The Experts below are selected from a list of 288 Experts worldwide ranked by ideXlab platform
Rudolf Amann - One of the best experts on this subject based on the ideXlab platform.
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optimization strategies for dna microarray based detection of bacteria with 16s rrna targeting Oligonucleotide Probes
Applied and Environmental Microbiology, 2003Co-Authors: Jorg Peplies, Frank Oliver Glockner, Rudolf AmannAbstract:The usability of the DNA microarray format for the specific detection of bacteria based on their 16S rRNA genes was systematically evaluated with a model system composed of six environmental strains and 20 Oligonucleotide Probes. Parameters such as secondary structures of the target molecules and steric hindrance were investigated to better understand the mechanisms underlying a microarray hybridization reaction, with focus on their influence on the specificity of hybridization. With adequate hybridization conditions, false-positive signals could be almost completely prevented, resulting in clear data interpretation. Among 199 potential nonspecific hybridization events, only 1 false-positive signal was observed, whereas false-negative results were more common (17 of 41). Subsequent parameter analysis revealed that this was mainly an effect of reduced accessibility of probe binding sites caused by the secondary structures of the target molecules. False-negative results could be prevented and the overall signal intensities could be adjusted by introducing a new optimization strategy called directed application of capture Oligonucleotides. The small number of false-positive signals in our data set is discussed, and a general optimization approach is suggested. Our results show that, compared to standard hybridization formats such as fluorescence in situ hybridization, a large number of Oligonucleotide Probes with different characteristics can be applied in parallel in a highly specific way without extensive experimental effort.
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In Situ Accessibility of Escherichia coli 23S rRNA to Fluorescently Labeled Oligonucleotide Probes
Applied and environmental microbiology, 2001Co-Authors: Bernhard M. Fuchs, Wolfgang Ludwig, Kazuaki Syutsubo, Rudolf AmannAbstract:CY3-labeled Oligonucleotide Probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows: class I, 3%; class II, 21%; class III, 35%; class IV, 18%; class V, 16%; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to Oligonucleotide Probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of Oligonucleotide Probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA of E. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future.
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fluorescence in situ hybridization fish with rrna targeted Oligonucleotide Probes
Methods in Microbiology, 2001Co-Authors: Jakob Pernthaler, Frank Oliver Glockner, Wilhelm Schonhuber, Rudolf AmannAbstract:Publisher Summary The chapter discusses the fluorescence in situ hybridization (FISH) with rRNA-targeted Oligonucleotide Probes, and describes probe designing and testing. Fluorescence FISH with rRNA-targeted Probes is a staining technique that allows phylogenetic identification of bacteria in mixed assemblages without prior cultivation by means of epifluorescence and confocal laser scanning microscopy, or by flow cytometry. FISH with Oligonucleotide Probes is for the purpose of bacterial identification that is to analyze bacterial community structure, and to follow the spatial and temporal dynamics of individual microbial populations in their habitat. Numerous aspects and applications of this method are discussed. FISH is successfully applied in freshwater, coastal, and offshore marine planktonic habitats, and in coastal sediments. It is shown that the fraction of bacteria detectable by FISH corresponds well with the abundance of active cells as determined by microautoradiography in coastal marine bacterioplankton.
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unlabeled helper Oligonucleotides increase the in situ accessibility to 16s rrna of fluorescently labeled Oligonucleotide Probes
Applied and Environmental Microbiology, 2000Co-Authors: Bernhard M. Fuchs, Frank Oliver Glockner, Jorg Wulf, Rudolf AmannAbstract:Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with Oligonucleotide Probes. Here, unlabeled Oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083T. The use of helpers enhanced the fluorescence signal of all six Probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled Oligonucleotide Probes available for E. coli. The temperature of dissociation and the mismatch discrimination of Probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with Oligonucleotide Probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.
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rRNA-Targeted Oligonucleotide Probes for the Identification of Genuine and Former Pseudomonads
Systematic and Applied Microbiology, 1996Co-Authors: Rudolf Amann, Wolfgang Ludwig, Renate Schulze, Stefan Spring, Edward R. B. Moore, Karl-heinz SchleiferAbstract:Summary The specificity of previously published Oligonucleotide Probes war re-evaluated based on the expanded 16S and 23S rRNA databases. Moreover, new genus- and (sub) species-specific 16S rRNA-targeted Oligonucleotide Probes were designed in a computer-assisted strategy using the PROBE DESIGN tool of the software package ARB. Probes for Pseudomonas stutzeri and for the genera Acidovorax, Comamonas and Hydrogenophaga were evaluated. Optimal hybridization conditions for the Probes have been determined. Using these conditions the Probes were tested for binding to 16S rRNA of reference cultures. Furthermore, fluorescently labeled derivatives of these Probes were tested for their applicability for whole cell hybridization. These rRNA-targeted Probes are the basis for high-resolution, rapid, automated microbial identification.
