How to detect three target nucleic acids in a sample

Abstract

The present invention can detect three types of target nucleic acids in one reaction vessel in real time by using the same fluorescent label and three detection temperatures. Conventional techniques detect multiple target nucleic acid sequences by melting analysis after target amplification. In contrast, the present invention does not require melting analysis after target amplification, dramatically shortening the analysis time.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting three target nucleic acids in a sample. [Background technology]

[0002] For detecting target nucleic acids, real-time detection methods are widely used to detect target nucleic acids while monitoring target amplification in real time. Real-time detection methods generally use labeled probes or primers that specifically hybridize with target nucleic acids. Examples of methods using hybridization between labeled probes and target nucleic acids include the molecular beacon method (Tyagi et al., Nature Biotechnology v. 14 MARCH 1996) using a dual-labeled probe with a hairpin structure, the HyBeacon method (French DJ et al., Mol. Cell Probes, 15(6):363-374 (2001)), the hybridization probe method (Bernad et al., 147-148 Clin Chem 2000; 46) using two probes labeled as donor and acceptor, respectively, and the Lux method (U.S. Patent No. 7,537,886) using a single-labeled oligonucleotide. The TaqMan method (U.S. Pat. Nos. 5,210,015 and 5,538,848), which uses a dual-labeled probe and cleavage of the probe by the 5'-nuclease activity of DNA polymerase, has been widely used in the art. Examples of methods using labeled primers include the Sunrise primer method (Nazarenko et al., pp. 2516-2521, Nucleic Acids Research, 1997, v. 25 no. 12, and U.S. Pat. No. 6,117,635), the Scorpion primer method (Whitcombe et al., pp. 804-807, Nature Biotechnology, v. 17 AUGUST 1999, and U.S. Pat. No. 6,326,145), and the TSG primer method (WO2011-078441).

[0003] As alternative approaches, real-time detection methods that utilize mers formed depending on the presence of target nucleic acids have been proposed: Invader analysis (U.S. Patent Nos. 5,691,142, 6,358,691, and 6,194,149), PTO cleavage and extension (PTOCE) method (WO 2012 / 096523), PTO cleavage and extension-dependent signaling oligonucleotide hybridization (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization (PCE-SH) method (WO 2013 / 115442), and PTO cleavage and extension-dependent non-hybridization (PCE-NH) method (PCT / KR2013 / 012312).

[0004] The conventional real-time detection techniques described above can detect only one target nucleic acid using one label, and therefore the number of target nucleic acids that can be simultaneously detected in one reaction is limited by the number of available labels (e.g., five or less).

[0005] Although melting analysis can be used to detect multiple target nucleic acids using one label, melting analysis has serious drawbacks in that it requires a longer run time than real-time techniques and the design of probes with different Tm values ​​becomes more difficult as the number of target nucleic acids increases.

[0006] Thus, conventional real-time detection techniques or melting assays have limitations in detecting multiple target nucleic acids.

[0007] Therefore, a real-time detection method that can simultaneously detect multiple target nucleic acids despite using limited labeling in a single reaction is desirable.

[0008] Throughout this specification, various patents and publications are referenced, the citations of which appear in parentheses. In order to more clearly describe the level of skill in the art to which this invention pertains, the publications of such patents and publications are hereby incorporated by reference in their entireties. Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have attempted to develop a real-time amplification method for detecting three target nucleic acids using a single type of label in one reaction vessel, and as a result, have developed a method for distinguishing and detecting three target nucleic acids per fluorescent label.

[0010] It is therefore an object of the present invention to provide a method and kit for detecting three target nucleic acids in a sample.

[0011] It is yet another object of the present invention to provide a computer readable medium containing instructions for implementing a processor to perform a method for detecting three target nucleic acids in a sample.

[0012] It is yet another object of the present invention to provide an apparatus for detecting three target nucleic acids in a sample.

[0013] It is yet another object of the present invention to provide a computer program storable on a computer-readable recording medium that embodies a processor for carrying out a method for detecting three target nucleic acids in a sample.

[0014] Other objects and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying claims and drawings. [Means for solving the problem]

[0015] According to one aspect of the present invention, there is provided a method for detecting a target nucleic acid in a sample, comprising the steps of:

[0016] (a) incubating a sample in a single reaction vessel with a first set of oligonucleotides directed to a first target nucleic acid, a second set of oligonucleotides directed to a second target nucleic acid, and a third set of oligonucleotides directed to a third target nucleic acid;

[0017] Each of the oligonucleotide sets comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that serves to generate a signal in the presence of the target nucleic acid and that is bound to a fluorescent label, wherein the amplification oligonucleotide and the signaling oligonucleotide are the same or different, and each oligonucleotide set commonly comprises a single type of fluorescent label;

[0018] the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid;

[0019] (b) measuring signals at a first detection temperature, a second detection temperature, and a third detection temperature; the first detection temperature, the second detection temperature, and the third detection temperature are different from each other;

[0020] (c) determining the presence of each target nucleic acid based on signals measured at the first detection temperature, the second detection temperature, and the third detection temperature;

[0021] The presence of the first target nucleic acid is determined by the difference between the signals measured at the first, second and third detection temperatures, the presence of the second target nucleic acid is determined by the difference between the signals measured at the second and third detection temperatures, and the presence of the third target nucleic acid is determined by the signal measured at the third detection temperature.

[0022] In one embodiment, the difference may include a difference obtained by mathematically processing signals measured at each temperature.

[0023] In one embodiment, the presence of the first target nucleic acid and the second target nucleic acid is determined using the difference and a reference value, and the reference value may reflect a change in the signal provided by each oligonucleotide set in the presence of each target nucleic acid at two temperatures selected from the first detection temperature, the second detection temperature, and the third detection temperature.

[0024] In one embodiment, the reference value may be obtained by (i) incubating at least one of the first oligonucleotide set, the second oligonucleotide set, and the third oligonucleotide set with at least one of the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid in a reaction vessel different from the reaction vessel in step (a), (ii) measuring signals at at least two temperatures selected from the first detection temperature, the second detection temperature, and the third detection temperature, and then (iii) calculating the difference between the signals measured at the selected two temperatures.

[0025] In one embodiment, at least one of three reference values ​​is used in step (c), and the three reference values ​​may be a reference value reflecting a signal generated by the third oligonucleotide set at the second detection temperature in the presence of the third target nucleic acid and a change in the signal generated at the third detection temperature, a reference value reflecting a signal generated by the third oligonucleotide set at the first detection temperature in the presence of the third target nucleic acid and a change in the signal generated at the third detection temperature, and a reference value reflecting a signal generated by the second oligonucleotide set at the first detection temperature in the presence of the second target nucleic acid and a change in the signal generated at the second detection temperature.

[0026] In one embodiment, the extracted signal provided using the reference value and the measured signal can be used to determine the presence of at least one target nucleic acid.

[0027] In one embodiment, the presence of a first target nucleic acid can be determined from an extracted signal for the first target nucleic acid at a first detection temperature, and the presence of a second target nucleic acid can be determined from an extracted signal for the second target nucleic acid at a second detection temperature.

[0028] In one embodiment, the extracted signal for the second target nucleic acid at the second detection temperature is

[0029] A third oligonucleotide set may be provided with a reference value reflecting the change in signal generated at the second temperature in the presence of a third target nucleic acid and at a third detection temperature.

[0030] In one embodiment, the extracted signal for the second target nucleic acid at the second detection temperature may be obtained by subtracting the signal for the third target nucleic acid at the second detection temperature from the signal measured at the second detection temperature, and the signal for the third target nucleic acid at the second detection temperature may be obtained by processing the signal measured at the third detection temperature with the second detection temperature and a reference value for the third target nucleic acid at the third detection temperature.

[0031] In one embodiment, the extracted signal for the first target nucleic acid at the first detection temperature may be obtained by subtracting (i) the signal for the third target nucleic acid at the first detection temperature and (ii) the signal for the second target nucleic acid at the first detection temperature from the signal measured at the first detection temperature.

[0032] In one embodiment, the extracted signal for the first target nucleic acid at the first detection temperature is provided using at least one of three reference values, and the three reference values ​​may be a reference value reflecting the signal generated by the third oligonucleotide set at the second detection temperature in the presence of the third target nucleic acid and a change in the signal generated at the third detection temperature; a reference value reflecting the signal generated by the third oligonucleotide set at the first detection temperature in the presence of the third target nucleic acid and a change in the signal generated at the third detection temperature; and a reference value reflecting the signal generated by the second oligonucleotide set at the first detection temperature in the presence of the second target nucleic acid and a change in the signal generated at the second detection temperature.

[0033] In one embodiment, the signal for the third target nucleic acid at the first detection temperature may be obtained using a signal measured at the third detection temperature and a reference value for the third target nucleic acid at the first detection temperature and the third detection temperature, and the signal for the second target nucleic acid at the first detection temperature may be obtained using an extracted signal for the second target nucleic acid at the second detection temperature and a reference value for the second target nucleic acid at the first detection temperature and the second detection temperature.

[0034] In one embodiment, when it is determined that the second target nucleic acid is not present, the extracted signal for the first target nucleic acid at the first detection temperature is obtained by subtracting the signal for the third target nucleic acid at the first detection temperature from the signal measured at the first detection temperature, and the signal for the third target nucleic acid at the first detection temperature may be obtained by processing the signal measured at the third detection temperature with reference values ​​for the third target nucleic acid at the first detection temperature and the third detection temperature.

[0035] In one embodiment, the first, second, and third detected temperatures may be spaced apart by 5 to 25°C and may be equal to or less than 90°C.

[0036] In one embodiment, (i) the first detected temperature may be lower than the second detected temperature, and the second detected temperature may be lower than the third detected temperature, or (ii) the first detected temperature may be higher than the second detected temperature, and the second detected temperature may be higher than the third detected temperature.

[0037] In one embodiment, the first detection temperature may be selected from 55°C to 70°C, the second detection temperature may be selected from 65°C to 80°C, and the third detection temperature may be selected from 75°C to 90°C.

[0038] In one embodiment, the first detection temperature may be selected from 75°C to 90°C, the second detection temperature may be selected from 65°C to 80°C, and the third detection temperature may be selected from 55°C to 70°C.

[0039] In one embodiment, the fluorescent label contained in the oligonucleotide set may be selected from the group consisting of FAM or a substitute thereof, CAL Fluor Orange 560 or a substitute thereof, CAL Fluor Red 610 or a substitute thereof, Quasar 670 or a substitute thereof, and Quasar 705 or a substitute thereof.

[0040] In one embodiment, the highest detected temperature among the first, second, and third detected temperatures may be selected from the range of 75°C to 90°C.

[0041] In one embodiment, the highest detected temperature among the first, second, and third detected temperatures may be selected from the range of 80°C to 86°C.

[0042] The fluorescent label may be a fluorescent label having an excitation wavelength in the range of 620 nm to 710 nm.

[0043] In one embodiment, the fluorescent label may be a carbopyronin-based compound.

[0044] In one embodiment, the compound may be represented by Formula 1 below:

[0045] [ka]

[0046] In the chemical formula 1, R1, R2, R3, R5, R6, R7, R8, R9, R 10 and R 11 are, independently of each other, hydrogen, C1 to C 10 Alkyl, C3-C7 cycloalkyl, C2-C 10 Alkenyl, C6-C 10 monocyclic or bicyclic aryl or heteroaryl containing heteroatoms selected from N, O and S, phenyl, benzyl, tolyl, halogen, hydroxyl, thiol, amino, sulfonyl, sulfoxide, sulfonic acid, nitro, carbonyl, carboxyl, carboxamide, aldehyde, phospho, nitrile, isonitrile, isocyanate, thiocyanate or isothiocyanate, or alternatively R1 and R8, R2 and R9, and / or R6 and R 10 taken together with the carbon or nitrogen atom to which they are attached form a ring which may be substituted or unsubstituted, optionally containing multiple bonds;

[0047] In this case, the alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, R1 and R8, R2 and R9, and / or R6 and R 10 the ring formed by may be unsubstituted or substituted with halogen, hydroxyl, thiol, amino, sulfo, nitro, carbonyl, carboxyl, carboxamide, aldehyde, phospho, nitrile, isonitrile, isocyanate, thiocyanate or isothiocyanate;

[0048] R4 is represented by the following chemical formula 2:

[0049] [ka]

[0050] In the above formula 2, X is hydrogen, C1 to C 10 saturated or unsaturated, substituted or unsubstituted, straight-chain, branched-chain or cyclic alkyl, C2-C 10 Substituted or unsubstituted, linear, branched or cyclic alkenyl, C(O)NR a R b , CH2OR a , (CH2) n NR a R b , (CH2) n NR a R b , OR a , N.R. a R b , SO2R a , SO2NH2, SO2NR a2 hydroxy or halogen;

[0051] R a is C1-C6 alkyl, C3-C7 cycloalkyl, or heterocyclyl containing a heteroatom selected from N, O, and S, wherein the alkyl, cycloalkyl is unsubstituted or substituted with halogen;

[0052] R b is hydrogen, halogen, hydroxyl, thiol, amino, sulfo, nitro, carbonyl, carboxyl, carboxamide, aldehyde, phospho, nitrile, isonitrile, isocyanate, thiocyanate or isothiocyanate;

[0053] n is 1 or 2,

[0054] m is an integer of 1 to 3.

[0055] In one embodiment, the compound may be any one selected from the group consisting of compounds represented by the following Formulas 3 to 5 and combinations thereof:

[0056] [ka]

[0057] [ka]

[0058] [ka]

[0059] In one embodiment, the fluorescent label exhibits stable fluorescent properties at pH 2-11.

[0060] In one embodiment, the fluorescent intensity of the fluorescent label at temperatures of 75°C to 95°C may be 1.1 to 10 times that of Quasar 670.

[0061] In one embodiment, the incubation may be by PCR, real-time PCR, or isothermal amplification.

[0062] In one embodiment, at least one of the oligonucleotide sets can generate a signal by forming or dissociating a duplex from the signaling oligonucleotide depending on the presence of the target nucleic acid.

[0063] In one embodiment, at least one of the oligonucleotide sets can generate a signal by cleavage of the signaling oligonucleotide after hybridization with the target nucleic acid, which occurs depending on the presence of the target nucleic acid.

[0064] In one embodiment, at least one of the oligonucleotide sets can generate a signal by forming or dissociating a complex between a cleavage product and a signaling oligonucleotide after cleavage of a mediation oligonucleotide hybridized to the target nucleic acid, which occurs depending on the presence of the target nucleic acid.

[0065] In one embodiment, at least one of the oligonucleotide sets can generate a signal by dissociation of the signaling oligonucleotide hybridized to the target nucleic acid after cleavage, which occurs depending on the presence of the target nucleic acid.

[0066] According to another aspect of the present invention, there is provided a kit for detecting a target nucleic acid in a sample, comprising:

[0067] (a) a first oligonucleotide set for a first target nucleic acid, a second oligonucleotide set for a second target nucleic acid, and a third oligonucleotide set for a third target nucleic acid;

[0068] Each of the oligonucleotide sets comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that serves to generate a signal in the presence of the target nucleic acid and that is bound to a fluorescent label, wherein the amplification oligonucleotide and the signaling oligonucleotide are the same or different, and each oligonucleotide set commonly comprises a single type of fluorescent label;

[0069] the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid;

[0070] (b) Instructions describing one embodiment of the method.

[0071] According to another aspect of the present invention, there is provided a computer readable medium containing instructions for implementing a processor to perform a method for detecting a sample target nucleic acid, the method comprising the steps of: (a) detecting a target nucleic acid in a sample;

[0072] (a) receiving signals measured at a first detection temperature, a second detection temperature, and a third detection temperature, wherein a first oligonucleotide set is directed to a first target nucleic acid, a second oligonucleotide set is directed to a second target nucleic acid, and a third oligonucleotide set is directed to a third target nucleic acid;

[0073] Each of the oligonucleotide sets comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that serves to generate a signal in the presence of the target nucleic acid and that is bound to a fluorescent label, wherein the amplification oligonucleotide and the signaling oligonucleotide are the same or different, and each oligonucleotide set commonly comprises a single type of fluorescent label;

[0074] the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid;

[0075] the first detected temperature, the second detected temperature, and the third detected temperature are different from each other;

[0076] (b) determining the presence of each target nucleic acid based on signals measured at the first detection temperature, the second detection temperature, and the third detection temperature,

[0077] the presence of the first target nucleic acid is determined by a difference between signals measured at the first detection temperature, the second detection temperature, and the third detection temperature, the presence of the second target nucleic acid is determined by a difference between signals measured at the second detection temperature and the third detection temperature, and the presence of the third target nucleic acid is determined by a signal measured at the third detection temperature;

[0078] According to another aspect of the present invention, there is provided an apparatus for detecting a target nucleic acid in a sample, comprising: (a) a computer processor; and (b) the aforementioned computer-readable recording medium coupled to the computer processor.

