AOAC RI ERP Final Action Recommendations

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

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

C for

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.

3.0

FDA-BAM (Orange Juice)

3.1

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

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.

Reporting Raw Data

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.

Collaborative Study Approved Protocol

Expert Review Panel Use Only