Detection of multiple target nucleic acids using multiple detection temperatures

Abstract

The present disclosure relates to a method for detecting multiple target nucleic acids in a single reaction vessel by using multiple detection temperatures with a single type of label alone, characterized in that the method provides a signal change dependent on the presence of corresponding target nucleic acids at the corresponding detection temperatures of each of the target nucleic acids. Conventional techniques using a single type of label require melting analysis after target amplification so that multiple target nucleic acids are detected. In contrast, the method of the present invention does not require melting curve analysis after target amplification even when using a single type of label, and therefore can significantly reduce analysis time.

Description

[Technical Field]

[0001] [1] The present invention relates to a method for detecting multiple target nucleic acids using multiple detection temperatures. [Background technology]

[0002] [2] Real-time detection methods are widely used to detect target nucleic acids, which can detect target nucleic acids by monitoring target amplification in real time. Real-time detection methods generally use labeled probes or primers that specifically hybridize with the target nucleic acid.

[0003] [3] Examples of methods using hybridization between a labeled probe and a target nucleic acid include the molecular beacon method, which uses a dual-labeled probe with a hairpin structure (Tyagi et al., Nature Biotechnology v.14, March 1996), the Hy beacon method (French DJ et al., Mol. Cell Probes, 15(6):363-374 (2001)), the hybridization probe method, which uses two probes labeled as donor and acceptor, respectively (Bernard et al., 147-148 Clin. Chem. 2000;46), and the Lux method, which uses a single labeled oligonucleotide (U.S. Patent No. 7,537,886). The TaqMan method, which uses cleavage of a dual-labeled probe by the 5'-nuclease activity of DNA polymerase (U.S. Patent Nos. 5,210,015 and 5,538,848), is also widely used in the technical field of the present invention.

[0004] [4] Examples of methods using labeled primers include the Sunrise primer method (Nazarenko et al., Nucleic Acids Research, 1997, Vol. 25, No. 12, pp. 2516-2521, and U.S. Pat. No. 6,117,635), the Scorpion primer method (Whitcombe et al., Nature Biotechnology, Vol. 17, August 1999, pp. 804-807, and U.S. Pat. No. 6,326,145), and the TSG primer method (WO 2011 / 078441).

[0005] [5] As an alternative approach, real-time detection methods using duplexes formed in response to the presence of target nucleic acids have been proposed: the Invader assay (U.S. Pat. Nos. 5,691,142, 6,358,691, and 6,194,149), the PTOCE (PTO cleavage and extension) method (WO 2012 / 096523), the PCE-SH (PTO cleavage and extension dependent signaling oligonucleotide hybridization) method (WO 2013 / 115442), and the PCE-NH (PTO cleavage and extension dependent dehybridization) method (PCT / KR2013 / 012312).

[0006] [6] Conventional real-time detection methods can only detect a single target nucleic acid with a single label, so the number of target nucleic acids that can be simultaneously detected in a single reaction is limited by the number of labels available (e.g., five or fewer).

[0007] [7] Melting analysis can potentially be used to detect multiple target nucleic acids using a single label, but has the disadvantage of requiring longer run times than real-time methods.

[0008] [8] Meanwhile, in recent years, methods have been proposed that enable real-time detection of multiple target nucleic acids with a single type of label using signal detection at different detection temperatures (WO 2015 / 147370, WO 2015 / 147377, WO 2015 / 147382, WO 2015 / 147412). However, among these different detection temperatures, these methods also enable target nucleic acids with a relatively higher detection temperature to be detected together with target nucleic acids with a relatively lower detection temperature, thus requiring a process for obtaining the difference between signals detected at different detection temperatures (e.g., the difference between a signal detected at a relatively lower detection temperature and a signal detected at a relatively higher detection temperature).

[0009] [9] Therefore, there is a need to develop novel methods or approaches for detecting multiple target nucleic acids with greatly improved convenience and high efficiency.

[10]

[0010]

[11] Throughout this application, various patents and publications are referenced, citations are provided within parentheses. The disclosures of these patents and publications in their entireties are hereby incorporated by reference into the present disclosure in order to more fully describe the present invention and the state of the art to which it pertains. Summary of the Invention [Problem to be solved by the invention]

[0011]

[12] The present inventors have endeavored to develop a method for detecting multiple target nucleic acids by using a single type of label and a single type of detector in a single reaction vessel. As a result, the present inventors have confirmed that it is possible to detect multiple target nucleic acids in a real-time manner by using a single type of label and a single type of detector in a single reaction vessel with improved convenience and high cost-effectiveness and efficiency, by adjusting the detection temperature and signal generation mechanism so that only a single signal indicating the presence of a single target nucleic acid sequence is provided at a single detection temperature, even while using multiple detection temperatures.

[0012]

[13] It is therefore an object of the present disclosure to provide a method for detecting n target nucleic acids in a sample.

[0013]

[14] Another object of the present disclosure is to provide a kit for detecting n target nucleic acids in a sample. [Means for solving the problem]

[0014]

[15] According to one aspect of the present disclosure, there is provided a method for detecting n target nucleic acids in a sample, comprising: (a) detecting signals at n detection temperatures while incubating a sample suspected of containing at least one of the n target nucleic acids in a reaction vessel with n compositions for detecting the target nucleic acid, wherein n is an integer of 2 or greater; the incubation includes a plurality of cycles; and the detection of the signal is performed in at least one of the plurality of cycles; each of the n compositions for detecting the target nucleic acid provides a signal change at a corresponding detection temperature among the n detection temperatures in the presence of a corresponding target nucleic acid; wherein the signal change indicates the presence of the corresponding target nucleic acid, and a composition for detecting an ith target nucleic acid among the n compositions for detecting target nucleic acids provides a signal change at the ith detection temperature among the n detection temperatures, and provides a constant signal at other detection temperatures in the presence of the ith target nucleic acid, the signal change indicating the presence of the ith target nucleic acid, i representing an integer from 1 to n, and the ith detection temperature being lower than the (i+1)th detection temperature; and (b) determining the presence of the n target nucleic acids from the signals detected in step (a), wherein the presence of the ith target nucleic acid is determined by the signal change detected at the ith detection temperature.

[0015]

[16] According to an embodiment of the present disclosure, in a temperature range covering all n detection temperatures, a composition for detecting an i-th target nucleic acid has a signal change temperature range (SChTR) in which the signal changes in response to the presence of the i-th target nucleic acid, and a signal constant temperature range (SCoTR) in which the signal is constant even in the presence of the i-th target nucleic acid.

[0016]

[17] According to an embodiment of the present disclosure, the composition for detecting the i-th target nucleic acid has one or two signal-constant temperature ranges.

[0017]

[18] According to an embodiment of the present disclosure, the composition for detecting the i-th target nucleic acid is any one of (i) an under-signal change (UnderSC) composition characterized in that the signal change temperature range is lower than the signal constant temperature range; (ii) an over-signal change (OverSC) composition characterized in that the signal change temperature range is higher than the signal constant temperature range; and (iii) an inter-signal change (InterSC) composition characterized in that the signal change temperature range is higher than one of two signal constant temperature ranges and lower than the other of the two signal constant temperature ranges.

[0018]

[19] According to an embodiment of the present disclosure, the i-th detection temperature is selected within the signal change temperature range of the composition for detecting the i-th target nucleic acid, and the i-th detection temperature is not included in the signal change temperature range of the composition for detecting any other target nucleic acid.

[0019]

[20] According to an embodiment of the present disclosure, the signal change temperature range of a composition for detecting an i-th target nucleic acid partially overlaps with the signal change temperature range of a composition for detecting a target nucleic acid having an adjacent detection temperature, and does not overlap with the signal change temperature range of a composition for detecting a target nucleic acid having a non-adjacent detection temperature.

[0020]

[21] According to an embodiment of the present disclosure, when n is 2, the composition for detecting a first target nucleic acid is an UnderSC composition or an InterSC composition, and the composition for detecting a second target nucleic acid is an InterSC composition or an OverSC composition.

[0021]

[22] According to an embodiment of the present disclosure, when n is 3 or greater, the composition for detecting the first target nucleic acid is an UnderSC composition or an InterSC composition, the composition for detecting the nth target nucleic acid is an InterSC composition or an OverSC composition, and each of the compositions for detecting target nucleic acids other than the first target nucleic acid and the nth target nucleic acid is an InterSC composition.

[0022]

[23] According to an embodiment of the present disclosure, a composition for detecting an i-th target nucleic acid includes a label that provides a signal dependent on the presence of the i-th target nucleic acid.

[0023]

[24] According to embodiments of the present disclosure, the label is either linked to the oligonucleotide or incorporated into the oligonucleotide during incubation.

[0024]

[25] According to an embodiment of the present disclosure, a composition for detecting an i-th target nucleic acid provides a duplex that provides a signal change.

[0025]

[26] According to an embodiment of the present disclosure, a composition for detecting an i-th target nucleic acid provides a duplex that provides a signal change, and when the duplex that provides the signal change is in an associated form, the composition for detecting an i-th target nucleic acid provides a signal from a label.

[0026]

[27] According to an embodiment of the present disclosure, the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and when the duplex that provides the signal change is in a dissociated form, the composition for detecting the i-th target nucleic acid provides a signal from the label.

[0027]

[28] According to an embodiment of the present disclosure, the duplex that provides the signal change is originally included in the composition for detecting the i-th target nucleic acid.

[0028]

[29] According to an embodiment of the present disclosure, the duplex that provides the signal change is generated by hybridization of a label-linked oligonucleotide with an oligonucleotide that is hybridizable to the label-linked oligonucleotide.

[0029]

[30] According to an embodiment of the present disclosure, duplexes that provide a signal change are generated during incubation.

[0030]

[31] According to an embodiment of the present disclosure, a duplex that provides a signal change is generated by hybridization of a label-linked oligonucleotide with a target nucleic acid.

[0031]

[32] According to an embodiment of the present disclosure, the duplex that provides the signal change is generated by a cleavage reaction that is dependent on the presence of the target nucleic acid.

[0032]

[33] According to an embodiment of the present disclosure, a composition for detecting a target nucleic acid comprises a tagging oligonucleotide that hybridizes to the target nucleic acid, and the cleavage reaction dependent on the presence of the target nucleic acid comprises cleavage of the tagging oligonucleotide.

[0033]

[34] According to embodiments of the present disclosure, the duplex that provides the signal change may be a single type of duplex or multiple types of duplexes.

[0034]

[35] According to an embodiment of the present disclosure, when the duplex that provides the signal change is a single type of duplex, the amount of the single type of duplex changes depending on the presence of the target nucleic acid, thereby changing the signal.

[0035]

[36] According to an embodiment of the present disclosure, when the duplexes that provide the signal change are multiple types of duplexes, the ratio of the amounts between the multiple types of duplexes changes depending on the presence of the target nucleic acid, thereby changing the signal.

[0036]

[37] According to an embodiment of the present disclosure, when the duplex is a multiple type duplex, the Tm values ​​of the duplexes are different from each other.

[0037]

[38] According to embodiments of the present disclosure, at least two of the multiple types of duplexes comprise the same single strand.

[0038]

[39] According to an embodiment of the present disclosure, the duplex that provides the signal change includes a label.

[0039]

[40] According to an embodiment of the present disclosure, a composition for detecting an i-th target nucleic acid provides a duplex that provides a signal change, and the signal change temperature range of the composition for detecting the i-th target nucleic acid is determined according to the length and / or sequence of the duplex.

[0040]

[41] According to an embodiment of the present disclosure, detecting the signal occurs in at least two of the multiple cycles.

[0041]

[42] According to an embodiment of the present disclosure, the signal change is measured using signals detected in at least two of the multiple cycles.

[0042]

[43] According to an embodiment of the present disclosure, the signal change at the ith detection temperature is measured using the signal detected in at least one of the plurality of cycles and a reference signal value.

[0043]

[44] According to an embodiment of the present disclosure, the reference signal value is obtained from a reaction in the absence of the i-th target nucleic acid.

[0044]

[45] According to an embodiment of the present disclosure, detection of the signal at each of the n detection temperatures is performed using a single type of detector.

[0045]

[46] According to an embodiment of the present disclosure, the signals detected at the n detection temperatures are not distinguished from one another by a single type of detector.

[0046]

[47] According to an embodiment of the present disclosure, the incubation comprises a nucleic acid amplification reaction.

[0047]

[48] ​​According to an embodiment of the present disclosure, the nucleic acid amplification reaction is a polymerase chain reaction (PCR).

[0048]

[49] According to another aspect of the present disclosure, there is provided a method for detecting two target nucleic acids in a sample, comprising:

[50] (a) detecting a signal at a first detection temperature and a second detection temperature while incubating a sample suspected of containing at least one of two target nucleic acids in a reaction vessel with a composition for detecting a first target nucleic acid and a composition for detecting a second target nucleic acid, wherein the incubation comprises a plurality of cycles, and the detection of the signal is performed in at least one of the plurality of cycles, wherein the composition for detecting the first target nucleic acid provides a signal change at the first detection temperature and a constant signal at the second detection temperature in the presence of the first target nucleic acid, and the signal change indicates the presence of the first target nucleic acid. a composition for detecting a second target nucleic acid, which, in the presence of the second target nucleic acid, provides a signal change at a second detection temperature and a constant signal at a first detection temperature, the signal change indicating the presence of the second target nucleic acid, the first detection temperature being lower than the second detection temperature; and (b) determining the presence of two target nucleic acids from the signals detected in step (a), wherein the presence of the first target nucleic acid is determined by the signal change detected at the first detection temperature and the presence of the second target nucleic acid is determined by the signal change detected at the second detection temperature.

[0049]

[51] According to another aspect of the present disclosure, there is provided a method for detecting three target nucleic acids in a sample, comprising:

[52] (a) detecting signals at a first detection temperature, a second detection temperature, and a third detection temperature while incubating a sample suspected of containing at least one of three target nucleic acids in a reaction vessel with a composition for detecting a first target nucleic acid, a composition for detecting a second target nucleic acid, and a composition for detecting a third target nucleic acid, wherein the incubation includes a plurality of cycles, and the detection of the signals is performed in at least one of the plurality of cycles, wherein the composition for detecting the first target nucleic acid provides a signal change at the first detection temperature in the presence of the first target nucleic acid and provides constant signals at the second detection temperature and the third detection temperature, the signal change indicating the presence of the first target nucleic acid; and the composition for detecting the second target nucleic acid provides a signal change at the second detection temperature in the presence of the second target nucleic acid and provides constant signals at the first detection temperature. and a third detection temperature, wherein the signal change indicates the presence of the second target nucleic acid; a composition for detecting the third target nucleic acid, in the presence of the third target nucleic acid, provides a signal change at the third detection temperature, and provides a constant signal at the first detection temperature and the second detection temperature, wherein the signal change indicates the presence of the third target nucleic acid, the first detection temperature being lower than the second detection temperature and the second detection temperature being lower than the third detection temperature; and (b) determining the presence of three target nucleic acids from the signals detected in step (a), wherein the presence of the first target nucleic acid is determined by the signal change detected at the first detection temperature, the presence of the second target nucleic acid is determined by the signal change detected at the second detection temperature, and the presence of the third target nucleic acid is determined by the signal change detected at the third detection temperature.

[0050]

[53] According to another aspect of the present disclosure, there is provided a kit comprising n compositions for detecting n target nucleic acids in a sample, where n is an integer of 2 or greater, each of the n compositions for detecting the n target nucleic acids provides a signal change at a corresponding detection temperature among n detection temperatures, where the signal change indicates the presence of the corresponding target nucleic acid, and a composition for detecting an i-th target nucleic acid among the n target nucleic acids provides a signal change at the i-th detection temperature among the n detection temperatures in the presence of the i-th target nucleic acid and provides a constant signal at other detection temperatures, where i represents an integer from 1 to n, and the i-th detection temperature is lower than the (i+1)-th detection temperature. [Effects of the Invention]

[0051]

[54] The features and advantages of the present disclosure are summarized as follows:

[0052]

[55] (a) The present disclosure relates to a method for detecting multiple target nucleic acids in a single reaction vessel by using multiple detection temperatures with a single type of label alone, characterized by providing a signal change dependent on the presence of the corresponding target nucleic acid at each corresponding detection temperature for the target nucleic acid.

[0053]

[56] (b) Interestingly, the present inventors have found that various signal generation mechanisms for detecting target nucleic acids known in the art include temperature ranges in which the signal changes in response to the presence of the target nucleic acid (i.e., signal change temperature ranges) and temperature ranges in which the signal does not change even in the presence of the target nucleic acid (i.e., signal constant temperature ranges), and can be categorized into three types according to the number and / or order of these signal change temperature ranges and signal constant temperature ranges. The present inventors have also found that an appropriate combination of these three types of signal generation mechanisms is useful in detecting multiple target nucleic acids.

[0054]

[57] (c) In particular, when a signal generation mechanism comprising one signal change temperature range and two signal constant temperature ranges (i.e., a signal generation mechanism that can be adopted by the InterSC composition for detecting target nucleic acids) is applied to the present disclosure, the number of target nucleic acids that can be detected by the method disclosed herein is further increased.

[0055]

[58] (d) By employing one or more of the three types of signal generating mechanisms described above, and further adjusting the signal change temperature range of one or more signal generating mechanisms so that only a signal indicating the presence of a single target nucleic acid is provided at each detection temperature, the present disclosure has the advantage that the presence of a specific target nucleic acid can be determined by a signal change measured at a specific detection temperature alone, without having to consider signal changes at other detection temperatures (e.g., detection temperatures other than the specific detection temperature, i.e., detection temperatures that show a signal change indicating the presence of other target nucleic acids). According to the present disclosure, a method according to the present disclosure utilizes n compositions for detecting n different target nucleic acids, each composition corresponding to a respective target nucleic acid. In one embodiment, each of the n compositions for detecting a target nucleic acid employs one of the three types of signal generating mechanisms so that a signal change indicating the presence of the corresponding target nucleic acid is provided only at the corresponding detection temperature among the n detection temperatures.

[0056]

[59] (e) The method disclosed herein enables the detection of multiple target nucleic acids in real time in a single reaction vessel by using a single type of label alone. Conventional techniques using a single type of label require target amplification followed by melting analysis so that multiple target nucleic acids can be detected. In contrast, the method of the present invention does not require melting curve analysis after target amplification, even when using a single type of label, thereby significantly reducing analysis time. [Brief explanation of the drawings]

[0057] [Figure 1a]1a-1h show examples of signal generation mechanisms that can be employed by the UnderSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 1b] 1a-1h show examples of signal generation mechanisms that can be employed by the UnderSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 1c] 1a-1h show examples of signal generation mechanisms that can be employed by the UnderSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 1d] 1a-1h show examples of signal generation mechanisms that can be employed by the UnderSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 1e] 1a-1h show examples of signal generation mechanisms that can be employed by the UnderSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 1f]1a-1h show examples of signal generation mechanisms that can be employed by the UnderSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 1g] 1a-1h show examples of signal generation mechanisms that can be employed by the UnderSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 1h] 1a-1h show examples of signal generation mechanisms that can be employed by the UnderSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 2a] 2a-2c show examples of signal generation mechanisms that can be employed by the OverSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 2b] 2a-2c show examples of signal generation mechanisms that can be employed by the OverSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 2c]2a-2c show examples of signal generation mechanisms that can be employed by the OverSC composition. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 3a] 3a-3b show examples of signal generation mechanisms that can be employed by InterSC compositions. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 3b] 3a-3b show examples of signal generation mechanisms that can be employed by InterSC compositions. In each figure, "(a)" represents pre-incubation in the presence or absence of target nucleic acid or post-incubation in the absence of target nucleic acid, and "(b)" represents post-incubation in the presence of target nucleic acid. [Figure 4] FIG. 4 shows temperature ranges that can be selected as detection temperatures in the cases where the SChTRs do not overlap each other or where the SChTRs partially overlap each other. [Figure 5] Figure 5 shows a schematic diagram of the PTOCE-based signal generation mechanism that can be employed by the UnderSC composition used in the examples. As depicted in the figure, the CTO has a reporter molecule and a quencher molecule in its templating portion. [Figure 6] Figure 6 shows a schematic diagram of the PTOCE-based signal generation mechanism that can be employed by the InterSC composition used in the examples. As depicted in the figure, the PTO has a quencher molecule in its 5'-tagging portion, and the CTO has a reporter molecule in its capture portion. [Figure 7]7 illustrates the signal generation mechanism of the dual quenching method that can be employed by the OverSC composition used in the examples. As depicted in the figure, the PTO has a first quencher molecule and a reporter molecule, and the CQO has a second quencher molecule. [Figure 8] FIG. 8 shows diagrammatically the signal generation mechanisms of the two compositions used in Combination 1 in the Examples, the UnderSC composition and the InterSC composition. [Figure 9] FIG. 9 illustrates the signal generation mechanism of the two compositions used in Combination 2 in the Examples, the UnderSC composition and the OverSC composition. [Figure 10] FIG. 10 shows diagrammatically the signal generation mechanisms of the two compositions used in Combination 3 in the Examples, the InterSC composition and the InterSC composition. [Figure 11] FIG. 11 illustrates the signal generation mechanism of the two compositions used in Combination 4 in the Examples, the InterSC composition and the OverSC composition. [Figure 12] FIG. 12 illustrates the signal generation mechanisms of the three compositions used in Combination 5 in the Examples: UnderSC composition, InterSC composition, and OverSC composition. [Figure 13] Figure 13 shows the results of real-time PCR for Combination 1 in this example. In the figure, "Target 1" represents the genomic DNA of Chlamydia trachomatis (CT), "Target 2" represents the genomic DNA of Neisseria gonorrhoeae (NG), "NTC" represents the no template control, and "RFU" represents relative fluorescence units. [Figure 14] Figure 14 shows the results of real-time PCR for Combination 2 in this example. In the figure, "Target 1" represents the genomic DNA of Chlamydia trachomatis (CT), "Target 2" represents the genomic DNA of Ureaplasma parvum (UP), "NTC" represents the no template control, and "RFU" represents relative fluorescence units. [Figure 15] Figure 15 shows the results of real-time PCR for Combination 3 in this example. In the figure, "Target 1" represents the genomic DNA of Chlamydia trachomatis (CT), "Target 2" represents the genomic DNA of Neisseria gonorrhoeae (NG), "NTC" represents the no template control, and "RFU" represents relative fluorescence units. [Figure 16] Figure 16 shows the results of real-time PCR for Combination 4 in this example. In the figure, "Target 1" represents genomic DNA of Neisseria gonorrhoeae (NG), "Target 2" represents genomic DNA of Ureaplasma parvum (UP), "NTC" represents no template control, and "RFU" represents relative fluorescence units. [Figure 17a] 17a and 17b show the results of real-time PCR for Combination 5 in this example. In each figure, "Target 1" represents the genomic DNA of Chlamydia trachomatis (CT), "Target 2" represents the genomic DNA of Neisseria gonorrhoeae (NG), "Target 3" represents the genomic DNA of Ureaplasma parvum (UP), "NTC" represents no template control, and "RFU" represents relative fluorescence units. [Figure 17b] 17a and 17b show the results of real-time PCR for Combination 5 in this example. In each figure, "Target 1" represents the genomic DNA of Chlamydia trachomatis (CT), "Target 2" represents the genomic DNA of Neisseria gonorrhoeae (NG), "Target 3" represents the genomic DNA of Ureaplasma parvum (UP), "NTC" represents no template control, and "RFU" represents relative fluorescence units. DETAILED DESCRIPTION OF THE INVENTION

[0058]

[84] The present inventors have found that various signal generation mechanisms for detecting target nucleic acids known in the art include temperature ranges in which the signal changes depending on the presence of the target nucleic acid (i.e., signal change temperature ranges) and temperature ranges in which there is no signal change even in the presence of the target nucleic acid (i.e., signal constant temperature ranges), and can be categorized into three types according to the number and / or order of these signal change temperature ranges and signal constant temperature ranges. By applying various combinations of these three types of signal generation mechanisms to the detection of target nucleic acids, the present inventors have developed a novel method for detecting multiple target nucleic acids by using a single type of label and a single type of detector in a single reaction vessel.

[85]

[0059]

[86] I. Process for detecting target nucleic acid

[87]

[0060]

[88] According to one aspect of the present disclosure, there is provided a method for detecting n target nucleic acids in a sample, comprising: (a) detecting signals at n detection temperatures while incubating a sample suspected of containing at least one of the n target nucleic acids in a reaction vessel with n compositions for detecting the target nucleic acid, wherein n is an integer of 2 or greater; the incubation includes a plurality of cycles; and the detection of the signal is performed in at least one of the plurality of cycles; each of the n compositions for detecting the target nucleic acid provides a signal change at a corresponding detection temperature among the n detection temperatures in the presence of a corresponding target nucleic acid; wherein the signal change indicates the presence of the corresponding target nucleic acid, and a composition for detecting an ith target nucleic acid among the n compositions for detecting target nucleic acids provides a signal change at the ith detection temperature among the n detection temperatures, and provides a constant signal at other detection temperatures in the presence of the ith target nucleic acid, the signal change indicating the presence of the ith target nucleic acid, i representing an integer from 1 to n, and the ith detection temperature being lower than the (i+1)th detection temperature; and (b) determining the presence of the n target nucleic acids from the signals detected in step (a), wherein the presence of the ith target nucleic acid is determined by the signal change detected at the ith detection temperature.

