Purpose: The purpose of this study is to reduce the turn around time of food pathogens detection methods. The benefits of a sample preparation containing a DNA concentration/purification method has been evaluated in association with FRET-based specific real-time PCR, which associate the specificity of both primers and probes sequences and the probe melting.
Methods: 375 g of ground beef were inoculated at a low level (target 1-5 CFU/375g) with E. coli O157:H7 or with Salmonella. After a 48 h equilibration at 4°C, samples were incubated in BPW at 42°C for various times and then analyzed by automated lysis/nucleic acid purification using the Nuclisens easyMAGTM instrument (bioMérieux) followed by a FRET-based real-time PCR. Inoculations were confirmed positive or negative with AOAC approved alternative methods. Results: Introduction of this simplified nucleic acids purification step upstream of PCR amplification enables to reduce the enrichment step by several hours; this protocol allows detection of E. coli O157:H7 after a 6-h enrichment and Salmonella after a 8-h enrichment in 100% of the confirmed positive samples.
Significance: This study suggests that the use of a very simple and automated nucleic acid purification step (Nuclisens easyMAGTM ) allows a turn around time of analysis of 7 h 30 min for E. coli O.157:H7 and 9 h 30 min for Salmonella sp. Same-day results for both E. coli O157:H7 and Salmonella in pooled samples enables rapid product release and quicker raw material orientation and therefore cost savings for food producers.