Karl-heinz Schleifer - One of the best experts on this subject based on the ideXlab platform.
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rRNA-Targeted Oligonucleotide Probes for the Identification of Genuine and Former Pseudomonads
Systematic and Applied Microbiology, 1996Co-Authors: Rudolf Amann, Wolfgang Ludwig, Renate Schulze, Stefan Spring, Edward R. B. Moore, Karl-heinz SchleiferAbstract:Summary The specificity of previously published Oligonucleotide Probes war re-evaluated based on the expanded 16S and 23S rRNA databases. Moreover, new genus- and (sub) species-specific 16S rRNA-targeted Oligonucleotide Probes were designed in a computer-assisted strategy using the PROBE DESIGN tool of the software package ARB. Probes for Pseudomonas stutzeri and for the genera Acidovorax, Comamonas and Hydrogenophaga were evaluated. Optimal hybridization conditions for the Probes have been determined. Using these conditions the Probes were tested for binding to 16S rRNA of reference cultures. Furthermore, fluorescently labeled derivatives of these Probes were tested for their applicability for whole cell hybridization. These rRNA-targeted Probes are the basis for high-resolution, rapid, automated microbial identification.
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Differentiation of Lactobacilli Occurring in Fermented Milk Products by Using Oligonucleotide Probes and Electrophoretic Protein Profiles
Systematic and Applied Microbiology, 1993Co-Authors: Christian Hertel, Wolfgang Ludwig, Karel Kersters, Karl-heinz SchleiferAbstract:Summary Rapid identification of Lactobacillus species that are used as starter organisms for fermented milk products was achieved by applying 23S rRNA-targeted Oligonucleotide Probes and numerical analysis of gel electrophoretic protein profiles. 23S rRNA genes of various lactobacilli were partially sequenced and complementary specific Oligonucleotide Probes were synthesised. Species specific Probes for Lactobacillus delbrueckii, L. helveticus , and L. paracasei and group specific Probes for L. caseil/L. rhamnosus and L. casei/L. paracasei/L. rhamnosus were designed. Moreover, the lactobacilli strains were examined by polyacrylamide gelelectrophoresis of sodium dodecylsulphate denatured whole-cell proteins. After numerical comparison of the resulting electrophoretic protein patterns, definite clusters could be determined, correlating well with the different species.
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23S rRNA-targeted Oligonucleotide Probes for the Rapid Identification of Meat Lactobacilli
Systematic and Applied Microbiology, 1991Co-Authors: Christian Hertel, Michaela Obst, Walter P. Hammes, Rudi F Vogel, Wolfgang Ludwig, Karl-heinz SchleiferAbstract:Summary A method for the rapid identification of Lactobacillus species that are used as starter organisms for fermented meat products was developed. Sequences of 23S rRNA specific for Lactobacillus curvatus, L. sake, and L. pentosus/L. plantarum were identified and complementary Oligonucleotides were synthesized. The specificity of the Probes was checked by dot blot and colony hybridizations. The Probes can be used for the analysis of mixed cultures. None of the other Gram-positive bacteria found in meat reacted with the specific Oligonucleotide Probes.
Wolfgang Ludwig - One of the best experts on this subject based on the ideXlab platform.
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Identification of Clostridium butyricum, Clostridium sporogenes and Clostridium tyrobutyricum by hybridization with 16S rRNA-targeted Oligonucleotide Probes
Systematic and Applied Microbiology, 2011Co-Authors: Stefan Knabel, Wolfgang Ludwig, Ralf Tatzel, Peter R. WallnöferAbstract:Summary A rapid method for the identification of bacteria, even usable in a routine laboratory was developed. After comparison with the sequences of 16S rRNA genes of a data-bank complementary Oligonucleotide Probes for Clostridium butyricum, Clostridium sporogenes and Clostridium tyrobutyricum were synthesized. The specificity of the Probes was tested by non-radioactive dot blot hybridization with nucleic acids, extracted from bacteria type strains typically occurring in milk and cheese and other related microorganisms. The Oligonucleotide Probes for Clostridium sporogenes and Clostridium tyrobutyricum showed specific signals whereas the probe for Clostridium butyricum could not distinguish between this organism and the closely related Clostridium kainantoi .