[0079] According to another aspect of the present invention, there is provided a computer program storable on a computer readable recording medium, embodying a processor for carrying out a method for detecting a target nucleic acid in a sample, the method comprising the steps of:

[0080] (a) receiving signals measured at a first detection temperature, a second detection temperature, and a third detection temperature, wherein a first oligonucleotide set is directed to a first target nucleic acid, a second oligonucleotide set is directed to a second target nucleic acid, and a third oligonucleotide set is directed to a third target nucleic acid;

[0081] Each of the oligonucleotide sets comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that serves to generate a signal in the presence of the target nucleic acid and that is bound to a fluorescent label, wherein the amplification oligonucleotide and the signaling oligonucleotide are the same or different, and each oligonucleotide set commonly comprises a single type of fluorescent label;

[0082] the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid;

[0083] the first detected temperature, the second detected temperature, and the third detected temperature are different from each other;

[0084] (b) determining the presence of each target nucleic acid based on signals measured at the first detection temperature, the second detection temperature, and the third detection temperature,

[0085] the presence of the first target nucleic acid is determined by a difference between signals measured at the first detection temperature, the second detection temperature, and the third detection temperature, the presence of the second target nucleic acid is determined by a difference between signals measured at the second detection temperature and the third detection temperature, and the presence of the third target nucleic acid is determined by a signal measured at the third detection temperature; [Effects of the Invention]

[0086] The present invention enables the real-time detection of three target nucleic acids in one reaction vessel by using a single fluorescent label and three detection temperatures. Conventional techniques detect multiple target nucleic acids by melting analysis after target amplification. Unlike conventional techniques, the present invention does not require melting analysis after target amplification, dramatically shortening analysis time.

[0087] In particular, the method of the present invention is an optimal method for determining the presence of each target nucleic acid using signals having the same fluorescent properties measured at different temperatures. While utilizing signals at detection temperatures at which signals of some target nucleic acids are measured together, false positive / false negative errors are minimized, analysis time is shortened, and it is cost-effective. [Brief explanation of the drawings]

[0088] [Figure 1] 1 shows the results of amplifying target nucleic acids from HPV 66, which has a low detection temperature (60°C), HPV 45, which has a medium detection temperature (72°C), and HPV 58, which has a high detection temperature (83°C), by incubating each target nucleic acid with three oligonucleotide sets that all contain a FAM fluorescent label, and then measuring the signals at 60°C (top), 72°C (middle), and 83°C (bottom). The signals for each target nucleic acid were generated by a PTOCE real-time PCR method (WO 2012 / 096523). As can be seen in the figure, the signal measured at 60°C contains all of the signals for the target nucleic acids from HPV 66, HPV 45, and HPV 58, the signal measured at 72°C contains the signals for the target nucleic acids from HPV 66 and HPV 45, and the signal measured at 83°C contains the signal for the target nucleic acid from HPV 58. Therefore, the signal intensity increases in the order of the signal measured at 60°C, the signal measured at 72°C, and the signal measured at 83°C.

[0089] [Figure 2] FIG. 2 shows the results of extracting only the signals for HPV 66 (top), HPV 45 (middle), and HPV 58 (bottom) from the signals measured at the three detection temperatures shown in FIG.

[0090] [Figure 3]3 shows the results of amplifying target nucleic acids from HPV 51, which has a low detection temperature (60°C), HPV 59, which has a medium detection temperature (72°C), and HPV 16, which has a high detection temperature (83°C), by incubating each target nucleic acid with three oligonucleotide sets that all contain a CAL Fluor Orange 560 fluorescent label, and then measuring the signals at 60°C (top), 72°C (middle), and 83°C (bottom). The signals for each target nucleic acid were generated using the PTOCE real-time PCR method (WO 2012 / 096523). As can be seen in the figure, the signal measured at 60°C contains all of the signals for the target nucleic acids from HPV 51, HPV 59, and HPV 16, the signal measured at 72°C contains the signals for the target nucleic acids from HPV 51 and HPV 59, and the signal measured at 83°C contains the signal for the target nucleic acid from HPV 16. Therefore, the signal intensity increases in the order of the signal measured at 60°C, the signal measured at 72°C, and the signal measured at 83°C.

[0091] [Figure 4] FIG. 4 shows the results of extracting only the signals for HPV 51 (top), HPV 59 (center), and HPV 16 (bottom) from the signals measured at the three detection temperatures shown in FIG.

[0092] [Figure 5]5 shows the results of amplifying target nucleic acids from HPV33, which has a low detection temperature (60°C), HPV39, which has a medium detection temperature (72°C), and HPV52, which has a high detection temperature (83°C), by incubating each target nucleic acid with three oligonucleotide sets that all contain a CAL Fluor Red 610 fluorescent label, and then measuring the signals at 60°C (top), 72°C (middle), and 83°C (bottom). The signals for each target nucleic acid were generated using a PTOCE real-time PCR method (WO2012 / 096523). As can be seen in the figure, the signal measured at 60°C contains all of the signals for the target nucleic acids from HPV33, HPV 39, and HPV 52, the signal measured at 72°C contains the signals for the target nucleic acids of HPV33 and HPV 39, and the signal measured at 83°C contains the signal for the target nucleic acid from HPV 39. Therefore, the signal intensity increases in the order of the signal measured at 60°C, the signal measured at 72°C, and the signal measured at 83°C.

[0093] [Figure 6] FIG. 6 shows the results of extracting only the signals for HPV33 (top), HPV39 (center), and HPV52 (bottom) from the signals measured at the three detection temperatures shown in FIG.

[0094] [Figure 7]7 shows the results of amplifying each target nucleic acid by incubating an internal control (IC) with a low detection temperature (60°C), a target nucleic acid from HPV 35 with a medium detection temperature (72°C), and a target nucleic acid from HPV 18 with a high detection temperature (83°C) with three oligonucleotide sets that all contain a Quasar 670 fluorescent label, and then measuring the signals at 60°C (top), 72°C (middle), and 83°C (bottom). The signals for each target nucleic acid were generated using the PTOCE real-time PCR method (WO 2012 / 096523). As can be seen in the figure, the signal measured at 60°C includes all of the signals for the target nucleic acids from the internal control group, HPV 35, and HPV 18, the signal measured at 72°C includes the signals for the target nucleic acids from HPV 35 and HPV 18, and the signal measured at 83°C includes the signal for the target nucleic acid from HPV 18. Therefore, the signal intensity increases in the order of the signal measured at 60°C, the signal measured at 72°C, and the signal measured at 83°C.

[0095] [Figure 8] FIG. 8 shows the results of extracting only the signals for the internal control group (top), HPV 35 (middle), and HPV 18 (bottom) from the signals measured at the three detection temperatures shown in FIG.

[0096] [Figure 9]9 shows the results of amplifying target nucleic acids from HPV56, which has a low detection temperature (60°C), HPV68, which has a medium detection temperature (72°C), and HPV31, which has a high detection temperature (83°C), by incubating them with three oligonucleotide sets that all contain a Quasar 705 fluorescent label, and then measuring the signals at 60°C (top), 72°C (middle), and 83°C (bottom). The signals for each target nucleic acid were generated using the PTOCE real-time PCR method (WO2012 / 096523). As can be seen in the figure, the signal measured at 60°C contains all of the signals for the target nucleic acids from HPV56, HPV68, and HPV31, the signal measured at 72°C contains the signals for the target nucleic acids from HPV68 and HPV31, and the signal measured at 83°C contains the signal for the target nucleic acid from HPV31. Therefore, the signal intensity increases in the order of the signal measured at 60°C, the signal measured at 72°C, and the signal measured at 83°C.

[0097] [Figure 10] FIG. 10 shows the results of extracting only the signals for HPV56 (top), HPV68 (center), and HPV31 (bottom) from the signals measured at the three detection temperatures shown in FIG.

[0098] [Figure 11]Figure 11 shows the results of amplifying target nucleic acids from HPV 18, which has a high detection temperature (83°C), by reacting them with an oligonucleotide set containing an ATTO 647N fluorescent label and an oligonucleotide set containing a Quasar 670 fluorescent label, respectively, and then measuring the signals at detection temperatures of 60°C (top), 72°C (middle), and 83°C (bottom). The signals for each target nucleic acid were generated using the PTOCE real-time PCR method (WO 2012 / 096523). As can be seen from the figure, the oligonucleotide set containing the Quasar 670 fluorescent label exhibited relatively low mean fluorescence intensity, and the signal decreased significantly as the temperature increased. The oligonucleotide set containing the ATTO 647N fluorescent label exhibited excellent mean fluorescence intensity and high long-wavelength fluorescence signal intensity even at high temperatures. DETAILED DESCRIPTION OF THE INVENTION

[0099] The greatest feature of the present invention is the simultaneous detection of three target nucleic acids by a single real-time amplification reaction. Conventional real-time amplification reactions detect multiple target nucleic acids by melting analysis after target amplification. In contrast, the method of the present invention can simultaneously detect three target nucleic acids by a real-time amplification reaction without performing melting analysis.

[0100] I. Methods for detecting target nucleic acids

[0101] According to one aspect of the present invention, there is provided a method for detecting a target nucleic acid in a sample, comprising the steps of:

[0102] (a) incubating a sample in a single reaction vessel with a first set of oligonucleotides directed to a first target nucleic acid, a second set of oligonucleotides directed to a second target nucleic acid, and a third set of oligonucleotides directed to a third target nucleic acid;

[0103] Each of the oligonucleotide sets comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that serves to generate a signal in the presence of the target nucleic acid and that is bound to a fluorescent label, wherein the amplification oligonucleotide and the signaling oligonucleotide are the same or different, and each oligonucleotide set commonly comprises a single type of fluorescent label;

[0104] the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid;

[0105] (b) measuring signals at a first detection temperature, a second detection temperature, and a third detection temperature; the first detection temperature, the second detection temperature, and the third detection temperature are different from each other;

[0106] (c) determining the presence of each target nucleic acid based on signals measured at the first detection temperature, the second detection temperature, and the third detection temperature;

[0107] The presence of the first target nucleic acid is determined by the difference between the signals measured at the first, second and third detection temperatures, the presence of the second target nucleic acid is determined by the difference between the signals measured at the second and third detection temperatures, and the presence of the third target nucleic acid is determined by the signal measured at the third detection temperature.

[0108] The present invention will be described in detail for each step as follows:

[0109] Step (a): Incubation of the target nucleic acid and the oligonucleotide set

[0110] First, in one reaction vessel, the sample is incubated with first, second, and third oligonucleotide sets designed to amplify or detect first, second, and third target nucleic acids, respectively.

[0111] As used herein, the term "sample" refers to cells, tissues, or fluids from a biological source, or any other medium that can be usefully evaluated by the present invention. Examples of such samples include viruses, bacteria, tissues, cells, blood, serum, plasma, lymph, milk, urine, feces, ocular 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). Samples also include naturally occurring nucleic acid molecules isolated from biological sources and synthetic nucleic acid molecules. Samples may also be lysates, extracts, or isolated target nucleic acids for a particular analyte. In certain embodiments, the sample is a cervical swab sample or a liquid-based cytology sample. The term "sample" includes samples collected from a subject or samples that have undergone additional processing, such as extraction.

[0112] The present invention is used to determine the presence or absence of one or more of a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid in a sample.

[0113] As used herein, the terms "target nucleic acid," "target nucleic acid sequence," or "target sequence" refer to a nucleic acid sequence to be detected or quantified. The target nucleic acid sequence includes sequences initially present in a nucleic acid sample as well as sequences newly generated in a reaction.

[0114] In one embodiment, the target nucleic acid refers to a target nucleic acid from HPV.

[0115] In one embodiment, the target nucleic acid is derived from three HPV types, and the target nucleic acid from the HPV types is in double-stranded form.

[0116] For example, when two or more types of detectors are applied to the method, four or more target nucleic acids, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 types of target nucleic acids can be detected.

[0117] In one embodiment, the target nucleic acid may include an internal control.

[0118] The target nucleic acid may be subjected to well-known strand separation methods to generate double-stranded single-stranded or partially single-stranded forms. These methods include, but are not limited to, heat, alkali, formamide, urea, and glycosyl treatment, enzymatic methods (e.g., helicase action), and binding proteins. For example, strand separation may be achieved by heating at temperatures ranging from 80°C to 105°C. General methods for achieving such treatments are provided in Joseph Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

[0119] According to the present invention, a first oligonucleotide set, a second oligonucleotide set, and a third oligonucleotide set are used to detect three different target nucleic acids.

[0120] The phrase "one oligonucleotide set is specific to one target nucleic acid" means that the oligonucleotide set contributes to detecting one target nucleic acid but does not contribute to detecting other target nucleic acids. Also, the phrase "one oligonucleotide set is specific to one target nucleic acid" means that the oligonucleotide set interacts with one target nucleic acid but does not interact with other target nucleic acids. The phrase does not mean that all oligonucleotides in the oligonucleotide set necessarily hybridize to the target nucleic acid.

[0121] Each of the oligonucleotide sets used in the present invention comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid of interest, and (ii) a signaling oligonucleotide that is conjugated to a fluorescent label and that serves to generate a signal in the presence of the target nucleic acid of interest, and the amplification oligonucleotide and the signaling oligonucleotide may be the same or different.

[0122] As an example, each of the oligonucleotide sets may be an amplification oligonucleotide or a signaling oligonucleotide, and may include oligonucleotides (e.g., probes and primers) that specifically hybridize to a target nucleic acid; capture oligonucleotides that specifically hybridize to a fragment when a probe or primer hybridized to a target nucleic acid is cleaved to release the fragment; oligonucleotides that specifically hybridize to an extended strand when a fragment hybridized to the capture oligonucleotide is extended to form the extended strand; oligonucleotides that specifically hybridize to the capture oligonucleotide; or combinations of these.

[0123] In this application, the term "amplification oligonucleotide" collectively refers to an oligonucleotide that serves to amplify a target nucleic acid.

[0124] In one embodiment, the amplification oligonucleotide is a "primer," as known in the art. As used herein, the term "primer" refers to an oligonucleotide that can act as a synthesis initiation point under conditions that induce synthesis of a primer extension product complementary to a target nucleic acid (template), i.e., the presence of nucleotides and a polymerizing agent, such as a DNA polymerase, at an appropriate temperature and pH. A primer must be sufficiently long to prime the synthesis of an extension product in the presence of a polymerizing agent. The appropriate length of a primer depends on several factors, including temperature, application, and primer source.

[0125] The primers may include a forward primer (also referred to as an upstream primer or upstream oligonucleotide), a reverse primer (also referred to as a downstream primer or downstream oligonucleotide), or both.

[0126] The amplification oligonucleotides may be oligonucleotides having structures known in the art and may be synthesized by methods known in the art.

[0127] As used herein, the term "signaling oligonucleotide" refers to an oligonucleotide that is involved in generating a signal to be detected. According to one embodiment of the present invention, the signaling oligonucleotide includes an oligonucleotide that actually participates in generating a signal. For example, hybridization or non-hybridization of the signaling oligonucleotide with another oligonucleotide (e.g., an oligonucleotide containing a nucleotide sequence complementary to the target nucleic acid or the signaling oligonucleotide) determines signal generation.

[0128] In one embodiment, the signaling oligonucleotide is a "probe," as known in the art. As used herein, the term "probe" refers to a single-stranded nucleic acid molecule containing a region or regions substantially complementary to a target nucleic acid. According to one embodiment of the present invention, the 3'-end of the probe is "blocked" to prevent its extension. Blocking can be achieved by conventional methods. For example, blocking can be achieved 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 can be achieved by removing the 3'-hydroxyl group of the last nucleotide or by using a nucleotide lacking a 3'-hydroxyl group, such as a dioxynucleotide.

[0129] In the present application, the signaling oligonucleotide comprises at least one fluorescent label, which together with the signaling oligonucleotide generates a fluorescent signal.

[0130] According to one embodiment of the present invention, the signaling oligonucleotide may be composed of at least one oligonucleotide. According to one embodiment of the present invention, when the signaling oligonucleotide is composed of multiple oligonucleotides, the signaling oligonucleotide may be labeled in various ways. For example, one of the multiple oligonucleotides may have at least one label, all of the multiple oligonucleotides may have at least one label, or some portions of the oligonucleotide may have at least one label and other portions may not have a label.

[0131] According to the present invention, the amplification oligonucleotides and signaling oligonucleotides contained in each of the oligonucleotide sets may be the same or different.

