Assays for resistance to echinocandin-class drugs

• US 9,657,334 B2
• Filed: 06/16/2014
• Issued: 05/23/2017
• Est. Priority Date: 07/26/2005
• Status: Active Grant

• Current Assignee: Merck & Co., Inc., Rutgers University
• Original Assignee: Merck & Co., Inc., Rutgers University
• Inventors: Perlin, David S., Kahn, Jennifer N., Park, Steven, Douglas, Cameron M., Parent, Stephen A., Kelly, Rosemarie
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US14/305,499
• Publication Number:
  US 20150125856A1
• Time in Patent Office: 1,072 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

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.

Claims

1. A nucleic acid assay for detecting a mutation in fungi susceptible to echinocandin drugs and containing the FKS1 gene corresponding to 1-3β

-D-glucan synthase subunit Fks1p comprising;

obtaining cells of a fungus;

lysing the cells to obtain nucleic acid molecules of the cells, said nucleic acid molecules comprising said FKS1 gene or a portion thereof;

contacting the nucleic acid molecules with primers for the FKS1 gene or a portion thereof, a polymerase, and dNTPs;

exponentially amplifying a first nucleic acid target sequence of the FKS1 gene corresponding to at least a portion of CaFks1p amino acids 636 to 654 that includes amino acids 641 to 649 to generate an amplified first target sequence,

contacting the amplified first target sequence with a first labeled hybridization probe specific for an amino acid mutation corresponding to one selected from the group consisting of S645P, S645Y, and S645F of CaFks1p, and

detecting whether the mutation is present by detecting hybridization between the amplified first target sequence and the first labeled hybridization probe.

2. The assay of claim 1 wherein the fungi are a Candida species.

3. The assay of claim 1, further comprising contacting the amplified first target sequence with an additional labeled hybridization probe specific for at least one amino acid mutation corresponding to one selected from the group consisting of F641L, F641S, D648Y and P649H of CaFks1p.

4. The assay according to claim 1 wherein the step of amplifying includes amplifying a second nucleic acid 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 to 1369 that includes amino acids 1357 to 1364 to generate an amplified second target sequence, and wherein the step of detecting includes detecting any difference from the wild-type allele in said amplified second target sequence.

5. The assay of claim 4 that is capable of detecting at least one amino acid change corresponding to one selected from the group consisting of R1361H and R1361G of CaFks1p.

6. The assay of claim 4 wherein amplification of the first target sequence and the second target sequence are performed in the same reaction mixture.

7. The assay of claim 1 wherein the step of detecting further comprises sequencing.

8. The assay of claim 4 wherein the step of detecting comprises contacting the amplified second target sequence with a second labeled hybridization probe specific for at least one amino acid mutation corresponding to one selected from the group consisting of R1361H and R1361G of CaFks1p.

9. The assay of claim 1 wherein detection is homogeneous end-point or real-time detection.

10. The assay according to claim 9, wherein the detection is a real-time detection.

11. The assay according to claim 1 wherein the step of amplifying includes an amplification method selected from the group consisting of PCR, NASBA and TMA.

12. The assay of claim 1, wherein the amplifying step is conducted in a sample obtained from a human or from a clinical isolate.

13. The assay of claim 1, further comprising culturing a plurality of cells of the fungus in presence of the echinocandin drugs to confirm a resistance phenotype.