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24 hours. HBO plates were examined for hemolysis reactions and well-isolated colonies were
1
transferred to BHI broth and incubated at 25°C for 24 ± 2 hours. Sample isolates from BHI broth
2
were analyzed for tumbling motility by preparing a wet mount, analyzed by a catalase test and
3
examined for morphology by preparing a Gram stain. Additionally, purified HBO isolates were
4
identified using the VITEK
®
GP Biochemical Identification following AOAC OMA 2013.02.
5
6
7
3M
™
Molecular Detection Assay 2 –Listeria monocytogenes
8
9
All 25 g samples were analyzed by the 3M
™
MDA2 -
Listeriamonocytogenes
were enriched
10
with 225 mL of Demi-Fraser Broth containing ferric ammonium citrate; all test portions were
11
then homogenized by stomaching thoroughly for 2 ± 0.2 minutes and incubated at 37 ±1 °C for
12
24-28 hours. For the 125 g deli turkey test portions, samples were enriched with 1125 mL of
13
Demi-Fraser Broth; test portions were then homogenized by stomaching thoroughly for 2 ± 0.2
14
minutes and incubated at 37 ±1 °C for 24 hours. For whole melons, test portions were enriched
15
with approximately 1.5 times the weight of the whole melon in Demi Fraser and incubated at 37
16
±1 °C for 26 hours. For stainless steel, sponge samples were enriched with 225 mL of Demi-
17
Fraser Broth; samples were homogenized by hand thoroughly for 2 ± 0.2 minutes and incubated
18
at 37 ±1 °C for 24-26 hours. Sealed concrete environmental surface sponge samples were
19
enriched with 100 mL of Demi-Fraser Broth; samples were homogenized by hand thoroughly for
20
2 ± 0.2 minutes and incubated at 37°C for 24-26 hours. Plastic environmental surface swab
21
samples were enriched with 10 mL of Demi-Fraser Broth; samples were homogenized by
22
vortexing thoroughly for 2 ± 0.2 minutes and incubated at 37 ±1 °C for 24-26 hours.
23
Prior to analysis, lysis tubes were brought to room temperature (20-25
o
C) by placing the
24
tubes on a sanitized bench top for 16-18 hours. Lysis tubes were inverted to mix up to four hours
25
before use. The lysis tubes were then de-capped and the rubber cap was discarded. A 20 µL
26
aliquot of each sample was transferred to separate lysis tubes; a new pipette tip was used after
27
each sample transfer. Uncovered samples were placed on a dry bath incubator for 15 ± 2minutes
28
at 100 ± 1
o
C. Following the heat lysis, samples were transferred to the sanitized
29
laboratorybench and were allowed to cool at 18-28 °C for 5-10 minutes.
30
A 20 µL aliquot of each lysed sample and control was added to separate reagent tubes, and
31
samples were mixed by pipetting up and down five times. A matrix control tube was analyzed
32
with the samples for each matrix to verify that no interference with the assay was caused by the
33
matrix. A sample of sterile Demi Fraser Broth was lysed for the kit Negative Control (NC). A
34
20 µL aliquot was transferred to the NC and the Reagent Control (RC) tubes. A 20 µL aliquot of
35
a randomly picked sample was added to the matrix control tube, mixed, and recapped. Using the
36
3M
™
software, prompts were followed to identify samples and controls. All samples were
37
loaded into the Speed Loader Tray, placed into the Molecular Detection System, and the
38
3M
™
MDA2
-
Listeria monocytogenes
assay was initiated and results were obtained within 75
39
minutes.
40
41
Matrix Study Results
42
43
For each method, the probability of detection (POD) was calculated as the number of positive
44
outcomes divided by the total number of trials. [12] The following were calculated
:
for
the
45
candidate presumptive results, POD
CP
; the candidate confirmatory results, POD
CC
; the difference
46