Detection of Salmonella spp. in Recreational Waters Using a Real-Time PCR Assay

The U.S. Environmental Protection Agency water quality guidelines for assessing bacteriological recreational water quality is currently based on Escherichia coli and enterococci counts as indicators of the presence of fecal pollution. Although these indicator organisms have been widely used, detection of specific pathogens rather than their surrogates may be preferable in the future for assessing health risks. This necessitates development of rapid, sensitive and reliable methods for pathogen detection in environmental waters. A quantitative PCR-based assay for detecting Salmonella spp., an important enteric pathogen, in recreational water and beach sediment samples was developed in the present study. The assay is based on the amplification of a 172 bp fragment in the invA gene which is quantified using a molecular beacon specific for Salmonella. The assay tests for the presence of Salmonella in 50 ml of environmental water samples and relies on the use of a 6 hr culture enrichment procedure. It has a lower detection limit of one CFU per mL and works within the 1 to 10 6 CFU per mL concentration range tested thus far. Due to the possible presence of PCR inhibitors in environmental samples, an internal amplification control, using the same primers but a different molecular beacon target sequence, was created to be included in each amplification reaction to distinguish true from false negative results. Preliminary studies reveal the presence of Salmonella in several coastal water samples along the northern Gulf of Mexico