Method for identifying 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 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 variable region of mycobacteria nucleic acid and at least two detectably distinguishable probe sets of a signaling probe and an associated quencher probe which hybridize to adjacent nucleic acid sequences in the amplified mycobacteria nucleic acid, the signaling probe emitting fluorescent signal when it hybridizes to a single stranded nucleic acid target sequence, wherein the signal is quenchable by the associated quencher probe and the signaling probe does not emit fluorescence above background when not hybridized to its target sequence, the quencher probe comprising a non-fluorescent quencher such that when both the signaling and quencher probes are hybridized to the adjacent nucleic acid sequences, the non-fluorescent quencher of the quencher probe quenches the fluorescent signal of the associated signaling probe, at least one probe being part of both of the two probe sets, said probe: being a quencher probe labeled with a quencher at each end, the ends interacting with different signaling probes, or having a fluorophore on one end and a quencher on the other end, the fluorophore interacting with a quencher probe, the quencher interacting with a 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. a) providing: i) a sample suspected of comprising one or more mycobacteria, and ii) detection reagents comprising at least one pair of primers 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 variable region of mycobacteria nucleic acid and at least two detectably distinguishable probe sets of a signaling probe and an associated quencher probe which hybridize to adjacent nucleic acid sequences in the amplified mycobacteria nucleic acid, the signaling probe emitting fluorescent signal when it hybridizes to a single stranded nucleic acid target sequence, wherein the signal is quenchable by the associated quencher probe and the signaling probe does not emit fluorescence above background when not hybridized to its target sequence, the quencher probe comprising a non-fluorescent quencher such that when both the signaling and quencher probes are hybridized to the adjacent nucleic acid sequences, the non-fluorescent quencher of the quencher probe quenches the fluorescent signal of the associated signaling probe, at least one probe being part of both of the two probe sets, said probe: being a quencher probe labeled with a quencher at each end, the ends interacting with different signaling probes, or having a fluorophore on one end and a quencher on the other end, the fluorophore interacting with a quencher probe, the quencher interacting with a signaling probe, i) a sample suspected of comprising one or more mycobacteria, and ii) detection reagents comprising at least one pair of primers 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 variable region of mycobacteria nucleic acid and at least two detectably distinguishable probe sets of a signaling probe and an associated quencher probe which hybridize to adjacent nucleic acid sequences in the amplified mycobacteria nucleic acid, being a quencher probe labeled with a quencher at each end, the ends interacting with different signaling probes, or having a fluorophore on one end and a quencher on the other end, the fluorophore interacting with a quencher probe, the quencher interacting with a 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.