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

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• 作者：G. Amagliani ; E. Omiccioli ; G. Br ; i ; I.J. Bruce ; M. Magnani
• 关键词：Magnetic capture hybridisation (MCH) ; Multiplex Real-Time PCR ; Seafood ; Listeria monocytogenes ; Salmonella spp.
• 刊名：Food Microbiology
• 出版年：2010
• 出版时间：August 2010
• 年：2010
• 卷：27
• 期：5
• 页码：580-585
• 全文大小：292 K

文摘

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.