Oligonucleotides useful in methods for detecting and characterizing Aspergillus fumigatus

Abstract

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.

Description

CROSS REFERENCES TO RELATED APPLICATIONS [0001] The present application claims benefit, under 35 U.S.C. 119(e), to U.S. Provisional Application No. 60 / 636,133, entitled “A Method for Detecting Triazole Resistant-Aspergillus Fumigatus,” filed on Dec. 15, 2004, the entire contents of which are hereby incorporated by reference.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention is broadly concerned with oligonucleotides useful in methods for detecting and characterizing Aspergillus fumigatus. More particularly, the present invention relates to oligonucleotides having nucleotide sequences derived from the gene encoding the cytochrome P450 14 alpha-sterol demethylase (i.e., the Cyp51A protein) of Aspergillus fumigatus, wherein these oligonucleotides are useful as forward and reverse primers for a polymerase chain reaction using nucleic acids from a biological sample as a template, as probes for detecting any resultant cyp51A-specific amplicon indicat...

AI Technical Summary

Benefits of technology

[0101] In more preferred embodiments, the first oligonucleotide probe is capable of hybridizing to at least a portion of a segment of the antisense strand of the amplicon, wherein the segment consists of nucleotides 274 through 295 of SEQ ID NO:15. Preferably, the first oligonucleotide probe is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or is 100% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, wherein the segment consists of nucleotides 274 through 295 of SEQ ID NO:15. More preferably, the first oligonucleotide probe comprises the nucleotide sequence of SEQ ID NO:48. Most preferably, the first oligonucleotide probe consists of the nucleotide sequence of SEQ ID NO:48. Advantageously, nucleotides 12, 13, and 14 of SEQ ID NO:48 are g, g, and t, respectively.

[0102] A

Problems solved by technology

Aspergillus fumigatus is the causative agent for medical conditions including invasive aspergillosis, which is a major cause of morbidity and mortality in immunocompromised patients.

Unfortunately, conventional laboratory tests for the presence of Aspergillus fumigatus, such as culture and galactomannan detection, lack sensitivity, and are rarely conclusive, resulting in true positive results only at advanced stages of infection or necessitating invasive procedures for formal microbiological evaluation (see Denning, 1998, Invasive aspergillosis.

Furthermore, the emergence of clinical resistance to azole-based drugs impedes successful treatment of infection by Aspergillus fumigatus (see Denning et al., 1997, Itraconazole resistance in Aspergillus fumigatus.

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