SOP: Detection of Shigella spp. in Foods by the Polymerase Chain Reaction (PCR)

GIFSQ — Global Institute for Food Safety & Quality

Principle

Following enrichment of samples in Shigella broth with novobiocin, a portion of the enrichment broth is treated and then subjected to a polymerase chain reaction (PCR) procedure which amplifies a 629-base-pair (bp) fragment within the 220-kbp invasive plasmids and chromosomal DNA of Shigella spp. The priming oligonucleotides (primers) used in the PCR are not only specific for Shigella spp. but also for enteroinvasive Escherichia coli (EIEC).

This method is applicable to the detection of viable Shigella spp. from fresh produce and commercial salads. It has been shown to give acceptable results with spinach, parsley, Greek salad, four bean salad, Greek pasta salad, Romaine lettuce, fresh mint, green onion, celery, softshell clams and strawberries. It can be used to determine compliance with Sections 4 and 7 of the Food and Drug Act. Where stipulated, the Official Methods and HPFB methods should be used. This revised method replaces MFLP-26, dated February 2004.

Initially, this method was shown to produce satisfactory results with artificially contaminated produce. The initial procedure was adapted and improved for the routine detection of Shigella spp. in diagnostic laboratories.

Apparatus

• Thermal cycler (Applied Biosystems Gene Amp 9700) or equivalent
• Microwave oven or hot plate
• Submarine gel casting tray and buffer reservoir, power pack and an appropriate comb
• Shortwave UV light table (transilluminator) to visualize stained DNA in agarose gels
• Photo documentation system (optional, for photographic records), including polaroid camera (hand-held or fixed), hood and Polaroid 667 film or equivalent
• Adjustable micropipettors to cover the range of volumes: 0.5 to 10 µL, 10 to 100 µL, and 200 to 1000 µL
• Filtered pipet tips for the above pipettors
• Thermomixer (Eppendorf), block heaters, or waterbath capable of accommodating 1.5 mL microfuge tubes and capable of maintaining a temperature of 56 °C and 100 °C
• Vortex mixer
• Stomacher bags
• Stomacher bags with filters
• Microfuge tubes (1.5 mL)
• Microcentrifuge, refrigerated
• Microfuge tubes for PCR — thin wall 0.2 mL or 0.5 mL capacity (depending on thermal cycler model)
• Regular incubator or CO₂ incubator, capable of maintaining 42 °C
• GasPak Jar systems (or equivalent) with CO₂ Gen (Oxoid), if CO₂ incubator is not available
• Magnetic stirrer
• Freezer capable of maintaining −20 °C

Reagents

• InstaGene Matrix, Bio-Rad (Cat. No 732-6030)
• Shigella broth (formulation in the media glossary SOP)
• To prepare: basal medium — 20.0 g tryptone, 2.0 g potassium phosphate, dibasic (K₂HPO₄), 2.0 g potassium phosphate, monobasic (KH₂PO₄), 5.0 g sodium chloride (NaCl), 1.0 g glucose and 1.5 mL Tween 80 per 1.0 L of distilled water; pH 7.0 ± 0.2. Suspend the basal ingredients in 1.0 L of distilled water, heat to boiling to dissolve completely, and autoclave at 121 °C for 15 min. Cool to 50 °C, then aseptically add 2.5 mL of the novobiocin solution to 225 mL of basal medium and mix thoroughly. Novobiocin solution: suspend 50 mg of novobiocin in 1.0 L of distilled water and sterilize by filtration through a 0.45 µm membrane filter.
• Novobiocin
• Tween 20 in 1% solution
• To prepare: add 1.0 mL of Tween 20 to distilled water and make up to 100 mL.
• ipaH (F) and ipaH (R) primers (see PCR Primers for the DNA sequences)
• Agarose
• Bromophenol blue (e.g. a tracking dye in loading buffer)
• dNTPs
• DNA Ladder 100 bp (or equivalent)
• Ethidium bromide
• EDTA disodium salt
• KCl
• Mineral oil (if required for some types of thermocycler)
• MgCl₂
• Sucrose
• PCR buffer 10X (concentrate)
• Taq DNA polymerase
• Xylene cyanol (the Tracking Dye recipe below prints “Xylene cyanole”)
• Tris[hydroxymethyl]aminomethane (e.g., Trizma base from Sigma)
• Boric acid
• Water (DNAse RNAse free)

PCR reaction mixture

PCR reaction mixtures can be made and 45 µL aliquots dispensed in 0.5 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 recipe for preparing a large batch equivalent to 20 reactions, and then aliquoting 45 µL portions into individual tubes.

