SOP: Detection of Listeria spp. from Environmental Surface Samples and Heat Processed Ready-to-Eat Meat and Poultry Using iQ-Check Listeria spp. Real-Time PCR Detection Kit

GIFSQ — Global Institute for Food Safety & Quality

Principle

This method is applicable to the rapid detection of Listeria spp. to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations. It has been validated for a variety of environmental surfaces and for the heat processed food type of the ready-to-eat meat and poultry food category. This revised method replaces MFLP-39, dated May 2013.

The method is approved only for environmental surfaces and heat processed ready-to-eat meat and poultry. Use with other commodities requires that the method be properly validated for those commodities following the criteria in the Compendium of Analytical Methods (refer to Development of Methods, Volume 1 of the Compendium of Analytical Methods).

The iQ-Check system is a real-time PCR based assay that involves simultaneous amplification and detection of the target organisms after a 25 ± 1 hr enrichment in Listeria Special Broth. The iQ-Check Listeria spp. kit detects all species of Listeria, including L. grayi. Results are automatically determined by the CFX Manager Software Industrial Diagnostic Edition (IDE) software.

The iQ-Check Listeria spp. kit is based on gene amplification and detection by real-time PCR. Ready-to-use PCR reagents contain DNA polymerase, nucleotides, DNA primers and a fluorescent DNA hybridization probe specific to a highly conserved region in the genome of all species of Listeria. PCR is a technique used to generate many copies of target DNA. In real-time PCR, specific oligonucleotide probes are used to detect the DNA during the amplification, by hybridizing to the amplicons. These probes are linked to a fluorophore which fluoresces only when hybridized to the target sequence. In the iQ-Check Listeria spp. kits, carboxyfluorescein (FAM) is the fluorophore linked to the probe hybridizing to the Listeria spp. specific DNA sequence. In the absence of target DNA, no fluorescence will be detected, and the sample is determined to be negative by the CFX Manager Software, IDE. As the amount of amplicons increases with each round of amplification, fluorescence intensity also increases. During each PCR cycle, at the annealing step, the real-time PCR system measures this fluorescence and the associated software plots the fluorescence intensity versus number of cycles. To monitor for a successful DNA amplification in each reaction well, a synthetic DNA “internal control” is included in the reaction mix. This control is amplified with a specific probe at the same time as the Listeria spp. target DNA sequence, and detected by a second fluorophore (HEX).

Apparatus

Supplies

Combitips pipette or equivalent repeat pipette
Combitips tips
Conical screw cap sterile tubes, 1.5 mL (for tube extraction only)
Deepwell microplates 12.5 cm × 8.5 cm × 4 cm (for deepwell extraction only) (catalog # 3594500)
Low-profile 0.2 mL PCR 8-tube strips without caps, thin-walled, white (Bio-Rad, catalog # TLS0851XTU)
Low-profile unskirted or skirted PCR plates, 48 or 96 well, white (Bio-Rad catalog # MLL4851XTU, MLL9651XTU, HSP9955XTU, HSP9655XTU)
Micropipettes — 20 µL, 200 µL and 1000 µL
Optical Flat 8-cap strips, ultraclear (Bio-Rad catalog # TCS0803XTU) or sealing film (Bio-Rad catalog # MSB1001XTU)
Pre-pierced sealing film (Bio-Rad catalog # 3600040) or equivalent (for deepwell extraction only)
Powder-free gloves
Sterile filter tips — adaptable to 20 µL, 200 µL and 1000 µL micropipettes
Stomacher bag with incorporated filter
Positive and negative control cultures

Equipment

Bench top centrifuge — maximum 10,000–12,000 × g, for 1.5 mL tubes (for tube extraction only)
Cell disruptor (Bio-Rad catalog # 3591456) (for tube extraction only)
Dry heat block, for 1.5 mL tubes — capable of maintaining 95–100 °C (for tube extraction only)
Incubator — capable of maintaining 30 °C for sample incubation
Magnetic stir plate
Real-time PCR system — CFX96 real-time PCR detection system (Bio-Rad cat. # 3600037) or MiniOpticon real-time PCR detection system
Stomacher, blender or equivalent
Thermoshaker (for deepwell extraction only)
Vortex apparatus