[0061]

[89] The present invention may be described in terms of each step as follows:

[90]

[91]

[0062]

[92] Step (a): Incubation and signal detection

[93] First, in a single reaction vessel, a sample suspected of containing at least one of n target nucleic acids is mixed and incubated with n compositions for detecting the target nucleic acid.

[0063]

[94] In one embodiment, n is an integer greater than or equal to 2. For example, n can be, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50.

[0064]

[95] The terms "target nucleic acid," "target nucleic acid sequence," or "target sequence," as used herein, refer to a nucleic acid sequence to be detected or quantified. Target nucleic acid sequences include double-stranded as well as single-stranded nucleic acid sequences. Target nucleic acid sequences include sequences originally present in a nucleic acid sample as well as sequences newly generated in a reaction.

[0065]

[96] Target nucleic acids include all DNA (gDNA and cDNA) and RNA molecules, as well as hybrids thereof (chimeric nucleic acids). The sequences may be in double-stranded or single-stranded form.

[0066]

[97] Target nucleic acids include any naturally occurring prokaryotic, eukaryotic (e.g., protozoa and parasites, fungi, yeast, higher plants, lower animals, and higher animals such as mammals and humans) or viral (e.g., herpes virus, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, polio virus, etc.) or viroid nucleic acid. In addition, the nucleic acid molecule may be any nucleic acid molecule that is or can be produced recombinantly, or any nucleic acid molecule that is or can be chemically synthesized. Thus, the nucleic acid sequence may be one found in nature or one that is not found in nature. The target nucleic acid sequence may be a known sequence or an unknown sequence.

[0067]

[98] In one embodiment, the n target nucleic acids may contain nucleotide variations. For example, one of the n target nucleic acids may contain one type of nucleotide variation, and another of the n target nucleic acids may contain a different type of nucleotide variation.

[0068]

[99] The term "nucleotide variation," as used herein, refers to any single or multiple nucleotide substitutions, deletions, or insertions in a DNA sequence at specific positions between contiguous DNA segments. Such contiguous DNA segments include genes or any other portions of chromosomes. These nucleotide variations may be variants or polymorphic allelic variations. For example, nucleotide variations detected in the present disclosure include single nucleotide polymorphisms (SNPs), mutations, deletions, insertions, substitutions, and translocations. Examples of nucleotide variations include numerous variations in the human genome (e.g., variations in the MTHFR (methylenetetrahydrofolate reductase) gene), variations involved in drug resistance in pathogens, and variations that cause tumorigenesis. The term "nucleotide variation," as used herein, includes any variation at a specific position in a nucleic acid sequence. That is, the term "nucleotide variation" includes both the wild type and any mutation at a specific position in the nucleic acid sequence.

[0069]

[0100] The term "sample," as used herein, refers to cells, tissues, or fluids from a biological source, or any other medium, that may prove useful in the present invention, including, for example, viruses, bacteria, tissues, cells, blood, serum, plasma, lymph, milk, urine, feces, intraocular fluid, saliva, semen, brain extracts, spinal fluid, appendix, spleen and tonsil tissue extracts, amniotic fluid, ascites, and non-biological samples (e.g., food and water). In addition, samples include naturally occurring nucleic acid molecules isolated from biological sources and synthetic nucleic acid molecules.

[0070]

[0101] The present invention can be used to determine whether at least one of n target nucleic acids is present in a sample. For example, when n is 2, the present invention can be used to determine whether at least one of a first target nucleic acid and a second target nucleic acid is present in a sample. As another example, when n is 3, the present invention can be used to determine whether at least one of a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid is present in a sample.

[0071]

[0102] In one embodiment, an incubation reaction refers to any reaction that induces a signal change in response to the presence of a corresponding target nucleic acid at a corresponding detection temperature when each of the target nucleic acids reacts with a corresponding composition for detecting the target nucleic acid.

[0072]

[0103] In one embodiment, the incubation comprises multiple cycles.

[0073]

[0104] In one embodiment, the incubation in step (a) may include an amplification reaction, and examples of the amplification reaction may include a signal amplification reaction and / or a nucleic acid amplification reaction.

[0074]

[0105] In one embodiment, the amplification reaction comprises multiple cycles.

[0075]

[0106] In one embodiment, the incubation in step (a) is carried out under conditions that allow target amplification and signal change by the composition for detecting the target nucleic acid, including the temperature, salt concentration, and pH of the solution.

[0076]

[0107] In one embodiment, the incubation in step (a) is carried out in a signal amplification process in the absence of nucleic acid amplification.

[0077]

[0108] In one embodiment, the signal may be amplified simultaneously with amplification of the target. Alternatively, the signal may be amplified without amplification of the target.

[0078]

[0109] In one embodiment, the signal change occurs during a process that includes signal amplification and target amplification.

[0079]

[0110] In one embodiment, amplification of the target nucleic acid may be performed by polymerase chain reaction (PCR). PCR is widely used in the art to amplify target nucleic acids and involves cycles of denaturing the target nucleic acid, annealing (hybridizing) the target nucleic acid with primers, and extending the primers (Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354).

[0080]

[0111] In one embodiment, amplification of the target nucleic acid is performed using techniques such as ligase chain reaction (LCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990)), strand displacement amplification (SDA) (Walker et al., Nucleic Acids Res. 20(7):1691-6 (1992); Walker PCR Methods Appl 3(1):1-6 (1993)), transcription-mediated amplification (Phyffer et al., J. Clin. Microbiol. 34:834-841 (1996); Vuorinen et al., J. Clin. Microbiol. 33:1856-1859 (1995)), helicase-dependent amplification (HAD) (M. Vincent, Y. Xu and H. Kong, EMBO Rep., 2004, 5, 795-800), nucleic acid sequence-based amplification (NASBA) (Compton, Nature 350(6313):91-2(1991)), rolling circle amplification (RCA) (Lisby, Mol. Biotechnol. 12(1):75-99(1999); Hatch et al., Genet. Anal. 15(2):35-40(1999)), Q-beta replicase (Lizardi et al., BiolTechnology 6:1197(1988)), loop-mediated isothermal amplification (LAMP) (Y. Mori, H. Kanda and T. Notomi, J. Infect. Chemother., 2013, 19, 404-411), or recombinase polymerase amplification (RPA) (J. Li, J. Macdonald and F. von Stetten, Analyst, 2018, 144, 31-67).

[0081]

[0112] A variety of DNA polymerases can be used in amplification reactions, including the "Klenow" fragment of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. In particular, the polymerases are thermostable DNA polymerases available from various bacteria, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Most of these polymerases can be isolated directly from bacteria or purchased commercially.

[0082]

[0113] The above-mentioned amplification methods can amplify target nucleic acids and / or signals by repeating a series of reactions with or without changing the temperature. A unit of amplification that includes repeating such a series of reactions is called a "cycle." A cycle can be expressed as the number of repetitions or the duration, depending on the amplification method used.

[0083]

[0114] In one embodiment, a series of reactions may be performed sequentially. For example, in the case of PCR, the target nucleic acid (i.e., template) may be denatured, followed by primer annealing and subsequent primer extension. In this case, the cycle may be expressed as the number of repetitions.

[0084]

[0115] In one embodiment, a series of reactions can be carried out simultaneously.For example, in isothermal amplification assay LAMP, primer annealing can occur for some of the templates, while primers can already be annealed and elongated for some other templates.In this case, cycles can be expressed as durations.In particular, one cycle can be 5 seconds, 10 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, or 2 hours.

[0085]

[0116] In one embodiment, incubation may be performed for multiple cycles that allow measurement of a signal change that depends on the presence of the target nucleic acid. For example, the multiple cycles may include 2 to 100 cycles, 2 to 90 cycles, 2 to 80 cycles, 2 to 70 cycles, 2 to 60 cycles, 2 to 50 cycles, 2 to 40 cycles, 2 to 30 cycles, 2 to 20 cycles, 2 to 10 cycles, 5 to 100 cycles, 5 to 90 cycles, 5 to 80 cycles, 5 to 70 cycles, 5 to 60 cycles, 5 to 50 cycles, 5 to 40 cycles, 5 to 30 cycles, 5 to 20 cycles, 5 to 10 cycles, 10 to 100 cycles, 10 to 90 cycles, 10 to 80 cycles, Examples of the number of cycles include 10 to 70 cycles, 10 to 60 cycles, 10 to 50 cycles, 10 to 40 cycles, 10 to 30 cycles, 10 to 20 cycles, 20 to 100 cycles, 20 to 90 cycles, 20 to 80 cycles, 20 to 70 cycles, 20 to 60 cycles, 20 to 50 cycles, 20 to 40 cycles, and 20 to 30 cycles, and particularly, 10 cycles, 15 cycles, 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles, and 50 cycles.

[0086]

[0117] In one embodiment, detection of a signal may occur at each cycle, at selected cycles, or at the endpoint of an incubation reaction comprising multiple cycles.

[0087]

[0118] In one embodiment, the target nucleic acid amplification reaction can be a multiple target nucleic acid amplification reaction.

[0088]

[0119] As used herein, the term "multiple target nucleic acid amplification reaction" refers to a reaction in which two or more nucleic acids are amplified as targets in a single reaction vessel. A multiple target nucleic acid amplification reaction refers to a reaction in which two or more nucleic acids are amplified together. For example, a multiple target nucleic acid amplification reaction can amplify two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, thirty or more, forty or more, or fifty or more target nucleic acids together in a single reaction.

[0089]

[0120] In one embodiment, the method of the present invention can detect 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 12, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 15, 3 to 12, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 12, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 types of target nucleic acids in a single reaction vessel using a single type of label.

[0090]

[0121] In one embodiment, the methods of the invention, when using multiple types of labels, can detect a greater number of target nucleic acids than can be detected using a single type of label according to the methods of the invention; for example, doubling the number of label types used can enable detection of more target nucleic acids.

[0091]

[0122] In accordance with the present invention, the method according to the present disclosure utilizes n different compositions corresponding to each of the n target nucleic acids to detect the n target nucleic acids.

[0092]

[0123] For example, when n is 2, a composition for detecting a first target nucleic acid and a composition for detecting a second target nucleic acid are used; when n is 3, a composition for detecting a first target nucleic acid, a composition for detecting a second target nucleic acid, and a composition for detecting a third target nucleic acid are used; when n is 4, a composition for detecting a first target nucleic acid to a fourth target nucleic acid are used.

[0093]

[0124] In one embodiment, each of the n compositions for detecting a target nucleic acid provides a signal change at a corresponding detection temperature among the n detection temperatures, wherein the signal change indicates the presence of the corresponding target nucleic acid.

[0094]

[0125] For example, a composition for detecting an ith target nucleic acid among n target nucleic acids provides a signal change at an ith detection temperature among n detection temperatures and provides a constant signal at other detection temperatures in the presence of the ith target nucleic acid.

[0095]

[0126] In one embodiment, a composition for detecting an i-th target nucleic acid provides a signal change when the target nucleic acid is amplified at an i-th detection temperature in the presence of the i-th target nucleic acid (i.e., an i-th signal change), but does not provide a signal change at other detection temperatures even if the target nucleic acid is amplified (i.e., the signal is constant).

[0096]

[0127] The term "ith signal," as used herein, refers to the signal provided by a composition for detecting the ith target nucleic acid at the ith detection temperature, and is used synonymously with "signal at the ith detection temperature."

[0097]

[0128] In one embodiment, the composition for detecting the i-th target nucleic acid provides no signal change, i.e., a constant signal, at the i-th detection temperature during an incubation reaction (e.g., a target nucleic acid amplification reaction) in the absence of the i-th target nucleic acid. In one embodiment, when detecting n target nucleic acids, the i-th signal may refer to the signal provided by the n compositions for detecting target nucleic acids, including the composition for detecting the i-th target nucleic acid, at the i-th detection temperature.

[0098]

[0129] In one embodiment, i represents an integer from 1 to n, and the i-th detected temperature is lower than the (i+1)-th detected temperature. In one embodiment, when i is n, the i+1-th detected temperature (i.e., the n+1-th detected temperature) does not exist. For example, when n is 3, i represents an integer from 1 to 3, and there are a first detected temperature, a second detected temperature, and a third detected temperature, the first detected temperature is lower than the second detected temperature, and the second detected temperature is lower than the third detected temperature.

[0099]

[0130] In certain embodiments, when n is 2, the composition for detecting a first target nucleic acid provides a signal change at a first detection temperature in the presence of the first target nucleic acid and a constant signal at another detection temperature, i.e., the second detection temperature; and the composition for detecting a second target nucleic acid provides a signal change at a second detection temperature in the presence of the second target nucleic acid and a constant signal at another detection temperature, i.e., the first detection temperature.

[0100]

[0131] In another specific embodiment, when n is 3, the composition for detecting a first target nucleic acid provides a signal change at a first detection temperature and, in the presence of the first target nucleic acid, provides a constant signal at other detection temperatures, i.e., the second detection temperature and the third detection temperature; the composition for detecting a second target nucleic acid provides a signal change at the second detection temperature in the presence of the second target nucleic acid and provides a constant signal at other detection temperatures, i.e., the first detection temperature and the third detection temperature; and the composition for detecting a third target nucleic acid provides a signal change at a third detection temperature in the presence of a third target nucleic acid and provides a constant signal at other detection temperatures, i.e., the first detection temperature and the second detection temperature.

[0101]

[0132] In one embodiment, a signal change includes "generation or disappearance of a signal" and "increase or decrease of a signal" from a label.

[0102]

[0133] In the present disclosure, a signal change refers to a significant "signal change," i.e., a significant change in signal, where the signal change indicates the presence of a target nucleic acid. For example, a significant signal change, i.e., a signal change indicating the presence of a target nucleic acid, may refer to the generation or disappearance of a signal having a distinct intensity compared to the background signal intensity or the intensity of the signal in the absence of the target nucleic acid, or may refer to a substantial increase or decrease in the intensity of the signal indicating the presence of the target nucleic acid when the target nucleic acid and / or signal is amplified during the incubation reaction in step (a).

[0103]

[0134] In one embodiment, the signal change may be generated in response to the presence of the target nucleic acid, for example, the signal change may be generated when a signal indicative of the presence of the target nucleic acid is generated or disappeared.

[0104]

[0135] In one embodiment, the signal change may occur upon amplification of the target nucleic acid. That is, the signal change may occur upon an increase in the amount of the target nucleic acid upon amplification of the target nucleic acid. For example, upon amplification of the target nucleic acid, the signal indicating the presence of the target nucleic acid may increase or decrease, thereby inducing a signal change.

[0105]

[0136] In one embodiment, the signal change may occur when a signal dependent on the target nucleic acid is amplified. That is, when a signal dependent on the target nucleic acid is amplified, the intensity of the signal changes, thereby changing the signal. For example, when a signal dependent on the presence of the target nucleic acid is amplified, the signal indicating the presence of the target nucleic acid may increase or decrease, thereby inducing a signal change.

[0106]

[0137] The term "constant signal" as used herein refers to the absence of substantial signal change during an incubation reaction (e.g., during a target nucleic acid amplification reaction). That is, the term "constant signal" refers to all or any signal pattern other than significant signal change caused by amplification of the target nucleic acid present. Specifically, a constant signal means that there is no signal change. For example, if the signal during the amplification reaction does not exceed the background signal intensity or the signal intensity in the absence of target nucleic acid, this can be expressed as "the signal is constant." In the present disclosure, a constant signal may be used synonymously with a signal that does not change or a signal that does not show change.

[0107]

[0138] In the present disclosure, the meaning of "signal change" and / or "constant signal" is based on a signal detected at the same temperature during a nucleic acid amplification reaction using the same composition for detecting a target nucleic acid. For example, the term "signal change" and / or "constant signal" is used based on the difference between signal values ​​detected at the same temperature using n compositions for detecting a target nucleic acid. More specifically, the term "signal change" and / or "constant signal" is based on (i) the difference between signal values ​​detected at the same temperature in multiple cycles, or (ii) the difference between the "reference signal value" described below and the signal value detected at the same temperature as the temperature at which the reference signal value is set. In other words, the term "signal change" and / or "constant signal" is not based on the difference between signal values ​​detected at different temperatures.

[0139]

[0108]

[0140] In the present disclosure, a composition for detecting target nucleic acids is used to provide a signal change for the target nucleic acids, each of which is detected by a corresponding composition for detecting target nucleic acids.

[0109]

[0141] In one embodiment, the composition for detecting the ith target nucleic acid comprises a label that provides a signal in response to the presence of the ith target nucleic acid.

[0110]

[0142] In one embodiment, the label is linked to the oligonucleotide or incorporated into the oligonucleotide during incubation (e.g., during a nucleic acid amplification reaction), i.e., the composition for detecting the target nucleic acid may initially include a label-linked oligonucleotide, or may provide a label-linked oligonucleotide when the label is incorporated into newly generated oligonucleotides (e.g., extended strands) during the incubation reaction.

[0111]

[0143] In one embodiment, the composition for detecting the ith target nucleic acid comprises an incorporation label that is incorporated into the oligonucleotide during incubation to provide a signal in response to the presence of the ith target nucleic acid.

[0112]

[0144] In one embodiment, a composition for detecting an i-th target nucleic acid provides an oligonucleotide linked to a label that serves to provide a signal in response to the presence of the i-th target nucleic acid.

[0113]

[0145] In one embodiment, a composition for detecting the ith target nucleic acid initially comprises an oligonucleotide linked to a label that serves to provide a signal in response to the presence of the ith target nucleic acid. Alternatively, a composition for detecting the ith target nucleic acid may comprise an oligonucleotide and a label that provides a signal in response to the presence of the target nucleic acid, or an oligonucleotide and a label that, when incorporated into the oligonucleotide during an incubation reaction (e.g., during a nucleic acid amplification reaction), provides an oligonucleotide linked to a label that serves to provide a signal in response to the presence of the target nucleic acid.

[0114]

[0146] The term "label-linked oligonucleotide," as used herein, refers to an oligonucleotide that participates in generating a signal that is detected.

[0115]

[0147] In one embodiment, the label-linked oligonucleotide may comprise an oligonucleotide that specifically hybridizes to a target nucleic acid (e.g., a probe or primer); if the probe or primer hybridized to the target nucleic acid is cleaved to release a fragment, the label-linked oligonucleotide may comprise a capture oligonucleotide that specifically hybridizes to the fragment; if the fragment hybridized to the capture oligonucleotide is extended to form an extended strand, the label-linked oligonucleotide may comprise an oligonucleotide that specifically hybridizes to the extended strand, an oligonucleotide produced by incorporating a label during fragment extension, an oligonucleotide that specifically hybridizes to a capture oligonucleotide, and combinations thereof.

[0116]

[0148] In one embodiment, the label-linked oligonucleotide includes an oligonucleotide that is involved in the actual signal generation, e.g., hybridization or non-hybridization of the label-linked oligonucleotide with another oligonucleotide (e.g., the label-linked oligonucleotide or an oligonucleotide comprising a nucleotide sequence complementary to the target nucleic acid) determines signal generation.

[0117]

[0149] In one embodiment, the label-linked oligonucleotide may be a "probe," as known in the art. The term "probe," as used herein, refers to a single-stranded nucleic acid molecule containing one or more portions substantially complementary to a target nucleic acid sequence. According to an embodiment of the present invention, the 3' end of the probe is "blocked" to prevent its extension. Blocking can be achieved according to conventional methods. For example, blocking may be performed by adding a chemical moiety, such as biotin, a label, a phosphate group, an alkyl group, a non-nucleotide linker, a phosphorothioate, or an alkane-diol residue, to the 3'-hydroxyl group of the last nucleotide. Alternatively, blocking may be performed by removing the 3'-hydroxyl group of the last nucleotide or by using a nucleotide lacking a 3'-hydroxyl group, such as a dideoxynucleotide.

[0118]

[0150] According to one embodiment, the label-linked oligonucleotide may be composed of at least one oligonucleotide. According to an embodiment of the present invention, when the label-linked oligonucleotide is composed of multiple oligonucleotides, the label-linked oligonucleotide can be labeled in various ways. For example, all or part of the multiple oligonucleotides may have at least one label.

[0119]

[0151] In one embodiment, the interactive label may be an interactive dual label comprising one reporter molecule and one quencher molecule.

[0120]

[0152] In one embodiment, the interactive label may be an interactive label comprising at least one reporter molecule and at least one quencher molecule. In particular, the interactive label may be an interactive dual label comprising one reporter molecule and one quencher molecule. Or, the interactive label may be an interactive label comprising one reporter molecule and two quencher molecules.

[0121]

[0153] In one embodiment, when the label is a single label, the single label may be linked to one oligonucleotide.

[0122]

[0154] In one embodiment, when the label is an interactive label, the interactive label may be an interactive label comprising at least one reporter molecule and at least one quencher molecule, and the interactive labels may all be linked to one oligonucleotide or may be linked to each of multiple oligonucleotides.

[0123]

[0155] For example, single labels include fluorescent labels, luminescent labels, chemiluminescent labels, electrochemical labels, and metal labels. In one embodiment, a single label provides a different signal (e.g., different signal intensity) depending on its presence in a double-stranded or single-stranded structure. In one embodiment, the single label is a fluorescent label. Preferred types and binding sites of single fluorescent labels used in the present disclosure are disclosed in U.S. Patent Nos. 7,537,886 and 7,348,141, the teachings of which are incorporated herein by reference in their entireties. For example, single fluorescent labels include JOE, FAM, TAMRA, ROX, and fluorescein-based labels. A single label may be linked to an oligonucleotide by various methods. For example, a label may be linked to a probe via a carbon atom-containing spacer (e.g., a 3-carbon spacer, a 6-carbon spacer, or a 12-carbon spacer).

[0124]

[0156] A typical example of an interactive labeling system is a FRET (fluorescence resonance energy transfer) labeling system, which comprises a fluorescent reporter molecule (donor molecule) and a quencher molecule (acceptor molecule). In FRET, the energy donor is fluorescent, while the energy acceptor may be fluorescent or non-fluorescent. In another form of interactive labeling system, the energy donor is non-fluorescent, e.g., a chromophore, and the energy acceptor is fluorescent. In yet another form of interactive labeling system, the energy donor is luminescent, e.g., bioluminescent, chemiluminescent, or electrochemiluminescent, and the acceptor is fluorescent. Interactive labeling systems include label pairs based on "contact-mediated quenching" (Salvatore et al., Nucleic Acids Research, 2002 (30) Vol. 21 e122 and Johansson et al., J. AM. CHEM. SOC 2002 (124) pp. 6950-6956). Interactive labeling systems include any and all labeling systems that induce a signal change through the interaction between at least two molecules (eg, dyes).

[0125]

[0157] Reporter and quencher molecules useful in the present invention can include any molecule known in the art, such as Cy2™ (506), YO-PRO™-1 (509), YOYO™-1 (509), calcein (517), FITC (518), FluorX™ (519), Alexa™ (520), rhodamine 110 (520), Oregon Green™ 500 (522), Oregon Green™ 488 (524), RiboGreen™ (525), Rhodamine Green™ (527), rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™ (533), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY530 / 550 (550), Dil (565), BODIPYTMR (568), BODIPY558 / 568 (568), BODIPY564 / 570 (570), Cy3™ (570), Alexa™ 546 (570), TRITC (572), Magnesium Orange™ (575), Phycoerythrin R&B (575), Rhodamine phalloidin (575), Calcium Orange™ (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red™ (590), Cy3.5™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594 (615), Texas Red (615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631), R-phycocyanin (642), C-phycocyanin (648), TO-PRO™-3 (660), TOTO3 (660), DiD DilC(5) (665), Cy5™ (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluoro Orange 560 (559), CAL Fluoro Red 590 (591), CAL Fluoro Red 610 (610), CAL Fluoro Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705), and Quasar 705 (610). The numbers in parentheses are the maximum emission wavelengths in nanometers. Preferred reporter and quencher molecules include JOE, FAM, TAMRA, ROX, and fluorescein-based labels.

[0126]

[0158] Suitable reporter-quencher pairs are disclosed in various publications, such as: Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color and Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, editors, Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); Haugland, R.P., Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996); U.S. Patent Nos. 3,996,345 and 4,351,760.

[0127]

[0159] In one embodiment, the incorporated label can be used in the process of incorporating a label during primer extension to generate a signal (e.g., Plexor technology, Sherrill CB et al., Journal of the American Chemical Society, 126:4550-45569 (2004)). In addition, the incorporated label can be used in signal generation by duplexes formed in a manner that depends on cleavage of an intermediary oligonucleotide hybridized to a target nucleic acid sequence.

[0128]

[0160] In one embodiment, the incorporated label may be generally linked to the nucleotide. Additionally, nucleotides with unnatural bases may be used.