[0132] The fact that the amplification oligonucleotide and the signaling oligonucleotide are the same means that one oligonucleotide not only serves as an amplification oligonucleotide that amplifies the target nucleic acid but also as a signaling oligonucleotide that generates a signal in the presence of the target nucleic acid. For example, an oligonucleotide having a fluorescent label can hybridize with the target nucleic acid, be extended, and generate a signal.

[0133] The amplification oligonucleotide and the signaling oligonucleotide are different, meaning that two oligonucleotides are used.

[0134] According to the present invention, the signaling oligonucleotide comprises a fluorescent label, which is attached to the signaling oligonucleotide.

[0135] In the present application, an oligonucleotide set including a signaling oligonucleotide can generate a signal in various ways known in the art.

[0136] (i) Signal generation by the formation or dissociation of a complex

[0137] In one embodiment, at least one of the oligonucleotide sets generates a signal by forming or dissociating a duplex from a signaling oligonucleotide depending on the presence of a target nucleic acid. In a specific embodiment, all of the oligonucleotide sets generate a signal by forming or dissociating a duplex from a signaling oligonucleotide depending on the presence of a target nucleic acid.

[0138] According to one embodiment, the complex comprises a double-stranded target nucleic acid.

[0139] As used herein in connection with an oligonucleotide set, the phrase "generating a signal by the formation or dissociation of a duplex from a signaling oligonucleotide that occurs depending on the presence of a target nucleic acid" means that a signal is provided depending on the formation of a duplex or the dissociation of a duplex resulting from binding of the signaling oligonucleotide to the target nucleic acid or a nucleic acid derived therefrom in the presence of the target nucleic acid. This phrase includes providing a signal by a duplex (e.g., a duplex between a labeled signaling oligonucleotide and a nucleic acid) formed depending on the presence of the target nucleic acid. This phrase also includes providing a signal by the inhibition of the formation of a duplex (e.g., a duplex between a labeled signaling oligonucleotide and a nucleic acid), where the inhibition occurs due to the formation of another duplex.

[0140] In particular, the signal is generated by duplex formation between a target nucleic acid and a signaling oligonucleotide that specifically hybridizes to the target nucleic acid.

[0141] Signals based on duplex formation between a target nucleic acid and a signaling oligonucleotide are transmitted 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 1999). 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).

[0142] According to one embodiment, at least one of the first, second, and third oligonucleotide sets generates a signal by forming or dissociating a duplex between a cleavage product and a signaling oligonucleotide after cleavage of a mediation oligonucleotide hybridized to the target nucleic acid, which occurs depending on the presence of the target nucleic acid. According to a specific embodiment, all of the first, second, and third oligonucleotide sets generate a signal by forming or dissociating a duplex between a cleavage product and a signaling oligonucleotide after cleavage of a mediation oligonucleotide hybridized to the target nucleic acid, which occurs depending on the presence of the target nucleic acid.

[0143] As used herein, the term "mediator oligonucleotide" refers to an oligonucleotide that mediates the formation of a duplex that does not involve a target nucleic acid.

[0144] According to one embodiment of the present invention, a signal is not generated by cleavage of the intermediate oligonucleotide itself, but after hybridization and cleavage of the intermediate oligonucleotide, the fragments formed by the cleavage participate in the subsequent reaction for signal generation.

[0145] According to one embodiment, no signal is generated by hybridization or cleavage of the intermediary oligonucleotide itself.

[0146] According to one embodiment of the present invention, the intermediate oligonucleotide mediates the formation of a duplex by hybridizing to the target nucleic acid and being cleaved to release fragments, and in particular, the fragments mediate the formation of a duplex by being extended on the capture oligonucleotide.

[0147] According to one embodiment of the present invention, the intermediary oligonucleotide comprises (i) a 3'-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid, and (ii) a 5'-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid.

[0148] According to one embodiment of the present invention, cleavage of the intermediate oligonucleotide releases fragments that specifically hybridize to capture oligonucleotides and are extended on the capture oligonucleotides by a nucleic acid polymerase.

[0149] According to one embodiment of the present invention, a mediator oligonucleotide hybridized to a target nucleic acid is cleaved to release fragments, which are then specifically hybridized to a capture oligonucleotide and extended to form an extension chain, which forms an extension complex with the capture oligonucleotide to provide a signal indicating the presence of the target nucleic acid. The capture oligonucleotide corresponds to the signaling oligonucleotide of this application.

[0150] According to one embodiment of the present invention, when a third oligonucleotide containing a hybridization nucleotide sequence complementary to the extended strand is used, the hybridization of the third oligonucleotide and the extended strand forms another type of duplex, providing a signal indicating the presence of the target nucleic acid. The third oligonucleotide corresponds to the signaling oligonucleotide of the present application.

[0151] According to one embodiment of the present invention, when a third oligonucleotide containing a hybridization nucleotide sequence complementary to the capture oligonucleotide is used, the duplex formation between the third oligonucleotide and the capture oligonucleotide is suppressed by the duplex formation between the extension strand and the capture oligonucleotide, thereby providing a signal indicating the presence of the target nucleic acid.

[0152] According to one embodiment of the present invention, the capture oligonucleotide, the third oligonucleotide, or a combination thereof may serve as a signaling oligonucleotide.

[0153] The signal generated by the duplex formed in a manner dependent on cleavage of the intermediate oligonucleotide may be generated by various methods, including the PTOCE (PTO cleavage and extension) method (WO2012 / 096523), the PCE-SH (PTO cleavage and extension-dependent signaling oligonucleotide hybridization) method (WO2013 / 115442), and the PCE-NH (PTO cleavage and extension-dependent non-hybridization) method (PCT / KR2013 / 012312).

[0154] In relation to the terms disclosed in the above-mentioned references, examples of corresponding oligonucleotides are as follows: the intermediary oligonucleotide corresponds to a probing and tagging oligonucleotide (PTO), the capture oligonucleotide corresponds to a capturing and templating oligonucleotide (CTO), and the third oligonucleotide corresponds to a signaling oligonucleotide (SO) or a hybridization oligonucleotide (HO). SO, HO, CTO, or a combination thereof can serve as a signaling oligonucleotide.

[0155] The signal from the duplex formed in a manner dependent on cleavage of the intermediary oligonucleotide includes a signal (e.g., PCE-NH) provided by the duplex formed in a manner dependent on cleavage of the intermediary oligonucleotide inhibiting the formation of other duplexes.

[0156] For example, when a signal is generated by the PTOCE method using a duplex formed in a manner dependent on cleavage of the intermediate oligonucleotide, the reaction includes an upstream oligonucleotide, a probing and tagging oligonucleotide (PTO) containing a hybridization nucleotide sequence complementary to the target nucleic acid, a capturing and templating oligonucleotide (CTO), an appropriate label, and a template-dependent nucleic acid polymerase having 5'-nuclease activity.

[0157] The PTO comprises (i) a 3'-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid, and (ii) a 5'-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid. The CTO comprises, in the 3'→5' direction, (i) a capturing 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 nucleotide sequences non-complementary to the 5'-tagging portion and 3'-targeting portion of the PTO.

[0158] A specific embodiment of signal generation by the PTOCE method comprises the following steps:

[0159] (a) hybridizing the target nucleic acid with an upstream oligonucleotide and a PTO; (b) contacting the result of step (a) with an enzyme having 5' nuclease activity under conditions for cleaving the PTO, wherein the upstream oligonucleotide or its extension induces cleavage of the PTO by the enzyme having 5' nuclease activity, and the cleavage releases a fragment containing the 5'-tagging portion or a part 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 capturing portion of the CTO; (d) analyzing the result of step (c) and extending the PTO using a template-dependent nucleic acid polymerase. performing an extension reaction, wherein the fragments hybridized to the capturing sites of the CTOs are extended to form extension complexes, the extension complexes having a Tm value adjustable by (i) the sequence and / or length of the fragments, (ii) the sequence and / or length of the CTO, or (iii) the sequence and / or length of the fragments and the sequence and / or length of the CTO, and the extension complexes provide a target signal via at least one label linked to the fragments and / or the CTO; and (e) detecting the extension complexes by measuring the target signal at a designated detection temperature at which the extension complexes retain their double-stranded form, the presence of the extension complexes indicating the presence of the target nucleic acid. In such cases, the method further comprises repeating all or part of steps (a) to (e), including denaturation between the repeated cycles.

[0160] In the phrase "denaturing during repeated cycles," the term "denaturation" refers to the separation of double-stranded nucleic acid molecules into single-stranded nucleic acid molecules.

[0161] In step (a) of the PTOCE method, a primer set for amplification of a target nucleic acid may be used instead of the upstream oligonucleotide, in which case the method further comprises repeating all or part of steps (a) to (e), including denaturation between repeated cycles.

[0162] The PTOCE method may be classified into a process in which a PTO fragment hybridized to a CTO is extended to form an extended chain, and then the extended chain is detected. The PTOCE method is characterized in that the formation of the extended chain is detected using a duplex between the extended chain and the CTO.

[0163] There are other approaches for detecting the formation of the extended chain. For example, the formation of the extended chain may be detected using an oligonucleotide that specifically hybridizes to the extended chain (e.g., the PCE-SH method). In such methods, the signal may be provided by (i) a label linked to the oligonucleotide that specifically hybridizes to the extended chain, or (ii) a label linked to the oligonucleotide that specifically hybridizes to the extended chain and a label linked to the PTO fragment.

[0164] Alternatively, the detection of the extension chain formation is carried out by other methods (e.g., the PCE-NH method) that detect the inhibition of hybridization between the CTO and an oligonucleotide specifically hybridizable to the CTO. Such inhibition is considered to indicate the presence of the target nucleic acid. The signal may be provided by (i) a label linked to the oligonucleotide hybridizable to the CTO, (ii) a label linked to the CTO, or (iii) a label linked to the oligonucleotide hybridizable to the CTO and a label linked to the CTO.

[0165] According to one embodiment, the oligonucleotide capable of specifically hybridizing to the CTO has an overlapping sequence with the PTO fragment.

[0166] According to one embodiment, the signaling oligonucleotide includes an oligonucleotide capable of specifically hybridizing to the extended strand (e.g., PCE-SH method) and an oligonucleotide capable of specifically hybridizing to the CTO (e.g., PCE-NH method).

[0167] 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 by cleavage and extension of PTO.

[0168] An example of signal generation using a PTOCE-based method includes the following steps: (a) hybridizing a target nucleic acid with an upstream oligonucleotide and a PTO; (b) contacting the result of step (a) with an enzyme having 5' nuclease activity under conditions for cleaving 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 a fragment containing 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 hybridizes to the capturing portion of the CTO; (d) performing an extension reaction using the result of step (c) and a template-dependent nucleic acid polymerase, wherein the fragment hybridized to the capturing portion of the CTO is extended to form an extended strand; and (e) detecting a signal generated depending on the presence of the extended strand to detect the formation of the extended strand. In step (a), a primer set for amplifying the target nucleic acid may be used instead 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.

[0169] According to one embodiment, the signal generated by duplex formation includes a signal induced by hybridization of the duplex (e.g., hybridization of the duplex itself or hybridization of a third oligonucleotide), or a signal induced by inhibition of hybridization of a third oligonucleotide by duplex formation.

[0170] According to one embodiment, at least one of the first oligonucleotide set, the second oligonucleotide set, and the third oligonucleotide set generates a signal by dissociation of the duplex after cleavage of the signaling oligonucleotide hybridized to the target nucleic acid, which occurs depending on the presence of the target nucleic acid.

[0171] According to a specific embodiment, the first oligonucleotide set, the second oligonucleotide set, and the third oligonucleotide set all generate a signal by dissociation of the duplex after cleavage of the signaling oligonucleotide hybridized to the target nucleic acid, which occurs depending on the presence of the target nucleic acid.

[0172] As an example, the signaling oligonucleotide may include a double-stranded tagging portion that includes a nucleotide sequence that is non-complementary to the target nucleic acid, a single-stranded targeting portion that includes a hybridizing nucleotide sequence that is complementary to the target nucleic acid, and a label.

[0173] In one embodiment, the label may be an interactive triple label comprising one reporter molecule and two quencher molecules.

[0174] According to one embodiment, in the presence of a target nucleic acid, the interactive triple label is configured such that at a first detection temperature, one or more of the two quenchers approach the reporter molecule, quenching the signal from the reporter molecule, and at a second detection temperature, both of the two quencher molecules separate from the reporter molecule, unquenching the signal from the reporter molecule.

[0175] According to one embodiment, one quencher molecule of the interactive triple label may be linked to a targeting portion of a signaling oligonucleotide, and the remaining quencher molecule and reporter molecule of the interactive triple label may be linked to a position where they can be released together with the cleaved tagging portion when the tagging portion of the signaling oligonucleotide is cleaved and released. In particular, the reporter molecule of the interactive triple label may be linked to a position where it can reversibly interact with the quencher molecule linked to the targeting portion.

[0176] According to a specific embodiment, one reporter molecule and one quencher molecule of the interactive triple label may be linked to the tagging portion of the double-stranded form of the signaling oligonucleotide, and the remaining quencher molecule may be linked to the targeting portion of the signaling oligonucleotide. In particular, the reporter molecule and quencher molecule linked to the tagging portion may be linked to the position of the tagging portion where they can reversibly interact (double-stranded form or single-stranded form).

[0177] For example, in the presence of a target nucleic acid, one reporter molecule and one quencher molecule of the interactive triple label are separated from the remaining quencher molecule by cleavage of the double-stranded tagging portion of the signaling oligonucleotide. The cleaved double-stranded tagging portion can either retain its double-stranded form or dissociate into a single-stranded form depending on the temperature. Specifically, (i) the cleaved double-stranded tagging portion retains its double-stranded form at temperatures lower than its Tm, such that the reporter molecule and quencher molecule linked to the tagging portion are close to each other, and the quencher molecule quenches the signal from the reporter molecule; and (ii) the cleaved double-stranded tagging portion dissociates into a single-stranded form at temperatures higher than its Tm, such that the reporter molecule and quencher molecule are separated from each other, and the quencher molecule unquenches the signal from the reporter molecule.

[0178] In one embodiment, cleavage of the tagging portion of the double-stranded form of the signaling oligonucleotide may be performed using, but is not limited to, a polymerase having nuclease activity or another nuclease, which may be naturally occurring, unmodified, or modified.

[0179] For an example of signal generation by dissociation of the duplex after cleavage of the signaling oligonucleotide hybridized to the target nucleic acid, which occurs depending on the presence of the target nucleic acid, see references [US11,034,997, US11,028,433, US10,590,469, US2020-0048682].

[0180] (ii) Signal generation by post-hybridization cleavage

[0181] According to one embodiment, at least one of the first, second, and third oligonucleotide sets generates a signal by cleavage after hybridization of the signaling oligonucleotide with the target nucleic acid, which occurs depending on the presence of the target nucleic acid.

[0182] After the signaling oligonucleotide is hybridized to the target nucleic acid, the signal generated by cleavage of the signaling oligonucleotide may be generated by various methods, including the TaqMan probe method (US Pat. Nos. 5,210,015 and 5,538,848).

[0183] When the signal is generated by the TaqMan probe method, the reaction includes a primer set for amplification of a target nucleic acid, a TaqMan probe with an appropriate label (e.g., an interactive dual label), and a nucleic acid polymerizing enzyme with 5'-nuclease activity. The TaqMan probe hybridized to the target nucleic acid is cleaved during target amplification, generating a signal indicative of the presence of the target nucleic acid.

[0184] A specific example of generating a signal using the TaqMan probe method includes the following steps: (a) hybridizing the target nucleic acid with a primer set and a TaqMan probe having an appropriate label (e.g., an interactive dual label); (b) amplifying the target nucleic acid using the result of step (a) and a nucleic acid polymerase having 5' nuclease activity, whereby the TaqMan probe is cleaved to release the label; and (c) detecting signal generation from the released label.

[0185] In particular, the signal is generated by cleavage of a signaling oligonucleotide in a manner that is dependent on cleavage of a mediator oligonucleotide that is specifically hybridized to the target nucleic acid.

[0186] According to one embodiment of the present invention, when a mediator oligonucleotide hybridized to a target nucleic acid is cleaved to release a fragment, the fragment specifically hybridizes to a signaling oligonucleotide, and the fragment induces cleavage of the signaling oligonucleotide.

[0187] According to one embodiment of the present invention, when the intermediary oligonucleotide hybridized to the target nucleic acid is cleaved to release a fragment, the fragment is extended and cleaves the signaling oligonucleotide containing a hybridized nucleotide sequence complementary to the capture oligonucleotide.

