SOP: Identification of Yersinia enterocolitica Colonies by Real-Time Polymerase Chain Reaction

GIFSQ — Global Institute for Food Safety & Quality

Principle

• This method is for the rapid identification of Yersinia enterocolitica colonies isolated by standard culture for Y. enterocolitica. It characterizes and identifies presumptive positive colonies isolated on validated plating media routinely used in the isolation of this pathogen, and can be used to determine compliance with Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations.
• It is a real-time multiplex polymerase chain reaction (qPCR) method for the rapid identification of presumptive Y. enterocolitica colonies isolated on validated agar media used in standard culture-based methods.
• Following pre-enrichment, presumptive positive colonies from selective/differential agars are subjected to a real-time multiplex PCR procedure which simultaneously amplifies two specific DNA sequences — the outL (outer membrane protein L) gene specific to Yersinia enterocolitica and the ail (attachment and invasion locus) gene that is a major virulence marker and second species marker for this organism. Specific TaqMan-style fluorogenic probes are used to detect the DNA during amplification. In addition, the multiplex PCR includes an internal amplification control (IAC) to determine if PCR inhibition has occurred.
• The entire procedure identifies presumptive colonies within 2 hours and can replace the biochemical assays used in conventional confirmation steps.

Apparatus required

• Thermal cycler (Roche LightCycler 96 or equivalent)
• Biohazard hood and/or PCR workstation with UV lights
• Block heater capable of shaking and accommodating 1.5 mL microcentrifuge tubes (Eppendorf ThermoMixer or equivalent)
• Adjustable micropipettors to cover a range of volumes from 1.0 µL to 1000 µL
• Freezer capable of maintaining -20°C
• Refrigerator capable of maintaining 2°C to 8°C
• Vortex mixer
• Microcentrifuge capable of spinning 1.5 mL microcentrifuge tubes
• Mini-centrifuge capable of spinning strips of 8 x 0.2 mL PCR tubes, or a centrifuge with 96-well plate adaptors
• Spectrophotometer (ThermoFisher NanoDrop One or equivalent) — for in-house generation of IAC DNA template
• Heat block or waterbath capable of maintaining 37°C and 65°C — for in-house generation of IAC DNA template
• Electrophoresis equipment and supplies — for in-house generation of IAC DNA template
• Gloves, disposable
• Ice and bucket or equivalent
• Microcentrifuge tubes, 1.5 mL capacity or equivalent
• Microtube racks
• Aerosol barrier filter pipet tips for the adjustable micropipettors above
• Thin-walled PCR tubes with optical caps/film, 0.2 mL capacity in strip or 96-well plate format
• Chiller blocks to accommodate 0.2 mL and 1.5 mL tubes
• EcoRI restriction endonuclease and buffer (ThermoFisher catalogue #: ER0275 or equivalent) — for in-house generation of IAC DNA template

Reagents

All reagents are commercially available. All reagents should be molecular-grade, free of nucleases, and prepared in a dedicated clean work area.

• Sterile, nuclease-free water (ultra-pure or molecular grade)
• FastStart Essential DNA Probes Master (Roche catalogue 06402682001) — no substitutions permitted
• Primers — sequences are given under Real-time multiplex PCR primers below
• Oligonucleotide probes — sequences are given under Real-time multiplex PCR probes below
• IAC template DNA (Integrated DNA Technologies) — sequence is given under IAC DNA below
• EcoRI restriction endonuclease
• 10 mM Tris-HCl (pH 8.0)
• Additional reagent requirements for in-house generation of IAC DNA template are given under Preparation of stock IAC solution below

Control cultures

• Positive control: Y. enterocolitica containing outL and ail genes (e.g., ATCC 23715)
• Negative control: Non-Y. enterocolitica

Real-time multiplex PCR primers

Primers are based on published nucleotide sequences and target two different sequences in the Yersinia enterocolitica genome. PCR primers can be obtained commercially as de-salted oligonucleotides and do not need to be HPLC purified for this method.

Real-time multiplex PCR probes

Probes are based on published nucleotide sequences and target two different sequences in the Yersinia enterocolitica genome.

Key: TxRED is Texas Red; FAM is 6-caboxyfluorescein (the source prints “6-caboxyfluorescein” — the missing “r” in “carboxy” is the source’s own spelling, confirmed on the rendered page); Cy5 is Cyanine-5; IABkFQ is Iowa Black FQ Dark Quencher; IAbRQSp is Iowa Black RQ Dark Quencher.