[0129]

[0161] The term "unnatural base," as used herein, refers to a derivative of a natural base, such as adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U), that can form hydrogen-bonded base pairs. The term "unnatural base," as used herein, includes bases that have a different base-pairing pattern from the parent natural base, as described, for example, in U.S. Patent Nos. 5,432,272, 5,965,364, 6,001,983, and 6,037,120. Base pairing between unnatural bases involves two or three hydrogen bonds, as with natural bases. Base pairing between unnatural bases also occurs in a specific manner. Specific examples of unnatural bases include the following bases in the form of base pair combinations: isoC / isoG, isodC / isodG, Z / P, V / J, K / X, H / J, Pa / Ds, Pa / Q, Pn / Ds, Pn / Dss, Px / Ds, NaM / 5SICS, 5FM / 5SICS, and M / N (see U.S. Pat. Nos. 5,432,272; 5,965,364; 6,001,983; 6,037,120; 6,140,496; 6,627,456; 6,617,106; and 7,422,850; and Filip Wojciechowski et al., Chem. Soc. Rev., 2011, 40, 5669-5679).

[0130]

[0162] Conventional methods for detecting multiple target nucleic acids require the use of different types of fluorescent labels for different target nucleic acids, or even if a single type of fluorescent label is used, these methods have the disadvantage that they require additional analysis such as melting curve analysis. Unlike such methods, by using a composition for detecting target nucleic acids that provides a duplex, the method disclosed herein can detect multiple target nucleic acids in a real-time manner using a single type of label (e.g., a single fluorescent label) without additional analysis such as melting analysis.

[0131]

[0163] In one embodiment, each of the n compositions for detecting a target nucleic acid provides one or more duplexes.

[0132]

[0164] The term "duplex" is used herein to encompass both associated and dissociated duplexes. That is, the term may refer to two single-stranded nucleic acid molecules that have partially or completely complementary sequences such that they can hybridize to each other under hybridization conditions to form a duplex structure. For example, depending on the detection temperature, all or part of the duplex may be in associated or dissociated form.

[0133]

[0165] The term "association or dissociation" has the same meaning as the term "hybridization or denaturation."

[0134]

[0166] The phrase "the composition for detecting a target nucleic acid provides a duplex" as used herein can mean providing a duplex in associated form and / or a dissociated form. Similarly, the phrase "the composition for detecting a target nucleic acid generates a duplex during incubation" as used herein can mean generating a duplex in associated form and / or a dissociated form during the incubation reaction.

[0135]

[0167] In one embodiment, at least one of the duplexes provided by the composition for detecting target nucleic acids is a duplex that provides a signal. In particular, the duplex is a duplex that provides a signal change. That is, the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal, and in particular, the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change in response to the presence of the i-th target nucleic acid.

[0136]

[0168] The term "signal-providing duplex," as used herein, refers to a duplex that is capable of providing a distinguishable signal depending on whether the duplex is in an associated or dissociated form. For example, this means that a duplex in its associated form generates (or quenches) a signal, and a duplex in its dissociated form quenches (or generates) a signal.

[0137]

[0169] In one embodiment, the signal-providing duplex may contain at least one label. In particular, the at least one label is linked to at least one of the two single strands constituting the duplex. For example, the signal-providing duplex may contain a single label, in which case the single label is linked to either one of the two single strands constituting the duplex. As another example, the signal-providing duplex may contain interactive labels, in which case all the interactive labels are linked to one of the two strands constituting the signal-providing duplex, or one of the interactive labels is linked to one of the two single strands and the other of the interactive labels is linked to the other of the two single strands.

[0138]

[0170] The term "duplex that provides a signal change," as used herein, refers to a duplex that provides a signal change indicative of the presence of a target nucleic acid when the amount of the duplex that provides a signal change changes in response to the presence of the target nucleic acid. In particular, in the PTOCE-based method described below, an extended duplex containing a label generated in response to the presence of a target nucleic acid is an example of a duplex that provides a signal change as described in this application.

[0139]

[0171] In one embodiment, the duplex that provides the signal change includes a label. In particular, at least one label is linked to at least one of the two single strands that make up the duplex. For example, the duplex that provides the signal change includes a single label, and in this case, the single label is linked to either one of the two single strands that make up the duplex. In another example, the duplex that provides the signal change includes interacting labels, and in this case, all of the interacting labels are linked to one of the two strands that make up the duplex that provides the signal change, or one of the interacting labels is linked to one of the two single strands and the other of the interacting labels is linked to the other of the two single strands.

[0140]

[0172] In one embodiment, the composition for detecting the ith target nucleic acid provides a duplex that provides a signal change.

[0141]

[0173] In one embodiment, the composition for detecting the ith target nucleic acid provides a signal from the label when the duplex that provides the signal change is present in an associated form, i.e., the composition for detecting the ith target nucleic acid provides a signal in response to the association of two single-stranded nucleic acid molecules that make up the duplex.

[0142]

[0174] In an alternative embodiment, the composition for detecting the ith target nucleic acid provides a signal from the label when the duplex that provides the signal change is present in a dissociated form, i.e., the composition for detecting the ith target nucleic acid provides a signal in response to dissociation of the two single-stranded nucleic acid molecules that make up the duplex.

[0143]

[0175] In one embodiment, the association or dissociation of the duplex may be triggered by temperature.

[0144]

[0176] In one embodiment, the duplex that provides the signal change may be the duplex that was originally included in the composition for detecting the target nucleic acid.

[0145]

[0177] In one embodiment, when a duplex that provides a signal change is included in a composition for detecting a target nucleic acid, the duplex may be generated by hybridization between a label-linked oligonucleotide and an oligonucleotide that can hybridize to the label-linked oligonucleotide. For example, the Yin-Yang probe described below is an example of a duplex that provides a signal change in response to the presence of a target nucleic acid, and is originally included in the composition for detecting a target nucleic acid.

[0146]

[0178] In one embodiment, when the duplex that provides a signal change is initially included in the composition for detecting a target nucleic acid, the amount of the duplex that provides a signal change varies, particularly decreases, depending on the presence of the target nucleic acid, thereby providing a signal change. For example, in the case of a Yin-Yang probe that is initially included in the composition for detecting a target nucleic acid, when the target nucleic acid is amplified, one of the two single strands that make up the Yin-Yang probe pairs with the amplified target nucleic acid to form a new duplex, and the amount of the Yin-Yang probe decreases, thereby providing a signal change depending on the presence of the target nucleic acid.

[0147]

[0179] In one embodiment, the duplex that provides the signal change may be a duplex newly provided by the composition for detecting the target nucleic acid during the incubation reaction.

[0148]

[0180] In one embodiment, the duplex that provides the signal change is one that is generated during the incubation reaction, which may be provided by hybridization of a label-linked oligonucleotide with a target nucleic acid.

[0149]

[0181] Signals generated by the formation of a duplex between a labeled oligonucleotide and a target nucleic acid can be detected by the Scorpion method (Whitcombe et al., Nature Biotechnology 17:804-807 (1999)), the Sunrise (or Amplifluor) method (Nazarenko et al., Nucleic Acids Research, 25(12):2516-2521 (1997) and U.S. Pat. No. 6,117,635), the LUX method (U.S. Pat. No. 7,537,886), the Plexor method (Sherrill CB et al., Journal of the American Chemical Society, 126:4550-4556 (2004)), the molecular beacon method (Tyagi et al., Nature Biotechnology v.14 March 1996), the Hybeacon method (French DJ et al., Mol. Cell. Probes, 15(6):363-374 (2001)), adjacent hybridization probe method (Bernard PS et al., Anal. Biochem., 273:221 (1999)), and LNA method (U.S. Pat. No. 6,977,295).

[0150]

[0182] In one embodiment, the duplex that provides the signal change is one that is generated during the incubation reaction, which may be a duplex generated by a cleavage reaction that is dependent on the presence of the target nucleic acid.

[0151]

[0183] In the above reaction, a 5'-nuclease and a 3'-nuclease, in particular a DNA polymerase having 5'-nuclease activity, a DNA polymerase having 3'-nuclease activity, or an FEN nuclease can be used.

[0152]

[0184] In one embodiment, the signal change is produced by a duplex created in a manner dependent on cleavage of an intermediary oligonucleotide specifically hybridized to the target nucleic acid.

[0153]

[0185] The term "mediator oligonucleotide," as used herein, refers to an oligonucleotide that mediates the formation of a duplex without involving a target nucleic acid.

[0154]

[0186] In one embodiment, cleavage of the intermediary oligonucleotide does not generate a signal by itself, but after hybridization and cleavage of the intermediary oligonucleotide, the fragments produced by cleavage (cleavage products) participate in a sequential reaction for signal generation.

[0155]

[0187] In one embodiment, hybridization or cleavage of the intermediary oligonucleotide does not alone generate a signal.

[0156]

[0188] In one embodiment, mediator oligonucleotides include oligonucleotides that hybridize to a target nucleic acid sequence and are cleaved to release fragments, thereby mediating the formation of a duplex.

[0157]

[0189] In one embodiment, the fragment mediates the generation of a duplex by extension of the fragment with a capture oligonucleotide.

[0158]

[0190] According to one embodiment, the intermediary oligonucleotide comprises (i) a targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence, and (ii) a tagging portion comprising a nucleotide sequence that is not complementary to the target nucleic acid sequence.

[0159]

[0191] In one embodiment, the composition for detecting a target nucleic acid may comprise a tagging oligonucleotide that hybridizes to the target nucleic acid, and the cleavage reaction dependent on the presence of the target nucleic acid may comprise cleavage of the tagging oligonucleotide, which corresponds to the example of the intermediary oligonucleotide described above.

[0160]

[0192] According to one embodiment, cleavage of the intermediary oligonucleotide releases fragments that are specifically hybridized to and extended with the capture oligonucleotide. If the capture oligonucleotide includes a label, the capture oligonucleotide corresponds to the examples of label-linked oligonucleotides described herein.

[0161]

[0193] According to one embodiment, the intermediary oligonucleotide hybridized to the target nucleic acid sequence is cleaved, releasing fragments, which are specifically hybridized to capture oligonucleotides, and the fragments are extended to form extended strands, which induce the formation of extended duplexes between the extended strands and the capture oligonucleotides, thereby providing a signal indicative of the presence of the target nucleic acid sequence.

[0162]

[0194] In one embodiment, a signal indicative of the presence of a target nucleic acid may be provided by (i) at least one label linked to a fragment and / or capture oligonucleotide, (ii) a label incorporated into the extended duplex during the extension reaction, and (iii) a label incorporated into the extended duplex during the extension reaction and a label linked to a fragment and / or capture oligonucleotide.

[0163]

[0195] According to one embodiment, when a third oligonucleotide containing a hybridizing nucleotide sequence complementary to the extended strand is used, hybridization of the extended strand with the third oligonucleotide forms another type of duplex, thereby providing a signal (e.g., PCE-SH) indicating the presence of the target nucleic acid. In this case, the another type of duplex is the duplex that provides the signal change.

[0164]

[0196] In one embodiment, the duplex formed by the cleavage reaction dependent on the presence of target nucleic acid is not the duplex that provides a signal change, but may be a duplex that changes the content of the duplex that provides a signal change. In this case, the duplex that provides a signal change was originally included in the composition for detecting the target nucleic acid. According to one embodiment, when an additional oligonucleotide containing a hybridizing nucleotide sequence complementary to the capture oligonucleotide is used, the duplex between the additional oligonucleotide and the capture oligonucleotide is the duplex that provides a signal change, and the amount of the duplex that provides a signal change is changed (e.g., decreased) by the generation of a duplex between the extended strand and the capture oligonucleotide, thereby providing a signal change indicating the presence of the target nucleic acid. For example, in the PCE-NH method, the duplex between the additional oligonucleotide and the capture oligonucleotide is the duplex that provides a signal change that was originally included in the composition for detecting the target nucleic acid, and the capture oligonucleotide that constitutes the duplex that provides a signal change is used to generate a new duplex (e.g., an extended duplex) by the cleavage reaction dependent on the presence of target nucleic acid, thereby decreasing the amount of the duplex that provides a signal change and providing a signal change.

[0165]

[0197] According to one embodiment, the fragment, the extended strand, the capture oligonucleotide, the additional oligonucleotide, or a combination thereof may act as the label-linked oligonucleotide.

[0166]

[0198] Signals from duplexes formed in a manner dependent on cleavage of an intermediary oligonucleotide can be generated by a variety of methods, including the PTOCE (PTO cleavage and extension) method (WO 2012 / 096523), the PCE-SH (PTO cleavage and extension dependent signaling oligonucleotide hybridization) method (WO 2013 / 115442), and the PCE-NH (PTO cleavage and extension dependent non-hybridization) method (WO 2014 / 104818).

[0167]

[0199] With respect to the terms disclosed in the above references, corresponding examples of oligonucleotides are as follows: the intermediary oligonucleotide corresponds to PTO (probing and tagging oligonucleotide), the capture oligonucleotide corresponds to CTO (capture and templating oligonucleotide), and the additional oligonucleotide corresponds to SO (signaling oligonucleotide) or HO (hybridization oligonucleotide). SO, HO, CTO, the extended chain or a combination thereof can serve as the label-linked oligonucleotide.

[0168]

[0200] In one embodiment, the signal from the duplex formed in a manner dependent on cleavage of the intermediary oligonucleotide includes a signal provided when the amount of other duplexes is reduced by the duplex formed in a manner dependent on cleavage of the intermediary oligonucleotide (e.g., PCE-NH).

[0201]

[0169]

[0202] In one embodiment, when a signal from a duplex generated in a manner dependent on cleavage of an intermediary oligonucleotide is generated by the PTOCE method, an upstream oligonucleotide, a probing and tagging oligonucleotide (PTO) comprising a hybridizing nucleotide sequence complementary to the target nucleic acid, a capture and templating oligonucleotide (CTO), an appropriate label, and a template-dependent DNA polymerase having 5'-nuclease activity may be included in the reaction, and a composition for detecting a target nucleic acid may contain these components.

[0170]

[0203] A PTO comprises (i) a 3'-targeting portion comprising a hybridizing nucleotide sequence complementary to a target nucleic acid sequence, and (ii) a 5'-tagging portion comprising a nucleotide sequence that is not complementary to the target nucleic acid sequence. A CTO comprises, in the 3' to 5' direction, (i) a capture portion comprising a nucleotide sequence complementary to the 5'-tagging portion or a portion of the 5'-tagging portion of the PTO, and (ii) a templating portion comprising a nucleotide sequence that is not complementary to the 5'-tagging portion and 3'-targeting portion of the PTO.

[0171]

[0204] A specific example of signal generation by the PTOCE method includes the following steps:

[0172]

[0205] (a) hybridizing a target nucleic acid with an upstream oligonucleotide and a PTO; (b) contacting the product of step (a) with an enzyme having 5' nuclease activity under conditions for cleavage of the PTO, wherein the upstream oligonucleotide or an extended strand thereof induces cleavage of the PTO by the enzyme having 5' nuclease activity, such that cleavage releases a fragment comprising the 5'-tagging portion or a portion of the 5'-tagging portion of the PTO; (c) hybridizing the fragment released from the PTO with a CTO, wherein the fragment released from the PTO is hybridized to the capture portion of the CTO; (d) hybridizing the result of step (c) and a template-dependent nucleic acid polymerase. performing an extension reaction using a probe enzyme, wherein the fragments hybridized to the capture portion of the CTO are extended to form an extended duplex; the extended duplex has a Tm value that can be adjusted by (i) the sequence and / or length of the fragment, (ii) the sequence and / or length of the CTO, or (iii) the sequence and / or length of the fragment and the sequence and / or length of the CTO; the extended duplex provides a target signal due to at least one label linked to the fragment and / or the CTO; and (e) detecting the extended duplex by measuring the target signal at a predetermined temperature at which the extended duplex retains its double-stranded form, wherein the presence of the extended duplex indicates the presence of the target nucleic acid. In this case, the method further comprises repeating all or part of steps (a) to (e), including denaturation between the repeated cycles.

[0173]

[0206] In the phrase "denaturing during repeated cycles," the term "denaturing" refers to the separation of double-stranded nucleic acid molecules into single-stranded nucleic acid molecules.

[0174]

[0207] In step (a) of the PTOCE method, a primer set for amplifying the target nucleic acid can be used instead of the upstream oligonucleotide. In this case, the method further comprises repeating all or part of steps (a) to (e), including denaturation between the repeated cycles.

[0175]

[0208] The PTOCE method can be classified as a process in which a PTO fragment hybridized to a CTO is extended to form an extended strand, and the extended strand is detected. The PTOCE method is characterized in that the formation of the extended strand is detected using a duplex between the extended strand and the CTO.

[0176]

[0209] There are other approaches for detecting the formation of extended strands. For example, the formation of extended strands can be detected using oligonucleotides specifically hybridized to the extended strands (e.g., PCE-SH assay). In such methods, the signal can be provided by (i) a label linked to the oligonucleotide specifically hybridized to the extended strand, or (ii) a label linked to the oligonucleotide specifically hybridized to the extended strand and a label linked to the PTO fragment.

[0177]

[0210] Alternatively, the formation of an extended strand can be detected by other methods (e.g., PCE-NH assay) to detect a change in the amount of duplex between the CTO and an oligonucleotide specifically hybridizable to the CTO. Such a change is considered to indicate the presence of the target nucleic acid. The signal can also be provided by (i) a label linked to an oligonucleotide hybridizable to the CTO, (ii) a label linked to the CTO, or (iii) a label linked to an oligonucleotide hybridizable to the CTO and the label linked to the CTO.

[0178]

[0211] According to one embodiment, the oligonucleotide capable of specifically hybridizing to the CTO has a sequence that overlaps with the PTO fragment.

[0179]

[0212] In one embodiment, the label-linked oligonucleotide includes an oligonucleotide that can specifically hybridize to the extended strand (e.g., PCE-SH assay) and an oligonucleotide that can specifically hybridize to CTO (e.g., PCE-NH assay).

[0180]

[0213] PTOCE-based methods generally involve the formation of an extended strand that is dependent on the presence of a target nucleic acid. The term "PTOCE-based method" is used herein to encompass a variety of methods for providing a signal, including the formation of an extended strand via PTO cleavage and extension.

[0181]

[0214] An example of signal generation by a PTOCE-based method includes the following steps: (a) hybridizing a target nucleic acid with an upstream oligonucleotide and a PTO; (b) contacting the product of step (a) with an enzyme having 5' nuclease activity under conditions for cleavage of the PTO, wherein the upstream oligonucleotide or its extended strand induces cleavage of the PTO by the enzyme having 5' nuclease activity, and the cleavage releases fragments containing the 5'-tagging portion or a portion of the 5'-tagging portion of the PTO; (c) hybridizing the fragments released from the PTO with a CTO, wherein the fragments released from the PTO are hybridized to the capture portion of the CTO; (d) performing an extension reaction using the product of step (c) and a template-dependent nucleic acid polymerase, wherein the fragments hybridized to the capture portion of the CTO are extended to form an extended duplex; and (e) detecting the formation of the extended duplex by measuring a signal generated depending on the presence of the extended strand. In step (a), a primer set for amplification of the target nucleic acid can be used in place of the upstream oligonucleotide, in which case the method further comprises repeating all or part of steps (a) to (e), including denaturation between the repeated cycles.

[0182]

[0215] Another example of a signal generation mechanism that relies on the formation of a duplex by cleavage of an intermediate oligonucleotide is the C-tag technology (Korean Patent No. 1961642). This method utilizes a primer (hereinafter referred to as a C-tag primer) having a structure that consecutively includes a random nucleic acid sequence that is not complementary to the target nucleic acid, a restriction enzyme recognition sequence, and a nucleic acid sequence complementary to the target nucleic acid sequence as the intermediate oligonucleotide. The C-tag primer is hybridized to the target nucleic acid and extended to form an amplification product that includes the restriction enzyme recognition sequence and a sequence complementary to the random nucleic acid sequence. Furthermore, when the restriction enzyme cleaves the amplification product, thereby releasing a tag fragment complementary to the random nucleic acid sequence, the tag fragment specifically hybridizes to a capture oligonucleotide to form a duplex, thereby providing a signal indicating the presence of the target nucleic acid sequence.

[0183]

[0216] In one embodiment, the signal change may be generated by dissociation of the formed duplex following cleavage of a labeled oligonucleotide hybridized to the target nucleic acid, which occurs in a manner dependent on the presence of the target nucleic acid.

[0184]

[0217] Another example of a signal generation mechanism that relies on the formation of a duplex by cleavage of an mediated oligonucleotide is the dual quench assay combined with melting analysis (WO 2016 / 101959).

[0185]

[0218] With respect to the terms disclosed in the above publications, corresponding examples of oligonucleotides are as follows: intermediary oligonucleotides correspond to probing and tagging oligonucleotides (PTOs), and capture oligonucleotides correspond to capture and quenching oligonucleotides (CQOs).

[0186]

[0219] In one embodiment, a particular embodiment of signal generation by dual quench assay comprises the following steps:

[0187]

[0220] (a) hybridizing a target nucleic acid sequence with a PTO, the PTO comprising (i) a targeting moiety comprising a nucleotide sequence substantially complementary to the target nucleic acid sequence, (ii) a melting temperature determining region (MTDR) comprising a nucleotide sequence that is not complementary to the target nucleic acid sequence, and (iii) at least one set of interactive labels comprising at least one fluorophore and at least one quencher;

[0188]

[0221] (b) hybridizing the PTO with the CQO,

[0222] the CQO comprises (i) a capture moiety comprising a nucleotide sequence reverse-complementary to the MTDR of the PTO, and (ii) at least one quencher molecule, wherein the MTDR is configured to hybridize with the capture moiety of the CQO to form a tag duplex;

[0189]

[0223] (c) contacting the tag duplex with an enzyme having nuclease activity, wherein the enzyme having nuclease activity induces cleavage of the tag duplex upon hybridization of the tag duplex with a target nucleic acid sequence, thereby releasing an activated tag duplex fragment comprising the MTDR hybridized to the capture portion of the CQO and a PTO fragment comprising at least one fluorophore;

[0190]

[0224] (d) melting and / or hybridizing the activated tag duplex fragments to obtain a signal from at least one fluorophore; and

[0191]

[0225] (e) detecting the activated tag duplex fragments by measuring a signal from at least one fluorophore, where the signal indicates the presence of the target nucleic acid sequence. In one embodiment, the order of steps (a) to (c) can be changed. For example, the steps may be performed in the following order: hybridizing a PTO with a CQO (step (b)), hybridizing a target nucleic acid sequence with a tag duplex (step (a)), cleaving the tag duplex hybridized to the target nucleic acid sequence with an enzyme having nuclease activity (step (c)); or hybridizing a PTO with a target nucleic acid sequence (step (a)), releasing an activated PTO fragment comprising at least one fluorophore and an MTDR with an enzyme having nuclease activity (step (c)), and hybridizing the activated PTO with a CQO (step (b)).

[0192]

[0226] In one embodiment, the duplex that provides the signal change may be a single type of duplex or multiple types of duplexes. Specifically, when the duplex that provides the signal change is a single type of duplex, the number of duplexes may be 1. When the duplex that provides the signal change is multiple types of duplexes, the number of duplexes may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20, more specifically 2, 3, or 4, and even more specifically 2 or 3.

[0193]

[0227] In one embodiment, a single type of duplex or every duplex among multiple types of duplexes comprises a label.

[0194]

[0228] In one embodiment, a duplex may exist in which two different single strands hybridize to different sites of the same single strand. For example, as shown in Figure 1c, an adjacent hybridization probe method using two probes is a method that provides a signal depending on whether the two probes hybridize to different adjacent sites of a single strand (e.g., target nucleic acid). In this case, the method disclosed in the present invention provides the same signal when at least one of the two probes dissociates, so that the duplex that provides the signal is one, and therefore the method disclosed in the present invention can be classified as a method that provides a single type of duplex.

[0195]

[0229] In one embodiment, when the duplexes providing the signal change are a single type of duplex, the amount of the single type of duplex varies depending on the presence of the target nucleic acid, thereby changing the signal. For example, referring to the drawings, the molecular beacon method in Figure 1a, the LUX probe method in Figure 1b, the hybrid probe method in Figure 1c, and the Yin-Yang probe method in Figure 1d, the PTOCE-based methods in Figures 1e, 1f, 1g, 1h, 2a, and 2c, and the dual quenching method in Figure 2b are examples of providing a single type of duplex.

[0196]

[0230] In particular, in the cases of Figures 1a, 1b, 1c, 1f, 1g, 1h, 2a, 2b, and 2c, a single type of duplex is generated during incubation, and the amount of the single type of duplex increases during incubation (e.g., during an amplification reaction), thereby providing a signal change. In the cases of Figures 1d and 1e, a single type of duplex is initially included in the composition for detecting a target nucleic acid, and the amount of the single type of duplex decreases during the incubation reaction, thereby providing a signal change.

[0197]

[0231] In one embodiment, when the duplex is a mixture of multiple types of duplexes, the ratio of the amounts of the multiple types of duplexes changes depending on the presence of the target nucleic acid, thereby changing the signal. For example, referring to the drawings, the PTOCE-based method in Figures 3a and 3b is an example of providing multiple types of duplexes (particularly, two types of duplexes). In Figures 3a and 3b, one of the two types of duplexes is initially included in the composition for detecting the target nucleic acid, and the other is generated during the incubation reaction. During the incubation reaction (e.g., during the amplification reaction), the ratio of the amounts of the two types of duplexes changes, thereby providing a signal change. In particular, the amount of the duplex initially included in the composition for detecting the target nucleic acid decreases, while the amount of the duplex newly generated during the incubation reaction increases, resulting in a change in the ratio of the amounts of these duplexes.