[0188] Signals generated by cleavage of the signaling oligonucleotide, which depend on cleavage of the intermediary oligonucleotide, can be generated by various methods, including Invader analysis (U.S. Pat. No. 5,691,142), PTO Cleavage and Extension-Dependent Cleavage (PCEC) method (WO 2012 / 134195), and the method described in U.S. Pat. No. 7,309,573. In particular, the method described in U.S. Pat. No. 7,309,573 can be considered a PTOCE-based method that uses cleavage to generate signals. In this method, the formation of the extended chain can be detected by detecting the cleavage of the oligonucleotide specifically hybridized to the CTO. Invader analysis generates fragments by cleavage of the intermediary oligonucleotide, and then induces a continuous cleavage reaction without fragment extension.

[0189] According to one embodiment of the present invention, when the signal is generated in a manner dependent on cleavage of a signaling oligonucleotide, cleavage of the signaling oligonucleotide induces a signal change or release of a labeled fragment that is detected.

[0190] When an oligonucleotide set simultaneously generates a signal by cleavage of a signaling oligonucleotide and by formation of a duplex, the oligonucleotide set may be considered to provide a signal by cleavage, so long as the oligonucleotide set is used to generate a signal by cleavage.

[0191] At least the first oligonucleotide set, the second oligonucleotide set, and the third oligonucleotide set used in the present application can employ the various signal generation methods described above.

[0192] As one example, the first oligonucleotide set, the second oligonucleotide set, and the third oligonucleotide set may all generate a signal by forming or dissociating a duplex from the signaling oligonucleotide depending on the presence of the target nucleic acid. As another example, some of the first oligonucleotide set, the second oligonucleotide set, and the third oligonucleotide set may generate a signal by forming or dissociating a duplex from the signaling oligonucleotide depending on the presence of the target nucleic acid, and the remaining oligonucleotides may generate a signal by cleavage of the signaling oligonucleotide after hybridization with the target nucleic acid depending on the presence of the target nucleic acid.

[0193] According to the method of the present application, in a first oligonucleotide set, a second oligonucleotide set, and a third oligonucleotide set that commonly contain any one type of fluorescent label, the first oligonucleotide set must be designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set must be designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set must be designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid.

[0194] Those skilled in the art can determine the signaling scheme of a set of oligonucleotides to meet such requirements.

[0195] For example, if an oligonucleotide set that generates a signal by cleavage of a signaling oligonucleotide after hybridization with a target nucleic acid, which occurs depending on the presence of a target nucleic acid, generates a signal at all of the first detection temperature, the second detection temperature, and the third detection temperature, the oligonucleotide set is used for the target nucleic acid having the third detection temperature, but is not used for the target nucleic acid having the first detection temperature or the target nucleic acid having the second detection temperature.

[0196] As another example, if an oligonucleotide set that generates a signal by dissociation of a duplex after cleavage of a signaling oligonucleotide hybridized to a target nucleic acid, which occurs depending on the presence of the target nucleic acid, generates a signal at a first detection temperature, the oligonucleotide set is used for a target nucleic acid having a first detection temperature; if an oligonucleotide set generates signals at the first detection temperature and a second detection temperature, the oligonucleotide set is used for a target nucleic acid having a second detection temperature; if an oligonucleotide set generates signals at the first detection temperature, the second detection temperature, and a third detection temperature, the oligonucleotide set is used for a target nucleic acid having a third detection temperature.

[0197] According to one embodiment, among three oligonucleotide sets each containing one type of fluorescent label, one oligonucleotide set generates a signal by hybridization of the signaling oligonucleotide with the target nucleic acid and subsequent cleavage, which occurs depending on the presence of the target nucleic acid.

[0198] According to one embodiment of the present invention, in three oligonucleotide sets each containing one type of fluorescent label in common, the oligonucleotide set for the third target nucleic acid is an oligonucleotide set that generates a signal by cleavage of a signaling oligonucleotide, and the oligonucleotide set for the second target nucleic acid and the oligonucleotide set for the first target nucleic acid are oligonucleotide sets that generate signals by duplex formation.

[0199] According to one embodiment of the present invention, in three oligonucleotide sets each containing one type of fluorescent label in common, the oligonucleotide set for the third target nucleic acid is an oligonucleotide set that generates a signal by cleavage of a signaling oligonucleotide, and the oligonucleotide set for the second target nucleic acid and the oligonucleotide set for the first target nucleic acid are oligonucleotide sets that utilize duplex formation in a manner dependent on cleavage of a mediator oligonucleotide specifically hybridized to the target nucleic acid.

[0200] In the method of the present invention, the first oligonucleotide set, the second oligonucleotide set, and the third oligonucleotide set all contain a single type of fluorescent label.

[0201] As used herein, a single or one type of fluorescent label refers to a fluorescent label having identical or substantially identical signal characteristics (e.g., optical properties, emission wavelength, and electrical signal). For example, FAM and CAL Fluor 610 provide different types of signals. Such a single or one type of fluorescent label does not depend on the chemical structure of the fluorescent label. Therefore, even if two fluorescent labels have different chemical structures, they are considered to be of the same type if they cannot be distinguished using a detection channel.

[0202] According to the present invention, signals generated from three oligonucleotide sets that share any one type of fluorescent label are indistinguishable by a single detection channel.

[0203] As used herein, the term "detection channel" refers to a means for detecting signals from a single type of fluorescent label. Thermocyclers available in the art, such as ABI 7500 (Applied Biosystems), QuantStudio (Applied Biosystems), CFX96 (Bio-Rad Laboratories), cobas Z 480 (Roche), and LightCycler (Roche), contain several channels (e.g., photodiodes) for detecting signals from several different types of fluorescent labels, and these channels are referred to as detection channels in this application.

[0204] One of the features of the present invention is that at least one detection temperature can be selected from the range of 75°C to 90°C while using a fluorescent label having an excitation wavelength selected from the range of 620 nm to 710 nm.

[0205] The appropriate selection of the fluorescent label contained in the oligonucleotide set is a key factor in the success of a method for determining the presence or absence of a target nucleic acid using an amplification reaction signal. Even if a signaling method of an oligonucleotide set suitable for the target nucleic acid is adopted, if the brightness or photostability of the signal expressed by the fluorescent label is reduced, the reliability of the signal analysis results may be significantly reduced.

[0206] In particular, in a method for detecting three target nucleic acids using the same fluorescent label, it is necessary that all signals measured at each of the three detection temperatures be stable. For example, if temperature-dependent changes in signal intensity occur due to the fluorescent label itself in addition to changes in signal intensity due to the oligonucleotide characteristics of the oligonucleotide set, it is not easy to extract signals for each target nucleic acid from signals at detection temperatures that include signals for multiple target nucleic acids.

[0207] Fluorescent labels in the relatively short wavelength range, such as FAM, CAL Fluor Orange 560, and CAL Fluor Red 610, generally do not suffer from temperature-dependent fluorescence intensity degradation, provided that they are compatible with each oligonucleotide set and the emission conditions of the dye itself are met. However, dyes in the long wavelength range, such as those with excitation wavelengths selected from the 620-710 nm range and emission wavelengths selected from the 650-720 nm range, suffer from a significant decrease in signal intensity at detection temperatures above 75°C, even in the presence of target nucleic acid. For example, even when an oligonucleotide set is designed to detect the presence of target nucleic acid at a detection temperature of 83°C, the signal detected at 83°C exhibits a significant decrease in fluorescence intensity to a level equivalent to or below the threshold RFU value, even in the presence of target nucleic acid.

[0208] Therefore, in order to realize a method for detecting three target nucleic acids, the inventors identified and applied a dye that can maintain a signal detected at a high detection temperature at a level similar to the fluorescence intensity expressed at a lower temperature.

[0209] In one embodiment, the excitation wavelength of the fluorescent label may be selected from the ranges of 630 nm to 660 nm, 635 nm to 655 nm, 638 nm to 650 nm, or 640 nm to 648 nm. In this case, the emission wavelength of the fluorescent label may be selected from the ranges of 650 nm to 680 nm, 655 nm to 670 nm, 660 nm to 670 nm, or 662 nm to 668 nm. By applying a detection temperature selected from the range of 75°C to 90°C to an oligonucleotide set containing fluorescent labels having such wavelength ranges, the presence of three target nucleic acids can be simultaneously determined using a single fluorescent label.

[0210] In one embodiment, the fluorescent label may be a carbopyronine-based compound.

[0211] Among fluorescent labels with red emission wavelength bands, carbopyronine-based compounds can generate stable signals at high temperatures. For example, cyanine-based compounds lose energy floating in rotational, transitional, and vibrational modes due to their central chain structure at temperatures above 75°C as energy other than fluorescent emission. Therefore, the fluorescence intensity is significantly lower at high detection temperatures compared to relatively low detection temperatures. In contrast, the present invention has the advantage of significantly improving red wavelength fluorescence efficiency at high detection temperatures by applying a carbopyronine-based dye with a robust fluorescent central structure to high detection temperatures.

[0212] In certain embodiments, the compound may be a carbopyronine compound represented by the following Formula 1:

[0213] [ka]

[0214] In the chemical formula 1, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 and R 11 are, independently of each other, hydrogen, C1 to C 10 Alkyl, C3-C7 cycloalkyl, C2-C 10 Alkenyl, C6-C 10 monocyclic or bicyclic aryl or heteroaryl containing heteroatoms selected from N, O and S, selected from among phenyl, benzyl, tolyl, halogen, hydroxyl, thiol, amino, sulfonyl, sulfoxide, sulfonic acid, nitro, carbonyl, carboxyl, carboxamide, aldehyde, phospho, nitrile, isonitrile, isocyanate, thiocyanate or isothiocyanate;

[0215] Or alternatively, R1 and R8, R2 and R9, and / or R6 and R 10 taken together with the carbon or nitrogen atom to which they are attached form a substituted or unsubstituted ring, optionally containing multiple bonds;

[0216] In this case, the alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, R1 and R8, R2 and R9, and / or R6 and R 10 The ring formed by may be unsubstituted or substituted with halogen, hydroxyl, thiol, amino, sulfo, nitro, carbonyl, carboxyl, carboxamide, aldehyde, phospho, nitrile, isonitrile, isocyanate, thiocyanate, or isothiocyanate.

[0217] In certain embodiments, R4 may be represented by Formula 2:

[0218] [ka]

[0219] In the formula 2, X is hydrogen, C1 to C 10 saturated or unsaturated, substituted or unsubstituted, straight-chain, branched-chain or cyclic alkyl, C2-C 10 substituted or unsubstituted, linear, branched or cyclic alkenyl, halogen, C(O)NR a R b , CH2OR a , (CH2) n NR a R b , (CH2) n NR a R b , OR a , N.R. a R b , SO2Ra, SO2NH2, SO2NR a2 hydroxy or halogen;

[0220] R a is C1-C6 alkyl, C3-C7 cycloalkyl, or heterocyclyl containing a heteroatom selected from N, O, and S, wherein the alkyl, cycloalkyl is unsubstituted or substituted with halogen;

[0221] R bis hydrogen, halogen, hydroxyl, thiol, amino, sulfo, nitro, carbonyl, carboxyl, carboxamide, aldehyde, phospho, nitrile, isonitrile, isocyanate, thiocyanate or isothiocyanate;

[0222] Each n is independently 1 or 2, and m is an integer of 1 to 3.

[0223] The compound is characterized in that it is any one selected from the group consisting of compounds represented by the following chemical formulas 3 to 5 and combinations thereof.

[0224] [ka]

[0225] [ka]

[0226] [ka]

[0227] In one embodiment, the carbopyronine compound may exhibit stable fluorescence properties at pH 2 to 11, pH 3 to 10, or pH 4 to 9. Therefore, even if the pH of the amplification reagent containing the sample changes depending on the selected amplification method, the compound may exhibit stable fluorescence.

[0228] Furthermore, when the fluorescent label is a carbopyronine dye, the fluorescent intensity of the fluorescent label at temperatures between 75°C and 95°C may be 1.01 to 100 times, 1.1 to 80 times, 1.2 to 50 times, 1.5 to 30 times, 2 to 20 times, or 3 to 10 times that of Quasar 670. The fluorescent intensity may be an average of the fluorescent intensity per cycle at any one temperature between 75°C and 95°C. The fluorescent intensity may also be the fluorescent intensity per same cycle at any one temperature between 75°C and 90°C.

[0229] Representative red fluorescent dyes, Quasar 670 and Quasar 705, are cyanine compounds, and therefore, when detecting signals at temperatures above 75°C, the fluorescence intensity at each cycle appears to be similar to or below the threshold for determining the presence of target nucleic acid. The fluorescent label of the present invention is a long-wavelength dye that is guaranteed to be bright and photostable at temperatures above 75°C, and therefore has superior average fluorescence intensity and / or fluorescence intensity at each cycle at high temperatures compared to Quasar 670.

[0230] Fluorescent labels useful in the present invention include various fluorescent labels known in the art, for example, fluorescent labels useful in the present invention include single fluorescent labels, interactive dual fluorescent labels, and interactive triple fluorescent labels.

[0231] According to one embodiment, the single fluorescent label provides a different signal (e.g., different signal intensity) depending on whether it is present in a double-stranded or single-stranded structure. Preferred types and binding sites of single fluorescent labels for use in the present invention 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 entirety. For example, the single fluorescent label includes JOE, FAM, TAMRA, ROX, and fluorescein-based labels. The single label may be linked to the oligonucleotide by various methods. For example, the label may be linked to the probe by a spacer containing carbon atoms (e.g., a 3-carbon spacer, a 6-carbon spacer, or a 12-carbon spacer).

[0232] A representative example of the interactive fluorescent labeling system is the FRET (fluorescence resonance energy transfer) fluorescent labeling system, which comprises a fluorescent reporter molecule (donor molecule) and a quencher molecule (acceptor molecule). In FRET, the energy donor is fluorescent, and 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 dual labeling based on "contact-mediated quenching" (Salvatore et al., Nucleic Acids Research, 2002(30) no.21 e122 and Johansson et al., J.AM.CHEM.SOC 2002(124) pp 6950-6956). The interactive labeling system includes any labeling system that induces a signal change through the interaction between a minimum of two molecules (eg, dyes).

[0233] Reporter and quencher molecules useful in the present invention may include any molecule known in the art, examples of which include: Cy2 TM (506), YO-PRO TM -1(509), YOYO TM -1(509), Calcein(517), FITC(518), FluorX TM (519),Alexa TM (520), Rhodamine 110(520), Oregon Green TM 500(522), Oregon Green TM 488(524), RiboGreen TM (525), Rhodamine Green TM(527)、Rhodamine 123(529)、Magnesium Green TM (531)、Calcium Green TM (533)、TO-PRO TM -1(533)、TOTO1(533)、JOE(548)、BODIPY530 / 550(550)、Dil(565)、BODIPY TMR(568)、BODIPY558 / 568(568)、BODIPY564 / 570(570)、Cy3 TM (570)、Alexa TM 546(570)、TRITC(572)、Magnesium Orange TM (575)、Phycoerythrin R&B(575)、Rhodamine Phalloidin(575)、Calcium Orange TM (576)、Pyronin Y(580)、Rhodamine B(580)、TAMRA(582)、Rhodamine Red TM (590)、Cy3.5 TM (596)、ROX(608)、Calcium Crimson TM (615)、Alexa TM 594(615)、Texas Red(615)、Nile Red(628)、YO-PRO TM -3(631)、YOYO TM -3(631)、Rphycocyanin(642)、C-Phycocyanin(648)、TO-PRO TM -3(660)、TOTO3(660)、DiD DilC(5)(665)、Cy5 TM(670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor 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.

[0234] Preferably, reporter and quencher molecules include JOE, FAM, TAMRA, ROX and fluorescein-based labels.

[0235] Suitable fluorescent molecules and 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, 2 ndEdition (Academic Press, New York, 1971); Griffiths, Color and Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, editor, 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, RP, Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996); U.S. Patent No. 3,996,345 and U.S. Patent No. 4,351,760.

[0236] In the present invention, non-fluorescent quencher molecules (eg, black quenchers or dark quenchers) that can quench fluorescence over a wide range of wavelengths or at specific wavelengths may be used.

[0237] In a signaling system comprising a reporter and a quencher molecule, the reporter comprises a FRET donor and the quencher comprises a FRET receptor partner (acceptor). For example, a fluorescein dye is used as the reporter and a rhodamine dye is used as the quencher.

[0238] The interactive dual-label may be linked to either strand of a duplex. When a strand containing the interactive dual-label exists in a single-stranded state, the strand forms a hairpin or random coil structure, inducing quenching between the interactive dual-labels. When the strand forms a duplex, the quenching is reduced. Alternatively, when the interactive dual-label is linked to nucleotides located adjacent to each other on the strand, quenching between the interactive dual-labels occurs. When the strand forms a duplex and is cleaved, the quenching is reduced.

