The Experts below are selected from a list of 16134 Experts worldwide ranked by ideXlab platform
Henrik Christensen - One of the best experts on this subject based on the ideXlab platform.
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specific detection of pasteurella multocida in chickens with fowl cholera and in pig lung tissues using fluorescent rrna in situ hybridization
Journal of Clinical Microbiology, 2001Co-Authors: P G Mbuthia, Henrik Christensen, Mette Boye, Kamille D Petersen, Magne Bisgaard, P N Nyaga, John Elmerdahl OlsenAbstract:A Pasteurella multocida species-specific Oligonucleotide Probe, pmhyb449, targeting 16S rRNA was designed and evaluated by whole-cell hybridization against 22 selected reference strains in animal tissues. It differentiated P. multocida from other bacterial species of the families Pasteurellaceae and Enterobacteriaceae and also from divergent species of the order Cytophagales (except biovar 2 strains of Pasteurella avium and Pasteurella canis, which have high 16S rRNA similarity to P. multocida). The potential of the Probe for specific identification and differentiation of P. multocida was further detected in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues P. multocida cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood vessels (wall and lumen). In pig lung, postmortem-injected P. multocida was detected in the alveoli (lumen and wall), and in both animals the bacterial cells were seen in the bronchi. The results showed that with the Oligonucleotide Probe pmhyb449, fluorescent in situ hybridization is a suitable and fast method for specific detection of P. multocida in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to P. multocida.
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counting and size classification of active soil bacteria by fluorescence in situ hybridization with an rrna Oligonucleotide Probe
Applied and Environmental Microbiology, 1999Co-Authors: Henrik Christensen, Michael Adsetts Edberg Hansen, Jan Tind SorensenAbstract:A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled Oligonucleotide Probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA Probe, EUB338, for the domain Bacteria was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 micrometer). A nonsense Probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of Probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the Probes and for binding by the rhodamine part of the Probe, control experiments in which excess unlabelled Probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 x 10(8) active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 x 10(8) active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 micrometer), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4', 6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic Probes and for automatic counting of soil bacteria.
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counting and size classification of active soil bacteria by fluorescence in situ hybridization with an rrna Oligonucleotide Probe
Applied and Environmental Microbiology, 1999Co-Authors: Henrik Christensen, Michael Hansen, Jan Tind SorensenAbstract:A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled Oligonucleotide Probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA Probe, EUB338, for the domain Bacteria was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 μm). A nonsense Probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of Probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the Probes and for binding by the rhodamine part of the Probe, control experiments in which excess unlabelled Probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 × 108 active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 × 108 active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 μm), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4′,6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic Probes and for automatic counting of soil bacteria.
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evaluation of a fluorescence labelled Oligonucleotide Probe targeting 23s rrna for in situ detection of salmonella serovars in paraffin embedded tissue sections and their rapid identification in bacterial smears
Journal of Clinical Microbiology, 1997Co-Authors: Steen Nordentoft, Henrik Christensen, Henrik Caspar WegenerAbstract:A method for the detection of Salmonella based on fluorescence in situ hybridization (FISH) has been developed and applied for the direct detection of Salmonella in pure cultures and in formalin-fixed, paraffin-embedded tissue sections. On the basis of the 23S rRNA gene sequences representing all of the S. enterica subspecies and S. bongori, an 18-mer Oligonucleotide Probe was selected. The specificity of the Probe was tested by in situ hybridization to bacterial cell smears of pure cultures. Forty-nine of 55 tested Salmonella serovars belonging to subspecies I, II, IIIb, IV, and VI hybridized with the Probe. The Probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The Probe was tested with sections of formalin-fixed, paraffin-embedded tissue from experimentally infected mice or from animals with a history of clinical salmonellosis. In these tissue sections the Probe hybridized specifically to Salmonella serovars, allowing for the detection of single bacterial cells. The development of a fluorescence-labelled specific Oligonucleotide Probe makes the FISH technique a promising tool for the rapid identification of S. enterica in bacterial smears, as well as for the detection of S. enterica in histological tissue sections.
Jan Tind Sorensen - One of the best experts on this subject based on the ideXlab platform.
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counting and size classification of active soil bacteria by fluorescence in situ hybridization with an rrna Oligonucleotide Probe
Applied and Environmental Microbiology, 1999Co-Authors: Henrik Christensen, Michael Hansen, Jan Tind SorensenAbstract:A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled Oligonucleotide Probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA Probe, EUB338, for the domain Bacteria was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 μm). A nonsense Probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of Probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the Probes and for binding by the rhodamine part of the Probe, control experiments in which excess unlabelled Probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 × 108 active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 × 108 active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 μm), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4′,6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic Probes and for automatic counting of soil bacteria.
