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David A Stahl - One of the best experts on this subject based on the ideXlab platform.
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DNA Microarray detection of nitrifying bacterial 16s rrna in wastewater treatment plant samples
Water Research, 2005Co-Authors: John J Kelly, Hidetoshi Urakawa, Slil Siripong, John L Mccormack, Lori R Janus, Said El Fantroussi, Peter A Noble, Laura Sappelsa, Bruce E Rittmann, David A StahlAbstract:A small scale DNA Microarray containing a set of oligonucleotide probes targeting the 16S rRNAs of several groups of nitrifying bacteria was developed for the monitoring of wastewater treatment plant samples. The Microarray was tested using reference rRNAs from pure cultures of nitrifying bacteria. Characterization of samples collected from an industrial wastewater treatment facility demonstrated that nitrifying bacteria could be detected directly by Microarray hybridization without the need for PCR amplification. Specifically, the Microarray detected Nitrosomonas spp. but did not detect Nitrobacter. The specificity and sensitivity of direct detection was evaluated using on-chip dissociation analysis, and by two independent analyses--an established membrane hybridization format and terminal restriction fragment length polymorphism fingerprinting (T-RFLP). The latter two analyses also revealed Nitrospira and Nitrobacter to be contributing populations in the treatment plant samples. The application of DNA Microarrays to wastewater treatment systems, which has been demonstrated in the current work, should offer improved monitoring capabilities and process control for treatment systems, which are susceptible to periodic failures.
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DNA Microarray detection of nitrifying bacterial 16s rrna in wastewater treatment plant samples
Water Research, 2005Co-Authors: John J Kelly, Hidetoshi Urakawa, Slil Siripong, John L Mccormack, Lori R Janus, Said El Fantroussi, Peter A Noble, Laura Sappelsa, Bruce E Rittmann, David A StahlAbstract:A small scale DNA Microarray containing a set of oligonucleotide probes targeting the 16S rRNAs of several groups of nitrifying bacteria was developed for the monitoring of wastewater treatment plant samples. The Microarray was tested using reference rRNAs from pure cultures of nitrifying bacteria. Characterization of samples collected from an industrial wastewater treatment facility demonstrated that nitrifying bacteria could be detected directly by Microarray hybridization without the need for PCR amplification. Specifically, the Microarray detected Nitrosomonas spp. but did not detect Nitrobacter. The specificity and sensitivity of direct detection was evaluated using on-chip dissociation analysis, and by two independent analyses—an established membrane hybridization format and terminal restriction fragment length polymorphism fingerprinting (T-RFLP). The latter two analyses also revealed Nitrospira and Nitrobacter to be contributing populations in the treatment plant samples. The application of DNA Microarrays to wastewater treatment systems, which has been demonstrated in the current work, should offer improved monitoring capabilities and process control for treatment systems, which are susceptible to periodic failures. r 2005 Elsevier Ltd. All rights reserved.
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parallel characterization of anaerobic toluene and ethylbenzene degrading microbial consortia by pcr denaturing gradient gel electrophoresis rna DNA membrane hybridization and DNA Microarray technology
Applied and Environmental Microbiology, 2002Co-Authors: Yoshikazu Koizumi, Hidetoshi Urakawa, John J Kelly, Tatsunori Nakagawa, Said Elfantroussi, Saleh Almuzaini, Manabu Fukui, Yoshikuni Urushigawa, David A StahlAbstract:A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA Microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the Microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA Microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA Microarrays for microbial community analysis.
Peter Slickers - One of the best experts on this subject based on the ideXlab platform.
