A method for detecting a plurality of target nucleic acid sequences in a sample, comprising: contacting in a single reaction vessel the sample to signal-generating compositions for detecting the plurality of target nucleic acid sequences; wherein the signal-generating compositions form a plurality of duplexes for the plurality of target nucleic acid sequences, each of the plurality of duplexes provides a signal indicating the presence or absence of each corresponding target nucleic acid sequence and the plurality of duplexes have T m values different from each other; performing a reaction for at least two cycles for (i) the formation of the plurality of duplexes and (ii) the dissociation of the duplexes; obtaining at all or partial cycles of the reaction signal values at at least two detection temperatures in each of a plurality of detection temperature ranges assigned to the plurality of target nucleic acid sequences; wherein each of the plurality of detection temperature ranges comprises a temperature range in which the amount of a duplex for each corresponding target nucleic acid sequence is changed but the amount of duplexes for other target nucleic acid sequences remains unchanged; obtaining a signal-change value of signal values between two reference temperatures selected from the at least two detection temperatures in each of the plurality of detection temperature ranges such that a data set of cycle/signal-change value for each of the plurality of target nucleic acid sequences is obtained; and determining the presence or absence of the plurality of target nucleic acid sequences in the sample by the data set of cycle/signal-change value for each of the plurality of target nucleic acid sequences. contacting in a single reaction vessel the sample to signal-generating compositions for detecting the plurality of target nucleic acid sequences; wherein the signal-generating compositions form a plurality of duplexes for the plurality of target nucleic acid sequences, each of the plurality of duplexes provides a signal indicating the presence or absence of each corresponding target nucleic acid sequence and the plurality of duplexes have T m values different from each other; performing a reaction for at least two cycles for (i) the formation of the plurality of duplexes and (ii) the dissociation of the duplexes; obtaining at all or partial cycles of the reaction signal values at at least two detection temperatures in each of a plurality of detection temperature ranges assigned to the plurality of target nucleic acid sequences; wherein each of the plurality of detection temperature ranges comprises a temperature range in which the amount of a duplex for each corresponding target nucleic acid sequence is changed but the amount of duplexes for other target nucleic acid sequences remains unchanged; obtaining a signal-change value of signal values between two reference temperatures selected from the at least two detection temperatures in each of the plurality of detection temperature ranges such that a data set of cycle/signal-change value for each of the plurality of target nucleic acid sequences is obtained; and determining the presence or absence of the plurality of target nucleic acid sequences in the sample by the data set of cycle/signal-change value for each of the plurality of target nucleic acid sequences. <EOS>