Reagents

iQ-Check Listeria spp. kit (Bio-Rad, catalog # 3578113), containing:
Reagent A — lysis reagent, 1 bottle (20 mL), contains magnetic stirbar
Reagent B — fluorescent probes, 1 tube (0.55 mL)
Reagent C — amplification mix, 2 tubes (2.2 mL each)
Reagent D — PCR negative control, 1 tube (0.5 mL)
Reagent E — PCR positive control, 1 tube (0.25 mL)
Reagent F — lysis beads, 1 bottle (17.6 g)
Listeria Special Broth (LSB, Bio-Rad catalog # 3555703 [225 mL × 6], 3564703 [500 g], 3564753 [5 kg]) — commercially available; prepare and sterilize according to the manufacturer’s instructions
Dey-Engley (D/E) neutralizing broth — prepare as follows:
Tryptone — 5.0 g
Yeast Extract — 2.5 g
Dextrose — 10.0 g
Sodium thioglycolate — 1.0 g
Sodium thiosulfate (Na₂S₂O₃) — 6.0 g
Sodium bisulfate (NaHSO₃) — 2.5 g
Polysorbate 80 (Tween 80) — 5.0 g
Lecithin (soybean) — 7.0 g
Bromocresol Purple — 0.02 g
Distilled water — 1.0 L
Dissolve ingredients with gentle heating, if necessary, and adjust pH to 7.6 ± 0.2. Dispense and autoclave for 15 min at 121 °C.

Sample preparation

Handling of sample units

In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen, depending on the nature of the product; thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death
Add Dey-Engley neutralizing broth to sterile sponges and swabs to moisten
The protocol of MFLP-41 (Environmental Sampling for the Detection of Microorganisms) should be followed for obtaining and transporting sponges and swabs
Analyze the sample units as soon as possible after receipt at the laboratory

Preparation of samples

Pre-warm Listeria Special Broth to 30 °C before use
To ensure a representative analytical unit, agitate liquids or free-flowing materials until the contents are homogeneous; if the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit
Prepare a 1-in-10 dilution of the food by aseptically adding 25 g or mL of the food (analytical unit) to 225 mL of LSB; for composite samples, analytical units may be combined up to 125 g or mL (e.g., 125 g or mL in 1125 mL of LSB); if alternate analytical units are required, maintain the 1-in-10 dilution
Add the environmental sponge to 100 mL of LSB, or composite up to 10 sponges with 100 mL of LSB per sponge; place environmental swabs (e.g., cotton tip) in 10 mL of LSB, or composite up to 10 swabs with 10 mL of LSB per swab
Blend, stomach or vortex as required for thorough mixing
Incubate all samples at 30 ± 1 °C for 25 ± 1 h

Procedure

Preparation for analysis

Turn on the heating block or thermoshaker in advance to reach 95–100 °C
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Turn on the thermal cycler and computer
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Carefully pour all the contents from Reagent F (lysis beads) into Reagent A (lysis reagent) to prepare the final lysis reagent
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Place the lysis reagent (A + F) on a magnetic stir plate and stir on medium speed
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Proceed to DNA extraction by the tube format or by the deepwell format

Note: A medium speed setting is used to ensure the lysis beads remain in suspension while dispensing. Using non-homogenous reagent could impact final results.

DNA extraction — tube format

Place the lysis reagent on a magnetic stir plate and stir on medium speed
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Add 100 µL of the lysis reagent to each 1.5 mL screw cap conical tube
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Collect a 100 µL aliquot of enriched sample and add to a tube containing lysis reagent, avoiding food particles; mix the solution by pipetting up and down in the tube a minimum of 3 times; store the remaining enrichment broth at 2–8 °C for later use in the confirmation of any positive assay results
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Place the tube in the cell disruptor for 3 ± 1 min
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Place the tube in the heat block at 95–100 °C for 15 min
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Vortex at high speed
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Centrifuge at 10,000–12,000 × g for 2 min

DNA extraction — deepwell format

Add 100 µL of the lysis reagent to each well of a deepwell microplate
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After sample enrichment, collect a 100 µL aliquot of each sample and add into the deepwell microplate, avoiding food particles
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Seal with pre-pierced sealing film
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Place in the thermoshaker instrument set at 95–100 °C at 1,300 rpm for 15–20 min
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Remove the plate from the thermoshaker and allow to cool in the refrigerator for 10 minutes