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DNA Microarray based genotyping of chlamydophila psittaci strains from culture and clinical samples
Veterinary Microbiology, 2009Co-Authors: Konrad Sachse, Karine Laroucau, Fabien Vorimore, Simone Magnino, Jens Feige, Wolfgang Muller, Steffen Kube, Helmut Hotzel, Evelyn Schubert, Peter SlickersAbstract:The avian and human pathogen Chlamydophila (C) psittaci represents a genetically heterogeneous species. To facilitate epidemiological surveys, more rapid yet highly specific molecular tests are needed. Currently used typing methods, i.e. serotyping and PCR-RFLP, have only limited sensitivity and are incapable of covering the wide spectrum of naturally occurring types of C psittaci strains. In the present study, a new DNA Microarray assay based on the ArrayTube(R) (AT) technology was used to genotype C. psittaci in 98 isolates and 23 clinical tissue samples. The present array carries 35 oligonucleotide probes derived from variable domains 2 and 4 of the ompA gene. The assay proved highly sensitive, allowing correct genotyping of DNA from 2 inclusion-forming units. The results of DNA Microarray genotyping of cultured strains proved highly concordant with the data from PCR-RFLP typing and serotyping. Sequencing of the ompA gene served as the reference test to verify the accuracy of AT genotyping results. In 15 instances (15.3%), strains were successfully typed by the AT assay, while serotyping and/or PCR-RFLP genotyping failed to produce unambiguous results. Eleven of these samples were ompA sequenced to confirm the AT findings. In addition to the currently accepted nine ompA genotypes, the Microarray test was shown to recognise new provisional genotypes, such as Mat116 and YP84. In conclusion, the new AT assay proved to be suitable for rapid, sensitive and reproducible genotyping of C psittaci strains and can be recommended for routine diagnosis. (C) 2008 Elsevier B.V. All rights reserved
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genotyping of chlamydophila psittaci using a new DNA Microarray assay based on sequence analysis of ompa genes
BMC Microbiology, 2008Co-Authors: Konrad Sachse, Karine Laroucau, Helmut Hotzel, Evelyn Schubert, Ralf Ehricht, Peter SlickersAbstract:The currently used genotyping system for the avian zoonotic pathogen Chlamydophila (C.) psittaci has evolved from serology and is based on ompA sequence variations. It includes seven avian and two non-avian genotypes. Restriction enzyme cleavage of the amplified ompA gene and, less frequently, ompA sequencing are being used for examination, but, beside methodological limitations, an increasing number of recently tested strains could not be assigned to any established genotype. Comprehensive analysis of all available ompA gene sequences has revealed a remarkable genetic diversity within the species C. psittaci, which is only partially covered by the present genotyping scheme. We suggest adjustments and extensions to the present scheme, which include the introduction of subgroups to the more heterogeneous genotypes A, E/B and D, as well as six provisional genotypes representing so far untypable strains. The findings of sequence analysis have been incorporated in the design of a new DNA Microarray. The ArrayTube™ Microarray-based ompA genotyping assay has been shown to discriminate among established genotypes and identify so far untyped strains. Its high specificity, which allows detection of single-nucleotide polymorphisms, is due to the parallel approach consisting in the use of 35 hybridization probes derived from variable domains 2 and 4 of the ompA gene. The traditional genotyping system does not adequately reflect the extent of intra-species heterogeneity in ompA sequences of C. psittaci. The newly developed DNA Microarray-based assay represents a promising diagnostic tool for tracing epidemiological chains, exploring the dissemination of genotypes and identifying non-typical representatives of C. psittaci.
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direct identification of chlamydiae from clinical samples using a DNA Microarray assay a validation study
Molecular and Cellular Probes, 2008Co-Authors: Nicole Borel, Peter Slickers, Helmut Hotzel, Evelyn Schubert, Ralf Ehricht, Evelyne Kempf, Paul R Torgerson, Taurai Tasara, Andreas Pospischil, Konrad SachseAbstract:Abstract While DNA Microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of Microarray platforms in direct testing of clinical samples. In the present study, a total of 339 samples, among them 293 clinical specimens from animals and humans, were examined by the ArrayTube™ (AT) DNA Microarray assay to detect chlamydial DNA and identify the species of Chlamydia and Chlamydophila involved. Samples included nasal and conjunctival swabs, formalin-fixed, paraffin-embedded and fresh organ tissue, milk, feces and cell culture. Notably, the AT test was shown to detect mixed infections in clinical samples. The calculated median sensitivity of 0.81 over the entire panel of clinical samples was comparable to conventional 16S PCR, but slightly lower than real-time PCR and other PCR assays. However, when a panel of long-time stored swab samples was excluded from the calculation, the sensitivity was clearly higher (0.87) and equivalent to that of real-time PCR. Altogether, the data demonstrate the suitability of this DNA Microarray assay for routine diagnosis.
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optimized DNA Microarray assay allows detection and genotyping of single pcr amplifiable target copies
Molecular and Cellular Probes, 2006Co-Authors: Ralf Ehricht, Peter Slickers, Helmut Hotzel, Stefanie Goellner, Konrad SachseAbstract:This study was conducted to determine the detection limit of an optimized DNA Microarray assay for detection and species identification of chlamydiae. Examination of dilution series of a plasmid standard carrying the target sequence from Chlamydia trachomatis and genomic DNA of this organism revealed that a single PCR-amplifiable target copy was sufficient to obtain a specific hybridization pattern. This performance renders the test suitable for routine testing of clinical samples.
