AOAC RI ERP Final Action Recommendations eBook

Collaborative Study Approved Protocol Expert Review Panel Use Only

FDA/BAM 2

BAX ® MP Media

Orange Juice

UPB

1 http://origin-www.fsis.usda.gov/PDF/MLG_4_05.pdf 2 http://www.fda.gov/downloads/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAn alyticalManualBAM/UCM244774.pdf

Hot Dogs (325 grams) – One test portion preparation

BAX ® System Real-Time PCR Assay for Salmonella vs. USDA-FSIS MLG Reference Method Comparison –

BAX ® System Real-Time PCR Assay for Salmonella and USDA/FSIS-MLG 1.1 To each 325g test portion, add one-third to one-half of 2925 mL pre-warmed (35± 1°C) BPW (approximately 975 mL to 1450 mL). Homogenize each sample for approximately two minutes. Add the remaining 2925 mL of BPW. Incubate for 18 - 24 hours at 35 ± 2°C. Note: Take care when enriching samples that pre-warmed media is not allowed to cool. Only remove as much primary enrichment as needed when preparing samples. Allowing the BPW to cool while enriching samples may impact the integrity of the test system. 1.2 Follow prompts in the Rack Wizard to enter identifying data on the entire rack and on the individual samples. 1.3 Place one cluster tube per sample in a cluster tube rack. Add 200 µ L of prepared lysis reagent to each tube (lysis tube). 1.4 Transfer 5 µ L of each pre-enriched sample to the corresponding lysis tube and ensure the tubes are capped. 1.5 Incubate lysis tubes at 37ºC for 20 minutes, then at 95ºC for 10 minutes in either individual heating blocks or the DuPont Thermal Block. Cool lysates for at least five minutes in either a refrigerated cooling block or the automated thermal block prior to the final transfer to PCR tubes. 1.6 Warm up the cycler/detector by selecting RUN FULL PROCESS from the menu bar of the application window. 1.7 Place a PCR tube holder on the PCR cooling block. Insert one PCR tube per sample into the holder and remove caps. Using a multi-channel pipette, transfer 30 µ L of sample lysate to a PCR tube. Cap PCR with the flat optical cap strips provided in the kit. 1.8 Follow screen prompts to load samples into the cycler/detector and begin the program. At the completion of the PCR and detection process, follow the screen prompts to remove samples and display results.

1.0

Test Result Confirmation 1.9

Using the primary enrichment (step 1.1 above), transfer 0.5 ± 0.05 mL enriched sample into 10 mL Tetrathionate (TT-Hajna) broth and 0.1 ± 0.02 mL into 10 mL modified

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