Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater
A plasmid-based 16S–23S rDNA intergenic spacer region (ISR) array was developed and optimized for analysis of microbial diversity within complex environmental samples. Plasmid probes with 16S–23S rDNA ISR inserts (800-1500 bp) from industrial wastewater treatment plant (WWTP) microorganisms were arr...
| Autores: | ; ; ; ; |
|---|---|
| Tipo de documento: | Artículo |
| Estado: | Published version |
| Fecha de publicación: | 2004 |
| País: | Argentina |
| Institución: | Consejo Nacional de Investigaciones Científicas y Técnicas |
| Repositorio: | CONICET Digital (CONICET) |
| Lenguaje: | Inglés |
| OAI Identifier: | oai:ri.conicet.gov.ar:11336/104169 |
| Acceso en línea: | http://hdl.handle.net/11336/104169 |
| Nivel de acceso: | Acceso abierto |
| Palabra clave: | INTERGENIC SPACER ARRAY MICROARRAY ISR ACTIVATED SLUDGE WASTEWATER https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| _version_ | 1878404081865719809 |
|---|---|
| author | Cook, Kimberly L. |
| author2 | Layton, Alice C. Dionisi, Hebe Monica Fleming, James T. Sayler, Gary S. |
| author2_role | author author author author |
| author_browse | Cook, Kimberly L. Dionisi, Hebe Monica Fleming, James T. Layton, Alice C. Sayler, Gary S. |
| author_facet | Cook, Kimberly L. Layton, Alice C. Dionisi, Hebe Monica Fleming, James T. Sayler, Gary S. |
| author_role | author |
| collection | CONICET Digital (CONICET) |
| description | A plasmid-based 16S–23S rDNA intergenic spacer region (ISR) array was developed and optimized for analysis of microbial diversity within complex environmental samples. Plasmid probes with 16S–23S rDNA ISR inserts (800-1500 bp) from industrial wastewater treatment plant (WWTP) microorganisms were arrayed onto glass slides. Hybridization of fluorescently labeled target sequences from two clones from the ISR WWTP library to arrayed probes showed that there was a good linear relationship between hybridization intensity and ISR similarity (r2 = 0.82). Hybridization was highly specific (average background from arrayed probes with less than 80% similarity in ISR sequence was less than 7%). Strong fluorescence intensity corresponded to near-perfect match clones (99% or greater similarity in ISR sequence). A majority of probes (79%) showed no background hybridization. However, weak background (less than 50% for arrayed probes with 90% and 95% similarity in the 16S rRNA genes) was observed from closely related microorganisms. Background fluorescence from the negative control (plasmid vector with no insert) was similar to water and dimethyl sulfoxide (DMSO)-negative controls. Hybridization using fluorescently labeled ISR sequences from a mixed community sample produced strong fluorescent signals with no background from negative controls. A Cy5-labeled reference standard, part of the vector and present in every spotted probe, was used to normalize hybridization values. These results indicate that arrayed plasmid containing ISR probe insert sequences provides specificity and sensitivity for microbial community analysis in a high-throughput array format. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | AR_f7dbadfa71af7ccc754286abc3205d7d |
| identifier_str_mv | Cook, Kimberly L.; Layton, Alice C.; Dionisi, Hebe Monica; Fleming, James T.; Sayler, Gary S.; Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater; Elsevier Science; Journal of Microbiological Methods; 57; 1; 4-2004; 79-93 0167-7012 1872-8359 CONICET Digital CONICET |
| institution | Consejo Nacional de Investigaciones Científicas y Técnicas |
| instname_str | Consejo Nacional de Investigaciones Científicas y Técnicas |
| language | eng |
| network_acronym_str | AR |
| network_name_str | Argentina |
| oai_identifier_str | oai:ri.conicet.gov.ar:11336/104169 |
| publishDate | 2004 |
| publishDateSort | 2004 |
| publisher | Elsevier Science |
| publisherStr | Elsevier Science |
| reponame_str | CONICET Digital (CONICET) |
| spelling | Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater Cook, Kimberly L. Layton, Alice C. Dionisi, Hebe Monica Fleming, James T. Sayler, Gary S. INTERGENIC SPACER ARRAY MICROARRAY ISR ACTIVATED SLUDGE WASTEWATER https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 A plasmid-based 16S–23S rDNA intergenic spacer region (ISR) array was developed and optimized for analysis of microbial diversity within complex environmental samples. Plasmid probes with 16S–23S rDNA ISR inserts (800-1500 bp) from industrial wastewater treatment plant (WWTP) microorganisms were arrayed onto glass slides. Hybridization of fluorescently labeled target sequences from two clones from the ISR WWTP library to arrayed probes showed that there was a good linear relationship between hybridization intensity and ISR similarity (r2 = 0.82). Hybridization was highly specific (average background from arrayed probes with less than 80% similarity in ISR sequence was less than 7%). Strong fluorescence intensity corresponded to near-perfect match clones (99% or greater similarity in ISR sequence). A majority of probes (79%) showed no background hybridization. However, weak background (less than 50% for arrayed probes with 90% and 95% similarity in the 16S rRNA genes) was observed from closely related microorganisms. Background fluorescence from the negative control (plasmid vector with no insert) was similar to water and dimethyl sulfoxide (DMSO)-negative controls. Hybridization using fluorescently labeled ISR sequences from a mixed community sample produced strong fluorescent signals with no background from negative controls. A Cy5-labeled reference standard, part of the vector and present in every spotted probe, was used to normalize hybridization values. These results indicate that arrayed plasmid containing ISR probe insert sequences provides specificity and sensitivity for microbial community analysis in a high-throughput array format. Fil: Cook, Kimberly L.. University of Tennessee; Estados Unidos. United States Department of Agriculture. Agricultural Research Service; Estados Unidos Fil: Layton, Alice C.. University of Tennessee; Estados Unidos Fil: Dionisi, Hebe Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico; Argentina. University of Tennessee; Estados Unidos Fil: Fleming, James T.. University of Tennessee; Estados Unidos Fil: Sayler, Gary S.. University of Tennessee; Estados Unidos Elsevier Science http://hdl.handle.net/11336/104169 Cook, Kimberly L.; Layton, Alice C.; Dionisi, Hebe Monica; Fleming, James T.; Sayler, Gary S.; Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater; Elsevier Science; Journal of Microbiological Methods; 57; 1; 4-2004; 79-93 0167-7012 1872-8359 CONICET Digital CONICET |
| spellingShingle | Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater Cook, Kimberly L. INTERGENIC SPACER ARRAY MICROARRAY ISR ACTIVATED SLUDGE WASTEWATER https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| status_str | publishedVersion |
| title | Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater |
| title_full | Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater |
| title_fullStr | Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater |
| title_full_unstemmed | Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater |
| title_short | Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater |
| title_sort | Evaluation of a plasmid-based 16S–23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater |
| topic | INTERGENIC SPACER ARRAY MICROARRAY ISR ACTIVATED SLUDGE WASTEWATER https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| topic_facet | INTERGENIC SPACER ARRAY MICROARRAY ISR ACTIVATED SLUDGE WASTEWATER https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| url | http://hdl.handle.net/11336/104169 |