IAC DNA

• The IAC DNA can be purchased in a lyophilized state from a supplier such as Integrated DNA Technologies in a pIDTSMART-KAN plasmid (with an EcoRI restriction site — total size is 2.3 kb), ThermoFisher Scientific in a pMX series cloning vector (with an EcoRI restriction site), or equivalent.
• Re-hydrate the lyophilized plasmid using 10 mM Tris-HCl (pH 8) to a concentration of approximately 50 to 100 ng/µL, then linearize with an EcoRI restriction endonuclease and dilute as described under Preparation of stock IAC solution below.
• Alternatively, the IAC can be propagated in-house using a competent E. coli via transformation using commercially available kits such as the One Shot TOP10 Electrocomp E. coli kit or an acceptable alternative.

IAC sequence (GenBank Accession No. FJ357008.1) (5′ → 3′):

TAC AGC ACC CTA GCT TGG TAG AAT CGA TCA GCT ACG TGA GGT CCT ACG ACG ATC
GCC AAG CAT GCC CTA GCT AAG ATG CAT CGA TTG CTC ATC ACG TAC GTT AGG TCG
ACT AGG AGG ACT GGA GTG CAT CGA CTA GCT AAG ATG GTT CGA TTG CTC ATC ACG
AAG GTT AGG TCG ACT ACG AAC GAG TCG TAC GTT AGG TT

Real-time multiplex PCR master mix

PCR master mixes may be prepared in larger quantities, aliquoted in portions of > 150 µL into sterile tubes, and stored at -20°C for up to 6 months and/or a maximum of 3 freeze thaws. All stock solutions should be stored at -20°C until use.

The following is the exact amount of PCR master mix required for a single PCR reaction (20 µL master mix + 5 µL template). When calculating volumes for batch preparation, multiply the reagent volume by the number of samples (including controls) and by 1.1 (a factor which will give the analyst a slight excess of master mix in case of pipetting loss or a reaction must be discarded or repeated).

Note: the IAC stock concentration should not be less than 650 fg/µL in 10 mM Tris-HCl (pH 8.0). The IAC should be diluted in ultra-pure nuclease-free sterile water immediately before the preparation of master mix to achieve a 40 fg/µL concentration. Weight is provided based on a 2.3 kbp linearized plasmid size. If needed, the final concentration of IAC DNA can be adjusted to achieve a Cq of approximately 25.

Thermal cycling parameters

Note: different brands/models of thermal cyclers may exhibit different performance characteristics; therefore, it may be necessary to optimize cycling parameters if models other than those specified are used.

Procedure

Presumptive positive Y. enterocolitica colonies isolated on the validated plating media listed below are sampled individually and subjected to the real-time multiplex PCR method as indicated below. A positive control consisting of a colony of a Y. enterocolitica strain is also processed along with each of the samples. In addition, a negative control (i.e., non-Y. enterocolitica) and a no-template/water control are also processed.

Cell lysis

• Pick an isolated bacterial colony with morphological characteristics typical of Y. enterocolitica from one of the following media: cefsulodin irgasan novobiocin (CIN) agar, xylose lysine desoxycholate (XLD) agar, CHROMagar Y. enterocolitica, or MacConkey agar
  ↓
• Inoculate 100 µL of sterile nuclease-free water
  ↓
• Vortex to resuspend the cells
  ↓
• Simultaneously set up the positive, negative, and no-template (use an un-inoculated tube of 100 µL of sterile nuclease-free water) controls
  ↓
• Heat the tube to 95°C for 5 minutes with constant shaking at approximately 400 rpm
  ↓
• Snap cool the tubes in an ice bath for 10 minutes
  ↓
• Store under refrigeration (i.e., 2°C to 8°C) until ready to proceed with the real-time multiplex PCR — if the PCR reaction cannot be completed within 24 hours, store the lysates at -20°C
  ↓
• Centrifuge the tubes to bring down condensate

Real-time multiplex PCR

• Pipet 20 µL of PCR master mix into a 0.2 mL thin-walled PCR tube or a 96-well plate
  ↓
• Add 5 µL of the cell lysate or negative control and pipet up and down once to mix
  ↓
• Carefully cap the tubes, using a PCR tube capping tool if necessary, to obtain a tight seal — alternatively, apply optical film to the 96-well plate
  ↓
• Gently vortex, then spin the PCR tubes/plates briefly to collect all the liquid at the bottom of the tube
  ↓
• Keep the tubes on ice or chilled until ready to place in the pre-heated thermal cycler
  ↓
• Place the tubes into the thermal cycler and start the amplification program using the thermal cycling parameters above

Interpretation of results

• A minimum endpoint fluorescence (EPF) value of 0.25 is used for the outL, ail, and IAC targets on the Roche LightCycler 96 platform.
• Note: setting the minimum endpoint fluorescence is required to reduce background noise of the assay. If a PCR platform other than the Roche LightCycler 96 (LC96) platform is to be used, the laboratory must ensure that the results obtained are equivalent to the LC96 platform using the EPF set at 0.25 for each target.
• Examine each individual real-time reaction amplification plot for its baseline, exponential, linear, and plateau phases to ensure proper amplification has occurred.

