SOP: Detection of Listeria Species in a Variety of Environmental Surface Samples Using the GENE-UP Listeria spp. 2 (LIS 2) Method

GIFSQ — Global Institute for Food Safety & Quality

Principle

This method detects Listeria species in a variety of environmental surface samples to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations. GENE-UP Listeria spp. 2 (LIS 2) is a real-time polymerase chain reaction (PCR) assay for Listeria spp. (the source prints “Listeria grayi” for the excluded species), and presumptive results can be obtained after a 24h enrichment of the environmental surface samples.

The chemistry of the kit method is based on Fluorescence Resonance Energy Transfer (FRET). Both the assay for the sample and the internal amplification control (IAC) use dual FRET hybridization probes: two different short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the reaction cycle. The first probe for the sample assay is labeled at the 3′ end with fluorescein; the second probe is labeled at the 5′ end with LC Red 640. FRET occurs only after the two probes come in close proximity from hybridizing to the template DNA.

The resulting fluorescent signal from the FRET interaction forms a real-time amplification curve, which is how the amplified target is detected by the GENE-UP Thermocycler. After the PCR cycling program finishes, the PCR product(s) are melted to determine the presence of the target DNA. The software uses both the real-time amplification curve (FRET) and the melt peak (MELT) to make a positive or negative call.

Apparatus

• GENE-UP Thermocycler System (ref. 4704266) – includes:
• GENE-UP thermocycler
• computer
• GENE-UP routine software
• MPS1000 Mini PCR Plate Spinner (110V)
• Troemner vortex mixer
• GENE-UP tips (Biotix)
• GENE-UP Lysis Tube Holder
• GENE-UP PCR Tube Holder
• GENE-UP Heavy Rack Holder
• GENE-UP Lysis Rack Adaptor
• GENE-UP Lysis Tube Remover
• GENE-UP System User Manual
• GENE-UP Listeria spp. 2 (LIS 2) Kit (ref. 423106) – includes:
• Ready-to-use freeze-dried pellets (contain sample-specific primers and probes and an internal amplification control)
• Strips tubes and caps
• 1 pouch with 1 control buffer vial – 850 μL
• GENE-UP Lysis Kit (ref. 414057) – includes:
• 2 pouches, each with 1 rack of 96 lysis tubes
• Calibrated adjustable, variable volume pipette (single or multichannel)
• Compatible sterile, filter pipette tips
• Stomacher bags
• Incubator capable of maintaining 35 ± 1.0°C
• Note: It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures. The following applies to steps of the method which apply to growth only. Where a temperature of ≤ 37°C is recommended in the text of the method, the temperature may be ± 1.0°C, e.g., 35 ± 1.0°C. However, where higher temperatures are recommended, it is imperative that the incubators or water baths be maintained within 0.5°C due to potential lethality of the higher temperatures on the microorganism(s) being isolated.

Reagents

The method formulates no media or reagents itself; the following are used as named:

• Enrichment Broth:
• Dehydrated LPT broth: 500 g (ref. 420984), 5 kg (ref. 420985)
• Ready to Use LPT broth: 6 x 100 mL (ref. 410846)
• Ready to Use LPT broth: 20 x 10 mL (ref. 410845)
• Ready to Use LPT broth: 4 x 3 L (Dilubag) (ref. 410849)
• Wetting Agents: Neutralizing buffer (NE), Dey and Engley or D/E neutralizing broth (D/E), Buffered Peptone Water, Letheen broth or HiCap neutralizing buffer (World Bioproducts) (the materials list prints “Dey and Engley or D/E neutralizing broth (D/E)”; the Collection of Samples section prints “Dey and Engley neutralizing broth (D/E)”)
• To prepare Buffered Peptone Water: dissolve 10.0 g of peptone, 5.0 g of sodium chloride (NaCl), 3.5 g of disodium phosphate and 1.5 g of potassium dihydrogen phosphate in 1.0 L of distilled water; final pH 7.2 ± 0.2. Dispense into appropriate glassware and autoclave at 121°C for 15 min.

Sample preparation

Follow MFLP-41 for collection and transportation of environmental surface samples. Moisten the environmental collection device with Neutralizing buffer (NE), Dey and Engley neutralizing broth (D/E), buffered peptone water, Letheen broth or HiCap neutralizing buffer.

• In the laboratory, prior to analysis, keep sample units refrigerated.
• Analyze the sample units as soon as possible after receipt at the laboratory.
• Acclimatize prepared LPT enrichment broth to room temperature (15-25°C) before use.
• For environmental surface sponges: place the sponge in a sterile stomacher bag; add 100 mL of LPT broth to each sponge, or composite up to 10 sponges with 100 mL of media per sponge ensuring complete coverage with the media; mix manually for 1 minute.
• For environmental surface swabs: place the swab in a 10 mL LPT broth tube, or composite up to 10 swabs with 10 mL per swab; rotate swabs by hand for 1 min.
• Incubate for 24h ± 2h at 35 ± 1°C.

