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Published on: October 23, 2011
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
Abstract:
Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100% with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1-10(3)cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1 cfu/g, in enriched samples, and higher sensitivity (10(2)-10(3)cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time.
Insights
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.
- 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.
