The Genequence Listeria monocytogenes assay for the detection of Listeria monocytogenes in foods

Laboratory Procedure MFLP-14
February 2011

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Don Warburton
Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Postal Locator: 2204E
HPFB, Ottawa, Ontario, K1A 0K9

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

1. Application

This method is applicable to the rapid detection of Listeria monocytogenes 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 raw meat. Insufficient validation data have been received for hot dogs, deli ham, deli turkey, ice cream, pasteurized milk, brie cheese, parmesan cheese, smoked salmon, cooked crab meat, lettuce, frozen peas, soy flour and environmental samples. This revised method replaces MFLP-14, dated November 2005.

NOTE: While this method is only approved for certain food products, as listed above, it is assumed that this method could be used with other commodities. To ensure the method is fit for purpose for commodities outside the application, it is imperative that other commodities be properly validated following the criteria in the Compendium of Analytical Methods. It is requested that these validation data be forwarded to the Microbiological Methods Committee so the Application Section can be expanded to include these new foods if the data fulfill MMC requirements (refer to Development of Methods in Volume 1 of the Compendium of Analytical Methods).

2. Description

This test is intended to qualitatively detect Listeria monocytogenes in foods, and should be used by personnel with appropriate laboratory training in microbiology. This DNA hybridization test employs L. monocytogenes-specific DNA probes directly labeled with horseradish peroxidase, and a colorimetric endpoint to detect as little as one CFU of L. monocytogenes in a 25 g food sample when the specified enrichment protocols are used.

3. Principle

The GeneQuence DNA hybridization test employs Listeria monocytogenes specific DNA probes labeled with horseradish peroxidase and a colorimetric endpoint for the detection of L. monocytogenes in food samples following broth culture enrichment. After sample pre-enrichment and a period of selective enrichment, target cells are lysed enzymatically at 37°C and L. monocytogenes-specific oligonucleotide probes are added for a 60 minute hybridization incubation at 45°C. If L. monocytogenes ribosomal RNA (rRNA) is present in the test sample, detector probe directly labeled with horseradish peroxidase (HRP) and polydeoxyadenylic acid (poly dA)-tailed capture probe hybridize to the target organism rRNA sequences. Concurrently, base pairing between the poly dA-tailed capture probe and polydeoxythymidylic acid (poly dT) coated polystyrene microwells facilitates solid phase capture of the probe-target hybrid molecules. Unbound probe is removed by washing and substrate-chromogen is added to each well. The reaction of the HRP with substrate-chromogen produces a blue color. The reaction is stopped with the addition of sulfuric acid, which changes the color of the substrate from blue to yellow. A microwell plate or microwell strip reader (A450) measures absorbance; absorbance in excess of the threshold value indicates the presumptive presence of L. monocytogenes in the test sample.

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

The media listed below are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 for the formula of 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 for Laboratory Supervisor to ensure equivalency.

6.1 Supplied with kit

(# 6708; 93 tests, Neogen Corp., Tel: 517-372-9200, fax: 517-372-0108, web:  http://www.neogen.com)

1 Microtiter plate, 96 coated wells in divisible strips
1 bottle of pretreatment concentrate (labeled 1a)
1 bottle of 12 mL pretreatment buffer (labeled 1b)
1 bottle of lysis reagent concentrate (labeled 2a)
1 bottle of 12 mL lysis reagent buffer (labeled 2b)
1 bottle of 18 mL hybridization solution (labeled 3)
1 bottle of 5 mL L. monocytogenes. probe solution (labeled 4)
1 bottle of 50 mL wash solution 20X concentrate (labeled 5)
1 bottle of 15 mL substrate chromogen solution (labeled 6)
1 bottle of 5 mL stop solution (labeled 7)
1 bottle of 5 mL positive control (labeled +)
1 bottle of 5 mL negative control (labeled -)
Store, microtiter plate, positive control, negative control, and reagents 1A, 2, 3, and 5 at 2-8ºC. Other reagents may be stored at 2-25 ºC. The entire kit may be stored at 2-8ºC. Once reagent 1A has been reconstituted, it must be stored at -20 ºC and is stable for 60 days in this form.