Shi-qiang Shang - One of the best experts on this subject based on the ideXlab platform.
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DNA Microarray technology for simultaneous detection and species identification of seven human herpes viruses
Journal of Medical Virology, 2008Co-Authors: Zhibei Zheng, Shi-qiang ShangAbstract:The aim of the study was to develop a multiplex PCR-based DNA Microarray technology for simultaneous detection and species identification of seven human herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to accurate diagnosis of herpesvirus-associated diseases. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. DNA Microarrays were made by printing the oligonucleotide probes onto special glass slides. After amplification and labeling with CY5, the PCR products were hybridized with the DNA Microarrays and species identified. Sixty-one cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this technique, and the results were compared with those of TaqMan PCR. Several specimens were sequenced further after cloning. The PCR products of the seven human herpes viruses ranged from 224 to 252 bp, and could be species identified with DNA Microarrays. The detection limits were 10(1) copies/microl for each virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E. coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and specificity of the DNA Microarray technology was 96.2% and 99.3%, respectively. This multiplex PCR-based DNA Microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses.
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DNA Microarray technology for simultaneous detection and species identification of seven human herpes viruses
Journal of Medical Virology, 2008Co-Authors: Zhibei Zheng, Shi-qiang ShangAbstract:The aim of the study was to develop a multiplex PCR-based DNA Microarray technology for simultaneous detection and species identification of seven human herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to accurate diagnosis of herpesvirus-associated diseases. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. DNA Microarrays were made by printing the oligonucleotide probes onto special glass slides. After amplification and labeling with CY5, the PCR products were hybridized with the DNA Microarrays and species identified. Sixty-one cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this technique, and the results were compared with those of TaqMan PCR. Several specimens were sequenced further after cloning. The PCR products of the seven human herpes viruses ranged from 224 to 252 bp, and could be species identified with DNA Microarrays. The detection limits were 101 copies/µl for each virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E. coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and specificity of the DNA Microarray technology was 96.2% and 99.3%, respectively. This multiplex PCR-based DNA Microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses. J. Med. Virol. 80:1042–1050, 2008. © 2008 Wiley-Liss, Inc.
Zhibei Zheng - One of the best experts on this subject based on the ideXlab platform.
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DNA Microarray technology for simultaneous detection and species identification of seven human herpes viruses
Journal of Medical Virology, 2008Co-Authors: Zhibei Zheng, Shi-qiang ShangAbstract:The aim of the study was to develop a multiplex PCR-based DNA Microarray technology for simultaneous detection and species identification of seven human herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to accurate diagnosis of herpesvirus-associated diseases. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. DNA Microarrays were made by printing the oligonucleotide probes onto special glass slides. After amplification and labeling with CY5, the PCR products were hybridized with the DNA Microarrays and species identified. Sixty-one cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this technique, and the results were compared with those of TaqMan PCR. Several specimens were sequenced further after cloning. The PCR products of the seven human herpes viruses ranged from 224 to 252 bp, and could be species identified with DNA Microarrays. The detection limits were 10(1) copies/microl for each virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E. coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and specificity of the DNA Microarray technology was 96.2% and 99.3%, respectively. This multiplex PCR-based DNA Microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses.
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DNA Microarray technology for simultaneous detection and species identification of seven human herpes viruses
Journal of Medical Virology, 2008Co-Authors: Zhibei Zheng, Shi-qiang ShangAbstract:The aim of the study was to develop a multiplex PCR-based DNA Microarray technology for simultaneous detection and species identification of seven human herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to accurate diagnosis of herpesvirus-associated diseases. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. DNA Microarrays were made by printing the oligonucleotide probes onto special glass slides. After amplification and labeling with CY5, the PCR products were hybridized with the DNA Microarrays and species identified. Sixty-one cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this technique, and the results were compared with those of TaqMan PCR. Several specimens were sequenced further after cloning. The PCR products of the seven human herpes viruses ranged from 224 to 252 bp, and could be species identified with DNA Microarrays. The detection limits were 101 copies/µl for each virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E. coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and specificity of the DNA Microarray technology was 96.2% and 99.3%, respectively. This multiplex PCR-based DNA Microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses. J. Med. Virol. 80:1042–1050, 2008. © 2008 Wiley-Liss, Inc.
