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3M MDA 2
Listeria
species and
L. monocytogenes
Collaborative Study
OMA-2016-MONTH-XXX
5/4/16
1.12
If suspect colonies are present on MOX from any source, streak for isolation on
1
one or more HL agar plates. Incubate the streaked HL at 35 ± 2°C for 22 ± 4 h.
2
1.13
After incubation, examine the HL plate(s) against backlight for translucent
3
colonies surrounded by a small zone of β-hemolysis.
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1.14
If at least one suspect colony is clearly isolated, proceed to confirmatory testing.
5
Hold all HL plates containing suspect colonies (room temperature or
6
refrigeration) until confirmatory testing is complete.
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1.15
If suspect colonies or β-hemolytic growth are present on HL but not clearly
8
isolated, re-streak representative suspect colonies/growth onto one or more fresh
9
HL plates and incubate at 35 ± 2°C for 22 ± 4 h.
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1.16
Confirm ALL test portions according to the USDA FSIS biochemical/serological
11
procedures. Biochemical tests will be performed using API or VITEK 2 GP.
12
Please follow manufacturer’s instructions when using any of these rapid methods.
13
14
Raw chicken breast
– use stomacher bag with filter for all 3M test portions.
15
16
2.0
3M MDA2 -
Listeria monocytogenes
and MDA2 -
Listeria
17
2.1
To each 25g test portion, add 475 mL Demi Fraser broth with ferric ammonium
18
citrate (FAC). Homogenize each sample for two minutes ± 30 seconds and
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incubate 28-32 h at 37 ± 1°C.
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2.2
Prepare the 3M MDS speed loader tray, chill block insert, heat block insert and
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instrument following the 3M MDA 2 -
Listeria monocytogenes
Instructions for
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Use [IFU] and 3M Molecular Detection Instrument manual.
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2.3
Equilibrate the LS tubes to room temperature (20-25 ºC) by setting LS tube
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overnight 16-18 hours. Or on the laboratory bench for at least 2 hours.
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2.4
Invert the capped LS tubes to mix, up to 4 hours before use.
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2.5
Transfer 20 µL enriched sample into a LS tube.
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2.6
Transfer 20 µL Negative Control [NC], sterile enrichment medium (e.g. Demi-
28
Fraser Broth) into a Lysis Solution [LS] tube after all enriched samples have been
29
completed. Do not use water as a NC.
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2.7
Place uncovered LS tubes in 3M Molecular detection Heat block, heat 15±1 min
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at 100±1°C. LS Solution will change from pink (cool) to yellow (hot).
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2.8
Place LS tubes (without rack lid) in chill block insert (at ambient temperature) for
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5-10 min. Lysis solution in LS tube will revert to pink color.
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2.9
Transfer 20 µL sample lysate from the upper portion of fluid in the LS tube into
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regent tube. Mix gently by pipetting up and down 5 times.
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2.10
Transfer 20 µL of NC lysate into a reagent tube.
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2.11
Transfer 20 µL of NC lysate into a Reagent Control (RC) tube.
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2.12
Load capped tubes into speed loader tray and close lid.
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2.13
Start assay.
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2.14
Validation Study Confirmation
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2.14.1
Confirm each test portion, regardless of presumptive result.
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2.14.2
Transfer 0.1 ± 0.02 ml of the enriched sample to 10 ± 0.5 ml of Fraser
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Broth (FB). As per media preparation instructions, be sure that
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appropriate supplements have been added to the FB prior to inoculation.
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Incubate inoculated FB tubes at 35 ± 2°C for 26 ± 2 h.
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AOAC Research Institute
Expert Review Panel Use Only
OMAMAN-29/OMAMAN-30 Combined Collaborative Study Protocol
OMA ERP - June 2016
ERP Use Only