AOAC RI ERP Final Action Recommendations

Rappaport-Vassiliadis (RV-R10) broth.

Pay special attention to the preparation of RV

and TT broths as USDA and FDA methods call for different formulations.

Incubate at

0.5

C for 22-24 h.

1.10

Streak both secondary enrichments onto Xylose Lysine Tergitol (XLT4) agar and Brilliant

Green Sulfa (BGS) agar. Use one loopful of inoculum for each plate. Incubate 35

for 18-24h. Select colonies. Re-incubate all plates for an additional 18-24 h. Reexamine

initially negative plates and pick colonies as above.

1.11

Transfer typical colonies to TSI/LIA slants. Incubate 35

C for 24

2h.

Confirm a minimum of one typical colony per sample with biochemical/serological

procedures prescribed by the USDA/FSIS 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.

Orange Juice– two test portion preparations

BAX

System Real-Time PCR Assay for

Salmonella

vs. FDA/BAM Reference Method Comparison –

Orange Juice

2.0

BAX

System Real-Time PCR Assay for

Salmonella

2.1

To each 25g test portions, add 225 mL BAX

MP broth (

pre-warmed to

39-42°C

). Mix each sample thoroughly. Incubate for 22-26 hours at 39-42°C.

Note: Take care when enriching samples that pre-warmed media is not allowed to

cool. Only remove as much primary enrichment as needed when preparing samples.

Allowing the BAX MP broth to cool while enriching samples may impact the integrity

of the test system.

2.2

Follow prompts in the Rack Wizard to enter identifying data on the entire rack and on

the individual samples.

2.3

Place one cluster tube per sample in a cluster tube rack. Add 200

L of prepared lysis

reagent to each tube (lysis tube).

2.4

Transfer 5

L of each pre-enriched sample to the corresponding lysis tube and ensure

the tubes are capped.

2.5

Incubate lysis tubes at 37ºC for 20 minutes, then at 95ºC for 10 minutes in either

individual heating blocks or the DuPont Thermal Block. Cool lysates for at least five

minutes in either a refrigerated cooling block or the automated thermal block prior to

the final transfer to PCR tubes.

2.6

Warm up the cycler/detector by selecting RUN FULL PROCESS from the menu bar of the

application window.

Collaborative Study Approved Protocol

Expert Review Panel Use Only