Detection of Salmonella in Foods and Environmental Surfaces - Assurance GDS™ for Salmonella Gene Detection System

Laboratory Procedure MFLP-36
June 2006

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The Microbiological Methods Committee
Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Postal Locator: 2204A1
HPFB, Ottawa, Ontario, K1A 0L2

E-mail: don_warburton@hc-sc.gc.ca

1. APPLICATION

This method is applicable to the detection of Salmonella in a variety of foods and environmental surfaces including raw beef, raw pork, ground poultry, poultry rinse, raw shrimp, nonfat dry milk, liquid milk, egg, stainless steel, rubber and concrete to determine compliance with the requirements of Section 4 and 7 of the Food and Drugs Act.

2. DESCRIPTION

Assurance GDS for Salmonella (GDS) is an automated gene-based assay that incorporates multiple levels of specificity to ensure highly accurate results. Specifically, the method utilizes proprietary probes and specific primers directed against a highly conserved DNA sequence of the target organism. GDS also utilizes a proprietary device and reagents that concentrate populations of target microorganisms and eliminate potential competitive microflora. The method is designed to be highly selective, and does not detect microorganisms that are potential cross reactors in antibody-based assays. The proof of the principle was previously demonstrated in the validations of the Assurance GDS for E. coli O157:H7 Method (MFLP-16, AOAC OMA 2005.04) and Assurance GDS for Shiga-like Toxin Genes Method (AOAC OMA 2005.05).

3. PRINCIPLE

The Assurance GDS system incorporates a sample concentration step, proprietary genetic detection reagents, and a multi-channel, rotary instrument platform to achieve fast and accurate results. After enrichment, populations of target microorganisms are concentrated using a proprietary concentration device and reagents or an automated sample concentration instrument. The step provides two benefits. First, the technique concentrates cell populations. Second, it physically segregates target microorganisms from both competitive microflora and the food matrix. This also affords easier identification of suspect colonies on agar plates.

Each concentrated sample is then transferred to a reaction tube containing amplification reagents. The reagent system utilizes specific primers and proprietary probes directed against a highly conserved DNA sequence in the target organism. The reaction tube is then sealed and placed in a proprietary multi-channel instrument which allows for simultaneous amplification and detection by on-line optical analysis. If the target sequence is present, a specific fluorescence will be generated and read automatically. A procedural control is also contained in every reaction tube and a different distinctive fluorescence is read simultaneously and separately by the instrument. All sample determinations, positive and negative, are indicated at the end of analysis.

4. DEFINITION OF TERMS

See Appendix A of Volume 3.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 3

6. MATERIALS AND SPECIAL EQUIPMENT

1. Assurance GDS for Salmonella kit - Catalog no. 61008-100 sufficient for 100 tests. ( BioControl Systems www.biocontrolsys.com, 800-245-0113) consisting of:
   
   Assurance GDS Reaction tubes containing lyophilized reagents needed for Salmonella genetic detection and an internal positive control, 25 strips of 4 reaction tubes.
   Assurance GDS Sample Concentration Reagent, 1 vial.
   Assurance GDS Resuspension Buffer, 1 vial.
   Assurance Polymerase Dilution Buffer, 1 vial.
   Assurance GDS Wash buffer, 1 vial.
   
   
2. Hot start polymerase enzyme. ( www.eppendorf.com) or other suitable source. To be added to Assurance Polymerase Dilution buffer from kit. Invert the tube 5 times to mix. Record the date on the tube. Prepared polymerase buffer solution can be stored up to 1 month at 2-8°C.
   
   
3. Enrichment media
   Buffered peptone water (BPW) for most foods. Refer to Figure 1 for additional information.
   Brain heart infusion (BHI)
   
   
4. Materials
   
   Equipment (provided with start-up package)
   
   Assurance GDS Rotor-Gene system, computer and software.
   Assurance GDS sample concentration device (PickPen™ or equivalent).
   Assurance GDS vortex mixer
   Various calibrated pipets
   Aluminum block
   
   Supplies
   
   Sample blocks
   Resuspension plates
   Tips for pipettes and PickPen
   Adhesive film