Precautions: Stop solution contains 4.0N sulfuric acid. Hybridization solution contains formamide. Avoid contact with skin and mucous membranes. Refer to  Material Safety Data Sheet for more information.

6.2. Enrichment broths (see MFHPB - 7)

UVM 2
Palcam Broth

6.3. Materials

6.3.1 Equipment

Blender or homogenizer
Incubator at 30 and 35ºC
Water bath or heat block at 37 ºC
96-well block or incubator at 45 ºC
Small orbital platform shaker capable of 150 rpm (optional)
Vacuum source
Microtiter plate washing device with 8 wells per strip orientation
Micropipette or repeater pipette to dispense 100 µL volume
8-channel pipette (recommened) or repeater pipette to dispense 50 µL, 125 µL, and 150 µL volumes
Microtiter plate or strip reader at 450nM with discrimination of at least 0.01 absorbance unit
500 mL or 1L graduated cylinder
Test tube rack
Timer

6.3.2. Supplies

Culture bottles for sample pre-enrichment
Culture tubes for 11 mL sample volume
Test tubes, glass, 12 X 75 mm, with caps
Petri plates, 100 X 15 mm
Sterile cotton swaps
Phosphate-buffered saline (PBS), 10 mM sodium phosphate, 0.85% sodium chloride, pH 7.4
Absorbent paper
10 or 25 mL disposable graduate pipettes

7. Procedure

7.1 Handling of Sample Units

Follow MFHPB-07

7.2 Preparation of Analysis

Follow MFHPB-07

7.3 Preparation of Samples

Follow MFHPB-07

7.4 Pre-Enrichment and Enrichment

Follow MFHPB-07

7.5 Test Procedure

7.5.1 Prior to starting the assay:

7.5.1.1 Allow the refrigerated reagents to equilibrate to room temperature.

7.5.1.2. Turn on the water bath or heater block and adjust to 37°C ± 1°. Fill a water bath to a level of approximately 1.5 inches, or fill the heater block wells about 1/3 with deionized water.

7.5.1.3. Prepare pretreatment reagent by adding 12 mL of pretreatment buffer (bottle 1b) directly to the pretreatment concentrate (bottle 1a). Dissolve contents by gently swirling.

7.5.1.4. Prepare lysis reagent by adding 12 mL of lysis reagent buffer (bottle 2b) directly to the bottle of lysis reagent concentrate (bottle 2a). Dissolve contents by gently swirling.

7.5.1.5. For each sample to be tested, label a 12 x 75 mm borosilicate glass test tube with the appropriate sample designation and place in a rack. Include tubes for 1 positive control and 1 negative control per experimental run.

7.5.1.6. Prepare the wash solution by mixing entire contents of wash solution concentrate (bottle 5) with 950 mL of distilled or deionized water (if washing manually with a 500 mL wash bottle, use 25 mL of concentrate with 475 mL of water). Fill the buffer reservoir of the plate-washing device (see manufacturer's instructions for set-up and use).

7.5.1.7. Prepare a 4:1 hybridization/probe mixture by mixing hybridization solution (bottle 3) and probe solution (bottle 4) in a plastic container. For mixture guidelines, refer to the L. mono. spp. mixing chart provided with this test kit or use the formula below. (N = number of test samples + controls)

Volume hybridization solution (bottle 3) = [(N x 0.1) + 1.6] mL
Volume probe solution (bottle 4) = [(N x 0.025) + 0.4] mL

7.5.1.8. Without touching the bottoms of the wells, place the appropriate number of microwells in the plate frame, filling the frame left to right and front to back in rows of 8. Include wells for the reagent blank, negative control and positive control.

7.5.2. Assay Procedure

7.5.2.1. Mix the test samples by gently shaking the test tubes. Shake the positive and negative control solutions by inverting the bottles several times. Add 0.2 mL of each control and test sample to the appropriately labeled tubes.

7.5.2.2. Add 0.05 mL of reconstituted pretreatment reagent (bottle 1a) to each tube and 0.05 mL of reconstituted lysis reagent (bottle 2a), or add 0.1 mL of the combined pretreatment/lysis solution (bottle 2a) to each tube. Mix by gently shaking the rack of tubes by hand for 5 seconds. The resulting solution should be green. If any tubes are not green, check for proper reagent addition. Incubate the rack of tubes in the 37°C water bath or heater block for 5 minutes.

