Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays

Citation
J. Small et al., Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays, APPL ENVIR, 67(10), 2001, pp. 4708-4716
Citations number
43
Categorie Soggetti
Biology,Microbiology
Journal title
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
ISSN journal
00992240 → ACNP
Volume
67
Issue
10
Year of publication
2001
Pages
4708 - 4716
Database
ISI
SICI code
0099-2240(200110)67:10<4708:DDO1RI>2.0.ZU;2-A
Abstract
We report on the development and validation of a simple microarray method f or the direct detection of intact 16S rRNA from unpurified soil extracts. T otal RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hyb ridized to an oligonucleotide array consisting of universal and species-spe cific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovib rio were easily and specifically detected under a range of hybridization ti mes, temperatures, and buffers. However, reproducible, specific hybridizati on and detection of intact rRNA could be accomplished only by using a chape rone-detector probe strategy. With this knowledge, assay conditions were de veloped for rRNA detection using a 2-h hybridization time at room temperatu re. Hybridization specificity and signal intensity were enhanced using frag mented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detec tion strategy, we were able to specifically hybridize and detect G. chapell ei 16S rRNA directly from a total-RNA soil extract, without further purific ation or removal of soluble soil constituents. The detection sensitivity fo r G. chapellei 16S rRNA in soil extracts was at least 0.5 mug of total RNA, representing approximately 7.5 X 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technolog y to the direct detection of microorganisms in environmental samples, witho ut using PCR.