[0239] The reporter molecule and quencher molecule of the interactive dual label may be linked to each of the two strands of the duplex. For example, the reporter molecule is linked to one strand of the duplex, and the quencher molecule is linked to the other strand of the duplex. Formation of the duplex induces quenching, and denaturation of the duplex induces unquenching. Alternatively, unquenching may be induced when one of the two strands is cleaved.

[0240] In one embodiment, the fluorescent label included in the oligonucleotide set may be selected from the group consisting of FAM or a substitute thereof, CAL Fluor Orange 560 or a substitute thereof, CAL Fluor Red 610 or a substitute thereof, Quasar 670 or a substitute thereof, and Quasar 705 or a substitute thereof.

[0241] In other embodiments, the fluorescent labels included in the oligonucleotide set are FAM or a substitute thereof, CAL Fluor Orange 560 or a substitute thereof, CAL Fluor Red 610 or a substitute thereof, Quasar 670 or a substitute thereof, and Quasar 705 or a substitute thereof. The five fluorescent labels may be used to detect up to 15 target nucleic acids, for example, 14 HPV types and one internal control.

[0242] The term "equivalent thereof" as used in the context of a fluorescent label means a fluorescent label that can be used in place of the referenced fluorescent label, including a fluorescent label that does not significantly affect the detection results when used in place of the referenced fluorescent label. As an example, still other fluorescent labels that exhibit signal properties (e.g., optical properties, emission wavelength, and electrical signal) that are identical or substantially identical to the referenced fluorescent label may be included in the definition.

[0243] Alternatives to the fluorescent labels used herein are known in the art. For example, alternatives to FAM fluorescent labels include Alexa Fluor 488, Cy2, Oregon Green, ATTO 465, etc.; alternatives to CAL Fluor Orange 560 include VIC, HEX, Yakima Yellow, ATTO 532, etc.; alternatives to CAL Fluor Red 610 include Pet, ROX, Cy3.5, ATTO 565, LC Red 610, Texas Red, Alexa Fluor 594, etc.; alternatives to Quasar 670 include Cy5, ATTO 647N, etc.; alternatives to Quasar 705 include Cy 5.5, ATTO 680, ATTO 700, etc.

[0244] According to one embodiment of the present invention, step (a) is performed in a signal amplification process accompanied by nucleic acid amplification.

[0245] In the present invention, the signal generated by the oligonucleotide set may be amplified simultaneously with target amplification. Alternatively, the signal may be amplified without target amplification.

[0246] According to one embodiment of the present invention, the signal generation is performed by a process including target amplification and signal amplification.

[0247] In one embodiment, the target amplification may be performed by polymerase chain reaction (PCR), real-time PCR, or isothermal amplification. PCR is widely used in the art for target amplification and involves cycles of target sequence denaturation, annealing (hybridization) between the target sequence and primers, and primer extension (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). Signals may be amplified by applying the above-mentioned signal generation methods (e.g., TaqMan and PTOCE-based methods) to the PCR process.

[0248] According to one embodiment, the amplification of the target nucleic acid can be performed by polymerase chain reaction (PCR), ligase chain reaction (LCR; see Wiedmann M, et al., "Ligase chain reaction (LCR) - overview and applications," PCR Methods and Applications, 1994 Feb;3(4):S51-64), gap filling LCR (GLCR; see WO90 / 01069, EP 439182, and WO93 / 00447), Q-beta replicase amplification (Q-beta; see Cahill P, et al., Clin Chem., 37(9):1482-5 (1991), U.S. Pat. No. 5,556,751), strand displacement amplification (SDA; see G.T. Walker et al., Nucleic Acids Res. 20(7):16911696 (1992); European Patent No. 497272), nucleic acid sequence-based amplification (NASBA, see Compton, J. Nature 350(6313):912 (1991)), transcription-mediated amplification (TMA, see Hofmann WP et al., J. Clin Virol. 32(4):289-93 (2005); U.S. Patent No. 5,888,779), rolling circle amplification (RCA, see Hutchison CA et al., Proc. Natl. Acad. Sci. USA. 102:1733217336 (2005)), loop-mediated isothermal amplification (LAMP, see Y. Mori, H. Kanda and T. Notomi, J. Infect. Chemother., 2013, 19, 404-411), recombinase polymerase amplification (RPA, J. Li, J.Macdonald and F. von Stetten, Analyst, 2018, 144, 31-67), or helicase-dependent amplification (HAD, M. Vincent, Y. Xu and H. Kong, EMBO Rep., 2004, 5, 795-800).

[0249] The above-mentioned amplification methods can amplify a target sequence by repeating a series of reactions with or without changing the temperature. A unit of amplification including the repetition of the series of reactions is referred to as a "cycle." The unit of cycle may be expressed as the number of repetitions or time, depending on the amplification method.

[0250] For example, detection of the signal may be performed at each cycle of amplification, at a selected subset of cycles, or at the end-point of the reaction.

[0251] According to an embodiment, when signals are detected in at least two cycles, the detection of signals in each cycle may be performed at all detection temperatures or at a selected portion of the detection temperatures.

[0252] According to one embodiment of the present invention, step (a) is performed under conditions that allow target amplification along with signal generation by the oligonucleotide set.

[0253] According to one embodiment of the present invention, step (a) is performed as a signal amplification process without nucleic acid amplification.

[0254] When a signal is generated by a method involving cleavage of an oligonucleotide, the signal may be amplified without target amplification. For example, the signal may be amplified by the CPT method (Duck P, et al., Biotechniques, 9:142-148 (1990)), Invader analysis (U.S. Patent No. 6,358,691 and U.S. Patent No. 6,194,149), a PTOCE-based method (e.g., PCE-SH method, PCE-NH method, and PCEC method), or a CER method (WO2011 / 037306), in which the signal is amplified but the target nucleic acid is not amplified, and step (a) is performed.

[0255] In one embodiment of the present invention, the amplification may be performed by PCR, real-time PCR, or isothermal amplification.

[0256] Amplification of the target nucleic acid is achieved by amplification oligonucleotides and nucleic acid polymerizing enzymes.

[0257] According to one embodiment of the present invention, a nucleic acid polymerizing enzyme having nuclease activity (e.g., 5' nuclease activity or 3' nuclease activity) may be used. According to one embodiment of the present invention, a nucleic acid polymerizing enzyme without nuclease activity may be used.

[0258] Nucleic acid polymerases useful in the present invention include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus litoralis, Thermococcus barossi, Thermococcus gorgonarius, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus and thermostable DNA polymerases obtained from various bacterial species, including Pyrodictium abyssi, Pyrodictium occultum, Aquifex pyrophilus, and Aquifex aeolieus. In particular, the thermostable DNA polymerase is Taq polymerase.

[0259] According to one embodiment of the present invention, the amplification of the target nucleic acid is achieved by asymmetric PCR.

[0260] The ratio of the amplification oligonucleotides may be selected to take into account cleavage or hybridization of the downstream oligonucleotide.

[0261] During or after incubation (reaction) of the sample with the oligonucleotide set to generate a signal, the signal generated in one reaction vessel is detected using a single type of detection channel.

[0262] According to one embodiment of the present invention, the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid. In the above description, the first oligonucleotide set is designed to generate a signal only at the first detection temperature in the presence of the first target nucleic acid but not at the second and third detection temperatures, the second oligonucleotide set is designed to generate a signal only at the first and second detection temperatures in the presence of the second target nucleic acid but not at the third detection temperature, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of the third target nucleic acid.

[0263] For example, if three oligonucleotide sets commonly contain an ATTO 647N fluorescent label, the first oligonucleotide set generates an ATTO 647N fluorescent signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set generates an ATTO 647N fluorescent signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set generates an ATTO 647N fluorescent signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid.

[0264] In the three oligonucleotide sets that commonly contain a single type of fluorescent label, the target nucleic acid that generates a signal at a first detection temperature has a first detection temperature determined by the corresponding oligonucleotide set, the target nucleic acid that generates signals at the first and second detection temperatures has a second detection temperature determined by the corresponding oligonucleotide set, and the target nucleic acid that generates signals at the first, second, and third detection temperatures has a third detection temperature determined by the corresponding oligonucleotide set.

[0265] As used herein, the expression "the target nucleic acid has a detection temperature determined by the corresponding oligonucleotide set" means that the target nucleic acid can be detected at a detection temperature pre-assigned to the target nucleic acid.

[0266] According to one embodiment of the present invention, one detection temperature determined by the corresponding oligonucleotide set is assigned to one target nucleic acid.

[0267] The first detection temperature is a temperature at which a signal for a target nucleic acid having the first detection temperature, a signal for a target nucleic acid having the second detection temperature, and a signal for a target nucleic acid having the third detection temperature can be generated, the second detection temperature is a temperature at which a signal for a target nucleic acid having the second detection temperature and a signal for a target nucleic acid having the third detection temperature can be generated, and the third detection temperature is a temperature at which a signal for a target nucleic acid having the third detection temperature can be generated. In other words, signals for all three target nucleic acids are detected at the first detection temperature, signals for only two target nucleic acids are detected at the second detection temperature, and signals for only one target nucleic acid are detected at the third detection temperature.

[0268] One of the features of the present invention is that the presence of three target nucleic acids can be distinguished and determined from signals measured at three detection temperatures.

[0269] According to one embodiment, the three detection temperatures are predetermined in consideration of the temperature range in which a signal can be generated by the oligonucleotide set.

[0270] The present invention takes advantage of the fact that there are specific temperature ranges that allow signal generation in an oligonucleotide set dependent manner.

[0271] For example, if an oligonucleotide set generates a signal upon duplex formation but not upon duplex dissociation, a signal will be generated at a temperature at which duplex formation occurs, but not at a temperature at which duplex formation does not occur. Thus, there is a specific temperature range in which signal generation (i.e., signal detection) is possible, and another temperature range in which no signal is generated. The temperature range is affected by the Tm value of the signaling oligonucleotide included in the oligonucleotide set.

[0272] When a signal generation method is used in which fluorescently labeled fragments are released after cleavage, the signal may theoretically be detected at any temperature (eg, 30-99°C).

[0273] The detection temperature is selected from the temperature range in which a signal can be generated by the oligonucleotide set.

[0274] The term "detection temperature range" is used herein to specifically describe the temperature range over which signal generation (ie, signal detection) is possible.

[0275] When the oligonucleotide sets for three target nucleic acids have different detection temperature ranges, the first detection temperature may be selected from a range where the detection temperature range in which a signal is generated from the first oligonucleotide set, the detection temperature range in which a signal is generated from the second oligonucleotide set, and the detection temperature range in which a signal is generated from the third oligonucleotide set overlap; the second detection temperature may be selected from a range where the detection temperature range in which a signal is generated from the second oligonucleotide set and the detection temperature range in which a signal is generated from the third oligonucleotide set overlap; and the third detection temperature may be selected from a detection temperature range in which a signal is generated only from the third oligonucleotide set.

[0276] According to one embodiment, the first, second, and third detection temperatures may be predetermined. For example, the first, second, and third detection temperatures may be predetermined to be 60° C., 72° C., and 83° C., respectively. Then, oligonucleotide sets suitable for the detection temperatures are constructed, followed by performing step (a).

[0277] According to one embodiment, oligonucleotide sets for three target nucleic acids are first constructed, and then detection temperatures for the three target nucleic acids are assigned, followed by carrying out step (a).

[0278] According to one embodiment of the present invention, when the oligonucleotide set generates a signal in a manner dependent on the formation of a duplex, the detection temperature is selected based on the Tm value of the duplex.

[0279] According to one embodiment of the present invention, when the oligonucleotide set generates a signal in a manner dependent on the formation of a duplex, the detection temperature can be adjusted by adjusting the Tm value of the duplex.

[0280] For example, when the signal is generated by a signaling oligonucleotide (e.g., a Lux probe, a molecular beacon probe, a HyBecon probe, or an adjacent hybridization probe) that specifically hybridizes to the target nucleic acid, detection of the signal can be successfully achieved at a predetermined temperature by adjusting the Tm value of the signaling oligonucleotide.

[0281] When Scorpion primers are used, the signal can be successfully detected at a predetermined temperature by adjusting the Tm value of the site hybridized to the extended strand.

[0282] When the signal is generated by a duplex formed in the presence of the target nucleic acid, the signal can be successfully detected at a predetermined temperature by adjusting the Tm value of the duplex. For example, when the signal is generated by the PTOCE method, the signal can be successfully detected at a predetermined temperature by adjusting the Tm value of the extended duplex formed by extension of the PTO fragment on the CTO.

[0283] The PTOCE-based method has the advantage that the Tm of the duplex or of a third hybrid whose hybridization is influenced by the duplex can be easily adjusted.

[0284] According to one embodiment of the present invention, when the oligonucleotide set generates a signal in a manner dependent on cleavage of the signaling oligonucleotide, the detection temperature can be arbitrarily selected. That is, any temperature can be selected as long as the signal generated by cleavage of the signaling oligonucleotide can be detected. As described above, when the signal is generated in a manner dependent on cleavage of the signaling oligonucleotide, the label released by cleavage can be detected at various temperatures.

[0285] According to one embodiment, when the signal is generated in a manner dependent on cleavage of the signaling oligonucleotide, the detection temperature may be selected to be the highest detection temperature.

[0286] As described above, the detection temperature is determined taking into consideration the detection temperature range depending on the oligonucleotide set. Therefore, the signal detection at a specific detection temperature can be explained as follows: detection at the first detection temperature is for obtaining a mixed signal from the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid, detection at the second detection temperature is for obtaining a mixed signal from the second target nucleic acid and the third target nucleic acid, and detection at the third detection temperature is for obtaining a signal from the third target nucleic acid.

[0287] One of the three target nucleic acids has a first detection temperature determined by a corresponding oligonucleotide set, another has a second detection temperature determined by a corresponding oligonucleotide set, and the remaining one has a third detection temperature determined by a corresponding oligonucleotide set.

[0288] For example, when a signal for a target nucleic acid having the first detection temperature, a signal for a target nucleic acid having the second detection temperature, and a signal for a target nucleic acid having the third detection temperature are all generated by the PTOCE method, the signal for the target nucleic acid having the first detection temperature is generated by an extended mer having a Tm value suitable for the first detection temperature, the signal for the target nucleic acid having the second detection temperature is generated by an extended mer having a Tm value suitable for the second detection temperature, and the signal for the target nucleic acid having the third detection temperature is generated by an extended mer having a Tm value suitable for the third detection temperature. For example, when the first detection temperature, the second detection temperature, and the third detection temperature are a low detection temperature, a middle detection temperature, and a high detection temperature, respectively, at the low detection temperature, an extended duplex having a Tm value suitable for the high detection temperature, an extended duplex having a Tm value suitable for the middle detection temperature, and an extended duplex having a Tm value suitable for the low detection temperature all have a duplex form, so that at the low detection temperature, a mixed signal for the target nucleic acid having a low detection temperature, a target nucleic acid having a middle detection temperature, and a target nucleic acid having a high detection temperature is detected; at the middle detection temperature, an extended duplex having a Tm value suitable for the high detection temperature and an extended duplex having a Tm value suitable for the middle detection temperature are detected. are in a duplex form, and the extended duplex having a Tm value suitable for the low detection temperature is dissociated, so that at the medium detection temperature, a mixed signal for the target nucleic acid having the high detection temperature and the target nucleic acid having the medium detection temperature is detected, and a signal for the target nucleic acid having the low detection temperature is not detected; at the high detection temperature, the extended duplex having a Tm value suitable for the high detection temperature is in a duplex form, and the extended duplex having a Tm value suitable for the medium detection temperature and the extended duplex having a Tm value suitable for the low detection temperature are dissociated into single strands, so that at the high detection temperature, only a signal for the target nucleic acid having the high detection temperature is detected.

[0289] In another example, when a signal for the target nucleic acid having a high detection temperature is generated by the TaqMan method and signals for the target nucleic acid having a medium detection temperature and the target nucleic acid having a low detection temperature are generated by the PTOCE method, the signal for the target nucleic acid having a high detection temperature is provided by a released fluorescent label, and the signal for the target nucleic acid having a medium detection temperature and the signal for the target nucleic acid having a low detection temperature are provided by an extended duplex having a Tm value suitable for the medium detection temperature and an extended duplex having a Tm value suitable for the low detection temperature, respectively. When a signal is detected at the high detection temperature, the extended duplex having a Tm value suitable for the medium detection temperature and the extended duplex having a Tm value suitable for the low detection temperature are dissociated into single strands, so no signal is generated, and therefore only the signal from the released fluorescent label for the target nucleic acid having a high detection temperature is detected. When a signal is detected at the mid-range detection temperature, a signal provided by an extended mer having a Tm value suitable for the mid-range detection temperature as well as a signal from the released fluorescent label is detected, thereby detecting a mixed signal for the target nucleic acid having the mid-range detection temperature and the target nucleic acid having the high detection temperature.When a signal is detected at the low detection temperature as well as a signal provided by an extended mer having a Tm value suitable for the low detection temperature as well as a signal provided by an extended mer having a Tm value suitable for the mid-range detection temperature and a signal from the released fluorescent label, thereby detecting a mixed signal for the target nucleic acid having the low detection temperature, the target nucleic acid having the mid-range detection temperature, and the target nucleic acid having the high detection temperature.

