Oligonucleotides and methods for using these oligonucleotides in the detection of
Aspergillus fumigatus are disclosed.
Aspergillus fumigatus is the causative agent for medical conditions including invasive
aspergillosis. The oligonucleotides of the invention have
nucleotide sequences derived from the
gene encoding the
cytochrome P450 14 alpha-
sterol demethylase (i.e., the Cyp51A
protein) of
Aspergillus fumigatus. The oligonucleotides of the invention include forward primers and reverse primers which in combination are capable of priming the synthesis of amplicons specific to cyp51A in
polymerase chain reactions using nucleic acids isolated from
Aspergillus fumigatus as templates. The oligonucleotides of the invention also include probes capable of detecting these cyp51A-specific amplicons. Thus, a biological sample is tested for the presence of
Aspergillus fumigatus by isolating
nucleic acid from the sample, conducting a
polymerase chain reaction in a mixture containing this
nucleic acid and these forward and reverse primers, and then determining, using an
oligonucleotide probe, whether an
amplicon is produced in the mixture, wherein detection of the
amplicon indicates the presence of
Aspergillus fumigatus in the sample. The oligonucleotides of the invention also include primers for
nucleotide sequencing reactions to determine whether an isolate of Aspergillus fumigatus is more tolerant than wild-type Aspergillus fumigatus to a
triazole, which is a compound commonly used as an
antifungal drug. Specifically, a strand of a cyp51A-specific
amplicon is at least partially sequenced using a
nucleotide sequencing primer in a
primer extension reaction. Identification of mutations giving rise to
amino acid substitutions at positions 54, 138, 220, and 448 of the
amino acid sequence of the wild-type Cyp51A
protein indicates that the isolate of Aspergillus fumigatus from which the amplicon is derived exhibits decreased susceptibility to at least one
triazole.