A multiplex magnetic capture hybridisation and multiplex Real-Time PCR protocol for pathogen detection in seafood |
| |
Authors: | G Amagliani E Omiccioli G Brandi IJ Bruce M Magnani |
| |
Affiliation: | 1. Dipartimento di Scienze Biomolecolari, Sez. di Scienze Tossicologiche, Igienistiche e Ambientali, Università di Urbino ‘‘Carlo Bo”, via S. Chiara 27, 61029 Urbino (PU), Italy;2. Diatheva srl, viale Piceno 137/F, 61032 Fano (PU), Italy;3. Department of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK;4. Dipartimento di Scienze Biomolecolari, Sez. di Biotecnologie, Università di Urbino ‘‘Carlo Bo''’, via Campanella 1, 61032 Fano (PU), Italy;5. Dipartimento di Scienze Biomolecolari, Sez. di Biochimica e Biologia Molecolare, Università di Urbino ‘‘Carlo Bo''’, via Saffi 2, 61029 Urbino (PU), Italy |
| |
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 (1–103 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 (102–103 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. |
| |
Keywords: | Magnetic capture hybridisation (MCH) Multiplex Real-Time PCR Seafood Listeria monocytogenes Salmonella spp |
本文献已被 ScienceDirect 等数据库收录! |
|