Home › Microbiology › Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis
Microbiology JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/59455-v
What you'll learn
  • ✓ Perform multiplex PCR and capillary electrophoresis for MLVA genotyping of Yersinia ruckeri
  • ✓ Calculate VNTR repeat counts and generate ten-loci MLVA profiles from electropherogram data
  • ✓ Construct and interpret minimum spanning trees for bacterial strain clustering and comparison
Protocol

Biopharma Insights The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay presented here enables inexpensive, robust and portable high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. Starting from pure cultures, the assay employs multiplex PCR and capillary electrophoresis to produce ten-loci MLVA profiles for downstream applications.

Difficulty
advanced
Total time
~4-6 hours per sample batch (including PCR cycling, electrophoresis, and data analysis)
Biosafety
BSL-2

Steps

1
Cultivate bacteria and extract genomic DNA

Grow Yersinia ruckeri from pure culture and perform DNA extraction from bacterial cells to obtain template for PCR amplification.

▶ 00:53
2
Set up multiplex PCR with optimized cycling conditions

Prepare multiplex PCR reaction mixtures targeting ten VNTR loci and execute thermocycler protocol with appropriate annealing temperatures and extension times.

▶ 01:54
3
Confirm PCR amplicons by agarose gel electrophoresis

Verify successful amplification and estimate product sizes by running PCR products on agarose gel and visualizing under UV light.

▶ 03:14
4
Perform capillary electrophoresis for precise sizing

Load PCR amplicons into capillary electrophoresis instrument with size standards and run under specified voltage and duration conditions to generate high-resolution electropherograms.

▶ 04:37
5
Calculate VNTR sizes and generate MLVA profiles

Determine allele sizes from electropherogram peaks, calculate repeat counts for each locus, and compile ten-loci MLVA genotype profiles for each strain.

▶ 06:06
6
Construct minimum spanning tree for strain clustering

Perform cluster analysis using MLVA profile data to generate a minimum spanning tree that visualizes evolutionary relationships and genetic distances between bacterial isolates.

▶ 07:36
7
Interpret gel electrophoresis and tree analysis results

Review representative gel images, electropherograms, and minimum spanning tree outputs to validate genotyping results and strain differentiation.

▶ 09:21
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