[0198]

[0232] In one embodiment, at least two of the multiple types of duplexes contain the same single strand. For example, referring to Figures 3a and 3b, in two different types of duplexes, one of the two single strands constituting each duplex is the same strand. In particular, a CTO and a PTO (i.e., an uncleaved PTO) constitute a duplex that provides a first signal change, and are initially included in a composition for detecting a target nucleic acid, where the PTO (or CTO) has an interactive dual label linked thereto, or the PTO has one of the interactive dual labels and the CTO has the other of the interactive dual labels. During the incubation reaction, the PTO is cleaved in response to the presence of the target nucleic acid to release a fragment, which hybridizes to the CTO to generate an extended strand, and the newly generated extended strand and the CTO constitute a duplex that provides a second signal change. In this case, the first duplex and the second duplex contain the same single strand (i.e., a CTO).

[0199]

[0233] In one embodiment, the Tm values ​​of the multiple types of duplexes differ from each other, for example, the Tm values ​​of the duplexes differ from each other by at least 2°C, 3°C, 4°C, 5°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, or 20°C.

[0200]

[0234] In one embodiment, the amount of a duplex refers to the sum of the amount of the duplex in a state where the two nucleic acid strands constituting the duplex are dissociated (i.e., the dissociated form of the duplex) and the amount of the duplex in a state where the two nucleic acid strands are hybridized (i.e., the associated form of the duplex).

[0201]

[0235] In one embodiment, when multiple types of duplexes contain the same single strand, the same single strand may be contained in the first duplex originally contained in the composition for detecting a target nucleic acid, and a new second duplex containing the same single strand may be generated during the incubation reaction. In this case, the same single strand contained in the first duplex is consumed while generating the second duplex during the incubation reaction, and as a result, the amount of the first duplex containing the same single strand decreases, and the amount of the second duplex containing the same single strand increases.

[0202]

[0236] In one embodiment, any one of the n compositions for detecting a target nucleic acid is capable of amplifying the corresponding target nucleic acid.

[0203]

[0237] In one embodiment, any one of the n compositions for detecting a target nucleic acid may include an amplification oligonucleotide that serves to amplify the corresponding target nucleic acid. In one embodiment, the amplification oligonucleotide may be the same as the oligonucleotide to which the label is linked.

[0204]

[0238] The term "amplification oligonucleotide," as used herein, refers to any oligonucleotide that serves to amplify a target nucleic acid.

[0205]

[0239] In one embodiment, the amplification oligonucleotide may be a "primer," as known in the art. The term "primer," as used herein, refers to an oligonucleotide that can act as a point of initiation of synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a target nucleic acid strand (template), i.e., in the presence of nucleotides and a polymerization agent, such as a DNA polymerase, at a suitable temperature and pH. A primer must be long enough to initiate the synthesis of an extension product in the presence of a polymerization agent. The appropriate length of a primer is determined by several factors, including temperature, the field of application, and the source of primer.

[0206]

[0240] Primers may include forward primers (also referred to as upstream primers or upstream oligonucleotides), reverse primers (also referred to as downstream primers or downstream oligonucleotides), or both. Amplification oligonucleotides may be oligonucleotides having structures known in the art or may be synthesized by methods known in the art.

[0207]

[0241] The amplification oligonucleotide and the label-linked oligonucleotide being the same means that a single oligonucleotide simultaneously functions as both an amplification oligonucleotide that amplifies a target nucleic acid and a label-linked oligonucleotide that generates a signal in the presence of the target nucleic acid. For example, the label-linked oligonucleotide can generate a signal by hybridizing to the target nucleic acid and being extended.

[0208]

[0242] The present inventors have found that various signal generation mechanisms that use duplexes include a temperature range in which the signal changes in response to the presence of target nucleic acid (i.e., a signal-changing temperature range), and a temperature range in which the signal does not change even in the presence of target nucleic acid (i.e., a signal-constant temperature range). Furthermore, the present inventors have found that methods that do not provide a signal-providing duplex, such as TaqMan probe assays, do not have a signal-constant temperature range.

[0209]

[0243] Furthermore, the inventors have found that known signal generation mechanisms can be classified into three types depending on the number and / or order of signal change temperature ranges and signal constant temperature ranges, and have further developed a novel method that uses various combinations of these three types of signal generation mechanisms to detect multiple target nucleic acids in a real-time manner using a single type of label without performing melting analysis after amplification.

[0210]

[0244] In one embodiment, in a temperature range covering all n detection temperatures, a composition for detecting an i-th target nucleic acid has a "signal changing temperature range" in which the signal changes in response to the presence of the i-th target nucleic acid, and a "signal constant temperature range" in which the signal remains constant even in the presence of the i-th target nucleic acid.

[0211]

[0245] In one embodiment, among n compositions for detecting target nucleic acids, the composition for detecting the i-th target nucleic acid may have a "signal change temperature range" in which the signal changes when the target nucleic acid is amplified, and a "constant signal temperature range" in which the signal remains constant even when the target nucleic acid is amplified.

[0212]

[0246] In one embodiment, the signal change temperature range is the temperature range over which a difference in signal value (eg, signal intensity) occurs in response to the presence of the target nucleic acid.

[0213]

[0247] In one embodiment, the signal change temperature range is a temperature range in which the signal value changes depending on the amplification level of the target nucleic acid (eg, the amount of amplified target nucleic acid).

[0214]

[0248] In one embodiment, the signal-constant temperature range is a temperature range in which the signal value does not change regardless of the presence of the target nucleic acid, i.e., the signal-constant temperature range is a temperature range in which there is no difference between the signal value in the presence of the target nucleic acid and the signal value in the absence of the target nucleic acid.

[0215]

[0249] In one embodiment, the composition for detecting the i-th target nucleic acid may have a signal change temperature range and a signal constant temperature range in consecutive or non-consecutive order.

[0216]

[0250] In one embodiment, the composition for detecting the ith target nucleic acid may have one or two signal constant temperature ranges.

[0217]

[0251] In one embodiment, the i-th detection temperature can be selected within the signal change temperature range of the composition for detecting the i-th target nucleic acid. In this case, in the present application, the composition for detecting the i-th target nucleic acid is referred to as having the i-th detection temperature. In addition, the i-th target nucleic acid corresponding to the composition for detecting the i-th target nucleic acid can also be referred to as the target nucleic acid having the i-th detection temperature.

[0218]

[0252] According to one embodiment, one target nucleic acid is assigned one detection temperature determined by a composition for detecting the corresponding target nucleic acid.

[0219]

[0253] In one embodiment, the signal change temperature ranges of the n compositions for detecting the target nucleic acid do not overlap with each other, and in this case, any temperature may be selected as the detection temperature for each of the n detection temperatures as long as it is within the corresponding signal change temperature range.

[0220]

[0254] In one embodiment, the i-th detection temperature is selected within the signal change temperature range of the composition for detecting the i-th target nucleic acid, and the i-th detection temperature is not included in the signal change temperature range of the composition for detecting any other target nucleic acid.

[0221]

[0255] In one embodiment, the signal change temperature range of any one of the compositions for detecting a target nucleic acid may overlap with the signal change temperature range of a composition for detecting a target nucleic acid having an adjacent detection temperature, while not overlapping with the signal change temperature range of a composition for detecting a target nucleic acid having a non-adjacent detection temperature. In this case, the detection temperature of a composition for detecting a target nucleic acid having an overlapping signal change temperature range with a composition for detecting another target nucleic acid is selected within a temperature range of signal change temperature ranges that do not overlap with the signal change temperature range of a composition for detecting another target nucleic acid. Therefore, by selecting a detection temperature within a temperature range that does not overlap between two signal change temperature ranges, only a signal indicating the presence of a single specific target nucleic acid can be provided at a single detection temperature (see Figure 4).

[0222]

[0256] In one embodiment, the signal change temperature range of any one of the compositions for detecting a target nucleic acid may overlap with the signal change temperature range of a composition for detecting a target nucleic acid having an adjacent detection temperature, but either of the two signal change temperature ranges is not entirely contained within the signal change temperature range of the other.

[0223]

[0257] The term "adjacent detected temperatures" is used in this specification to refer to consecutive detected temperatures among n detected temperatures, for example, an adjacent detected temperature of the i-th detected temperature is the (i-1)-th detected temperature or the (i+1)-th detected temperature.

[0224]

[0258] In one embodiment, the signal change temperature range of a composition for detecting an i-th target nucleic acid may partially overlap with the signal change temperature range of a composition for detecting a target nucleic acid having an adjacent detection temperature, while not overlapping with the signal change temperature range of a composition for detecting a target nucleic acid having a non-adjacent detection temperature.

[0225]

[0259] In one embodiment, a composition for detecting an i-th target nucleic acid may have one signal change temperature range and one signal constant temperature range.

[0226]

[0260] In one embodiment, a composition for detecting an i-th target nucleic acid may have one signal change temperature range and two signal constant temperature ranges.

[0227]

[0261] In one embodiment, the composition for detecting the i-th target nucleic acid may be any one of the following: (i) an under-signal change (UnderSC) composition characterized in that the signal change temperature range is lower than the signal constant temperature range; (ii) an over-signal change (OverSC) composition characterized in that the signal change temperature range is higher than the signal constant temperature range; and (iii) an inter-signal change (InterSC) composition characterized in that the signal change temperature range is higher than one of two signal constant temperature ranges and lower than the other of the two signal constant temperature ranges.

[0228]

[0262] In one embodiment, the expression "one temperature range is lower than the other temperature range" used with respect to the signal change temperature range and the signal constant temperature range of a composition for detecting a target nucleic acid means that the highest temperature in one temperature range is lower than the lowest temperature in the other temperature range. Furthermore, the expression "one temperature range is higher than the other temperature range" means that the lowest temperature in one temperature range is higher than the highest temperature in the other temperature range. For example, when the signal constant temperature range is higher than the signal change temperature range, it means that the lowest temperature in the signal constant temperature range is higher than the highest temperature in the signal change temperature range.

[0229]

[0263] In one embodiment, the signal change temperature range of the composition for detecting the ith target nucleic acid can be determined depending on the length and / or sequence of the duplex that provides the signal change.

[0230]

[0264] In one embodiment, the composition for detecting the i-th target nucleic acid provides a single type of duplex, and the composition for detecting the target nucleic acid may have one signal change temperature range and one signal constant temperature range, which can be determined according to the length and / or sequence of the single type of duplex.

[0231]

[0265] In one embodiment, when the composition for detecting the i-th target nucleic acid provides multiple types of duplexes, particularly two types of duplexes, the composition for detecting the target nucleic acid may have one signal change temperature range and two signal constant temperature ranges, which can be determined according to the length and / or sequence of the two types of duplexes.

[0232]

[0266] Any one of the n compositions for detecting a target nucleic acid used herein may employ the various signal generation mechanisms described above.

[0233]

[0267] In one embodiment, the various signal generation mechanisms described above can be used in any one of the UnderSC, InterSC, and OverSC compositions.

[0234]

[0268] In one embodiment, compositions for detecting target nucleic acids that contain oligonucleotides with different sequences can be considered to be different from each other even if the signal generating mechanisms of the compositions for detecting target nucleic acids are the same. Different compositions for detecting nucleic acids have different detection temperatures.

[0235]

[0269] More specifically, with reference to the Figures, non-limiting examples of signal generation mechanisms that can be employed by the UnderSC, InterSC, and OverSC compositions are described.

[0236]

[0270] (i) Signal generation mechanisms that can be employed by the UnderSC composition to detect target nucleic acids

[0271] Figures 1a-h illustrate various signal generation mechanisms that can be employed by UnderSC compositions.

[0237]

[0272] The signal generation mechanism in Figures 1a and 1h has one signal change temperature range in which the signal changes when the target nucleic acid is amplified, and one signal constant temperature range in which the signal is constant even when the target nucleic acid is amplified, and the signal change temperature range is lower than the signal constant temperature range.

[0238]

[0273] In one embodiment, similar to the molecular beacon method in Figure 1a, the LUX method in Figure 1b, and the hybridization probe method in Figure 1c, various signal generation mechanisms can be utilized in the UnderSC composition for detecting target nucleic acids, in which hybridization of an oligonucleotide linked to a label (e.g., a single label or an interactive label) with a target nucleic acid provides a signal change from a duplex (i.e., a duplex that provides a signal change). In this case, the duplex that provides the signal change is a duplex generated during the incubation reaction.

[0239]

[0274] In one embodiment, various signal generation mechanisms may be utilized as the signal generation mechanism for the UnderSC composition for detecting a target nucleic acid, such as those shown in the Yin-Yang probe method in FIG. 1d and the PCE-NH method in FIG. 1e, in which the composition for detecting a target nucleic acid initially comprises a duplex that provides a signal change, and one of the two single strands constituting the duplex that provides a signal change pairs with the target nucleic acid, thereby forming a new duplex, or a new duplex is formed when one of the two single strands constituting the duplex that provides a signal change pairs with one of the two strands constituting the duplex formed by the cleavage reaction that depends on hybridization of the target nucleic acid with an intermediary oligonucleotide, such that the amount of the duplex that provides a signal change changes (particularly decreases).

[0240]

[0275] In one embodiment, various signal generation mechanisms can be used in the UnderSC composition, such as those shown in Figures 1f, 1g, and 1h, in which a signal change is provided from a duplex that provides a signal change formed by a cleavage reaction that relies on hybridization of an intermediary oligonucleotide with a target nucleic acid. In this case, the duplex that provides the signal change is a duplex generated during the incubation reaction, and in particular, the label of the duplex that provides the signal change in Figure 1f (i.e., the extended duplex) is incorporated during the incubation reaction.

[0241]

[0276] (ii) a signal generation mechanism that can be employed by the OverSC composition to detect target nucleic acids

[0242]

[0277] 2a-2c illustrate various signal generation mechanisms that can be employed by OverSC compositions.

[0243]

[0278] The signal generation mechanism in Figures 2a and 2c has one signal change temperature range in which the signal changes when the target nucleic acid is amplified, and one signal constant temperature range in which the signal is constant even when the target nucleic acid is amplified, and the signal change temperature range is higher than the signal constant temperature range.

[0244]

[0279] In one embodiment, various signal generation mechanisms can be used in the UnderSC composition, such as the PTOCE-based method in Figures 2a and 2c and the dual quenching method in Figure 2b, in which the signal change is provided by a duplex that provides a signal change formed by a cleavage reaction that relies on hybridization of an intermediary oligonucleotide with a target nucleic acid. In this case, the duplex that provides the signal change is a duplex generated during the incubation reaction, and in particular, the label of the duplex that provides the signal change in Figure 2c (i.e., the extended duplex) is incorporated during the incubation reaction.

[0245]

[0280] (iii) Signal generation mechanisms that can be employed by the InterSC composition to detect target nucleic acids

[0246]

[0281] 3a-b illustrate various signal generation mechanisms that can be employed by InterSC compositions.

[0247]

[0282] The signal generation mechanism in Figures 3a and 3b has one signal change temperature range in which the signal changes when the target nucleic acid is amplified, and two signal constant temperature ranges in which the signal is constant even when the target nucleic acid is amplified, and the signal change temperature range is higher than one of the two signal constant temperature ranges and lower than the other of the two signal constant temperature ranges.

[0248]

[0283] In one embodiment, a variety of signal generating mechanisms that provide multiple types of signal changes from duplexes can be used in the InterSC composition.

[0249]

[0284] For example, as in the PTOCE-based method shown in Figures 3a and 3b, a mechanism can be used in which the signal change is provided by two types of duplexes that provide signal change. In particular, in the method shown in Figures 3a and 3b, one of the two types of duplexes is initially included in the composition for detecting the target nucleic acid, and the other is generated by a cleavage reaction depending on the hybridization of the intermediary oligonucleotide with the target nucleic acid. As the incubation reaction proceeds, the ratio of the amounts of these two types of duplexes changes, thereby providing the signal change. In this case, the two types of duplexes have different Tm values.

[0250]

[0285] In one embodiment, the multiple duplexes may have different Tm values ​​from each other.

[0251]

[0286] In one embodiment, the InterSC composition has one signal change temperature range in which the signal changes depending on the presence of the target nucleic acid, and two signal constant temperature ranges in which the signal is constant even in the presence of the target nucleic acid, and the signal change temperature range is higher than one of the two signal constant temperature ranges and lower than the other of the two signal constant temperature ranges.

[0252]

[0287] In one embodiment, the InterSC composition provides duplexes that provide multiple types of signal changes.

[0253]

[0288] In one embodiment, the multiple types of duplexes have different Tm values ​​from each other.

[0254]

[0289] In one embodiment, the signal change temperature range and signal constant temperature range of the InterSC composition can be adjusted by adjusting the Tm values ​​of multiple types of duplexes, and the target nucleic acid can be detected using a detection temperature selected from the signal change temperature range.

[0255]

[0290] In one embodiment, the ratio of amounts between the types of duplexes changes in response to the presence of the target nucleic acid, thereby altering the signal.

[0256]

[0291] In one embodiment, the InterSC composition provides two signal-changing duplexes. Depending on the presence of target nucleic acid, the ratio of the amounts of the two duplexes changes, resulting in a signal change. The two duplexes have different Tm values.

[0257]

[0292] In one embodiment, one of the two signal change-providing duplexes (e.g., the duplex with a relatively low Tm) is included in the InterSC composition from the beginning, and the other (e.g., the duplex with a relatively high Tm) is generated during the incubation reaction.

[0258]

[0293] In one embodiment, one of the two single strands constituting each of the two double strands is the same strand. In particular, the amount of the double strand originally included in the composition for detecting a target nucleic acid decreases, and the amount of the newly generated double strand increases during the incubation reaction, so the ratio of the amounts of these double strands changes. That is, of the two double strands containing the same strand, the amount of the first double strand originally included in the InterSC composition decreases due to the second double strand newly generated during the incubation reaction, and the amount of the second double strand increases during the incubation, so the ratio of the amounts between them changes.

[0259]

[0294] In one embodiment, one of the duplexes provided by the InterSC composition is a duplex provided by a cleavage reaction that depends on the presence of a target nucleic acid. For example, the duplex newly generated during the incubation reaction can be generated by a cleavage reaction that depends on the presence of a target nucleic acid (e.g., a PTOCE-based method).

[0260]

[0295] In one embodiment, the two duplexes each comprise an interactive dual label (e.g., a reporter molecule and a quencher molecule). In particular, (i) when the two duplexes are in an associated form, the quencher molecule is in close proximity to the reporter molecule, thereby quenching the signal from the reporter molecule, and when the two duplexes are in a dissociated form, the quencher molecule is separated from the reporter molecule, thereby not quenching the signal from the reporter molecule (see Figure 3a), or (ii) when the two duplexes are in an associated form, the quencher molecule is separated from the reporter molecule, thereby not quenching the signal from the reporter molecule, and when the two duplexes are in a dissociated form, the quencher molecule is in close proximity to the reporter molecule, thereby quenching the signal from the reporter molecule (see Figure 3b).

[0261]

[0296] The signal generation mechanism that can be employed by the InterSC composition can be used to detect a single target nucleic acid, and in addition, can be used to detect multiple target nucleic acids by using multiple InterSC compositions and adjusting the signal change temperature ranges of each of the multiple InterSC compositions.

[0262]

[0297] As described above, compositions for detecting target nucleic acids may have the same signal generation mechanism but may have different orders of signal change temperature ranges and signal constant temperature ranges depending on (i) the type of label (e.g., single label, interactive label, etc.), (ii) the type of oligonucleotide to which the label is linked (e.g., PTO, CTO, etc.), and (iii) the position at which the label is linked and / or the manner in which the label is linked (e.g., whether the label is linked to the oligonucleotide from the beginning before the incubation reaction, or whether the label is linked to the oligonucleotide by being incorporated therein during the incubation reaction, etc.). Thus, the same signal generation mechanism may be used for any one of the UnderSC composition, InterSC composition, and OverSC composition. For example, in PTOCE-based methods, the mechanism by which the presence of a target nucleic acid is determined by signals from PTO extension and cleavage may be the same, but the cases in which all interactive double labels are linked to CTOs (e.g., Figures 1g and 1h) and the cases in which one of the interactive labels is linked to a PTO and the other to a CTO (e.g., Figure 3a) may be applied as signal generation mechanisms for the UnderSC composition and the InterSC composition, respectively. In addition, the cases in which interactive double labels are incorporated (e.g., Figure 2c) and the cases in which interactive double labels are linked to oligonucleotides from the beginning (e.g., Figure 3b) may be applied as signal generation mechanisms for the OverSC composition and the InterSC composition, respectively, for detecting a target nucleic acid.

[0263]

[0298] In certain embodiments, as in the PTOCE-based method described above, specifically, a PTOCE-based method in which a CTO having an interactive dual label linked thereto forms a duplex with an extended strand of a cleaved PTO fragment in response to the presence of a target nucleic acid to provide a signal, can be employed as a signal generation mechanism for the UnderSC composition (see FIG. 5). In this case, the signal change temperature range and signal constant temperature range of the UnderSC composition can be controlled by adjusting the Tm value of the extended duplex (i.e., the duplex between the CTO linked with the interactive dual label and the extended strand).

[0264]

[0299] In certain embodiments, the dual quenching method described above can be employed as a signal generation mechanism for the OverSC composition (see FIG. 7). In this case, the signal change temperature range and signal constant temperature range of the OverSC composition can be controlled by adjusting the Tm value of the tag duplex (i.e., the activated tag duplex fragment).

[0265]

[0300] In certain embodiments, the PTOCE-based method described above, specifically using a PTO having one of two interactive dual labels linked thereto and a CTO having the other of two interactive dual labels linked thereto, can be employed as a signal generation mechanism for an InterSC composition (see FIG. 6). In this case, the signal change temperature range and signal constant temperature range of the InterSC composition can be controlled by adjusting the Tm value of the duplex between the CTO and the tagging portion of the uncleaved PTO, and the Tm value of the extended duplex between the CTO and the extended strand of the PTO fragment that is cleaved depending on the presence of target nucleic acid.

[0266]

[0301] In one embodiment, when n is 2, the composition for detecting a first target nucleic acid may be an UnderSC composition or an InterSC composition, and the composition for detecting a second target nucleic acid may be an InterSC composition or an OverSC composition.

[0267]

[0302] In certain embodiments, when n is 2, the composition for detecting a first target nucleic acid may be an UnderSC composition, and the composition for detecting a second target nucleic acid may be an InterSC composition. For example, as shown in Figure 8, the signal generation mechanism of the composition for detecting a first target nucleic acid may be a PTOCE-based method using a CTO linked to an interactive dual label, and the signal generation mechanism of the composition for detecting a second target nucleic acid may be a PTOCE-based method in which one of the interactive dual labels is linked to a PTO and the other is linked to a CTO. In one embodiment, the first detection temperature can be selected to be a temperature at which the duplex provided by the composition for detecting a first target nucleic acid (i.e., the extended duplex) and the two types of duplex provided by the composition for detecting a second target nucleic acid (i.e., the uncleaved PTO-CTO duplex and the extended duplex) are all in an associated form, and the second detection temperature can be selected to be a temperature at which the extended duplex provided by the composition for detecting a first target nucleic acid and the uncleaved PTO-CTO duplex provided by the composition for detecting a second target nucleic acid are in a dissociated form, and the extended duplex provided by the composition for detecting a second target nucleic acid is in an associated form.The composition for detecting a first target nucleic acid provides a signal change in the presence of the first target nucleic acid at a first detection temperature (i.e., when extended duplexes in associated form are generated when the target nucleic acid is amplified, such that the quencher molecule is separated from the reporter molecule, thereby not quenching the signal from the reporter molecule), and a constant signal at a second detection temperature (i.e., when the extended duplexes, even if generated, are all in dissociated form, such that the quencher molecule is in close proximity to the reporter molecule, thereby quenching the signal from the reporter molecule); The composition for detecting the second target nucleic acid provides a constant signal in the presence of the second target nucleic acid at a first detection temperature (i.e., both types of duplexes are in associated form such that the quencher molecule is in proximity to the reporter molecule, thereby quenching the signal from the reporter molecule) and a signal change at a second detection temperature (uncleaved PTO and CTO duplexes are consumed to generate an extended duplex in associated form such that the quencher molecule is in proximity to the reporter molecule, thereby quenching the signal from the reporter molecule).

