PNA probes, probe sets, methods and kits pertaining to the detection of candida

• US 20030175727A1
• Filed: 05/17/2002
• Published: 09/18/2003
• Est. Priority Date: 05/18/2001
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Boston Probes, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Hyldig-Nielsen, Jens J., Oliveira, Kenneth M., Stender, Henrik, Rigby, Susan
• Granted Patent: US 8,026,051 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid

Abstract

3This invention is related to novel PNA probes, probe sets, methods and kits pertaining to the detection of one or more species of Candida yeast. Non-limiting examples of probing nucleobase sequences that can be used for the probes of this invention can be selected from the group consisting of: AGA-GAG-CAG-CAT-GCA (Seq. Id. No. 1), AGA-GAG-CAA-CAT-GCA (Seq. Id. No. 2), ACA-GCA-GAA-GCC-GTG (Seq. Id. No. 3), CAT-AAA-TGG-CTA-CCA-GA (Seq. Id. No. 4), CAT-AAA-TGG-CTA-CCC-AG (Seq. Id. No. 5), ACT-TGG-AGT-CGA-TAG (Seq. Id. No. 6), CCA-AGG-CTT-ATA-CTC-GC (Seq. Id. No. 7), CCC-CTG-AAT-CGG-GAT (Seq. Id. No. 8), GAC-GCC-AAA-GAC-GCC (Seq. Id. No. 9), ATC-GTC-AGA-GGC-TAT-AA (Seq. Id. No. 10), TAG-CCA-GAA-GAA-AGG (Seq. Id. No. 11), CAT-AAA-TGG-CTA-GCC-AG (Seq. Id. No. 12), CTC-CGA-TGT-GAC-TGC-G (Seq. Id. No. 13), TCC-CAG-ACT-GCT-CGG (Seq. Id. No. 14), TCC-AAG-AGG-TCG-AGA (Seq. Id. No. 15), GCC-AAG-CCA-CAA-GGA (Seq. Id. No. 16), GCC-GCC-AAG-CCA-CA (Seq. Id. No. 17), GGA-CTT-GGG-GTT-AG (Seq. Id. No. 18), CCG-GGT-GCA-TTC-CA (Seq. Id. No. 19), ATG-TAG-AAC-GGA-ACT-A (Seq. Id. No. 20), GAT-TCT-CGG-CCC-CAT-G (Seq. Id. No. 21), CTG-GTT-CGC-CAA-AAA-G (Seq. Id. No. 22) and AGT-ACG-CAT-CAG-AAA (Seq. Id. No. 23).

Claims

1. A PNA probe comprising a probing nucleobase sequence suitable for detecting, identifying and/or quantitating one or more species of Candida yeast in a sample.

2. The PNA probe of claim 1, wherein at least a portion of the probing nucleobase sequence is at least ninety percent homologous to the nucleobase sequences, or their complements, selected from the group consisting of:

3. The PNA probe of claim 1, wherein the probe is unlabeled.

4. The PNA probe of claim 1, wherein the probe is labeled with at least one detectable moiety.

5. The PNA probe of claim 4, wherein the detectable moiety or moieties are selected from the group consisting of:

a dextran conjugate, a branched nucleic acid detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester and a chemiluminescent compound.

6. The PNA probe of claim 1, wherein the probe is labeled with at least two independently detectable moieties.

7. The PNA probe of claim 6, wherein the two or more independently detectable moieties are independently detectable fluorophores.

8. The PNA probe of claim 1, wherein the probe is support bound.

9. The PNA probe of claim 1, wherein the probe is self-indicating.

10. The PNA probe of claim 1, wherein in situ hybridization is used to detect, identify or quantitate one or more organisms of Candida in the sample.

11. A PNA probe set suitable for detecting, identifying or quantitating one or more species of Candida yeast in a sample wherein at least one species of interest is selected from the group consisting of:

C. albicans, C. dubliniensis, C. krusei, C. glabrata, C. parapsilosis and C. tropicalis.

12. The probe set of claim 11, wherein at least one PNA probe of the set comprises a probing nucleobase sequence wherein at least a portion is at least ninety percent homologous to the nucleobase sequences, or their complements, selected from the group consisting of:

AGA-GAG-CAG-CAT-GCA (Seq. Id. No.

     1), AGA-GAG-CAA-CAT-GCA (Seq. Id. No.

     2), ACA-GCA-GAA-GCC-GTG (Seq. Id. No.

     3), CAT-AAA-TGG-CTA-CCA-GA (Seq. Id. No.

     4), CAT-AAA-TGG-CTA-CCC-AG (Seq. Id. No.

     5), ACT-TGG-AGT-CGA-TAG (Seq. Id. No.

     6), CCA-AGG-CTT-ATA-CTC-GC (Seq. Id. No.

     7), CCC-CTG-AAT-CGG-GAT (Seq. Id. No.

     8), GAC-GCC-AAA-GAC-GCC (Seq. Id. No.

     9), ATC-GTC-AGA-GGC-TAT-AA (Seq. Id. No.

     10), TAG-CCA-GAA-GAA-AGG (Seq. Id. No.

     11), CAT-AAA-TGG-CTA-GCC-AG (Seq. Id. No.

     12), CTC-CGA-TGT-GAC-TGC-G (Seq. Id. No.

     13), TCC-CAG-ACT-GCT-CGG (Seq. Id. No.

     14), TCC-AAG-AGG-TCG-AGA (Seq. Id. No.

     15), GCC-AAG-CCA-CAA-GGA (Seq. Id. No.

     16), GCC-GCC-AAG-CCA-CA (Seq. Id. No.

     17), GGA-CTT-GGG-GTT-AG (Seq. Id. No.

     18), CCG-GGT-GCA-TTC-CA (Seq. Id. No.

     19), ATG-TAG-AAC-GGA-ACT-A (Seq. Id. No.

     20), GAT-TCT-CGG-CCC-CAT-G (Seq. Id. No.

     21), CTG-GTT-CGC-CAA-AAA-G (Seq. Id. No.

     22) and AGT-ACG-CAT-CAG-AAA (Seq. Id. No.

     23).

13. The probe set of claim 11, wherein a PNA probe for C. albicans is selected from the group consisting of:

AGA-GAG-CAG-CAT-GCA (Seq. Id. No.

     1), AGA-GAG-CAA-CAT-GCA (Seq. Id. No.

     2), ACA-GCA-GAA-GCC-GTG (Seq. Id. No.

     3), CAT-AAA-TGG-CTA-CCA-GA (Seq. Id. No.

     4), CAT-AAA-TGG-CTA-CCC-AG (Seq. Id. No.

     5), ACT-TGG-AGT-CGA-TAG (Seq. Id. No.

     6), CCA-AGG-CTT-ATA-CTC-GC (Seq. Id. No.

     7) and CCC-CTG-AAT-CGG-GAT (Seq. Id. No.

     8).

14. The probe set of claim 11, wherein a PNA probe for C. dubliniesis is selected from the group consisting of:

TAG-CCA-GAA-GAA-AGG (Seq. Id. No.

     11), CAT-AAA-TGG-CTA-GCC-AG (Seq. Id. No.

     12), CTC-CGA-TGT-GAC-TGC-G (Seq. Id. No.

     13) and TCC-CAG-ACT-GCT-CGG (Seq. Id. No.

     14).

15. The probe set of claim 11, wherein a PNA probe for C. glabrata is selected from the group consisting of:

TCC-AAG-AGG-TCG-AGA (Seq. Id. No.

     15), GCC-AAG-CCA-CAA-GGA (Seq. Id. No.

     16), GCC-GCC-AAG-CCA-CA (Seq. Id. No.

     17), GGA-CTT-GGG-GTT-AG (Seq. Id. No.

     18) and CCG-GGT-GCA-TTC-CA (Seq. Id. No.

     19).

16. The probe set of claim 11, wherein a PNA probe for C. krusei is selected from the group consisting of:

ATG-TAG-AAC-GGA-ACT-A (Seq. Id. No.

     20) and GAT-TCT-CGG-CCC-CAT-G (Seq. Id. No.

     21).

17. The probe set of claim 11, wherein at least one probe of the set is unlabeled.

18. The probe set of claim 11, wherein all probes of the set are unlabeled.

19. The probe set of claim 11, wherein at least one probe is labeled with a detectable moiety.

20. The probe set of claim 11, wherein all probes of the set are labeled with one or more detectable moieties.

21. The probe set of claim 19, wherein the detectable moiety or moieties are selected from the group consisting of:

a dextran conjugate, a branched nucleic acid detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester and a chemiluminescent compound.

