All qPCR assays were performed on an iCycler iQ™5 Real-Time

PCR Detection System (Bio-rad) with iCycler iQ™ PCR plates, 96 wells (Bio-rad) closed with the PCR Sealers Microseal B films (Bio-rad). All qPCR assay reactions were performed according to the same protocol: the reactions were performed in a final volume of 25 μl containing 5 μl of the diluted DNA extract (1/2 for Listeria qPCR assays and 1/1000 for Salmonella qPCR assays), 1X SYBR®Green PCR Mastermix (DMSG-2X-A300, Diagenode), and the appropriate concentration of each primer ( Barbau-Piednoir et al., 2013a and Barbau-Piednoir et al., 2013b). Primers were purchased from Eurogentec (Belgium). The following thermal programme was applied: a single cycle Sorafenib of DNA polymerase activation for 10 min at 95 °C followed by 40 amplification cycles of 15 s at 95 °C (denaturing step) and 1 min at 60 °C (annealing–extension step). Subsequently, CHIR-99021 mw melting temperature analysis of the amplification products was performed by gradually increasing the temperature from 60 °C to 95 °C over 20 min (± 0.6 °C/20 s). The fluorescent reporter signal was normalized against the internal reference dye (ROX) signal and the threshold limit was set manually at the beginning of the exponential amplification phase. “No Template” Controls (NTC) using DNase and RNase free water

were included in each reaction to assess primer dimer formation or non-specific amplification. A positive control using 104 copies of gDNA of L. monocytogenes 1/2a strain ATCC 51772 or S. enterica subsp. enterica Enteritidis (Belgian CNR Salmonella ref H.V.6.32) from pure strains extracted with the DNeasy® Blood and tissue Extraction kit (Qiagen) was included in each qPCR reaction. For the interpretation PDK4 of a SYBR®Green qPCR assay, two criteria

were analysed: the quantification cycle (Cq) value, and the melting temperature of the amplicon (Tm). The Cq-value represents the fractional cycle at which the PCR amplification reaches the threshold level for the reaction (Bustin, 2000). Since it is a screening assay, only a qualitative response is required. To be considered as positive, a signal generated in the CoSYPS Path Food detection system should display an (exponential) amplification above the limit of detection of each qPCR determined previously, with the expected Tm-value (Barbau-Piednoir et al., 2013a and Barbau-Piednoir et al., 2013b). The combination of positive assays generates the list of bacteria possibly present into the sample (presumptive positive) according to the decision tree presented in Fig. 2. The selective enrichment, isolation and the confirmation were performed only if a presumptive positive result was obtained. All these steps were performed as previously described in the ISO reference methods section. The complete CoSYPS Path Food workflow was validated for the enrichment, detection, isolation and confirmation of the presence of Listeria spp. and Salmonella spp. in beef carcass swab samples.