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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

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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 × 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.

La spécificité ultime d’essais de détection moléculaires de pathogènes repose sur la planification des amorces et des sondes. Leur hybridation à des séquences d’ADN trouvées uniquement chez les pathogènes peut être obtenue en concevant des amorces et des sondes complémentaires à des gènes de virulence spécifiques aux pathogènes. Cette étude a évalué la potentiel de détection et d’énumération de la PCR en temps réel (qPCR) et de l’hybridation in situ fluorescente (FISH) de pathogènes de l’eau sélectionnés, ainsi que leur applicabilité dans un contexte de surveillance. Les limites de détection calculées de la qPCR étaient de 150 copies du gène tir (codant le récepteur de l’intimine) chez Escherichia coli O157:H7 et de 2 × 103 copies du gène invA (codant la protéine de membrane interne envahissantes) chez Salmonella enterica sérovar Typhimurium. Les limites de détection étaient cependant au moins 100 fois moins sensibles dans des extraits d’eaux usées, en partie à cause des effets inhibiteurs des eaux usées elles-mêmes. Les signaux fluorescents de cellules cibles entières hybridées étaient sous la limite de détection du FISH. Alors que ce travail de recherche démontre le potentiel de détection de la qPCR, il met en évidence le besoin d’obtenir des ensembles d’amorces et de sondes hautement fiables pour la PCR et la FISH, ainsi que le besoin d’obtenir une amplification de signal plus élevée en FISH pour détecter l’ADN de gènes à simple copie présents dans des échantillons environnementaux.

Document Type: Research Article

Publication date: March 1, 2010

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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