Identification of Presumptive Positive Listeria monocytogenes from Foods and Environmental Samples by the Polymerase Chain Reaction (PCR)

Laboratory Procedure MFLP-78
February 2011

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Health Products and Food Branch
Ottawa

Identification of Listeria monocytogenes from foods
and environmental samples by the Polymerase Chain Reaction (PCR)

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

1. Application

This method is applicable to the rapid identification of Listeria monocytogenes to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. This method has been validated for use on isolates from foods, as well as environmental and clinical samples using Health Canada's methods, MFHPB-30 (7.5), MFHPB-07 (7.6), and MFLP-74 (7.4). This revised method replaces MFLP-78, dated January 2006.

2. Principle

Following pre-enrichment and selective enrichment, a number of presumptive positive colonies identified by plating on selective agars are subjected to a polymerase chain reaction (PCR) procedure which amplifies a specific DNA sequence of the listeriolysin (hlyA) gene. The priming oligonucleotides (primers) used in the PCR are highly specific for Listeria monocytogenes and do not amplify DNA present in any other Listeria sp. or non-Listeria organisms. The resulting amplified hlyA DNA fragment has a specific molecular size, defined by the primers, and is readily identified by agarose gel electrophoresis. The entire procedure identifies presumptive positive colonies within 4 h, and in most instances, can replace the usual battery of biochemical screening and confirmation tests, thus providing considerable savings in time, labour and cost of analysis. This PCR technique has proven to be a highly specific and sensitive method for the identification of L. monocytogenes (5.1) isolated from a variety of products, including dairy, egg, cheese and environmental samples (5.2). The polymerase chain reaction (PCR) process is covered by U.S. patents owned by Hoffman-LaRoche.

3. Definition of terms

See Appendix A of Volume 3.

4. Collection of samples

See Appendix B of Volume 3.

5. Materials and special equipment

1. Thermocycler
2. Microwave oven or hot plate
3. Submarine gel casting tray and buffer reservoir, power pack, and an appropriate mini-comb.
4. Shortwave UV light table (transilluminator) to visualize stained DNA in agarose gels
5. Photodocumentation system (optional, for photographic records), including polaroid camera (hand-held or fixed), hood and Polaroid 667 film
6. Adjustable micropipettors: to cover a range of volumes: 0.5-10 μL,10-100 μL, and 200-1000 μL
7. Pipet tips for above pipettors (filtered tips)
8. Block heater with block capable of accommodating 1.5 mL microfuge tubes
9. Vortex mixer
10. Microfuge tubes - 1.5 mL capacity or equivalent
11. Microfuge tubes - 0.2 mL (200μL) capacity or equivalent
12. DNA molecular size marker
13. In addition, the following chemicals and reagents should be on hand. See Section 8 for the list of individual buffers and reagents and their formulas:
    • Agarose
    • Boric acid
    • Bromophenol blue
    • dATP
    • dCTP
    • dGTP
    • dTTP
    • Ethidium bromide, SYBR Save, SYBR green or equivalent
    • EDTA
    • Taq DNA polymerase
    • MgCl2
    • KCl
    • Sucrose
    • PCR buffer 10X
    • Triton X100
    • Tris[hydroxymethyl]aminomethane (e.g., Trizma7 base from Sigma)
    • Mineral oil (optional depends on method used)

6. Procedure

Prepare and enrich food samples according to MFHPB-30 (7.5), MFHPB-07 (7.6) or MFLP-74 (7.4) for isolation of Listeria monocytogenes. At the stage where presumptive positive colonies are observed on plating media, (typical colonies are described in MFHPB-30, section 6) a number of the colonies can be sampled (in addition to the mandatory biochemical tests) and each individual colony is subjected to the PCR procedure, which is carried out in accordance with the instructions below. Note that this PCR procedure is an optional step in MFHPB-30 and MFHPB-07 and can not be used as a replacement for biochemical confirmation steps.

6.1 Handling of samples

Sample units are handled and subjected to the enrichment, isolation and plating procedures according to MFHPB-30, MFHPB-07 or MFLP-74. Presumptive positive (L. monocytogenes) colonies isolated on the selective agar plates (e.g., PAL, OX and/or MOX) are then sampled individually and subjected to the PCR method as indicated below. A positive control consisting of a colony or broth culture (Trypticase Soy Broth) of a L. monocytogenes laboratory strain is also processed along with each set of samples.

6.2 Cell lysis

6.2.1 Pick a small portion of a suspect colony (lightly touching the surface with a loop or needle is sufficient) and suspend in 50 μL of 1 X PCR buffer in a sterile 1.5 mL microfuge tube. For a negative control, leave one tube of 50 μL of 1 X PCR buffer devoid of cells.

