Schematic real-time PCR results for the second step reaction. Representative real-time PCR results as established by the second step multiplex reaction (described in Materials and Methods). The plots show average normalised linear amplification of representative samples shown for demonstration of typical results obtained from S. Typhimurium, S. Enteritidis and other Salmonella samples. With DNA from S. Typhimurium strains, only fliC-specific, HEX-labelled molecular beacons hybridise to the amplicons, generating pink fluorescence, whereas the prot6E-specific, TET-labelled molecular beacons retain their stem-and-loop structure and cannot produce an orange fluorescent signal. With DNA from S. Enteritidis strains, the prot6E-specific, TET-labelled molecular beacons hybridise to their target amplicons and produce an orange fluorescent signal, whereas the fliC-specific, HEX-labelled molecular beacons remain dark. With DNA from other Salmonella serotypes, no target amplicons are detected and both molecular beacons remain dark. The dashed line on the plots represents the normalised threshold for detection of fluorescence, the baseline above which fluorescence increases significantly on amplification and detection of the target sequence.