[0268]

[0303] In certain embodiments, when n is 2, the composition for detecting a first target nucleic acid may be an UnderSC composition, and the composition for detecting a second target nucleic acid may be an OverSC composition. For example, as shown in Figure 9, the signal generation mechanism of the composition for detecting a first target nucleic acid may be a PTOCE-based method using a CTO linked to an interactive dual label, and the signal generation mechanism of the composition for detecting a second target nucleic acid may be a dual-quenching method. In one embodiment, the first detection temperature may be selected to be a temperature at which the duplex provided by the composition for detecting a first target nucleic acid (i.e., an extended duplex) and the duplex provided by the composition for detecting a second target nucleic acid (i.e., a tag duplex) are in an associated form, and the second detection temperature may be selected to be a temperature at which both the extended duplex and the tag duplex are in a dissociated form.In this case, the composition for detecting the first target nucleic acid provides a signal change in the presence of the first target nucleic acid at a first detection temperature (i.e., when extended duplexes in associated form are generated when the target nucleic acid is amplified, such that the quencher molecule is separated from the reporter molecule, thereby not quenching the signal from the reporter molecule) and a constant signal at a second detection temperature (i.e., when the extended duplexes, even if generated, are all in dissociated form, such that the quencher molecule is in close proximity to the reporter molecule, thereby quenching the signal from the reporter molecule); In the presence of a second target nucleic acid, the composition provides a constant signal at a first detection temperature (i.e., in the associated form of the tag duplex (uncleaved tag duplex and activated tag duplex fragment), at least one of the two quenchers is in close proximity to the reporter molecule, thereby quenching the signal from the reporter molecule), and a signal change at a second detection temperature (i.e., in the dissociated form of the activated tag duplex fragment produced when the target nucleic acid is amplified, such that both of the two quencher molecules are separated from the reporter molecule, thereby unquenching the signal from the reporter molecule).

[0269]

[0304] In certain embodiments, when n is 2, the composition for detecting the first target nucleic acid may be an InterSC composition, and the composition for detecting the second target nucleic acid may be an InterSC composition. For example, as shown in Figure 10, a PTOCE-based method can be used in which one of the interactive dual labels is linked to a PTO and the other is linked to a CTO, as the signal generation mechanism of the composition for detecting the first target nucleic acid and the composition for detecting the second target nucleic acid. In one embodiment, the first detection temperature can be selected to be a temperature at which, among the two types of duplexes provided by the composition for detecting a first target nucleic acid (i.e., the extended duplex and the uncleaved PTO-CTO duplex) and the two types of duplexes provided by the composition for detecting a second target nucleic acid (i.e., the extended duplex and the uncleaved PTO-CTO duplex), all other duplexes are in an associated form except for the uncleaved PTO-CTO duplex provided by the composition for detecting a first target nucleic acid, and the uncleaved PTO-CTO duplex provided by the composition for detecting a first target nucleic acid is in a dissociated form; and the second detection temperature can be selected to be a temperature at which, among the two types of duplexes provided by the composition for detecting a first target nucleic acid and the two types of duplexes provided by the composition for detecting a second target nucleic acid, only the extended duplex provided by the composition for detecting a second target nucleic acid is in an associated form, and the rest of the other duplexes are all in a dissociated form.The composition for detecting a first target nucleic acid provides a signal change at a first detection temperature in the presence of the first target nucleic acid (i.e., when an extended duplex in associated form is generated when the target nucleic acid is amplified, the quencher molecule is in proximity to the reporter molecule, thereby quenching the signal from the reporter molecule) and a constant signal at a second detection temperature (i.e., such that the quencher molecule is separated from the reporter molecule, the extended duplex is all in dissociated form even when generated, thereby not quenching the signal from the reporter molecule); The composition for detecting two target nucleic acids provides, in the presence of the second target nucleic acid, a constant signal at a first detection temperature (i.e., both types of duplexes are in an associated form such that the quencher molecule is in proximity to the reporter molecule, thereby quenching the signal from the reporter molecule) and a signal change at a second detection temperature (when the uncleaved PTO and CTO duplex is consumed and an extended duplex in an associated form is produced, the quencher molecule is in proximity to the reporter molecule, thereby quenching the signal from the reporter molecule).

[0270]

[0305] In certain embodiments, when n is 2, the composition for detecting the first target nucleic acid may be an InterSC composition, and the composition for detecting the second target nucleic acid may be an OverSC composition. For example, as shown in Figure 11, the signal generation mechanism of the composition for detecting the first target nucleic acid may be a PTOCE-based method in which one of the dual labels is linked to a PTO and the other is linked to a CTO, and the signal generation mechanism of the composition for detecting the second target nucleic acid may be a dual-quenching method. In one embodiment, the first detection temperature can be selected so that all other duplexes among the two types of duplexes provided by the composition for detecting a first target nucleic acid (i.e., the extended duplex and the uncleaved PTO-CTO duplex) and the activated tag duplex fragments provided by the composition for detecting a second target nucleic acid are in associated forms, except for the uncleaved PTO-CTO duplex provided by the composition for detecting a first target nucleic acid, and the uncleaved PTO-CTO duplex provided by the composition for detecting a first target nucleic acid is in dissociated form; the second detection temperature can be selected so that all two types of duplexes provided by the composition for detecting a first target nucleic acid (i.e., the extended duplex and the uncleaved PTO-CTO duplex) and the activated tag duplex fragments provided by the composition for detecting a second target nucleic acid are in dissociated forms.In this case, the composition for detecting the first target nucleic acid provides a signal change at a first detection temperature in the presence of the first target nucleic acid (i.e., when extended duplexes in associated form are generated when the target nucleic acid is amplified, the quencher molecule is in proximity to the reporter molecule, thereby quenching the signal from the reporter molecule) and a constant signal at a second detection temperature (i.e., such that the quencher molecule is separated from the reporter molecule, the extended duplexes, even when generated, are all in dissociated form, thereby not quenching the signal from the reporter molecule); In the presence of a second target nucleic acid, the composition provides a constant signal at a first detection temperature (i.e., in the associated form of the tag duplex (uncleaved tag duplex and activated tag duplex fragment), at least one of the two quenchers is in close proximity to the reporter molecule, thereby quenching the signal from the reporter molecule), and a signal change at a second detection temperature (i.e., in the dissociated form of the activated tag duplex fragment produced when the target nucleic acid is amplified, such that both of the two quencher molecules are separated from the reporter molecule, thereby unquenching the signal from the reporter molecule).

[0271]

[0306] In one embodiment, when n is 3 or greater, the composition for detecting the first target nucleic acid may be an UnderSC composition or an InterSC composition, the composition for detecting the nth target nucleic acid may be an InterSC composition or an OverSC composition, and the composition(s) for detecting target nucleic acids other than the first target nucleic acid and the nth target nucleic acid may be an InterSC composition.

[0272]

[0307] In certain embodiments, when n is 3, the composition for detecting the first target nucleic acid may be an UnderSC composition, the composition for detecting the second target nucleic acid may be an InterSC composition, and the composition for detecting the third target nucleic acid may be an OverSC composition. For example, as shown in Figure 12, the signal generation mechanism of the composition for detecting the first target nucleic acid can be a PTOCE-based method using a CTO linked to an interactive dual label, the signal generation mechanism of the composition for detecting the second target nucleic acid can be a PTOCE-based method in which one of the interactive dual labels is linked to a PTO and the other is linked to a CTO, and the signal generation mechanism of the composition for detecting the third target nucleic acid can be a dual-quenching method. In one embodiment, the first detection temperature can be selected so that the duplexes provided by the compositions for detecting the first to third target nucleic acids (particularly the duplexes that provide a signal change) are all in an associated form; the second detection temperature can be selected so that the extended duplex provided by the composition for detecting the first target nucleic acid and the uncleaved PTO-CTO duplex provided by the composition for detecting the second target nucleic acid are in a dissociated form; the other duplexes (i.e., the extended duplex provided by the composition for detecting the second target nucleic acid and the tag duplex provided by the composition for detecting the third target nucleic acid) are in an associated form; and the third detection temperature can be selected so that all duplexes are in a dissociated form.In such a case, the composition for detecting the first target nucleic acid provides a signal change at the first detection temperature in the presence of the first target nucleic acid (i.e., extended duplexes in associated form are generated when the target nucleic acid is amplified such that the quencher molecules are separated from the reporter molecules, thereby not quenching the signal from the reporter molecules), and a constant signal at the second and third detection temperatures (i.e., extended duplexes, even when generated, are all in dissociated form such that the quencher molecules are in proximity to the reporter molecules, thereby quenching the signal from the reporter molecules); the composition for detecting the second target nucleic acid provides a constant signal at the first and third detection temperatures in the presence of the second target nucleic acid (i.e., both types of duplexes are in associated form such that the quencher molecules are in proximity to the reporter molecules, thereby quenching the signal from the reporter molecules, or both types of duplexes are in dissociated form such that the quencher molecules are in proximity to the reporter molecules, thereby quenching the signal from the reporter molecules). the quencher molecule is in proximity to the reporter molecule, thereby quenching the signal from the reporter molecule; and the composition for detecting a third target nucleic acid provides a constant signal in the presence of the third target nucleic acid at the first and second detection temperatures (i.e., in the associated form of the tag duplex (uncleaved tag duplex and activated tag duplex fragment), at least one of the two quenchers is in proximity to the reporter molecule, thereby quenching the signal from the reporter molecule), and provides a signal change at the third detection temperature (i.e., the activated tag duplex produced from the amplification of the target nucleic acid is in a dissociated form such that both of the two quencher molecules are separated from the reporter molecule, thereby not quenching the signal from the reporter molecule).

[0273]

[0308] In certain embodiments, when n is 3, the composition for detecting a first target nucleic acid may be an UnderSC composition, the composition for detecting a second target nucleic acid may be an InterSC composition, and the composition for detecting a third target nucleic acid may be an InterSC composition.

[0274]

[0309] In certain embodiments, when n is 3, the composition for detecting a first target nucleic acid may be an InterSC composition, the composition for detecting a second target nucleic acid may be an InterSC composition, and the composition for detecting a third target nucleic acid may be an OverSC composition.

[0275]

[0310] In certain embodiments, when n is 3, the composition for detecting a first target nucleic acid may be an InterSC composition, the composition for detecting a second target nucleic acid may be an InterSC composition, and the composition for detecting a third target nucleic acid may be an InterSC composition.

[0276]

[0311] In certain embodiments, when n is 4, the composition for detecting a first target nucleic acid may be an UnderSC or InterSC composition, the composition for detecting a second target nucleic acid and the composition for detecting a third target nucleic acid may both be InterSC compositions, and the composition for detecting a fourth target nucleic acid may be an InterSC or OverSC composition. The method according to the present disclosure takes advantage of the fact that there is a signal change temperature range depending on the signal generation mechanism of the composition for detecting a target nucleic acid.

[0277]

[0312] In one embodiment, the detection temperatures according to the present disclosure can be predetermined in terms of the signal change temperature ranges of each of the n compositions for detecting the target nucleic acid.

[0278]

[0313] In one embodiment, the signal change temperature range of any one of the n compositions for detecting a target nucleic acid can be determined according to the length and / or sequence of the duplex, i.e., by adjusting the Tm value of the duplex, the signal change temperature range can be predetermined.

[0279]

[0314] In one embodiment, when the signal change is generated by a label-linked oligonucleotide (e.g., a LUX probe, a molecular beacon probe, a Hy beacon probe, an adjacent hybridization probe, etc.) that specifically hybridizes to a target nucleic acid, detection of the signal can be successfully achieved at a predetermined detection temperature by adjusting the Tm value of the label-linked oligonucleotide.

[0280]

[0315] In one embodiment, when a Scorpion primer is used as a label-linked oligonucleotide, signal detection is conveniently performed at a predetermined temperature by adjusting the Tm value of the moiety that hybridizes to the extended strand.

[0281]

[0316] In one embodiment, when a signal is generated by a duplex formed based on the presence of a target nucleic acid sequence, detection of the signal is conveniently performed at a predetermined temperature by adjusting the Tm value of the duplex. For example, when a signal is generated by the PTOCE method, detection of the signal is conveniently achieved at a predetermined temperature by adjusting the Tm value of the extended duplex formed by extension of the PTO fragment with the CTO.

[0282]

[0317] PTOCE-based methods have the advantage that it is easy to control the Tm of the duplex or of a third hybridization product whose hybridization is influenced by the duplex.

[0283]

[0318] As described above, the detection temperature is determined by taking into consideration the temperature range of signal change that varies depending on the duplex provided by the composition for detecting the target nucleic acid.

[0284]

[0319] In one embodiment, the detection temperature of any one of the n compositions for detection of the n target nucleic acids can be predetermined within a signal change temperature range that does not overlap with the signal change temperature range of the other compositions (see Figure 4).

[0285]

[0320] In one embodiment, the detection temperatures assigned to compositions for detecting target nucleic acids differ from each other by at least 2°C, 3°C, 4°C, 5°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 15°C, or 20°C or more.

[0286]

[0321] In one embodiment, the n detection temperatures are 45°C to 97°C, 45°C to 96°C, 45°C to 95°C, 45°C to 94°C, 45°C to 93°C, 45°C to 92°C, 45°C to 91°C, 45°C to 90°C, 46°C to 97°C, 46°C to 96°C, 46°C to 95°C, 46°C to 94°C, 46°C to 93°C, 46°C to 92°C, 46°C to 91°C, 46°C to 90°C, 47°C to 97°C, 47°C to 96°C, 47°C to 95°C, 47°C to 94°C, 47°C to 93°C, 47°C to 92°C, 47°C to 91°C, 47°C to 90°C, The temperature range can be selected from 48°C to 97°C, 48°C to 96°C, 48°C to 95°C, 48°C to 94°C, 48°C to 93°C, 48°C to 92°C, 48°C to 91°C, 48°C to 90°C, 49°C to 97°C, 49°C to 96°C, 49°C to 95°C, 49°C to 94°C, 49°C to 93°C, 49°C to 92°C, 49°C to 91°C, 49°C to 90°C, 50°C to 97°C, 50°C to 96°C, 50°C to 95°C, 50°C to 94°C, 50°C to 93°C, 50°C to 92°C, 50°C to 91°C, or 50°C to 90°C.

[0287]

[0322] For example, the highest detection temperature among the above detection temperatures (i.e., the nth detection temperature) is 70°C to 97°C, 70°C to 95°C, 70°C to 93°C, 70°C to 90°C, 73°C to 97°C, 73°C to 95°C, 73°C to 93°C, 73°C to 90°C, 75°C to 97°C, 75°C to 95°C, 75°C to 93°C, 75°C to 90°C, 78°C to 99°C, The temperature range can be selected from 7°C, 78°C to 95°C, 78°C to 93°C, 78°C to 90°C, 80°C to 97°C, 80°C to 95°C, 80°C to 93°C, 80°C to 90°C, 83°C to 97°C, 83°C to 95°C, 83°C to 93°C, 83°C to 90°C, 85°C to 97°C, 85°C to 95°C, 85°C to 93°C, or 85°C to 90°C.

[0288]

[0323] For example, the lowest detection temperature among the n detection temperatures (i.e., the first detection temperature) can be selected from the temperature ranges of 45°C to 70°C, 45°C to 68°C, 45°C to 65°C, 45°C to 63°C, 45°C to 60°C, 45°C to 58°C, 45°C to 55°C, 48°C to 70°C, 48°C to 68°C, 48°C to 65°C, 48°C to 63°C, 48°C to 60°C, 48°C to 58°C, 48°C to 55°C, 50°C to 70°C, 50°C to 68°C, 50°C to 65°C, 50°C to 63°C, 50°C to 60°C, 50°C to 58°C, or 50°C to 55°C.

[0289]

[0324] For example, among the n detected temperatures (for example, from the second detected temperature to the (n-1)th detected temperature), the intermediate detected temperatures are 55°C to 85°C, 55°C to 83°C, 55°C to 80°C, 55°C to 78°C, 55°C to 75°C, 55°C to 73°C, 55°C to 70°C, 55°C to 68°C, 55°C to 65°C, 55°C to 63°C, 55°C to 6 ... 8℃~85℃, 58℃~83℃, 58℃~80℃, 58℃~78℃, 58℃~75℃, 58℃~73℃, 58℃~70℃, 58℃~68℃, 58℃~65℃, 58℃~63℃, 58℃~60℃, 60℃~85℃, 60℃~83℃, 60℃~80℃, 60℃~78℃, 60℃~75℃, 60℃~73℃, 60℃~70℃ , 60℃~68℃, 60℃~65℃, 60℃~63℃, 63℃~85℃, 63℃~83℃, 63℃~80℃, 63℃~78℃, 63℃~75℃, 63℃~73℃, 63℃~70℃, 63℃~68℃, 63℃~65℃, 65℃~85℃, 65℃~83℃, 65℃~80℃, 65℃~78℃, 65℃~75℃, 65℃~7 The temperature range can be selected from the following: 3°C, 65°C to 70°C, 65°C to 68°C, 68°C to 85°C, 68°C to 83°C, 68°C to 80°C, 68°C to 78°C, 68°C to 75°C, 68°C to 73°C, 68°C to 70°C, 70°C to 85°C, 70°C to 83°C, 70°C to 80°C, 70°C to 78°C, 70°C to 75°C, or 70°C to 73°C.

[0290]

[0325] According to one embodiment, n target nucleic acids are respectively assigned to n detection temperatures, and n compositions for detecting the n target nucleic acids appropriate for the detection temperatures are prepared, and then step (a) may be performed.

[0291]

[0326] In one embodiment, when n is 3, the first detection temperature can be selected from a temperature range of 50°C to 60°C, the second detection temperature can be selected from a temperature range of 65°C to 75°C, and the third detection temperature can be selected from a temperature range of 80°C to 95°C.

[0292]

[0327] In step (a), signals are detected at n detection temperatures during incubation.

[0293]

[0328] In one embodiment, detection of the signal may occur at each cycle, or at selected cycles, or at the end point of the reaction.

[0294]

[0329] In one embodiment, signal detection may be performed in at least one cycle. For example, signals may be detected at n detection temperatures in one selected cycle, or may be detected at n detection temperatures in each of two selected cycles. For example, if n is 3 and signals are detected in cycle 1 and cycle 30, signals (i.e., the first signal, the second signal, and the third signal) are detected at the first detection temperature, the second detection temperature, and the third detection temperature in cycle 1, and signals are detected at the first detection temperature, the second detection temperature, and the third detection temperature in cycle 30.

[0295]

[0330] In one embodiment, the detection of the signal may be carried out in at least two cycles.

[0296]

[0331] In one embodiment, signal changes can be measured using signals detected in at least two cycles.For example, nucleic acid amplification can be carried out over 30, 40, 45, or 50 cycles of PCR, and signals can be measured at n detection temperatures in each cycle.The signal values ​​detected at each detection temperature in multiple cycles can then be represented as an amplification curve (a collection of data points of cycles and RFUs in cycles) at each detection temperature.As a specific example, when n is 3, when each target nucleic acid is present, an amplification curve at the first detection temperature, an amplification curve at the second detection temperature, and an amplification curve at the third detection temperature can be obtained, and signal changes can be identified from the amplification curves.

[0297]

[0332] As used herein, the term "amplification curve" refers to a curve obtained from a signal generation reaction, particularly an amplification reaction of a target analyte (particularly a target nucleic acid). Amplification curves include curves obtained from a reaction in the presence of the target nucleic acid in a sample, and curves or lines obtained from a reaction in the absence of the target nucleic acid in a sample.

[0298]

[0333] In one embodiment, the signal change and / or constant signal can be measured from an indicator that indicates amplification of the target nucleic acid.

[0299]

[0334] The term "indicator of amplification," as used herein, refers to any indicator closely related to the appearance of amplification of the target nucleic acid obtainable from the signal provided in step (a). An indicator may also refer to a value generated dependent on the amplification of the target nucleic acid. The indicator may be an indicator that provides a greater value as the amplification of the target nucleic acid increases (i.e., as the amount of the target nucleic acid increases), or an indicator that provides a lesser value as the amplification increases. The indicator may be any indicator as long as it indicates amplification.

[0300]

[0335] In one embodiment, the indicator of amplification can be obtained from an amplification curve or melting curve. In particular, the indicator can include the signal value (e.g., RFU) at a specific cycle, the signal value at each cycle, the difference in signal values ​​between specific cycles, or the difference between a reference signal value and a specific cycle in the amplification curve, or the height, width, or area of ​​the maximum melting peak in the melting curve. In one embodiment, the indicator can be, but is not limited to, the Ct (cycle threshold) value, ΔRFU (e.g., the difference in RFU between two cycles, the difference between the reference RFU and the RFU at a specific cycle, etc.), the ratio of RFU (e.g., the ratio of RFU between two cycles, or the ratio between the reference RFU and the RFU at a specific cycle, etc.), and the height / area / width of the melting peak (e.g., the height / area / width of the maximum peak in the melting curve).

[0301]

[0336] According to one embodiment, the indicator of amplification is a Ct value. The Ct value may be the intersection of the amplification curve with a threshold line. The concept of a Ct value is well known in the art.

[0302]

[0337] According to one embodiment, the indicator of amplification is a delta RFU or RFU ratio between the RFU values ​​obtained in the amplification reaction, for example, the indicator is the difference (subtraction) or ratio between the RFU in two cycles, or the difference (subtraction) or ratio between the RFU in a particular cycle and a reference RFU.

[0303]

[0338] In one embodiment, the indicator of amplification is the area or width of the melting peak. The area or width of the melting peak refers to the area or width of the largest peak in the derivative of the melting curve obtained by melting analysis. The area or width of the melting peak is widely known in the art.

[0304]

[0339] In one embodiment, signal detection may be performed in one cycle. In this case, it is difficult to measure the signal change by the signal value detected in one cycle alone. Therefore, the signal change may be detected using another reference signal value.

[0305]

[0340] The method according to the present disclosure utilizes the fact that a composition for detecting a target nucleic acid provides a signal change in response to the presence of the target nucleic acid only at a corresponding detection temperature. According to one embodiment, the method according to the present disclosure can measure a signal change using signal values ​​detected at the detection temperature in at least two cycles. In another embodiment, the method according to the present disclosure can measure a signal change by using a signal value at the detection temperature detected in one cycle (i.e., the signal value detected in step (a)) and a reference signal value.

[0306]

[0341] In one embodiment, when a signal is detected at multiple cycles in step (a), the first and last cycles at which the signal is detected may be selected to be separated by at least 1 to 20 cycles, particularly the first and last cycles at which the signal is detected may be selected to be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more cycles, more particularly 5, 10, 15, 20, or 30 or more cycles.

[0307]

[0342] In one embodiment, the signal may be detected in either the middle cycle including the logarithmic phase region or in either the later cycle including the plateau phase region. For example, the signal may be detected in two cycles, one of which is the early cycle including the baseline phase region and the other is either the middle or later cycle, or the signal may be detected in two cycles, one of which is the middle cycle and the other is either the middle or later cycle.

[0308]

[0343] In one embodiment, the early cycles include cycles up to and including the cycle adjacent to the value obtained by dividing the final cycle by 3. For example, if the final cycle is 45, 45 divided by 3 equals 15, and therefore the early cycles can be determined to be cycles 1 to 20, cycles 1 to 15, cycles 1 to 10, or cycles 1 to 5. The intermediate cycles can be determined to be cycles adjacent to the value obtained by dividing the final cycle by 2. For example, if the final cycle is 45, 45 divided by 2 equals 22.5, and therefore the intermediate cycles can be determined to be cycles 16 to 30, cycles 18 to 30, cycles 20 to 30, cycles 16 to 27, cycles 18 to 27, cycles 20 to 27, cycles 16 to 25, cycles 18 to 25, or cycles 20 to 25. The late cycles can be determined to be the final cycle of the amplification reaction or a cycle adjacent to the final cycle. For example, if the final cycle is cycle 45, the later cycles can be determined to be cycles 31 to 45, cycles 35 to 45, cycles 38 to 45, cycles 40 to 45, or cycles 43 to 45. The initial, intermediate, and later cycles can be varied depending on the final number of cycles in the amplification reaction. In particular, in step (a), the cycle at which a signal is detected may be one of the later cycles (e.g., the final cycle), or a cycle at which a reference signal is detected may be one of the early cycles of the positive control reaction (e.g., cycle 1).

[0309]

[0344] In one embodiment, the signal change can be measured using a signal detected in at least one cycle and a "reference signal value." The reference signal value may also refer to a value at which a signal change can be determined depending on the presence of the target nucleic acid through a separate reaction.

[0310]

[0345] In one embodiment, the reference signal value may be obtained from a reaction in the absence of the corresponding target nucleic acid at the corresponding detection temperature. For example, the reference signal value may be the "signal value at the detection temperature" in the absence of the target nucleic acid.

[0311]

[0346] In one embodiment, there are n reference signal values ​​for n detected temperatures.

[0312]

[0347] In one embodiment, the "signal value at the detection temperature (e.g., the i-th detection temperature)" detected in the absence of the target nucleic acid (e.g., the i-th target nucleic acid) can be obtained via a separate negative control reaction.

[0313]

[0348] In one embodiment, the reference signal value may be obtained by running a negative control reaction simultaneously with or separately from the method according to the present disclosure.

[0314]

[0349] In one embodiment, the reference signal value can be obtained through a negative control reaction. According to one specific embodiment, the reference signal value at the i-th detection temperature can be obtained by mixing a sample (e.g., distilled water) that does not contain the i-th target nucleic acid with n compositions for detecting the target nucleic acid and detecting a signal at the i-th detection temperature while amplifying the nucleic acid. In this case, signal detection can be performed at any cycle. In particular, a signal value detected in any of the early cycles of the negative control reaction can be used as the reference signal value, or a signal value detected in any of the later cycles of the negative control reaction can be used as the reference signal value. More specifically, a signal value detected in the same cycle as the cycle in which the signal is detected in step (a) can be used as the reference signal value.

