SOP: The Genequence Listeria spp. assay for the detection of Listeria spp. in foods and environmental surfaces

GIFSQ — Global Institute for Food Safety & Quality

Application

This method applies to the rapid detection of Listeria spp. for checking compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. It has been validated for raw meat, raw unprocessed seafood, raw frozen vegetables, cheese made from unpasteurized milk, and environmental samples, including swabs or sponge samples taken from stainless steel, plastic, concrete, cast iron, ceramic, or painted wood surfaces. Not enough validation data have been received for heat processed meat, heat processed poultry, heat processed dairy, and frozen dairy. This method replaces the November 2005 version of MFLP-13.

• Although this method is approved only for the food products listed above, it is assumed that it could be used with other foods. For commodities outside this application, the method must be properly validated as fit for purpose following the criteria in the Compendium of Analytical Methods, and the validation data should be forwarded to the Microbiological Methods Committee so the application can be expanded if the data meet the Committee’s requirements.

Principle

The GeneQuence DNA hybridization test (the method’s printed title spells it “Genequence”) is a DNA probe-based diagnostic supplied in kit format, which permits rapid and accurate detection of Listeria spp. in foods and on environmental surfaces, with results in approximately 30–48 hours. Its sensitivity is equivalent to that of reference culture procedures, and it can detect as few as 1 Listeria cell in a 25 g food sample when the specific enrichment protocols are used. The method is designed for use by qualified laboratory personnel who follow standard microbiological practices.

Listeria spp. specific DNA probes labeled with horseradish peroxidase and a colorimetric endpoint are used after broth culture enrichment. After sample pre-enrichment and selective enrichment, target cells are lyzed enzymatically at 37 °C, and Listeria-specific oligonucleotide probes (the source prints “Listeria B specific”) are added for a 60 minute hybridization incubation at 45 °C. If Listeria ribosomal RNA (rRNA) is present in the test sample, the detector probe — directly labeled with horseradish peroxidase (HRP) — and the polydeoxyadenylic acid (poly dA)-tailed capture probe hybridize to the target organism’s rRNA sequences. At the same time, base pairing between the poly dA-tailed capture probe and polydeoxythymidylic acid (poly dT) coated polystyrene microwells captures the probe–target hybrid molecules on the solid phase. Unbound probe is removed by washing, and substrate-chromogen is added to each well. The reaction of HRP with the substrate-chromogen produces a blue color. Adding sulfuric acid stops the reaction and changes the color of the substrate from blue to yellow. A microwell plate or microwell strip reader (A450) measures absorbance; absorbance above the threshold value indicates the presence of Listeria spp. in the test sample.

Apparatus

Equipment

• Blender or homogenizer
• Incubator at 30 and 35 °C
• Water bath or heat block at 37 °C
• 96-well block or incubator at 45 °C
• 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 (recommended) or repeater pipette to dispense 50 µL, 125 µL, and 150 µL volumes
• Microtiter plate or strip reader at 450 nm with discrimination of at least 0.01 absorbance unit
• 500 mL or 1 L graduated cylinder
• Test tube rack

• Timer

• Each laboratory is responsible for keeping the incubators or water baths at the recommended temperatures. Where 30 °C is recommended in the text of the method, the incubator may be 30 ± 2.0 °C.

Supplies

• Culture bottles for sample pre-enrichment
• Culture tubes for 11 mL sample volume
• Test tubes, glass, 12 × 75 mm, with caps
• Petri plates, 100 × 15 mm
• Sterile cotton swabs (the source prints “swaps”)
• Absorbent paper
• 10 or 25 mL disposable graduated pipettes (the source prints “graduate”)

Reagents

• GeneQuence Listeria spp. test kit (# 6708; 93 tests, Neogen Corporation), containing:
• Microtiter plate, 96 coated wells in divisible strips
• Pretreatment concentrate (bottle 1a)
• Pretreatment buffer, 12 mL (bottle 1b)
• Lysis reagent concentrate (bottle 2a)
• Lysis reagent buffer, 12 mL (bottle 2b)
• Hybridization solution, 18 mL (bottle 3)
• Listeria probe solution, 5 mL (bottle 4)
• Wash solution 20× concentrate, 50 mL (bottle 5)
• Substrate chromogen solution, 15 mL (bottle 6)
• Stop solution, 5 mL (bottle 7)
• Positive control, 5 mL (bottle +)
• Negative control, 5 mL (bottle −)
• Store the microtiter plate, the positive control, the negative control, and reagents 1A, 2, 3, and 5 at 2–8 °C. The other reagents may be stored at 2–25 °C. The entire kit may be stored at 2–8 °C.
• Once reagent 1A has been reconstituted, store it at −20 °C; it is stable for 60 days in this form.
• Enrichment broths (see MFHPB-07): Palcam Broth and UVM
• Phosphate-buffered saline (PBS): 10 mM sodium phosphate, 0.85% sodium chloride, pH 7.4

Sample preparation

Handling of sample units, preparation of analysis, preparation of samples, and pre-enrichment and enrichment all follow MFHPB-07.

Procedure

Make all pipetting transfers with a disposable pipette and pipetting bulb, or with a micropipette fitted with disposable tips. Do not mouth pipette. Perform the assay in a normal laboratory environment with respect to humidity, lighting, and so on. Carry out steps requiring room temperature incubation at 18–30 °C. Equilibrate refrigerated kit reagents to room temperature before use, but do not leave them out unrefrigerated for long periods.

