Multiplex detection of nucleic acids

A method for determining the presence or absence of first and second targets in a sample, the method comprising:

• (a) preparing a reaction mixture by contacting the sample or a derivative thereof putatively comprising the first and/or second targets or amplicons thereof with:
• first and second closed stem-loop oligonucleotides, wherein each of the closed stem-loop oligonucleotides comprise a double-stranded stem portion of hybridised nucleotides, connected to a closed single-stranded loop portion of unhybridised nucleotides, a fluorophore being connected to one strand of the double-stranded portion and a quencher to the other strand, wherein:
• the number of hybridised nucleotides and/or the sequence of hybridised nucleotides in the stem portion of the first and second closed stem-loop oligonucleotide differs, and
• enzymes capable of cleaving or degrading the single-stranded loop portion of the first and second closed stem-loop oligonucleotides only when in contact with the first or second target or an amplicon of the first or second target;
• (b) treating the reaction mixture:
• under conditions suitable for the enzymes to induce cleavage or degradation of the loop portion of the first and second closed stem-loop oligonucleotides if the first and second targets are present in the reaction mixture to thereby produce first and second open stem-loop oligonucleotides;
• at a first temperature at or above which strands of the double-stranded stem portion of the first open stem-loop oligonucleotide disassociate to thereby facilitate spatial separation of the fluorophore and quencher molecules of the stem portion of the first open stem-loop oligonucleotide and provide a first detectable fluorescent signal, and
• at a second temperature at or above which strands of the double-stranded stem portion of the second open stem-loop oligonucleotide disassociate to thereby facilitate spatial separation of the fluorophore and quencher molecules of the stem portion of the second open stem-loop oligonucleotide and provide a second detectable fluorescent signal;
• wherein:
• the first temperature is lower than the second temperature, and
• the fluorophore of the first open stem-loop oligonucleotide and the fluorophore of the second open stem-loop oligonucleotide emit in the same colour region of the visible spectrum;
• and
• (c) detecting levels of said first and second fluorescent signals at one or more temperatures comprising or consisting of a temperature equal to or above the second temperature, to thereby determine the presence or absence of said targets in the sample.