Campylobacter MBiT

MLPA probes have been designed for a selection of 18 genes exhibiting variable carriage in Campylobacter jejuni and hypothesized to be associated with pathogenicity or survival. The system is also applicable for subtyping C. coli. The assay has been designed as two probemixes that can be analysed either separately or together allowing for detection by multiple platforms including gel electrophoresis (as two reactions), chip-based micro-fluid electrophoresis (such as the MultiNA Microchip Electrophoresis System from Shimadzu Corporation and QIAxcel from Qiagen) and capillary electrophoresis (as one reaction).

MBiT typing is possible from PFGE plugs, colonies and extracted DNA making it rapid and versatile. This also offers the ability to rapidly and accurately generate back-compatible data as it is not necessary to re-extract DNA for isolates already tested by methods such as PFGE or MLST.

Campylobacter MBiT genes and amplicon sizes

Laboratory Protocol

Below is a detailed laboratory protocol using the MLPA kit supplied by MRC Holland and probe mix specific for Campylobacter MBiT;

Prepare DNA from a selection of methods, for example CHELEX extractions, Qiagen DNeasy Blood and Tissue Kit extractions and heatblast methods from bacterial cells and agarose embedded bacterial cells.

Step One

DNA Denaturation:

1. Transfer 5 µL of each DNA sample to a separate 0.2 mL PCR tube.
2. Heat for 5 min at 98°C in a thermocycler with a heated lid and then cool to 25°C before opening the thermocycler.

Step Two

Hybridisation of the MLPA Probes:

1. While the samples are denaturing bring probemixes X054 and X055 (black caps) and MLPA Dilution Buffer (yellow cap), as appropriate for the run, to room temperature and vortex to mix. If using both probemixes together transfer an equal volume of each probemix allowing enough for 1.5 µL of each solution per sample and mix by pipetting or vortexing. If using only one probemix replace the volume of the second probemix with MLPA Dilution Buffer.
2. Stop the run and take the tubes out of the thermocycler.
3. Add 3 µL of the appropriate synthetic probemix-MLPA Buffer solution to each sample and mix by repeated pipetting.
4. Incubate for 1 min at 95°C, followed by 1-2 hr at 60°C in a thermocycler with a heated lid.

Step Three

Ligation Reaction:

1. Prepare sufficient Ligase mastermix for all samples – below is the volumes required per sample. Keep the solution on ice and use within 1 hr of preparation.
   1. 3 µL Ligase-65 Buffer A (transparent cap)
   2. 3 µL Ligase-65 Buffer B (white cap)
   3. 25 µL water (vortex and spin briefly before adding Ligase-65)
   4. 1 µL Ligase-65 (green cap) (mix by pipetting).
2. Reduce the temperature on the thermocycler to 54°C.
3. Once the temperature has reached 54°C dispense 32 µL of the Ligase mastermix to all samples and mix well by repeated pipetting.
4. Once Ligase mastermix has been added to all of the tubes close the thermocycler lid and incubate for 15 min at 54°C.
5. Heat for 5 min at 98°C then cool to 4°C.
6. Samples can be stored at 4°C for up to one week or -20°C if longer storage is required.

Step Four

PCR:

1. Prepare enough Polymerase mastermix for all samples – below is the volumes required per sample. Keep the solution on ice and use within 1 hr of preparation.
   1. 7.5 μL water
   2. 2 µL SALSA PCR primer mix (brown cap)
   3. 0.5 µL SALSA Polymerase (orange cap).
2. Dispense 10 µL volumes into PCR tubes containing all of the ligation products.
3. Run the following thermal cycling conditions in a thermocycler with a heated lid:
   1. 35 cycles of 30 sec 95°C, 30 sec 60°C, 60 sec 72°C
   2. followed by 20 min 72°C
   3. hold 15°C
4. PCR products can be stored at 4°C for up to 1 week or -20°C if longer storage is required. Store in a dark box or covered in foil.

Step Five

Product Detection:

1. Capillary Electrophoresis:
   1. dilute each MLPA product 1 in 100 in sterile water.
2. MultiNA:
   1. use the MLPA products directly.
3. Gel Electrophoresis:
   1. use the MLPA products directly.
   2. prepare a 2% agarose gel in 1 X TBE (or suitable equivalent).
   3. load 10 µL of MLPA product per lane. For ease of interpretation load the control plasmid (positive control) at least every 6 samples.
   4. run at 110 V for 70 min.
   5. stain using ethidium bromide or a suitable replacement.
   6. visualise the DNA bands and capture the gel image.
   7. score the presence (1) or absence (0) of each target band.
   8. combine the scores for the two probemixes to produce a full MBiT code.