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9
Foods
1
(a)
Allow the modified tryptic soy broth enrichment medium (includes casamino acids) to
2
equilibrate to 42 ± 1 °C.
3
(b)
Aseptically combine the enrichment medium and sample according to Table A. For all
4
meat and highly particulate samples, the use of filter bags is recommended.
5
(c)
Homogenize thoroughly by blending, stomaching, or hand mixing for 2 ±0.2 minutes.
6
Incubate at 42 ± 1 °C according to Table A.
7
8
Table A: Sample Enrichment and Incubation
9
10
E.
Preparing Samples
11
(a)
Manual mericonDNA Extraction Method
12
13
1)
Gently mix all enriched samples to disperse
E. coli
organisms throughout the
14
sample. Transfer 1 mL of enriched sample to a sterile 2 mL screw cap tube and
15
centrifuge for 5 minutes at 13,000 x
g
.
16
17
2)
Discard the supernatant with a pipettor, taking care not to disrupt the pellet.
18
19
3)
Add 200 µL Fast Lysis Buffer to the bacterial pellet and vortex the tube to
20
resuspend the pellet.
21
22
4)
Place the microcentrifuge tube into a dry bath incubator (100 ± 1°C) for 10 minutes.
23
24
5)
Remove the sample and allow it to cool to room temperature (15-24 °C) for 2
25
minutes.
26
27
6)
Centrifuge the tube at 13,000 x
g
for 5 minutes.
28
29
7)
For raw ground beef (73% lean), dilute the extracted DNA 1:10 using sterile
30
nuclease-free water.
31
32
8)
Prepare the reconstituted assay by adding 1040 µL (96 sample kit) or 130 µl (24
33
sample kit) of the Multiplex PCR master mix to a vial of
mericon
®
Assay and mix
34
by pipetting up and down 2-3 times.
35
36
9)
Prepare a positive control by re-suspending the lyophilized Positive Control DNA
37
in 200 µL of QuantiTect
®
Nucleic Acid Dilution Buffer and mix.
38
Matrix
Sample Size
Enrichment Media
and Volume
Incubation
Conditions
Fresh Spinach
25 g
mTSB/225 mL
42 ± 1
o
C for 10 hours
Raw Ground Beef
(73% Lean)
325 g
mTSB/975 mL
42 ± 1
o
C for 10 hours
Raw Beef Trim
325 g
mTSB/975 mL
42 ± 1
o
C for 10 hours
OMAMAN-36 A : Collaborative Study Manuscript
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only