• The IAC was incorporated into the master mix at a low concentration to ensure it does not interfere with the other targets (i.e., outL and/or ail) of this assay. Consequently, the IAC amplification signal may disappear in positive samples where target DNA is present in high amounts.
• Negative test results are considered valid if the IAC target has a Cq within two cycles of the negative control IAC (approximately 25). Absence or delay of the IAC reaction is evidence of inhibition of the PCR, failure of one of the reagents, or omission of a critical component in the procedure.
• Isolates are confirmed as Y. enterocolitica when positive reactions occur with the outL and/or the ail gene targets. Report results for all gene targets detected.

Calculation

• Determine the copy number per µL using the formula below:

Copy number per µL = [(Concentration of Linearized Plasmid (g/µL)) ÷ (Molar Mass of IAC Plasmid (g/mole))] × 6.023 × 10²³

• Where,
• Concentration of Linearized Plasmid is the DNA concentration of the digested plasmid determined with the small-volume spectrophotometer, in g/µL
• Molar Mass of IAC Plasmid is the molar mass of the IAC plasmid provided by the IAC plasmid supplier, in g/mole
• 6.023 × 10²³ is the constant multiplier used in the calculation
• Worked example (from the method): for a DNA concentration of 8.1 × 10⁻⁹ g/µL and a molar mass of 1.3 × 10⁶ g/mole:

Copy number per µL = [(8.1 × 10⁻⁹ g/µL) ÷ (1.3 × 10⁶ g/mole)] × 6.023 × 10²³ = 3.75 × 10⁹

Preparation of stock IAC solution

Plasmid linearization

Note: the protocol for plasmid linearization outlined below is based on the manufacturer’s instructions provided with an EcoRI restriction endonuclease. It is recommended to follow the manufacturer’s instructions as they will contain the most up-to-date protocol.

• Add 40 µL of purified plasmid DNA, 12 µL of restriction endonuclease buffer (provided with the restriction endonuclease), 60 µL of RNase/DNase-free water, and 10 µL of EcoRI (10 U/µL) restriction endonuclease in a microcentrifuge tube
  ↓
• Incubate 4 hours in a waterbath or heating block at 37°C
  ↓
• Inactivate the restriction endonuclease by incubating 20 minutes in a waterbath or heating block at 65°C
  ↓
• Recommended: after the reaction has been completed, use electrophoresis to verify the plasmid was successfully linearized by comparing it against the undigested plasmid (from the IAC DNA preparation above). Both the digested and the undigested plasmid may be diluted (e.g., 1 in 10) using DNase-free water to minimize the amount of end-product utilized. The DNA fragment generated by the EcoRI restriction endonuclease is 2.3 kbp (2.1 kbp plasmid + 200 bp IAC) in molecular size. Only one band should appear for the linearized plasmid. In addition, the undigested plasmid should appear as a larger band due to supercoiling.

Quantification and stock solution preparation

• Determine the DNA concentration (in g/µL) of the digested plasmid using a small-volume spectrophotometer, following the manufacturer’s instructions for spectrophotometer use
  ↓
• Determine the molar mass (in g/mole) of the IAC plasmid — this information is provided by the IAC plasmid supplier
  ↓
• Determine the copy number per µL using the formula given under Calculation above
  ↓
• Prepare the stock IAC solution by diluting the digested plasmid in 10 mM Tris-HCl (pH 8.0) — the stock IAC concentration should not be less than 650 fg/µL, should be aliquoted in small volumes (e.g., 20 µL) to reduce the number of freeze thaws, and can be stored at -20°C

Note: the IAC stock solution will need to be diluted in ultra-pure nuclease-free sterile water immediately before the preparation of master mix (see the master mix preparation 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. Identification of Yersinia enterocolitica colonies by real-time polymerase chain reaction (MFLP-118). In: Compendium of Analytical Methods, Volume 3, March 2024. Health Products and Food Branch.
• Deer, D.M. et al. 2010. A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays. Letters in Applied Microbiology, 50 366-372.
• Health Canada. 2012. Isolation of Yersinia enterocolitica from foods and environmental samples (MFLP-48). In: Volume 3. Compendium of Analytical Methods.
• International Organization for Standardization (ISO) Technical Specification 18867. 2015. Microbiology of the food chain – Polymerase chain reaction (PCR) for the detection of food-borne pathogens – Detection of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis.
• Mäde, D. et al. 2008. A real-time PCR for detection of pathogenic Yersinia enterocolitica in food combined with an universal internal amplification system. Journal of Consumer Protection and Food Safety, 3: 141-151.
• Thomas, M.C. et al. 2017. Development of a novel multiplexed qPCR and pyrosequencing method for the detection of human pathogenic yersiniae. International Journal of Food Microbiology. 257: 247-253.
• US Food and Drug Administration. 2017. Bacteriological Analytical Manual. Chapter 8: Yersinia enterocolitica.