DuPont Qualicon BAX(R) System Real-Time PCR Assay for Salmonella - AOAC 2013.02

SCOPE

This method is applicable to the analysis of:

-  Meat and meat products (including carcase swabs)

PRINCIPLES

The BAX System rapidly amplifies specific DNA fragments unique to Salmonella using polymerase chain reaction (PCR). DuPont Qualicon BAX System Real-Time Assay for Salmonella test kits must be used. All samples identified as potentially positive for Salmonella using this test must be confirmed using AS 5013.10.

The detection of Salmonella spp. is broken down into the following stages:

§  Pre-enrichment in non-selective liquid medium

A 1:10 dilution of the sample is enriched in pre-warmed (35°C) buffered peptone water at 35°C for 20-24h. For carcass sponges, buffered peptone is added to the moistened sponge to bring the total volume to 60-100 mL and the sample incubated at 35°C for 20-24 h. In the case of carcase sponges the BPW only needs to be pre-warmed to room temperature before use.

Note: This method has also been validated for the analysis of 375 g of ground beef/trim sample in 1.5 L pre-warmed TSB with 2 mg/L novobiocin and incubated at 41°C for 24 hours.

§  BAX system for screening Salmonella

The BAX® System Real-Time Assay for Salmonella is an automated method that uses polymerase chain reaction (PCR) technology for the detection of Salmonella in foods. The system identifies specific DNA fragments, unique to Salmonella. BAX® System method uses lysis reagent which is prepared by adding 150 μL protease to 12 mL of lysis buffer. For each sample, 5 μL of the enrichment broth is added to 200 μL of prepared lysis reagent in cluster tubes. Tubes are heated for 20 minutes at 37°C and 10 minutes at 95°C and then cooled for at least 5 minutes at 4°C. PCR tablets in tubes are hydrated with 30 μL of lysate and run in a BAX® System Q7 machine.

§  Confirmation

In all cases of BAX-positive, BAX-indeterminate or a BAX-signal-error the BPW must be tested using AS 5013.10 (starting at the selective enrichment stage of the analysis). Confirmation must be carried out at a Department approved laboratory.

Issue 2017 01 10 | Approved Methods Manual

Export Standards Branch | Exports Division Page 2 of 2

Department of Agriculture and Water Resources

BAX(R) System Real-Time PCR for Salmonella - AOAC 2013.02

CHECKLIST

Pre-enrichment / Is the buffered peptone water warmed to room temperature (35°C)?
Is the correct amount of enrichment broth used for the weight of sample analysed?
Is a positive control run with each batch of samples analysed?
Are reference cultures inoculated into primary enrichment broth at a level of 10 to 100 cells?
Is enrichment carried out at 35°C for 20-24 h?
BAX screening / Are the manufacturer’s instructions available?
Is the shelf-life of media and kits controlled?
Confirmation / Are all suspect Salmonella sent to a reference Laboratory (see AS 5013.10) to be serotyped?
BPW should be supplied to off-site laboratories for confirmation following AS 5013.10
If Salmonella confirmation is done in-house refer to AS 5013.10.

Issue 2017 01 10 | Approved Methods Manual

Export Standards Branch | Exports Division Page 2 of 2

Department of Agriculture and Water Resources