[0315]

[0350] In one embodiment, the reference signal value can be obtained through a positive control reaction. According to a specific embodiment, the reference signal value can be obtained by mixing a sample containing the i-th target nucleic acid with a composition for detecting the i-th target nucleic acid, and detecting a signal at the i-th detection temperature while amplifying the nucleic acid.

[0316]

[0351] When the reference signal value is obtained through a positive control reaction, the cycle at which the signal value is detected may be a cycle in the baseline region of the positive control reaction. The baseline region refers to the region in which the signal (e.g., fluorescent signal) remains substantially constant during the initial cycle of an amplification reaction (e.g., PCR). In this region, the level of the amplification product is not sufficient to be detectable, and most of the fluorescent signal in this region is due to background signals, including the fluorescent signal inherent to the reaction sample and the fluorescent signal of the measurement system itself. In other words, the signal value detected in the cycle in the baseline region of the positive control reaction is substantially identical to the reference signal value obtained from a reaction in the absence of target nucleic acid (e.g., a negative control reaction).

[0317]

[0352] In one embodiment, the signal change can be measured via the difference between a reference signal value and the signal value detected in step (a).

[0318]

[0353] In one embodiment, the reference signal value may be a threshold value previously determined from a negative control reaction by taking into consideration the background signal and the sensitivity of the detector or the characteristics of the label used. The threshold value can be used to determine the significance of a signal change. The threshold value can be determined by a known threshold setting method. For example, the threshold value can be determined by taking into consideration the background signal, the sensitivity, the characteristics of the label, the signal fluctuation of the detector, the tolerance for error, etc.

[0319]

[0354] In one embodiment, when a threshold value is used as the reference signal value, it can be determined that the signal has changed if the signal value detected in step (a) is equal to or greater than the threshold value.

[0320]

[0355] In one embodiment, detection of the signal at each of the n detection temperatures may be performed using a single type of detector.

[0321]

[0356] In one embodiment, the single type of detector is one detector. In one embodiment, the signals from the labels of the label-linked oligonucleotides included in the n compositions for detecting target nucleic acids are indistinguishable from each other by the single type of detector for each target nucleic acid.

[0322]

[0357] A single or one type of fluorescent label, as used herein, refers to fluorescent labels that have identical or substantially identical signal characteristics (e.g., optical characteristics, emission wavelength, and electrical signal).

[0323]

[0358] For example, FAM and CAL Fluor 610 provide different types of signals. In this application, a single or one type of fluorescent label means that the signals from the fluorescent labels cannot be distinguished from each other using a detection channel. Such a single or one type of fluorescent label does not depend on the chemical structure of the fluorescent label, and therefore, even if two fluorescent labels have different chemical structures from each other, they are considered to be of one type if they cannot be distinguished from each other using a detection channel.

[0324]

[0359] According to the present disclosure, signals generated from n compositions for detecting target nucleic acids that commonly contain one type of fluorescent label are not distinguished by one detection channel.

[0325]

[0360] The term " detection channel " as used herein refers to the means for detecting the signal from a single type of fluorescent label.Thermocycler suitable for use in the field, for example, ABI7500 (Applied Biosystems), QuantStudio (Applied Biosystems), CFX96 (Bio-Rad Laboratories), Cobas z 480 (Roche), LightCycler (Roche) etc., comprises multiple channels (for example, optical diodes) for detecting the signals from several different types of fluorescent labels, and these channels correspond to the detection channel used herein.

[0326]

[0361] The detection channel used in the present invention includes a means for detecting a signal. For example, the detection channel may be an optical diode capable of detecting a fluorescent signal at a particular wavelength.

[0327]

[0362] According to one embodiment, the signals detected at the n detection temperatures are not distinguishable from one another by a single type of detector.

[0363]

[0328]

[0364] Step (b): Determining the presence of the target nucleic acid

[0329]

[0365] After detection of the signals, the presence of the n target nucleic acids is determined from the signals detected in step (a).

[0330]

[0366] In one embodiment, the presence of the i-th target nucleic acid is determined by a signal change detected at the i-th detection temperature. For example, a signal change can be measured from the signal detected at the i-th detection temperature to determine the presence of the i-th target nucleic acid.

[0331]

[0367] In one embodiment, if a change in signal at the ith detection temperature is measured, it can be determined that the ith target nucleic acid is present.

[0332]

[0368] In one embodiment, if the signal is constant at the i-th detection temperature, it can be determined that the i-th target nucleic acid is not present.

[0333]

[0369] In one embodiment, the signal change can be measured using a signal detected in at least two cycles, or a signal detected in at least one cycle, and a "reference signal value."

[0334]

[0370] Determining the presence of the target nucleic acid from the signal detected at each detection temperature may be accomplished by the processes described in step (a) for measuring signal changes, such as by using a label that indicates amplification, and by various other methods known in the art.

[0335]

[0371] In certain embodiments, when n is 3 and signal detection occurs at cycle 10, cycle 20, and cycle 30, the presence of the first target nucleic acid can be determined from signals detected at the first detection temperature (first signal at cycle 10, first signal at cycle 20, and first signal at cycle 30), the presence of the second target nucleic acid can be determined from signals detected at the second detection temperature (second signal at cycle 10, second signal at cycle 20, and second signal at cycle 30), and the presence of the third target nucleic acid can be determined from signals detected at the third detection temperature (third signal at cycle 10, third signal at cycle 20, and third signal at cycle 30).

[0336]

[0372] In certain embodiments, when n is 4 and signal detection is performed at cycle 30, the presence of the first target nucleic acid is determined from the signal detected at the first detection temperature (i.e., the first signal at cycle 30) and the reference signal value, the presence of the second target nucleic acid is determined from the signal detected at the second detection temperature (i.e., the second signal at cycle 30) and the reference signal value, and the presence of the third target nucleic acid is determined from the signal detected at the third detection temperature (i.e., the third signal at cycle 30) and the reference signal value.

[0337]

[0373] In one embodiment, the reference signal value can be obtained via a separate negative or positive control reaction.

[0338]

[0374] In one embodiment, the method according to the present disclosure may be carried out with a negative control reaction. The signal value detected in the negative control reaction may be used as a reference signal value. For example, in a reaction containing a composition for detecting the i-th target nucleic acid, the signal detected in one cycle (e.g., the final cycle) at the i-th detection temperature can be compared with the signal detected in the same cycle (e.g., the final cycle) at the same detection temperature (i.e., the i-th detection temperature) in the negative control reaction to determine whether the signal has changed.

[0339]

[0375] In certain embodiments, when n is 3 and a signal is detected at cycle 30, the presence of a first target nucleic acid can be determined from the signal detected at the first detection temperature (i.e., the first signal at cycle 30) and a first reference signal value (e.g., the signal at the first detection temperature detected at cycle 30 of a negative control reaction), the presence of a second target nucleic acid can be determined from the signal detected at the second detection temperature (i.e., the second signal at cycle 30) and a second reference signal value (e.g., the signal at the second detection temperature detected at cycle 30 of a negative control reaction), and the presence of a third target nucleic acid can be determined from the signal detected at the third detection temperature (i.e., the third signal at cycle 30) and a third reference signal value (e.g., the signal at the third detection temperature detected at cycle 30 of a negative control reaction).

[0340]

[0376] In one embodiment, the method according to the present disclosure may be performed with a positive control reaction. The signal value detected in the positive control reaction may be used as a reference signal value. For example, the signal detected in one cycle at the i-th detection temperature, e.g., cycle 30, the first signal detected in cycle 30, can be compared with the signal detected in the positive control reaction at the i-th detection temperature in the cycle before cycle 30, e.g., cycle 1, to determine whether the signal has changed.

[0341]

[0377] In one embodiment, when signal detection is performed in one cycle in step (a) and the signal change is measured using a reference signal value obtained via a positive control reaction, signal detection in the positive control reaction to obtain the reference signal value may be performed at least 30 cycles, 20 cycles, 10 cycles, or 5 cycles before the cycle in which signal detection is performed in step (a).

[0342]

[0378] In certain embodiments, when n is 3 and signal detection is performed at cycle 30, the presence of the first target nucleic acid can be determined from the signal detected at the first detection temperature (i.e., the first signal at cycle 30) and the first reference signal value (e.g., the signal at the first detection temperature detected at cycle 1 of a positive control reaction of the first target nucleic acid), the presence of the second target nucleic acid can be determined from the signal detected at the second detection temperature (i.e., the second signal at cycle 30) and the second reference signal value (e.g., the signal at the second detection temperature detected at cycle 1 of a positive control reaction of the second target nucleic acid), and the presence of the third target nucleic acid can be determined from the signal detected at the third detection temperature (i.e., the third signal at cycle 30) and the third reference signal value (e.g., the signal at the third detection temperature detected at cycle 1 of a positive control reaction of the third target nucleic acid).

[0379]

[0343]

[0380] According to another aspect of the present disclosure, there is provided a method for detecting two target nucleic acids in a sample, comprising:

[0381] (a) detecting a signal at a first detection temperature and a second detection temperature while incubating a sample suspected of containing at least one of two target nucleic acids in a reaction vessel with a composition for detecting a first target nucleic acid and a composition for detecting a second target nucleic acid, wherein the incubation comprises a plurality of cycles, and the detection of the signal is performed in at least one of the plurality of cycles, and the composition for detecting the first target nucleic acid provides a signal change at the first detection temperature and a constant signal at the second detection temperature in the presence of the first target nucleic acid, and the signal change is indicative of a change in the signal of the first target nucleic acid. a composition for detecting a second target nucleic acid, wherein the composition provides a signal change at the second detection temperature in the presence of the second target nucleic acid and provides a constant signal at the first detection temperature, the signal change indicating the presence of the second target nucleic acid, the first detection temperature being lower than the second detection temperature; and (b) determining the presence of the two target nucleic acids from the signals detected in step (a), wherein the presence of the first target nucleic acid is determined by the signal change detected at the first detection temperature and the presence of the second target nucleic acid is determined by the signal change detected at the second detection temperature.

[0344]

[0382]

[0383] According to another aspect of the present disclosure, there is provided a method for detecting three target nucleic acids in a sample, comprising:

[0384] (a) detecting signals at a first detection temperature, a second detection temperature, and a third detection temperature while incubating a sample suspected of containing at least one of three target nucleic acids in a reaction vessel with a composition for detecting a first target nucleic acid, a composition for detecting a second target nucleic acid, and a composition for detecting a third target nucleic acid, wherein the incubation includes a plurality of cycles, and the detection of the signals is performed in at least one of the plurality of cycles, wherein the composition for detecting the first target nucleic acid provides a signal change at the first detection temperature in the presence of the first target nucleic acid and provides constant signals at the second detection temperature and the third detection temperature, the signal change indicating the presence of the first target nucleic acid; and the composition for detecting the second target nucleic acid provides a signal change at the second detection temperature in the presence of the second target nucleic acid and provides constant signals at the first detection temperature and the third detection temperature. providing a constant signal at a third detection temperature, wherein the signal change indicates the presence of a second target nucleic acid; a composition for detecting the third target nucleic acid, in the presence of the third target nucleic acid, providing a signal change at the third detection temperature, wherein the composition for detecting the third target nucleic acid provides a constant signal at the first detection temperature and the second detection temperature, wherein the signal change indicates the presence of the third target nucleic acid, wherein the first detection temperature is lower than the second detection temperature and the second detection temperature is lower than the third detection temperature; and (b) determining the presence of three target nucleic acids from the signals detected in step (a), wherein the presence of the first target nucleic acid is determined by the signal change detected at the first detection temperature, the presence of the second target nucleic acid is determined by the signal change detected at the second detection temperature, and the presence of the third target nucleic acid is determined by the signal change detected at the third detection temperature.

[0385]

[0345]

[0386] The second and third embodiments of the present disclosure follow the same principles as the first embodiment of the present disclosure described above, and therefore, common features between these embodiments will not be redundantly described for the sake of clarity of this application.

[0387]

[0346]

[0388] II. Kits for detecting target nucleic acids

[0347]

[0389] According to another aspect of the present disclosure, there is provided a kit comprising n compositions for detecting n target nucleic acids in a sample, where n is an integer of 2 or greater, each of the n compositions for detecting the n target nucleic acids provides a signal change at a corresponding detection temperature among n detection temperatures, where the signal change indicates the presence of the corresponding target nucleic acid, and a composition for detecting an i-th target nucleic acid among the n target nucleic acids provides a signal change at the i-th detection temperature among the n detection temperatures in the presence of the i-th target nucleic acid and provides a constant signal at other detection temperatures, where i represents an integer from 1 to n, and the i-th detection temperature is lower than the (i+1)-th detection temperature.

[0390]

[0348]

[0391] Because the kits of the present disclosure are prepared to enable the methods of the present disclosure, common features shared by the two will not be redundantly described for the sake of clarity of this application.

[0349]

[0392]

[0393] According to one embodiment, in a temperature range covering all n detection temperatures, a composition for detecting an i-th target nucleic acid has a "signal changing temperature range" in which the signal changes in response to the presence of the i-th target nucleic acid, and a "signal constant temperature range" in which the signal remains constant even in the presence of the i-th target nucleic acid.

[0350]

[0394] According to one embodiment, the composition for detecting the ith target nucleic acid has one or two signal-constant temperature ranges.

[0351]

[0395] According to one embodiment, the composition for detecting the i-th target nucleic acid is any one of (i) an under-signal change (UnderSC) composition characterized in that the signal change temperature range is lower than the signal constant temperature range; (ii) an over-signal change (OverSC) composition characterized in that the signal change temperature range is higher than the signal constant temperature range; and (iii) an inter-signal change (InterSC) composition characterized in that the signal change temperature range is higher than one of two signal constant temperature ranges and lower than the other of the two signal constant temperature ranges.

[0352]

[0396] According to one embodiment, the i-th detection temperature is selected within the signal change temperature range of the composition for detecting the i-th target nucleic acid, and the i-th detection temperature is not included in the signal change temperature range of the composition for detecting any other target nucleic acid.

[0353]

[0397] According to one embodiment, the signal change temperature range of the composition for detecting the i-th target nucleic acid may partially overlap with the signal change temperature range of a composition for detecting a target nucleic acid having an adjacent detection temperature, but does not overlap with the signal change temperature range of a composition for detecting a target nucleic acid having a non-adjacent detection temperature.

[0354]

[0398] According to one embodiment, when n is 2, the composition for detecting the first target nucleic acid is an UnderSC composition or an InterSC composition, and the composition for detecting the second target nucleic acid is an InterSC composition or an OverSC composition.

[0355]

[0399] According to one embodiment, when n is 3 or greater, the composition for detecting the first target nucleic acid is an UnderSC composition or an InterSC composition, the composition for detecting the nth target nucleic acid is an InterSC composition or an OverSC composition, and the composition for detecting target nucleic acids other than the first target nucleic acid and the nth target nucleic acid is an InterSC composition.

[0356]

[0400] According to one embodiment, the composition for detecting the ith target nucleic acid comprises a label that provides a signal in response to the presence of the ith target nucleic acid.

[0357]

[0401] According to one embodiment, the label is linked to the oligonucleotide or is incorporated into the oligonucleotide during incubation of the kit with the sample.

[0358]

[0402] According to one embodiment, the composition for detecting the ith target nucleic acid provides a duplex that provides a signal change.

[0359]

[0403] According to one embodiment, the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and when the duplex is present as an associated or dissociated duplex, the composition for detecting the i-th target nucleic acid provides a signal from a label.

[0360]

[0404] According to one embodiment, the signals provided by the n compositions for detecting the n target nucleic acids are indistinguishable from one another by a single type of detector.

[0361]

[0405] In one embodiment, the signals indicative of the presence of each target nucleic acid provided by the n compositions for detecting the target nucleic acid are indistinguishable from one another by a single type of detector, e.g., all of the labels included in the n compositions for detecting the target nucleic acid are the same type of label (e.g., one fluorescent label).

[0362]

[0406] In one embodiment, the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and the signal change temperature range of the composition for detecting the i-th target nucleic acid is determined according to the length and / or sequence of the duplex.

[0363]

[0407] According to one embodiment, the kit further comprises instructions describing the methods of the invention.

[0364]

[0408] According to one embodiment, the instructions for describing or practicing the methods of the invention may be recorded on a suitable storage medium. For example, the instructions may be printed on a substrate such as paper or plastic. In other embodiments, the instructions may be present as an electronic storage data file present on a suitable computer-readable storage medium, such as a CD-ROM or diskette. In yet other embodiments, the actual instructions may not be present in the kit, but means for obtaining the instructions from a remote source, for example, via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded.

[0365]

[0409] All of the kits described above may optionally include reagents necessary to perform a target nucleic acid amplification reaction (e.g., a PCR reaction), such as buffers, DNA polymerase cofactors, and deoxyribonucleotide-5-triphosphates. Optionally, the kit may also include various polynucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. The kit may also include reagents necessary to perform positive and negative control reactions. The optimal amounts of reagents expected to be used in a given reaction can be readily determined by one of skill in the art having the benefit of the present disclosure. The above-described components of the kit may be present in separate containers, or multiple components may be present in a single container.

[0410]

[0366]

[0411] The present invention will be described in more detail below through embodiments. The following embodiments are provided to further describe the present invention, and it will be apparent to those skilled in the art to which the present invention pertains that the scope of the present invention as suggested by the appended claims is not limited by the following embodiments.

[0412]

[0413] [Example]

[0367]

[0414]

[0415] The inventors have confirmed whether multiple target nucleic acids can be detected in real time using the same type, i.e., a single type, of label by using various combinations of an UnderSC composition for detecting a target nucleic acid, an InterSC composition for detecting a target nucleic acid, and an OverSC composition for detecting a target nucleic acid according to the methods disclosed herein.

[0368]

[0416] In particular, among the various signal generation mechanisms that can be adopted by compositions for detecting three types of target nucleic acids, a PTOCE-based method (hereinafter referred to as PTOCE-based 1) using a CTO to which all of the interactive double labels are linked, as in the case of Figure 5, was used for the UnderSC composition; a PTOCE-based method (hereinafter referred to as PTOCE-based 2) in which one of the interactive double labels is linked to the 5'-tagging portion of the PTO and the other is linked to the capture portion of the CTO, as in the case of Figure 6, was used for the InterSC composition; and a double quenching method as shown in Figure 7 was used for the OverSC composition.

[0369]

[0417] The templates for multiple target nucleic acids were Chlamydia trachomatis (CT) genomic DNA (accession number: ATCC VR-1500, Koram Deo Lab), Neisseria gonorrhoeae (NG) (accession number: ATCC700825, Koram Deo Lab), and Ureaplasma parvum (UP) (accession number: ATCC27815, Koram Deo Lab). As shown in Table 1, the three target nucleic acids were detected by various combinations of the three types of compositions and detection temperatures. Combinations 1 to 4 are cases where the number of target nucleic acids (n) is 2, and combination 5 is a case where the number of target nucleic acids (n) is 3.

[0418]

[0419]

[0370] [Table 1]

[0371]

[0420]

[0421] Example 1: Preparation of oligonucleotides for compositions for detecting target nucleic acids

[0422]

[0372]

[0423] <Combination 1>

[0424] The first detection temperature for detecting a first signal change indicating the presence of the first target nucleic acid, CT, was set to 50° C., and the second detection temperature for detecting a second signal change indicating the presence of the second target nucleic acid, NG, was set to 72° C. Then, oligonucleotides for a composition for detecting the CT target nucleic acid and a composition for detecting the NG target nucleic acid were prepared as shown in Table 2 below.

[0373]

[0425] The composition for detecting the CT target nucleic acid comprises a primer pair, a PTO, and a CTO linked to a reporter molecule (CAL Fluoro Red 610) and a quencher molecule (BHQ-2), and the sequences of the PTO and CTO were designed so that the duplex between the PTO (i.e., uncleaved PTO) and the CTO is in a dissociated form at both the first and second detection temperatures, and the extended duplex formed in response to the presence of CT is in an associated form at the first detection temperature and in a dissociated form at the second detection temperature (see Figure 8).

[0374]

[0426] The composition for detecting the NG target nucleic acid comprises a primer pair, a PTO linked to its tagging portion with a quencher molecule (BHQ-2), and a CTO linked to its capture portion with a reporter molecule (CAL Fluoro Red 610), and the sequences of the PTO and CTO were designed such that the duplex between the PTO (i.e., uncleaved PTO) and the CTO is in an associated form at the first detection temperature and in a dissociated form at the second detection temperature, and the extended duplex formed in response to the presence of NG is in an associated form at both the first and second detection temperatures (see Figure 8).

[0427]

[0375]

[0428] <Combination 2>

[0429] The first detection temperature for detecting a first signal change indicating the presence of the first target nucleic acid, CT, was set to 57° C., and the second detection temperature for detecting a second signal change indicating the presence of the second target nucleic acid, UP, was set to 85° C. Then, oligonucleotides for a composition for detecting the CT target nucleic acid and a composition for detecting the UP target nucleic acid were prepared as shown in Table 2 below.

[0376]

[0430] The composition for detecting the CT target nucleic acid comprises a primer pair, a PTO, and a CTO linked to a reporter molecule (CAL Fluoro Red 610) and a quencher molecule (BHQ-2), and the sequences of the PTO and CTO were designed so that the duplex between the PTO (i.e., uncleaved PTO) and the CTO is in a dissociated form at both the first and second detection temperatures, and the extended duplex formed in response to the presence of CT is in an associated form at the first detection temperature and in a dissociated form at the second detection temperature (see Figure 9).

[0377]

[0431] The composition for detecting a UP target nucleic acid comprises a primer pair, a reporter molecule (CAL Fluoro Red 610), a PTO linked to a first quencher molecule (BHQ-2), and a CQO linked to a second quencher molecule (BHQ-2), and the sequences of the PTO and CQO were designed such that a tag duplex between the PTO (i.e., uncleaved PTO) and the CQO is in an associated form at a first detection temperature and in a dissociated form at a second detection temperature. That is, the sequences of the PTO and CQO were designed such that the tag duplex between the PTO and the CQO is cleaved in response to the presence of the UP target nucleic acid to generate an activated tag duplex fragment, and the activated tag duplex fragment is in an associated form at the first detection temperature and in a dissociated form at a second detection temperature (see FIG. 9 ).

[0432]

[0378]

[0433] <Combination 3>

[0434] The first detection temperature for detecting a first signal change indicating the presence of the first target nucleic acid, CT, was set at 57° C., and the second detection temperature for detecting a second signal change indicating the presence of the second target nucleic acid, NG, was set at 72° C. Then, oligonucleotides for a composition for detecting the CT target nucleic acid and a composition for detecting the NG target nucleic acid were prepared as shown in Table 2 below.

[0379]

[0435] The composition for detecting the CT target nucleic acid comprises a primer pair, a PTO linked to its tagging portion with a quencher molecule (BHQ-2), and a CTO linked to its capture portion with a reporter molecule (CAL Fluoro Red 610), and the sequences of the PTO and CTO were designed such that the duplex between the PTO (i.e., uncleaved PTO) and the CTO is in a dissociated form at the first and second detection temperatures, and the extended duplex formed in response to the presence of CT is in an associated form at the first detection temperature and in a dissociated form at the second detection temperature (see Figure 10).

[0380]

[0436] The composition for detecting the NG target nucleic acid comprises a primer pair, a PTO linked to its tagging portion with a quencher molecule (BHQ-2), and a CTO linked to its capture portion with a reporter molecule (CAL Fluoro Red 610), and the sequences of the PTO and CTO were designed such that the duplex between the PTO (i.e., uncleaved PTO) and the CTO is in an associated form at the first detection temperature and in a dissociated form at the second detection temperature, and the extended duplex formed in response to the presence of NG is in an associated form at both the first and second detection temperatures (see Figure 10).

[0437]

[0381]

[0438] <Combination 4>

[0439] The first detection temperature for detecting a first signal change indicating the presence of the first target nucleic acid, NG, was set at 72° C., and the second detection temperature for detecting a second signal change indicating the presence of the second target nucleic acid, UP, was set at 95° C. Then, oligonucleotides for the composition for detecting the NG target nucleic acid and the composition for detecting the UP target nucleic acid were prepared as shown in Table 2 below.

[0382]

[0440] The composition for detecting the NG target nucleic acid comprises a primer pair, a PTO linked to a quencher molecule (BHQ-2) at its tagging portion, and a CTO linked to a reporter molecule (CAL Fluoro Red 610) at its capture portion, and the sequences of the PTO and CTO were designed so that the duplex between the PTO (i.e., uncleaved PTO) and the CTO is in a dissociated form at the first and second detection temperatures, and the extended duplex formed in response to the presence of NG is in an associated form at the first detection temperature and in a dissociated form at the second detection temperature (see Figure 11).

[0383]

[0441] The composition for detecting a UP target nucleic acid includes a primer pair, a reporter molecule (CAL Fluoro Red 610), and a PTO linked to a first quencher molecule (BHQ-2), and a CQO linked to a second quencher molecule (BHQ-2). The sequences of the PTO and CQO were designed so that a tag duplex between the PTO (i.e., uncleaved PTO) and the CQO is in an associated form at a first detection temperature and a dissociated form at a second detection temperature. That is, the tag duplex between the PTO and the CQO is cleaved in response to the presence of the UP target nucleic acid to generate an activated tag duplex fragment, and the activated tag duplex fragment is in an associated form at the first detection temperature and a dissociated form at a second detection temperature (see FIG. 11).

