AOAC RI ERP Final Action Recommendations eBook

FINAL (Version 4)

DuPont™ BAX ® System Real-Time PCR Assay for Salmonella : Collaborative Study

E. Re-Growth 1 After enrichment, transfer 10 µL of the enrichment to 500 µL BHI broth, C(b). Incubate, B(m), at 37°C 2 for 3 hours. 3 Re-growth is required for orange juice, nonfat dry milk, peanut butter and dry pet food samples. For 4 all other matrices, re-growth is either optional or not required. 5 F. Assay 6 1. After enriching the sample, turn on the heating blocks, B(e), and set temperatures to 37°C and 7 95°C. Make sure that the cooling blocks have been refrigerated overnight or otherwise chilled at 8 2-8°C. 9 2. Create a rack file by follow prompts in the Rack Wizard, B(b) to enter identifying data on the 10 entire rack and on the individual samples. 11 3. Label and arrange cluster tubes, B(c), in the cluster tube rack, according to the rack file. 12 4. Prepare the lysis reagent by adding 150 µ L protease, B(l) to one 12-mL bottle lysis buffer, B(k). 13 Transfer 200 µ L prepared lysis reagent to each of the cluster tubes. 14 5. Transfer 5 µ L enriched sample to the corresponding cluster tubes. Secure caps with the 15 capping/de-capping tool, B(d). 16 6. Heat cluster tubes at 37°C for 20 minutes. 17 7. Heat cluster tubes at 95° for 10 minutes. 18 8. Cool cluster tubes at 2-8° for at least 5 minutes. 19 9. Warm up the cycler/detector, B(a), by selecting RUN FULL PROCESS from the Operations menu 20 of the application window, B(b). 21 10. Place a PCR tube holder, B(h) on the PCR cooling block, B(e). Insert one PCR tube, B(i) per 22 sample into the holder and remove caps with the capping/de-capping tool, B(d). 23 11. Using a multi-channel pipette, B(f), transfer 30 µL of sample lysate to PCR tubes, B(i). Seal with 24 flat optical caps, B(j) with the capping/de-capping tool, B(d). 25 12. Follow screen prompts, B(b), to load samples into the cycler/detector, B(a), and begin the 26 program. At the completion of the PCR and detection process, follow the screen prompts to 27 remove samples and display results. 28 G. Assay Results 29 The results are recorded on the rack display or from a spreadsheet printout of the results (called Detail 30 View). Negative results are indicated by the green circle with (-) symbol, positive results are indicated by 31 the red circle with (+) symbol, and indeterminate results are indicated with a yellow circle with (?) 32 symbol. A yellow circle with a (?) symbol and a red slash indicated a low signal or signal error. 33 BAX® System results were displayed as follows: 34

Negative for Salmonella

Green (-)

Yellow (?)

Indeterminate result

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