7.5.2.3. Remove the tubes from heat source. Transfer 0.150 mL of each lysed sample, including the controls, to a designated microwell. The first well should be reserved for the reagent blank and receives no sample. The second well should be used for the negative control, and the third for the positive control.

7.5.2.4. Vigorously mix the hybridization/probe solution prepared earlier. Add 0.125 mL to each microwell, and mix each well 5 times with the pipettor. Do not add hybridization/probe solution to the reagent blank microwell.

7.5.2.5. Incubate the plate at 45°C for 60 minutes.

7.5.2.6. Wash the wells using one of the following procedures:

1. Plate-washing device: Wash 5 times at room temperature. For each wash, process one 8-well strip at a time by aspirating the liquid, filling the wells, and then proceeding to the next strip. After the last wash, aspirate the liquid from the wells, then remove any residual liquid by inverting the plate and tapping it onto absorbent paper. Hold the plate by gently squeezing on the sides of the frame to keep the strips in place.
2. Manual: Thoroughly wash the microwells at least 5 times using a plastic wash bottle filled with the wash solution prepared earlier. Wash by emptying the wells into a suitable container, tapping the inverted plate on absorbent paper, completely filling the wells with wash solution, and vigorously shaking out the contents.

7.5.2.7. Add 0.150 mL of substrate chromogen solution (bottle 6) to each microwell, including the reagent blank microwell. Incubate the plate at room temperature for 20 minutes.

7.5.2.8. Add 0.050 mL of stop solution (bottle 7) to each microwell, including the blank microwell.

7.5.2.9. Gently tap the side of frame a few times to ensure mixing.

7.5.2.10. Read absorbance at 450 nm using a plate or strip reader according to the manufacturer's instructions. Blank using the first microwell that contains the mixture of substrate chromogen and stop solution (do not blank with air).

7.6 Interpretation of Results

7.6.1 Control Values

The absorbance value for the Negative Control must be < 0.15. Otherwise, the assay is invalid and should be repeated.

The absorbance value for the Positive Control must be > 1.00. Otherwise, the assay is invalid and should be repeated.

7.6.2 Negative Criterion

Assays producing an absorbance value < 0.10 are indicative of the absence of Listeria monocytogenes in the test sample.

7.6.3 Positive Criterion

Assays producing an absorbance value > 0.10 are indicative of the presence of Listeria monocytogenes in the test sample. These samples must be confirmed by standard culture procedures.

7.7 Confirmation

Using the refrigerated secondary enrichment broth, proceed with plating and confirmation steps of a cultural method (i.e., MFHPB-07 or MFHPB-30).

8. References

8.1 Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.

8.2 Pagotto, F., K. Hébert and J. Farber. 2011. Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples (MFHPB-30). Volume 2. Compendium of Analytical Methods. http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index_e.html

8.3 USDA-FSIS. 1998. Isolation and identification of Listeria monocytogenes from meat, poultry and egg products. Microbiology Laboratory Guidebook, 3rd ed. Government Printing Office, Washington, DC.  http://www.fsis.usda.gov/Ophs/Microlab/Mlgbook.pdf

8.4 US Food & Drug Administration, Center for Food Safety & Applied Nutrition. Bacteriological Analytical Manual Online. Washington: Chapter 10, Listeria monocytogenes. Available from  http://www.cfsan.fda.gov/~ebam/bam-10.html

8.5 US Food & Drug Administration, Center for Food Safety & Applied Nutrition. Bacteriological Analytical Manual Online. Washington: Chapter 24 Identification of Foodborne Bacterial Pathogens by Gene Probes  http://www.cfsan.fda.gov/~ebam/bam-24.html

8.6 Warburton, D., Boville, A., Pagotto, F., Daley, E. and Chow, C. 2011. The Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples using palcam broth (MFHPB-07). In: Volume 2. The Compendium of Analytical Methods. http://www.hc-sc.gc.ca/food-aliment

End of Document