The utility and application of real-time PCR and FISH in the detection of single-copy gene targets inEscherichia coliO157:H7 andSalmonellaTyphimurium

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 × 103invA (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.

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Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents

Microorganisms ◽

10.3390/microorganisms9081610 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1610

Author(s):

Christian Klotz ◽

Elke Radam ◽

Sebastian Rausch ◽

Petra Gosten-Heinrich ◽

Toni Aebischer

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Gastrointestinal Disease ◽

Single Copy ◽

Marker Genes ◽

Small Rodent ◽

Analytical Sensitivity ◽

Single Copy Gene ◽

Wild Rodents ◽

Copy Gene

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

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Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

Journal of Periodontal Research ◽

10.1034/j.1600-0765.2003.00684.x ◽

2003 ◽

Vol 38 (5) ◽

pp. 518-524 ◽

Cited By ~ 33

Author(s):

Juan M Morillo ◽

Laura Lau ◽

Mariano Sanz ◽

David Herrera ◽

Augusto Silva

Keyword(s):

Real Time ◽

Porphyromonas Gingivalis ◽

Real Time Pcr ◽

Gene Sequence ◽

Single Copy ◽

Actinobacillus Actinomycetemcomitans ◽

Single Copy Gene ◽

Quantitative Real Time Pcr ◽

Copy Gene

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Detection and quantification of five major periodontal pathogens by single copy gene-based real-time PCR

Innate Immunity ◽

10.1177/1753425908101920 ◽

2009 ◽

Vol 15 (4) ◽

pp. 195-204 ◽

Cited By ~ 53

Author(s):

Kati Hyvärinen ◽

Saara Laitinen ◽

Susanna Paju ◽

Anne Hakala ◽

Liisa Suominen-Taipale ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Single Copy ◽

Single Copy Gene ◽

Periodontal Pathogens ◽

Copy Gene ◽

Detection And Quantification

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Detection of Bifidobacterium animalis subsp. lactis (Bb12) in the Intestine after Feeding of Sows and Their Piglets

Applied and Environmental Microbiology ◽

10.1128/aem.00309-08 ◽

2008 ◽

Vol 74 (20) ◽

pp. 6338-6347 ◽

Cited By ~ 24

Author(s):

Gloria Solano-Aguilar ◽

Harry Dawson ◽

Marta Restrepo ◽

Kate Andrews ◽

Bryan Vinyard ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Protein Biosynthesis ◽

Bifidobacterium Longum ◽

Elongation Factor ◽

Single Copy ◽

Bifidobacterium Animalis ◽

Gene Copies ◽

Intestinal Contents ◽

High Level

ABSTRACT A real-time PCR method has been developed to distinguish Bifidobacterium animalis subspecies in the gastrointestinal tracts of pigs. Identification of a highly conserved single-copy tuf gene encoding the elongation factor Tu involved in bacterial protein biosynthesis was used as a marker to differentiate homologous Bifidobacterium animalis subsp. lactis (strain Bb12) from Bifidobacterium animalis subsp. animalis, as well as Bifidobacterium suis, Bifidobacterium breve, Bifidobacterium longum, several species of Lactobacillus, and Enterococcus faecium. Real-time PCR detection of serially diluted DNA extracted from a pure culture of Bb12 was linear for bacterial numbers ranging from 10 to 10,000 tuf gene copies per PCR (r 2 = 0.99). Relative differences in Bb12 bacterial numbers in pigs fed daily with Bb12 were determined after detection of Bb12 tuf gene copies in DNA extracted from the intestinal contents. Piglets treated with Bb12 immediately after birth maintained a high level of Bb12 in their large intestines with continuous daily administration of Bb12. Piglets born to Bb12-treated sows during the last third of their gestation and also treated with Bb12 at birth (T/T group) had a higher number of Bb12 organisms per gram of intestinal contents compared to placebo-treated piglets born to placebo-treated sows (C/C group), Bb12-treated sows (T/C group), or piglets born to placebo sows but treated with Bb12 immediately after birth (C/T group). In addition, there was a significant increase in gene expression for Toll-like receptor 9 (TLR9) in piglets from the T/T group, with no change in TLR2 and TLR4. These findings suggest that the tuf gene represents a specific and functional marker for detecting Bifidobacterium animalis subsp. lactis strain Bb12 within the microbiota of the intestine.

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Comparative detection and enumeration strengths of quantitative real-time PCR & FISH for waterborne bacterial pathogens in municipal wastewater

10.32920/ryerson.14655012.v1 ◽

2021 ◽

Author(s):

Merriam Haffar

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Municipal Wastewater ◽

Bacterial Pathogens ◽

Virulence Gene ◽

Target Cells ◽

Rt Pcr ◽

Hybridization Probes ◽

Gene Copies ◽

Pure Cultures

This study comparatively evaluates the detection and enumeration strengths of Real-Time PCR (RT PCR) and FISH, for selected bacterial pathogens in municipal wastewater. Both assays were performed using three primer and probe sets complementary to the same chromosomal virulence gene sequences. Primer & probe specificity was confirmed with DNA & fixed cells from pure bacterial cultures as well as seeded wastewater samples. Detection limits calculated for the RT PCR assay were 25 to 3030 tir gene copies for Escherichia coli O157:H7 and 3 x 10⁴ to 293 x10⁷ invA gene copies for Salmonella enterica, using pure cultures and seeded wasewater samples, respectively. In spite of the confirmed specificity of the DNA hybridization probes with target nucleic acids, fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay, and consequently were not quantified. This research demonstrates both the utility of RT PCR in detecting bacterial pathogens and the need for further optimization with DNA-targeted FISH, using environmental samples.

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