Methods and compositions for detecting and identifying species of Candida

• US 20080102449A1
• Filed: 12/29/2005
• Published: 05/01/2008
• Est. Priority Date: 01/06/2005
• Status: Active Grant

• Current Assignee: Medical Diagnostic Laboratories LLC
• Original Assignee: Medical Diagnostic Laboratories LLC
• Inventors: Mordechai, Eli, Adelson, Martin E., Trama, Jason
• Granted Patent: US 8,501,408 B2
• 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

Abstract

Methods and compositions useful in the detection and identification of species of Candida are disclosed. These species include Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis, each of which is a causative agent for vaginal candidiasis. The compositions of the invention are combinations of oligonucleotides. These oligonucleotides include pairs of forward and reverse primers for polymerase chain reactions, wherein each primer pair is capable of priming the synthesis of an amplicon specific to one of Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis, but preferably is not capable of priming the synthesis of an amplicon specific to any of the other three species. In preferred embodiments, the forward primers of the primer pairs have identical sequences, while each reverse primer of the primer pairs has a unique sequence relative to all of the other reverse primers; or the reverse primers of the primer pairs have identical sequences, while each forward primer of the primer pairs has a unique sequence relative to all of the other forward primers. These unique primer sequences account for the species specificity of the resultant amplicons. The oligonucleotides also include probes capable of detecting these amplicons, and sequencing primers for determining, in primer extension reactions, the nucleotide sequences contained within the amplicons. In preferred methods of the invention, a biological sample is tested for the presence of at least one isolate of Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis by isolating nucleic acid from the sample, attempting a polymerase chain reaction in a mixture containing this nucleic acid and a plurality of these primer pairs, ascertaining whether any amplicon is produced in the mixture using an oligonucleotide probe, and determining the sequence of any resultant amplicon using the sequencing primers. The detection of an amplicon indicates that the sample contains at least one isolate of Candida albicans, Candida glabrata, Candida parapsilosis, or Candida tropicalis, and the nucleotide sequence data is used to determine which of these four Candida species is present.

Claims

1. A method for determining whether a sample contains an isolate of Candida albicans, Candida glabrata, Candida parapsilosis, or Candida tropicalis, comprising

(a) providing a vessel containing (1) a nucleic acid from the sample, (2) at least two primers selected from the group consisting of first, second, third, and fourth primers, and (3) a fifth primer,

wherein the first and fifth primers are capable of priming, in a polymerase chain reaction, the synthesis of a first amplicon specific to the isolate of Candida albicans, and wherein the first and fifth primers are not capable of priming, in the polymerase chain reaction, the synthesis of an amplicon specific to the isolate of Candida glabrata, Candida parapsilosis, or Candida tropicalis,

wherein the second and fifth primers are capable of priming, in the polymerase chain reaction, the synthesis of a second amplicon specific to the isolate of Candida glabrata, and wherein the second and fifth primers are not capable of priming, in the polymerase chain reaction, the synthesis of an amplicon specific to the isolate of Candida albicans, Candida parapsilosis, or Candida tropicalis,

wherein the third and fifth primers are capable of priming, in the polymerase chain reaction, the synthesis of a third amplicon specific to the isolate of Candida parapsilosis, and wherein the third and fifth primers are not capable of priming, in the polymerase chain reaction, the synthesis of an amplicon specific to the isolate of Candida albicans, Candida glabrata, or Candida tropicalis,

wherein the fourth and fifth primers are capable of priming, in the polymerase chain reaction, the synthesis of a fourth amplicon specific to the isolate of Candida tropicalis, and wherein the fourth and fifth primers are not capable of priming, in the polymerase chain reaction, the synthesis of an amplicon specific to the isolate of Candida albicans, Candida glabrata, or Candida parapsilosis,

wherein the nucleotide sequences of the first, second, third, and fourth amplicons diverge from each other,

