SOP: The Genequence Listeria monocytogenes assay for the detection of Listeria monocytogenes in raw meat and stainless steel surfaces

GIFSQ — Global Institute for Food Safety & Quality

Note: The method’s title block prints “Genequence”; the Principle section prints “GeneQuence”. Both are carried as printed.

Principle

This qualitative test detects Listeria monocytogenes in foods by DNA hybridization. Specific DNA probes for L. monocytogenes, labeled directly with horseradish peroxidase, give a colorimetric endpoint after broth-culture enrichment. After pre-enrichment and selective enrichment, target cells are lysed enzymatically at 37 °C, and L. monocytogenes B-specific oligonucleotide probes (as the source prints it) are added for a 60-minute hybridization at 45 °C. Where L. monocytogenes ribosomal RNA (rRNA) is present, the peroxidase-labeled detector probe and a poly dA-tailed capture probe hybridize to the target rRNA, while base pairing between the poly dA tail and poly dT-coated polystyrene microwells captures the probe–target hybrids on the solid phase. Unbound probe is washed away and substrate–chromogen is added; the peroxidase reaction produces a blue colour, stopped by sulfuric acid, which turns the colour from blue to yellow. Absorbance measured at 450 nm in excess of the threshold value indicates the presumptive presence of L. monocytogenes in the test sample.

The test can detect as little as one CFU of L. monocytogenes in a 25 g food sample when the specified enrichment protocols are used, and should be performed by personnel with appropriate laboratory training in microbiology. Positive results must be confirmed with a cultural method.

This revised method replaces MFLP-14, dated February 2011. It is validated for raw meat and stainless steel surfaces. The method is assumed usable with other foods, but any commodity outside the listed application must be properly validated for fitness for purpose following the criteria in the Compendium of Analytical Methods, with the validation data forwarded to the Microbiological Methods Committee.

Apparatus

• Blender or homogenizer
• Incubator at 30 °C 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 (the source prints “450nM” — rendered from the printed value)
• 500 mL or 1 L graduated cylinder
• Test tube rack
• Timer

Note: Where 30 °C is recommended in the method, the incubator may be 30 ± 2.0 °C.

Reagents

Supplied with the kit (#6708; 93 tests, Neogen Corp.)

• 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; the source prints “L. monocytogenes.”)
• 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 the microtiter plate, positive control, negative control, and reagents 1A, 2, 3, and 5 at 2–8 °C. 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, it must be stored at −20 °C and is stable for 60 days in this form.

Enrichment broths

• Palcam Broth
• To prepare: basal medium — 23.0 g peptone, 5.0 g yeast extract, 10.0 g lithium chloride (LiCl), 0.8 g esculin, 0.5 g ammonium iron(III) citrate, 5.0 g D(-) mannitol, 0.08 g phenol red, 1.0 g soybean lecithin and 2.0 g Tween 80 per 1.0 L of distilled water; final pH 7.2 ± 0.2. Supplements (per 500 mL basal medium): 5.0 mg polymyxin-B-sulphate, 10.0 mg ceftazidime and 2.5 mg acriflavine dissolved in 5.0 mL distilled water and filter sterilized. Suspend the basal ingredients in 1.0 L of distilled water, adjust the pH if necessary, heat to boiling to dissolve completely, and autoclave at 121 °C for 15 min; cool to 50 °C, aseptically add the supplement solution to 500 mL of the cooled base (double the amount for 1.0 L), mix and dispense. Store under refrigeration in the dark, since acriflavine can photo-oxidize to form compounds inhibitory to Listeria spp.
• UVM 2

The sample handling, preparation, enrichment, and the formulations of these broths follow MFHPB-07, “Isolation of Listeria monocytogenes and Other Listeria spp. from Foods and Environmental Samples Using Palcam Broth” (see the GIFSQ SOP page: https://gifsq.com/food-analysis/sop-isolation-of-listeria-monocytogenes-and-other-listeria-spp-from-foods-and-environmental-samples-using-palcam-broth/). Media are commercially available and are prepared and sterilized according to the manufacturer’s instructions. Formulas of individual media are given in Appendix G of Volume 1 of the Compendium.

Note: It is the responsibility of the laboratory to ensure equivalency if any variations of the media formulations listed here are used (either product that is commercially available or made from scratch).

