 -
Listeria
assay was initiated and results were obtained within 75 minutes.
46
AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-29 D/ PTM Validation Report 111501
OMA ERP - June 2016
ERP Use Only