Detection of Fish Pathogens Using Variable Number Tandem Repeat (VNTR) Analysis

Begin with a PCR plate containing a formamide-based denaturing solution.

Add the diluted PCR amplicons derived from a fish-pathogenic bacterium into each well.

These amplicons contain fluorescently tagged variable number tandem repeats (VNTRs), short repetitive sequences used as genetic markers for bacterial identification.

Seal the plate and centrifuge to eliminate air bubbles.

Heat the plate in a thermal cycler to denature the double-stranded VNTRs.

Formamide enhances strand separation and inhibits re-annealing.

Cool the plate rapidly to stabilize the single-stranded DNA.

Centrifuge to collect the sample volume.

Remove the seal, and secure the plate with a frame.

Place the capillary tube and perform capillary electrophoresis.

During electrophoresis, VNTR fragments migrate through the capillary under an electric field and separate by size.

A laser excites the fluorescent tags on each fragment, producing color-coded signals.

The resulting electropherogram shows VNTR peak patterns that correspond to the pathogenic bacterium.

Following confirmation of PCR amplicons, use new PCR strips or plates to dilute PCR products 1 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 to 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 4 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.