Equilibrate the refrigerated reagents to room temperature
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Turn on the water bath or heater block and adjust to 37 ± 1 °C — fill the water bath to about 1.5 inches, or fill the heater block wells about one-third with deionized water
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Prepare the pretreatment reagent: add 12 mL of pretreatment buffer (bottle 1b) directly to the pretreatment concentrate (bottle 1a) — dissolve by gently swirling
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Prepare the lysis reagent: add 12 mL of lysis reagent buffer (bottle 2b) directly to the lysis reagent concentrate (bottle 2a) — dissolve by gently swirling
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Note: the prepared pretreatment reagent and the prepared lysis reagent may be combined in bottle 2a — the separate reagents, or the combined reagents in bottle 2a, are stable in the reconstituted form for 60 days when stored at −20 °C; thaw them at room temperature, gently swirl when thawed, and return the reconstituted reagents to −20 °C storage immediately after each use
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For each sample to be tested, label a 12 × 75 mm borosilicate glass test tube with the sample designation and place it in a rack — include tubes for 1 positive control and 1 negative control per experimental run
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Prepare the wash solution: mix the entire contents of the 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 — and fill the buffer reservoir of the plate-washing device (see the manufacturer’s instructions for set-up and use)
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Note: the wash solution can be stored in a closed bottle at room temperature for up to 60 days
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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, use the Listeria spp. mixing chart provided with the kit or the formula below (N = number of test samples + controls):
– Volume of hybridization solution (bottle 3) = [(N × 0.1) + 1.6] mL
– Volume of probe solution (bottle 4) = [(N × 0.025) + 0.4] mL
– The formula provides for an excess volume of 2.0 mL; a different excess volume may be appropriate depending on the type of container used to prepare the mixture
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Without touching the bottoms of the wells, place the needed 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, the negative control, and the positive control
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Mix the test samples by gently shaking the test tubes — shake the positive and negative control solutions by inverting the bottles several times — and add 0.2 mL of each control and test sample to the appropriately labeled tubes
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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, and if any tube is not green, check that the reagents were added properly — incubate the rack of tubes in the 37 °C water bath or heater block for 5 minutes
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Remove the tubes from the heat source — transfer 0.150 mL of each lyzed sample, including the controls, to a designated microwell — reserve the first well for the reagent blank (it receives no sample), use the second well for the negative control, and the third for the positive control
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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
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Incubate the plate at 45 °C for 60 minutes
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Wash the wells using one of the following procedures:
– (a) 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, 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 the sides of the frame to keep the strips in place
– (b) Manual: thoroughly wash the microwells at least 5 times using a plastic wash bottle filled with the wash solution prepared earlier — empty the wells into a suitable container, tap the inverted plate on absorbent paper, completely fill the wells with wash solution, and vigorously shake out the contents
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Note: all air bubbles must be removed before proceeding to the next step — if air bubbles remain, repeat vigorous striking of the wells onto an absorbent paper towel on a flat surface until they are gone
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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
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Add 0.050 mL of stop solution (bottle 7) to each microwell, including the blank microwell
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Gently tap the side of the frame a few times to ensure mixing
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Read absorbance at 450 nm using a plate or strip reader according to the manufacturer’s instructions — blank using the first microwell, which contains the mixture of substrate chromogen and stop solution (do not blank with air)

Interpretation of results

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.

Negative criterion

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

Positive criterion

• Assays producing an absorbance value > 0.10 are indicative of the presumptive presence of Listeria spp. in the test sample.

Confirmation

Using the refrigerated secondary enrichment broth, presumptive positive results may be confirmed with the plating and confirmation steps of a cultural method (MFHPB-07 or MFHPB-30), as appropriate.

• If the results indicate that Listeria spp. have not been detected, it is understood that the presence of L. monocytogenes has also not been detected, since no species belonging to the Listeria genus were detected. However, if Listeria spp. are detected in environmental samples, regardless of the amount of colony screening or additional confirmation steps done, these results cannot be used to state that L. monocytogenes is absent. Report all species of Listeria that are identified for environmental samples.

Precautions

• The Laboratory Supervisor must ensure that the analysis described in this method is carried out in accordance with the International Standard ISO/IEC 17025:1999 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories.
• The stop solution contains 4.0 N sulfuric acid. The hybridization solution contains formamide. Avoid contact with skin and mucous membranes.
• If the analyst uses any variations of the media listed here (either a commercially available product or media made from scratch), it is the analyst’s or Laboratory Supervisor’s responsibility to ensure equivalency.

References

• Health Canada, Compendium of Analytical Methods, Volume 3 — Laboratory Procedure MFLP-13, February 2011. The Genequence Listeria spp. assay for the detection of Listeria spp. in foods and environmental surfaces.
• Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.
• Pagotto, F., Hébert, K., Farber, J. 2011. Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples (MFHPB-30). In: Volume 2, The Compendium of Analytical Methods.
• Health Canada, Health Protection and Food Branch, Food Directorate. 2010. Policy on Listeria monocytogenes in Ready-to-Eat Foods.
• 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.
• US Food and Drug Administration, Center for Food Safety and Applied Nutrition. Bacteriological Analytical Manual Online. Washington: Chapter 10, Listeria monocytogenes.
• US Food and Drug Administration, Center for Food Safety and Applied Nutrition. Bacteriological Analytical Manual Online. Washington: Chapter 24, Identification of Foodborne Bacterial Pathogens by Gene Probes.
• 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.