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

机译：用于海鲜中病原体检测的多重磁捕获杂交和多重实时PCR协议

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摘要

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.
机译：通过暴露于污染的水中或通过加工做法，海鲜可能成为细菌病原体的来源，从而构成公共健康危害。传统的基于培养物的分析方法需要几天才能完成，而细菌病原体的分子快速鉴定对于有效控制疾病至关重要。开发的应用程序包括用于同时分离沙门氏菌的多重磁捕获杂交（mMCH）分析。和海鲜中的单核细胞增生李斯特氏菌DNA，使用具有捕获寡核苷酸的顺磁性氨基修饰的纳米粒子，以及具有内部扩增对照（IAC）的三重实时PCR，根据ISO22174。检测概率为100％，具有10个基因组当量每个目标物种在同一反应中共同扩增。直接或在培养富集后，对被已知量的一种或两种目标细菌（1-10〜3 cfu / g）人工污染的生鱼和烟熏三文鱼柳进行完整的分子程序测试，并与标准方法进行比较。结果显示两种方法之间完全吻合，富集样品的灵敏度为1 cfu / g，而培养物富集前检测的分子方法的灵敏度更高（10〜2-10〜3 cfu / g）。所提议的程序还能够在相当短的时间内识别烟熏鲑鱼中单核细胞增生李斯特氏菌的自然污染。

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  • 来源
    《Food microbiology》 |2010年第5期|580-585|共6页
  • 作者

    G. Amagliani; E. Omiccioli; G. Brandi; I. J. Bruce; M. Magnani;

  • 作者单位

    Dipanimento di Scienze Biomolecolari, Sez. di Scienze Tossicologiche, Igienistiche e Ambientali, Universita di Urbino 'Carlo Bo', via S. Chiara 27, 61029 Urbino (PU), Italy;

    Diatheva srl, viale Piceno 137/F, 61032 Fano (PU), Italy;

    Dipanimento di Scienze Biomolecolari, Sez. di Scienze Tossicologiche, Igienistiche e Ambientali, Universita di Urbino 'Carlo Bo', via S. Chiara 27, 61029 Urbino (PU), Italy;

    Department of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK;

    Dipanimento di Scienze Biomolecolari, Sez. di Biotecnologie, Universita di Urbino 'Carlo Bo', via Campanella 1, 61032 Fano (PU), Italy Dipanimento di Scienze Biomolecolari, Sez. di Biochimica e Biologia Molecolare, Universita di Urbino 'Carlo Bo', via Saffi 2, 61029 Urbino (PU), Italy;

  • 收录信息 美国《科学引文索引》(SCI);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    magnetic capture hybridisation (MCH); multiplex real-time PCR; seafood; listeria monocytogenes; salmonella spp.;

    机译：磁捕获杂交（MCH）;多重实时PCR海鲜;李斯特菌;沙门氏菌;

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