Vibrio vulnificus is an opportunistic human pathogen that may cause gastroenteritis, severe necrotizing soft-tissue infections and primary septicaemia, with a high lethality rate. Illness is associated to ingestion of seafood or to the exposure of contaminated water. The aim of this work was to determine the occurrence of V. vulnificus in water and seafood samples from a coastal area near the Mediterranean (Valencia, Spain). A TaqMan probe-based real-time PCR assay was optimised and applied to 22 sea water, 42 raw sewage and 40 seafood samples. Results were compared with those obtained for culture isolation. The detection level of the PCR assay was 10 CFU g⁻¹ in inoculated samples. Seven seawater, four shellfish and six wastewater samples were positive by real time PCR. V. vulnificus was isolated from two oyster, three sea water and two wastewater samples. All the strains were obtained after 20 h enrichment, except for wastewater strains, which were isolated directly from the sample. To our knowledge, this is the first report on the isolation of V. vulnificus from sewage in Spain. Our results about the presence of V. vulnificus in food and environmental samples are strong enough to consider that the organism may represent a human health hazard in our geographical area.

Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the β-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower C t values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier C t values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.