ASSAYS FOR FUNGAL INFECTION

• US 20100221710A1
• Filed: 11/01/2007
• Published: 09/02/2010
• Est. Priority Date: 11/02/2006
• Status: Active Grant

• Current Assignee: Rutgers University
• Original Assignee: University of Manchester
• Inventors: Denning, David Wemyss, Park, Steven, Perlin, David S.
• Granted Patent: US 9,657,355 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

Methods and kits are described for testing for the presence or absence of any fungus in a sample. Examples of fungi that can be detected include, but are not limited to, those belonging to the genera Candida, Aspergillus and Pneumocystis. The methods include obtaining a sample suspected of containing fungal nucleic acid, including at least one universal region of fungal nucleic acid, and testing for the presence or absence in the sample of the at least one universal region of fungal nucleic acid. Samples may be biological or non-biological.

Claims

1. A method of testing for the presence or absence of any fungus in a sample, comprising obtaining a sample suspected of containing fungal nucleic acid, including at least one universal region of fungal nucleic acid, and testing for the presence or absence in the sample of the at least one universal region of fungal nucleic acid, in which the at least one universal region of fungal nucleic acid includes SEQ ID NOS:

1, 52 or 53, a complement or transcript of SEQ ID NOS;

1, 52 or 53, or a sequence having 80% or more sequence homology with SEQ ID NOS;

1, 52 or 53, or complements or transcripts thereof.

2. The method of claim 1 in which the sample is obtained from a biological source.

3. The method of claim 2 in which the sample comprises a biological fluid, tissue, or combination thereof.

4. The method of claim 1 in which the sample is obtained from a non-biological source.

5. The method of claim 4 in which the sample comprises a piece of a vehicle, watercraft, aircraft, building, or dwelling.

6. The method of claim 1 in which the sample is obtained from an environment.

7. The method of claim 6 in which the sample comprises an air sample, a water sample, a soil sample, or combinations thereof.

8. The method of claim 1 in which the testing step includes contacting the sample with an oligonucleotide probe comprising a nucleic acid capable of hybridizing to the at least one universal region of fungal nucleic acid under stringent conditions.

9. The method of claim 8 in which the probe includes a detectable label.

10. The method of claim 8 in which the probe includes SEQ ID NO:

4, complement or transcript thereof, or a sequence having 90% or more sequence homology with SEQ ID NO;

4, complement or transcript thereof.

11. The method of claim 8 in which the probe includes SEQ ID NO:

44, its complement, or a sequence having 90% or more sequence homology with SEQ ID NO;

44 or its complement.

12. The method of claim 8 in which the probe includes SEQ ID NO:

45, its complement, or a sequence having 90% or more sequence homology with SEQ ID NO;

45 or its complement

13. The method of claim 8, further comprising testing for the presence or absence in the sample of at least one region of fungal nucleic acid characteristic of the genus Candida wherein the at least one region of fungal nucleic acid characteristic of the genus Candida includes SEQ ID NO:

5, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

5, complement or transcript thereof; and

testing for the presence or absence in the sample of at least one region of fungal nucleic acid characteristic of the genus Aspergillus wherein the at least one region of fungal nucleic acid characteristic of the genus Aspergillus includes SEQ ID NO;

14, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

14, complement or transcript thereof.

14. The method of claim 13, further comprising amplifying the regions of fungal nucleic acid and wherein said testing comprises the use of (A) at least two amplification primers selected from the group consisting of SEQ ID NO:

48, SEQ ID NO;

49, SEQ ID NO;

50 and SEQ ID NO;

51; and

(B) at least one oligonucleotide probe selected from the group consisting of SEQ ID NO;

44 and SEQ ID NO;

45.

15. The method of claim 14, further comprising the use of amplification primers having the sequences of SEQ ID NO:

6, SEQ ID NO;

7, SEQ ID NO;

46 and SEQ ID NO;

47, and oligonucleotides probes having the sequences of SEQ ID NO;

8 and SEQ ID NO;

17.

16. The method of claim 1 further comprising amplifying the at least one universal region of fungal nucleic acid.

17. The method of claim 16 in which the amplifying step includes contacting the sample with a pair of primers.

18. The method of claim 17 wherein the pair of primers includes SEQ ID NO:

2 and SEQ ID NO;

3.

