Compositions, methods, and kits for detecting and identifying mycobacteria

Patent metadata

Field	Value
Publication number	US-10273525-B2
Application number	US-201113884873-A
Country	US
Kind code	B2
Filing date	Nov 10, 2011
Priority date	Nov 10, 2010
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

Abstract

Provided herein are methods for detecting and identifying strains of mycobacteria, and compositions and kits for performing such methods. In particular, nucleic acid amplification and fluorescence detection methods are provided for the detection and differentiation of mycobacteria based on, for example, pathogenicity, species, and antibiotic resistance or sensitivity. Compositions and methods are provided herein to identify and differentiate mycobacteria in mixtures of different mycobacteria and mycobacteria and non-mycobacteria.

First claim

We claim:

1. Reagents for amplification and detection of a type of mycobacterium in a sample comprising:
a) at least one pair of primers, wherein said primers are configured to hybridize to regions of mycobacteria nucleic acid conserved between two or more types of mycobacteria, and wherein said primers are configured to amplify a region of mycobacteria nucleic acid that varies between the two or more types of mycobacteria; and
b) at least two detectably distinguishable probe sets, wherein each set of the at least two detectably distinguishable probe sets comprises a signaling probe and an associated quencher probe which hybridize to adjacent nucleic acid sequences in an amplified region of mycobacteria nucleic acid amplified by the primers in a), wherein each set of the at least two detectably distinguishable probe sets contains at least one identical probe such that the at least one identical probe is shared between each set of the at least two detectably distinguishable probe sets, and the at least one identical probe is adjacent to two other probes in the at least two detectably distinguishable probe sets on the amplified region of the mycobacteria nucleic acid amplified by the primers in a) when the at least two detectably distinguishable probe sets hybridize to the mycobacteria nucleic acid amplified by the primers in a) such that there are no unhybridized nucleic acid bases between the at least one identical probe and the two other probes on the amplified region of mycobacteria nucleic acid amplified by the primers in a);
the signaling probe comprising a fluorescence-emitting fluorophore with a quencher of the fluorescence-emitting fluorophore adjacent to the fluorescence-emitting fluorophore on the signaling probe such that said signaling probe does not emit a fluorescent signal above background fluorescence when not hybridized to its target sequence, the quencher probe comprising a non-fluorescent quencher such that when both the quencher probe and the signaling probe are hybridized to the adjacent nucleic acid sequences in the amplified region of the mycobacteria nucleic acid amplified by the primers in a), the non-fluorescent quencher of the quencher probe quenches the fluorescent signal emitted by the fluorescence-emitting fluorophore of the signaling probe,
wherein said at least one identical probe is the quencher probe, wherein each end of the quencher probe is labeled with a non-fluorescent quencher, the non-fluorescent quencher on one end of the quencher probe interacts with the fluorescence-emitting fluorophore of said signaling probe from one set of the at least two detectably distinguishable probe sets and the non-fluorescent quencher on other end of the quencher probe interacts with said fluorescence-emitting fluorophore of the signaling probe from another set of the at least two detectably distinguishable probe sets when the at least two detectably distinguishable probe sets hybridize to the amplified mycobacteria nucleic acid amplified by the primers in a), or
said at least one identical probe is either the signal probe or the quencher probe, said at least one identical probe has a fluorophore on its one end and a fluorophore quencher on its other end, the fluorophore of said at least one identical probe interacts with the non-fluorescent quencher of said quencher probe from one set of the at least two detectably distinguishable probe sets and the fluorophore quencher of said at least one identical probe interacts with the fluorescence-emitting fluorophore of said signaling probe from another set of the at least two detectably distinguishable probe sets when the at least two detectably distinguishable probe sets hybridize to the amplified region of mycobacteria nucleic acid amplified by the primers in a).

2. The reagents of claim 1 , wherein, in each set of the at least two detectably distinguishable probe sets, the melting temperature of the signaling probe is higher than the melting temperature of the associated quencher probe.

3. The reagents of claim 1 , wherein the at least two detectably distinguishable probe sets comprise 5 or more probe sets.

4. The reagents of claim 1 , wherein said at least one pair of primers comprises a Limiting Primer and an Excess Primer, wherein the Limiting Primer and Excess Primer have initial concentrations and melting temperatures that allow amplification of a region of mycobacteria nucleic acid that varies between the two or more types of mycobacteria by Linear-After-The-Exponential-PCR.

5. The reagents of claim 1 , wherein one or more of the two detectably distinguishable probe sets are configured to hybridize to a region sequence of mycobacteria nucleic acid to differentiate between non-tuberculosis mycobacteria (NTM) and Mycobacterium tuberculosis complex (MTBC).

6. The reagents of claim 5 , wherein said at least one pair of primers is configured to amplify a mycobacterial nucleic acid sequence that codes for a ribosomal RNA.

7. The reagents of claim 6 , wherein the ribosomal RNA is 16S ribosomal RNA.

8. The reagents of claim 7 , wherein said reagents comprise one or more of SEQ ID NOS.:51-54.

9. The reagents of claim 1 , wherein one or more sets of the at least two detectably distinguishable probe sets are configured to hybridize to a region of mycobacteria nucleic acid to differentiate between different species of MTBC.

10. The reagents of claim 9 , wherein one or more primer pairs of the at least one pair of primers are configured to amplify a region of mycobacteria gyrB gene.

11. The reagents of claim 10 , wherein said reagents comprise one or more of SEQ ID NOS.:58-62.

12. The reagents of claim 1 , wherein three or more detectably distinguishable probe sets of the at least two detectably distinguishable probe sets are configured to hybridize to a region of mycobacteria nucleic acid to differentiate between rifampin-resistant mycobacteria and rifampin-sensitive mycobacteria.

13. The reagents of claim 12 , wherein one or more primer pairs of the at least one pair of primers are configured to amplify a region of mycobacteria rpoB gene.

14. The reagents of claim 13 , wherein said three or more detectably distinguishable probe sets comprise SEQ ID NO.:30-35.

15. The reagents of claim 14 , wherein said at least one pair of primers comprise SEQ ID NO.:28 and SEQ ID NO.:29.

16. The reagents of claim 1 , wherein one or more detectably distinguishable probe sets of the at least two detectably distinguishable probe sets are configured to amplify a region of mycobacteria nucleic embB gene to differentiate between ethambutol-resistant mycobacteria and ethambutol-sensitive mycobacteria.

17. The reagents of claim 1 , wherein one or more detectably distinguishable probe sets of the at least two detectably distinguishable probe sets are configured to hybridize to a region of mycobacteria nucleic acid to differentiate between isoniazid-resistant mycobacteria and isoniazid-sensitive mycobacteria.

18. The reagents of claim 17 , wherein one or more primer pairs of the at least one pair of primers are configured to amplify a region of mycobacteria mabA promoter region.

19. The reagents of claim 18 , wherein said reagents comprise one or more of SEQ ID NOS.:45-48.

20. The reagents of claim 17 , wherein one or more primer pairs of the at least one pair of primers are configured to amplify a region of mycobacteria ahpC gene.

21. The reagents of claim 17 , wherein one or more primer pairs of the at least one pair of primers are configured to amplify a region of mycoba

Assignees

• Wangh Lawrence J
• Rice John
• Univ Brandeis

Inventors

• Wangh Lawrence J
• Rice John

Classifications

• C12Q1/689 Primary

  for bacteria · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2527/107

  Temperature of melting, i.e. Tm · CPC title

• C12Q2600/106

  Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism · CPC title

• C12Q2537/101

  Homogeneous assay format, e.g. one pot reaction · CPC title