The Qualicon Bax® System Method for the detection of Salmonella in a variety of food and environmental samples

Laboratory Procedure
MFLP-29
June 2012

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Health Products and Food Branch
Ottawa

Microbiological Methods Committee
Microbiology Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Health Products and Food Branch, Health Canada
Postal Locator: 2204E
Ottawa, Ontario K1A 0K9

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

1. Application

This method is applicable to the detection of Salmonella species to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. Positive results must be confirmed with a cultural method. This method has been validated for use in a wide variety of food, including meat, poultry, fish and seafood, fruits and vegetables, dairy, miscellaneous food products and environmental samples. This revised method replaces MFLP-29, dated October 2011.

2. Description

The BAX® system is a yes/no screening tool for detecting Salmonella in a variety of foods using Polymerase Chain Reaction (PCR) technology for rapid amplification and fluorescent detection. After a 22-26 hour pre-enrichment, followed by a 3 hour regrowth step or overnight secondary enrichment in TT/RVS in some foods, sample preparation involves about 1 hour of user time. The automated procedure then delivers reliable results about 4 hours later.

The test kit has been modified to include hot start technology, but the remainder of the assay remains the same. The hot start version of the test kit, BAX® system PCR assay for Salmonella 2, is interchangeable with the original BAX® System PCR assay for Salmonella. The BAX® system is designed for use by qualified lab personnel who follow standard microbiology practices.

3. Principle

The BAX® System uses the Polymerase Chain Reaction (PCR) to amplify three specific fragments of bacterial DNA, which are stable and unaffected by growth environment. The three fragments are genetic sequences that are unique to Salmonella, thus providing a highly reliable indicator that the organism is present. The automated BAX® system then uses fluorescent detection to analyze PCR product for positive or negative results. PCR is a means for quickly providing millions of copies of a specific DNA fragment. In a typical application, sample DNA is combined with polymerase, nucleotides and primers that are specific for a given nucleotide sequence. This mixture then undergoes a series of timed heating and cooling cycles. Heating denatures or separates the DNA into single strands, then as the mixture cools, primers recognize and anneal to the target DNA sequence. DNA polymerase then uses nucleotides to extend the primers, thereby creating two copies of the target DNA fragment. Repeated cycles of denaturing, annealing, and extending produce exponential increases in the number of target DNA fragments within a matter of hours. If the target sequence is not present, no detectable amplification takes place.

The BAX® system combines the primers, polymerase, nucleotides and positive control into a single sample tablet that is packaged inside PCR tubes. Additionally, the automated fluorescent detection allows for closed-tube testing, eliminating the potential for carry-over contamination with amplified DNA.

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

Note: The Laboratory Supervisor must ensure that the analysis described in this method is carried out in accordance with the International Standard referred to as AISO/IEC 17025:1999 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories".

The media and reagents listed below are commercially available and are to be used, prepared and/or sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 for the formula of most of the individual media.

Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.

1. Supplied with kit - (Salmonella no. D11000133, Salmonella 2 no. D14368501; each sufficient for 96 tests B DuPont Qualicon, phone: 1-800-863-6842, fax: 302-695-5301)

   2 bags

   PCR sample tablets, packaged 1 tablet per PCR tube in 12 strips of 8 tubes. The tablets include the reagents needed for the test reaction as well as an internal positive control (which eliminates the need to run a separate QC reaction). Tablets are 7.6 ± 0.1 mg.

   1 bag

   Optical caps, 12 strips of 8 caps.

   2 bottles

   Lysis buffer, pH 8.35 ± 0.05 @ 25°C. 12 ml/bottle. Used to prepare working lysis reagent.

   1 vial

   Protease solution, 400 μL/vial. Used to prepare working lysis reagent

   
   BAX® system User Guide
2. Enrichment/regrowth broths - vary by food type (see Section 7)
   • Buffered Peptone Water broth
   • Buffered Peptone Water + Novobiocin broth (for sprouts)
   • Lactose broth
   • BHI broth
   • Brilliant Green Water
   • Nonfat dry milk
3. Equipment
   • Stomacher
   • Incubator capable of maintaining 35 and 42°C

Note: It is the responsibility of each laboratory to ensure that incubators and waterbaths are maintained at the recommended temperatures. Where 35°C is recommended in the text of the method, the incubators and waterbaths may be at 35 ± 1.0° C. Similarly, lower temperatures of 30 or 25 may be ± 1.0°C. However, where higher temperatures are recommended, such as 43 or 45.5°C, it is imperative that the incubators and waterbaths be maintained within 0.5°C due to potential lethality of higher temperatures on the microorganism being isolated.

