SOP: Specific Detection of Vibrio parahaemolyticus Strains Using a Multiplex Polymerase Chain Reaction (PCR) Based on the R72H Taxonomic Marker and the Hemolysin Genes TDH and TRH

GIFSQ — Global Institute for Food Safety & Quality

Principle

This method identifies strains of Vibrio parahaemolyticus by a multiplex polymerase chain reaction (PCR) that amplifies three DNA sequences at once: the r72h fragment of the V. parahaemolyticus genome (the species identifier), the tdh gene (thermostable direct hemolysin), and the trh gene (tdh-related hemolysin). The priming oligonucleotides (primers) used are highly specific for V. parahaemolyticus and do not amplify DNA from any other Vibrio species or non-Vibrio organisms under the reaction conditions described. The amplified DNA fragments have specific molecular sizes defined by the primers and are identified by agarose gel electrophoresis. The entire procedure identifies presumptive colonies within four hours and, in most instances, can replace the usual battery of biochemical assays used in downstream confirmation, saving time, labour and cost of analysis.

The method applies to the rapid identification of V. parahaemolyticus strains in foods and seawater, as well as environmental and clinical samples, following the enrichment, isolation and plating procedures of MFLP-37 (reference 8.2) or other equivalent methodologies. It is applied to the characterization and identification of presumptive positive colonies isolated on selective/differential agar medium routinely used for this pathogen; used as the identification method in quantitative procedures such as the Most Probable Number (MPN) method, it generates quantitative results for V. parahaemolyticus strains.

Apparatus

• Thermal cycler (Stratagene Robocycler or Applied Biosystems GeneAmp 9700) or equivalent
• Microwave oven or hot plate
• Submarine gel casting tray and buffer reservoir, power pack, and appropriate mini-combs or equivalent
• Shortwave UV light table (transilluminator) to visualize stained DNA in agarose gels
• Photodocumentation system or equivalent digital imaging system
• Adjustable micropipettors covering the ranges 0.5–10 µL, 10–100 µL, 20–200 µL, and 200–1000 µL
• Pipet tips for the above pipettors (aerosol barrier filter tips are required)
• Block heater capable of shaking and accommodating 1.5 mL microcentrifuge tubes (VWR Thermomixer) or equivalent
• Vortex mixer
• Microcentrifuge tubes — 1.5 mL capacity or equivalent
• Thin-walled PCR strip tubes with caps and/or individual tubes with caps — 0.2 mL (200 µL) capacity or equivalent
• Micro-centrifuge
• Strip-fuge to centrifuge PCR tubes
• DNA molecular weight marker (50 bp DNA ladder recommended)
• Culture controls: positive control — Vibrio parahaemolyticus (a well-characterized strain, such as ATCC 49529); negative controls — non-Vp

Reagents

• Agarose
• Ultrapure nuclease-free, pyrogen-free sterile water or equivalent
• Orthophosphoric acid (85%, 1.679 g/mL)
• Used as supplied.
• Orange G or equivalent loading buffer
• Glycerol
• 100 mM dATP, dCTP, dGTP, dTTP
• Ethidium bromide (10 mg/mL)
• EDTA
• Taq DNA polymerase (5 units/µL)
• 50 mM MgCl₂
• 10X PCR buffer, minus MgCl₂
• Tris[hydroxymethyl]aminomethane
• Tris hydrochloride
• De-ionized water supply for electrophoresis

PCR master mix

PCR master mix (minus Taq DNA polymerase) can be made well ahead of time and stored at −20 °C until use (stable for up to one year); Taq is added immediately prior to use. All stock solutions are also stored at −20 °C until use. Once thawed, PCR reagents (except Taq DNA polymerase) are stored at 4 °C; the long-term stability of PCR reagents at refrigeration temperatures is unknown. Reagents are aliquoted into smaller working volumes to minimize extended storage at refrigeration temperatures.

The amounts below prepare the exact PCR master mix for one reaction (23 µL master mix + 2 µL template). For batch preparation, multiply the per-reaction volume by the number of analytes (including controls) and by 1.1, which gives a slight excess of master mix in case a reaction must be discarded or repeated.

• Ultrapure nuclease-free sterile water or equivalent — 7.22 µL per reaction tube
• 10X PCR Buffer — 2.50 µL per reaction tube
• 50 mM MgCl₂ — 1.25 µL per reaction tube
• 10 mM dNTPs (dCTP, dATP, dTTP, dGTP) — 0.63 µL per reaction tube
• 5 µM VP-32 primer — 2.50 µL per reaction tube
• 5 µM VP-33 primer — 2.50 µL per reaction tube
• 5 µM TRH-L primer — 2.00 µL per reaction tube
• 5 µM TRH-R primer — 2.00 µL per reaction tube
• 5 µM TDH-L primer — 1.00 µL per reaction tube
• 5 µM TDH-R primer — 1.00 µL per reaction tube
• Taq DNA polymerase (5 units/µL) — 0.40 µL per reaction tube

The PCR primers are based on published nucleotide sequences and target three different sequences in the V. parahaemolyticus genome. They can be obtained commercially as de-salted oligonucleotides and do not need to be HPLC purified for this method.