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
• Phosphate-buffered saline (PBS), 10 mM sodium phosphate, 0.85% sodium chloride, pH 7.4
• Absorbent paper
• 10 or 25 mL disposable graduated pipettes

Sample preparation

Handling of sample units, preparation for analysis, preparation of samples, and pre-enrichment and enrichment all follow MFHPB-07, “Isolation of Listeria monocytogenes and Other Listeria spp. from Foods and Environmental Samples Using Palcam Broth” (see the GIFSQ SOP page: https://gifsq.com/food-analysis/sop-isolation-of-listeria-monocytogenes-and-other-listeria-spp-from-foods-and-environmental-samples-using-palcam-broth/).

Procedure

Note: Perform the assay in a normal laboratory environment with respect to humidity, lighting, etc. Steps requiring room temperature incubation are performed at 18–30 °C. Kit reagents that have been refrigerated are equilibrated to room temperature before use, but are not left out unrefrigerated for long periods of time.

Preparing the reagents

Allow the refrigerated reagents to equilibrate to room temperature
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Turn on the water bath or heater block and adjust to 37 ± 1 °C
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Fill a water bath to a level of approximately 1.5 inches, or fill the heater block wells about 1/3 with deionized water
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Prepare the pretreatment reagent by adding 12 mL of pretreatment buffer (bottle 1b) directly to the pretreatment concentrate (bottle 1a); dissolve the contents by gently swirling
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Prepare the lysis reagent by adding 12 mL of lysis reagent buffer (bottle 2b) directly to the bottle of lysis reagent concentrate (bottle 2a); dissolve the contents by gently swirling

Note: The pretreatment reagent prepared in step 3 and the lysis reagent prepared in step 4 may be combined in bottle 2a. Separate pretreatment and lysis reagents, or the reagents combined in bottle 2a, are stable in the reconstituted form for 60 days when stored at −20 °C. To thaw, place bottles at room temperature. When thawed, gently swirl the contents. Return the reconstituted reagents to storage at −20 °C immediately after each use.

For each sample to be tested, label a 12 × 75 mm borosilicate glass test tube with the appropriate 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 by mixing 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); fill the buffer reservoir of the plate-washing device (see the manufacturer’s instructions for set-up and use; the source prints “manufacturer=s”)

Note: Wash solution can be stored in a closed bottle at room temperature for up to 60 days.

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. monocytogenes mixing chart provided with the test kit or use the formulas in the Calculation section
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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

Assay

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
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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
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The resulting solution should be green; if any tubes are not green, check for proper reagent addition
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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 lysed 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:
– 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
– 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

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 eliminated.

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
• Control values: 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 monocytogenes in the test sample
• Positive criterion: assays producing an absorbance value > 0.10 are indicative of the presumptive presence of Listeria monocytogenes in the test sample; these samples must be confirmed by standard culture procedures

Confirmation

Using the secondary enrichment broth, proceed with the plating and confirmation steps of a cultural method (MFHPB-07 or MFHPB-30, “Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples”; see the GIFSQ SOP page: https://gifsq.com/food-analysis/sop-isolation-of-listeria-monocytogenes-and-other-listeria-spp-from-foods-and-environmental-samples/).

Calculation

Volume of hybridization solution (bottle 3) = [(N × 0.1) + 1.6] mL

Volume of probe solution (bottle 4) = [(N × 0.025) + 0.4] mL

Where,

• N = number of test samples + controls

Note: The formulas provide 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.

Precautions

• The laboratory Supervisor must ensure that the analysis is carried out in accordance with ISO/IEC 17025:2005 (or the latest version): General Requirements for the Competence of Testing and Calibration Laboratories
• Stop solution contains 4.0 N sulfuric acid; hybridization solution contains formamide; avoid contact with skin and mucous membranes; refer to the Material Safety Data Sheet for more information
• It is the responsibility of each laboratory to ensure that the temperature of the incubators or water baths is maintained at the recommended temperatures
• Perform the assay in a normal laboratory environment with respect to humidity, lighting, etc.; steps requiring room temperature incubation are performed at 18–30 °C
• Kit reagents that have been refrigerated must be equilibrated to room temperature before use, but must not be left out unrefrigerated for long periods of time
• Reconstituted reagents are stable for 60 days at −20 °C; return them to storage at −20 °C immediately after each use
• All air bubbles must be removed from the wells before proceeding to the substrate step; if air bubbles remain, repeat vigorous striking of the wells onto an absorbent paper towel on a flat surface until eliminated
• It is the responsibility of the laboratory to ensure equivalency if any variations of the media formulations listed here are used (either product that is commercially available or made from scratch)

References

• Health Canada, Compendium of Analytical Methods, Volume 3 — Laboratory Procedure MFLP-14, October 2013: “The Genequence Listeria monocytogenes assay for the detection of Listeria monocytogenes in raw meat and stainless steel surfaces”
• 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.
• 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). Volume 2. The Compendium of Analytical Methods.