(b) incubating the vessel under conditions allowing production of (1) the first amplicon if the sample contains the isolate of Candida albicans, (2) the second amplicon if the sample contains the isolate of Candida glabrata, (3) the third amplicon if the sample contains the isolate of Candida parapsilosis, or (4) the fourth amplicon if the sample contains the isolate of Candida tropicalis, and

(c) determining that the sample contains (1) the isolate of Candida albicans if the first amplicon is produced in (b), (2) the isolate of Candida glabrata if the second amplicon is produced in (b), (3) the isolate of Candida parapsilosis if the third amplicon is produced in (b), or (4) the isolate of Candida tropicalis if the fourth amplicon is produced in (b);

or determining that the sample does not contain any of the isolates of Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis if none of the first, second, third, and fourth amplicons are produced in (b).

2. The method of claim 1,

wherein, in (b), the first primer is capable of hybridizing to at least a portion of a segment of the plus strand of the first amplicon, and wherein the segment consists of nucleotides 240-261 of SEQ ID NO:

1,

wherein, in (b), the second primer is capable of hybridizing to at least a portion of a segment of the plus strand of the second amplicon, and wherein the segment consists of nucleotides 268-298 of SEQ ID NO;

3,

wherein, in (b), the third primer is capable of hybridizing to at least a portion of a segment of the plus strand of the third amplicon, and wherein the segment consists of nucleotides 227-251 of SEQ ID NO;

5,

wherein, in (b), the fourth primer is capable of hybridizing to at least a portion of a segment of the plus strand of the fourth amplicon, and wherein the segment consists of nucleotides 223-247 of SEQ ID NO;

7, and

wherein, in (b), the fifth primer is capable of hybridizing to at least a portion of a segment of the minus strand of each of the first, second, third, and fourth amplicons, and wherein the segment consists of nucleotides 242-261 of SEQ ID NO;

2.

3. The method of claim 2,

wherein the first primer is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 240-261 of SEQ ID NO:

1,

wherein the second primer is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 268-298 of SEQ ID NO;

3,

wherein the third primer is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 227-251 of SEQ ID NO;

5,

wherein the fourth primer is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 223-247 of SEQ ID NO;

7, and

wherein the fifth primer is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 242-261 of SEQ ID NO;

2.

4. The method of claim 1,

wherein the plus strand of the first amplicon comprises the nucleotide sequence of SEQ ID NO:

1 and the minus strand of the first amplicon comprises the nucleotide sequence of SEQ ID NO;

2,

wherein the plus strand of the second amplicon comprises the nucleotide sequence of SEQ ID NO;

3 and the minus strand of the second amplicon comprises the nucleotide sequence of SEQ ID NO;

4,

wherein the plus strand of the third amplicon comprises the nucleotide sequence of SEQ ID NO;

5 and the minus strand of the third amplicon comprises the nucleotide sequence of SEQ ID NO;

6, and

wherein the plus strand of the fourth amplicon comprises the nucleotide sequence of SEQ ID NO;

7 and the minus strand of the fourth amplicon comprises the nucleotide sequence of SEQ ID NO;

8.

5. The method of claim 1, wherein each of the first, second, third, and fourth amplicons is specific to a ribosomal RNA gene.

6. The method of claim 5, wherein the ribosomal RNA gene encodes a 5.8S ribosomal RNA.

7. The method of claim 6, wherein each of the first, second, third, and fourth amplicons is specific to an internal transcribed spacer of the ribosomal RNA gene.

8. The method of claim 7, wherein the internal transcribed spacer is internal transcribed spacer 2.

9. The method of claim 1, wherein each of the first, second, third, fourth, and fifth primers is from 8 to 50 nucleotides long.

10. The method of claim 1, wherein the first primer comprises the nucleotide sequence of SEQ ID NO:

9, wherein the second primer comprises the nucleotide sequence of SEQ ID NO;

10, wherein the third primer comprises the nucleotide sequence of SEQ ID NO;

11, wherein the fourth primer comprises the nucleotide sequence of SEQ ID NO;

12, and wherein the fifth primer comprises the nucleotide sequence of SEQ ID NO;

13.

