An homogeneous fluorescence polymerase chain reaction assay to identify Salmonella

We have developed a semiquantitative homogeneous fluorescence assay that combines polymerase chain reaction (PCR) amplification with direct fluorescence detection (HF-PCR). The assay eliminates the need to perform gel electrophoresis on test samples. Using a set of Salmonella-specific primers, this system was used to verify suspect colonies from culture plates as Salmonella. The fluorescence signal is generated by a nucleic acid dye, YO-PRO-1, that is included in the amplification reaction. This homogeneous PCR assay was used to test 84 Salmonella strains picked from selective culture plates. All data indicated positive results when compared with 17 non-Salmonella strains (in general, Citrobacter, Hafnia, Proteus, and Escherichia). The HF-PCR assay is a sensitive, simple, accurate, and reproducible method that correlates well with size-exclusion high-performance liquid chromatography and gel electrophoresis techniques as a means to monitor PCR-mediated DNA amplification. This assay can confirm suspect colonies within 2.5 h.