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counting and size classification of active soil bacteria by fluorescence in situ hybridization with an rrna Oligonucleotide Probe
Applied and Environmental Microbiology, 1999Co-Authors: Henrik Christensen, Michael Adsetts Edberg Hansen, Jan Tind SorensenAbstract:A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled Oligonucleotide Probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA Probe, EUB338, for the domain Bacteria was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 micrometer). A nonsense Probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of Probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the Probes and for binding by the rhodamine part of the Probe, control experiments in which excess unlabelled Probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 x 10(8) active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 x 10(8) active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 micrometer), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4', 6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic Probes and for automatic counting of soil bacteria.
Chan-gi Pack - One of the best experts on this subject based on the ideXlab platform.
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poly a sensing of hybridization sensitive fluorescent Oligonucleotide Probe characterized by fluorescence correlation methods
International Journal of Molecular Sciences, 2021Co-Authors: Bjorn Paulson, Yeonhee Shin, Akimitsu Okamoto, Jun Ki Kim, Chan-gi PackAbstract:Ribonucleic acid (RNA) plays an important role in many cellular processes. Thus, visualizing and quantifying the molecular dynamics of RNA directly in living cells is essential to uncovering their role in RNA metabolism. Among the wide variety of fluorescent Probes available for RNA visualization, exciton-controlled hybridization-sensitive fluorescent Oligonucleotide (ECHO) Probes are useful because of their low fluorescence background. In this study, we apply fluorescence correlation methods to ECHO Probes targeting the poly(A) tail of mRNA. In this way, we demonstrate not only the visualization but also the quantification of the interaction between the Probe and the target, as well as of the change in the fluorescence brightness and the diffusion coefficient caused by the binding. In particular, the uptake of ECHO Probes to detect mRNA is demonstrated in HeLa cells. These results are expected to provide new insights that help us better understand the metabolism of intracellular mRNA.
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Poly(A)+ Sensing of Hybridization-Sensitive Fluorescent Oligonucleotide Probe Characterized by Fluorescence Correlation Methods
'MDPI AG', 2021Co-Authors: Bjorn Paulson, Yeonhee Shin, Akimitsu Okamoto, Jun Ki Kim, Chan-gi PackAbstract:Ribonucleic acid (RNA) plays an important role in many cellular processes. Thus, visualizing and quantifying the molecular dynamics of RNA directly in living cells is essential to uncovering their role in RNA metabolism. Among the wide variety of fluorescent Probes available for RNA visualization, exciton-controlled hybridization-sensitive fluorescent Oligonucleotide (ECHO) Probes are useful because of their low fluorescence background. In this study, we apply fluorescence correlation methods to ECHO Probes targeting the poly(A) tail of mRNA. In this way, we demonstrate not only the visualization but also the quantification of the interaction between the Probe and the target, as well as of the change in the fluorescence brightness and the diffusion coefficient caused by the binding. In particular, the uptake of ECHO Probes to detect mRNA is demonstrated in HeLa cells. These results are expected to provide new insights that help us better understand the metabolism of intracellular mRNA
Elisabeth R. M. Tillier - One of the best experts on this subject based on the ideXlab platform.
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design and verification of a pangenome microarray Oligonucleotide Probe set for dehalococcoides spp
Applied and Environmental Microbiology, 2011Co-Authors: Laura A. Hug, Maryam Salehi, Paulo A. S. Nuin, Elisabeth R. M. TillierAbstract:Dehalococcoides spp. are an industrially relevant group of Chloroflexi bacteria capable of reductively dechlorinating contaminants in groundwater environments. Existing Dehalococcoides genomes revealed a high level of sequence identity within this group, including 98 to 100% 16S rRNA sequence identity between strains with diverse substrate specificities. Common molecular techniques for identification of microbial populations are often not applicable for distinguishing Dehalococcoides strains. Here we describe an Oligonucleotide microarray Probe set designed based on clustered Dehalococcoides genes from five different sources (strain DET195, CBDB1, BAV1, and VS genomes and the KB-1 metagenome). This "pangenome" Probe set provides coverage of core Dehalococcoides genes as well as strain-specific genes while optimizing the potential for hybridization to closely related, previously unknown Dehalococcoides strains. The pangenome Probe set was compared to Probe sets designed independently for each of the five Dehalococcoides strains. The pangenome Probe set demonstrated better predictability and higher detection of Dehalococcoides genes than strain-specific Probe sets on nontarget strains with <99% average nucleotide identity. An in silico analysis of the expected Probe hybridization against the recently released Dehalococcoides strain GT genome and additional KB-1 metagenome sequence data indicated that the pangenome Probe set performs more robustly than the combined strain-specific Probe sets in the detection of genes not included in the original design. The pangenome Probe set represents a highly specific, universal tool for the detection and characterization of Dehalococcoides from contaminated sites. It has the potential to become a common platform for Dehalococcoides-focused research, allowing meaningful comparisons between microarray experiments regardless of the strain examined.
