BROAD RANGE PCR-BASED COMPOSITIONS AND METHODS FOR THE DETECTION AND IDENTIFICATION OF FUNGAL PATHOGENS

• US 20100129821A1
• Filed: 11/24/2009
• Published: 05/27/2010
• Est. Priority Date: 11/26/2008
• Status: Active Grant

• Current Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Original Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Inventors: Ko, Daisy L., Fredricks, David N., Khot, Prasanna D.
• Granted Patent: US 9,803,250 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Disclosed herein are methods for detecting a fungal pathogen in a patient sample, involving isolating the sample, carrying out a PCR reaction on the sample to generate an amplicon that includes a region of the fungal 28S ribosomal RNA gene, and detecting the PCR amplicon. Also disclosed are sequences of primers for specifically detecting a broad range of fungal pathogens in the presence of human ribosomal DNA. In certain embodiments, the amplicon is detected by sequencing or by two-dimensional melt-curve analysis. In yet other embodiments, more than one fungal pathogen is detected in a sample using the methods disclosed herein.

Claims

1. A method for detecting a fungal pathogen in a patient sample, said method comprising the steps of:

(a) isolating a patient sample,

(b) carrying out a PCR reaction on said patient sample to generate a PCR amplicon that comprises a region of a fungal ribosomal RNA (rRNA) gene, wherein said PCR reaction uses a primer set comprising a forward primer and a reverse primer and wherein at least one of said forward primer and said reverse primer is complementary to said fungal rRNA gene, and

(c) detecting said PCR amplicon.

2. The method of claim 1 further comprising the step of extracting DNA from said isolated patient sample.

3. The method of claim 1 wherein said PCR amplicon comprises a region of a fungal internal transcribed spacer 1 (ITS-1) region and wherein at least one of said forward and said reverse primer is complementary to said fungal ITS-1 region.

4. The method of claim 1 wherein said PCR amplicon comprises a region of a fungal 28S rRNA gene and wherein at least one of said forward and said reverse primer is complementary to said fungal 28S rRNA gene.

5. The method of claim 4 wherein said region of a fungal 28S rRNA gene comprises a sequence that is 3′

to a D1-D2 highly variable region.

6. The method of claim 1, further comprising the step of sequencing said PCR amplicon.

7. The method of claim 1 wherein said PCR amplicon is between 50 and 1000 base pairs.

8. The method of claim 7 wherein said PCR amplicon is between 75 and 400 base pairs.

9. The method of claim 1 wherein said PCR reaction is a quantitative PCR reaction.

10. The method of claim 1 wherein said forward primer is complementary to a fungal 18S rRNA gene and said reverse primer is complementary to a fungal 28S rRNA gene.

11. The method of claim 10 wherein said forward primer comprises the nucleotide sequence 5′

-GTAAAAGTCGTAACAAGGTTTC-3′

(SEQ ID NO;

     1).

12. The method of claim 1 wherein said forward primer is complementary to a fungal 5.8S rRNA gene and said reverse primer is complementary to a fungal 28S rRNA gene.

13. The method of claim 12 wherein said forward primer comprises the nucleotide sequence 5′

-GTGAATCATCGARTCTTTGAAC-3′

(SEQ ID NO;

     2).

14. The method of claim 1 wherein said forward primer and said reverse primer are both complementary to a fungal 28S rRNA gene.

15. The method of claim 1 wherein said forward primer is selected from the group consisting of:

16. The method of claim 1 wherein said reverse primer is selected from the group consisting of:

17. The method of claim 1 wherein said primer set comprises a forward and reverse primer pair selected from the group consisting of (SEQ ID NO:

2 and SEQ ID NO;

     31), (SEQ ID NO;

2 and SEQ ID NO;

     32), (SEQ ID NO;

11 and SEQ ID NO;

     41), (SEQ ID NO;

1 and SEQ ID NO;

     29), (SEQ ID NO;

2 and SEQ ID NO;

     30), (SEQ ID NO;

12 and SEQ ID NO;

     41), (SEQ ID NO;

12, SEQ ID NO;

41, and SEQ ID NO;

     108), (SEQ ID NO;

14 and SEQ ID NO;

     42), (SEQ ID NO;

17 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     52), and (SEQ ID NO;

25 and SEQ ID NO;

     54).

18. The method of claim 1 wherein said primer set comprises a forward and reverse primer pair selected from the group consisting of (SEQ ID NO:

11 and SEQ ID NO;

     41), (SEQ ID NO;

12 and SEQ ID NO;

     41), (SEQ ID NO;

12, SEQ ID NO;

41, and SEQ ID NO;

     108), (SEQ ID NO;

14 and SEQ ID NO;

     42), (SEQ ID NO;

17 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     52), and (SEQ ID NO;

25 and SEQ ID NO;

     54).