[0290] In one embodiment, the first detected temperature is a temperature selected from 57 to 63° C., the second detected temperature is a temperature selected from 69 to 75° C., and the third detected temperature is a temperature selected from 80 to 86° C. In a specific embodiment, the first detected temperature is 60° C., the second detected temperature is 72° C., and the third detected temperature is 83° C.

[0291] Such detection temperatures may be used when all oligonucleotide sets generate signals by the formation or dissociation of a duplex between the signaling oligonucleotide and a cleavage product after cleavage of a mediation oligonucleotide hybridized to the target nucleic acid, which occurs depending on the presence of the target nucleic acid, or when the oligonucleotide set for the target nucleic acid having the third detection temperature generates a signal by the cleavage of the signaling oligonucleotide after hybridization to the target nucleic acid, which occurs depending on the presence of the target nucleic acid, and the oligonucleotide sets for the target nucleic acids having the first and second detection temperatures generate signals by the formation or dissociation of a duplex between the signaling oligonucleotide and a cleavage product after cleavage of the mediation oligonucleotide hybridized to the target nucleic acid, which occurs depending on the presence of the target nucleic acid.

[0292] In another embodiment, the first detected temperature is a temperature selected from 80 to 86°C, the second detected temperature is a temperature selected from 69 to 75°C, and the third detected temperature is a temperature selected from 57 to 63°C. In a specific embodiment, the first detected temperature is 83°C, the second detected temperature is 72°C, and the third detected temperature is 60°C.

[0293] Such detection temperatures may be used when all oligonucleotide sets generate signals by dissociation of a duplex after cleavage of a signaling oligonucleotide hybridized to a target nucleic acid, which occurs depending on the presence of the target nucleic acid, or when an oligonucleotide set for a target nucleic acid having a third detection temperature generates a signal by cleavage of a signaling oligonucleotide after hybridization with the target nucleic acid, which occurs depending on the presence of the target nucleic acid, and oligonucleotide sets for target nucleic acids having a first detection temperature and a second detection temperature generate signals by dissociation of a duplex after cleavage of a signaling oligonucleotide hybridized to a target nucleic acid, which occurs depending on the presence of the target nucleic acid.

[0294] A detection channel as used in the present invention includes any means capable of detecting a signal. For example, a detection channel may be a photodiode capable of detecting a fluorescent signal of a particular wavelength.

[0295] According to one embodiment, the generation of a signal includes "signal generation or disappearance" and "signal increase or decrease" from the label. Specifically, the generation of a signal refers to the generation of a significant signal, and a significant signal refers to a signal that can be distinguished from a background signal or a signal generated from a reaction using a sample that does not contain the target nucleic acid (i.e., a negative sample). That is, if the specific intensity of the generated signal is the same as or similar to the intensity of the background signal or a signal generated from a reaction using a sample that does not contain the target nucleic acid, it is considered that no signal is generated for the sample of interest.

[0296] Step (b): Measurement of signals at three temperatures

[0297] In this step, signals are measured at a first detection temperature, a second detection temperature, and a third detection temperature, and the first detection temperature, the second detection temperature, and the third detection temperature are different from each other.

[0298] In one embodiment, signals are measured at three temperatures selected at intervals of 5°C to 25°C from each other within a range not exceeding 90°C.

[0299] One of the features of the present invention is that in the method for detecting three target nucleic acids using one detection channel, the temperature range does not exceed 90° C. In this case, the detection temperatures may be selected at intervals of 5° C. to 25° C., 7° C. to 20° C., 8° C. to 18° C., 10° C. to 16° C., 11° C. to 15° C., or 12° C. to 13° C. to prevent interference between the fluorescent signals detected in one detection channel in the presence of each target nucleic acid.

[0300] When preparing primers and / or probes for detecting a target nucleic acid, designing the target nucleic acid to have a detection temperature above 90°C poses problems such as excessively long oligonucleotides, a high GC content, or a significant decrease in the stability of the fluorescent label. Therefore, general methods for detecting target nucleic acids do not use detection temperatures above 90°C.

[0301] In contrast, the present invention detects the first target nucleic acid at the first detection temperature, the second target nucleic acid at the second detection temperature, and the third target nucleic acid at the third detection temperature, and by deriving the optimal reference value that can be applied when using the signal difference, the presence of three target nucleic acids can be clearly determined even if all detection temperatures are designed to be below 90°C.

[0302] Specifically, the detection temperature may be selected from the ranges of 55°C to 90°C, 56°C to 89°C, 56°C to 89°C, 56°C to 90°C, 55°C to 88°C, 57°C to 87°C, 57°C to 89°C, 57°C to 90°C, 58°C to 86°C, 58°C to 87°C, or 58°C to 86°C.

[0303] For example, the highest detection temperature among the first, second, and third detection temperatures may be selected from within the ranges of 76°C to 89°C, 77°C to 88°C, 78°C to 87°C, 79°C to 86°C, or 80°C to 86°C. The middle detection temperature among the first, second, and third detection temperatures may be selected from within the ranges of 65°C to 80°C, 66°C to 79°C, 67°C to 78°C, 68°C to 77°C, 69°C to 76°C, or 69°C to 75°C. The lowest detection temperature among the first, second, and third detection temperatures may be selected from within the ranges of 55°C to 70°C, 55°C to 69°C, 55°C to 68°C, 56°C to 67°C, 56°C to 65°C, 57°C to 64°C, or 57°C to 63°C.

[0304] In one embodiment, (i) the first detected temperature may be lower than the second detected temperature, and the second detected temperature may be lower than the third detected temperature, or (ii) the first detected temperature may be higher than the second detected temperature, and the second detected temperature may be higher than the third detected temperature.

[0305] In one embodiment, the first detection temperature may be a temperature selected from 55°C to 70°C, the second detection temperature may be a temperature selected from 65°C to 80°C, and the third detection temperature may be a temperature selected from 75°C to 90°C, and the first detection temperature may be lower than the second detection temperature, and the second detection temperature may be lower than the third detection temperature.

[0306] In a specific embodiment, the first detection temperature may be a temperature selected from 57°C to 63°C, 58°C to 62°C, 59°C to 61°C, or 59°C to 60°C; the second detection temperature may be a temperature selected from 69°C to 75°C, 70°C to 74°C, 71°C to 73°C, or 72°C to 73°C; and the third detection temperature may be a temperature selected from 80°C to 86°C, 81°C to 85°C, 82°C to 84°C, or 83°C to 84°C.

[0307] In one embodiment, the first detection temperature may be a temperature selected from 75°C to 90°C, the second detection temperature may be a temperature selected from 65°C to 80°C, and the third detection temperature may be a temperature selected from 55°C to 70°C, and the first detection temperature may be higher than the second detection temperature, and the second detection temperature may be higher than the third detection temperature.

[0308] In a specific embodiment, the first detection temperature may be a temperature selected from 80°C to 86°C, 81°C to 85°C, 82°C to 84°C, or 83°C to 84°C; the second detection temperature may be a temperature selected from 69°C to 75°C, 70°C to 74°C, 71°C to 73°C, or 72°C to 73°C; and the third detection temperature may be a temperature selected from 57°C to 63°C, 58°C to 62°C, 59°C to 61°C, or 59°C to 60°C.

[0309] In specific embodiments, signals are measured using detection channels for FAM or a substitute thereof, CAL Fluor Orange 560 or a substitute thereof, CAL Fluor Red 610, Quasar 670 or a substitute thereof, or Quasar 705 or a substitute thereof at detection temperatures of 60°C, 72°C, and 83°C.

[0310] The measurement of signals at the three detection temperatures may be performed for each cycle of amplification. For example, nucleic acid amplification may be performed for 40, 45, or 50 cycles of PCR, and signals may be measured at the three detection temperatures for each cycle.

[0311] In one embodiment, measuring the signals at the three detection temperatures generates three amplification curves consisting of an amplification curve (a collection of data points consisting of RFU and cycles) at the first detection temperature, an amplification curve at the second detection temperature, and an amplification curve at the third detection temperature.

[0312] According to the present invention, three oligonucleotide sets are used to obtain three amplification curves per detection channel.

[0313] Step (c): Determining the presence of the target nucleic acid

[0314] After signal detection, the presence of each target nucleic acid is determined from the signal measured in step (b).

[0315] The presence of the first target nucleic acid can be determined from signals measured at the first, second, and third detection temperatures. The presence of the second target nucleic acid can be determined from signals measured at the second and third detection temperatures. The presence of the third target nucleic acid can be determined from a signal measured at the third detection temperature. Specifically, the presence of the first target nucleic acid is determined by the difference between two signals among the signals measured at the first, second, and third detection temperatures. The presence of the second target nucleic acid is determined by the difference between the signals measured at the second and third detection temperatures. The presence of the third target nucleic acid is determined by the signal measured at the third detection temperature.

[0316] As described above, the first oligonucleotide set generates a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set generates a signal at the first and second detection temperatures in the presence of a corresponding second target nucleic acid, and the third oligonucleotide set generates a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid.

[0317] Therefore, depending on the relationship between each target nucleic acid and each oligonucleotide and whether or not each target nucleic acid is contained in the sample, the signal measured at the first detection temperature may include any one selected from the group consisting of a signal generated by the first oligonucleotide set at the first detection temperature, a signal generated by the second oligonucleotide set at the first detection temperature, a signal generated by the third oligonucleotide set at the first detection temperature, and combinations thereof. In this case, the signal generated by each oligonucleotide set means a signal with detectable significance.

[0318] For example, if a sample contains a first target nucleic acid, no second target nucleic acid, and no third target nucleic acid, the signal measured at the first detection temperature will include the signal generated by the first oligonucleotide set, no signal generated by the second oligonucleotide set, and no signal generated by the third oligonucleotide set.

[0319] Furthermore, when the sample does not contain the first target nucleic acid, but does contain the second target nucleic acid, and does not contain the third target nucleic acid, the signal measured at the first detection temperature does not include a signal generated by the first oligonucleotide set, but does include a signal generated by the second oligonucleotide set, and does not include a signal generated by the third oligonucleotide set.

[0320] Furthermore, when the sample does not contain the first target nucleic acid, does not contain the second target nucleic acid, but does contain the third target nucleic acid, the signal measured at the first detection temperature will not include the signal generated by the first oligonucleotide set, will not include the signal generated by the second oligonucleotide set, and will include the signal generated by the third oligonucleotide set.

[0321] Furthermore, when a sample contains a first target nucleic acid, a second target nucleic acid, and no third target nucleic acid, the signal measured at the first detection temperature includes a signal generated by the first oligonucleotide set, a signal generated by the second oligonucleotide set, and no signal generated by the third oligonucleotide set.

[0322] Furthermore, when the sample does not contain the first target nucleic acid, but does contain the second target nucleic acid and does contain the third target nucleic acid, the signal measured at the first detection temperature does not include a signal generated by the first oligonucleotide set, but does include a signal generated by the second oligonucleotide set and does include a signal generated by the third oligonucleotide set.

[0323] Furthermore, when a sample contains a first target nucleic acid, does not contain a second target nucleic acid, and contains a third target nucleic acid, the signal measured at the first detection temperature will include a signal generated by the first oligonucleotide set, will not include a signal generated by the second oligonucleotide set, and will include a signal generated by the third oligonucleotide set.

[0324] Furthermore, when a sample contains all of the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid, the signal measured at the first detection temperature includes a signal generated by the first oligonucleotide set, a signal generated by the second oligonucleotide set, and a signal generated by the third oligonucleotide set.

[0325] Furthermore, when a sample does not contain any of the first, second, and third target nucleic acids, the signal measured at the first detection temperature does not contain any of the signals generated by the first, second, and third oligonucleotide sets. That is, in this case, no signal is measured at the first detection temperature. Therefore, when the signal measured at the first detection temperature is insignificant, it can be determined that none of the first, second, and third target nucleic acids is present.

[0326] In addition, the signal measured at the second detection temperature may include any one selected from the group consisting of a signal generated by the second oligonucleotide set at the second detection temperature, a signal generated by the third oligonucleotide set at the second detection temperature, and a combination thereof, depending on the relationship characteristics between each target nucleic acid and each oligonucleotide and whether or not each target nucleic acid is contained in the sample.

[0327] In other words, if the sample does not contain any of the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid, or if the sample contains the first target nucleic acid but does not contain the second target nucleic acid and does not contain the third target nucleic acid, no signal may be measured at the second detection temperature. Therefore, if the signal measured at the second detection temperature is insignificant, it can be determined that the second target nucleic acid and the third target nucleic acid are not present.

[0328] In the present application, a signal generated by a first oligonucleotide set is generated by the interaction between the first oligonucleotide set and a first target nucleic acid, and therefore the signal can be referred to as a signal generated by the first target nucleic acid, a signal generated from the first target nucleic acid, a signal generated in the presence of the first target nucleic acid, a signal provided by the first target nucleic acid, or a signal relative to the first target nucleic acid. This also applies to signals generated by a second oligonucleotide set or a third oligonucleotide set in the same manner.

[0329] In this application, the signal generated by the oligonucleotide set should be distinguished from the signal measured at the first detection temperature, the second detection temperature, or the third detection temperature. As mentioned above, the signal measured at each detection temperature may include a combination of signals for multiple targets, whereas the signal generated by the oligonucleotide set is a signal for one target.

[0330] According to one embodiment, in step (c), the presence of the first target nucleic acid may be determined by the difference between signals measured at the first detection temperature, the second detection temperature, and the third detection temperature, the presence of the second target nucleic acid may be determined by the difference between signals measured at the second detection temperature and the third detection temperature, and the presence of the third target nucleic acid may be determined by the signal measured at the third detection temperature.

[0331] The signal used to determine the presence of the target nucleic acid includes various signal characteristics obtained from signal detection, such as signal intensity (e.g., RFU (relative fluorescence unit) value or, in the case of amplification, RFU value at a specific cycle, selected cycle or end point), signal variation pattern (or pattern) or Ct value, or values ​​obtained by mathematically processing the above characteristics.

[0332] According to one embodiment of the present invention, when an amplification curve is obtained by real-time PCR, various signal values ​​(or characteristics) from the amplification curve may be selected for use in determining the presence of a target nucleic acid.

[0333] The characteristics of the signal obtained at each detection temperature may itself be used to determine the presence of each of the target nucleic acids.

[0334] Alternatively, modified signals provided by mathematically processing the signal characteristics may be used to determine the presence of each target nucleic acid.

[0335] The characteristics of the signal measured at the first detection temperature, the characteristics of the signal measured at the second detection temperature and the characteristics of the signal measured at the third detection temperature may themselves be used to obtain the difference between the signals at each detection temperature.

[0336] Alternatively, one or more of the signals at the first, second and third detection temperatures may be transformed by mathematically processing the characteristics of the signal and used to obtain the difference between the signals at two temperatures selected from the first, second and third detection temperatures.

[0337] According to one embodiment, the term "signal" used in connection with a signal measured at a first, second, and / or third detection temperature includes not only the signal itself obtained at the detection temperature, but also a modified signal obtained by mathematically processing the signal. Furthermore, "a signal measured" or "measuring" a signal at a first, second, and / or third detection temperature refers to measuring and / or detecting a signal generated from a corresponding oligonucleotide set at the detection temperature, and may be used interchangeably with "a detected signal" or "detection" of a signal, respectively.

[0338] According to one embodiment, when the mathematical processing is performed, the characteristics of the signal must be such that the mathematical processing can be performed. In a specific embodiment, the mathematical processing includes calculations using the signal (e.g., addition, multiplication, subtraction, and division).

[0339] In the present invention, the signal used to determine the presence of a target nucleic acid is generally a significant signal. That is, the signal is generated depending on the presence of the target nucleic acid. Meanwhile, when calculating the difference between signals measured at two selected temperatures from the first, second, and third detection temperatures, an insignificant signal, such as a background signal, may be used to calculate the difference. In this regard, the signal used to determine the presence of a target nucleic acid should be understood to encompass both significant and insignificant signals, as long as they can be used to calculate the difference or be involved in the determination process.

