Oligonucleotides useful in methods for detecting and characterizing Aspergillus fumigatus

• US 20060127934A1
• Filed: 12/09/2005
• Published: 06/15/2006
• Est. Priority Date: 12/15/2004
• Status: Abandoned Application

• Current Assignee: Medical Diagnostic Laboratories LLC
• Original Assignee: Medical Diagnostic Laboratories LLC
• Inventors: Mordechai, Eli, Adelson, Martin E., Trama, Jason
• Application Number:
  US11/299,362
• Publication Number:
  US 20060127934A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Oligonucleotides and methods for using these oligonucleotides in the detection of Aspergillus fumigatus are disclosed. Aspergillus fumigatus is the causative agent for medical conditions including invasive aspergillosis. The oligonucleotides of the invention have nucleotide sequences derived from the gene encoding the cytochrome P450 14 alpha-sterol demethylase (i.e., the Cyp51A protein) of Aspergillus fumigatus. The oligonucleotides of the invention include forward primers and reverse primers which in combination are capable of priming the synthesis of amplicons specific to cyp51A in polymerase chain reactions using nucleic acids isolated from Aspergillus fumigatus as templates. The oligonucleotides of the invention also include probes capable of detecting these cyp51A-specific amplicons. Thus, a biological sample is tested for the presence of Aspergillus fumigatus by isolating nucleic acid from the sample, conducting a polymerase chain reaction in a mixture containing this nucleic acid and these forward and reverse primers, and then determining, using an oligonucleotide probe, whether an amplicon is produced in the mixture, wherein detection of the amplicon indicates the presence of Aspergillus fumigatus in the sample. The oligonucleotides of the invention also include primers for nucleotide sequencing reactions to determine whether an isolate of Aspergillus fumigatus is more tolerant than wild-type Aspergillus fumigatus to a triazole, which is a compound commonly used as an antifungal drug. Specifically, a strand of a cyp51A-specific amplicon is at least partially sequenced using a nucleotide sequencing primer in a primer extension reaction. Identification of mutations giving rise to amino acid substitutions at positions 54, 138, 220, and 448 of the amino acid sequence of the wild-type Cyp51A protein indicates that the isolate of Aspergillus fumigatus from which the amplicon is derived exhibits decreased susceptibility to at least one triazole.

Claims

1. An isolated oligonucleotide capable of hybridizing, under highly stringent hybridization conditions, to at least a portion of a segment of a polynucleotide, wherein the segment consists of:

(a) nucleotides 720 through 743, nucleotides 515 through 537, nucleotides 662 through 686, nucleotides 903 through 923, nucleotides 838 through 854, nucleotides 846 through 877, nucleotides 1212 through 1235, nucleotides 1107 through 1129, nucleotides 1085 through 1099, nucleotides 1070 through 1095, nucleotides 1784 through 1804, nucleotides 1769 through 1783, or nucleotides 1754 through 1775 of SEQ ID NO;

6, or

(b) nucleotides 1551 through 1574, nucleotides 1538 through 1559, nucleotides 1363 through 1387, nucleotides 1304 through 1327, nucleotides 1257 through 1277, nucleotides 1216 through 1235, nucleotides 1172 through 1203, nucleotides 1062 through 1084, nucleotides 983 through 1002, nucleotides 972 through 989, nucleotides 954 through 979, nucleotides 352 through 374, nucleotides 288 through 304, or nucleotides 274 through 295 of SEQ ID NO;

7,

wherein the nucleotide sequence of SEQ ID NO;

6 is a sense-strand nucleotide sequence encoding a Cyp51A protein of Aspergillus fumigatus, and

wherein the nucleotide sequence of SEQ ID NO;

7 is the reverse complement of the nucleotide sequence of SEQ ID NO;

6.

2. The oligonucleotide of claim 1, wherein the oligonucleotide is from 8 to 50 nucleotides long.

3. The oligonucleotide of claim 2, wherein the oligonucleotide is from 12 to 24 nucleotides long.

4. The oligonucleotide of claim 1, wherein the oligonucleotide is from 10 to 30 nucleotides long.

5. The oligonucleotide of claim 4, wherein the oligonucleotide is from 15 to 25 nucleotides long.

6. The oligonucleotide of claim 1, wherein the oligonucleotide is from 15 to 50 nucleotides long.

7. The oligonucleotide of claim 6, wherein the oligonucleotide is from 25 to 35 nucleotides long.

8. The oligonucleotide of claim 1, wherein the oligonucleotide comprises the nucleotide sequence of SEQ ID NO:

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, or SEQ ID NO;

49.