Procedure

The test shall be carried out in accordance with the following instructions:

Sample lysis

• After incubation, manually mix the samples.
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• Use the plate map created in the GENE-UP Routine software to determine the number of lysis tubes required from the GENE-UP Lysis kit. Prepare blank, positive and negative controls as per laboratory requirements.
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• Place the required number of lysis tubes in the GENE-UP Lysis Tube Holder. The strip can be cut apart, if less than 8 tubes are required.
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• Clip the GENE-UP Lysis Tube Holder on the GENE-UP Heavy Rack Holder.
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• Transfer 20 μL of enriched sample into the lysis tube (buffer is red). Use the plate map from the GENE-UP Routine software to pipette each sample into the correct plate position.
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• Remove the GENE-UP Lysis Tube Holder from the GENE-UP Heavy Rack Holder.
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• Clip the GENE-UP Lysis Tube Holder on the GENE-UP Lysis Rack Adaptor.
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• Run the vortex mixer at 2000 – 2200 rpm (depending on the max speed of the vortex mixer) for 5 minutes.
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• When the lysis is complete, remove the GENE-UP Lysis Tube Holder from the GENE-UP Lysis Rack Adaptor.
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• Clip the GENE-UP Lysis Tube Holder into the GENE-UP Heavy Rack Holder, and proceed to Final Setup for PCR.

Final setup for PCR

• Use the plate map in the GENE-UP Routine software to determine the number of PCR tubes needed from the GENE-UP LIS 2 kit. Place the required number of PCR tubes in the GENE-UP PCR Tube Holder. The strip can be cut apart, if less than 8 tubes are required.
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• Remove the transportation caps from the strips. First tap the strips on the bench to ensure the pellets are at the bottom of the tubes. Then, carefully open the caps and visually check the bottom of each tube for the freeze-dried pellets.
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• Using the 10 μL Biotix filter pipette tip with a single or multichannel pipette, transfer 10 μL of lysed sample (samples are red) to the corresponding PCR tube. For a negative control, use 10 μL of control buffer instead of lysed sample.
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• Do NOT agitate the lysate before aspirating the sample. The solid material must stay at the bottom of the tube.
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• Visually check the tips to confirm the absence of beads, bubbles, and for correct volume of lysate.
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• Place the strip caps onto each strip tube using the GENE-UP Lysis Tube Remover Tool. The strips can be cut apart, if less than 8 caps are required.
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• Place the GENE-UP PCR Tube Holder containing the PCR tubes in the plate centrifuge. Balance the centrifuge and spin for 10 seconds. The samples are ready to be processed in the GENE-UP instrument.

Amplification and detection

• Follow the screen prompts on the GENE-UP instrument to start a run, and view results. Refer to the appropriate GENE-UP Instrument User Manual for instructions, and for use of the GENE-UP Routine software.

Interpretation

Results are automatically interpreted once the GENE-UP LIS 2 real-time PCR run is completed. The GENE-UP Routine software interprets data of both amplification and melting curves for each sample and gives a positive, negative or invalid result as indicated in the following table:

Confirmation of presumptive positive results

• Using the enrichment broth, proceed with the plating and confirmation steps of a cultural method (i.e., MFHPB-30), as appropriate.
• Note that to isolate Listeria monocytogenes or other Listeria spp. from the enrichment broth, at least one chromogenic agar listed in MFHPB-30 must be used in addition to Oxford agar.

Negative and invalid results

• Negative results can be reported as Listeria spp. Not Detected.
• Invalid results must be treated as presumptive positive. Proceed with confirmation as described in the Confirmation of presumptive positive results section above.

Precautions

• The laboratory supervisor must ensure that completion of the analysis described in this method is done in accordance with the International Standards reference ISO/IEC 17025 (latest version): “General Requirements for the Competence of Testing and Calibration Laboratories”.
• 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. 2021. MFLP-112: Detection of Listeria Species in a Variety of Environmental Surface Samples Using GENE-UP Listeria spp. 2 (LIS 2) Method. Volume 3. Compendium of Analytical Methods. Health Products and Food Branch.
• Microbiological Methods Committee Evaluation Division, Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch (HPFB). 2011. Environmental sampling for the detection of microorganisms (MFLP-41). In: Volume 3, Compendium of Analytical Methods.
• Pagotto, F., Hébert, K., and J. Farber. 2011. Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples (MFHPB-30). In: Volume 2, The Compendium of Analytical Methods.