Assays for fungal infection

Patent metadata

Field	Value
Publication number	US-11230742-B2
Application number	US-201916542782-A
Country	US
Kind code	B2
Filing date	Aug 16, 2019
Priority date	Nov 2, 2006
Publication date	Jan 25, 2022
Grant date	Jan 25, 2022

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.

First claim

We claim:

1. A method for detecting the presence or absence of any species of a fungus belonging to the genus Aspergillus in a human clinical sample, comprising:
i. obtaining a human clinical sample suspected of containing fungal nucleic acid from the genus Aspergillus , and
ii. testing for the presence or absence in the human clinical sample of an identifying region of fungal nucleic acid comprising SEQ ID NO: 14, the complement or transcript thereof, or a sequence having 80% or more sequence identity with SEQ ID NO: 14, the complement or transcript thereof, said identifying region being present in species of the genus Aspergillus but not those of the genus of Candida or Pneumocystis , wherein the testing includes contacting the human clinical sample with a molecular beacon probe comprising SEQ ID NO: 17 and
wherein the presence of said identifying region of fungal nucleic acid indicates the presence of any species of a fungus belonging to the genus Aspergillus in the human clinical sample, and the absence of said identifying region of fungal nucleic acid in the human clinical sample indicates the absence of any species of a fungus belonging to the genus Aspergillus in the human clinical sample.

2. The method of claim 1 which further comprises amplifying the identifying region of fungal nucleic acid by contacting the human clinical sample with a pair of primers including SEQ ID NO: 15 and SEQ ID NO: 16.

3. The method of claim 1 which further comprises amplifying the identifying region of fungal nucleic acid by contacting the human clinical sample with a pair of primers including SEQ ID NO: 46 and SEQ ID NO: 47.

4. The method of claim 1 , further comprising testing for the presence or absence in the human clinical sample of at least one region of fungal nucleic acid characteristic of the genus Pneumocystis jirovecii.

5. The method of claim 4 , wherein the testing comprises the use of amplification primers having the sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20 and oligonucleotide probes having the sequences of SEQ ID NO: 17 and SEQ ID NO: 21.

6. The method of claim 2 in which the amplifying step is carried out in the presence of one or more internal PCR amplification controls.

7. The method of claim 6 in which the one or more internal PCR amplification controls comprise a non-fungal sequence.

8. The method of claim 6 in which the amplifying 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.

9. The method of claim 8 in which the tRNA-LEU intron region comprises a portion of the Maize ( Zea mays ) tRNA-LEU intron region.

10. The method of claim 9 in which the Maize ( Zea mays ) tRNA-LEU intron region includes SEQ ID NO: 9.

11. The method of claim 10 further comprising detecting the presence of a nucleic acid including SEQ ID NO: 10.

12. The method of claim 11 in which the detecting step comprises contacting the human clinical sample with a pair of oligonucleotides primers including SEQ ID NO: 11 and SEQ ID NO: 12 and a molecular beacon probe including SEQ ID NO: 13.

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

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

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

16. The method of claim 15 in which the human clinical sample comprises a biological fluid, tissue, or combination thereof.

17. The method of claim 1 in which the testing step further comprises contacting the human clinical sample with an oligonucleotide probe comprising a nucleic acid capable of hybridizing to 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 Aspergillus in a patient, and the human clinical sample is from said patient.

Assignees

• Univ Rutgers

Inventors

• Perlin David S
• Park Steven
• Denning David W

Classifications

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/6895 Primary

  for plants, fungi or algae · CPC title