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Development of a rapid assay for determining the relative abundance of bacteria

Lookup NU author(s): Dr Arlene Rowan, Professor Russell Davenport, Dr Michael Barer, Professor Thomas Curtis, Professor Ian Head

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Abstract

A sandwich hybridization assay for high-throughput, rapid, simple, and inexpensive quantification of specific microbial populations was evaluated. The assay is based on the hybridization of a target rRNA with differentially labeled capture and detector probes. Betaproteobacterial ammonia-oxidizing bacteria (AOB) were selected as the target group for the study, since they represent a phylogenetically coherent group of organisms that perform a well-defined geochemical function in natural and engineered environments. Reagent concentrations, probe combinations, and washing, blocking, and hybridization conditions were optimized to improve signal and reduce background. The detection limits for the optimized RNA assay were equivalent to approximately 10(3) to 10(4) and 10(4) to 10(5) bacterial cells, respectively, for E. coli rRNA and RNA extracted from activated sludge, by using probes targeting the majority of bacteria. Furthermore, the RNA assay had good specificity, permitted discrimination of rRNA sequences that differed by a 2-bp mismatch in the probe target region, and could distinguish the sizes of AOB populations in nitrifying and nonnitrifying wastewater treatment plants.


Publication metadata

Author(s): Rowan AK, Davenport RJ, Snape JR, Fearnside D, Barer MR, Curtis TP, Head IM

Publication type: Article

Publication status: Published

Journal: Applied and Environmental Microbiology

Year: 2005

Volume: 71

Issue: 12

Pages: 8481-8490

ISSN (print): 0099-2240

ISSN (electronic): 1098-5336

Publisher: American Society for Microbiology

URL: http://dx.doi.org/10.1128/AEM.71.12.8481-8490.2005

DOI: 10.1128/AEM.71.12.8481-8490.2005


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