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Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis
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A novel high-resolution melting analysis-based method for Yersinia pseudotuberculosis genotyping.

Roberto A Souza1, Juliana P Falcão

  • 1Brazilian Reference Center on Yersinia spp. other than Y. pestis, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto, SP, Brazil.

Journal of Microbiological Methods
|October 9, 2012
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Summary

A new genotyping method using high-resolution melting analysis (HRMA) efficiently differentiates Yersinia pseudotuberculosis strains. This rapid, cost-effective approach aids epidemiological studies of this widespread enteric pathogen.

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Area of Science:

  • Microbiology
  • Genetics
  • Epidemiology

Background:

  • Yersinia pseudotuberculosis is a widespread enteric pathogen causing human and animal infections.
  • Effective epidemiological studies require fast and inexpensive Yersinia pseudotuberculosis typing systems.

Purpose of the Study:

  • To develop a rapid and cost-effective Yersinia pseudotuberculosis genotyping method using high-resolution melting analysis (HRMA).
  • To screen for single-nucleotide polymorphisms (SNPs) in key genetic loci to differentiate strains.

Main Methods:

  • Screened ten single-nucleotide polymorphisms (SNPs) across nine primer pairs in 16S rRNA, glnA, gyrB, and recA genes.
  • Utilized high-resolution melting analysis (HRMA) for genotyping 40 clinical Yersinia pseudotuberculosis strains.
  • Compared HRMA results with multilocus sequence typing (MLST) for validation.

Main Results:

  • Identified distinct melting profiles in five out of nine analyzed fragments using HRMA.
  • Constructed a phylogenetic tree showing Yersinia pseudotuberculosis strains separated into two distinct clusters corresponding to O:1a and O:3 serogroups.
  • Demonstrated concordance between HRMA-based genotyping and MLST results.

Conclusions:

  • The developed HRMA assay is a simple, reproducible, rapid, and cost-effective method for genotyping Yersinia pseudotuberculosis strains.
  • This HRMA method effectively differentiates strains belonging to O:1 and O:3 serogroups.
  • The HRMA assay can complement sequence-based methods, significantly aiding epidemiological investigations of Yersinia pseudotuberculosis infections.