10X PCR buffer

• 200 mM Tris-HCl (pH 8.4)
• 500 mM KCl

The PCR buffer is supplied as a 10X concentrate and is diluted for use.

10X TPE buffer (or 10X TBE buffer or 10X TAE buffer)

Prepare the 10X concentrated TPE buffer (Tris-Phosphate-EDTA) as follows:

• Tris[hydroxymethyl]aminomethane — 108 g
• Orthophosphoric acid (85%, 1.679 g/mL) — 15.5 g
• 0.5 M EDTA (pH 8) — 40.0 mL

Add de-ionized water to a final volume of 1 litre. Store at room temperature. For use, dilute 1:10 with de-ionized water for agarose gel electrophoresis. The 10X concentrated TBE (Tris-Borate-EDTA) and 10X concentrated TAE (Tris-Acetate-EDTA) buffers are commercially available; for use, dilute 1:10 with de-ionized water for agarose gel electrophoresis.

10X Orange G

• Ultrapure nuclease-free sterile water or equivalent — 60.0 mL
• Glycerol — 40.0 mL
• Orange G — 0.25 g

Sterilize and aliquot in 1 mL portions in sterile 1.5 mL microcentrifuge tubes. Store at −20 °C for long-term storage; once thawed, store at 4 °C.

50 bp ladder (DNA molecular weight marker)

• 50 bp DNA ladder (1 µg/µL) — 50 µL
• 10X Orange G — 50 µL
• Ultrapure nuclease-free sterile water or equivalent — 400 µL

Sample preparation

Sample units are handled and subjected to the enrichment, isolation and plating procedures according to MFLP-37 (reference 8.2) or other equivalent methodologies. At the stage where presumptive positive colonies are observed on the plating media, sample the number of colonies as specified in the method and subject each individual colony to the multiplex PCR procedure described below.

Procedure

Handling of samples

• Sample the presumptive positive (V. parahaemolyticus) colonies individually from the selective/differential agar plates (e.g. TCBS) and subject each to the multiplex PCR method
• Process a positive control consisting of a colony of a V. parahaemolyticus laboratory strain along with each of the samples
• Also process a negative control (non-Vp) and a no-template/water control

Cell lysis

• Pick an isolated bacterial colony and inoculate 100 µL of sterile nuclease-free water
• Vortex to resuspend the cells
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• Simultaneously set up the positive, negative, and no-template controls (for the no-template control, use an uninoculated tube of 100 µL of sterile nuclease-free water)
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• Heat the tube to 95 °C for 5 minutes
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• Snap cool the tube on wet ice for 10 minutes and store under refrigeration until ready to proceed with PCR
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• If the PCR reaction cannot be completed within 24 hours, store the lysates at −20 °C

PCR

• Pipet 23.0 µL of PCR master mix (see the PCR master mix section) into a 0.2 mL thin-walled PCR tube
• Add 2.0 µL of the cell lysate or negative control and pipet up and down once to mix
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• Carefully cap the tubes, using a PCR tube capping tool if necessary, to obtain a tight seal
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• Vortex and then spin the PCR tubes in a strip-fuge to mix the contents thoroughly
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• Keep the tubes on ice or at 4 °C until ready to place them in the programmed and preheated thermal cycler
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• Place the tubes into the thermal cycler and start the temperature cycling program

Temperature cycling program

• Initial denaturation: 5 minutes at 94 °C
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• PCR amplification — 40 cycles:
• Denaturation: 30 seconds at 94 °C
• Annealing: 45 seconds at 58 °C
• Extension: 75 seconds at 68 °C
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• Final extension: 7 minutes at 68 °C
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• If necessary, after the PCR is complete, store the amplicons under refrigeration until ready to proceed with agarose gel electrophoresis

Note: Different makes of thermal cyclers may exhibit different performance characterisitcs; therefore, it may be necessay to optimize cycling parameters if models other than those specified are used.