• Other (included with BAX® system start-up package):
  • Cycler/detector with verification plates
  • Computer workstation with Microsoft Windows 7 operating system, BAX® system application, and printer
  • Dry block heaters with thermometers inserts for lysis tubes
  • Capping/decapping tools
  • Various pipettes for reagent and sample transfers
  • Cooling block with inserts for lysis tubes and PCR tubes
  • PCR tube holders
• Supplies
  • Lysis tubes with caps and rack
  • Tips for pipettes
  • Powder-free nitrile gloves

7. Procedure

7.1 Collect and enrich samples

Prepare samples according to a standard method for the food type, as follows:

Table 1. Sample Pre-enrichment and Enrichment or Regrowth Steps

Sample type	Pre-enrichment	Enrichment or Regrowth
Table 1 footnotes Table 1 footnote 1 raw meat: any raw meat with NO spices, fillers, or flavours. Table 1 footnote 2 any processed meats and raw meats containing spices, fillers and flavours.
Raw meat and poultry	Blend 25 g sample in 225 mL buffered peptone water broth. Incubate at 42°C for 24 hours.	Add 10 μL enriched sample to 500 μL BHI broth (room temperature). Incubate at 37°C for 3 hours. Proceed to step 7.3.1.4
Raw processed meats	Blend 25 g sample in 225 mL buffered peptone water broth. Incubate at 35°C for 20-24 hours.	Add 10 μL enriched sample to 500 μL BHI broth (room temperature). Incubate at 37°C for 3 hours. Proceed to step 7.3.1.4
Environmentals, such as swabs, sponges, and meat trays	Incubate sample in buffered peptone water broth at 35°C for 20-24 h.	Add 10 μL enriched sample to 500 μL BHI broth (room temperature). Incubate at 37°C for 3 hours. Proceed to step 7.3.1.4
Eggs	Blend 25 g sample in 225 mL buffered peptone water broth. Incubate at 35°C for 20-24 hours.	Add 10 μL enriched sample to 500 μL BHI broth (room temperature). Incubate at 37°C for 3 hours. Proceed to step 7.3.1.4
Sprouts	Blend 25 g sample in 225 mL buffered peptone water with novobiocin. Incubate at 42°C for 20-24 hours. Novobiocin: Prepare novobiocin as a 20 mg/mL solution in distilled water. Filter sterilize through a 0.2 µm filter, then transfer 1 mL novobiocin solution to 1 litre of the cooled (<50°C) buffered peptone water broth. Final pH 7.2± 0.2.	Add 10 μL enriched sample to 500 μL BHI (room temperature). Incubate at 37°C for 3 hours. Proceed to step 7.3.1.4
Dairy products	For fluid milk, ice cream, powdered products (e.g. whey and buttermilk) and skim milk powder, suspend 25 g in 225 mL brilliant green water. For cheese and all other dairy products, suspend 25 g in 225 mL buffered peptone water. Adjust pH to 6.0 - 7.0 Incubate raw milk cheese at 42°C and other dairy products at 35°C, for 22-26 hours.	Transfer 0.1 mL and 1.0 mL of the buffered peptone broth into 9.0 mL of RVS and TBG broths. Incubate for 24 ± 2 h at 42.5°C. Combine 2 mL from each broth and mix. Proceed to step 7.3.1.4
Fish and seafood	Blend 25 g sample in 225 mL lactose broth. Let stand at room temperature for 55-65 minutes. Adjust pH to 6.8±0.2. Incubate at 35°C for 22-26 hours.	No enrichment nor regrowth. Proceed to step 7.3.1.4
Candy and candy coating (including chocolate)	Blend 25 g sample with 225 mL Skim Milk Medium. Let stand at room temperature for 55-65 minutes. Adjust pH to 6.8±0.2. Add 0.45 ml brilliant green dye (1.0% w/v) and incubate at 35°C for 22-26 hours.	Add 10 μL enriched sample to 500 μL BHI (room temperature). Incubate at 37°C for 3 hours. Proceed to step 7.3.1.4
Cantaloupes	Place entire cantaloupe into a stomacher bag and add up to 1.5 times the weight of the cantaloupe of buffered peptone broth. When the analysis is done on a larger cantaloupe (~2 kg), a lesser volume of buffered peptone broth may be used provided there is sufficient volume to totally submerge the fruit. Mix for 30 seconds by manually rubbing the rind. Submerge and hold at room temperature for 30 minutes and again manually rub the rind. Adjust pH to 6.0 - 7.0 Incubate at 35°C for 20-24 hours. Add a weight to the outside of the stomacher bag to ensure that the fruit remains submerged during incubation.	Transfer 0.1 mL and 1.0 mL of the buffered peptone broth into 9.0 mL of RVS and TBG broths. Incubate for 24 ± 2 h at 42.5°C. Combine 2 mL from each broth and mix. Proceed to step 7.3.1.4
Other foods	Prepare 25 g sample in 225 mL appropriate enrichment broth for the food type according to specifications in MFHPB-20 or FDA-BAM Chapter 5.	Add 10 μL enriched sample to 500 μL BHI (room temperature). Incubate at 37°C for 3 hours. Proceed to step 7.3.1.4
Alternative: all foods except any listed above	Prepare 25 g sample in 225 mL appropriate enrichment broth for the food type according to specifications of ISO 6579 method.	(Exceptions: meat, poultry, fish and seafood) Add 10 μL enriched sample to 500 μL BHI (room temperature). Incubate at 37°C for 3 hours. Proceed to step 7.3.1.4