19. The method of claim 17 wherein the pair of primers includes SEQ ID NO:

48 and SEQ ID NO;

49.

20. The method of claim 19 wherein amplicons from said amplification step are detected by hybridizing with a labeled oligonucleotide of SEQ ID NO:

44 or SEQ ID NO;

45.

21. The method of claim 17 in which the pair of primers includes SEQ ID NO:

50 and SEQ ID NO;

51.

22. The method of claim 21 wherein amplicons from said amplification step are detected by hybridizing with a labeled oligonucleotide of SEQ ID NO:

44 or SEQ ID NO;

45.

55. The method of claim 13 in which the amplifying step is carried out in the presence of one or more internal PCR amplification controls to ensure appropriate amplification of any fungal nucleic acid present in the sample.

56. The method of claim 55 in which the one or more internal PCR amplification controls comprise a non-fungal sequence.

57. The method of claim 55 in which the amplifying step is carried out in the presence of a cloned or synthesized tRNA-LEU intron region, which is added to the amplification mixture in a predetermined amount to rule out the presence of inhibitors or other defective amplification steps.

58. The method of claim 57 in which the tRNA-LEU intron region comprises a portion of the Maize (Zea mayis) tRNA-LEU intron region.

59. The method of claim 58 in which the Maize (Zea mayis) tRNA-LEU intron region includes SEQ ID NO:

9.

60. The method of claim 59 further comprising detecting the presence of a nucleic acid including SEQ ID NO:

10.

61. The method of claim 60 in which the detecting step comprises contacting the sample with a pair of oligonucleotides primers including SEQ ID NO:

11 and SEQ ID NO;

12 and a molecular beacon probe including SEQ ID NO;

13.

62. The method of claim 1 in which the nucleic acid comprises DNA.

63. The method of claim 1 in which the nucleic acid comprises RNA.

23. A method of testing for the presence or absence of a fungus belonging to the genus Aspergillus in a sample, comprising obtaining a sample suspected of containing fungal nucleic acid, including at least one region of fungal nucleic acid characteristic of the genus Aspergillus, and testing for the presence or absence in the sample of the at least one region of fungal nucleic acid characteristic of the genus Aspergillus.

24. The method of claim 23 in which the at least one region of fungal nucleic acid characteristic of the genus Aspergillus includes SEQ ID NO:

14, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

14, complement or transcript thereof.

25. The method of claim 24 in which the testing step includes contacting the sample with a probe including SEQ ID NO:

17.

26. The method of claim 25 which further comprises amplifying the at least one region of fungal nucleic acid characteristic of the genus Aspergillus by contacting the sample with a pair of primers including SEQ ID NO:

15 and SEQ ID NO;

16.

27. The method of claim 23 which further comprises amplifying the at least one region of fungal nucleic acid characteristic of the genus Aspergillus by contacting the sample with a pair of primers including SEQ ID NO:

46 and SEQ ID NO;

47.

28. The method of claim 23, further comprising testing for the presence or absence in the sample of at least one region of fungal nucleic acid characteristic of the genus Pneumocystis jirovecii.

29. The method of claim 28, wherein the testing comprises the use of amplification primers having the sequences of SEQ ID NO:

15, SEQ ID NO;

16, SEQ ID NO;

19, and SEQ ID NO;

20 and oligonucleotide probes having the sequences of SEQ ID NO;

17 and SEQ ID NO;

21.

30. A method of testing for the presence or absence of a fungus belonging to the genus Candida in a sample, comprising obtaining a sample suspected of containing fungal nucleic acid, including at least one region of fungal nucleic acid characteristic of the genus Candida, and testing for the presence or absence in the sample of the at least one region of fungal nucleic acid characteristic of the genus Candida.

31. The method of claim 30 in which the at least one region of fungal nucleic acid characteristic of the genus Candida includes SEQ ID NO:

5, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

5, complement or transcript thereof.

32. The method of claim 31 in which the testing step includes contacting the sample with a probe including SEQ ID NO:

8.