19. The method of claim 17 wherein said primer set comprises a forward and reverse primer pair selected from the group consisting of (SEQ ID NO:

1 and SEQ ID NO;

     29), (SEQ ID NO;

2 and SEQ ID NO;

     30), (SEQ ID NO;

12 and SEQ ID NO;

     41), (SEQ ID NO;

12, SEQ ID NO;

41, and SEQ ID NO;

     108), (SEQ ID NO;

14 and SEQ ID NO;

     42), (SEQ ID NO;

17 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     52), and (SEQ ID NO;

25 and SEQ ID NO;

     54).

20. The method of claim 19 wherein said primer set comprises a forward and reverse primer pair selected from the group consisting of (SEQ ID NO:

2 and SEQ ID NO;

     30), (SEQ ID NO;

12, SEQ ID NO;

41, and SEQ ID NO;

     108), and (SEQ ID NO;

12 and SEQ ID NO;

     41).

21. The method of claim 1 wherein said fungal pathogen is selected from the group consisting of Absidia corymbifera;

Cunninghamella bertholletiae;

Fusarium solani;

Mucor racemosus;

Paecilomyces variotii;

Penicillium chrysogenum;

Rhizomucor miehei;

Rhodotorula glutinis;

Scedosporium apiospermum;

Antrodia vaillantii;

Aspergillus fumigatus;

Aspergillus niger;

Aspergillus oryzae;

Aspergillus terreus;

Batrachochytrium dendrobatidis;

Botrytis cinerea;

Candida albicans;

Candida dublineinsis;

Candida glabrata;

Candida gulliermundei;

Candida kefyr;

Candida krusei;

Candida lipolytica;

Candida lusitaniae;

Candida parapsilosis;

Candida tropicalis;

Chaetomium globosum;

Coccidioides immitis;

Coccidioides posadasii;

Cryptococcus neoformans;

Fusarium graminearum;

Fusarium oxysporum;

Histoplasma capsulatum;

Hypocrea jecorina;

Lodderomyces elongisporus;

Magnaporthe grisea;

Metarhizium anisopliae;

Microsporum gypseum;

Mucor racemosus;

Neurospora crassa;

Paracoccidioides brasiliens;

Pneumocystis carinii;

Penicillium verrucosum;

Pichia stipitis;

Rhizomucor miehei;

Rhizopus oryzae;

Saccharomyces cerevisiae;

Schizosaccharomyces japonicus;

Schizosaccharomyces pombe;

Sclerotinia sclerotiorum;

Stagonospora nodorum;

Umbilicaria esculenta; and

Uncinocarpus reesii.

22. The method of claim 1 wherein each primer of the primer set specifically binds only to a fungal DNA.

23. The method of claim 1 wherein each primer of the primer set specifically binds only to a fungal DNA in the presence of a non-fungal DNA.

24. The method of claim 23 wherein the non-fungal DNA is mammalian DNA.

25. The method of claim 24 wherein the mammalian DNA is human DNA.

26. The method of claim 23 wherein the non-fungal DNA is in greater than 1,000,000-fold mass excess of the fungal DNA.

27. The method of claim 23 wherein the non-fungal DNA is in greater than 5,000,000-fold excess of the fungal DNA.

28. The method of claim 23 wherein the non-fungal DNA is in greater than 30,000,000-fold excess of the fungal DNA.

29. The method of claim 1 wherein said patient sample is selected from the group consisting of a blood sample, a sputum sample, a lung lavage fluid sample, and a tissue biopsy sample.

30. The method of claim 29 wherein said blood sample is selected from the group consisting of whole blood, plasma, serum, and a white blood cell fraction.

31. The method of claim 1 wherein said fungal pathogen causes a fungal infection selected from the group consisting of aspergillosis, candidiasis, zygomycosis, scedosporiosis, fusariosis, cryptococcosis, histoplasmosis, coccidioidomycosis, and blastomycosis.

68. The method of claim 1 wherein said primer set further comprises a second reverse primer, the primer set consisting of SEQ ID NO:

12, SEQ ID NO;

41, and SEQ ID NO;

108.

32. A primer set for detecting a fungal DNA, said primer set comprising a forward primer and a reverse primer wherein at least one of said forward primer and said reverse primer is complementary to a fungal 28S ribosomal RNA (rRNA) gene.

33. The primer set of claim 32 wherein at least one of said forward primer and said reverse primer is complementary to a sequence that is 3′

to a D1-D2 highly variable region in said fungal 28S ribosomal rRNA gene.

34. The primer set of claim 32 wherein said forward primer is complementary to a fungal 18S rRNA gene and said reverse primer is complementary to a fungal 28S rRNA gene.