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In Situ Accessibility of Escherichia coli 23S rRNA to Fluorescently Labeled Oligonucleotide Probes
Applied and environmental microbiology, 2001Co-Authors: Bernhard M. Fuchs, Wolfgang Ludwig, Kazuaki Syutsubo, Rudolf AmannAbstract:CY3-labeled Oligonucleotide Probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows: class I, 3%; class II, 21%; class III, 35%; class IV, 18%; class V, 16%; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to Oligonucleotide Probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of Oligonucleotide Probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA of E. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future.
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rRNA-Targeted Oligonucleotide Probes for the Identification of Genuine and Former Pseudomonads
Systematic and Applied Microbiology, 1996Co-Authors: Rudolf Amann, Wolfgang Ludwig, Renate Schulze, Stefan Spring, Edward R. B. Moore, Karl-heinz SchleiferAbstract:Summary The specificity of previously published Oligonucleotide Probes war re-evaluated based on the expanded 16S and 23S rRNA databases. Moreover, new genus- and (sub) species-specific 16S rRNA-targeted Oligonucleotide Probes were designed in a computer-assisted strategy using the PROBE DESIGN tool of the software package ARB. Probes for Pseudomonas stutzeri and for the genera Acidovorax, Comamonas and Hydrogenophaga were evaluated. Optimal hybridization conditions for the Probes have been determined. Using these conditions the Probes were tested for binding to 16S rRNA of reference cultures. Furthermore, fluorescently labeled derivatives of these Probes were tested for their applicability for whole cell hybridization. These rRNA-targeted Probes are the basis for high-resolution, rapid, automated microbial identification.
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Differentiation of Lactobacilli Occurring in Fermented Milk Products by Using Oligonucleotide Probes and Electrophoretic Protein Profiles
Systematic and Applied Microbiology, 1993Co-Authors: Christian Hertel, Wolfgang Ludwig, Karel Kersters, Karl-heinz SchleiferAbstract:Summary Rapid identification of Lactobacillus species that are used as starter organisms for fermented milk products was achieved by applying 23S rRNA-targeted Oligonucleotide Probes and numerical analysis of gel electrophoretic protein profiles. 23S rRNA genes of various lactobacilli were partially sequenced and complementary specific Oligonucleotide Probes were synthesised. Species specific Probes for Lactobacillus delbrueckii, L. helveticus , and L. paracasei and group specific Probes for L. caseil/L. rhamnosus and L. casei/L. paracasei/L. rhamnosus were designed. Moreover, the lactobacilli strains were examined by polyacrylamide gelelectrophoresis of sodium dodecylsulphate denatured whole-cell proteins. After numerical comparison of the resulting electrophoretic protein patterns, definite clusters could be determined, correlating well with the different species.
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23S rRNA-targeted Oligonucleotide Probes for the Rapid Identification of Meat Lactobacilli
Systematic and Applied Microbiology, 1991Co-Authors: Christian Hertel, Michaela Obst, Walter P. Hammes, Rudi F Vogel, Wolfgang Ludwig, Karl-heinz SchleiferAbstract:Summary A method for the rapid identification of Lactobacillus species that are used as starter organisms for fermented meat products was developed. Sequences of 23S rRNA specific for Lactobacillus curvatus, L. sake, and L. pentosus/L. plantarum were identified and complementary Oligonucleotides were synthesized. The specificity of the Probes was checked by dot blot and colony hybridizations. The Probes can be used for the analysis of mixed cultures. None of the other Gram-positive bacteria found in meat reacted with the specific Oligonucleotide Probes.
Michael Wagner - One of the best experts on this subject based on the ideXlab platform.
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a straightforward dope double labeling of Oligonucleotide Probes fish fluorescence in situ hybridization method for simultaneous multicolor detection of six microbial populations
Applied and Environmental Microbiology, 2012Co-Authors: Faris Behnam, Michael Wagner, Andreas Vilcinskas, Kilian StoeckerAbstract:Fluorescence in situ hybridization (FISH) with rRNA-targeted Oligonucleotide Probes is an essential tool for the cultivationindependent identification of microbes within environmental and clinical samples. However, one of the major constraints of conventional FISH is the very limited number of different target organisms that can be detected simultaneously with standard epifluorescence or confocal laser scanning microscopy. Recently, this limitation has been overcome via an elegant approach termed combinatorial labeling and spectral imaging FISH (CLASI-FISH) (23). This technique, however, suffers compared to conventional FISH from an inherent loss in sensitivity and potential probe binding biases caused by the competition of two differentially labeled Oligonucleotide Probes for the same target site. Here we demonstrate that the application of multicolored, double-labeled Oligonucleotide Probes enables the simultaneous detection of up to six microbial target populations in a straightforward and robust manner with higher sensitivity and less bias. Thus, this newly developed technique should be an attractive option for all researchers interested in applying conventional FISH methods for the study of microbial communities.