9. The oligonucleotide of claim 8, wherein nucleotides 12, 13, and 14 of SEQ ID NO:

41 are a, t, and g, respectively, wherein nucleotides 13, 14, and 15 of SEQ ID NO;

42 are c, a, and t, respectively, wherein nucleotides 12, 13, and 14 of SEQ ID NO;

48 are g, g, and t, respectively, and wherein nucleotides 9, 10, and 11 of SEQ ID NO;

49 are a, c, and c, respectively.

10. The oligonucleotide of claim 8, wherein the oligonucleotide consists of the nucleotide sequence of SEQ ID NO:

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, or SEQ ID NO;

49.

11. The oligonucleotide of claim 10, wherein nucleotides 12, 13, and 14 of SEQ ID NO:

41 are a, t, and g, respectively, wherein nucleotides 13, 14, and 15 of SEQ ID NO;

42 are c, a, and t, respectively, wherein nucleotides 12, 13, and 14 of SEQ ID NO;

48 are g, g, and t, respectively, and wherein nucleotides 9, 10, and 11 of SEQ ID NO;

49 are a, c, and c, respectively.

12. An isolated oligonucleotide, wherein, based on the Clustal V or W alignment method using the default parameters, the oligonucleotide is at least 50% identical to the reverse complement of a segment of a polynucleotide, wherein the segment consists of:

(a) nucleotides 720 through 743, nucleotides 515 through 537, nucleotides 662 through 686, nucleotides 903 through 923, nucleotides 838 through 854, nucleotides 846 through 877, nucleotides 1212 through 1235, nucleotides 1107 through 1129, nucleotides 1085 through 1099, nucleotides 1070 through 1095, nucleotides 1784 through 1804, nucleotides 1769 through 1783, or nucleotides 1754 through 1775 of SEQ ID NO;

6, or

(b) nucleotides 1551 through 1574, nucleotides 1538 through 1559, nucleotides 1363 through 1387, nucleotides 1304 through 1327, nucleotides 1257 through 1277, nucleotides 1216 through 1235, nucleotides 1172 through 1203, nucleotides 1062 through 1084, nucleotides 983 through 1002, nucleotides 972 through 989, nucleotides 954 through 979, nucleotides 352 through 374, nucleotides 288 through 304, or nucleotides 274 through 295 of SEQ ID NO;

7,

wherein the nucleotide sequence of SEQ ID NO;

6 is a sense-strand nucleotide sequence encoding a Cyp51A protein of Aspergillus fumigatus, and

wherein the nucleotide sequence of SEQ ID NO;

7 is the reverse complement of the nucleotide sequence of SEQ ID NO;

6.

13. The oligonucleotide of claim 12, wherein the oligonucleotide is from 8 to 50 nucleotides long.

14. The oligonucleotide of claim 13, wherein the oligonucleotide is from 12 to 24 nucleotides long.

15. The oligonucleotide of claim 12, wherein the oligonucleotide is from 10 to 30 nucleotides long.

16. The oligonucleotide of claim 15, wherein the oligonucleotide is from 15 to 25 nucleotides long.

17. The oligonucleotide of claim 12, wherein the oligonucleotide is from 15 to 50 nucleotides long.

18. The oligonucleotide of claim 17, wherein the oligonucleotide is from 25 to 35 nucleotides long.

19. The oligonucleotide of claim 12, wherein the oligonucleotide comprises the nucleotide sequence of SEQ ID NO:

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, or SEQ ID NO;

49.

20. The oligonucleotide of claim 19, wherein nucleotides 12, 13, and 14 of SEQ ID NO:

41 are a, t, and g, respectively, wherein nucleotides 13, 14, and 15 of SEQ ID NO;

42 are c, a, and t, respectively, wherein nucleotides 12, 13, and 14 of SEQ ID NO;

48 are g, g, and t, respectively, and wherein nucleotides 9, 10, and 11 of SEQ ID NO;

49 are a, c, and c, respectively.

21. The oligonucleotide of claim 19, wherein the oligonucleotide consists of the nucleotide sequence of SEQ ID NO:

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, or SEQ ID NO;

49.

