A multiplex magnetic capture hybridization and multiplex Real-Time PCR protocol for pathogen detection in seafood.
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.
Dipartimento 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
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Amagliani
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Omiccioli
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Brandi
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Bruce
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Magnani
D000818:Animals; D005506:Food Contamination / Q000032:analysis; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D008280:Magnetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012474:Salmon / Q000382:microbiology; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D017747:Seafood / Q000382:microbiology*