Assays for Resistance to Echinocandin-Class Drugs

• US 20100075302A1
• Filed: 07/26/2006
• Published: 03/25/2010
• Est. Priority Date: 07/26/2005
• Status: Active Grant

• Current Assignee: Merck & Co., Inc., Rutgers University
• Original Assignee: Merck & Co., Inc., University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Inventors: Parent, Stephen A., Douglas, Cameron M., Kahn, Jennifer Nielsen, Park, Steven, Perlin, David S., Kelly, Rosemarie
• Granted Patent: US 8,753,819 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 echinocandin drug resistance in fungi susceptible to echinocandin drugs and containing the FKS1 gene corresponding to 1-3β

-D-glucan synthase subunit Fks1p comprising amplifying a first nucleic acid target sequence of the FKS1 corresponding to at least a portion of CaFks1p amino acids 636 to 654 that includes amino acids 641 to 649, and detecting any difference from the wild-type allele in said amplified first target sequence.

2. The assay of claim 1 wherein the fungus is a Candida species.

3. The assay of claim 1 that is capable of detecting at least one amino acid mutation selected from the group consisting of F641L, F641S, S645P, S645Y, S645F, 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, 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 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 comprises sequencing.

8. The assay of claim 1 wherein the step of detecting comprises contacting the amplified first and second target sequences with labeled hybridization probes.

9. The assay of claim 8 wherein detection is homogeneous end-point or real-time detection.

10. A real-time assay according to claim 9.

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.

18. A kit of reagents for performing an assay according to claim 1.

20. The kit of reagents according to claim 18 further including reagents for sample preparation and DNA extraction.

12. An oligonucleotide set that includes a first pair of forward and reverse primers for amplifying said first target sequence and forward and reverse primers for amplifying a first 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 636-654 that includes amino acids 641-649.

13. The oligonucleotide set of claim 12 that includes 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 that includes amino acids 1357-1364.

14. The oligonucleotide set of claim 12 wherein said first pair of forward and reverse primers also encompasses the amino acids corresponding to CaFks1p amino acids 1357-1364.

15. The oligonucleotide set of claim 12 further including labeled, allele-discriminating hybridization probes that selectively hybridize to at least one mutation selected from the group consisting of T1921C, T9122C, G1932T, T1933C, C1934A, C1934T, C1934G, C1942T, and C1946A of CaFKS1.

16. The oligonucleotide set of claim 13 further including labeled, allele-discriminating hybridization probes that selectively hybridize to at least one mutation selected from the group consisting of C4081G and G4082A of CaFKS1.

17. The oligonucleotide set of claim 12 further comprising a sequencing primer for sequencing the amplified product defined by said forward and reverse primers.

19. The kit of reagents of claims-18 that includes an oligonucleotide set of claim 12.