AOAC-RI ERP Micro June 2016

OMAMAN-29/OMAMAN-30 Combined Collaborative Study Protocol OMA ERP - June 2016 ERP Use Only

3M MDA 2 Listeria species and L. monocytogenes Collaborative Study OMA-2016-MONTH-XXX

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

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13 14 Raw chicken breast – use stomacher bag with filter for all 3M test portions.

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2.0

2.13 Start assay.

2.14 Validation Study Confirmation

2.14.1 Confirm each test portion, regardless of presumptive result. 2.14.2 Transfer 0.1 ± 0.02 ml of the enriched sample to 10 ± 0.5 ml of Fraser Broth (FB). As per media preparation instructions, be sure that appropriate supplements have been added to the FB prior to inoculation.

Incubate inoculated FB tubes at 35 ± 2°C for 26 ± 2 h.

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