22. The oligonucleotide of claim 21, wherein nucleotides 12, 13, and 14 of SEQ ID NO:

41 are a, t, and g, respectively, wherein nucleotides 13, 14, and 15 of SEQ ID NO;

42 are c, a, and t, respectively, wherein nucleotides 12, 13, and 14 of SEQ ID NO;

48 are g, g, and t, respectively, and wherein nucleotides 9, 10, and 11 of SEQ ID NO;

49 are a, c, and c, respectively.

23. A method for determining whether a sample contains Aspergillus fumigatus comprising:

(a) providing a vessel containing a composition,

wherein the composition contains a forward primer, a reverse primer, and a nucleic acid from the sample,

wherein the composition is capable of amplifying, by a polymerase chain reaction, a segment of the nucleic acid to produce an amplicon,

wherein production of the amplicon is primed by the forward primer and the reverse primer,

wherein the amplicon encodes at least a portion of a Cyp51A protein,

(b) incubating the vessel under conditions allowing production of the amplicon if the sample contains Aspergillus fumigatus,

wherein, during production of the amplicon, the forward primer is capable of hybridizing to at least a portion of a segment of the antisense strand of the amplicon, and the reverse primer is capable of hybridizing to at least a portion of a segment of the sense strand of the amplicon,

wherein the segment of the antisense strand of the amplicon consists of nucleotides 1551 through 1574, nucleotides 1304 through 1327, nucleotides 1257 through 1277, nucleotides 1062 through 1084, nucleotides 983 through 1002, or nucleotides 352 through 374 of SEQ ID NO;

7, and the segment of the sense strand of the amplicon consists of nucleotides 720 through 743, nucleotides 903 through 923, nucleotides 1212 through 1235, nucleotides 1107 through 1129, or nucleotides 1784 through 1804 of SEQ ID NO;

6, and

(c) determining that the sample contains Aspergillus fumigatus if the amplicon is detected, or determining that the sample does not contain Aspergillus fumigatus if the amplicon is not detected.

24. The method of claim 23, wherein, in (b), the vessel contains an oligonucleotide probe capable of detecting the amplicon if the amplicon is produced in (b).

25. A method for determining whether an isolate of Aspergillus fumigatus is more tolerant to a triazole than wild-type Aspergillus fumigatus comprising:

(a) providing a vessel containing a composition,

wherein the composition contains a forward primer, a reverse primer, and a nucleic acid isolated from the isolate,

wherein the composition is capable of amplifying, by a polymerase chain reaction, a segment of the nucleic acid to produce an amplicon,

wherein production of the amplicon is primed by the forward primer and the reverse primer,

wherein the amplicon encodes at least a portion of a Cyp51A protein,

wherein the sense strand of the amplicon contains at least the first nucleotide of the codon corresponding to the codon encoding amino acid 54 of SEQ ID NO;

2,

wherein the codon encoding amino acid 54 of SEQ ID NO;

2 consists of nucleotides 512, 513, and 514 of SEQ ID NO;

8,

wherein the reverse complement of the codon encoding amino acid 54 of SEQ ID NO;

2 consists of nucleotides 1535, 1536, and 1537 of SEQ ID NO;

9,

(b) incubating the vessel under conditions allowing production of the amplicon,

(c) isolating the antisense strand of the amplicon or the sense strand of the amplicon produced in (b),

(d) if the antisense strand of the amplicon is isolated in (c), then (1) providing a first sequencing primer capable of hybridizing to the antisense strand and capable of being extended during a first nucleotide sequencing reaction, and (2) identifying, by conducting the first nucleotide sequencing reaction, the nucleotide corresponding to nucleotide 512 or 513 of SEQ ID NO;

8, or

if the sense strand of the amplicon is isolated in (c), then (1) providing a second sequencing primer capable of hybridizing to the sense strand and capable of being extended during a second nucleotide sequencing reaction, and (2) identifying, by conducting the second nucleotide sequencing reaction, the nucleotide corresponding to nucleotide 1536 or 1537 of SEQ ID NO;

9, and

(e) if the first nucleotide sequencing reaction is conducted in (d), then determining that the isolate is more tolerant to the triazole if the nucleotide corresponding to nucleotide 512 or 513 of SEQ ID NO;

8 is not g, or determining that the isolate is not more tolerant to the triazole if the nucleotides corresponding to nucleotides 512 and 513 of SEQ ID NO;

8 are both g, or

if the second nucleotide sequencing reaction is conducted in (d), then determining that the isolate is more tolerant to the triazole if the nucleotide corresponding to nucleotide 1536 or 1537 of SEQ ID NO;

9 is not c, or determining that the isolate is not more tolerant to the triazole if the nucleotides corresponding to nucleotides 1536 and 1537 of SEQ ID NO;

9 are both c.