22. The probe set of claim 11, wherein at least one probe of the set is labeled with at least two independently detectable moieties.

23. The probe set of claim 22, wherein the two or more independently detectable moieties are independently detectable fluorophores.

24. The probe set of claim 11, wherein in situ hybridization is used to detect, identify or quantitate one or more organisms in the sample.

25. The probe set of claim 11, wherein two or more probes of the set are independently detectable.

26. The probe set of claim 11, wherein at least one probe of the set is self-indicating.

27. The probe set of claim 11, wherein at least one probe of the set is support bound.

28. A method for detecting, identifying and/or quantitating one or more species of Candida yeast in a sample;

said method comprising the steps of;

a) contacting the sample with one or more PNA probes, wherein the one or more PNA probes have a probing nucleobase sequence that is at least ninety percent homologous to the nucleobase sequences, or their complements, selected from the group consisting of;

AGA-GAG-CAG-CAT-GCA,(Seq. Id. No.

     1) AGA-GAG-CAA-CAT-GCA,(Seq. Id. No.

     2) ACA-GCA-GAA-GCC-GTG,(Seq. Id. No.

     3) CAT-AAA-TGG-CTA-CCA-GA,(Seq. Id. No.

     4) CAT-AAA-TGG-CTA-CCC-AG,(Seq. Id. No.

     5) ACT-TGG-AGT-CGA-TAG,(Seq. Id. No.

     6) CCA-AGG-CTT-ATA-CTC-GC,(Seq. Id. No.

     7) CCC-CTG-AAT-CGG-GAT,(Seq. Id. No.

     8) GAC-GCC-AAA-GAC-GCC,(Seq. Id. No.

     9) ATC-GTC-AGA-GGC-TAT-AA,(Seq. Id. No.

     10) TAG-CCA-GAA-GAA-AGG,(Seq. Id. No.

     11) CAT-AAA-TGG-CTA-GCC-AG,(Seq. Id. No.

     12) CTC-CGA-TGT-GAC-TGC-G,(Seq. Id. No.

     13) TCC-CAG-ACT-GCT-CGG,(Seq. Id. No.

     14) TCC-AAG-AGG-TCG-AGA,(Seq. Id. No.

     15) GCC-AAG-CCA-CAA-GGA,(Seq. Id. No.

     16) GCC-GCC-AAG-CCA-CA,(Seq. Id. No.

     17) GGA-CTT-GGG-GTT-AG,(Seq. Id. No.

     18) CCG-GGT-GCA-TTC-CA,(Seq. Id. No.

     19) ATG-TAG-AAC-GGA-ACT-A,(Seq. Id. No.

     20) GAT-TCT-CGG-CCC-CAT-G,(Seq. Id. No.

     21) CTG-GTT-CGC-CAA-AAA-G(Seq. Id. No.

     22)and AGT-ACG-CAT-CAG-AAA;

(Seq. Id. No.

     23)and

b) detecting, identify and/or quantitating hybridization of the probing nucleobase sequence of a PNA probe to the target sequence of an organism of interest in the sample, under suitable hybridization conditions or suitable in-situ hybridization conditions, and correlating the result with the presence, absence or quantity of Candida yeast in the sample.

29. The method of claim 28, wherein at least one of the PNA probes is unlabeled.

30. The method of claim 28, wherein the one or more PNA probes are all unlabeled.

31. The method of claim 28, wherein at least one PNA probe is labeled with a detectable moiety.

32. The method of claim 28, wherein all probes of the set are labeled with one or more detectable moieties.

33. The method of claim 31, wherein the detectable moiety or moieties are selected from the group consisting of:

a dextran conjugate, a branched nucleic acid detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester and a chemiluminescent compound.

34. The method of claim 28, wherein at least one PNA probe is labeled with at least two independently detectable moieties.

35. The method of claim 34, wherein the two or more independently detectable moieties are independently detectable fluorophores.

36. The method of claim 29, wherein in situ hybridization using a fluorophore or enzyme-linked probe is used to determine the presence, absence or number of organisms of Candida yeast in the sample.

37. The method of claim 29, wherein a set of at least two PNA probes is used in the assay.

38. The method of claim 37, wherein two or more PNA probes are independently detectable.

39. The method of claim 29, wherein one or more probes of the set are labeled with two or more independently detectable moieties.