[0340] According to one embodiment, the significance of the measured signal may be determined using a threshold value. For example, a threshold value may be determined in advance from a negative control group, taking into account the background signal of the detector, sensitivity, or the label used, and then the significance of the signal from the sample may be determined.

[0341] If a signal (ie, a significant signal) is detected at the third detection temperature, the third target nucleic acid is determined to be present.

[0342] An insignificant signal may also be referred to herein as "absence of signal" or "undetected signal."

[0343] The term "by a signal" as used herein in connection with determining the presence of a target nucleic acid means that the presence of the target nucleic acid is determined by directly or indirectly utilizing or modifying a signal generated from an oligonucleotide set, including utilizing the numerical value of the signal or modifications thereof, utilizing the presence / absence of the signal, and comparing the signal to a threshold value.

[0344] The term "determination by a signal" as used herein in connection with determining the presence of a target nucleic acid may include determining the presence of a third target nucleic acid based on the significance of a signal measured at a third detection temperature, determining the absence of a second target nucleic acid and a third target nucleic acid based on the significance of a signal measured at a second detection temperature, or determining the absence of a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid based on the significance of a signal measured at a first detection temperature.

[0345] In the present invention, the presence of the first target nucleic acid is determined by analyzing a signal measured at a first detection temperature, a signal measured at a second detection temperature, and a signal measured at a third detection temperature.

[0346] When signals are measured at the first detection temperature, the second detection temperature, and the third detection temperature, the presence of the first target nucleic acid and / or the presence of the second target nucleic acid cannot be determined by the measured signal itself, because the signal for the third target nucleic acid may also be detected at the first detection temperature and the second detection temperature, and the signal for the second target nucleic acid may also be detected at the first detection temperature.

[0347] One feature of the present invention is that a signal measured at a second detection temperature and a signal measured at a third detection temperature are used to analyze a signal measured at the first detection temperature, and a signal measured at a third detection temperature is used to analyze a signal measured at the second detection temperature.

[0348] Interestingly, the inventors have confirmed that when a signal indicating the presence of a single target nucleic acid is detected in one reaction vessel at three predetermined detection temperatures, there is a specific pattern (rule) of signal changes, particularly changes in signal intensity.

[0349] For example, for each target nucleic acid, the signal change (change in signal intensity) between the signal measured at the third detection temperature and the signal measured at the second detection temperature, the signal change between the signal measured at the third detection temperature and the signal measured at the first detection temperature, and the signal change between the signal measured at the second detection temperature and the signal measured at the first detection temperature show a specific pattern (rule). For example, the signal intensities at two temperatures selected from the first detection temperature, the second detection temperature, and the third detection temperature may be identical or substantially identical to each other, or the signal intensities may be different from each other but within a specific range.

[0350] A feature of the present invention is the application of the above results to the detection of target nucleic acids.

[0351] Since the signal for the target nucleic acid in one reaction vessel is detected simply by changing the detection temperature (for example, without changing the target content or buffer conditions), there is a specific pattern (rule) in the signal change between the two detection temperatures. Based on the specific pattern (rule) in the signal change, the signal measured at the second detection temperature and the signal measured at the third detection temperature can be used to analyze the signal measured at the first detection temperature, and the signal measured at the third detection temperature can be used to analyze the signal measured at the second detection temperature.

[0352] According to one embodiment, the method of the present invention is carried out under conditions where there is a specific pattern (rule) of signal change between two detection temperatures for a target nucleic acid.

[0353] According to one embodiment, the presence of the second target nucleic acid is determined by analyzing the signal measured at the second detection temperature using the signal measured at the third detection temperature to determine whether the signal measured at the second detection temperature includes a signal provided by the second target nucleic acid.

[0354] According to one embodiment, the presence of the first target nucleic acid is determined by analyzing the signal measured at the first detection temperature using the signal measured at the third detection temperature and the signal measured at the second detection temperature to determine whether the signal measured at the first detection temperature includes a signal provided by the first target nucleic acid.

[0355] The analysis of the signal measured at the second detection temperature with the signal measured at the third detection temperature may be performed by obtaining and analyzing the difference between the signal measured at the third detection temperature and the signal detected at the second detection temperature.

[0356] According to one embodiment of the present invention, the magnitude (or portion) of the signal for the second target nucleic acid among the signals measured at the second detection temperature may be obtained by using the signal measured at the third detection temperature. Furthermore, analysis of the signal measured at the first detection temperature using the signal measured at the third detection temperature and the signal measured at the second detection temperature may be performed by obtaining and analyzing a portion of the difference between the signal measured at the third detection temperature, the signal measured at the second detection temperature, and the signal measured at the first detection temperature.

[0357] According to one embodiment of the present invention, the magnitude (or portion) of the signal for the first target nucleic acid among the signals measured at the first detection temperature may be obtained by using the signal measured at the third detection temperature and the signal measured at the second detection temperature.

[0358] According to one embodiment, the presence of the second target nucleic acid may be determined by the difference between the signal measured at the third detection temperature and the signal measured at the second detection temperature, and the presence of the first target nucleic acid may be determined by the difference between the signals measured at the first detection temperature, the second detection temperature, and the third detection temperature.

[0359] For example, if a sample contains a first target nucleic acid, but does not contain a second target nucleic acid, and does not contain a third target nucleic acid, a signal will be detected at the first detection temperature but not at the second and third detection temperatures. Since the absence of a signal at the second and third detection temperatures indicates the absence of the second and third target nucleic acids, the signal measured at the first detection temperature may be recognized as being due to the first target nucleic acid, thereby determining the presence of the first target nucleic acid.

[0360] Alternatively, the difference may be obtained using an insignificant (e.g., background) signal measured at the second or third detection temperature, and in this alternative, the difference is highly likely to be different from the differences in the other cases below, thereby determining the presence of the first target nucleic acid.

[0361] Furthermore, if a sample does not contain the first target nucleic acid, but does contain the second target nucleic acid and does not contain the third target nucleic acid, a signal will not be detected at the third detection temperature, but will be detected at the second detection temperature and the first detection temperature. The signal measured at the second detection temperature may differ from the signal measured at the first detection temperature. Since all conditions except for the detection temperature are the same, such a difference is highly likely to fall within a specific range. If the difference calculated for a sample falls within the specific range, the signals detected at the first detection temperature and the second detection temperature are solely due to the second target nucleic acid. In other words, it may be determined that the first target nucleic acid and the third target nucleic acid are absent from the sample.

[0362] Alternatively, if a sample does not contain the first target nucleic acid, does not contain the second target nucleic acid, but does contain the third target nucleic acid, a signal will be detected at all of the third detection temperature, the second detection temperature, and the first detection temperature. The signal measured at the third detection temperature may differ from the signal measured at the second detection temperature and / or the signal measured at the first detection temperature. Since all conditions except for the detection temperature are the same, such a difference is highly likely to fall within a specific range. If the difference calculated for the sample falls within the specific range, the signals detected at the first and second detection temperatures are solely due to the third target nucleic acid. In other words, it may be determined that the first and second target nucleic acids are absent from the sample.

[0363] Alternatively, if a sample contains a first target nucleic acid, a second target nucleic acid, and no third target nucleic acid, a signal may be detected at the second detection temperature and the first detection temperature, but not at the third detection temperature. Because the first target nucleic acid is present, the difference between the signals may be more distinguishable than if only the second target nucleic acid were present. The presence of the first target nucleic acid may be determined using the difference.

[0364] Furthermore, when a sample does not contain a first target nucleic acid but does contain a second target nucleic acid and a third target nucleic acid, a signal may be significantly measured at all of the first, second, and third detection temperatures. Because the second target nucleic acid is present, the difference in signal between the second and third detection temperatures can be more clearly distinguished than when only the third target nucleic acid is present. Meanwhile, because the second and third target nucleic acids generate signals at both the first and second detection temperatures, the difference in signal between the second and third detection temperatures can be used to determine the presence of the second target nucleic acid as well as when utilizing the difference between the first and second detection temperatures, and the absence of the first target nucleic acid can be determined from the signal measured at the first detection temperature.

[0365] Furthermore, when a sample contains a first target nucleic acid, but not a second target nucleic acid, and contains a third target nucleic acid, a significant signal may be measured at any of the first, second, and third detection temperatures. Because the first target nucleic acid is present, the difference in signal between the first and third detection temperatures can be more clearly distinguished than when only the third target nucleic acid is present. Meanwhile, because both the first and third target nucleic acids generate signals at the first detection temperature, the difference in signal between the first and third detection temperatures can be used to determine the presence of the second target nucleic acid as well as when the difference between the first and second detection temperatures is utilized, and the absence of the first target nucleic acid can be determined from the signal measured at the first detection temperature.

[0366] Furthermore, when a sample contains all of the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid, the signal may be significantly measured at any of the first detection temperature, the second detection temperature, and the third detection temperature, and the presence of the first target nucleic acid and the second nucleic acid may be determined using the difference.

[0367] The difference between two of the signals measured at the first, second and third detection temperatures may be obtained by a wide variety of approaches.

[0368] The term "difference" used herein in connection with "difference between signals (or using the difference between signals)" includes not only differences obtained by mathematically processing the signals themselves or modified signals, but also differences due to the presence or absence of signals. For example, the difference may be obtained by calculating the ratio or subtraction between two signals among the signals measured at the first, second, and third detection temperatures. Alternatively, the difference may be obtained by modifying a signal at one detection temperature and comparing it with a signal at another detection temperature. The difference between two signals among the signals measured at the first, second, and third detection temperatures may be expressed in various aspects. For example, the difference may be expressed as a numerical value, the presence / absence of a signal, or a plot of signal characteristics.

[0369] According to one embodiment of the present invention, the difference between two signals among the signals measured at the first detection temperature, the second detection temperature, and the third detection temperature includes a difference obtained by mathematically processing two signals among the signals measured at the first detection temperature, the second detection temperature, and the third detection temperature.

[0370] According to one embodiment of the present invention, when no signal is detected at the third detection temperature, the presence of the first and second target nucleic acids is determined based on the signals detected at the first and second detection temperatures, taking into account that no signal is detected at the third detection temperature. Also, when no signal is detected at the second detection temperature, the presence of the first target nucleic acid is determined based on the signal detected at the first detection temperature, taking into account that no signal is detected at the second detection temperature. This embodiment demonstrates that the presence of the first target nucleic acid can ultimately be determined by utilizing the difference between the presence and absence of signals at each detection temperature.

[0371] According to one embodiment, in order to calculate the difference, if no signal is detected at the third detection temperature or no signal is detected at the second detection temperature, the background signal measured at each temperature at that time may be treated as "0" or "1."

[0372] According to one embodiment, if a negative value is obtained during the calculation, the negative value may be converted to an absolute value and used to obtain the difference.

[0373] According to one embodiment of the present invention, the signal measured at the third detection temperature is a calculation parameter for analyzing the signals of the second target nucleic acid and the first target nucleic acid, and the signal measured at the second detection temperature is a calculation parameter for analyzing the signal of the first target nucleic acid, i.e., two or more parameters are required to analyze the signal of the first target nucleic acid.

[0374] The difference between two of the signals measured at the first detection temperature, the second detection temperature, and the third detection temperature and the signal for determining the presence of the third target nucleic acid may have different dimensions or units, or may have the same dimensions or units.

[0375] As used herein, the term "determined by a difference" includes determining by the occurrence / non-occurrence of a difference, determining by a numerical value or range of the difference, and determining by plotting the difference. Furthermore, "determined by a difference" includes obtaining a value (e.g., CT) for a target nucleic acid having a first detection temperature and a second detection temperature based on the difference.

[0376] The term "by a difference" used herein in connection with determining the presence of a target nucleic acid means that the presence of the target nucleic acid is determined by directly or indirectly using or modifying the difference, including using the numerical value of the difference or a modification thereof, using the presence / absence of a signal, and comparing the difference to a threshold. There is no difference between the terms "by a difference" and "by using a difference" and they may be used interchangeably.

[0377] The mathematical processing of the signals may be performed by various computational methods and their variations.

[0378] According to one embodiment of the present invention, the mathematical processing of the signals to obtain the difference between the signals is calculating the ratio of the signal measured at the second detection temperature to the signal measured at the third detection temperature, calculating the ratio of the signal measured at the first detection temperature to the signal measured at the third detection temperature, and / or calculating the ratio of the signal measured at the first detection temperature to the signal measured at the second detection temperature.

[0379] The term "ratio" as used herein refers to the relationship between two numbers. The presence of the first target nucleic acid and the second target nucleic acid may be determined by using the ratio. If the ratio of the signal detected at the second detection temperature to the signal detected at the third detection temperature is significant, this is an indicator of the presence of the second target nucleic acid. Also, if at least one of the ratio of the signal detected at the first detection temperature to the signal detected at the third detection temperature, the ratio of the signal detected at the second detection temperature to the signal detected at the third detection temperature, and the ratio of the signal detected at the first detection temperature to the signal detected at the second detection temperature is significant, this is an indicator of the presence of the first target nucleic acid.

[0380] For example, if the ratio of the endpoint intensity measured at the second detection temperature to the endpoint intensity of the signal measured at the third detection temperature is significant (i.e., an increase in endpoint intensity), this indicates the presence of a second target nucleic acid. Also, if the ratio of the endpoint intensity measured at the second detection temperature to the endpoint intensity of the signal measured at the third detection temperature is significant, and the ratio of the endpoint intensity measured at the first detection temperature to the endpoint intensity of the signal measured at the second detection temperature is significant, this indicates the presence of both the second target nucleic acid and the first target nucleic acid.

[0381] The mathematical processing may be performed in a variety of ways.

[0382] The mathematical processing may be performed using a machine. For example, the signals may be mathematically processed by a processor in a detector or real-time PCR device. Alternatively, the signals may be mathematically processed manually, in particular by a predetermined algorithm.

[0383] According to one embodiment of the present invention, a threshold value may be used to analyze whether the difference obtained by the approach for obtaining the difference indicates the presence of the first target nucleic acid and the second target nucleic acid. For example, the threshold value may be predetermined by taking into account the difference obtained from a reference sample containing the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid. A negative control group, sensitivity, or the label used may also be considered in determining the threshold value.

[0384] According to one embodiment of the present invention, the presence of the first target nucleic acid and / or the second target nucleic acid may be determined using the obtained difference itself by the approach for obtaining the difference. For example, after applying a threshold to the signal at the third detection temperature, the difference between the applied signal and the signal at the second detection temperature can be obtained. Alternatively, after applying a threshold to the signal at the third detection temperature and the signal at the second detection temperature, the difference between the applied signal and the signal at the first detection temperature can be obtained. In particular, the threshold is predetermined taking into account the difference obtained from a reference sample containing the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid.

[0385] According to one embodiment of the present invention, the threshold value is determined by a user or automatically.

[0386] In one embodiment, when the difference between the signal at the first detection temperature, the signal at the second detection temperature, and the signal at the third detection temperature for the third target nucleic acid becomes larger, the threshold value can be used to reduce detection errors.

[0387] In one embodiment, if the signal provided by the third target nucleic acid has a pattern (or rule) showing little or no difference between the first detection temperature, the second detection temperature, and the third detection temperature, or if the signal provided by the second target nucleic acid has a pattern (or rule) showing little or no difference between the first detection temperature and the second detection temperature, the signal detected at the third detection temperature or the signal detected at the second detection temperature may be used without additional modification to calculate or use the difference to determine the presence of the first target nucleic acid or the second target nucleic acid.

[0388] In one embodiment, if the signal has a pattern (or rule) that shows a difference within a specific range, in determining the presence of the target nucleic acid, the signal at the third detection temperature and the signal at the second detection temperature may be modified to reflect the difference.

[0389] A value that reflects the pattern (rule) of change between the signals detected at the detection temperature is the reference value.

[0390] According to one embodiment of the present invention, the reference value may reflect a change in a signal provided by each oligonucleotide set that generates a signal in the presence of each target nucleic acid at two temperatures selected from the first detection temperature, the second detection temperature, and the third detection temperature.

[0391] For example, the reference value may be a change in a signal generated by a first oligonucleotide set in the presence of a first target nucleic acid at a first detection temperature and a change in a signal generated by a second oligonucleotide set in the presence of a first target nucleic acid at a second detection temperature and a change in a signal generated by a first oligonucleotide set in the presence of a first target nucleic acid at a third detection temperature, a change in a signal generated by a first oligonucleotide set in the presence of a first target nucleic acid at a second detection temperature and a change in a signal generated by a second oligonucleotide set in the presence of a second target nucleic acid at a first detection temperature and a second detection temperature, ... third detection temperature, or a change in a signal generated by a second oligonucleotide set in the presence of a second target nucleic acid at a first detection temperature and a second detection temperature. and a change in the signal generated at the third detection temperature; a signal generated by the second oligonucleotide set in the presence of the second target nucleic acid at the second detection temperature and a change in the signal generated at the third detection temperature; a signal generated by the third oligonucleotide set in the presence of the third target nucleic acid at the first detection temperature and a change in the signal generated at the second detection temperature; a signal generated by the third oligonucleotide set in the presence of the third target nucleic acid at the first detection temperature and a change in the signal generated at the third detection temperature; a signal generated by the third oligonucleotide set in the presence of the third target nucleic acid at the first detection temperature and a change in the signal generated at the third detection temperature;

[0392] According to an embodiment of the present invention, the presence of the first target nucleic acid and the second target nucleic acid can be determined using the difference and / or the reference value.

