Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

This MLB genotyping protocol has greatly increased our understanding of the epidemiology and global population structure of Yersinia ruckeri, an internationally important fish pathogenic bacterium. Compared to previously established Yersinia ruckeri typing schemes, it offers a very high strain resolution. The procedure is simple, cheap, and awards portable results that can be readily compared across laboratories.

The technique greatly improves diagnostic specificity and enables infection tracing. For instance, we can now often distinguish virulent Yersinia ruckeri clones existing against the backdrop of non-virulent environmental strains. Demonstrating the procedure will be Saima Nasrin Mohammad, a technician in our laboratory.

To begin, use sterile inoculation loops to sow out Yersinia ruckeri pure cultures on any suitable agar type on Petri dishes. Incubate at 22 degrees Celsius for one to two days or 15 degrees Celsius for three to four days. After that, using inoculation loops, pick a single representative colony from each Petri dish and transfer to 1.5-milliliter centrifuge tubes, containing 50 microliters of ultra purified water to resuspend.

Vortex briefly and incubate for seven minutes on a heating block at 100 degrees Celsius. Then, centrifuge at 16, 000 g for three minutes and use a pipette to carefully transfer the supernatant template DNA into empty 1.5-milliliter centrifuge tubes. Proceed to the next step or store the DNA at 20 degrees Celsius.

To prepare master mixes, first calculate reagent volumes according to the number of bacterial samples to be analyzed, also accounting for one negative and one positive control, and a final 10%surplus volume, as detailed in the manuscript. Note that different primer concentrations are used. In two separate centrifuge tubes, one per PCR assay, add multiplex PCR plus master mix, primers, and RNase-free water.

Vortex the prepared master mixes gently at low speed and then, distribute 22 microliters of each master mix separately into individual wells on either PCR strips or plates. Then, add three microliters of the prepared DNA template to each well containing master mix;two wells per bacteria sample, one for each assay. Use DNA from a verified Yersinia ruckeri strain for a positive control, and ultra purified water for a negative control.

Seal and centrifuge briefly. Load the prepared reactions onto a PCR thermo cycler and set up the program according to the manuscript. The program will complete in less than three hours.

According to the manufacturer's recommendations, add agarose gel powder in one times TBE buffer to reach 1.5 weight/volume percent and heat to dissolve. Prior to casting, chill the dissolved gel solution briefly under running water, add 5 microliters of fluorescent nucleic acid dye per 50 microliters of gel solution and mix. Assemble and level the casting tray.

Add cones as appropriate for the number of samples. Pour the gel solution into the cast and wait for it to solidify. After that, submerge the cast containing the gel in one times TBE buffer in a gel electrophoresis system.

Mix five microliters of the PCR products together with two microliters of loading dye in new PCR strips. Transfer five microliters of the mixtures to the wells in the gel. Save at least one well empty.

Add five microliters of DNA ladder in the empty well for reference. Plug in the electrodes and run the gel at 110 volts per 15 centimeters for approximately one hour. Use a UV-based gel imaging system to verify the presence of multiple bands representing PRC amplicons.

Following confirmation of PCR amplicons, use new PCR strips or plates to dilute PCR products one to 10 in purified water. Vortex and centrifuge briefly. While working in a fume cupboard, prepare a master mix in a centrifuge tube consisting of formamide and reference-size standard solution.

According the number of PCR products to be analyzed, use reagent volumes as detailed in the manuscript. Allow a 10%surplus volume. Vortex briefly and distribute 9.5 microliters into individual wells on a PCR plate appropriate for the available capillary electrophoresis system.

Then, add 0.5 microliters of diluted PCR product. Seal and centrifuge briefly. Then, load the plate containing the samples into a PCR thermo cycler and denature the samples at 95 degrees Celsius for three minutes before cooling to four degrees Celsius indefinitely.

Centrifuge at 1000 g for one minute, carefully remove the seal, and load the plate onto a calibrated capillary electrophoresis system according to the manufacturer's instructions. Run fragment analysis capillary electrophoresis, using reagents as appropriate for the apparatus of choice, and adjust the run conditions according to the description in the manuscript. For Yersinia ruckeri VNTR capillary electrophoresis size calling, first import capillary electrophoresis result files into Gene Mapper 5 software.

Set the analysis method to, Microsatellite Default, select the appropriate product choice under size standard, then press the Analyze button. Through the size match editor, verify the correct identification of size standard fragments and rectify any visibly erroneous allocations. Then, select the sample to be read, hit the Display Plot button, and press Control A to enable view of the sizing table.

While in the top panel, hold down Control, while clicking on the five peaks representing the VNTR amplicons. Press Control G to filter the sizing table, showing only characteristics of the five highlighted peaks, and record the size calls for each VNTR locus for downstream application. In order to account for biased amplicon mobility patterns during capillary electrophoresis, calculate accurate VNTR repeat counts by employing the acquired size calls in combination with the locus specific variables.

The formula may be implemented in a spreadsheet template for automation. In the spreadsheet, round calculated VNRT repeat counts off to the nearest integer prior to downstream analysis. In the BioNumerics software, create a new database, and opt to activate the MLVA plugin.

In the experiment type panel, press Create new experiment type, choose Character type, and use the default settings when asked. Then select the new experiment and add new characters for each of the 10 VNTR loci, employing zero to 100 as the value range. Back in the main panel, import Yersinia ruckeri VNTR repeat counts and method data by selecting import fields and characters.

Locate the file where this is stored and make selections in the Destination type column. For the VNTRs, this means selecting the appropriate character value, while the miscellaneous metadata is classified as entry info fields. The choices made may be stored as a template in order to ease the process for later.

Select imported samples destined for MST cluster analysis, then click the Create new comparison button in the Comparisons panel. If desired for the visual presentation of the MST, allocate the samples to colored groups, for example, according to a particular metadata feature. Then, select Advanced cluster analysis and MST for categorical data to generate an MST diagram based on the chosen samples.

Further modify the visual presentation of the MST by adding partition parameters, cross lengths, node branch labeling, etc. If needed, export the finalized MST in a desired format using the Export image selection. Following multiplex PCR, the gel electrophoresis image verifies the presence of multiple amplicons in all 12 lanes containing samples, with the first lane representing the DNA ladder used.

In electropherograms resulting from capillary electrophoresis, the different VNTR loci can be identified according to a combination of size and dye labeling. Orange peaks represent the size standard employed. An example minimum spanning tree, based on MLVA profiles from Yersinia Ruckeri isolates recovered from Atlantic salmon in five different Norwegian farms is shown.

A clear clustering tendency linked to farm origin can be observed. The identification by MLVA of different Yersinia ruckeri clones dominating, for instance, in particular geographic regions of fish species, has already been exploited for improving vaccination strategies against this pathogen.