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A multiplex magnetic capture hybridisation and multiplex Real-Time PCR protocol for pathogen detection in seafood

Amagliani, G., Omiccioli, E., Brandi, E., Bruce, L.J., Magnani, M.
Food microbiology 2010 v.27 no.5 pp. 580-585
salmon, polymerase chain reaction, food pathogens, food microbiology, bacterial contamination, Listeria monocytogenes, Salmonella, genome, smoked fish, raw fish, detection, genomics, food contamination
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.