6.2.2 Add 50 μL of 2% (w/v) Triton X-100 to tubes containing cell suspension or negative control and vortex.

6.2.3 Incubate at 100°C for 10 minutes. Cool to room temperature and centrifuge the tube at 2000 rpm for a few seconds.

6.3 PCR method

Oligonucleotide primers for the PCR are based on the published nucleotide sequence of the hlyA gene (7.3). For the amplification of a 730 bp hlyADNA fragment, the following primer sequences are required: 5'-CATTAGTGGAAAGATGGAATG-3' (primer A), and 5'-GTATCCTCCAGAGTGATCGA-3' (primer B). PCR primers need not be of high purity. They can be synthesized using a suitable oligonucleotide synthesis system and following the manufacturer's instructions. Synthesis of oligonucleotide primers can also be contracted out to a university or biotechnology company with expertise in this area.

6.3.1 Add 5 μL of the cell lysate or negative control to 44.5 μL of PCR reaction mixture (8.1) in a microfuge tube suitable for thermocycler use and vortex.

6.3.2 Optional:

If the thermocycler is not equipped with a heated lid, add 2 drops (100 μL) of sterile mineral oil to each tube, and place 2 drops of mineral oil in each well of the thermocycler.

6.3.3 Place tubes into thermocycler and start temperature cycling program.

6.4 Temperature cycling program

Note: Different makes of thermal cyclers may exhibit different performance characteristics; therefore, it may be necessary to optimize cycling parameters for each model.

Note: The number of enzyme units required may be variable and depends on the Taq being used. Follow manufacture's instructions for use.

Note: If a hot start Taq polymerase is used, follow the manufacturer's protocol in place of the manual hot start procedure.

6.4.1 An example of a typical thermocycling program includes the following parameters:

Version a	Version ba,b
Manual "hot start"...10 min, 80°C	Use of "Hot start" Taq (or other similar delayed activity Taq)
a) denaturation... 5 min, 94°C	a) denaturation... 10 min, 94°C
b) add 0.5 units of Taq	b) add 0.5 units of Taq
c) denaturation...3 min, 94°C	c) denaturation...3 min, 94°C
d) followed by 30 cycles of: denaturation...1 min, 94°C annealing......1 min, 55°C extension......2 min, 72°C	d) followed by 30 cycles of: denaturation...1 min, 94°C annealing......1 min, 55°C extension......2 min, 72°C
e) followed by: final extension...2 min, 72°C	e) followed by: final extension...2 min, 72°C

Note^a: If a hot start Taq polymerase is used (version b), protocol may need to be optimized (especially annealing temperatures) to reflect the conditions obtained using standard protocol (version a).

Note^b: If using version b, a 0.2 ml tube will be used. A typical reaction volume will be 22.5 or 45 μL of PCR reaction mixture with 2.5 or 5 μL, respectively, of template.

6.4.2 If using mineral oil (6.3.2) carefully insert the tip under the mineral oil layer.

6.4.3 Return tube to the machine immediately after addition of the Taq enzyme.

6.4.4 Use a separate pipet tip for each addition of Taq.

6.4.5 After the PCR is completed, remove tubes and analyze by agarose gel electrophoresis. If unable to analyse immediately, the tubes can be stored at -20°C.

6.5 Agarose gel electrophoresis

6.5.1 Prepare a 1.2% (w/v) agarose gel in 0.5 X TBE (Tris-Borate-EDTA) buffer. The agarose can be dissolved by stirring on a hot plate or by microwaving for 1-2 minutes using high power. Ensure that the agarose is completely dissolved (i.e., clear liquid with no particles in suspension). Make up volume with 0.5 X TBE buffer if volume has decreased due to evaporation.

6.5.2 Cool agarose to 50°C, then pour into a gel tray. Add a well-forming comb and allow gel to set for about 30 min.

6.5.3 Prepare samples for electrophoresis: in clean microfuge tubes, mix 5 μL of tracking dye with 10 μL of PCR sample. If using oil, the sample should be taken from under the oil layer. The pipet tip should be wiped with a tissue after taking the sample.

6.5.4 When the agarose gel has set, remove the comb and place the tray with gel in the electrophoresis apparatus. Fill the reservoir with 0.5 X TBE buffer, covering the gel with buffer to a depth of 5 mm. Gently pipet 10-15 μL of sample (6.5.3) into the wells of the submerged gel. Pipet 10-15 μL of a suitable DNA molecular size marker (i.e., a marker that will allow differentiation of PCR products within the expected range for L. monocytogenes) into an empty well.