26. The method of claim 25, wherein, in (b), the vessel contains an oligonucleotide probe capable of detecting the amplicon if the amplicon is produced in (b).

27. A method for determining whether an isolate of Aspergillus fumigatus is more tolerant to a triazole than wild-type Aspergillus fumigatus comprising:

(a) providing a vessel containing a composition,

wherein the composition contains a forward primer, a reverse primer, and a nucleic acid isolated from the isolate,

wherein the composition is capable of amplifying, by a polymerase chain reaction, a segment of the nucleic acid to produce an amplicon,

wherein production of the amplicon is primed by the forward primer and the reverse primer,

wherein the amplicon encodes at least a portion of a Cyp51A protein,

wherein the sense strand of the amplicon contains at least the first nucleotide of the codon corresponding to the codon encoding amino acid 138 of SEQ ID NO;

3,

wherein the codon encoding amino acid 138 of SEQ ID NO;

3 consists of nucleotides 835, 836, and 837 of SEQ ID NO;

10,

wherein the reverse complement of the codon encoding amino acid 138 of SEQ ID NO;

3 consists of nucleotides 1212, 1213, and 1214 of SEQ ID NO;

11,

(b) incubating the vessel under conditions allowing production of the amplicon,

(c) isolating the antisense strand of the amplicon or the sense strand of the amplicon produced in (b),

(d) if the antisense strand of the amplicon is isolated in (c), then (1) providing a first sequencing primer capable of hybridizing to the antisense strand and capable of being extended during a first nucleotide sequencing reaction, and (2) identifying, by conducting the first nucleotide sequencing reaction, the nucleotide corresponding to nucleotide 835 or 836 of SEQ ID NO;

10, or

if the sense strand of the amplicon is isolated in (c), then (1) providing a second sequencing primer capable of hybridizing to the sense strand and capable of being extended during a second nucleotide sequencing reaction, and (2) identifying, by conducting the second nucleotide sequencing reaction, the nucleotide corresponding to nucleotide 1213 or 1214 of SEQ ID NO;

11, and

(e) if the first nucleotide sequencing reaction is conducted in (d), then determining that the isolate is more tolerant to the triazole if the nucleotide corresponding to nucleotide 835 or 836 of SEQ ID NO;

10 is not g, or determining that the isolate is not more tolerant to the triazole if the nucleotides corresponding to nucleotides 835 and 836 of SEQ ID NO;

10 are both g, or

if the second nucleotide sequencing reaction is conducted in (d), then determining that the isolate is more tolerant to the triazole if the nucleotide corresponding to nucleotide 1213 or 1214 of SEQ ID NO;

11 is not c, or determining that the isolate is not more tolerant to the triazole if the nucleotides corresponding to nucleotides 1213 and 1214 of SEQ ID NO;

11 are both c.

28. The method of claim 27, wherein, in (b), the vessel contains an oligonucleotide probe capable of detecting the amplicon if the amplicon is produced in (b).

29. A method for determining whether an isolate of Aspergillus fumigatus is more tolerant to a triazole than wild-type Aspergillus fumigatus comprising:

(a) providing a vessel containing a composition,

wherein the composition contains a forward primer, a reverse primer, and a nucleic acid isolated from the isolate,

wherein the composition is capable of amplifying, by a polymerase chain reaction, a segment of the nucleic acid to produce an amplicon,

wherein production of the amplicon is primed by the forward primer and the reverse primer,

wherein the amplicon encodes at least a portion of a Cyp51A protein,

wherein the sense strand of the amplicon contains at least the first nucleotide of the codon corresponding to the codon encoding amino acid 220 of SEQ ID NO;

4,

wherein the codon encoding amino acid 220 of SEQ ID NO;

4 consists of nucleotides 1081, 1082, and 1083 of SEQ ID NO;

12,

wherein the reverse complement of the codon encoding amino acid 220 of SEQ ID NO;

4 consists of nucleotides 966, 967, and 968 of SEQ ID NO;

13,

(b) incubating the vessel under conditions allowing production of the amplicon,

(c) isolating the antisense strand of the amplicon or the sense strand of the amplicon produced in (b),

