Method for detecting target nucleic acid

A method for detecting one or more target nucleic acids present in a sample, comprising the following steps:

• (1) adding, to the sample,
• a fluorophore-labeled primer or probe, wherein the fluorophore-labeled primer or probe is a fluorophore-labeled oligonucleotide having complementarity to a target nucleic acid, and
• a quencher-labeled probe, wherein the quencher-labeled probe is a quencher-labeled oligonucleotide having complementarity to the fluorophore-labeled primer or probe and having a melting temperature (Tm) lower than that of the fluorophore-labeled primer or probe;
• (2) incubating the sample at a temperature equal to or lower than the melting temperature (Tm) of the fluorophore-labeled primer or probe and higher than the melting temperature (Tm) of the quencher-labeled probe;
• (3) incubating the sample at a temperature equal to or lower than the melting temperature (Tm) of the quencher-labeled probe;
• and
• (4) measuring fluorescence of the fluorophore-labeled primer or probe bound with the target nucleic acid.