Technical Stuff

List of reference tests used in our laboratory :

Polymerase Chain Reaction (PCR)


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Appendices


1- Aim

Animal Pathogenic and Zoonotic E. coli (APZEC) are E. coli carrying genes encoding virulence factors enabling them to cause disease in animals or humans. APZEC are subdivided into the ETEC, EPEC, STEC/VTEC, and ExPEC pathotypes. ETEC carry one or more of the genes encoding enterotoxins LT, STa, and STb. ETEC in pigs mostly carry the genes encoding F4 or F18 fimbriae. EPEC carry the eae gene encoding Eae or Intimin. STEC/VTEC carry genes encoding VT1/Stx1 and/or VT2/Stx2 and STEC/VTEC potentially pathogenic to humans often carry the eae gene. Most ExPEC carry the genes encoding aerobactin, and often carry the genes encoding Tsh, CNF, or P fimbriae.

The aim of this procedure is to detect by multiplex PCR the presence of genes in E. coli cultures for their identification as ETEC, EPEC, STEC, or ExPEC. In addition, the determination of the presence of the genes encoding F4 or F18 in ETEC will permit their assignation as definitive agents of diarrhea in pigs. This procedure can be applied to the identification of APZEC in LB enrichment broth cultures originating from clinical samples or colonies isolated on MacConkey or other solid medium.

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2- Principle

The method is based on PCR amplification of specific DNA regions from a template DNA, with oligonucleotides triggering the start of the PCR reaction. The PCR reaction is carried out using specific primers (Appendix 1).

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3- Methodology

3.1- Template preparation

Cultures streaked onto solid media (e.g. TSA, blood Agar or MacConkey Agar) are processed as follows:

  • Take a streak from the first quadrant with a loop and inoculate in a tube of 5 ml of LB broth.
  • Swabs from feces or tissues are placed in a tube of 5 ml of LB broth.
  • Incubate overnight at 37±1oC.

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3.2- DNA template

(Adapted from the method used by the Laboratory for Foodborne Zoonoses MFLP-86)

  • Identify the sample by a code on the cap of the 1.5 ml tube, centrifuge 1 ml of LB broth culture incubated overnight at 37±1oC, at 12 000 RPM for 2 minutes.
  • Discard the supernatant in a Corning bottle reserved for this use.
  • Add 1 ml PBS or FA buffer (Difco) to the pellet, mix well (vortex or up and down with pipetter).
  • Centrifuge at 12 000 RPM for 2 minutes, remove the supernatant and resuspend the pellet in 0.5 ml of sterile water Milli-Q (vortex or up and down with pipetter).
  • Heat at 100oC for 10 minutes, centrifuge at 12 000 RPM for 2 minutes. Transfer the supernatant to another tube, and identify the tube. When preparing a large number of tubes, ensure that the tubes containing DNA are kept on ice or cold.
  • Conserve sample DNA tubes at -20oC if not tested immediately.

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3.3- Setting up the PCR reaction

For each sample, set up a 25 μl PCR reaction mixture (Appendix 2). The volume of the reagents can be scaled according to the final volume of reaction. MilliQ water must be used for PCR reactions. In each PCR assay, a positive and two negative controls must be included. The positive controls are DNA templates obtained from E. coli strains possessing the virulence genes tested, whereas one negative control is the DNA from a non-pathogenic E. coli isolate (no virulence genes harbored) and the other consists of a sample without template added. The PCR reaction mixtures are incubated in a thermal cycler programmed with the thermal profile described (Appendix 2).

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3.4- Agarose gel electrophoresis

Prepare a 1.8% (w/v) agarose gel in 1X Tris/EDTA (Appendix 3). Each well of the gel is loaded with 15 μl of appropriate PCR reaction products with loading dye added at 1X final concentration. Run the samples in 1X running buffer (Tris/EDTA) at constant voltage (96V). Use a molecular weight marker suitable for assignment of the correct molecular weights to the amplicons produced (refer to the table in Appendix 1). Take into consideration that a correct band assignment is a crucial point in the assessment of the presence of the virulence genes. Make sure that the bands produced by the reference strains match exactly the expected molecular weight.

Ethidium Bromide should be added to agarose gels to allow the visualisation of DNA. This reagent is a DNA intercalating agent commonly used as a nucleic acid stain in molecular biology laboratories. When exposed to ultraviolet light, it will fluoresce with a red-orange color. EtBr should be added at a final concentration of 0.5 μg/ml before pouring the agarose gel in the electrophoresis gel cast. Alternatively, the agarose gel can be stained after electrophoresis in a 0.5 μg/ml ethidium bromide aqueous solution.

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4- Equipment and instruments

  • Laminar flow hood for PCR
  • Bacteriology loops
  • 100 ml Corning bottles
  • 37oC+/-1oC incubator
  • Micropipettes
  • Sterile micropipette tips
  • 1.5 ml microcentrifuge tubes
  • 0.2 or 0.5 ml PCR tubes
  • Thermal cycler
  • MilliQ deionizer
  • Electrophoresis apparatus
  • U.V. transilluminator
  • Microwave oven

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5- Reagents and media

  • Agar plates (blood Agar and MacConkey Agar)
  • Luria-Bertani Broth
  • FA buffer or PBS
  • PCR Reaction Mixture (Appendix 2)
    • dNTP mix stock solution
    • 10X PCR buffer stock solution (BIOTOOLS or equivalent)
    • MgCl2 stock solution 50 mM (BIOTOOLS or equivalent)
    • Taq DNA polymerase (BIOTOOLS or equivalent)
  • Synthetic oligonucleotide (primer)
  • Electrophoresis running buffer
  • Molecular weight DNA marker
  • Agarose
  • Loading buffer
  • EtBr solution

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6- Safety and protection devices

Some STEC/VTEC strains can infect humans at a very low infectious dose and can cause severe disease. Laboratory acquired infections have been reported. Consequently, working with VTEC requires good laboratory practices and the use of protection devices. EtBr is a mutagen and toxic agent; therefore it should be used in compliance with the safety sheet provided and with protection devices (lab-coats and nitrile gloves). The U.V. light may cause damage to the eyes, thus, the use of plexiglass shields and protective glasses is mandatory.

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7- Reference strains

The control templates can be prepared in advance and stored in 50 μl ready to use aliquots at −20oC for eight months. Data sheets for the reference strains used at the EcL are found in Appendix 4.

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8- Interpretation of the results

Samples showing amplification fragments of the expected size (Appendix 1) are considered as positive for appropriate target genes. Positive and negative controls must be included in each reaction and give positive and negative results, respectively.

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