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Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres
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A multiplex magnetic capture hybridization and multiplex Real-Time PCR protocol for pathogen detection in seafood.

G Amagliani1, E Omiccioli, G Brandi

  • 1Dipartimento di Scienze Biomolecolari, Sez. di Scienze Tossicologiche, Igienistiche e Ambientali, Università di Urbino Carlo Bo, via S. Chiara 27, 61029 Urbino (PU), Italy. giulia.amagliani@uniurb.it

Food Microbiology
|June 1, 2010
PubMed
Summary

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Rapid molecular methods for seafood safety are essential. A new multiplex magnetic capture hybridization assay combined with real-time PCR accurately detects Salmonella spp. and Listeria monocytogenes DNA, significantly reducing analysis time.

Area of Science:

  • Food Microbiology
  • Molecular Biology
  • Public Health

Background:

  • Seafood can harbor bacterial pathogens like Salmonella spp. and Listeria monocytogenes, posing public health risks.
  • Traditional culture-based methods for pathogen detection are time-consuming, delaying effective control measures.

Purpose of the Study:

  • To develop and validate a rapid molecular method for simultaneous detection of Salmonella spp. and Listeria monocytogenes DNA in seafood.
  • To compare the performance of the molecular method against standard culture-based techniques.

Main Methods:

  • A multiplex magnetic capture hybridization (mMCH) assay using paramagnetic nanoparticles for DNA isolation.
  • A triplex Real-Time PCR with an Internal Amplification Control (IAC) for simultaneous pathogen detection.

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  • Testing on artificially and naturally contaminated raw and smoked salmon fillets.

    • Main Results:

    • The molecular assay achieved 100% detection probability with 10 genome equivalents of target DNA.
    • Complete agreement was found between the molecular method and standard methods, with a sensitivity of 1 cfu/g in enriched samples.
    • The molecular method demonstrated higher sensitivity in non-enriched samples (10^2–10^3 cfu/g) and detected natural L. monocytogenes contamination in smoked salmon.

    Conclusions:

    • The developed mMCH and Real-Time PCR assay provides a rapid, accurate, and sensitive tool for detecting Salmonella spp. and Listeria monocytogenes in seafood.
    • This molecular approach significantly reduces analysis time compared to conventional methods, enhancing seafood safety and public health protection.