PCR

Prepare the PCR mix combining the amplification solution (Reagent C) and the fluorescent probes (Reagent B) according to the chart in the package insert; the PCR mix (Reagent B + C) must be used immediately, otherwise it is stable for 1 hour at 4 °C
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Add 45 µL of PCR mix to each well of a 48- or 96-well PCR plate, pipetting carefully to avoid bubbles
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Add 5 µL of each sample or control to the appropriate well; if the DNA extract was obtained using the tube format, carefully pipette the supernatant, being careful not to disturb the pellet
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One positive control (Reagent E) and one negative control (Reagent D) should be run with each assay, in addition to the laboratory’s standard broth/enrichment controls; controls are provided as ready-to-use solutions in the kit
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Seal the plate with the optical caps
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Place the plate into the reaction module of the thermal cycler and close the lid
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Verify the plate setup and protocol selection are correct
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Click the “run” button in the software program
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When the window appears allowing the data to be saved in a specific location, select “save” — the PCR protocol is then started
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A report is generated at the end of the run

Interpretation of results

The CFX Manager IDE Software allows complete automated analysis for Bio-Rad real-time PCR detection systems. Before releasing results, verify the typical characteristics of the amplification curves: a flat baseline followed by a rapid exponential increase of fluorescence and then flattening out (as shown in the method’s Figure 1). If the curves do not have these characteristics, repeat the PCR reaction from the PCR mix preparation step.
If amplification curves remain atypical upon repeat analysis, treat the sample as presumptive positive and proceed with confirmation of presumptive positive results.

Controls

Before interpreting sample results, verify the positive and negative controls. For the experiment to be valid, the controls must give the following results; otherwise repeat the PCR reaction from the PCR mix preparation step.

*The software indicates a Cq value (the cycle at which the amplification curve crosses the threshold) of N/A (not applicable) when the fluorescence of a sample does not rise significantly above the background noise, and hence does not cross the threshold.

Samples

For inhibition results, repeat analysis beginning with the PCR mix preparation step.

Confirmation of presumptive positive results

Using the refrigerated enrichment broth, proceed with the plating and confirmation steps of a cultural method — MFHPB-07: The Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples using palcam broth or MFHPB-30: Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples, as appropriate
Use at least one chromogenic agar listed in the cultural method in addition to Oxford agar

Note: For the heat processed food type of the ready-to-eat meat and poultry food category, 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 heat processed ready-to-eat meat and poultry, regardless of the amount of colony screening or additional confirmation steps done, these results cannot be used to state that L. monocytogenes is absent. For heat processed ready-to-eat meat and poultry, report all species of Listeria that are identified.

Negative and inhibition results

Negative results can be reported as Listeria spp. Not Detected
Repeated inhibition samples (i.e., more than one) or repeated atypical curves (i.e., more than one) must be treated as presumptive positive; proceed with confirmation as described under Confirmation of presumptive positive results

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)
The following applies to steps of the method which apply to growth only. It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures. 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). Where higher temperatures are recommended, the incubators or water baths must be maintained within 0.5 °C, due to potential lethality of the higher temperatures on the microorganism(s) being isolated.

References

Health Canada, Health Products and Food Branch. Detection of Listeria spp. from Environmental Surface Samples and Heat Processed Ready-to-Eat Meat and Poultry Using iQ-Check Listeria spp. Real-Time PCR Detection Kit. Compendium of Analytical Methods, Volume 3, Laboratory Procedure MFLP-39, March 2023.
Bio-Rad. iQ-Check Listeria spp. Kit User Guide 3578113. Test for the real-time PCR detection of Listeria spp. in food and environmental samples.
Microbiological Methods Committee. 2010. MFLP-41: Environmental sampling for the detection of microorganisms. Volume 3. The Compendium of Analytical Methods.
Pagotto, F., Hébert, K., J. Farber. 2011. MFHPB-30: Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples. Volume 2. The Compendium of Analytical Methods.
Warburton, D., Boville, A., Pagotto, F., Daley, E. and Chow, C. 2011. MFHPB-07: The Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples using palcam broth. Volume 2. The Compendium of Analytical Methods.

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