Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR Andrea Germini a, * , Annalisa Masola a , Paola Carnevali b , Rosangela Marchelli a a University of Parma, Department of Org. and Ind. Chemistry, Viale G.P. Usberti 17/A, 43100 Parma, Italy b Barilla G&R f.lli SpA, Food Microbiology and Bioprocess Research, Via Mantova 166, 43100 Parma, Italy article info Article history: Received 7 February 2008 Received in revised form 1 September 2008 Accepted 11 September 2008 Keywords: Foodborne pathogens PCR Egg Multiplex Salmonella Listeria Escherichia Food 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 detec- tion 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 sys- tem 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. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Foodborne diseases are a widespread and growing public health problem affecting both developed and developing countries, microbiologically contaminated food and water being the major causes of diarrhoeal diseases. Although their global incidence is difficult to estimate, authors generally agree in estimating that the percentage of the population suffering from foodborne diseases each year could be up to 30% in industrialized countries and figures could be even worse in developing countries (Mead et al., 1999; WHO, 2007). The microbiological safety of food production is a significant concern of regulatory agencies due to the potential consequences to human health, and the food industry due to the important impli- cations in terms of the risk of recalls of products placed on the mar- ket, and the potential economic loss as well as the loss of consumer confidence. Classical microbiological methods offer reliable and standard- ized procedures for the detection of foodborne pathogens (e.g. ISO standards), but because of the time-to-result needed for the different operations (enrichment, isolation of colonies, and identi- fication) they often result in time consuming analyses not always compatible with the need for rapid results. In the last few years molecular techniques have appeared as promising alternatives in food microbiology. Nucleic acid based amplification techniques, of which the polymerase chain reaction (PCR) has been so far the most extensively employed, offer several advantages over the classical microbiological methods such as shorter time of analysis, low detection limits, specificity and poten- tial for automation. During the last few years international standards have been agreed on the use of PCR for the detection of foodborne pathogens (ISO 22174:2005 general requirements and definitions; ISO/TS 20836:2005 performance criteria for thermocyclers; ISO/TS 20837:2006 sample preparation; ISO 20838:2006 amplification and detection for qualitative methods) and legislations are imple- menting new types of analyses as the accepted official methods. For example, European regulation EC 2073/2005 allows for the use of alternative detection methods if based on certified analyses according to international standards (EU, 2005). Although several etiological agents can be transmitted through food and water consumption, Salmonella enterica, Listeria monocyt- ogenes and Escherichia coli O157:H7 are considered among the most relevant foodborne pathogens. 0956-7135/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2008.09.010 * Corresponding author. Tel.: +39 0521 905676; fax: +39 0521 905472. E-mail address: andrea.germini@unipr.it (A. Germini). Food Control 20 (2009) 733–738 Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont