AOAC RI ERP Final Action Recommendations eBook

Collaborative Study Approved Protocol Expert Review Panel Use Only

2.7 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. 2.8 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. Test Result Confirmation 2.8.1 Using the primary enrichment (step 2.1 above), follow steps starting at step 3.2 below (secondary enrichments) to confirm each test portion, regardless of presumptive result. 2.8.2 Transfer typical colonies to TSI/LIA slants. Incubate 35 ± 1 ° C for 24 ± 2h. 2.8.3 Confirm a minimum of one typical colony per test portion with biochemical/serological procedures prescribed by the FDA/BAM. Either the API20E (Official Method 978.24) or the VITEK GN (Official Method 2011.17) will be used as an alternative to the conventional biochemical tests. The somatic (O) and flagellar (H) tests will also be performed. To each 25g test portion, add 225 mL Universal Pre-enrichment broth in a sterile, wide mouth, screw-capped jar or other appropriate container. Swirl the flask contents thoroughly. Cap jar securely and let stand 60 ± 5 min at room temperature. Do not adjust pH. Incubate loosely capped container for 24 ± 2h at 35°C. 3.2 Transfer 0.1 ml to 10 ml Rappaport-Vassiliadis (RV) broth (prepared from individual ingredients) and 1 mL to 10 mL TT broth. Pay special attention to the preparation of RV and TT broths as USDA and FDA methods call for different formulations. Incubate the RV at 42 ± 0.2°C for 24 ± 2 h in a circulating water bath. Incubate the TT broth at 35 ± 2.0 ° C for 24 ± 2 h. 3.3 Mix and streak 3 mm loopful (10 µ L) RV broth onto BS, XLD and HE agars. Repeat from TT broth. Incubate at 35 ° C for 24 ± 2 h. Follow isolation procedure according to FDA- BAM. 3.4 Transfer typical colonies to TSI and LIA. Incubate at 35 ° C for 24 ± 2hr. Confirm 1 typical colony per test portion with biochemical/serological procedures prescribed by the FDA-BAM method. Either the API20E (Official Method 978.24) or the VITEK GN (Official Method 2011.17) will be used as an alternative to the conventional biochemical tests. The somatic (O) and flagellar (H) tests will also be performed. FDA-BAM (Orange Juice) 3.1

3.0

Reporting Raw Data

1. All results are to be recorded on the data sheets provided. For each test portion, record the results reported by the BAX ® System along with the confirmation results for both the BAX ® System and reference methods.

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