Assays for fungal infection

• US 9,657,355 B2
• Filed: 11/01/2007
• Issued: 05/23/2017
• Est. Priority Date: 11/02/2006
• Status: Active Grant

• Current Assignee: Rutgers University
• Original Assignee: Rutgers University
• Inventors: Denning, David Wemyss, Park, Steven, Perlin, David S.
• Primary Examiner(s): SALMON, KATHERINE D
• Application Number:
  US12/447,606
• Publication Number:
  US 20100221710A1
• Time in Patent Office: 3,491 Days
• 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 for detecting the presence or absence of any species of a fungus belonging to the genus Pneumocystis in a sample, comprising

i. obtaining a sample suspected of containing fungal nucleic acid from the genus Pneumocystis,

ii. contacting said sample with a forward primer consisting of SEQ ID NO:

19 and a reverse primer consisting of SEQ ID NO;

20 in the presence of one or more internal PCR amplification controls under conditions under which PCR may occur; and

iii. detecting the presence or absence of an amplified identifying region of fungal nucleic acid in the sample,

wherein said identifying region of fungal nucleic acid comprises SEQ ID NO;

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

18, the complement or transcript thereof, said identifying region being present in species of the genus Pneumocystis but not those of the genus of Candida or Aspergillus, and

wherein the presence of said amplified identifying region of fungal nucleic acid in the sample indicates the presence of any species of a fungus belonging to the genus Pneumocystis in the sample, and the absence of said amplified identifying region of fungal nucleic acid in the sample indicates the absence of any species of a fungus belonging to the genus Pneumocystis in the sample.

2. The method of claim 1 in which the detecting step includes contacting the sample with a probe comprising SEQ ID NO:

21.

3. The method of claim 1 in which the one or more internal PCR amplification controls comprise a non-fungal sequence.

4. The method of claim 1 in which the contacting 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.

5. The method of claim 4 in which the tRNA-LEU intron region comprises a portion of the Maize (Zea mayis) tRNA-LEU intron region.

6. The method of claim 5 in which the Maize (Zea inayis) tRNA-LEU intron region includes SEQ ID NO:

9.

7. The method of claim 6 further comprising detecting the presence of a nucleic acid including SEQ ID NO:

10.

8. The method of claim 7 in which the detecting step comprises contacting the sample with a pair of oligonucleotide primers including SEQ ID NO:

11 and SEQ ID NO;

12 and a molecular beacon probe including SEQ ID NO;

13.

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

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

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

12. The method of claim 11 in which the sample comprises a biological fluid, tissue, or combination thereof.

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

14. The method of claim 13 in which the source comprises a piece of a vehicle, watercraft, aircraft, building, or dwelling.

15. The method of claim 1 in which the source is obtained from an environment.

16. The method of claim 15 in which the sample comprises an air sample, a water sample, a soil sample, or combinations thereof.

17. The method of claim 1 in which the detecting 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.

18. The method of claim 17 in which the probe includes a detectable label.

19. The method of claim 1, wherein said method is used for the diagnosis of a fungal infection belonging to the genus Pneumocystis in a patient, and the sample is from said patient.