(*) Certain commercially available buffers contain 15 mM MgCl₂, other buffers may not; therefore add MgCl₂ to a final concentration of 2.5 mM.

Note: All buffers, distilled water, pipet tips and other materials coming into contact with samples or PCR reagents should be sterile and free of 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 Shigella spp. or PCR products. To avoid any non-specific amplifications, the mix should be prepared by putting all the reagents on ice or on a refrigerated rack. Also, for convenience with some thermocyclers and reducing the cost of reagents, 20 µL of PCR mix in 200 µL tubes may be used.

10X PCR buffer

Commercially available; generally comes with the Taq polymerase kit. The 10X concentrated PCR buffer usually consists of 500 mM KCl, 200 mM Tris-HCl (pH 8.4).

Mineral oil

For some thermal cyclers. Autoclave in a glass bottle (cloudy appearance on cooling is normal), then aliquot in 1 mL portions in sterile 1.5 mL microfuge tubes. Store at room temperature.

10X Tris-Borate-EDTA (TBE) buffer

Commercially available.

• Trizma Base — 108.0 g
• Boric Acid — 55.0 g
• EDTA disodium — 7.5 g
• Add distilled water to a volume of 800 mL, dissolve, complete to 1.0 L
• This buffer is used at a 1:20 dilution (TBE 0.5X Buffer) in distilled water
• The pH of the 0.5X buffer should be 8.3. Do not adjust the pH.

Tracking dye / Loading buffer

Or commercially available prepared.

• Bromphenol blue — 0.025 g (0.25% (w/v))
• Xylene cyanole — 0.025 g (0.25% (w/v); the materials list prints “Xylene cyanol”)
• Sucrose — 4.0 g (40% (w/v))
• Sterile distilled water — 10 mL
• Mix all the ingredients thoroughly, filter and store at 4 °C

DNA molecular size marker

The 100 bp ladder DNA marker provides a useful range of DNA fragment sizes and facilitates the “sizing” of PCR amplicons. A stock preparation of this marker is prepared as follows: combine 50 µL of 100 bp ladder DNA with 41.5 µL of Tracking Dye and 158 µL of TBE buffer 0.5X. Store at −20 °C. Use 5 µL per gel.

Taq DNA polymerase

Good quality Taq DNA polymerase can be purchased from Qiagen or Invitrogen Molecular Biology. Taq DNA polymerase from other suppliers may also be suitable, but has not been tested in the laboratory that developed the method, and their use may affect the performance of the PCR. In the latter case, the user may have to provide some adjustments that may be needed. The “Hot Start” Taq polymerase has been shown to produce good quality results in routine use and may prevent the formation of “primer-dimers” or non-specific amplified products.

Note: Synthesis of oligonucleotide primers can usually be contracted out to a local university or, alternatively, many biotechnology firms offer a custom synthesis service.

Sample preparation

• During storage and transport keep the sample units refrigerated
• Analyse the sample units as soon as possible after receipt at the laboratory
• Prepare all buffers and solutions as described under Reagents
• Prepare and verify the suitability of all controls. Include positive and reagent controls. Shigella sonnei (ATCC 29930), Shigella flexneri (ATCC 29903), Shigella dysenteriae (ATCC 13313) or Shigella boydii (ATCC 8700) should be included as a positive control
• Note: ATCC numbers have been included as reference; equivalent Shigella strains may also be used
• Each sample unit may be analysed individually or the analytical units may be combined
• To ensure a truly 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. To reduce the workload the analytical units may be combined for analysis. It is recommended that a composite contain no more than five analytical units
• Prepare a 1:10 dilution of the food, in a filter stomacher bag, by aseptically adding 25 g of analytical unit into 225 mL of Shigella broth containing 0.5 µg/mL of novobiocin
• Gently massage the sample, and hold the suspension 10 minutes at room temperature. Shake periodically by hand
• Note: In order to avoid the formation of excessive food particles that may interfere with the PCR reaction, do not use a stomacher or blender
• Pour the supernatant into a new sterile stomacher bag
• Adjust pH, if necessary, to 7.0 ± 0.2 with sterile 1.0 N NaOH or HCl
• Include positive and reagent controls
• Place the bag, with opening loosely closed, in a jar system supplied with CO₂ or in a CO₂ incubator. Incubate at 42 °C for 20 ± 2 hours

Procedure

Prepare the DNA template using the InstaGene Matrix kit:

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Transfer 100 µL of the sample (culture or enriched broth) to 900 µL of RNAse DNAse free water (for a total volume of 1 mL), or directly 1 mL of the sample into a sterile 1.5 mL microfuge tube

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Centrifuge at 4 °C for 3 minutes at 9,000 × g to 13,000 × g

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Remove the supernatant and resuspend the pellet in 1 mL of sterile Tween-20 1% solution. Vortex at high speed for 10 seconds

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Centrifuge at 4 °C for 3 minutes at 9,000 × g to 13,000 × g

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Remove the supernatant, add 200 µL of InstaGene matrix to the pellet and vortex for at least 10 seconds (the InstaGene matrix should be mixed at moderate speed on a magnetic stirrer to maintain the matrix in suspension; use a pipet tip with a large bore, such as a 1000 µL pipet tip)

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Transfer the tube to a heating block or bath and incubate at 56 °C for 15 to 30 minutes

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Mix contents by vortexing for 10 seconds and place the tube in a 100 °C heating block or boiling water bath for 8 minutes

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Cool on ice. Vortex for 10 seconds, then centrifuge at 4 °C for 3 minutes at 9,000 × g to 13,000 × g. Proceed with PCR amplification or store at −20 °C until needed

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Add 5 µL of the prepared template to 45 µL of PCR reaction mixture (other volumes may be used providing that the ratio 1:10 is maintained)

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Insert the microfuge tubes in the thermal cycler and proceed with PCR amplification according to the temperature cycling program (some older thermal cycler models will require the use of sterile mineral oil in each well (2 drops) and on the surface of each PCR reaction mixture in each tube; use a different pipet tip for each addition)

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After the PCR is completed, analyse the PCR product (or amplicon) by agarose gel electrophoresis. If necessary, the amplicons can be stored at 4 °C until analysis

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Prepare a 2.0% (w/v) agarose gel in 0.5X TBE (Tris-Borate-EDTA) buffer. Dissolve the agarose by stirring on a hot plate or by microwaving for 1 to 2 minutes using high power. Ensure the agarose is completely dissolved (clear liquid with no particles in suspension) (as a guide to volume: an average gel is 5–10 mm thick, and the combs should be two-thirds covered, or refer to the manufacturer’s instructions)

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Cool the agarose and add 2 µL of Ethidium Bromide Solution (10 mg/mL) per 40 mL of agarose. Mix gently, avoiding bubble formation (the addition of EtBr to the gel is optional if the gel is submerged in EtBr solution after migration)

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Pour into the gel tray avoiding bubble formation or bubble trapping. Add a well-forming comb and allow the gel to set for about 20 to 30 min

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Prepare samples for electrophoresis: in clean microfuge tubes, mix 4 µL of tracking dye (loading buffer 5x concentrated) with 25 µL of PCR product (for smaller PCR reaction volumes (200 µL PCR tubes) where 2 µL of the prepared template is added to 20 µL of reaction mixture (or 2.5 µL in 22.5 µL), use 3 µL of tracking dye with 10 µL of PCR sample)
– Note: If samples were taken from under the mineral-oil layer of the PCR tubes, wipe the pipet tip with a tissue after taking the sample (for thermocyclers using oil)

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When the gel has set, remove the comb, place the tray with gel in the electrophoresis apparatus and fill the reservoir with 0.5X TBE buffer to cover the gel to a depth of 4 mm. Gently pipet approximately 10 µL of samples into the wells of the submerged gel. Pipet a sample of DNA molecular size marker (e.g., 100 bp ladder DNA) into an empty well. Include positive and reagent controls (the volume of the well may vary — 10 to 20 µL — with different combs and gel set-up)

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Connect the apparatus to the power supply with cathode (−, black) at the top (near the sample wells) and anode (+, red) at the bottom. Apply approximately 100 volts to the gel (approximately 30 mA per gel for mini-gels) and run for about 50 minutes or until the tracking dye has spread a distance of approximately two-thirds the length of the gel

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Remove the gel from the tray and visualize the DNA bands by exposure to ultraviolet light (shortwave) using a transilluminator. Gels may be photographed on Polaroid 667 film to facilitate analysis and for record keeping purposes

PCR Primers

Screening PCR: Oligonucleotide primers for the PCR were designed from the published nucleotide sequence of the ipaH gene, coding for the invasion plasmid and chromosome, of Shigella. For the amplification of the 629 bp fragment, the following primer sequences are required:

• ipaH (F) — forward — 28 bases = 5′-GTT CCT TGA CCG CCT TTC CGA TAC CGT C-3′
• ipaH (R) — reverse — 26 bases = 5′-GCC GGT CAG CCA CCC TCT GAG AGT AC-3′

Temperature cycling program

• Step 1 — Denaturation: 5 minutes at 95 °C (for regular Taq Polymerase); 15 minutes at 95 °C (for “Hot Start” Taq Polymerase)
• Step 2 — 35 cycles of:
• Melting: 15 seconds at 95 °C
• Annealing: 30 seconds at 68 °C
• Extension: 30 seconds at 72 °C
• Step 3 — Final elongation: 10 minutes at 72 °C

Note: The use of thermal cyclers other than the models stated above may alter the performance of the PCR, and it may be necessary, for the user, to optimize cycling parameters for different models.

Interpretation of results

• The amplicon generated from Shigella spp. ipaH gene sequences by this PCR method is a double stranded DNA fragment of 629 bp length. Therefore, a positive PCR test will yield a 629 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 (e.g., 100 bp ladder DNA) run on the same gel
• Note: A positive reaction (i.e. 629 bp amplicon) might also be generated by bacterial species belonging to the Enteroinvasive Escherichia coli (EIEC) group
• 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 629 bp amplicon (non-specific amplification products) is considered to be negative
• A 629 bp band should appear for the positive control(s). Absence of a positive control band invalidates the test and the samples should be re-analyzed
• Any band corresponding to the 629 bp amplicon occurring in the reagent control indicates contamination problems with either mineral oil, if used, or the PCR reaction mixture, and the whole batch is considered suspect and should be discarded. The samples should be reanalysed using new reagent batches
• Any test sample showing a distinct band at 629-bp is considered as a presumptive positive. Isolation from the enrichment broth should be attempted by the use of the culture method (see MFLP-25)

Precautions

• The Laboratory Supervisor must ensure that completion of the analysis, described in this method, is done in accordance with the International Standard referred to as “ISO/IEC 17025:2005 (or latest version). General requirements for the competence of testing and calibration laboratories”
• It is the responsibility of each laboratory to ensure that the incubators or water baths are maintained at the recommended temperatures. Where 35 °C is recommended the incubator may be at 35 °C ± 1 °C. Similarly, lower temperatures of 30 °C or 25 °C may be ± 1 °C. However, where higher temperatures are required, such as 42.0 °C or 45.5 °C, it is imperative that the incubators or water baths be maintained within 0.5 °C due to potential lethality of higher temperatures on the microorganisms being isolated
• EtBr IS A POTENT MUTAGEN: USE DOUBLE GLOVES WHEN HANDLING
• UV LIGHT CAN CAUSE EYE DAMAGE: WEAR SAFETY GOGGLES
• The use of a refrigerated microcentrifuge is strongly recommended to avoid the spreading of DNA
• If the EtBr has not been added directly to the gel, the gel must be removed from the tray and DNA stained by placing in EtBr solution (10 µg/mL) for 15 min. Remove the gel from the EtBr using a gel scoop, rinse briefly with tap water, and visualize the DNA bands by exposure to UV light
• When a DNA preparation stored in the freezer is reused, repeat the final template-preparation centrifugation step. After the DNA extraction, condensation on the microfuge cap can be eliminated by centrifuging the tube at 9,000 × g for a few seconds

References

• Health Canada. Detection of Shigella spp. in Foods by the Polymerase Chain Reaction (PCR). Compendium of Analytical Methods, Volume 3, Laboratory Procedure MFLP-26, February 2006. Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada.
• Lampel, K. A., J.A. Jagow, M. Trucksess and W. E. Hill. 1990. Polymerase chain reaction for Detection of Invasive Shigella flexneri in Foods. Appl. Environ. Microbiol. 56: 1536-1540.
• Lampel, K. CFSAN, FDA Survey of Domestic Fresh Produce, Domestic Produce Sampling Assignment — FY 00/01 DOEP # 00-16. United States Food and Drug Administration, Office of Plant, Dairy Foods and Beverages. Division of Virulence Assessment, Virulence Mechanisms Branch.
• Andrews, Wallace H. 1998. U.S. FDA Chap VI in “Bacteriological Analytical Manual”, 8th ed. Food and Drug Administration, Washington, D.C.
• Hartman, A. B., M. Venkatesan, E. V. Oaks, and J.M. Busyee. 1990. Sequence and and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri. J. Bacteriol. 172:1905-1915.