11. The method of claim 10, wherein the first primer consists of the nucleotide sequence of SEQ ID NO:

9, wherein the second primer consists of the nucleotide sequence of SEQ ID NO;

10, wherein the third primer consists of the nucleotide sequence of SEQ ID NO;

11, wherein the fourth primer consists of the nucleotide sequence of SEQ ID NO;

12, and wherein the fifth primer consists of the nucleotide sequence of SEQ ID NO;

13.

12. The method of claim 1, further comprising detecting the first, second, third, or fourth amplicon using an oligonucleotide probe.

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

14 or SEQ ID NO;

15.

14. The method of claim 13, wherein the oligonucleotide probe consists of the nucleotide sequence of SEQ ID NO:

14 or SEQ ID NO;

15.

15. The method of claim 1, further comprising isolating the plus or minus strand of the first, second, third, or fourth amplicon.

16. The method of claim 15, further comprising determining the nucleotide sequence of at least a portion of the plus or minus strand of the first, second, third, or fourth amplicon.

17. The method of claim 16, further comprising determining the nucleotide sequence by conducting a nucleotide sequencing reaction using a sequencing primer capable of hybridizing, in the nucleotide sequencing reaction, to the plus or minus strand of the first, second, third, or fourth amplicon, wherein the sequencing primer is extended during the nucleotide sequencing reaction.

18. The method of claim 17, further comprising determining the nucleotide sequence of at least the portion of the minus strand of the first, second, third, or fourth amplicon using a first, second, third, or fourth sequencing primer, respectively.

19. The method of claim 17, wherein the sequencing primer is from 8 to 30 nucleotides long.

20. The method of claim 18,

wherein the first sequencing primer is capable of hybridizing, in the nucleotide sequencing reaction, to at least a portion of a segment of the plus strand of the first amplicon, and wherein the segment consists of nucleotides 240-259 of SEQ ID NO:

1,

wherein the second sequencing primer is capable of hybridizing, in the nucleotide sequencing reaction, to at least a portion of a segment of the plus strand of the second amplicon, and wherein the segment consists of nucleotides 278-297 of SEQ ID NO;

3,

wherein the third sequencing primer is capable of hybridizing, in the nucleotide sequencing reaction, to at least a portion of a segment of the plus strand of the third amplicon, and wherein the segment consists of nucleotides 227-251 of SEQ ID NO;

5, and

wherein the fourth sequencing primer is capable of hybridizing, in the nucleotide sequencing reaction, to at least a portion of a segment of the plus strand of the fourth amplicon, and wherein the segment consists of nucleotides 227-247 of SEQ ID NO;

7.

21. The method of claim 20,

wherein the first sequencing primer is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 240-259 of SEQ ID NO:

1,

wherein the second sequencing primer is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 278-297 of SEQ ID NO;

3,

wherein the third sequencing primer is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 227-251 of SEQ ID NO;

5, and

wherein the fourth sequencing primer is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 227-247 of SEQ ID NO;

7.

22. The method of claim 18, wherein the first sequencing primer comprises the nucleotide sequence of SEQ ID NO:

16, wherein the second sequencing primer comprises the nucleotide sequence of SEQ ID NO;

17, wherein the third sequencing primer comprises the nucleotide sequence of SEQ ID NO;

18, and wherein the fourth sequencing primer comprises the nucleotide sequence of SEQ ID NO;

19.

23. The method of claim 22, wherein the first sequencing primer consists of the nucleotide sequence of SEQ ID NO:

16, wherein the second sequencing primer consists of the nucleotide sequence of SEQ ID NO;

17, wherein the third sequencing primer consists of the nucleotide sequence of SEQ ID NO;

18, and wherein the fourth sequencing primer consists of the nucleotide sequence of SEQ ID NO;

19.