35. The primer set of claim 34 wherein said forward primer comprises the nucleotide sequence 5′

-GTAAAAGTCGTAACAAGGTTTC-3′

(SEQ ID NO;

     1).

36. The primer set of claim 32 wherein said forward primer is complementary to a fungal 5.8S rRNA gene and said reverse primer is complementary to a fungal 28S rRNA gene.

37. The primer set of claim 36 wherein said forward primer comprises the nucleotide sequence 5′

-GTGAATCATCGARTCTTTGAAC-3′

(SEQ ID NO;

     2).

38. The primer set of claim 32 wherein said forward primer and said reverse primer are both complementary to a fungal 28S rRNA gene.

39. The primer set of claim 32 wherein said forward primer is selected from the group consisting of:

40. The primer set of claim 32 wherein said reverse primer is selected from the group consisting of:

41. The primer set of claim 32 wherein said primer set comprises a forward and reverse primer pair selected from the group consisting of (SEQ ID NO:

2 and SEQ ID NO;

     31), (SEQ ID NO;

2 and SEQ ID NO;

     32), (SEQ ID NO;

11 and SEQ ID NO;

     41), (SEQ ID NO;

1 and SEQ ID NO;

     29), (SEQ ID NO;

2 and SEQ ID NO;

     30), (SEQ ID NO;

12 and SEQ ID NO;

     41), (SEQ ID NO;

12, SEQ ID NO;

41, and SEQ ID NO;

     108), (SEQ ID NO;

14 and SEQ ID NO;

     42), (SEQ ID NO;

17 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     52), and (SEQ ID NO;

25 and SEQ ID NO;

     54).

42. The primer set of claim 32 wherein said primer set comprises a forward and reverse primer pair selected from the group consisting of (SEQ ID NO:

1 and SEQ ID NO;

     29), (SEQ ID NO;

2 and SEQ ID NO;

     30), (SEQ ID NO;

12 and SEQ ID NO;

     41), (SEQ ID NO;

12, SEQ ID NO;

41, and SEQ ID NO;

     108), (SEQ ID NO;

14 and SEQ ID NO;

     42), (SEQ ID NO;

17 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     52), and (SEQ ID NO;

25 and SEQ ID NO;

     54).

43. The primer set of claim 32 wherein said primer set comprises a forward and reverse primer pair selected from the group consisting of (SEQ ID NO:

11 and SEQ ID NO;

     41), (SEQ ID NO;

12 and SEQ ID NO;

     41), (SEQ ID NO;

12, SEQ ID NO;

41, and SEQ ID NO;

     108), (SEQ ID NO;

14 and SEQ ID NO;

     42), (SEQ ID NO;

17 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     51), (SEQ ID NO;

20 and SEQ ID NO;

     52), and (SEQ ID NO;

25 and SEQ ID NO;

     54).

44. The primer set of claim 32 wherein said primer set comprises a forward and reverse primer pair selected from the group consisting of (SEQ ID NO:

2 and SEQ ID NO;

     30), (SEQ ID NO;

12, SEQ ID NO;

41, and SEQ ID NO;

     108), and (SEQ ID NO;

12 and SEQ ID NO;

     41).

45. The primer set of claim 32 wherein said fungal DNA is from a fungal species selected from the group consisting of Absidia corymbifera;

Cunninghamella bertholletiae;

Fusarium solani;

Mucor racemosus;

Paecilomyces variotii;

Penicillium chrysogenum;

Rhizomucor miehei;

Rhodotorula glutinis;

Scedosporium apiospermum;

Antrodia vaillantii;

Aspergillus fumigatus;

Aspergillus niger;

Aspergillus oryzae;

Aspergillus terreus;

Batrachochytrium dendrobatidis;

Botrytis cinerea;

Candida albicans;

Candida dublineinsis;

Candida glabrata;

Candida gulliermundei;

Candida kefyr;

Candida krusei;

Candida lipolytica;

Candida lusitaniae;

Candida parapsilosis;

Candida tropicalis;

Chaetomium globosum;

Coccidioides immitis;

Coccidioides posadasii;

Cryptococcus neoformans;

Fusarium graminearum;

Fusarium oxysporum;

Histoplasma capsulatum;

Hypocrea jecorina;

Lodderomyces elongisporus;

Magnaporthe grisea;

Metarhizium anisopliae;

Microsporum gypseum;

Mucor racemosus;

Neurospora crassa;

Paracoccidioides brasiliens;

Pneumocystis carinii;

Penicillium verrucosum;

Pichia stipitis;

Rhizomucor miehei;

Rhizopus oryzae;

Saccharomyces cerevisiae;

Schizosaccharomyces japonicus;

Schizosaccharomyces pombe;

Sclerotinia sclerotiorum;

Stagonospora nodorum;

Umbilicaria esculenta; and

Uncinocarpus reesii.

