P3-151 An Effective Real-Time Quantitative PCR Protocol for Quantification of Pathogens in Foodstuffs

Wednesday, July 25, 2012
Exhibit Hall (Rhode Island Convention Center)
Ysabelle Adolphe, University of Liege, Liege, Belgium
Sebastien Crevecoeur, University of Liege, Liege, Belgium
Perrine Duval, University of Liege, Liege, Belgium
Georges Daube, University of Liege, Liege, Belgium
Antoine Clinquart, University of Liege, Liege, Belgium
Introduction: Several methods of real-time quantitative PCR have been developed for the detection and quantification of pathogens in food products. All molecular techniques needed an enrichment step which could hedge the quantification. Furthermore, most of those techniques are not available on a large range of foodstuffs.

Purpose: The behavior of our study was to develop an effective molecular quantification for pathogens, as Listeria monocytogenes, Escherichia coli O157:H7 or Salmonella spp. To present an interest for food industries, our technique should be efficient on several foodstuffs.

Methods: Experiments were carried out on pure culture, pork minced meat, white pudding, ham, and different field pancakes. Food matrices have been artificially contaminated with 4 to 100 cfu/g of Listeria monocytogenes, Escherichia coli O157:H7 or Salmonella spp. During the same period, to numerate pathogens, classical microbial analysis and molecular quantification by real-time quantitative PCR had been carried out at + 20 °C and with aging tests. Taqman technology was used for pathogens quantification.

Results: Different standard curves were established for each pathogen group. Those were carried out and validated in pure culture, and also in the different foodstuffs. Statistical analyses between classical and molecular quantifications allowed the validation of standard curves. The lower threshold for quantification was about 5 ufc/g. Under this threshold, detection was still possible.

Significance: Many studies have confirmed the value of qPCR as a rapid and reliable method that could be used in food industries for detecting organisms of interest, but there are still reservations about its routine use in food analysis. Commercial kits have been developed for the detection, but not for precise quantification of the main pathogens. Our technique allowed an effective quantification of three groups of pathogens, at least for now. This new technique could be used directly on food matrix, without enrichment step, with a vey interesting lower quantification threshold.