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Applied and Environmental Microbiology, October 2001, p. 4708-4716, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4708-4716.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

Jack Small,1 Douglas R. Call,2 Fred J. Brockman,3 Timothy M. Straub,1 and Darrell P. Chandler1,*

Analytical Microbiology1 and Environmental Microbiology Group,3 Pacific Northwest National Laboratory, Richland, Washington 99352, and Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 991642

Received 18 June 2001/Accepted 24 July 2001

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 µg of total RNA, representing approximately 7.5 × 106 Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.

* Corresponding author. Mailing address: Analytical Microbiology, Pacific Northwest National Laboratory, 900 Battelle Blvd., Mail Stop P7-50, Richland, WA 99352. Phone: (509) 376-8644. Fax: (509) 376-1321. E-mail: dp.chandler{at}pnl.gov.

Applied and Environmental Microbiology, October 2001, p. 4708-4716, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4708-4716.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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