Journal article
Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays
Applied and environmental microbiology, Vol.67(10), pp.4708-4716
10/2001
Handle:
https://hdl.handle.net/2376/103033
PMCID: PMC93223
PMID: 11571176
Abstract
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 × 10
6
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.
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Details
- Title
- Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays
- Creators
- Jack Small - Analytical MicrobiologyDouglas R Call - Analytical MicrobiologyFred J Brockman - Analytical MicrobiologyTimothy M Straub - Analytical MicrobiologyDarrell P Chandler - Analytical Microbiology
- Publication Details
- Applied and environmental microbiology, Vol.67(10), pp.4708-4716
- Academic Unit
- Paul G. Allen School for Global Animal Health
- Publisher
- American Society for Microbiology
- Identifiers
- 99900546524601842
- Language
- English
- Resource Type
- Journal article