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double labeling of Oligonucleotide Probes for fluorescence in situ hybridization dope fish improves signal intensity and increases rrna accessibility
Applied and Environmental Microbiology, 2010Co-Authors: Kilian Stoecker, Christiane Dorninger, Holger Daims, Michael WagnerAbstract:Fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted Oligonucleotide Probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled Oligonucleotide Probes with 5'-, 3'-doubly labeled Probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled Probes strongly increase in situ accessibility of rRNA target sites and thus provide more flexibility for probe design.
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probebase an online resource for rrna targeted Oligonucleotide Probes
Nucleic Acids Research, 2003Co-Authors: Matthias Horn, Michael WagnerAbstract:Ribosomal RNA-(rRNA)-targeted Oligonucleotide Probes are widely used for culture-independent identification of microorganisms in environmental and clinical samples. ProbeBase is a comprehensive database containing more than 700 published rRNA-targeted Oligonucleotide probe sequences (status August 2002) with supporting bibliographic and biological annotation that can be accessed through the internet at http://www.probebase.net. Each Oligonucleotide probe entry contains information on target organisms, target molecule (small- or large-subunit rRNA) and position, G+C content, predicted melting temperature, molecular weight, necessity of competitor Probes, and the reference that originally described the Oligonucleotide probe, including a link to the respective abstract at PubMed. In addition, Probes successfully used for fluorescence in situ hybridization (FISH) are highlighted and the recommended hybridization conditions are listed. ProbeBase also offers difference alignments for 16S rRNA-targeted Probes by using the probe match tool of the ARB software and the latest small-subunit rRNA ARB database (release June 2002). The option to directly submit probe sequences to the probe match tool of the Ribosomal Database Project II (RDP-II) further allows one to extract supplementary information on probe specificities. The two main features of probeBase, ‘search probeBase’ and ‘find probe set’, help researchers to find suitable, published Oligonucleotide Probes for microorganisms of interest or for rRNA gene sequences submitted by the user. Furthermore, the ‘search target site’ option provides guidance for the development of new FISH Probes.
Christian Hertel - One of the best experts on this subject based on the ideXlab platform.
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Differentiation of Lactobacilli Occurring in Fermented Milk Products by Using Oligonucleotide Probes and Electrophoretic Protein Profiles
Systematic and Applied Microbiology, 1993Co-Authors: Christian Hertel, Wolfgang Ludwig, Karel Kersters, Karl-heinz SchleiferAbstract:Summary Rapid identification of Lactobacillus species that are used as starter organisms for fermented milk products was achieved by applying 23S rRNA-targeted Oligonucleotide Probes and numerical analysis of gel electrophoretic protein profiles. 23S rRNA genes of various lactobacilli were partially sequenced and complementary specific Oligonucleotide Probes were synthesised. Species specific Probes for Lactobacillus delbrueckii, L. helveticus , and L. paracasei and group specific Probes for L. caseil/L. rhamnosus and L. casei/L. paracasei/L. rhamnosus were designed. Moreover, the lactobacilli strains were examined by polyacrylamide gelelectrophoresis of sodium dodecylsulphate denatured whole-cell proteins. After numerical comparison of the resulting electrophoretic protein patterns, definite clusters could be determined, correlating well with the different species.
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23S rRNA-targeted Oligonucleotide Probes for the Rapid Identification of Meat Lactobacilli
Systematic and Applied Microbiology, 1991Co-Authors: Christian Hertel, Michaela Obst, Walter P. Hammes, Rudi F Vogel, Wolfgang Ludwig, Karl-heinz SchleiferAbstract:Summary A method for the rapid identification of Lactobacillus species that are used as starter organisms for fermented meat products was developed. Sequences of 23S rRNA specific for Lactobacillus curvatus, L. sake, and L. pentosus/L. plantarum were identified and complementary Oligonucleotides were synthesized. The specificity of the Probes was checked by dot blot and colony hybridizations. The Probes can be used for the analysis of mixed cultures. None of the other Gram-positive bacteria found in meat reacted with the specific Oligonucleotide Probes.