[0442]

[0384]

[0443] <Combination 5>

[0444] The first detection temperature for detecting a first signal change indicating the presence of the first target nucleic acid, CT, was set to 50° C., the second detection temperature for detecting a second signal change indicating the presence of the second target nucleic acid, NG, was set to 72° C., and the third detection temperature for detecting a third signal change indicating the presence of the third target nucleic acid, UP, was set to 95° C. Then, oligonucleotides for a composition for detecting the CT target nucleic acid, a composition for detecting the NG target nucleic acid, and a composition for detecting the UP target nucleic acid were prepared as shown in Table 2 below.

[0385]

[0445] The composition for detecting the CT target nucleic acid comprises a primer pair, a PTO, and a CTO linked to a reporter molecule (CAL Fluoro Red 610) and a quencher molecule (BHQ-2). The sequences of the PTO and CTO were designed so that the duplex between the PTO (i.e., uncleaved PTO) and the CTO is in a dissociated form at all of the first to third detection temperatures, and the extended duplex formed in response to the presence of CT is in an associated form at the first detection temperature and in a dissociated form at the second and third detection temperatures (see Figure 12).

[0386]

[0446] The composition for detecting the NG target nucleic acid comprises a primer pair, a PTO linked to a quencher molecule (BHQ-2) at its tagging portion, and a CTO linked to a reporter molecule (CAL Fluoro Red 610) at its capture portion, and the sequences of the PTO and CTO were designed such that the duplex between the PTO (i.e., uncleaved PTO) and the CTO is in an associated form at the first detection temperature and in a dissociated form at the second and third detection temperatures, and the extended duplex formed in response to the presence of NG is in an associated form at the first and second detection temperatures and in a dissociated form at the third detection temperature (see Figure 12).

[0387]

[0447] The composition for detecting a UP target nucleic acid includes a primer pair, a reporter molecule (CAL Fluoro Red 610), and a PTO linked to a first quencher molecule (BHQ-2), and a CQO linked to a second quencher molecule (BHQ-2). The sequences of the PTO and CQO were designed so that a tag duplex between the PTO (i.e., uncleaved PTO) and the CQO is in an associated form at the first and second detection temperatures and in a dissociated form at the third detection temperature. That is, the sequences of the PTO and CQO were designed so that the tag duplex between the PTO and the CQO is cleaved in response to the presence of the UP target nucleic acid to generate an activated tag duplex fragment, and the activated tag duplex fragment is in an associated form at the first and second detection temperatures and in a dissociated form at the third detection temperature (see FIG. 12).

[0448]

[0388]

[0449] The 3' ends of the PTO and CTO in PTOCE base 1 were blocked by spacer C3 to prevent extension reaction by DNA polymerase.

[0450]

[0451]

[0389] [Table 2] TIFF0007737474000003.tif212149 TIFF0007737474000004.tif109149 TIFF0007737474000005.tif167149

[0390]

[0452]

[0453]

[0454] Example 2: Preparation of the reaction mixture

[0455] Using the oligonucleotides of Combinations 1 to 5 prepared in Example 1 above, reaction mixtures were prepared as follows: Taq DNA polymerase with 5' nuclease activity was included in the reaction mixture for extension of the forward and reverse primers and for cleavage of the oligonucleotides.

[0456]

[0391]

[0457] <Reaction mixture of combination 1>

[0458] The target nucleic acids (Tube 1: 500 pg of CT genomic DNA; Tube 2: 500 pg of NG genomic DNA; Tube 3: a mixture of 500 pg of CT genomic DNA and 500 pg of NG genomic DNA; and Tube 4: distilled water (negative control)) were mixed with 5 pmoles of forward primer (SEQ ID NO: 1), 5 pmoles of reverse primer (SEQ ID NO: 2), 3 pmoles of PTO (SEQ ID NO: 3), and 1 pmole of CTO (SEQ ID NO: 4) as oligonucleotides for the CT target nucleic acid; and 5 pmoles of forward primer (SEQ ID NO: 5), 5 pmoles of reverse primer (SEQ ID NO: 6), 1 pmole of PTO (SEQ ID NO: 7), and 1 pmole of CTO (SEQ ID NO: 8) as oligonucleotides for the NG target nucleic acid. The oligonucleotides were then combined with 5 μL of 4x master mix (final, 200 μM dNTPs, 2 mM MgCl2, 2 U of Taq DNA polymerase) (Enzynomics, Korea) to prepare a reaction mixture in a final volume of 20 μL.

[0459]

[0392]

[0460] <Reaction mixture of combination 2>

[0461] The target nucleic acids (Tube 1: 50 pg of CT genomic DNA; Tube 2: 50 pg of UP genomic DNA; Tube 3: a mixture of 50 pg of CT genomic DNA and 50 pg of UP genomic DNA; and Tube 4: distilled water (negative control)) were mixed with 5 pmoles of forward primer (SEQ ID NO: 1), 5 pmoles of reverse primer (SEQ ID NO: 2), 3 pmoles of PTO (SEQ ID NO: 3), and 1 pmole of CTO (SEQ ID NO: 4) as oligonucleotides for the CT target nucleic acid; and 5 pmoles of forward primer (SEQ ID NO: 9), 5 pmoles of reverse primer (SEQ ID NO: 10), 1 pmole of PTO (SEQ ID NO: 11), and 5 pmoles of CQO (SEQ ID NO: 12) as oligonucleotides for the UP target nucleic acid. Then, the mixture was combined with 5 μL of 4× master mix (final: 200 μM dNTPs, 2 mM MgCl2, 2 U of Taq DNA polymerase) (Enzynomics, Korea) to prepare a reaction mixture in a final volume of 20 μL.

[0462]

[0393]

[0463] <Reaction mixture of combination 3>

[0464] The target nucleic acids (Tube 1: 500 pg of CT genomic DNA; Tube 2: 500 pg of NG genomic DNA; Tube 3: a mixture of 500 pg of CT genomic DNA and 500 pg of NG genomic DNA; and Tube 4: distilled water (negative control)) were mixed with 5 pmoles of forward primer (SEQ ID NO: 1), 5 pmoles of reverse primer (SEQ ID NO: 2), 5 pmoles of PTO (SEQ ID NO: 13), and 2 pmoles of CTO (SEQ ID NO: 14) as oligonucleotides for the CT target nucleic acid; and 5 pmoles of forward primer (SEQ ID NO: 5), 5 pmoles of reverse primer (SEQ ID NO: 6), 3 pmoles of PTO (SEQ ID NO: 7), and 1 pmole of CTO (SEQ ID NO: 8) as oligonucleotides for the NG target nucleic acid. Then, the mixture was combined with 5 μL of 4× master mix (final: 200 μM dNTPs, 2 mM MgCl2, 2 U of Taq DNA polymerase) (Enzynomics, Korea) to prepare a reaction mixture in a final volume of 20 μL.

[0465]

[0394]

[0466] <Reaction mixture of combination 4>

[0467] The target nucleic acids (Tube 1: 500 pg of NG genomic DNA; Tube 2: 500 pg of UP genomic DNA; Tube 3: a mixture of 500 pg of NG genomic DNA and 500 pg of UP genomic DNA, and Tube 4: distilled water (negative control)) were mixed with 5 pmoles of forward primer (SEQ ID NO: 5), 5 pmoles of reverse primer (SEQ ID NO: 6), 1 pmole of PTO (SEQ ID NO: 7), and 1 pmole of CTO (SEQ ID NO: 8) as oligonucleotides for the NG target nucleic acid; and 5 pmoles of forward primer (SEQ ID NO: 9), 5 pmoles of reverse primer (SEQ ID NO: 10), 0.5 pmoles of PTO (SEQ ID NO: 11), and 3 pmoles of CQO (SEQ ID NO: 12) as oligonucleotides for the UP target nucleic acid, and then 5 μL of 4× master mix (final: 200 μM dNTPs, 2 mM MgCl, 2 U of Taq DNA polymerase) (Enzynomics, Korea) to prepare a reaction mixture in a final volume of 20 μL.

[0468]

[0395]

[0469] <Reaction mixture of combination 5>

[0470] The target nucleic acid (Tube 1: 50 pg of CT genomic DNA; Tube 2: 50 pg of NG genomic DNA; Tube 3: 50 pg of UP genomic DNA; Tube 4: a mixture of 50 pg of CT genomic DNA and 50 pg of NG genomic DNA; Tube 5: a mixture of 50 pg of CT genomic DNA and 50 pg of UP genomic DNA; Tube 6: a mixture of 50 pg of NG genomic DNA and 50 pg of UP genomic DNA; Tube 7: a mixture of 50 pg of CT genomic DNA, 50 pg of NG genomic DNA, and 50 pg of UP genomic DNA; Tube 8: distilled water (negative control)) was mixed with the oligonucleotides for the CT target nucleic acid, including 5 pmoles of forward primer (SEQ ID NO: 1), 5 pmoles of reverse primer (SEQ ID NO: 2), 3 pmoles of ribosomal RNA, and 5 pmoles of ribosomal RNA. 1 pmole of PTO (SEQ ID NO: 3) and 1 pmole of CTO (SEQ ID NO: 4); 5 pmole of forward primer (SEQ ID NO: 5), 5 pmole of reverse primer (SEQ ID NO: 6), 1 pmole of PTO (SEQ ID NO: 7), and 1 pmole of CTO (SEQ ID NO: 8) as oligonucleotides for the NG target nucleic acid; and 5 pmole of forward primer (SEQ ID NO: 9), 5 pmole of reverse primer (SEQ ID NO: 10), 0.5 pmole of PTO (SEQ ID NO: 11), and 3 pmole of CQO (SEQ ID NO: 12) as oligonucleotides for the UP target nucleic acid were mixed and then combined with 5 μL of 4x master mix (final: 200 μM dNTPs, 2 mM MgCl, 2 U of Taq DNA polymerase) (Enzynomics, Korea) to prepare a reaction mixture in a final volume of 20 μL.

[0471]

[0396]

[0472] Example 3: Real-time PCR

[0473] Next, real-time PCR was carried out using the reaction mixtures of Combination 1 to Combination 5 prepared in Example 2.

[0474]

[0397]

[0475] <Real-time PCR using the reaction mixture of Combination 1>

[0476] The test tube containing the reaction mixture of Combination 1 was placed in a real-time thermal cycler (CFX96 Real-Time Cycler, Bio-Rad), incubated at 50°C for 4 minutes, denatured at 95°C for 15 minutes, and then subjected to 50 cycles of 57°C for 15 seconds, 50°C for 1 second, 72°C for 10 seconds, 85°C for 1 second, and 95°C for 10 seconds. Signal detection was carried out at 50°C (first detection temperature) and 72°C (second detection temperature) in each cycle.

[0398]

[0477] The signal change was measured by comparing the presence and absence of the target nucleic acid. In this regard, based on the signal value of the negative control (i.e., RFU is 0), the signal was considered to have changed if the signal value at the first detection temperature was greater than or equal to a set threshold of 300, and the signal was considered to have changed if the signal value at the second detection temperature was less than or equal to a set threshold of -300.

[0399]

[0478] As a result, as shown in Figure 13, a signal change was identified at the first detection temperature in test tube 1 containing the CT target nucleic acid, a signal change was identified at the second detection temperature in test tube 2 containing the NG target nucleic acid, and a signal change was identified at the first and second detection temperatures in test tube 3 containing the CT and NG target nucleic acids, respectively.

[0400]

[0479] Meanwhile, the negative control, test tube 4, provided a constant signal during the amplification reaction at both the first and second detection temperatures, i.e., no signal change was identified.

[0480]

[0481]

[0401] [Table 3]

[0402]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488] As also shown in Table 3, there was no difference between the Ct (cycle threshold) value (28.53) indicating the presence of CT in test tube 1, which contained only the CT target nucleic acid, and the Ct value (28.27) indicating the presence of CT in test tube 3, which contained both the CT and NG target nucleic acids, and there was no difference between the Ct value (29.59) indicating the presence of NG in test tube 2, which contained only the NG target nucleic acid, and the Ct value (29.28) indicating the presence of NG in test tube 3, which contained both the CT and NG target nucleic acids.

[0403]

[0489] These results indicate that at each detection temperature, only a signal change dependent on the presence of the corresponding target nucleic acid is provided, and no signal change dependent on the presence of other target nucleic acids is provided. That is, these results indicate that multiple different target nucleic acids can be independently detected at their corresponding detection temperatures. Therefore, the method disclosed herein has the advantage that the presence of a specific target nucleic acid can be determined solely by the signal change detected at a specific detection temperature, and there is no need to consider signal changes detected at detection temperatures other than the specific detection temperature (e.g., detection temperatures other than the specific detection temperature, i.e., detection temperatures that provide signal changes indicating the presence of other target nucleic acids).

[0490]

[0404]

[0491] <Real-time PCR using the reaction mixture of Combination 2>

[0492] The test tube containing the reaction mixture of Combination 2 was placed in a real-time thermal cycler (CFX96 Real-Time Cycler, Bio-Rad), incubated at 50°C for 4 minutes, denatured at 95°C for 15 minutes, and then subjected to 50 cycles of 57°C for 15 seconds, 50°C for 1 second, 72°C for 10 seconds, 85°C for 1 second, and 95°C for 10 seconds. Signal detection was carried out at 57°C (first detection temperature) and 85°C (second detection temperature) in each cycle.

[0405]

[0493] The signal change was measured by comparing the presence and absence of the target nucleic acid, and in this regard, based on the signal value of the negative control (i.e., RFU is 0), the signal was considered to have changed if the signal value at the first detection temperature and the second detection temperature was greater than or equal to a set threshold of 300.

[0406]

[0494] As a result, as shown in Figure 14, a signal change was identified at the first detection temperature in test tube 1 containing the CT target nucleic acid, a signal change was identified at the second detection temperature in test tube 2 containing the UP target nucleic acid, and a signal change was identified at the first and second detection temperatures in test tube 3 containing the CT and UP target nucleic acids, respectively.

[0407]

[0495] Meanwhile, the negative control test tube 4 showed a constant signal during the amplification reaction at both the first and second detection temperatures, i.e., no signal change was detected.

[0496]

[0497]

[0408] [Table 4]

[0409]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504] As also shown in Table 4, there was no difference between the Ct (cycle threshold) value (34.29) indicating the presence of CT in test tube 1, which contained only the CT target nucleic acid, and the Ct value (33.83) indicating the presence of CT in test tube 3, which contained both the CT and UP target nucleic acids, and there was no difference between the Ct value (33.93) indicating the presence of UP in test tube 2, which contained only the UP target nucleic acid, and the Ct value (33.47) indicating the presence of UP in test tube 3, which contained both the CT and UP target nucleic acids.

[0410]

[0505] These results demonstrate that, as described above, multiple different target nucleic acids can be detected independently at their respective detection temperatures.

[0506]

[0411]

[0507] <Real-time PCR using the reaction mixture of Combination 3>

[0508] The test tube containing the reaction mixture of Combination 3 was placed in a real-time thermal cycler (CFX96 Real-Time Cycler, Bio-Rad), incubated at 50°C for 4 minutes, denatured at 95°C for 15 minutes, and then subjected to 50 cycles of the following reaction process: 57°C for 15 seconds, 50°C for 1 second, 72°C for 10 seconds, 85°C for 1 second, and 95°C for 10 seconds. Signal detection was performed at 57°C (first detection temperature) and 72°C (second detection temperature) in each cycle.

[0412]

[0509] The signal change was measured by comparing the presence and absence of the target nucleic acid, and in this regard, based on the signal value of the negative control (i.e., RFU is 0), the signal was considered to have changed if the signal value at the first detection temperature and the second detection temperature was less than or equal to a set threshold of −300.

[0413]

[0510] As a result, as shown in Figure 15, a signal change was identified at the first detection temperature in test tube 1 containing the CT target nucleic acid, a signal change was identified at the second detection temperature in test tube 2 containing the NG target nucleic acid, and a signal change was identified at the first and second detection temperatures in test tube 3 containing the CT and NG target nucleic acids, respectively.

[0414]

[0511] Meanwhile, the negative control test tube 4 showed a constant signal during the amplification reaction at both the first and second detection temperatures, i.e., no signal change was detected.

[0512]

[0513]

[0415] [Table 5]

[0416]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520] As also shown in Table 5 above, there was no difference between the Ct (cycle threshold) value (30.62) indicating the presence of CT in test tube 1 containing only the CT target nucleic acid and the Ct value (30.33) indicating the presence of CT in test tube 3 containing both the CT and NG target nucleic acids, and there was no difference between the Ct value (28.33) indicating the presence of NG in test tube 2 containing only the NG target nucleic acid and the Ct value (28.17) indicating the presence of NG in test tube 3 containing both the CT and NG target nucleic acids.

[0417]

[0521] These results demonstrate that, as described above, multiple different target nucleic acids can be detected independently at their respective detection temperatures.

[0522]

[0418]

[0523] <Real-time PCR using the reaction mixture of Combination 4>

[0524] The test tube containing the reaction mixture of Combination 4 was placed in a real-time thermal cycler (CFX96 Real-Time Cycler, Bio-Rad), incubated at 50°C for 4 minutes, denatured at 95°C for 15 minutes, and then subjected to 50 cycles of the following reaction process: 57°C for 15 seconds, 50°C for 1 second, 72°C for 10 seconds, 85°C for 1 second, and 95°C for 10 seconds. Signal detection was performed at 72°C (first detection temperature) and 95°C (second detection temperature) in each cycle.

[0419]

[0525] The signal change was measured by comparing the presence and absence of the target nucleic acid. In this regard, based on the signal value of the negative control (i.e., RFU is 0), the signal was considered to have changed if the signal value at the first detection temperature was less than or equal to the set threshold of -300, and the signal was considered to have changed if the signal value at the second detection temperature was greater than or equal to the set threshold of 300.

[0420]

[0526] As a result, as shown in Figure 16, a signal change was identified at the first detection temperature in test tube 1 containing the NG target nucleic acid, a signal change was identified at the second detection temperature in test tube 2 containing the UP target nucleic acid, and a signal change was identified at the first and second detection temperatures in test tube 3 containing the NG and UP target nucleic acids, respectively.

[0421]

[0527] Meanwhile, the negative control test tube 4 showed a constant signal during the amplification reaction at both the first and second detection temperatures, i.e., no signal change was detected.

[0528]

[0529]

[0422] [Table 6]

[0423]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536] As also shown in Table 6, there was no difference between the Ct (cycle threshold) value (29.62) indicating the presence of NG in tube 1 containing only the NG target nucleic acid and the Ct value (30.72) indicating the presence of NG in tube 3 containing both the NG and UP target nucleic acids, and there was no difference between the Ct value (33.42) indicating the presence of UP in tube 2 containing only the UP target nucleic acid and the Ct value (32.68) indicating the presence of UP in tube 3 containing both the NG and UP target nucleic acids.

[0424]

[0537] These results demonstrate that, as described above, multiple different target nucleic acid sequences can be detected independently at their respective detection temperatures.

[0538]

[0425]

[0539] <Real-time PCR using the reaction mixture of Combination 5>

[0540] The test tube containing the reaction mixture of Combination 5 was placed in a real-time thermal cycler (CFX96 Real-Time Cycler, Bio-Rad), incubated at 50°C for 4 minutes, denatured at 95°C for 15 minutes, and then subjected to 50 cycles of the following reaction process: 57°C for 15 seconds, 50°C for 1 second, 72°C for 10 seconds, 85°C for 1 second, and 95°C for 10 seconds. Signal detection was performed at 50°C (first detection temperature), 72°C (second detection temperature), and 95°C (third detection temperature) in each cycle.

[0426]

[0541] The signal change was measured by comparing the presence and absence of the target nucleic acid. In this regard, based on the signal value of the negative control (i.e., RFU is 0), the signal was considered to have changed if the signal value at the first detection temperature and the third detection temperature was greater than or equal to the set threshold of 300, and the signal was considered to have changed if the signal value at the second detection temperature was less than or equal to the set threshold of -300.

[0427]

[0542] As a result, as shown in Figures 17a and 17b, a signal change was identified at the first detection temperature in test tube 1 containing the CT target nucleic acid, a signal change was identified at the second detection temperature in test tube 2 containing the NG target nucleic acid, and a signal change was identified at the third detection temperature in test tube 3 containing the UP target nucleic acid.

[0428]

[0543] In test tubes 4 to 7, each containing at least two of CT, NG, and UP, signal changes were also identified at the corresponding detection temperatures depending on the target nucleic acid present in the test tube.

[0429]

[0544] Meanwhile, the negative control test tube 8 showed a constant signal at all of the first, second, and third detection temperatures during the amplification reaction, i.e., no signal change was detected.

[0545]

[0546]

[0430] [Table 7]

[0431]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557] As also shown in Table 7 above, there was no difference in Ct values ​​between the presence of a specific target nucleic acid alone and its presence in combination with other target nucleic acids (CT: 32.92 vs. 33.03 vs. 32.47 vs. 33.34; NG: 33.00 vs. 33.23 vs. 33.99 vs. 34.12; UP: 35.46 vs. 34.99 vs. 35.91 vs. 35.32).

[0432]

[0558] These results demonstrate that, as described above, multiple different target nucleic acids can be detected independently at their respective detection temperatures.

[0559]

[0433]

[0560] Meanwhile, as described above in the present application, the detection temperature can be selected within the signal change temperature range of the composition for detecting the target nucleic acid. Furthermore, the detection temperature may be selected taking into consideration the signal change temperature ranges of other compositions for detecting the target nucleic acid that are used together.

[0434]

[0561] To confirm that the detection temperature can be selected at various points within the signal change temperature range, the inventors set different detection temperatures for each CT in Combination 2 and Combination 5 using the same composition as for detecting CT. That is, the detection temperature for CT in Combination 2 was set to 57°C, and the detection temperature for CT in Combination 5 was set to 50°C. As a result, it was found that both Combination 2 and Combination 5 provided a signal change indicating the presence of the CT target nucleic acid only at their respective detection temperatures (i.e., 57°C and 50°C). In Combination 2 and Combination 5, the same composition as for detecting UP was also used, but the detection temperatures for UP were set differently. That is, the detection temperature for UP in Combination 2 was set to 85°C, and the detection temperature for UP in Combination 5 was set to 95°C. As a result, it was also found that a signal change indicating the presence of the UP target nucleic acid was only provided at the respective corresponding detection temperatures (85°C and 95°C).

[0435]

[0562] Additionally, as described above in this specification, the signal change temperature range and the signal constant temperature range can be controlled by adjusting the length and / or sequence of the duplex provided by the composition for detecting a target nucleic acid (e.g., by adjusting the Tm of the duplex).

[0436]

[0563] To confirm that the signal change temperature range and signal constant temperature range of a composition for detecting a target nucleic acid can be controlled by adjusting the Tm of the duplex, the inventors used an InterSC composition in combination 3 using the same signal generation mechanism for different target nucleic acids (CT and NG), and designed the oligonucleotide sequences so that CT provides a duplex with a relatively low Tm and NG provides a duplex with a relatively high Tm, thereby enabling detection to be performed at different detection temperatures (i.e., at the first detection temperature and the second detection temperature) even when using compositions with the same signal generation mechanism.

[0437]

[0564] As a result, as shown in the results of real-time PCR of combination 3, it was found that each detection temperature provided only a signal change indicative of its corresponding target nucleic acid.

[0438]

[0565] Therefore, according to the present disclosure, by applying at least one of the three types of signal generation mechanisms described above and adjusting the signal change temperature range so that each detection temperature provides only a signal indicative of the presence of a single target nucleic acid, multiple target nucleic acids can be detected in a real-time manner using a single type of label.