(d) if the antisense strand of the amplicon is isolated in (c), then (1) providing a first sequencing primer capable of hybridizing to the antisense strand and capable of being extended during a first nucleotide sequencing reaction, and (2) identifying, by conducting the first nucleotide sequencing reaction, the nucleotide corresponding to nucleotide 1081, 1082, or 1083 of SEQ ID NO;

12, or

if the sense strand of the amplicon is isolated in (c), then (1) providing a second sequencing primer capable of hybridizing to the sense strand and capable of being extended during a second nucleotide sequencing reaction, and (2) identifying, by conducting the second nucleotide sequencing reaction, the nucleotide corresponding to nucleotide 966, 967, or 968 of SEQ ID NO;

13, and

(e) if the first nucleotide sequencing reaction is conducted in (d), then determining that the isolate is more tolerant to the triazole if the nucleotide corresponding to nucleotide 1081, 1082, or 1083 of SEQ ID NO;

12 is not a, t, or g, respectively, or determining that the isolate is not more tolerant to the triazole if the nucleotides corresponding to nucleotides 1081, 1082, and 1083 of SEQ ID NO;

12 are a, t, and g, respectively, or

if the second nucleotide sequencing reaction is conducted in (d), then determining that the isolate is more tolerant to the triazole if the nucleotide corresponding to nucleotide 966, 967, or 968 of SEQ ID NO;

13 is not c, a, or t, respectively, or determining that the isolate is not more tolerant to the triazole if the nucleotides corresponding to nucleotides 966, 967, and 968 of SEQ ID NO;

13 are c, a, and t, respectively.

30. The method of claim 29, wherein, in (b), the vessel contains an oligonucleotide probe capable of detecting the amplicon if the amplicon is produced in (b).

31. A method for determining whether an isolate of Aspergillus fumigatus is more tolerant to a triazole than wild-type Aspergillus fumigatus comprising:

(a) providing a vessel containing a composition,

wherein the composition contains a forward primer, a reverse primer, and a nucleic acid isolated from the isolate,

wherein the composition is capable of amplifying, by a polymerase chain reaction, a segment of the nucleic acid to produce an amplicon,

wherein production of the amplicon is primed by the forward primer and the reverse primer,

wherein the amplicon encodes at least a portion of a Cyp51A protein,

wherein the sense strand of the amplicon contains at least the first nucleotide of the codon corresponding to the codon encoding amino acid 448 of SEQ ID NO;

5,

wherein the codon encoding amino acid 448 of SEQ ID NO;

5 consists of nucleotides 1765, 1766, and 1767 of SEQ ID NO;

14,

wherein the reverse complement of the codon encoding amino acid 448 of SEQ ID NO;

5 consists of nucleotides 282, 283, and 284 of SEQ ID NO;

15,

(b) incubating the vessel under conditions allowing production of the amplicon,

(c) isolating the antisense strand of the amplicon or the sense strand of the amplicon produced in (b),

(d) if the antisense strand of the amplicon is isolated in (c), then (1) providing a first sequencing primer capable of hybridizing to the antisense strand and capable of being extended during a first nucleotide sequencing reaction, and (2) identifying, by conducting the first nucleotide sequencing reaction, the nucleotide corresponding to nucleotide 1765 or 1766 of SEQ ID NO;

14, or

if the sense strand of the amplicon is isolated in (c), then (1) providing a second sequencing primer capable of hybridizing to the sense strand and capable of being extended during a second nucleotide sequencing reaction, and (2) identifying, by conducting the second nucleotide sequencing reaction, the nucleotide corresponding to nucleotide 283 or 284 of SEQ ID NO;

15, and

(e) if the first nucleotide sequencing reaction is conducted in (d), then determining that the isolate is more tolerant to the triazole if the nucleotide corresponding to nucleotide 1765 or 1766 of SEQ ID NO;

14 is not g, or determining that the isolate is not more tolerant to the triazole if the nucleotides corresponding to nucleotides 1765 and 1766 of SEQ ID NO;

14 are both g, or

if the second nucleotide sequencing reaction is conducted in (d), then determining that the isolate is more tolerant to the triazole if the nucleotide corresponding to nucleotide 283 or 284 of SEQ ID NO;

15 is not c, or determining that the isolate is not more tolerant to the triazole if the nucleotides corresponding to nucleotides 283 and 284 of SEQ ID NO;

15 are both c.

32. The method of claim 31, wherein, in (b), the vessel contains an oligonucleotide probe capable of detecting the amplicon if the amplicon is produced in (b).