Rapid and sensitive method for detecting histoplasma capsulatum

• US 6,469,156 B1
• Filed: 01/12/2001
• Issued: 10/22/2002
• Est. Priority Date: 04/20/1999
• Status: Active Grant

• Current Assignee: Technology Transfer Partners, LLC
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Reid, Thomas M., Schafer, Millie P.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Spiegler, Alexander H.
• Application Number:
  US09/673,298
• Time in Patent Office: 648 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.1, 536/24.33
• US Class Current: 536/24.32
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae

Abstract

Oligonucleotides are provided which are useful as primers to initiate the amplification of a segment of Histoplasma capsulatum DNA that is specific to H. capsulatum, using the polymerase chain reaction. A method of detecting the presence of H. capsulatum in a sample using a nested, or two-stage, PCR assay is also provided. The outer pair of primers, for use in the first stage of the assay, are fungal-specific oligonucleotides; the inner pair of primers, for use in the second stage, are oligonucleotides specific for H. capsulatum.

Claims

1. An oligonucleotide primer pair comprising sequences specific for the 5.8S rRNA gene of Histoplasma capsulatum, wherein both primers of said primer pair are specific for 5.8s rRNA, and wherein both primers of said pair specifically amplify 5.8S rRNA gene sequence, or an oligonucleotide comprising a sequence complementary to one or the other of the primer pair specific for the H. capsulatum 5.8S rRNA gene.

2. An oligonucleotide comprising a sequence given by SEQ ID NO:

3 or a sequence complementary to that given by SEQ ID NO;

3, wherein the sequence of SEQ ID NO;

3 defines the 3′

end of the oligonucleotide or wherein the sequence complementary to SEQ ID NO;

3 defines the 3′

end of the oligonucleotide.

3. An oligonucleotide comprising a sequence given by SEQ ID NO:

4 or a sequence complementary to that given by SEQ ID NO;

4, wherein the sequence of SEQ ID NO;

4 defines the 3′

end of the oligonucleotide or wherein the sequence complementary to SEQ ID NO;

4 defines the 3′

end of the oligonucleotide.

4. A method of detecting H. capsulatum in a sample, comprising:

(a) contacting the sample with an oligonucleotide primer pair specific for a fungal nucleic acid sequence that comprises the coding sequence for the 5.8S rRNA gene;

(b) amplifying a first amplified nucleic acid by a DNA polymerase chain reaction, wherein both primers of the oligonucleotide primer pair defines the limits of the first amplified nucleic acid and wherein the first amplified nucleic acid comprises fungal nucleic acid sequence that comprises the coding sequence for the 5.8S rRNA gene;

(c) contacting the first amplified nucleic acid from step (b) with a second oligonucleotide primer pair, wherein the second oligonucleotide primer pair are specific for the H. capsulatum 5.8S rRNA gene, and wherein each second oligonucleotide primer is specific for a nucleotide sequence of the first amplified nucleic acid;

(d) amplifying a second amplified nucleic acid by a DNA polymerase chain reaction, wherein the second oligonucleotide pair defines the limits of the second amplified nucleic acid; and

(e) detecting an amplification product of step (d), whereby detecting the amplification product of step (d) indicates the presence of H. capsulatum.

5. The method of claim 4 wherein the sample is an environmental sample comprising soil or a clinical sample from a human subject.

6. The method of claim 4 wherein before step (a) the sample is subjected to partial purification comprising separation of any soil or contaminants contained in the sample from the H. capsulatum nucleic acid.

7. The method of claim 4 wherein the first amplified nucleic acid is amplified in a solution comprising bovine serum albumin.

8. The method of claim 4 wherein the detecting step comprises performing gel electrophoresis of the second amplified nucleic acid and detecting any H. capsulatum-specific DNA.

9. The method of claim 4 wherein the detecting step further comprises contacting the second amplified nucleic acid with a labeled probe specific to H. capsulatum DNA and detecting the label.

10. The method of claim 4 wherein the oligonucleotide primer pair employed in step (a) is specific for the fungal internal transcribed spacer regions flanking the coding sequence for the 5.8S rRNA gene.

11. The method of claim 4 wherein the sample is an environmental sample comprising soil or a clinical sample from a human subject.

12. The method of claim 4 wherein before step (a) the sample is subjected to partial purification comprising separation of any soil or contaminants contained in the sample from the H. capsulatum nucleic acid.

13. The method of claim 4 wherein the first amplified nucleic acid is amplified in a solution comprising bovine serum albumin.

14. The method of claim 4 wherein the detecting step (e) comprises performing gel electrophoresis of the second amplified nucleic acid and detecting any H. capsulatum-specific DNA.

15. The method of claim 4 wherein the detecting step (e) comprises contacting the second amplified nucleic acid with a labeled probe specific to H. capsulatum DNA and detecting the label.

16. The method of claim 4 wherein the detecting step (e) comprises performing gel electrophoresis of the second amplified nucleic acid and detecting any H. capsulatum-specific DNA.

17. The method of claim 4 wherein the detecting step (e) comprises contacting the second amplified nucleic acid with a labeled probe specific to H. capsulatum DNA and detecting the label.

18. A method of detecting H. capsulatum in a sample, comprising:

(a) contacting the sample with a first oligonucleotide primer comprising the sequence of SEQ ID NO;

1 and a second oligonucleotide primer comprising the sequence of SEQ ID NO;

2;

(b) amplifying a first amplified nucleic acid by a DNA polymerase chain reaction, wherein the first and second oligonucleotide primers define the limits of the first amplified nucleic acid;

(c) contacting the first amplified nucleic acid from step (b) with a third oligonucleotide primer comprising the sequence of SEQ ID NO;

3 and a fourth oligonucleotide comprising the sequence of SEQ ID NO;

4;

(d) amplifying a second amplified nucleic acid by a DNA polymerase chain reaction, wherein the third and fourth oligonucleotide primers define the limits of the second amplified nucleic acid; and

(e) detecting an amplification product of step (d), whereby detecting the amplification product of step (d) indicates the presence of H. capsulatum.