[0566]

[0439]

[0567] While preferred embodiments of the present invention have been described, it will be understood that variations and modifications thereof which fall within the true spirit of the invention may become apparent to those skilled in the art. Therefore, the scope of the present invention should be determined by the appended claims and their equivalents. Further embodiments are as follows. [Embodiment 1] 1. A method for detecting n target nucleic acids in a sample, comprising: (a) detecting signals at n detection temperatures while incubating a sample suspected of containing at least one of the n target nucleic acids with n compositions for detecting the target nucleic acid in a reaction vessel; n is an integer of 2 or greater; the incubation comprises a plurality of cycles, and the detection of the signal occurs during at least one of the plurality of cycles; each of the n compositions for detecting the target nucleic acid provides a signal change at a corresponding detection temperature among the n detection temperatures in the presence of a corresponding target nucleic acid, the signal change indicating the presence of the corresponding target nucleic acid; a composition for detecting an i-th target nucleic acid among the n compositions for detecting the target nucleic acids provides a signal change at an i-th detection temperature among the n detection temperatures, and provides a constant signal at other detection temperatures in the presence of the i-th target nucleic acid, the signal change indicating the presence of the i-th target nucleic acid; i represents an integer from 1 to n, and the i-th detected temperature is lower than the (i+1)-th detected temperature; and (b) determining the presence of the n target nucleic acids from the signals detected in step (a), wherein the presence of the i target nucleic acid is determined by a signal change detected at the i detection temperature. A method comprising: [Embodiment 2] 2. The method of embodiment 1, wherein, in a temperature range covering all of the n detection temperatures, the composition for detecting the i-th target nucleic acid has a signal change temperature range (SChTR) in which a signal changes in response to the presence of the i-th target nucleic acid, and a signal constant temperature range (SCoTR) in which a signal is constant even in the presence of the i-th target nucleic acid. [Embodiment 3] 3. The method of embodiment 2, wherein the composition for detecting the i target nucleic acid has one or two signal-constant temperature ranges. [Embodiment 4] The composition for detecting the i target nucleic acid comprises: (i) an under-signal change (UnderSC) composition characterized in that the signal change temperature range is lower than the signal constant temperature range; (ii) an over-signal change (OverSC) composition characterized in that the signal change temperature range is higher than the signal constant temperature range; and (iii) an inter-signal change (InterSC) composition characterized in that the signal change temperature range is higher than one of the two signal constant temperature ranges and lower than the other of the two signal constant temperature ranges; 3. The method of embodiment 2, wherein the method is any one of [Embodiment 5] 3. The method of embodiment 2, wherein the i detection temperature is selected within a signal change temperature range of a composition for detecting the i target nucleic acid, and the i detection temperature is not included in the signal change temperature range of a composition for detecting any other target nucleic acid. [Embodiment 6] 3. The method of embodiment 2, wherein the signal change temperature range of the composition for detecting the i-th target nucleic acid partially overlaps with the signal change temperature range of a composition for detecting a target nucleic acid having an adjacent detection temperature, but does not overlap with the signal change temperature range of a composition for detecting a target nucleic acid having a non-adjacent detection temperature. [Embodiment 7] The method of embodiment 4, wherein when n is 2, the composition for detecting the first target nucleic acid is an UnderSC composition or an InterSC composition, and the composition for detecting the second target nucleic acid is an InterSC composition or an OverSC composition. [Embodiment 8] The method of embodiment 4, wherein when n is 3 or greater, the composition for detecting the first target nucleic acid is an UnderSC composition or an InterSC composition, the composition for detecting the nth target nucleic acid is an InterSC composition or an OverSC composition, and each of the compositions for detecting target nucleic acids other than the first target nucleic acid and the nth target nucleic acid is an InterSC composition. [Embodiment 9] 2. The method of embodiment 1, wherein the composition for detecting the i target nucleic acid comprises a label that provides a signal dependent on the presence of the i target nucleic acid. [Embodiment 10] 10. The method of embodiment 9, wherein said label is linked to or incorporated into the oligonucleotide during said incubation. [Embodiment 11] 2. The method of embodiment 1, wherein the composition for detecting the i target nucleic acid provides a duplex that provides a signal change. [Embodiment 12] 10. The method of embodiment 9, wherein the composition for detecting the i target nucleic acid provides a duplex that provides a signal change, and when the duplex that provides the signal change is in an associated form, the composition for detecting the i target nucleic acid provides a signal from the label. [Embodiment 13] 10. The method of embodiment 9, wherein the composition for detecting the i target nucleic acid provides a duplex that provides a signal change, and when the duplex that provides the signal change is in a dissociated form, the composition for detecting the i target nucleic acid provides a signal from the label. [Embodiment 14] 12. The method of embodiment 11, wherein the duplex that provides the signal change was originally included in the composition for detecting the i target nucleic acid. [Embodiment 15] 15. The method of embodiment 14, wherein the duplex that provides the signal change is generated by hybridization of a label-linked oligonucleotide with an oligonucleotide that is hybridizable to the label-linked oligonucleotide. [Embodiment 16] 12. The method of embodiment 11, wherein the duplex that provides the signal change is generated during incubation. [Embodiment 17] 17. The method of embodiment 16, wherein the duplex that provides the signal change is generated by hybridization of a label-linked oligonucleotide with the target nucleic acid. [Embodiment 18] 17. The method of embodiment 16, wherein the duplex that provides the signal change is generated by a cleavage reaction that is dependent on the presence of the target nucleic acid. [Embodiment 19] 19. The method of embodiment 18, wherein the composition for detecting the target nucleic acid comprises a tagging oligonucleotide that hybridizes to the target nucleic acid, and the cleavage reaction dependent on the presence of the target nucleic acid comprises cleavage of the tagging oligonucleotide. [Embodiment 20] 12. The method of embodiment 11, wherein the duplex that provides the signal change is a single type of duplex or multiple types of duplex. [Embodiment 21] 21. The method of embodiment 20, wherein when the duplex that provides the signal change is a single type of duplex, the amount of the single type of duplex changes in response to the presence of the target nucleic acid, thereby changing the signal. [Embodiment 22] 21. The method of embodiment 20, wherein when the duplex that provides the signal change is a plurality of types of duplex, the ratio of the amounts between the plurality of types of duplex changes in response to the presence of the target nucleic acid, thereby changing the signal. [Embodiment 23] 21. The method of embodiment 20, wherein when the duplex is a plurality of types of duplex, the Tm values ​​of the duplexes are different from each other. [Embodiment 24] 21. The method of embodiment 20, wherein at least two of said multiple types of duplexes comprise the same single strand. [Embodiment 25] 12. The method of embodiment 11, wherein the duplex that provides the signal change comprises a label. [Embodiment 26] 6. The method of embodiment 5, wherein the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and the signal change temperature range of the composition for detecting the i-th target nucleic acid is determined depending on the length and / or sequence of the duplex. [Embodiment 27] 2. The method of embodiment 1, wherein said detecting a signal occurs in at least two of said plurality of cycles. [Embodiment 28] 28. The method of embodiment 27, wherein said signal change is measured using signals detected in said at least two of said plurality of cycles. [Embodiment 29] 2. The method of embodiment 1, wherein the signal change at the i detection temperature is measured using the signal detected in the at least one of the plurality of cycles and a reference signal value. [Embodiment 30] 30. The method of embodiment 29, wherein said reference signal value is obtained from a reaction in the absence of said i target nucleic acid. [Embodiment 31] 2. The method of embodiment 1, wherein the detection of the signal at each of the n detection temperatures is performed using a single type of detector. [Embodiment 32] 32. The method of embodiment 31, wherein the signals detected at said n detection temperatures are indistinguishable from one another by said single type of detector. [Embodiment 33] 2. The method of embodiment 1, wherein said incubation comprises a nucleic acid amplification reaction. [Embodiment 34] 34. The method of embodiment 33, wherein the nucleic acid amplification reaction is a polymerase chain reaction (PCR). [Embodiment 35] 1. A method for detecting two target nucleic acids in a sample, comprising: (a) detecting signals at a first detection temperature and a second detection temperature while incubating a sample suspected of containing at least one of the two target nucleic acids in a reaction vessel with a composition for detecting the first target nucleic acid and a composition for detecting the second target nucleic acid; the incubation comprises a plurality of cycles, and the detection of the signal occurs during at least one of the plurality of cycles; the composition for detecting the first target nucleic acid provides a signal change at the first detection temperature in the presence of the first target nucleic acid and a constant signal at the second detection temperature, the signal change indicating the presence of the first target nucleic acid; the composition for detecting the second target nucleic acid provides a signal change at the second detection temperature in the presence of the second target nucleic acid and a constant signal at the first detection temperature, the signal change indicating the presence of the second target nucleic acid; the first detected temperature is lower than the second detected temperature; and (b) determining the presence of the two target nucleic acids from the signals detected in step (a), wherein the presence of the first target nucleic acid is determined by a signal change detected at the first detection temperature and the presence of the second target nucleic acid is determined by a signal change detected at the second detection temperature. A method comprising: [Embodiment 36] 1. A method for detecting three target nucleic acids in a sample, comprising: (a) detecting signals at a first detection temperature, a second detection temperature, and a third detection temperature while incubating a sample suspected of containing at least one of the three target nucleic acids in a reaction vessel with a composition for detecting a first target nucleic acid, a composition for detecting a second target nucleic acid, and a composition for detecting a third target nucleic acid; the incubation comprises a plurality of cycles, and the detection of the signal occurs during at least one of the plurality of cycles; the composition for detecting the first target nucleic acid provides a signal change at the first detection temperature in the presence of the first target nucleic acid and a constant signal at the second detection temperature and the third detection temperature, the signal change indicating the presence of the first target nucleic acid; the composition for detecting the second target nucleic acid provides a signal change at the second detection temperature in the presence of the second target nucleic acid and a constant signal at the first detection temperature and the third detection temperature, the signal change indicating the presence of the second target nucleic acid; the composition for detecting the third target nucleic acid provides a signal change at the third detection temperature in the presence of the third target nucleic acid and a constant signal at the first detection temperature and the second detection temperature, the signal change indicating the presence of the third target nucleic acid; the first detected temperature is lower than the second detected temperature, and the second detected temperature is lower than the third detected temperature; and (b) determining the presence of the three target nucleic acids from the signals detected in step (a), wherein the presence of the first target nucleic acid is determined by a signal change detected at the first detection temperature, the presence of the second target nucleic acid is determined by a signal change detected at the second detection temperature, and the presence of the third target nucleic acid is determined by a signal change detected at the third detection temperature. A method comprising: [Embodiment 37] 1. A kit comprising n compositions for detecting n target nucleic acids in a sample, n is an integer of 2 or greater; each of the n compositions for detecting the n target nucleic acids provides a signal change at a corresponding detection temperature among the n detection temperatures, the signal change indicating the presence of the corresponding target nucleic acid; a composition for detecting an ith target nucleic acid among the n target nucleic acids, which, in the presence of the ith target nucleic acid, provides a signal change at the ith detection temperature among the n detection temperatures and provides a constant signal at other detection temperatures; The letter i represents an integer of 1 to n, and the i-th detected temperature is lower than the (i+1)-th detected temperature. [Embodiment 38] 38. The kit of embodiment 37, wherein, in a temperature range covering all of the n detection temperatures, the composition for detecting the i-th target nucleic acid has a signal change temperature range (SChTR) in which the signal changes in response to the presence of the i-th target nucleic acid, and a signal constant temperature range (SCoTR) in which the signal is constant even in the presence of the i-th target nucleic acid. [Embodiment 39] 39. The kit of embodiment 38, wherein the composition for detecting the i target nucleic acid has one or two signal-constant temperature ranges. [Embodiment 40] The composition for detecting the i target nucleic acid comprises: (i) an under-signal change (UnderSC) composition characterized in that the signal change temperature range is lower than the signal constant temperature range; (ii) an over-signal change (OverSC) composition characterized in that the signal change temperature range is higher than the signal constant temperature range; and (iii) an inter-signal change (InterSC) composition characterized in that the signal change temperature range is higher than one of the two signal constant temperature ranges and lower than the other of the two signal constant temperature ranges; 39. The kit of embodiment 38, wherein the kit is any one of: [Embodiment 41] 39. The kit of embodiment 38, wherein the i-th detection temperature is selected within the signal change temperature range of a composition for detecting the i-th target nucleic acid, and the i-th detection temperature is not included in the signal change temperature range of a composition for detecting any other target nucleic acid. [Embodiment 42] 39. The kit of embodiment 38, wherein the signal change temperature range of the composition for detecting the i-th target nucleic acid partially overlaps with the signal change temperature range of a composition for detecting a target nucleic acid having an adjacent detection temperature, but does not overlap with the signal change temperature range of a composition for detecting a target nucleic acid having a non-adjacent detection temperature. [Embodiment 43] The kit of embodiment 40, wherein when n is 2, the composition for detecting the first target nucleic acid is an UnderSC composition or an InterSC composition, and the composition for detecting the second target nucleic acid is an InterSC composition or an OverSC composition. [Embodiment 44] The kit of embodiment 40, wherein when n is 3 or greater, the composition for detecting the first target nucleic acid is an UnderSC composition or an InterSC composition, the composition for detecting the nth target nucleic acid is an InterSC composition or an OverSC composition, and each of the compositions for detecting target nucleic acids other than the first target nucleic acid and the nth target nucleic acid is an InterSC composition. [Embodiment 45] 38. The kit of embodiment 37, wherein the composition for detecting the i target nucleic acid comprises a label that provides a signal dependent on the presence of the i target nucleic acid. [Embodiment 46] 46. ​​The kit of embodiment 45, wherein the label is linked to or incorporated into the oligonucleotide during incubation of the kit and the sample. [Embodiment 47] 38. The kit of embodiment 37, wherein the composition for detecting the i target nucleic acid provides a duplex that provides a signal change. [Embodiment 48] 46. ​​The kit of embodiment 45, wherein the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and when the duplex that provides the signal change is in an associated form, the composition for detecting the i-th target nucleic acid provides a signal from the label. [Embodiment 49] 46. ​​The kit of embodiment 45, wherein the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and when the duplex that provides the signal change is in a dissociated form, the composition for detecting the i-th target nucleic acid provides a signal from the label. [Embodiment 50] 39. The kit of embodiment 38, wherein the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and the signal change temperature range of the composition for detecting the i-th target nucleic acid is determined according to the length and / or sequence of the duplex. [Embodiment 51] 38. The kit of embodiment 37, wherein the signals provided by the n compositions for detecting the n target nucleic acids are indistinguishable from one another by a single type of detector.

Claims

1. 1. A method for detecting n target nucleic acids in a sample, comprising: (a) detecting signals at n detection temperatures while incubating a sample suspected of containing at least one of the n target nucleic acids with n compositions for detecting the target nucleic acid in a reaction vessel; n is an integer of 2 or greater; the incubation comprises a plurality of cycles, and the detection of the signal occurs during at least one of the plurality of cycles; each of the n compositions for detecting the target nucleic acid provides a signal change at a corresponding detection temperature among the n detection temperatures in the presence of a corresponding target nucleic acid, the signal change indicating the presence of the corresponding target nucleic acid; a composition for detecting an i-th target nucleic acid among the n compositions for detecting the target nucleic acids provides a signal change at an i-th detection temperature among the n detection temperatures, and provides a constant signal at other detection temperatures in the presence of the i-th target nucleic acid, the signal change indicating the presence of the i-th target nucleic acid; i represents an integer from 1 to n, and the i-th detected temperature is lower than the (i+1)-th detected temperature; and (b) determining the presence of the n target nucleic acids from the signals detected in step (a), wherein the presence of the i target nucleic acid is determined by a signal change detected at the i detection temperature. A method comprising:

2. 2. The method of claim 1, wherein, in a temperature range covering all of the n detection temperatures, the composition for detecting the i-th target nucleic acid has a signal change temperature range (SChTR) in which the signal changes in response to the presence of the i-th target nucleic acid, and one or two signal constant temperature ranges (SCoTR) in which the signal is constant even in the presence of the i-th target nucleic acid.

3. The composition for detecting the i-th target nucleic acid comprises: (i) an under-signal change (Under-SC) composition characterized in that the signal change temperature range is lower than the signal constant temperature range; (ii) an over-signal change (OverSC) composition characterized in that the signal change temperature range is higher than the signal constant temperature range; and (iii) An inter-signal change (InterSC) composition characterized in that the signal change temperature range is higher than one of the two signal constant temperature ranges and lower than the other of the two signal constant temperature ranges.

3. The method of claim 2, wherein the method is any one of:

4. The method of claim 2, wherein the i-th detection temperature is selected within the signal change temperature range of a composition for detecting the i-th target nucleic acid, and the i-th detection temperature is not included in the signal change temperature range of a composition for detecting another target nucleic acid.

5. 3. The method of claim 2, wherein the signal change temperature range of the composition for detecting the i-th target nucleic acid partially overlaps with the signal change temperature range of a composition for detecting a target nucleic acid having an adjacent detection temperature, but does not overlap with the signal change temperature range of a composition for detecting a target nucleic acid having a non-adjacent detection temperature.

6. (i) when n is 2, the composition for detecting a first target nucleic acid is an Under-SC composition or an Inter-SC composition, and the composition for detecting a second target nucleic acid is an Inter-SC composition or an Over-SC composition; or (ii) when n is 3 or greater, the composition for detecting a first target nucleic acid is an Under-SC composition or an Inter-SC composition, the composition for detecting an nth target nucleic acid is an Inter-SC composition or an Over-SC composition, and each of the compositions for detecting target nucleic acids other than the first target nucleic acid and the nth target nucleic acid is an Inter-SC composition; The method of claim 3.

7. 2. The method of claim 1, wherein the composition for detecting the i-th target nucleic acid comprises a label that provides a signal dependent on the presence of the i-th target nucleic acid.

8. 8. The method of claim 7, wherein the label is linked to or incorporated into the oligonucleotide during the incubation.

9. 2. The method of claim 1, wherein the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and the duplex that provides the signal change comprises a label.

10. 8. The method of claim 7, wherein the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and wherein the composition for detecting the i-th target nucleic acid provides a signal from the label when the duplex that provides the signal change is in an associated or dissociated form.

11. The method of claim 9, wherein the duplex that provides the signal change was originally included in the composition for detecting the i-th target nucleic acid.

12. The method of claim 11, wherein the duplex that provides the signal change is generated by hybridization of a label-linked oligonucleotide with an oligonucleotide that is hybridizable to the label-linked oligonucleotide.

13. The method of claim 9, wherein the duplex that provides the signal change is generated during incubation.

14. the duplex that provides the signal change is (i) produced by hybridization of a label-linked oligonucleotide with the target nucleic acid; or (ii) produced by a cleavage reaction dependent on the presence of the target nucleic acid; The method of claim 13.

15. 15. The method of claim 14, wherein the composition for detecting the target nucleic acid comprises a tagging oligonucleotide that hybridizes to the target nucleic acid, and the cleavage reaction dependent on the presence of the target nucleic acid comprises cleavage of the tagging oligonucleotide.

16. 10. The method of claim 9, wherein the duplex that provides the signal change is a single type of duplex or multiple types of duplex.

17. (i) if the duplex that provides the signal change is a single type of duplex, the amount of the single type of duplex changes in response to the presence of the target nucleic acid, thereby changing the signal; or (ii) when the duplex that provides the signal change is a plurality of types of duplex, the ratio of the amounts of the plurality of types of duplex changes in response to the presence of the target nucleic acid, thereby changing the signal; 17. The method of claim 16.

18. 17. The method of claim 16, wherein when the duplex is a plurality of types of duplex, the Tm values ​​of the duplexes are different from each other.

19. The method of claim 4, wherein the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and the signal change temperature range of the composition for detecting the i-th target nucleic acid is determined depending on the length and / or sequence of the duplex.

20. The method of claim 1 , wherein the detection of the signal occurs in at least two of the plurality of cycles.

21. 21. The method of claim 20, wherein the signal change is measured using signals detected in the at least two of the plurality of cycles.

22. 2. The method of claim 1, wherein the signal change at the i detection temperature is measured using the signal detected in the at least one of the plurality of cycles and a reference signal value.

23. 23. The method of claim 22, wherein the reference signal value is obtained from a reaction in the absence of the i target nucleic acid.

24. The method of claim 1 , wherein the detection of the signal at each of the n detection temperatures is performed using a single type of detector.

25. 25. The method of claim 24, wherein the signals detected at the n detection temperatures are indistinguishable from one another by the single type of detector.

26. The method of claim 1 , wherein the incubation comprises a nucleic acid amplification reaction.

27. 1. A method for detecting two target nucleic acids in a sample, comprising: (a) detecting signals at a first detection temperature and a second detection temperature while incubating a sample suspected of containing at least one of the two target nucleic acids with a composition for detecting a first target nucleic acid and a composition for detecting a second target nucleic acid in a reaction vessel; the incubation comprises a plurality of cycles, and the detection of the signal occurs during at least one of the plurality of cycles; the composition for detecting the first target nucleic acid provides a signal change at the first detection temperature in the presence of the first target nucleic acid and a constant signal at the second detection temperature, the signal change indicating the presence of the first target nucleic acid; the composition for detecting the second target nucleic acid provides a signal change at the second detection temperature in the presence of the second target nucleic acid and a constant signal at the first detection temperature, the signal change indicating the presence of the second target nucleic acid; the first detected temperature is lower than the second detected temperature; and (b) determining the presence of the two target nucleic acids from the signals detected in step (a), wherein the presence of the first target nucleic acid is determined by a signal change detected at the first detection temperature and the presence of the second target nucleic acid is determined by a signal change detected at the second detection temperature. A method comprising:

28. 1. A method for detecting three target nucleic acids in a sample, comprising: (a) detecting signals at a first detection temperature, a second detection temperature, and a third detection temperature while incubating a sample suspected of containing at least one of the three target nucleic acids in a reaction vessel with a composition for detecting a first target nucleic acid, a composition for detecting a second target nucleic acid, and a composition for detecting a third target nucleic acid; the incubation comprises a plurality of cycles, and the detection of the signal occurs during at least one of the plurality of cycles; the composition for detecting the first target nucleic acid provides a signal change at the first detection temperature in the presence of the first target nucleic acid and a constant signal at the second detection temperature and the third detection temperature, the signal change indicating the presence of the first target nucleic acid; the composition for detecting the second target nucleic acid provides a signal change at the second detection temperature in the presence of the second target nucleic acid and a constant signal at the first detection temperature and the third detection temperature, the signal change indicating the presence of the second target nucleic acid; the composition for detecting the third target nucleic acid provides a signal change at the third detection temperature in the presence of the third target nucleic acid and a constant signal at the first detection temperature and the second detection temperature, the signal change indicating the presence of the third target nucleic acid; the first detected temperature is lower than the second detected temperature, and the second detected temperature is lower than the third detected temperature; and (b) determining the presence of the three target nucleic acids from the signals detected in step (a), wherein the presence of the first target nucleic acid is determined by a signal change detected at the first detection temperature, the presence of the second target nucleic acid is determined by a signal change detected at the second detection temperature, and the presence of the third target nucleic acid is determined by a signal change detected at the third detection temperature. A method comprising:

29. 1. A kit comprising n compositions for detecting n target nucleic acids in a sample, n is an integer of 2 or greater; each of the n compositions for detecting the n target nucleic acids provides a signal change at a corresponding detection temperature among the n detection temperatures, the signal change indicating the presence of the corresponding target nucleic acid; a composition for detecting an i-th target nucleic acid among the n target nucleic acids, which, in the presence of the i-th target nucleic acid, provides a signal change at the i-th detection temperature among the n detection temperatures and provides a constant signal at other detection temperatures; The kit, wherein i represents an integer from 1 to n, and the i-th detected temperature is lower than the (i+1)-th detected temperature.

30. 30. The kit of claim 29, wherein, in a temperature range covering all of the n detection temperatures, the composition for detecting the i-th target nucleic acid has a signal change temperature range (SChTR) in which the signal changes in response to the presence of the i-th target nucleic acid, and one or two signal constant temperature ranges (SCoTR) in which the signal is constant even in the presence of the i-th target nucleic acid.

31. The composition for detecting the i-th target nucleic acid comprises: (i) an under-signal change (Under-SC) composition characterized in that the signal change temperature range is lower than the signal constant temperature range; (ii) an over-signal change (OverSC) composition characterized in that the signal change temperature range is higher than the signal constant temperature range; and (iii) An inter-signal change (InterSC) composition characterized in that the signal change temperature range is higher than one of the two signal constant temperature ranges and lower than the other of the two signal constant temperature ranges.

31. The kit of claim 30, wherein the kit is any one of:

32. The kit of claim 30, wherein the i-th detection temperature is selected within the signal change temperature range of a composition for detecting the i-th target nucleic acid, and the i-th detection temperature is not included in the signal change temperature range of a composition for detecting any other target nucleic acid.

33. 31. The kit of claim 30, wherein the signal change temperature range of the composition for detecting the i-th target nucleic acid partially overlaps with the signal change temperature range of a composition for detecting a target nucleic acid having an adjacent detection temperature, but does not overlap with the signal change temperature range of a composition for detecting a target nucleic acid having a non-adjacent detection temperature.

34. (i) when n is 2, the composition for detecting a first target nucleic acid is an Under-SC composition or an Inter-SC composition, and the composition for detecting a second target nucleic acid is an Inter-SC composition or an Over-SC composition; or (ii) when n is 3 or greater, the composition for detecting a first target nucleic acid is an Under-SC composition or an Inter-SC composition, the composition for detecting an nth target nucleic acid is an Inter-SC composition or an Over-SC composition, and each of the compositions for detecting target nucleic acids other than the first target nucleic acid and the nth target nucleic acid is an Inter-SC composition; 32. The kit of claim 31.

35. 30. The kit of claim 29, wherein the composition for detecting the i target nucleic acid comprises a label that provides a signal dependent on the presence of the i target nucleic acid.

36. 36. The kit of claim 35, wherein the label is linked to or incorporated into the oligonucleotide during incubation of the kit and the sample.

37. 30. The kit of claim 29, wherein the composition for detecting the i target nucleic acid provides a duplex that provides a signal change.

38. 36. The kit of claim 35, wherein the composition for detecting the i target nucleic acid provides a duplex that provides a signal change, and wherein the composition for detecting the i target nucleic acid provides a signal from the label when the duplex that provides the signal change is in an associated or dissociated form.

39. The kit of claim 30, wherein the composition for detecting the i-th target nucleic acid provides a duplex that provides a signal change, and the signal change temperature range of the composition for detecting the i-th target nucleic acid is determined according to the length and / or sequence of the duplex.

40. 30. The kit of claim 29, wherein the signals provided by the n compositions for detecting the n target nucleic acids are indistinguishable from one another by a single type of detector.

Images