40. The method of claim 39, wherein the two or more independently detectable moieties are independently detectable fluorophores.

41. The method of claim 29, wherein at least one PNA probe is self-indicating.

42. The method of claim 29, wherein at least one PNA probe is support bound.

43. A kit suitable for performing an assay that determines the presence, absence or quantity of one or more species of Candida yeast in a sample, wherein said kit comprises:

a) one or more PNA probes, wherein the PNA probes comprise a probing nucleobase sequence such that at least a portion is at least ninety percent homologous to the nucleobase sequences, or their complements, selected from the group consisting of;

AGA-GAG-CAG-CAT-GCA,(Seq. Id. No.

     1) AGA-GAG-CAA-CAT-GCA,(Seq. Id. No.

     2) ACA-GCA-GAA-GCC-GTG,(Seq. Id. No.

     3) CAT-AAA-TGG-CTA-CCA-GA,(Seq. Id. No.

     4) CAT-AAA-TGG-CTA-CCC-AG,(Seq. Id. No.

     5) ACT-TGG-AGT-CGA-TAG,(Seq. Id. No.

     6) CCA-AGG-CTT-ATA-CTC-GC,(Seq. Id. No.

     7) CCC-CTG-AAT-CGG-GAT,(Seq. Id. No.

     8) GAC-GCC-AAA-GAC-GCC,(Seq. Id. No.

     9) ATC-GTC-AGA-GGC-TAT-AA,(Seq. Id. No.

     10) TAG-CCA-GAA-GAA-AGG,(Seq. Id. No.

     11) CAT-AAA-TGG-CTA-GCC-AG,(Seq. Id. No.

     12) CTC-CGA-TGT-GAC-TGC-G,(Seq. Id. No.

     13) TCC-CAG-ACT-GCT-CGG,(Seq. Id. No.

     14) TCC-AAG-AGG-TCG-AGA,(Seq. Id. No.

     15) GCC-AAG-CCA-CAA-GGA,(Seq. Id. No.

     16) GCC-GCC-AAG-CCA-CA,(Seq. Id. No.

     17) GGA-CTT-GGG-GTT-AG,(Seq. Id. No.

     18) CCG-GGT-GCA-TTC-CA,(Seq. Id. No.

     19) ATG-TAG-AAC-GGA-ACT-A,(Seq. Id. No.

     20) GAT-TCT-CGG-CCC-CAT-G,(Seq. Id. No.

     21) CTG-GTT-CGC-CAA-AAA-G(Seq. Id. No.

     22)and AGT-ACG-CAT-CAG-AAA;

(Seq. Id. No.

     23)and

b) other reagents or compositions necessary to perform the assay.

44. The kit of claim 43, wherein the kit is designed for the determination of one or more species of Candida selected from the group consisting of:

C. albicans, C. dubliniensis, C. krusei, C. glabrata, C. parapsilosis and C. tropicalis.

45. The kit of claim 43, wherein the probes of the kit are unlabeled.

46. The kit of claim 43, wherein at least one probe is labeled with a detectable moiety.

47. The kit of claim 43, wherein two or more probes are labeled with independently detectable moieties.

48. The kit of claim 47, wherein the independently detectable moieties are independently detectable fluorophores.

49. The kit of claim 43, wherein at least one probe is labeled with at least two independently detectable moieties.

50. The kit of claim 49, wherein the two or more independently detectable moieties are independently detectable fluorophores.

51. The kit of claim 45, wherein hybridization of the probing nucleobase sequence of the probe to the nucleic acid of the organism of interest is detected using an antibody or antibody fragment, wherein the antibody or antibody fragment specifically binds to the PNA/nucleic acid complex.

52. The kit of claim 51, further comprising an antibody labeled with a detectable moiety.

53. The kit of claim 52, wherein the detectable moiety is selected from the group consisting of a dextran conjugate, a branched nucleic acid detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester and a chemiluminescent compound.

54. The kit of claim 43, further comprising buffers and/or other reagents suitable for performing a PNA-ISH or PNA-FISH assay.

55. The kit of claim 43, further comprising buffers and/or other reagents suitable for performing a nucleic acid amplification reaction.

56. The kit of claim 43, wherein at least one PNA probe is self-indicating.