Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR |
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| Authors: | Andrea Germini Annalisa Masola Paola Carnevali Rosangela Marchelli |
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| Affiliation: | 1. Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy;2. Instituto Tecnológico Agrario de Castilla y León (ITACyL), Valladolid, Spain;3. Microbiology Section, Faculty of Sciences, University of Burgos, Burgos, Spain;4. Istituto Zooprofilattico Sperimentale delle Venezie, SCS 8 — Valorizzazione delle Produzioni Alimentari, Padua, Italy;5. Alma Mater Studiorum, Università di Bologna, Dip. Scienze e Tecnologie Agro-Alimentari Ozzano dell''Emilia (BO), Italy;7. National Food Chain Safety Office, Food and Feed Safety Directorate, Food Microbiological National Reference Laboratory, Mester, Budapest, Hungary;8. CNTA, Centro Nacional de Tecnología y Seguridad Alimentaria, San Adrian, Navarra, Spain;9. University of Zagreb, Faculty of Veterinary Medicine, Dpt. of Hygiene, Technology and Food Safety, Zagreb, Croatia;10. Section for Bacteriology — Food and GMO, Norwegian Veterinary Institute, Oslo, Norway;11. Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark;12. Direzione Operativa Controllo degli Alimenti, Istituto Zooprofilattico Sperimentale del Lazio e Toscana, Rome, Italy;13. Istituto Zooprofilattico Sperimentale della Lombardia e dell''Emilia Romagna, Brescia, Italy |
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| Abstract: | The wide application of nucleic acid amplification techniques and the increasing industrial interest toward rapid methods has led to the development and application of PCR based methods for the detection of microbial pathogens in food. In the present paper we describe the development of a multiplex PCR method for simultaneous detection of Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7 in a complex food matrix (liquid whole egg).Four different DNA extraction procedures were evaluated for their application on food and, among these, Chelex resin combined with a DNA purification step were found to better perform on the food system considered.A multiplex PCR system was developed, based on the evaluation and combination of published primer sets, and applied to the simultaneous detection of the target pathogens plus an internal amplification control, both in culture media and in a model food system.The overall system proposed, based on an overnight enrichment step followed by DNA isolation and multiplex PCR, was satisfactorily tested for its specificity and sensitivity and allowed the detection of the presence of bacterial DNA and the identification of the target pathogens down to 10 cells/25 g liquid whole egg. |
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