A method for reducing fluorescent signal variations in detecting a target nucleic acid sequence in a sample using two different detection temperatures, comprising: (a) incubating a reaction mixture containing the sample and a signal-generating means for detection of the target nucleic acid sequence in a reaction vessel and detecting fluorescent signals generated at a high detection temperature and at a low detection temperature; wherein the incubating and detecting are performed by a real-time amplification reaction; wherein the signal-generating means comprises an oligonucleotide labeled with a fluorescent label; wherein the signal-generating means generates a fluorescent signal (i) by a process comprising cleaving a mediation oligonucleotide specifically hybridized with the target nucleic acid sequence to release a fragment, hybridizing the fragment with a capture oligonucleotide, and extending the fragment to form an extended strand; wherein the formation of the extended strand is detected by detecting fluorescent signal generated dependent on the presence of the extended strand, or (ii) by a process comprising cleaving a detection oligonucleotide specifically hybridized with the target nucleic acid sequence; wherein the high detection temperature is temperature for generating a fluorescent signal indicating the presence of the target nucleic acid sequence and the low detection temperature is temperature for generating a fluorescent signal indicating the presence of the target nucleic acid sequence which has a different intensity from the fluorescent signal generated at the high detection temperature; and (b) determining the presence of the target nucleic acid sequence by a processed fluorescent signal obtained from a difference between the fluorescent signal intensity detected at the high detection temperature and the fluorescent signal intensity detected at the low detection temperature. (a) incubating a reaction mixture containing the sample and a signal-generating means for detection of the target nucleic acid sequence in a reaction vessel and detecting fluorescent signals generated at a high detection temperature and at a low detection temperature; wherein the incubating and detecting are performed by a real-time amplification reaction; wherein the signal-generating means comprises an oligonucleotide labeled with a fluorescent label; wherein the signal-generating means generates a fluorescent signal (i) by a process comprising cleaving a mediation oligonucleotide specifically hybridized with the target nucleic acid sequence to release a fragment, hybridizing the fragment with a capture oligonucleotide, and extending the fragment to form an extended strand; wherein the formation of the extended strand is detected by detecting fluorescent signal generated dependent on the presence of the extended strand, or (ii) by a process comprising cleaving a detection oligonucleotide specifically hybridized with the target nucleic acid sequence; wherein the high detection temperature is temperature for generating a fluorescent signal indicating the presence of the target nucleic acid sequence and the low detection temperature is temperature for generating a fluorescent signal indicating the presence of the target nucleic acid sequence which has a different intensity from the fluorescent signal generated at the high detection temperature; and (b) determining the presence of the target nucleic acid sequence by a processed fluorescent signal obtained from a difference between the fluorescent signal intensity detected at the high detection temperature and the fluorescent signal intensity detected at the low detection temperature. <EOS>