Oligonucleotides useful in methods for detecting and characterizing
Methods for using oligonucleotides in the detection of Aspergillus fumigatus are disclosed. The oligonucleotides of the invention have nucleotide sequences derived from the gene encoding the cytochrome P450 14 alpha-sterol demethylase (the cyp51A protein) of Aspergillus fumigatus . The oligonucleotides include primers capable of producing amplicons specific to cyp51A in polymerase chain reactions using nucleic acids isolated from Aspergillus fumigatus as templates. The oligonucleotides also include probes capable of detecting these cyp51A-specific amplicons. 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.
1. A method for determining the presence of Aspergillus fumigatus in whole blood of a human and whether said Aspergillus fumigatus is more tolerant to triazole than wild-type Aspergillus fumigatus , comprising the steps of:
(a) obtaining whole blood from a human subject, wherein said human subject is suspected of infection with triazole-resistant Aspergillus fumigatus;
(b) extracting DNA from said whole blood;
(c) performing a real-time PCR on said extracted DNA using a forward primer consisting of the nucleotide sequence SEQ ID NO: 16, a reverse primer consisting of the nucleotide sequence SEQ ID NO: 17 and a probe consisting of the nucleotide sequence SEQ ID NO: 26 to generate an amplicon, wherein the generation of said amplicon in said real-time PCR is indicative of the presence of Aspergillus fumigatus;
(d) isolating said generated amplicon;
(e) performing a pyrosequencing reaction on said isolated amplicon using a sequencing primer consisting of the nucleotide sequence SEQ ID NO: 18; and
(f) determining the identity of the nucleotides encoding the amino acid at position 54 of the Cyp51A protein, wherein the presence of an amino acid other than glycine at said position 54 is indicative of the presence of said Aspergillus fumigatus that is more tolerant to triazole than wild-type Aspergillus fumigatus.
2. The method of claim 1 , wherein said probe is dual-florescence labeled to detect said generated amplicon.
3. The method of claim 1 , wherein said reverse primer is biotinylated at its 5′ end to facilitate said isolation of said generated amplicon.