GARGAS, ANDREA1* and PAULA T. DEPRIEST2. 1Department of Botany, 132 Birge Hall, 430 Lincoln Dr, University of Wisconsin, Madison, WI 53706; 2Department of Systematic Biology, NHB-166, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560. - Diagnosis of cryptic microbes based on ITS2 rRNA secondary structure information.
Conventional techniques of isolating and identifying fungi or other
cryptic microbes are time-consuming and expensive, limiting their
utility for rapid and widespread surveys. We invented a novel method
for detection and identification of microbes including those never
before isolated or identified. This approach relies on analyses of the
internal transcribed spacer 2 (ITS2) rDNA sequences - highly variable
regions flanked by the 5.8S and 28S ribosomal subunits within nuclear
DNA. After determining secondary structures of ITS2 rRNAs we identify
nucleotide motifs between 20 and 50 nt in length within each primary
sequence. These signature sequences, readily defined and present in
high copy number, allow taxonomic diagnoses of known and unknown
microbes from various substrates including agricultural, medical and
ecological samples. Such sequences are readily adapted for use with
probing techniques including PCR, microarray assays or other molecular
detection methods.
Key words: detection, fungi, ribosomal DNA, secondary structure