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Design and verification of a pangenome microarray Oligonucleotide Probe set for Dehalococcoides spp.
Applied and environmental microbiology, 2011Co-Authors: Laura A. Hug, Maryam Salehi, Paulo A. S. Nuin, Elisabeth R. M. TillierAbstract:Dehalococcoides spp. are an industrially relevant group of Chloroflexi bacteria capable of reductively dechlorinating contaminants in groundwater environments. Existing Dehalococcoides genomes revealed a high level of sequence identity within this group, including 98 to 100% 16S rRNA sequence identity between strains with diverse substrate specificities. Common molecular techniques for identification of microbial populations are often not applicable for distinguishing Dehalococcoides strains. Here we describe an Oligonucleotide microarray Probe set designed based on clustered Dehalococcoides genes from five different sources (strain DET195, CBDB1, BAV1, and VS genomes and the KB-1 metagenome). This "pangenome" Probe set provides coverage of core Dehalococcoides genes as well as strain-specific genes while optimizing the potential for hybridization to closely related, previously unknown Dehalococcoides strains. The pangenome Probe set was compared to Probe sets designed independently for each of the five Dehalococcoides strains. The pangenome Probe set demonstrated better predictability and higher detection of Dehalococcoides genes than strain-specific Probe sets on nontarget strains with
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a fast and flexible approach to Oligonucleotide Probe design for genomes and gene families
Bioinformatics, 2007Co-Authors: Shengzhong Feng, Elisabeth R. M. TillierAbstract:Motivation: With hundreds of completely sequenced microbial genomes available, and advancements in DNA microarray technology, the detection of genes in microbial communities consisting of hundreds of thousands of sequences may be possible. The existing strategies developed for DNA Probe design, geared toward identifying specific sequences, are not suitable due to the lack of coverage, flexibility and efficiency necessary for applications in metagenomics. Methods: ProDesign is a tool developed for the selection of Oligonucleotide Probes to detect members of gene families present in environmental samples. Gene family-specific Probe sequences are generated based on specific and shared words, which are found with the spaced seed hashing algorithm. To detect more sequences, those sharing some common words are re-clustered into new families, then Probes specific for the new families are generated. Results: The program is very flexible in that it can be used for designing Probes for detecting many genes families simultaneously and specifically in one or more genomes. Neither the length nor the melting temperature of the Probes needs to be predefined. We have found that ProDesign provides more flexibility, coverage and speed than other software programs used in the selection of Probes for genomic and gene family arrays. Availability: ProDesign is licensed free of charge to academic users. ProDesign and Supplementary Material can be obtained by contacting the authors. A web server for ProDesign is available at http://www.uhnresearch.ca/labs/tillier/ProDesign/ProDesign.html Contact:e.tillier@utoronto.ca or fsz@ncic.ac.cn Supplementary information: Supplementary data are available at Bioinformatics online.
John Quackenbush - One of the best experts on this subject based on the ideXlab platform.
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assessing unmodified 70 mer Oligonucleotide Probe performance on glass slide microarrays
Genome Biology, 2003Co-Authors: Hongying Wang, Renae L Malek, Anne E Kwitek, Andrew S Greene, Babak Behbahani, Bryan Frank, John QuackenbushAbstract:Background: Long Oligonucleotide microarrays are potentially more cost- and managementefficient than cDNA microarrays, but there is little information on the relative performance of these two Probe types. The feasibility of using unmodified Oligonucleotides to accurately measure changes in gene expression is also unclear. Results: Unmodified sense and antisense 70-mer Oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two Probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with Probe type but depended on the extent of UV irradiation. To determine the effect of Oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 M). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between Oligonucleotide and cDNA Probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two Probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR. Conclusions: Microarrays of UV cross-linked unmodified Oligonucleotides provided sensitive and specific measurements for most of the genes studied.
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assessing unmodified 70 mer Oligonucleotide Probe performance on glass slide microarrays
Genome Biology, 2003Co-Authors: Hongying Wang, Renae L Malek, Anne E Kwitek, Andrew S Greene, Babak Behbahani, John Quackenbush, Truong V Luu, Bryan C Frank, Norman H LeeAbstract:Long Oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two Probe types. The feasibility of using unmodified Oligonucleotides to accurately measure changes in gene expression is also unclear. Unmodified sense and antisense 70-mer Oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two Probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with Probe type but depended on the extent of UV irradiation. To determine the effect of Oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 μm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between Oligonucleotide and cDNA Probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two Probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR. Microarrays of UV cross-linked unmodified Oligonucleotides provided sensitive and specific measurements for most of the genes studied.
