title: The utility and application of real-time PCR and FISH in the detection of single-copy gene targets in Escherichia coli O157:H7 and Salmonella Typhimurium.
abstract: The ultimate specificity in molecular-based assays for pathogen detection relies on the design of the primers and probes. Their ability to hybridize to DNA sequences found only in pathogens can be realized by designing primers and probes that are complementary to pathogen-specific virulence genes. This study evaluates the detection and enumeration strengths of real-time PCR (qPCR) and fluorescent in situ hybridization (FISH) for selected waterborne pathogens and their ultimate applicability within a monitoring framework. Detection limits calculated in the qPCR assay were 150 tir (intimin protein receptor) gene copies for Escherichia coli O157:H7 and 2 x 103 invA (inner membrane invasive protein) gene copies for Salmonella enterica serovar Typhimurium. Detection limits were, however, at least 100-fold less sensitive in wastewater extracts, partly because of the inhibitory effect of the wastewater itself. Fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay. While this research demonstrates the potential detection strength of qPCR, it highlights the need for strong dependable primer and probe sets among PCR and FISH methodologies as well as the need for further signal amplification with DNA-targeted FISH for single-copy gene targets within environmental samples.
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, ON, Canada.
authors.forename: Merriam
authors.initials: M
authors.lastname: Haffar
authors.forename: Kimberley A
authors.initials: KA
authors.lastname: Gilbride
mesh_terms: D017931:DNA Primers / Q000592:standards; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification; D017404:In Situ Hybridization, Fluorescence; D016133:Polymerase Chain Reaction; D012486:Salmonella typhimurium / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014865:Waste Disposal, Fluid; D014871:Water Microbiology
chemical_list: D017931:DNA Primers