24. The method of claim 16, further comprising determining that the first, second, third, or fourth amplicon is respectively specific to an isolate of Candida albicans, Candida glabrata, Candida parapsilosis, or Candida tropicalis, based upon the nucleotide sequence of at least the portion of the plus or minus strand of the first, second, third, or fourth amplicon.

25. The method of claim 15, further comprising nucleotide sequencing at least a portion of the plus or minus strand of each of at least two amplicons selected from the group consisting of the first, second, third, and fourth amplicons.

26. The method of claim 25, further comprising conducting at least two nucleotide sequencing reactions in a single vessel.

27. The method of claim 26, further comprising extending a sequencing primer in each of the nucleotide sequencing reactions.

28. The method of claim 26, further comprising generating a composite nucleotide sequence from all of the nucleotide sequencing reactions.

29. The method of claim 28, wherein the composite nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, or SEQ ID NO;

29.

30. The method of claim 29, wherein the composite nucleotide sequence consists of the nucleotide sequence of SEQ ID NO:

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, or SEQ ID NO;

29.

31. The method of claim 28, further comprising determining from the composite nucleotide sequence that the sample contains at least two species of Candida selected from the group consisting of Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis.

32. The method of claim 31, further comprising identifying each of the species of Candida present in the sample.

33. A method for determining whether a sample contains an isolate of Candida albicans, Candida glabrata, Candida parapsilosis, or Candida tropicalis, comprising

(a) providing a vessel containing (1) a nucleic acid from the sample, (2) a primer selected from the group consisting of a first, second, third, or fourth primer, and (3) a fifth primer,

wherein the first and fifth primers are capable of priming, in a polymerase chain reaction, the synthesis of a first amplicon specific to the isolate of Candida albicans,

wherein the second and fifth primers are capable of priming, in the polymerase chain reaction, the synthesis of a second amplicon specific to the isolate of Candida glabrata,

wherein the third and fifth primers are capable of priming, in the polymerase chain reaction, the synthesis of a third amplicon specific to the isolate of Candida parapsilosis,

wherein the fourth and fifth primers are capable of priming, in the polymerase chain reaction, the synthesis of a fourth amplicon specific to the isolate of Candida tropicalis,

wherein the nucleotide sequences of the first, second, third, and fourth amplicons diverge from each other,

wherein the first primer is capable of hybridizing, in the polymerase chain reaction, to at least a portion of a segment of the plus strand of the first amplicon, and wherein the segment consists of nucleotides 240-261 of SEQ ID NO:

1,

wherein the second primer is capable of hybridizing, in the polymerase chain reaction, to at least a portion of a segment of the plus strand of the second amplicon, and wherein the segment consists of nucleotides 268-298 of SEQ ID NO;

3,

wherein the third primer is capable of hybridizing, in the polymerase chain reaction, to at least a portion of a segment of the plus strand of the third amplicon, and wherein the segment consists of nucleotides 227-251 of SEQ ID NO;

5,

wherein the fourth primer is capable of hybridizing, in the polymerase chain reaction, to at least a portion of a segment of the plus strand of the fourth amplicon, and wherein the segment consists of nucleotides 223-247 of SEQ ID NO;

7,

wherein the fifth primer is capable of hybridizing, in the polymerase chain reaction, to at least a portion of a segment of the minus strand of each of the first, second, third, and fourth amplicons, and wherein the segment consists of nucleotides 242-261 of SEQ ID NO;

2,

(b) incubating the vessel under conditions allowing production of (1) the first amplicon if the sample contains the isolate of Candida albicans, (2) the second amplicon if the sample contains the isolate of Candida glabrata, (3) the third amplicon if the sample contains the isolate of Candida parapsilosis, or (4) the fourth amplicon if the sample contains the isolate of Candida tropicalis, and

(c) determining that the sample contains (1) the isolate of Candida albicans if the first amplicon is produced in (b), (2) the isolate of Candida glabrata if the second amplicon is produced in (b), (3) the isolate of Candida parapsilosis if the third amplicon is produced in (b), or (4) the isolate of Candida tropicalis if the fourth amplicon is produced in (b);

or determining that the sample does not contain any of the isolates of Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis if none of the first, second, third, and fourth amplicons are produced in (b).