Agarose gel electrophoresis

• Prepare a 2.0% (w/v) agarose gel in 1X TPE (Tris-Phosphate-EDTA) or 1X TBE (Tris-Borate-EDTA) or 1X TAE (Tris-Acetate-EDTA) buffer
• Dissolve the agarose by stirring on a hot plate or by microwaving for 1–2 minutes using high power; if using a microwave, ensure that excessive boiling of the suspension does not occur by stopping and starting the microwave when boiling is observed
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• Ensure the agarose is completely dissolved (a clear liquid with no particles in suspension)
• Make up the volume gravimetrically with de-ionized water if the volume has decreased due to evaporation
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• Cool the agarose to 60 °C, then add sufficient ethidium bromide (EtBr) to obtain a final 0.2 µg/mL concentration
• Swirl gently to mix and pour into a gel tray
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• Add a well-forming comb and allow the gel to set for approximately 20 to 30 minutes
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• Prepare the samples for electrophoresis by adding 3 µL of 10X Orange G (or equivalent loading buffer) to 25 µL of PCR sample, to give a final approximate 1X concentration
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• When the agarose gel has set, remove the comb and place the tray with the gel in the electrophoresis apparatus
• Fill the reservoir with 1X TPE or 1X TBE or 1X TAE buffer (the same buffer used to prepare the agarose gel), containing ethidium bromide (0.2 µg/mL final concentration), covering the gel with buffer to a depth of approximately 3–5 mm
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• Gently pipet 15 µL of sample into the wells of the submerged gel
• Pipet 7.5 µL of a 50 bp DNA ladder (0.1 µg/µL) into wells on either side of the PCR samples to allow differentiation of the PCR products within the expected range for V. parahaemolyticus; with gels having more than 20 wells, consider loading additional molecular weight markers in the middle gel lanes
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• Connect the apparatus to the power supply with the cathode (−, black) situated at the top (near the sample wells) and the anode (+, red) at the bottom (the end) of the gel
• Run the gel between 5–10 volts/cm of the gel length (for example, a 12 × 14 cm gel run at 70–140 volts)
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• Run the gel for a sufficient amount of time to resolve the nucleic acids of interest (for example, a 12 × 14 cm gel running at 75 volts takes approximately 1 hour)
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• Remove the gel from the tray and visualize the DNA bands by exposure to ultraviolet light (shortwave) using a transilluminator
• Document results using suitable digital imaging systems or other suitable methods, to facilitate analysis and for record keeping purposes

Interpretation of results

The amplicons generated by this multiplex PCR method from the V. parahaemolyticus trh, r72h and tdh DNA sequences are double-stranded DNA fragments of 486, 387 or 320, and 270 bp in length, respectively. A positive PCR test yields DNA fragments that appear as an intense band on an EtBr-stained agarose gel; the molecular size of the band is verified by comparing its migration to that of a DNA molecular size marker run on the same gel. A negative PCR test normally produces no visible bands in an EtBr-stained agarose gel.

Expected results and their interpretation:

For results not consistent with this table, repeat the multiplex PCR and confirm with biochemical tests as outlined in MFLP-37 (reference 8.2) or an equivalent method.

• Any sample giving bands not corresponding to the 486, 387 or 320, and/or 270 bp amplicons (non-specific amplification products, an extremely rare occurrence) is considered negative
• Any band corresponding to the 486, 387 or 320, and/or 270 bp amplicons occurring in the negative control indicates possible contamination problems: the reagents are considered as possible sources of contamination and must be discarded; samples must be re-analyzed with new reagents; also review all possible sources where aerosol contamination may have occurred

A companion gel image (“Figure 1. Gel Image of a Typical Vp Multiplex PCR”, issued with this method as separate material) shows a typical result, with a 50 bp molecular weight ladder in the outer lanes, one lane positive for R72H and tdh, one lane positive for trh, R72H and tdh, and one lane negative for all three targets; the image itself is not reproduced here.

Biochemical confirmation (optional)

API 20E strips or equivalent rapid identification kits may be used as an alternative to conventional tube media for biochemical tests. However, some Vibrio spp. will not grow in commercial test strip media when physiological saline (0.85% NaCl) is used as the diluent; use 2% NaCl as the diluent. If commercial strips do not allow identification, continue with conventional tests using an HPFB method (reference 8.2) or equivalent.

Precautions

• All buffers, de-ionized water, pipets, pipet tips and other materials to be used in sample preparation, PCR set-up and post-PCR manipulations (excluding the agarose gel electrophoresis procedure) 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, prepare all reagents in a dedicated work area
• Ethidium bromide is a potent mutagen: use medical grade non-latex gloves when handling
• UV light can cause eye damage: wear UV-blocking safety glasses
• The polymerase chain reaction (PCR) process is covered by U.S. patents owned by Hoffman-LaRoche

References

• Health Canada. Specific Detection of Vibrio parahaemolyticus Strains Using a Multiplex Polymerase Chain Reaction (PCR) Based on the R72H Taxonomic Marker and the Hemolysin Genes TDH and TRH. Compendium of Analytical Methods, Volume 3, Laboratory Procedure MFLP-23, March 2006. Health Products and Food Branch, Health Canada.
• Bej, A., D. Patterson, C. Brasher, M. Vickery, D. Jones, and C. Kaysner. 1999. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh, and trh. Journal of Microbiological Methods 36: 215-225.
• Health Canada. 2004. Detection of Vibrio. Part 1: Detection of Halophilic Vibrio Species in Seafood. Part 2: Detection of Vibrio Cholerae. In: Volume 3. Compendium of Analytical Methods.
• Joint FAO/WHO Food Standards Programme. Codex Committee on the Fish and Fishery Products. Matters arising from the FAO and WHO: Microbiological Risk Assessment of Vibrio Spp. 26th sess., 2003. CX/FPP 03/2-Add.1. Alesund Norway, 13-17 October 2003.
• Lee, C-Y, S-F. Pan, and C-H. Chen. 1995. Sequence of a Cloned pR72H Fragment and its Use for Detection of Vibrio parahaemolyticus in Shellfish with the PCR. Applied and Environmental Microbiology 61: 1311-1317.
• Robert-Pillot, A., A. Guenole, and J.M. Fournier. 2002. Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA-DNA hybridization. FEMS Microbiology Letters 215: 1-6.