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Applied and Environmental Microbiology, December 2005, p. 8481-8490, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8481-8490.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development of a Rapid Assay for Determining the Relative Abundance of Bacteria

Arlene K. Rowan,1 Russell J. Davenport,1* Jason R. Snape,2 David Fearnside,3 Michael R. Barer,4 Thomas P. Curtis,1 and Ian M. Head1

School of Civil Engineering and Geosciences, University of Newcastle, Newcastle upon Tyne NE1 7RU,1 Brixham Environmental Laboratory, AstraZeneca Global SHE, Brixham, Devon TQ5 8BA,2 Yorkshire Water Services, Western House, Halifax Road, Bradford, West Yorkshire BD6 2LZ,3 Department of Microbiology and Immunology, University of Leicester, Leicester LE1 7RH, United Kingdom4

Received 3 June 2005/ Accepted 8 September 2005

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 103 to 104 and 104 to 105 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.

* Corresponding author. Mailing address: School of Civil Engineering and Geosciences, Cassie Building, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, United Kingdom. Phone: 44 191 222 5544. Fax: 44 191 222 6502. E-mail: r.j.davenport{at}ncl.ac.uk.

Applied and Environmental Microbiology, December 2005, p. 8481-8490, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8481-8490.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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Copyright © 2005 by the American Society for Microbiology. All rights reserved.