34. A composition comprising (a) at least two oligonucleotides selected from the group consisting of first, second, third, and fourth oligonucleotides, and (b) a fifth oligonucleotide,

wherein the first and fifth oligonucleotides are capable of priming, in a polymerase chain reaction, the synthesis of a first amplicon specific to the isolate of Candida albicans, and wherein the first and fifth oligonucleotides are not capable of priming, in the polymerase chain reaction, the synthesis of an amplicon specific to the isolate of Candida glabrata, Candida parapsilosis, or Candida tropicalis,

wherein the second and fifth oligonucleotides are capable of priming, in the polymerase chain reaction, the synthesis of a second amplicon specific to the isolate of Candida glabrata, and wherein the second and fifth oligonucleotides are not capable of priming, in the polymerase chain reaction, the synthesis of an amplicon specific to the isolate of Candida albicans, Candida parapsilosis, or Candida tropicalis,

wherein the third and fifth oligonucleotides are capable of priming, in the polymerase chain reaction, the synthesis of a third amplicon specific to the isolate of Candida parapsilosis, and wherein the third and fifth oligonucleotides are not capable of priming, in the polymerase chain reaction, the synthesis of an amplicon specific to the isolate of Candida albicans, Candida glabrata, or Candida tropicalis,

wherein the fourth and fifth oligonucleotides are capable of priming, in the polymerase chain reaction, the synthesis of a fourth amplicon specific to the isolate of Candida tropicalis, and wherein the fourth and fifth oligonucleotides are not capable of priming, in the polymerase chain reaction, the synthesis of an amplicon specific to the isolate of Candida albicans, Candida glabrata, or Candida parapsilosis.

35. A composition comprising (a) a first, second, third, or fourth oligonucleotide, and (b) a fifth oligonucleotide,

wherein the first oligonucleotide is capable of hybridizing, under highly stringent hybridization conditions, to at least a portion of a segment of the plus strand of the first amplicon, and wherein the segment consists of nucleotides 240-261 of SEQ ID NO:

1,

wherein the second oligonucleotide is capable of hybridizing, under highly stringent hybridization conditions, to at least a portion of a segment of the plus strand of the second amplicon, and wherein the segment consists of nucleotides 268-298 of SEQ ID NO;

3,

wherein the third oligonucleotide is capable of hybridizing, under highly stringent hybridization conditions, to at least a portion of a segment of the plus strand of the third amplicon, and wherein the segment consists of nucleotides 227-251 of SEQ ID NO;

5,

wherein the fourth oligonucleotide is capable of hybridizing, under highly stringent hybridization conditions, to at least a portion of a segment of the plus strand of the fourth amplicon, and wherein the segment consists of nucleotides 223-247 of SEQ ID NO;

7, and

wherein the fifth oligonucleotide is capable of hybridizing, under highly stringent hybridization conditions, to at least a portion of a segment of the minus strand of each of the first, second, third, and fourth amplicons, and wherein the segment consists of nucleotides 242-261 of SEQ ID NO;

2.

36. A composition comprising (a) a first, second, third, or fourth oligonucleotide, and (b) a fifth oligonucleotide,

wherein the first oligonucleotide is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 240-261 of SEQ ID NO:

1,

wherein the second oligonucleotide is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 268-298 of SEQ ID NO;

3,

wherein the third oligonucleotide is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 227-251 of SEQ ID NO;

5,

wherein the fourth oligonucleotide is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 223-247 of SEQ ID NO;

7, and

wherein the fifth oligonucleotide is at least 50% identical to the reverse complement of a segment of a polynucleotide based on the Clustal V or W alignment method using the default parameters, and wherein the segment consists of nucleotides 242-261 of SEQ ID NO;

2.