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

作者：Amagliani G^*; Omiccioli E; Brandi G; Bruce I J; Magnani M

来源：Food Microbiology, 2010, 27(5): 580-585.

DOI：10.1016/j.fm.2010.01.007

• Magnetic capture hybridisation (MCH)
• Multiplex Real-Time PCR
• Seafood
• Listeria monocytogenes
• Salmonella spp.

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.

• 出版日期2010-8