46. The primer set of claim 32 wherein said forward primer has the sequence set forth in SEQ ID NO:

1 and wherein said reverse primer has the sequence set forth in SEQ ID NO;

30.

47. The primer set of claim 32 wherein said forward primer has the sequence set forth in SEQ ID NO:

12 and wherein said reverse primer has the sequence set forth in SEQ ID NO;

41.

48. The primer set of claim 32 wherein said primer set comprises an additional reverse primer, the primer set consisting of SEQ ID NO:

12, SEQ ID NO;

41, and SEQ ID NO;

108.

49. A method for determining the identity of a fungal species in a patient sample, said method comprising the steps of:

(a) isolating a patient sample;

(b) carrying out a first PCR reaction to generate a first PCR amplicon, wherein said first PCR reaction comprises a first primer set capable of amplifying a region in a fungal ribosomal RNA (rRNA) gene comprising an internal transcribed spacer 2 (ITS-2) sequence;

(c) carrying out a second PCR reaction to generate a second PCR amplicon, wherein said second PCR reaction comprises a second primer set capable of amplifying a region in a fungal ribosomal 28S rRNA gene; and

(d) determining the melting temperature of said first PCR amplicon and of said second PCR amplicon, wherein the identity of said fungal species is determined by comparing the melting point of said first PCR amplicon and of said second PCR amplicon to known standards using a dye that binds to double stranded DNA and fluoresces preferentially in that state.

50. The method of claim 49 further comprising the step of extracting DNA from said isolated patient sample.

51. The method of claim 49 wherein said first PCR reaction and said second PCR reaction are each quantitative PCR (qPCR) reactions.

52. The method of claim 49 wherein said first primer set comprises a forward primer sequence as set forth in SEQ ID NO:

2 and a reverse primer sequence as set forth in SEQ ID NO;

30 and wherein said second primer set comprises a forward primer sequence as set forth in SEQ ID NO;

12 and a reverse primer sequence as set forth in SEQ ID NO;

41.

53. A method for identifying a primer set capable of detecting a fungal pathogen in a sample, said method comprising the steps of:

(a) obtaining the nucleic acid sequence of at least the 28S region of a fungal rRNA operon, (b) designing a forward primer capable of hybridizing with said nucleic acid sequence at a specific site in said 28S region, (c) designing a reverse primer capable of hybridizing with said nucleic acid sequence at a region in said sequence that is 3′

to the region to which the forward primer is capable of hybridizing, and (d) determining whether said forward primer and said reverse primer are capable of generating a PCR amplicon that is useful for identifying fungal DNA in a PCR reaction containing a specific fungal DNA.

54. The method of claim 53 wherein the method further comprises the step of resolving said PCR amplicon on an agarose gel to determine the analytical sensitivity of said forward primer and said reverse primer.

55. The method of claim 54 wherein said agarose gel is stained with ethidium bromide and said PCR amplicon is visualized by ultraviolet light.

56. The method of claim 53 wherein the method further comprises the step of determining the cross-reactivity of said forward primer and said reverse primer with non-fungal DNA.

57. The method of claim 56 wherein the non-fungal DNA is mammalian DNA.

58. The method of claim 57 wherein the mammalian DNA is human DNA.

59. The method of claim 53 wherein the method further comprises the step of determining the species resolution of said forward primer and said reverse primer, wherein said forward primer and said reverse primer comprise a primer set.

60. The method of claim 59 wherein an ability of said primer set to resolve a species is determined by a method comprising the following steps:

(a) sequencing said PCR amplicon, (b) comparing the sequence of said PCR amplicon with a sequence of a second PCR amplicon generated using said forward and reverse primers in a PCR reaction containing DNA from a different fungal species, and (c) repeating steps (a) and (b) using fungal DNA from at least 30 different fungal species to determine sequences of amplicons for at least 31 different fungal species, and (d) comparing the sequences of each amplicon.

61. The method of claim 60 wherein the sequences of each amplicon are compared to each other by generating a multiple sequence alignment of said sequences.

62. The method of claim 61 wherein the method further comprises the step of generating a distance matrix for each amplicon from said multiple sequence alignment.

63. The method of claim 62 wherein the distance matrix of each amplicon is compared to the distance matrix of each other amplicon, and wherein the comparison is used to determine which of said primer sets are capable of resolving a fungal species.

64. The method of claim 62 wherein the distance matrix is generated using the Tajima-Nei algorithm.

65. The method of claim 61, further comprising the step of assembling a phylogenetic tree.

66. The method of claim 65 wherein said phylogenetic tree is assembled using the Neighbor-Joining tree building method.

67. The method of claim 53 wherein said sample comprises 1,000,000-fold mass excess of non-fungal DNA to fungal DNA.