AOAC RI ERP Final Action Recommendations eBook

Dupont BAX Salmonella PTM Report Modification Approved 2012 / PTM Certification No. 081201 For Expert Review Panel Use Only Do Not Distribute

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day of testing) and incubated at 42°C for 22-26 hours. An additional 1 mL LB enrichment was added to

10 mL TT broth and incubated at 35±2°C for 22-26 hours.

For dry pet food tested with the reference method, 20 spiked and 5 unspiked samples were

stomached for 2 minutes with approximately 750 mL pre-warmed (35°C) buffered peptone water

(BPW), then additional pre-warmed media was added to reach a total volume of 3375 mL. Samples

were incubated at 35±1 o C for 18-26 hours. After this primary enrichment, 1 mL of each sample was

added to 9 mL RVS broth. An additional 1 mL of each sample was added to 9 mL TBG broth. All

secondary enrichments were incubated at 42.5°C for 22-26 hours.

For dry pet food tested with the BAX® System method, 20 spiked and 5 unspiked samples were

stomached for 2 minutes with approximately 750 mL pre-warmed (35°C) lactose broth (LB), then

additional pre-warmed media was added to reach a total volume of 3375 mL. Samples were incubated

at 35±1°C for 22-26 hours. After this primary enrichment, 10 µL aliquots of each sample were added to

500 µL brain-heart infusion (BHI) broth and incubated at 37°C for 3 hours. For reference method

confirmation from the alternative enrichment, 1 mL of each primary enrichment was added to 9 mL

RVS broth. An additional 1 mL of each sample was added to 9 mL TBG broth. All secondary

enrichments were incubated at 42.5°C for 22-26 hours.

BAX® System Method

All samples were prepared for processing in the BAX® System according to the instructions found

in the BAX® System User Guide. A rack file was created using the Rack Wizard to enter identifying

AOAC INTERNATIONAL data on the entire rack and on the individual samples. BAX® System lysis reagent was prepared by adding 150 µL protease to 12 mL lysis buffer, and 200 µL of prepared lysis reagent was added to each cluster (lysis) tube. Sample enrichment was added in 5 µL aliquots to cluster tubes, and all tubes were heated at 37ºC for 20 minutes, then at 95ºC for 10 minutes. Lysates were cooled at 2-8°C for at least 5

minutes, then 30 µL sample lysate was transferred to a PCR tube for the BAX® System real-time

Salmonella assay to hydrate the PCR tablet.