Assays for resistance to echinocandin-class drugs
Nucleic acid amplification assays for mutations to two short sections of the fungal gene FKS1. Mutations in these target sequences have been shown to correlate with resistance to echinocandin-class drugs. Assays may include detection by sequencing or by labeled hybridization probes. Also, primers, probes and reagent kits for performing such assays.
1. An oligonucleotide set that includes a first pair of forward and reverse primers for amplifying a first target sequence of the FK506-supersensitive (FKS1) gene corresponding to 1-3-β-D-glucan synthase subunit Fks1p corresponding to at least a portion of Candida albicans FK506-supersensitive (CaFks1p) amino acids 636-654 present within SEQ ID NO: 2 that includes amino acids 641-649 present within SEQ ID NO: 2, and further comprising labeled, allele-discriminating hybridization probes that selectively hybridize to at least one mutation in an amplified first target sequence, the at least one mutation selected from the group consisting of: S645P, S645Y, and S645F of CaFks1p, wherein each of the allele-discriminating hybridization probes comprises a single-stranded loop flanked by complementary 3′ and 5′ arm sequences that form a stem, and wherein the allele-discriminating hybridization probes are capable of hybridizing with the amplified first target sequence at a temperature between 62.7° C. and 64° C.
2. The oligonucleotide set of claim 1 further comprising a second pair of forward and reverse primers for amplifying a second target sequence of the FKS1 gene corresponding to 1-3-β-D-glucan synthase subunit Fks1p corresponding to at least a portion of CaFks1p amino acids 1345-1369 present within SEQ ID NO: 2 that includes amino acids 1357-1364 present within SEQ ID NO: 2.
3. The oligonucleotide set of claim 1 further comprising a sequencing primer for sequencing the amplified product defined by said forward and reverse primers.
4. A kit of reagents for performing a nucleic acid assay for detecting a mutation in fungi not susceptible to echinocandin drugs and containing the FKS1 gene corresponding to 1-3-β-D-glucan synthase subunit Fks1p comprising the oligonucleotide set of claim 1 .
5. The kit of reagents of claim 4 , wherein the hybridization probes are labeled with a fluorophore.
6. The oligonucleotide set of claim 1 , wherein the hybridization probes are labeled with a fluorophore.
7. The oligonucleotide set of claim 1 , wherein the stem is a 6-nucleotide stem.
8. The oligonucleotide set of claim 1 , wherein the complementary 3′ and 5′ arm sequences of the stem respectively comprise the nucleotide sequences of 5′-CGCGAG and 5′-CTCGCG.