6.5.5 Connect the apparatus to the power supply with cathode (-, black) situated at the top (i.e., near sample wells) and anode (+, red) at the bottom (i.e., the end) of the gel. Apply 80-100 volts (approximately 30 mA) to each gel and run for about 1 hour or until the tracking dye has spread a distance of approximately two-thirds the length of the gel.

6.5.6 Remove gel from tray and stain the DNA by placing in ethidium bromide (EtBr) solution (10 μg/mL) for 15 min. Remove the gel from EtBr using a gel scoop, rinse briefly with tap water, and visualize DNA bands by exposure to ultraviolet light (shortwave) using a transilluminator. Gels may be photographed using Polaroid 667 film or other suitable methods, to facilitate analysis and for record keeping purposes.

Note: UV LIGHT CAN CAUSE EYE DAMAGE: WEAR SAFETY GOGGLES.

6.6 Interpretation of results

6.6.1 The amplicon (PCR product) generated from L. monocytogenes hlyAgene sequences by this PCR method is a double stranded DNA fragment of 730 bp in length. Therefore, a positive PCR test will yield a 730 bp DNA fragment which will appear as an intense band on an EtBr-stained agarose gel. The molecular size of the band can be verified by comparing its migration to that of a DNA molecular size marker run on the same gel. A negative PCR test will normally not produce any visible bands in an EtBr-stained agarose gel. Although an extremely rare occurrence, any sample giving bands not corresponding to the 730 bp amplicon (non-specific amplification products) is considered to be negative. Any band corresponding to the 730 bp amplicon occurring in the negative control (section 6.2.1) indicates contamination problems with either Triton X-100, mineral oil, the PCR reaction mixture or bacteria in the form of aerosols. The above reagents are considered as possible sources of contamination and have to be discarded. Samples must be re-analyzed with new reagents. Also review all possible sources where aerosol contamination may have occurred.

7. References

7.1 Blais, B.W. and L.M. Phillippe. 1993. A simple RNA probe system for analysis of Listeria monocytogenes polymerase chain reaction products. Appl. Environ. Microbiol., 59: 2795-2800.

7.2 Blais, B.W. and L.M. Phillippe. 1994. Applicability of the Listeria monocytogenes polymerase chain reaction in the routine analytical laboratory. Safety Watch, Foodborne Disease Bulletin, No. 32. Agriculture and Agri-Food Canada, Ottawa.

7.3 Mengaud, J., M. Vicente, J. Chenevert, J.M. Pereira, C. Geoffroy, B. Gicquel-Sanzey, F. Baquero, J. Perez-Diaz and P. Cossart.1988. Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes. Infect. Immun. 56: 766-772.

7.4 Pagotto, F., Trottier, Y.L., Upham, J., and Iugovaz, I. 2011. Enumeration of Listeria monocytogenes in Foods (MFLP-74). The Compendium of Analytical Methods. Volume 3. http://www.hc-sc.gc.ca/food-aliment

7.5 Pagotto, F., Hébert, K., J. Farber. 2011. Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples (MFHPB-30). Volume 2. The Compendium of Analytical Methods. http://www.hc-sc.gc.ca/food-aliment

7.6 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). Volume 2. The Compendium of Analytical Methods. http://www.hc-sc.gc.ca/food-aliment

8. Reagents

All buffers, distilled water, pipets, pipet tips and other materials coming into contact with samples or PCR reagents should be autoclaved prior to use to remove any DNases and/or other contaminants. To avoid contamination problems, all reagents should be prepared in a laminar flow cabinet which has never been exposed to L. monocytogenes or PCR products.

8.1 PCR reaction mixture

PCR reaction mixtures can be made and 44.5 μL aliquots dispensed into 0.2 mL microfuge tubes well ahead of time and stored at -20°C until use (stable for up to one year). All stock solutions are also stored at -20°C until use. The following is a formula for preparing single 44.5 μL reaction tubes, though in practice it may be more convenient to prepare a large batch equivalent to 100 reactions ahead of time, and then aliquoting 44.5 μL portions into individual tubes.

Stock solutions	per reaction tube
Distilled H20 (sterile)	32.5 μL
10x PCR buffer (Sec. 7.2)	5.0 μL
25 mM MgCl2	4.0 μL
100 μM primer A	0.5 μL
100 μM primer B	0.5 μL
20 mM dNTPs (dCTP, dATP, dTTP, dGTP)	0.5 μL each
Final volume	44.5 μL

8.2 10 X PCR buffer

(commercially available; generally comes with the Taq solution)

The 10 X concentrated PCR buffer usually consists of 500 mM KCl, 100 mM Tris-HCl (pH 9.0), and 1% (w/v) Triton X-100.