33. The method of claim 32 further comprising amplifying the at least one region of fungal nucleic acid characteristic of the genus Candida by contacting the sample with a pair of primers including SEQ ID NO:

6 and SEQ ID NO;

7.

34. A method of testing for the presence or absence of a fungus belonging to the genus Pneumocystis in a sample, comprising obtaining a sample suspected of containing fungal nucleic acid, including at least one region of fungal nucleic acid characteristic of the genus Pneumocystis, and testing for the presence or absence in the sample of the at least one region of fungal nucleic acid characteristic of the genus Pneumocystis.

35. The method of claim 34 in which the at least one region of fungal nucleic acid characteristic of the genus Pneumocystis includes SEQ ID NO:

18, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

18, complement or transcript thereof.

36. The method of claim 35 in which the testing step includes contacting the sample with a probe including SEQ ID NO:

21.

37. The method of claim 36 further comprising amplifying the at least one region of fungal nucleic acid characteristic of the genus Pneumocystis by contacting the sample with a pair of primers including SEQ ID NO:

19 and SEQ ID NO;

20.

38. A method of testing for the presence or absence of a fungus belonging to the species Candida tropicalis in a sample, comprising obtaining a sample suspected of containing fungal nucleic acid, including at least one region of fungal nucleic acid characteristic of the species Candida tropicalis, and testing for the presence or absence in the sample of the at least one region of fungal nucleic acid characteristic of the species Candida tropicalis.

39. The method of claim 38 in which the at least one region of fungal nucleic acid characteristic of the species Candida tropicalis includes SEQ ID NO:

30, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

30, complement or transcript thereof.

40. The method of claim 39 in which the testing step includes contacting the sample with a probe including SEQ ID NO:

33.

53. The method of claim 38, which further comprises amplifying the at least one region of fungal nucleic acid characteristic of the Candida species by contacting the sample with a pair of primers including SEQ ID NO:

31 and SEQ ID NO;

32.

41. A method of testing for the presence or absence of a fungus belonging to the species Candida parapsilosis in a sample, comprising obtaining a sample suspected of containing fungal nucleic acid, including at least one region of fungal nucleic acid characteristic of the species Candida parapsilosis, and testing for the presence or absence in the sample of the at least one region of fungal nucleic acid characteristic of the species Candida parapsilosis.

42. The method of claim 41 in which the at least one region of fungal nucleic acid characteristic of the species Candida parapsilosis includes SEQ ID NO:

34, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

34, complement or transcript thereof

43. The method of claim 42 in which the testing step includes contacting the sample with a probe including SEQ ID NO:

35.

75. The method of claim 41, which further comprises amplifying the at least one region of fungal nucleic acid characteristic of the Candida species by contacting the sample with a pair of primers including SEQ ID NO:

31 and SEQ ID NO;

32.

44. A method of testing for the presence or absence of a fungus belonging to the species Candida albicans in a sample, comprising obtaining a sample suspected of containing fungal nucleic acid, including at least one region of fungal nucleic acid characteristic of the species Candida albicans, and testing for the presence or absence in the sample of the at least one region of fungal nucleic acid characteristic of the species Candida albicans.

45. The method of claim 44 in which the at least one region of fungal nucleic acid characteristic of the species Candida albicans includes SEQ ID NO:

36, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

36, complement or transcript thereof.

46. The method of claim 45 in which the testing step includes contacting the sample with a probe including SEQ ID NO:

37.

76. The method of claim 44, which further comprises amplifying the at least one region of fungal nucleic acid characteristic of the Candida species by contacting the sample with a pair of primers including SEQ ID NO:

31 and SEQ ID NO;

32.

47. A method of testing for the presence or absence of a fungus belonging to the species Candida glabrata in a sample, comprising obtaining a sample suspected of containing fungal nucleic acid, including at least one region of fungal nucleic acid characteristic of the species Candida glabrata, and testing for the presence or absence in the sample of the at least one region of fungal nucleic acid characteristic of the species Candida glabrata.

48. The method of claim 47 in which the at least one region of fungal nucleic acid characteristic of the species Candida glabrata includes SEQ ID NO:

38, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

38, complement or transcript thereof.