7. PROCEDURE

7.1 Enrichment

For most foods, add 25 g of sample to 225 mL of Buffered Peptone Water (BPW) with the exceptions indicated in Figure 1. If larger test portion sizes are analyzed, proportionately increase the volume of media to maintain a 1:9 ratio. For environmental sponges, pre-moisten sterile dehydrated sponges with Dey-Engley Neutralizing (D/E) broth or equivalent. Sample a surface area of 100 cm² (4" X 4"). Place sponge into a sterile sample bag, then add 100 mL of BPW and mix well. For environmental swabs, pre-moisten sterile swabs with D/E broth or equivalent. Swab an area of 5 cm² (1" X 1"). Place swab into a tube with 10 mL of BPW and mix. Incubate all enrichments (foods and environmental surface swabs) for 18 - 24 h at 35-37°C.

Figure 1 - Recommended Enrichments

Food Type	Enrichment
Non-Fat Dry Milk	Brilliant Green Water for 20-24 hr @ 35-37°C
Raw Seafood	BPW w/ novobiocin for 18-24 hr@35-37°C)

7.2 Performing the Assurance GDS for Salmonella Assay

7.2.1. Vortex concentration reagent. Transfer 20 μL to each of the required number of wells on the sample block.

7.2.2 Continue with one of the following:

7.2.2.1. Low Microbial and Environmental Samples - Transfer 1.0 mL of Wash Solution to each of the required number of wells of a second or an unused portion of the first sample block (1 well per sample).

7.2.2.2. High Microbial Samples - Transfer 0.5 ml of sterile BHI broth to each of the required number of wells of a second or an unused portion of the first sample block (1 well/sample).

7.2.3. Transfer 35 μL of Resuspension Buffer to the sample wells in the resuspension plate.

7.2.4. Add 1 mL of incubated sample to each well in sample block containing Sample Concentration Reagent. Immediately return samples to 35-37°C incubator. Seal sample block with adhesive film and place on the vortex mixer and vortex at 600 rpm for 10 min. Remove sealing film after vortex mixing is complete.

7.2.5. Load tips onto the PickPen. Extend the magnetic tips of the PickPen and insert into the first row of the sample block. Stir gently for 30 secs.

7.2.6 Continue with one of the following:

7.2.6.1 Low Microbial and Environmental Samples - Transfer PickPen to corresponding sample block wells containing Wash Solution and gently swirl for 10 sec (do not release particles into solution). Transfer PickPen to the corresponding row of the prepared resuspension plate. With tips submerged, retract the PickPen magnets and release particles into the Resuspension Buffer.

7.2.6.2 High Microbial Samples - Transfer PickPen to corresponding sample block wells containing BHI. With tips submerged, retract the PickPen magnets and tap gently to release particles into the BHI. Incubate BHI sample block and particles for 2 h at 35-37°C. Following incubation, transfer the particles from the BHI sample block to the corresponding row of the prepared resuspension plate using the PickPen. With tips submerged, retract the PickPen magnets and release particles into the Resuspension Buffer.

7.2.7. Repeat steps in 7.2.5 through 7.2.6 for all samples using new tips for each row of samples.

7.2.8. Cool aluminum block at 2-8°C for at least 20 min prior to use. Place the required number of Assurance GDS reaction tubes in the aluminum block.

7.2.9. Open caps of reaction tubes in the aluminum block. Transfer 10 μL prepared polymerase buffer solution to each reaction tube.

7.2.10. Transfer 20 μL sample from each resuspension plate well into each reaction tube. Firmly press down on each reaction tube lid to close.

7.2.11. Place reaction tubes into Assurance Rotor-Gene™ in sequential order beginning with position #1 and run program.

7.3. Assay Results

7.3.1. Upon completion of the run, the Assurance Rotor-Gene program will provide a results table. Each sample will be identified as "Positive" indicating that the sample is positive for Salmonella, "Negative" indicating that the sample is negative for Salmonella, or "No Amp" indicating that amplification did not occur. Confirm GDS positive samples culturally following MFHPB-20. For "No Amp" results, contact BioControl Systems Technical Service 800245 0113

7.3.2. When the assay is completed, handle and dispose of all waste as a biohazard. Never open reaction tubes after amplification has started under any circumstances.