Compositions, methods, and kits for detecting and identifying mycobacteria

Reagents for amplification and detection of one or more types of mycobacterium in a sample comprising:

• a) at least one pair of primers, wherein said primers are configured to bind to regions of mycobacteria nucleic acid conserved between two or more types of mycobacteria, and wherein said primers are configured to amplify a variable region of mycobacteria nucleic acid;
• and
• b) at least one detectably distinguishable probe set of two hybridization probes which hybridize to adjacent target nucleic acid sequences within said variable region of mycobacteria nucleic acid, comprising:
• i) a quencher probe labeled with a non-fluorescent quencher, and
• ii) a signaling probe labeled with a fluorescence-emitting fluorophore and a non-fluorescent quencher, wherein said signal probe does not emit fluorescence above background when not hybridized to its target sequence, but emits a fluorescence signal above background upon hybridization to its target sequence in the absence of bound quencher probe, wherein, if both signaling and quencher probes are hybridized to their adjacent target nucleic acid sequences, the non-fluorescent quencher of the quencher probe quenches the signal from the signaling probe.

A method for amplification and identification of one or more types of mycobacteria in a sample, comprising:

• a) providing:
• i) a sample suspected of comprising one or more mycobacteria, and
• ii) detection reagents comprising at least one pair of primers and at least one detectably distinguishable probe set of two hybridization probes which hybridize to adjacent target nucleic acid sequences in one or more mycobacteria, each probe set comprising:
• A) a quencher probe labeled with a non-fluorescent quencher, and
• B) a signaling probe labeled with a fluorescence-emitting fluorphore and a non-fluorescent quencher, wherein said signal probe does not emit fluorescence above background when not hybridized to its target sequence, but emits a fluorescence signal above background upon hybridization to its target sequence in the absence of bound quencher probe, wherein, if both signaling and quencher probes are hybridized to their adjacent target nucleic acid sequences, the non-fluorescent quencher of the quencher probe quenches the signal from the signaling probe;
• b) amplifying nucleic acid from said one or more mycobacteria with said primers;
• c) detecting the fluorescence of said fluorescence-emitting fluorophore from each detectably distinguishable probe set at a range of temperatures;
• d) generating temperature-dependent fluorescence signatures for each fluorescence-emitting fluorophore;
• and
• e) analyzing said temperature-dependent fluorescence signatures to identify one or more mycobacterium in said sample.