Hidetoshi Urakawa - One of the best experts on this subject based on the ideXlab platform.
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DNA Microarray mediated transcriptional profiling of nitrosomonas europaea in response to linear alkylbenzene sulfonates
Fems Microbiology Letters, 2008Co-Authors: Hidetoshi Urakawa, Junpei Matsumoto, Kazuho Inaba, Satoshi TsunedaAbstract:Linear alkylbenzene sulfonates (LAS) constitute, quantitatively, the most important group of synthetic surfactants used today. We studied the gene expression of Nitrosomonas europaea in response to LAS using a DNA Microarray because ammonia-oxidizers are thought to be more sensitive to LAS than other microorganisms. Our objective was to elucidate which genes are expressed for N. europaea in response to LAS exposure. Microarray analysis and real-time PCR assay revealed that c. 30 genes were significantly expressed after LAS exposure, in particular genes associated with energy production and conversion. Our findings demonstrate that physical disruption of membrane structures, which contain enzymes associated with energy production and conversion, might be an important explanation for the high sensitivity of N. europaea to LAS exposure.
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DNA Microarray detection of nitrifying bacterial 16s rrna in wastewater treatment plant samples
Water Research, 2005Co-Authors: John J Kelly, Hidetoshi Urakawa, Slil Siripong, John L Mccormack, Lori R Janus, Said El Fantroussi, Peter A Noble, Laura Sappelsa, Bruce E Rittmann, David A StahlAbstract:A small scale DNA Microarray containing a set of oligonucleotide probes targeting the 16S rRNAs of several groups of nitrifying bacteria was developed for the monitoring of wastewater treatment plant samples. The Microarray was tested using reference rRNAs from pure cultures of nitrifying bacteria. Characterization of samples collected from an industrial wastewater treatment facility demonstrated that nitrifying bacteria could be detected directly by Microarray hybridization without the need for PCR amplification. Specifically, the Microarray detected Nitrosomonas spp. but did not detect Nitrobacter. The specificity and sensitivity of direct detection was evaluated using on-chip dissociation analysis, and by two independent analyses--an established membrane hybridization format and terminal restriction fragment length polymorphism fingerprinting (T-RFLP). The latter two analyses also revealed Nitrospira and Nitrobacter to be contributing populations in the treatment plant samples. The application of DNA Microarrays to wastewater treatment systems, which has been demonstrated in the current work, should offer improved monitoring capabilities and process control for treatment systems, which are susceptible to periodic failures.
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DNA Microarray detection of nitrifying bacterial 16s rrna in wastewater treatment plant samples
Water Research, 2005Co-Authors: John J Kelly, Hidetoshi Urakawa, Slil Siripong, John L Mccormack, Lori R Janus, Said El Fantroussi, Peter A Noble, Laura Sappelsa, Bruce E Rittmann, David A StahlAbstract:A small scale DNA Microarray containing a set of oligonucleotide probes targeting the 16S rRNAs of several groups of nitrifying bacteria was developed for the monitoring of wastewater treatment plant samples. The Microarray was tested using reference rRNAs from pure cultures of nitrifying bacteria. Characterization of samples collected from an industrial wastewater treatment facility demonstrated that nitrifying bacteria could be detected directly by Microarray hybridization without the need for PCR amplification. Specifically, the Microarray detected Nitrosomonas spp. but did not detect Nitrobacter. The specificity and sensitivity of direct detection was evaluated using on-chip dissociation analysis, and by two independent analyses—an established membrane hybridization format and terminal restriction fragment length polymorphism fingerprinting (T-RFLP). The latter two analyses also revealed Nitrospira and Nitrobacter to be contributing populations in the treatment plant samples. The application of DNA Microarrays to wastewater treatment systems, which has been demonstrated in the current work, should offer improved monitoring capabilities and process control for treatment systems, which are susceptible to periodic failures. r 2005 Elsevier Ltd. All rights reserved.
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parallel characterization of anaerobic toluene and ethylbenzene degrading microbial consortia by pcr denaturing gradient gel electrophoresis rna DNA membrane hybridization and DNA Microarray technology
Applied and Environmental Microbiology, 2002Co-Authors: Yoshikazu Koizumi, Hidetoshi Urakawa, John J Kelly, Tatsunori Nakagawa, Said Elfantroussi, Saleh Almuzaini, Manabu Fukui, Yoshikuni Urushigawa, David A StahlAbstract:A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA Microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the Microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA Microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA Microarrays for microbial community analysis.