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

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.