The most pressing problem in clinical mycology is the need for an methods for the early diagnosis opportunistic candidiasis and cryptococcosis. This investigation will produce oligonucleotide sequences that are specific for the most significant fungal pathogens and evaluate their ability to amplify fungal DNA in clinical specimens to diagnose mycotic infection. In addition, the taxonomic relationships in medical fungi will be examined by comparative analysis of the ribosomal RNA genes (rDNA) and by surveying representative isolates of pathogenic fungi for phylogenetically informative sequence variations within the major ribosomal subunit RNAs (5.8S, 17S and 25S RNAs) and the internal transcribed spacer region (ITS). The methods will include reverse transcriptase sequencing of ribosomal RNA and dideoxy-sequencing of single stranded DNA templates that are amplified from genomic DNA using the polymerase chain reaction (PCR). Studies of restriction fragment length polymorphisms (RFLP) will also be undertaken to investigate these relationships. The results of the taxonomic survey will complement the application of DNA technology to the clinical identification of pathogenic fungi. Taxon-specific "signature sequences" will be serve as probes and markers for the identification of pathogens, and the PCR will be used to amplify specific fungal genes in clinical specimens as a diagnostic tool for the rapid identification of fungi associated with human disease.
NIH ExPorter
NIH ExPorter