AOAC RI ERP Final Action Recommendations eBook

Collaborative Study Approved Protocol Expert Review Panel Use Only

1 Follow prompts in the Rack Wizard to enter identifying data on the entire rack and on the individual 2 samples. 3 Place one cluster tube per sample in a cluster tube rack. Add 200 µ L of prepared lysis reagent to 4 each tube (lysis tube). Transfer 5 µ L of each pre-enriched sample to the corresponding lysis tube and 5 ensure the tubes are capped. Incubate lysis tubes at 37ºC for 20 minutes, then at 95ºC for 10 minutes in 6 either individual heating blocks or the DuPont Thermal Block. Cool lysates for at least five minutes in 7 either a refrigerated cooling block or the automated thermal block prior to the final transfer to PCR 8 tubes. 9 Warm up the cycler/detector by selecting RUN FULL PROCESS from the menu bar of the application 10 window. 11 Place a PCR tube holder on the PCR cooling block. Insert one PCR tube per sample into the holder 12 and remove caps. Using a multi-channel pipette, transfer 30 µ L of sample lysate to a PCR tube. Cap PCR 13 with the flat optical cap strips provided in the kit. 14 Follow screen prompts to load samples into the cycler/detector and begin the program. At the 15 completion of the PCR and detection process, follow the screen prompts to remove samples and display 16 results.

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F. Interpretation of Test Result

The results are recorded on the rack display or from a spreadsheet printout of the results 21 (called Detail View). Negative results are indicated by the green circle with (-) symbol, positive 22 results are indicated by the red circle with (+) symbol, and indeterminate results are indicated with 23 a yellow circle with (?) symbol. A yellow circle with a (?) symbol and a red slash indicated a low 24 signal or signal error. 25 BAX® System results were displayed as follows: 26

Indeterminate result

Negative for Salmonella

Green (-)

Yellow (?)

Yellow (?) with red slash

Positive for Salmonella

Signal error

Red (+)

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G. Confirmation

Presumptive positive samples must be confirmed culturally as described in AOAC Official Method 31 967.26 , AOAC Official Method 2000.06 (see http://www.eoma.aoac.org/), the U.S. FDA Bacteriological 32 Analytical Manual (see 33 http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/u 34 cm070149.htm) the USDA-FSIS Microbiology Laboratory Guidebook (see 35 http://www.fsis.usda.gov/PDF/MLG_4_05.pdf), or the Health Canada Compendium of Analytical 36

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