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Simultaneous detection of Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes using multiplex PCR

Identification

Key words multiplex real-time PCR, food-borne pathogen, simultaneous detection, salmonella, escherichia coli, listeria monocytogenes, multiple platform
Latest version 2010/12/22
Completed by TTZ

How does it work?

Primary objective analytical tool for simultaneous detection of the pathogenic microorganisms Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes
Working principle Samples are mixed with enrichment medium and incubated. After enrichment of the target organisms bacterial DNA is extracted and amplified with the multiplex PCR. The detection of PCR products can be done in real-time via fluorescent signals either via intercalating dyes within the bacterial DNA, followed by high resolution melting analysis for specification or with dual-labeled probes. Alternatively to real-time detection of PCR products, PCR can be followed by an electrophoresis with ethidium bromide staining and UV detection.
Images
Additional effects time saving process (48 h instead of 7 days required in the standard culture method)

automatized high-throughput process higher sensitivity than conventional methods allows quantification of pathogens reduced risk of cross-contamination during analysis

Important process parameters suitable broth enabling simultaneous enrichment of the three bacteria (e.g. No17 enrichment broth according to Kawasaki et al. 2005)

enrichment time (15-20 h depending on food matrix) DNA extraction method (depending on food matrix)

Important product parameters

What can it be used for?

Products relevant for all products exposed to contamination by the mentioned microorganisms
Operations quality assurance, pathogen analytic
Solutions for short comings The method enables inexpensive screening of several samples for more than one pathogen with low workload.

appropriate to support ISO culture methods with preliminary screening

What can it NOT be used for?

Products no restricted products reported yet
Operations
Other limitations The multiplex real-time PCR is not valid as an official reference method.

sensitivity: down to 1 cell of each species in 25 g sample enrichment media and PCR parameters have to be adapted for different pathogens

Risks or hazards no risks

Implementation

Maturity assays for single detection: commercially available

assays for multiple detection: lab scale, comparison with conventional methods showed reliability of the multiplex PCR

Modularity /Implementation PCR instrument and fluorescence or UV detector is needed
Consumer aspects Intoxications and infections caused by food-borne pathogens represent an increasing public health problem. The three discussed pathogens account for about 40 % of pathogen intoxications in the EU. Suitable analytical methods are necessary for industry to combat this problem by detecting contaminations of their products at an early stage.
Legal aspects limits for pathogens in food according to (EC) No 2073/2005 on microbiological criteria for foodstuffs
Environmental aspects Rapid control and analysis of raw material is an important tool to ensure cleanability and to minimize the potential for cross-contamination and recontamination.

Further Information

Institutes University of Parma, Urbino University, CTC
Companies Barilla, Diatheva, Roche
References 1. Bhagwat A. A. (2002) Simultaneous detection of Escherichia coli O157:H7,Listeria monocytogenes and Salmonella strains by real-time PCR. International Journal of Food Microbiology 84, 2, 217– 224.

2. Elizaquível, P., Gabaldón, J.A., Aznar, R. (2010) Quantification of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 in non-spiked food products and evaluation of real-time PCR as a diagnostic tool in routine food analysis. Food Control, 22, 2, 158-164.

3. Germini, A., Masola, A., Carnevali, P., Marchelli, R. (2008) Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR. Food Control, 20, 8, 733-738.

4. Kawasaki, S., Horikoshi, N., Okada, Y., Takeshita, K., Sameshima, T., Kawamoto, S. (2005) Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:next termH7 in meat samples, J. Food Prot. 68, pp. 551–556.

5. Kawasaki, S., Fratamico, P., Horikoshi, N., Okada, Y., Takeshita, K., Sameshima, T., Kawamoto, S. (2009) Evaluation of a Multiplex PCR System for Simultaneous Detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in Foods and in Food Subjected to Freezing. Foodborne Pathogens and Disease, 6, 1, 81-89.

6. Kim, J., Demeke, T., Clear, R., Patrick, S. (2006) Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain. International Journal of Food Microbiology, 111, 1, 21-25.

7. Omiccioli E., Amagliani G., Brandi G., Magnani M. (2009) A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk, Food Microbiology 26, 615–622.

8. European Commission. Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union 2005; L338:1-26 (as amended by Regulation (EC) No 365/2010).

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Created by WikiSysop on 29 January 2011, at 01:42