The shipment included a positive MM-102 datasheet DNA control (1 μg/ml S. Typhimurium CCUG 31369) and a negative DNA control (1 μg/ml Escherichia coli O157 (Sample ID 077,
Institute for Reference Materials and Measurements, Geel, Belgium)), a ready-to-use PCR mixture with added IAC, reagents for the magnetically based DNA extraction and the consumables for the DNA extraction and PCR analysis. To minimize any inter-laboratory variability (not attributable to the method performance), all the reagents necessary were supplied by the expert laboratory. At the participating laboratories, DNA extraction and PCR analysis were performed as described above. Real-time PCR at the participating laboratories was performed on an Mx3000 or Mx4000 real-time PCR system (Stratagene, La Jolla, CA). Each participant received a detailed protocol describing the DNA extraction, real-time PCR setup, real-time PCR run, and data analysis as well as a reporting form to record the obtained PCR results to return to the expert laboratory. The participants were also asked to return a file containing the real-time PCR runs. The participating laboratories were asked to use the negative template control (NTC), the process blank (a Salmonella-negative sample processed throughout the entire protocol) and the negative control to assign the threshold.
External validation Slices of pork filet buy Epacadostat were obtained from a local supermarket, and aseptically cut into pieces of 25 grams. Thirty-nine pieces of pork filet were inoculated by adding 0.5 ml of an appropriate Citarinostat dilution of Salmonella cells (see “”Preparation of inoculum”") onto the surface of the meat resulting in the following estimated inoculation levels for each of the three strains: one sample containing
approximately 1000 CFU/25 g, one sample containing approximately 100 CFU/25 g, three samples containing approximately 10 CFU/25 g, four samples containing approximately 5 CFU/25 g and four samples containing approximately 2 CFU/25 g. After inoculation, the meat the samples were placed in a stomacher bag and frozen at -18°C for 24 hours in order to induce a slight freezing stress to the Salmonella, resembling the stress during blast-cooling as used by the Danish abattoir. All 39 samples were analyzed by the real-time PCR method and the BAX Salmonella Detection System (BAX, DuPont Qualicon, Oxoid) using the following protocol. The 25-g sample was thawed overnight at 4°C, 225 ml pre-warmed BPW (37°C, Oxoid) was added, and the samples were then incubated at 37°C. After 10 hours, a 5-ml aliquot was drawn for DNA extraction and subsequent real-time PCR analysis as described above. The remaining BPW was further incubated at 37°C for an additional 8 hours, and samples were thereafter treated according to the manufacturer’s instructions.