Examples of processed meat: chicken nuggets, sausages, bacon bits, chicken with BBQ spices, and pre-made hamburgers.

7.2 Prepare equipment (Refer to BAX® System User Guide for details)

• 7.2.1 Make sure cooling blocks have been refrigerated overnight.
• 7.2.2 Warm up heating blocks. Check that temperatures are set to 37°C and 95°C.
• 7.2.3 Initialize BAX® system cycler/detector (perform verification, if prompted).
• 7.2.4 Create a rack file.
• 7.2.5 Select RUN FULL PROCESS in the menu bar to warm up the cycler/detector.

7.3 Prepare samples

• 7.3.1 Lyse samples
  • 7.3.1.1 Arrange required number of lysis tubes (one for each sample and one for the "blank"; see the BAX® manual for explanation of "blank") in the rack according to the rack file.
  • 7.3.1.2 Prepare lysis reagent by pipetting 150 µL of protease into one 12-mL bottle of lysis buffer. Prepared lysis reagent can be stored in the refrigerator for up to two weeks.
  • 7.3.1.3 Add 200 µL of lysis reagent to each lysis tube.
  • 7.3.1.4 After the appropriate pre-enrichment, and enrichment or regrowth step listed in Table 1 (7.1), transfer 5 µL of enriched sample to the corresponding lysis tube.
  • 7.3.1.5 Secure the caps and heat the tubes at 37°C for 20 minutes, then at 95°C for 10 minutes.
  • 7.3.1.6 Place the lysis tubes in cooling block for at least 5 minutes.
• 7.3.2 Prepare samples for PCR
  • 7.3.2.1 Place PCR tube holder onto PCR cooling block.
  • 7.3.2.2 Arrange one PCR tube per sample in the holder.
  • 7.3.2.3 Remove and discard lid from one strip of tubes at a time.
  • 7.3.2.4 Using multi-channel pipette, transfer 50 µL of each lysed sample into a corresponding PCR tube.
  • 7.3.2.5 Cover tubes with a new optical cap strip and secure tightly. Repeat for all samples.
  • 7.3.2.6 Take the entire cooling block to the cycler/detector. Samples should remain in the cooling block until the cycler/detector is ready for loading, but no more than 30 minutes after tablet hydration.

7.4 Process samples

Follow the screen prompts of the PCR Wizard to load your samples, run the program and unload your samples, as specified in the User Guide.

7.5 Review results

7.6 Confirm positive results

Positive samples must be confirmed culturally as describe in MFHPB-20. Spread plate to specified selective agars and confirm biochemically and serologically.

8.0 References

• 8.1 Health Canada, 1998. MFHPB-20. Methods for the Isolation and Identification of Salmonella from Foods. In: Volume 2. Compendium of Analytical Methods, Ottawa.
• 8.2 Technical Committee 34. 2002. ISO 6579.  Horizontal method for the detection of Salmonella spp. International Organization for Standardization. Microbiology of food and animal feeding stuffs. 4th ed. International Organization of Standards: Geneva.
• 8.3 US Department of Agriculture 2001.  Chapter 4, Isolation and Identification of Salmonella from Meat, Poultry, and Egg Products. Microbiology Laboratory Guidebook. Washington.
• 8.4 US Food & Drug Administration, Center for Food Safety & Applied Nutrition. 2001. Chapter 5,  Salmonella. Bacteriological Analytical Manual. Washington.

End of Document