49. The method of claim 48 in which the testing step includes contacting the sample with a probe including SEQ ID NO:

39.

77. The method of claim 47, which further comprises amplifying the at least one region of fungal nucleic acid characteristic of the Candida species by contacting the sample with a pair of primers including SEQ ID NO:

31 and SEQ ID NO;

32.

50. A method of testing for the presence or absence of a fungus belonging to the species Candida krusei in a sample, comprising obtaining a sample suspected of containing fungal nucleic acid, including at least one region of fungal nucleic acid characteristic of the species Candida krusei, and testing for the presence or absence in the sample of the at least one region of fungal nucleic acid characteristic of the species Candida krusei.

51. The method of claim 50 in which the at least one region of fungal nucleic acid characteristic of the species Candida krusei includes SEQ ID NO:

40, a complement or transcript thereof, or a sequence having 80% or more sequence homology with SEQ ID NO;

40, complement or transcript thereof.

52. The method of claim 51 in which the testing step includes contacting the sample with a probe including SEQ ID NO:

43.

54. The method of claim 50 further comprising amplifying the at least one region of fungal nucleic acid characteristic of the Candida species by contacting the sample with a pair of primers including SEQ ID NO:

41 and SEQ ID NO;

42.

78. The method of claim 50, which further comprises amplifying the at least one region of fungal nucleic acid characteristic of the Candida species by contacting the sample with a pair of primers including SEQ ID NO:

31 and SEQ ID NO;

32.

64. A panfungal nucleic acid testing kit comprising a molecular beacon probe including SEQ ID NO:

4 and a pair of primers including SEQ ID NO;

2 and SEQ ID NO;

3.

69. The kit of claim 64 further comprising one or more internal PCT amplification controls.

65. A kit for testing for the presence or absence of at least one region of fungal nucleic acid characteristic of the genus Candida in a sample comprising a molecular beacon probe including SEQ ID NO:

8 and a pair of primers including SEQ ID NO;

6 and SEQ ID NO;

7.

66. A kit for testing for the presence or absence of at least one region of fungal nucleic acid characteristic of the genus Aspergillus in a sample comprising a molecular beacon probe including SEQ ID NO:

17 and a pair of primers including SEQ ID NO;

15 and SEQ ID NO;

16.

67. A kit for testing for the presence or absence of at least one region of fungal nucleic acid characteristic of the genus Pneumocystis in a sample comprising a molecular beacon probe including SEQ ID NO:

21 and a pair of primers including SEQ ID NO;

19 and SEQ ID NO;

20.

68. A kit for testing for the presence or absence of at least one region of fungal nucleic acid characteristic of a species of Candida in a sample comprising one or more molecular beacon probes including SEQ ID NO:

33, SEQ ID NO;

35, SEQ ID NO;

37, SEQ ID NO;

39, SEQ ID NO;

43, or combinations thereof and a pair of primers including SEQ ID NO;

31 and SEQ ID NO;

32, SEQ ID NO;

41 and SEQ ID NO;

42, or both pairs.

70. An isolated polynucleotide having the sequence of SEQ ID NOS:

44-45.

71. An isolated polynucleotide having the sequence of SEQ ID NOS:

46-51.

72. A fungal detection kit comprising (A) at least two amplification primers selected from the group consisting of SEQ ID NO:

48, SEQ ID NO;

49, SEQ ID NO;

50 and SEQ ID NO;

51; and

(B) at least one oligonucleotide probe selected from the group consisting of SEQ ID NO;

44 and SEQ ID NO;

45.

73. The fungal detection kit of claim 72, further comprising amplification primers SEQ ID NO:

6, SEQ ID NO;

7, SEQ ID NO;

46, and SEQ ID NO;

47, and oligonucleotide probes SEQ ID NO;

8, and SEQ ID NO;

17.

74. A fungal detection kit comprising (A) amplification primers having the sequences of SEQ ID NO:

15, SEQ ID NO;

16, SEQ ID NO;

19, and SEQ ID NO;

20 and (B) oligonucleotide probes having the sequences of SEQ ID NO;

17 and SEQ ID NO;

21.