Detection of echinocandin-resistant
View Patent ↗Probes and primers are disclosed for detecting a C. glabrata resistant to an echinocandin in a sample. Method are also disclosed that utilize these probes and primers, wherein the methods can be used to detect a C. glabrata resistant to an echinocandin in a sample.
1. A method for detecting a C. glabrata resistant to an echinocandin in a sample, comprising
contacting a biological sample comprising DNA with unequal amounts of a forward primer and a reverse primer that can be used to amplify a FKS1-hot spot 1 (HS1) nucleic acid and unequal amounts of a forward and a reverse primer that can be used to amplify a FKS2-HS1 nucleic acid;
performing asymmetric polymerase chain reaction (PCR) to form amplified DNA product; and
contacting the amplified DNA product with a set of probes, wherein each probe in the set of probes specifically binds FKS1-HS1 DNA mutation or specifically binds a FKS2-HS1 DNA mutation, wherein each of the probes is labeled with a unique label such that the identity of each probe is known by detecting the label;
detecting amplified DNA product hybridized to the set of probes; and
determining if a probe in the set of probes specific for the FKS1-HS1 DNA mutation or a probe in the set of probe specific for an FKS2-HS1 DNA mutation is bound to the amplified DNA product, wherein binding of the probe specific for the FKS1-HS1 DNA or the probe specific for an FKS2-HS1 DNA mutation indicates that the C. glabrata is resistant to the echinocandin is present in the sample.
2. The method of claim 1 , wherein the method further comprises contacting the amplified DNA product with a probe that specifically binds a FKS1-HS1 wild type nucleic acid comprising the nucleic acid sequence set forth as SEQ ID NO: 19.
3. The method of claim 1 , wherein the method further comprises contacting the amplified DNA product with a probe that specifically binds a FKS2-HS1 wild type nucleic acid comprising the nucleic acid sequence set forth as SEQ ID NO: 20 or SEQ ID NO: 21.
4. The method of claim 1 , wherein the set of probes specific for the FKS1-HS1 mutation comprises:
a) a probe that specifically binds FKS1-HS1 encoding FKS1 with a 629P mutation;
b) a probe that specifically binds FKS1-HS1 encoding FKS1 with a 631G mutation; and
c) a probe that specifically binds FKS1-HS1 encoding FKS1 with a 632V mutation, wherein each capture probe in labeled with a unique detectable label.
5. The method of claim 4 , wherein:
a) the probe that specifically binds FKS1-HS1 encoding FKS1 with a 629P mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 5;
b) the probe that specifically binds FKS1-HS1 encoding FKS1 with a 631G mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 6; and
c) the probe that specifically binds FKS1-HS1 encoding FKS1 with a 632V mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 7.
6. The method of claim 1 , wherein the method further comprises contacting the amplified DNA product with
a) a probe that specifically binds FKS1-HS1 DNA encoding FKS1 with a smC mutation; and/or
b) a probe that specifically binds FKS1-HS1 encoding FKS1 with a smT mutation.
7. The method of claim 6 , wherein:
a) the probe that specifically binds FKS1-HS1 DNA encoding FKS1 with a smC mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 3; and/or
b) the probe that specifically binds FKS1-HS1 encoding FKS1 with a smT mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 4.
8. The method of claim 1 , wherein the set of probes specific for the FKS2-HS1 mutation comprises:
a) a probe that specifically binds FKS2-HS1 DNA encoding FKS2 with a 659Y mutation;
b) a probe that specifically binds FKS2-HS1 encoding FKS2 with a 659S mutation;
c) a probe that specifically binds FKS2-HS1 encoding FKS2 with a 663P mutation;
d) a probe that specifically binds FKS2-HS1 encoding FKS2 with a 663F mutation;
e) a probe that specifically binds FKS2-HS1 encoding FKS2 with a 665G mutation;
f) a probe that specifically binds FKS2-HS1 encoding FKS2 with a 666V mutation; and
g) a probe that specifically binds FKS2-HS1 encoding FKS2 with a 667H mutation.
9. The method of claim 6 , wherein:
a) the probe that specifically binds FKS2-HS1 DNA encoding FKS1 with a 659Y mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 8;
b) the probe that specifically binds FKS2-HS1 encoding FKS1 with a 659S mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 9;
c) a probe that specifically binds FKS2-HS1 encoding FKS1 with a 663P mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 10;
d) a probe that specifically binds FKS2-HS1 encoding FKS1 with a 663P mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 11;
e) a probe that specifically binds FKS2-HS1 encoding FKS1 with a 665G mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 12;
f) a probe that specifically binds FKS2-HS1 encoding FKS1 with a 666V mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 13; and
g) a probe that specifically binds FKS2-HS1 encoding FKS1 with a 667H mutation comprises the nucleic acid sequence set forth as SEQ ID NO: 14.
10. The method of claim 1 , wherein the forward primer that can be used to amplify a FKS1-HS1 nucleic acid comprises the nucleic acid sequence set forth as SEQ ID NO: 15, and the reverse primer that can be used to amplify a FKS1-HS1 nucleic acid comprises the nucleic acid sequence set forth as SEQ ID NO: 16.
11. The method of claim 10 , wherein either the forward primer that can be used to amplify a FKS1-HS1 nucleic acid or the reverse primer that can be used to amplify the FKS1-HS1 nucleic acid is labeled.
12. The method of claim 11 , wherein the label is biotin.
13. The method of claim 12 , further comprising
contacting the amplified DNA product with streptavidin coupled to a unique label, and
detecting the unique label, wherein detection of the label indicates the presence of FKS1-HS1 amplified DNA.
14. The method of claim 1 , wherein the forward primer that can be used to amplify a FKS2-HS1 nucleic acid comprise the nucleic acid sequence set forth as SEQ ID NO: 17, and the reverse primer that can be used to amplify a FKS2-HS1 nucleic acid comprise the nucleic acid sequence set forth as SEQ ID NO: 18.
15. The method of claim 14 , wherein either the forward primer that can be used to amplify a FKS2-HS1 nucleic acid or the reverse primer that can be used to amplify the FKS2-HS1 nucleic acid is labeled with a unique label.
16. The method of claim 15 , wherein the label is biotin.
17. The method of claim 16 , further comprising
contacting the amplified DNA product with streptavidin coupled to a unique label, and
detecting the unique label, wherein detection of the label indicates the presence of amplified FKS2-HS1 DNA.
18. The method of claim 2 , wherein the probe specific for wild-type FKS1-HS1 DNA is labeled with a unique label.
19. The method of claim 18 , wherein the probe specific for wild-type FKS1-HS1 DNA comprises the nucleic acid sequence set forth as SEQ ID NO: 19.
20. The method of claim 3 , wherein the probe specific for wild-type FKS2-HS1 DNA is labeled with a unique label.
21. The method of claim 20 , wherein the probe specific for wild-type FKS2-HS1 DNA comprises the nucleic acid sequence set forth as SEQ ID NO: 20.
22. The method of claim 20 , further comprising contacting the amplified DNA product with a second probe specific for wild-type FKS2-HS1 DNA labeled with a unique label.
23. The method of claim 22 , wherein the second probe specific for wild-type FKS2-HS1 DNA comprises the nucleic acid sequence set forth as SEQ ID NO: 21.
24. The method of claim 1 , wherein performing asymmetric PCR comprises performing quantitative PCR.
25. The method of claim 1 , wherein each of the probes in the set of probes specific for the FKS1-HS1 mutation and each of the probes in the set of probes specific for the FKS1-HS2 mutation is also labeled with a magnetic label.