Molecular beacon-based real-time PCR detection of primary isolates of Salmonella Typhimurium and Salmonella Enteritidis in environmental and clinical samples

Abstract

Background: A fast and simple two-step multiplex real-time PCR assay has been developed to replace the traditional, laborious Salmonella serotyping procedure. Molecular beacons were incorporated into the assay as probes for target DNA. Target sequences were regions of the invA, prot6E and fliC genes specific for Salmonella spp. Salmonella Enteritidis and Salmonella Typhimurium, respectively, the two most clinically relevant serotypes. An internal amplification positive control was included in the experiment to ensure the optimal functioning of the PCR and detect possible PCR inhibition. Three sets of primers were used for the amplification of the target sequences. The results were compared to those of the Kauffmann-White antigenic classification scheme.

Results: The assay was 100% sensitive and specific, correctly identifying all 44 Salmonella strains, all 21 samples of S. Enteritidis and all 17 samples of S. Typhimurium tested in this work. Therefore, the entire experiment had specificity and sensitivity of 100%. The detection limit was down to 10 copies of DNA target per 25 microl reaction.

Conclusion: The assay can amplify and analyse a large number of samples in approximately 8 hours, compared to the 4 to 5 days conventional identification takes, and is thus considered a very promising method for detecting the two major serotypes of Salmonella quickly and accurately from clinical and environmental samples.

Figure 1

Thermal denaturation profiles of the molecular beacons. Thermal denaturation profiles of the molecular beacons used in this study as established by melting curve analysis (described in Materials and Methods). The figure shows normalised fluoresence thermal transitions of molecular beacon plotted in pink circles and beacon-target complexes plotted in blue squares.

Figure 2

Standard curves for targets invA, fliC and prot6E. Standard curves for molecular beacon-based real-time PCR detection of targets invA, fliC and prot6E. The plots illustrate the relationship of known number of target DNA copies per reaction to the threshold cycle of detection (C_T) for each molecular beacon reaction. The C_T is directly proportional to the log of the input copy equivalents, as demonstrated by the standard curves generated.

Figure 3

Schematic real-time PCR results for the first step reaction. Representative real-time PCR results as established by the first 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 Salmonella and non-Salmonella bacteria. With DNA from non-Salmonella bacterial samples, only the IAC-specific, ROX-labelled molecular beacons hybridise to the IAC amplicons, generating violet fluorescence, whereas the invA-specific, FAM-labelled molecular beacons retain their stem-and-loop structure and cannot produce a green fluorescent signal. With DNA from Salmonella samples, both molecular beacons hybridise to their respective target amplicons and generate both green and violet fluorescence. 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.

Figure 4

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.