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Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray.

Santiago F González1, Melissa J Krug, Michael E Nielsen

  • 1Department of Microbiology and Parasitology, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain.

Journal of Clinical Microbiology
|April 9, 2004
PubMed
Summary

This study developed a sensitive and specific DNA microarray assay for simultaneously detecting five marine fish pathogens. The assay uses multiplex PCR to identify bacteria causing diseases in both fish and humans.

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Area of Science:

  • Marine microbiology
  • Molecular diagnostics
  • Aquatic animal health

Background:

  • Marine fish are susceptible to various bacterial pathogens.
  • Accurate and rapid detection of these pathogens is crucial for aquaculture and public health.
  • Existing diagnostic methods may lack the capacity for simultaneous detection of multiple species.

Purpose of the Study:

  • To develop and validate a multiplex assay for the simultaneous detection of five key marine fish pathogens.
  • To assess the sensitivity and specificity of the developed assay.
  • To provide a tool for identifying virulent strains of specific pathogens.

Main Methods:

  • Coupling multiplex PCR with a DNA microarray for simultaneous pathogen detection.
  • Designing oligonucleotide probes targeting chromosomal and plasmid-borne loci.

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  • Amplifying target DNA fragments using multiplex PCR and labeling for hybridization.
  • Testing the assay against target species and related bacterial species.

    • Main Results:

    • The assay successfully detected all five target marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus).
    • High sensitivity (up to 100%) and specificity (100%) were achieved for most probes.
    • The assay demonstrated high sensitivity, detecting as little as 20 fg of genomic DNA, and was more sensitive than gel electrophoresis.
    • Virulent strains of Listonella anguillarum serotype O1 were tentatively identified using the fatA gene probe.

    Conclusions:

    • The developed multiplex PCR and DNA microarray assay is a sensitive and specific tool for simultaneous detection of multiple marine fish pathogens.
    • This assay can aid in disease management in aquaculture and contribute to food safety.
    • The method offers a significant advancement in the rapid and accurate diagnosis of bacterial fish diseases.