title: Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis.
abstract: A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16-18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.
authors.affiliation: Southern African Young Water Professionals, East Rand Water Care Company, Kempton Park, South Africa E-mail: nicovanblerk@erwat.co.za
authors.forename: G N
authors.initials: GN
authors.lastname: van Blerk
authors.forename: L
authors.initials: L
authors.lastname: Leibach
authors.forename: A
authors.initials: A
authors.lastname: Mabunda
authors.forename: A
authors.initials: A
authors.lastname: Chapman
authors.forename: D
authors.initials: D
authors.lastname: Louw
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods; D009691:Nucleic Acid Denaturation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012475:Salmonella / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D004269:DNA, Bacterial