[0393] For example, when the signals at the third detection temperature and the second detection temperature by the third oligonucleotide set are identical or substantially identical and the degree of difference between the signals at the two detection temperatures is calculated by subtraction of the signals, the reference value for the signals at the two detection temperatures by the third oligonucleotide set is "0." As another example, when the degree of difference between the signals at the two detection temperatures is calculated by division of the signals, the reference value for the signals at the two detection temperatures by the third oligonucleotide set is "1."

[0394] On the other hand, when the signals at the two temperatures for the specific target nucleic acid are different from each other and the degree of difference between the two signals is calculated by subtracting the signals, the reference value for the signals at the two detection temperatures for the specific target nucleic acid is a positive value other than "0" or a negative value. As another example, when the degree of difference between the signals at the two detection temperatures is calculated by dividing the signals, the reference value for the signals at the two detection temperatures for the specific target nucleic acid is a value other than "1", greater than 1 or less than 1.

[0395] In certain embodiments, the difference between the signal provided by the third oligonucleotide in the presence of the third target nucleic acid at the second detection temperature and the third detection temperature may be expressed as a reference value.

[0396] In certain embodiments, the reference value for when the signals provided by the third oligonucleotide in the presence of the third target nucleic acid at the second detection temperature and the third detection temperature are different from each other may differ by 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 12%, 15%, 20%, or 30% or more compared to the reference value for when the two signals are identical.

[0397] In certain embodiments, when the reference value for the third target nucleic acid calculated from the signals at two detection temperatures differs by 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 12%, 15%, 20%, or 30% or more compared to the reference value when the two signals are identical, the reference value for the third target nucleic acid can be used to extract the signal from the first target nucleic acid and determine the presence of the first target nucleic acid.

[0398] According to one embodiment, the reference value is relevant to determining the presence of the first target nucleic acid and the second target nucleic acid.

[0399] According to one embodiment of the present invention, the reference value may be used to analyze whether the obtained difference indicates the presence of the first target nucleic acid and / or the second target nucleic acid.

[0400] According to one embodiment of the present invention, the reference value may be used to obtain the difference between a signal generated by the third oligonucleotide in the presence of the third target nucleic acid at the second detection temperature and a signal generated by the third oligonucleotide in the presence of the third target nucleic acid at the third detection temperature. For example, the signal measured at the third detection temperature may be multiplied or divided by the reference values ​​of the third target nucleic acid at the second and third detection temperatures, and then the difference between the multiplied or divided signal and the signal measured at the second detection temperature may be obtained. As another example, the signal measured at the second detection temperature may be multiplied or divided by the reference values ​​of the third target nucleic acid at the second and third detection temperatures, and then the difference between the multiplied or divided signal and the signal measured at the third detection temperature may be obtained.

[0401] According to one embodiment of the present invention, the reference value is used to determine a threshold value. According to one embodiment of the present invention, the reference value is used as a threshold value after modifying or unmodifying the value. The terms "threshold" and "reference value" used herein to determine the presence of a target nucleic acid by analyzing the difference between signals may be the same value or have the same meaning.

[0402] Alternatively, when the reference value is used to obtain the difference between the signal measured at the third detection temperature and the signal measured at the second detection temperature, an additional threshold value may be used to determine the significance of the difference, i.e., to determine whether the difference indicates the presence of the second target nucleic acid. Also, when the reference value is used to obtain the difference between the signal measured at the third detection temperature, the signal measured at the second detection temperature, and the signal measured at the first detection temperature, an additional threshold value may be used to determine the significance of the difference, i.e., to determine whether the difference indicates the presence of the first target nucleic acid.

[0403] According to one embodiment of the present invention, to determine the presence of the first target nucleic acid and the second target nucleic acid, the method uses a reference value, and the reference value may be obtained by (i) incubating at least one of the first oligonucleotide set, the second oligonucleotide set, and the third oligonucleotide set with at least one of the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid in a reaction vessel different from the reaction vessel in step (a), (ii) detecting signals at at least two temperatures selected from the first detection temperature, the second detection temperature, and the third detection temperature, and then (iii) calculating the difference between the signals detected at the selected two temperatures.

[0404] In one embodiment, the signal difference obtained by the method is a numerical value, which is used as a reference value in a modified or unmodified state.

[0405] For example, a first oligonucleotide set can be reacted with a sample containing only the first target nucleic acid, and signals can be detected at the first, second, and third detection temperatures to determine the difference between the signals. Alternatively, a second oligonucleotide set can be reacted with a sample containing only the second target nucleic acid, and signals can be detected at the first, second, and third detection temperatures to determine the difference between the signals. Alternatively, a third oligonucleotide set can be reacted with a sample containing only the third target nucleic acid, and signals can be detected at the first, second, and third detection temperatures to determine the difference between the signals. Alternatively, the first and second oligonucleotide sets can be reacted with a sample containing the first and second target nucleic acids, and signals can be detected at the first, second, and third detection temperatures to determine the difference between the signals when two targets are present. Alternatively, the first and third oligonucleotide sets can be reacted with a sample containing the first and third target nucleic acids, and signals can be detected at the first, second, and third detection temperatures to determine the difference between the signals when two targets are present. Alternatively, the second and third oligonucleotide sets may be reacted with a sample containing the second and third target nucleic acids and signals may be detected at the first, second, and third detection temperatures to determine the difference between the signals when two targets are included. Alternatively, the first, second, and third oligonucleotide sets may be reacted with a sample containing the first, second, and third target nucleic acids and signals may be detected at the first, second, and third detection temperatures to determine the difference between the signals at each detection temperature when three targets are included.

[0406] According to one embodiment, the difference between the signals obtained by the method is a value, which is used as a reference value either without modification or with modification.

[0407] According to one embodiment, when deriving the difference between the signals, the reference value can be obtained by calculating a ratio or subtraction. According to one embodiment of the present invention, the reference value is obtained by calculating a ratio of signals detected at two temperatures selected from the first detection temperature, the second detection temperature, and the third detection temperature.

[0408] According to one embodiment, the calculation method for the signal difference from the sample and the calculation method for the difference to obtain the reference value may be the same or different. For example, the signal difference from the sample may be obtained by subtracting two signals, and the difference to obtain the reference value may be obtained by dividing the two signals. Alternatively, both the signal difference from the sample and the difference to obtain the reference value may be obtained by dividing the two signals to obtain a ratio.

[0409] For a target nucleic acid, the reference value can be obtained under various reaction conditions, including the amount of components (e.g., target nucleic acid, signal generating means, enzyme, or dNTPs), buffer pH, or reaction time. According to one embodiment of the present invention, the reference value can be obtained under reaction conditions sufficient to provide a saturated signal upon completion of the reaction. According to one embodiment of the present invention, the difference between signals obtained during calculation of the reference value has a specific range, and the reference value is selected within or with reference to the specific range. According to one embodiment of the present invention, the reference value can be selected as the maximum or minimum value of the specific range, or with reference to the maximum or minimum value of the specific range. In particular, the reference value can be modified taking into account the standard variation, acceptable error ranges, specificity, or sensitivity of the reference value obtained under various conditions.

[0410] According to one embodiment of the present invention, the reference value can be obtained under the same reaction conditions used in the sample, including components (enzymes or amplification primers, if used), buffer pH, and reaction process. According to one embodiment of the present invention, the reference value can be obtained using a signal amplification process with or without nucleic acid amplification.

[0411] According to one embodiment of the present invention, if there is a significant difference between the reference value and the difference obtained to determine the presence of the first or second target nucleic acid, it is determined that the first or second target nucleic acid is present.

[0412] The second target nucleic acid is determined to be present if there is a significant difference between the reference value reflecting the signal change at the third detection temperature and the second detection temperature for the third target nucleic acid and the difference obtained to determine the presence of the second target nucleic acid.

[0413] Furthermore, the first target nucleic acid is determined to be present when there is a significant difference between a reference value reflecting a signal change at the second and first detection temperatures for the second target nucleic acid, a reference value reflecting a signal change at the third and first detection temperatures for the third target nucleic acid, and the difference obtained to determine the presence of the first target nucleic acid. The reference value may be expressed as a value of the same type as the difference obtained to determine the presence of the corresponding target nucleic acid (e.g., a ratio of endpoint values ​​of signal intensity).

[0414] Alternatively, the reference value may be used to calculate the difference between the signals obtained at the first, second, and third detection temperatures. For example, the difference for determining the presence of a first target nucleic acid may be calculated by multiplying (or dividing) the signal (e.g., RFU) detected at the third detection temperature by a reference value reflecting the signal change for the third target nucleic acid at the third and first detection temperatures, subtracting the result from the signal (e.g., RFU) detected at the first detection temperature, multiplying (or dividing) the signal at the third detection temperature by a reference value reflecting the signal change for the third target nucleic acid at the third and second detection temperatures in the same manner, subtracting the result from the signal detected at the second detection temperature, multiplying (or dividing) this result by a reference value reflecting the signal change for the second target nucleic acid at the second and first detection temperatures, and then subtracting the result from the signal detected at the first detection temperature. If the difference is greater (or less) than "0" or a predetermined value, it may be determined that the first target nucleic acid is present.

[0415] According to one embodiment, the predetermined value may serve as the threshold value.

[0416] According to an embodiment, when a threshold value is obtained using a reference value for determining the presence of a specific target nucleic acid, the threshold value may be obtained using a specific reference value or some of the reference values ​​obtained from various combinations of target nucleic acids. For example, when the third target nucleic acid is analyzed to be absent, a reference value obtained from a combination of target nucleic acids that does not include the third target nucleic acid is used to determine the threshold.

[0417] In one embodiment, to confirm the presence of a specific target nucleic acid, a standard sample containing a combination of target nucleic acids other than the specific target nucleic acid is prepared in advance, and a reference value is obtained. Taking the reference value into consideration, a threshold value is obtained for determining the presence of the target nucleic acid having the specific detection temperature.

[0418] According to one embodiment, if the signal measured at the third detection temperature shows significance, or if the difference between the signals detected at each detection temperature is obtained by a mathematical process, the reference value is used to determine the presence of the first target nucleic acid and the second target nucleic acid.

[0419] According to one embodiment, when a signal is generated in real time in association with target amplification by PCR, mathematical processing of the signal includes calculating the ratio between the signal intensities measured at each detection temperature in every amplification cycle, and the result of the calculation is plotted against cycles and used to determine the presence of the first and second target nucleic acids.

[0420] According to one embodiment, the Ct value is the signal for target detection, where the signal is generated in real time in conjunction with target amplification by PCR.

[0421] The Ct value for the first target nucleic acid generated by the first oligonucleotide may be determined using the signal measured at the first detection temperature, the signal measured at the second detection temperature, and the signal measured at the third detection temperature, which can be explained by the following example: first, real-time PCR is performed on the sample to be analyzed to obtain signals at each detection temperature, and then amplification curves for the three detection temperatures are obtained.

[0422] For example, if there is no Ct value for the third target nucleic acid in detection at a third detection temperature, it can be determined that the third target nucleic acid is not present. Then, the Ct value for the second target nucleic acid is calculated from the amplification curve obtained at the second detection temperature. If the second target nucleic acid is also not present, there is no Ct value for the second target nucleic acid. Then, the Ct value for the first target nucleic acid is calculated from the amplification curve obtained at the first detection temperature. If the first target nucleic acid is also not present, there is no Ct value for the first target nucleic acid. The confirmation order may vary depending on the signal generation characteristics due to temperature changes.

[0423] On the other hand, if a Ct value for the third target nucleic acid is present, the ratio of the RFU values ​​obtained at the first and second detection temperatures to the RFU value obtained at the third detection temperature in the cycle showing the Ct value is calculated. The ratio of the RFU values ​​obtained in cycles after the cycle showing the Ct value is also calculated. For example, if the ratio of all RFU values ​​is smaller than a reference value (e.g., the value obtained using the third nucleic acid sequence as described above), it is determined that the first and second target nucleic acids are not present. Therefore, there are no Ct values ​​for the first and second target nucleic acids.

[0424] If the calculated ratio is equal to the reference value, the determination may be made arbitrarily. For example, the above example states that the determination is made considering whether the ratio is less than or greater than the reference value. Alternatively, the determination may be made considering whether the ratio is below or above the reference value.

[0425] Alternatively, the Ct values ​​of the first target nucleic acid and the second target nucleic acid may be calculated as follows: after calculating the ratio between the RFU values ​​obtained at the first detection temperature, the second detection temperature and the third detection temperature in every cycle; the Ct value is calculated taking into account a threshold value.

[0426] Alternatively, the Ct value of the second target nucleic acid may be calculated as follows: the RFU value obtained at the third detection temperature for each cycle is modified using the reference value for each cycle; and then the ratio of the RFU value obtained at the second detection temperature to the modified RFU value for each cycle is calculated; and then the Ct value is calculated.

[0427] When calculating the Ct value of the first target nucleic acid in this manner, it is necessary to take into account both the RFU value obtained at the third detection temperature and the RFU value obtained at the second detection temperature, modify the RFU value obtained at the third detection temperature, and calculate the ratio of the RFU values ​​obtained at the first detection temperature and the second detection temperature.

[0428] According to one embodiment of the present invention, using the signals and differences measured at the first detection temperature, the second detection temperature, and the third detection temperature includes obtaining a qualifying value for determining the presence of each target nucleic acid.

[0429] According to one embodiment of the present invention, utilizing the difference includes obtaining a qualitative value for determining the presence of the first target nucleic acid and / or the second target nucleic acid, and the qualitative value is obtained by (i) mathematically processing the signals measured at the third detection temperature, the second detection temperature, and the first detection temperature, or (ii) using the detected signals themselves.

[0430] The qualitative value may be further processed mathematically to obtain a transformed value, which is used to determine the presence of the target nucleic acid in the sample.

[0431] According to one embodiment of the present invention, step (c) may utilize at least one reference value to determine the presence of at least one target nucleic acid, for example, the reference value may be utilized to determine the presence of the first target nucleic acid and / or the second target nucleic acid.

[0432] As described above, the reference value may be a value calculated in various ways. For example, the reference value may be a change in a signal generated by a first oligonucleotide set at a first detection temperature and a signal generated at a second detection temperature in the presence of a first target nucleic acid, a change in a signal generated by a first oligonucleotide set at a first detection temperature and a signal generated at a third detection temperature in the presence of a first target nucleic acid, a change in a signal generated by a first oligonucleotide set at a second detection temperature and a signal generated at a third detection temperature in the presence of a first target nucleic acid, a change in a signal generated by a second oligonucleotide set at a first detection temperature and a signal generated at a second detection temperature in the presence of a second target nucleic acid, a change in a signal generated by a second oligonucleotide set at a first detection temperature and a signal generated at a third detection temperature in the presence of a second target nucleic acid, or a change in a signal generated by a second oligonucleotide set at a first detection temperature and a signal generated at a third detection temperature in the presence of a second target nucleic acid. The reference value may be any one of the following values ​​reflecting a change in the signal generated by the third oligonucleotide set at the first detection temperature and the signal generated by the second oligonucleotide set at the third detection temperature in the presence of the second target nucleic acid; a change in the signal generated by the third oligonucleotide set at the first detection temperature and the signal generated by the third oligonucleotide set at the third detection temperature in the presence of the third target nucleic acid; a change in the signal generated by the third oligonucleotide set at the first detection temperature and the signal generated by the third oligonucleotide set at the third detection temperature in the presence of the third target nucleic acid; and a change in the signal generated by the third oligonucleotide set at the second detection temperature and the signal generated by the third oligonucleotide set at the third detection temperature in the presence of the third target nucleic acid. Some of these reference values ​​can be selected and used depending on the method for determining the presence of the target nucleic acid.

[0433] A feature of the present invention is that it utilizes a reference value that reflects the change in the signal generated by the third oligonucleotide set at the second detection temperature and the signal generated at the third detection temperature in the presence of the third target nucleic acid, a reference value that reflects the change in the signal generated by the third oligonucleotide set at the first detection temperature and the signal generated at the third detection temperature in the presence of the third target nucleic acid, and a reference value that reflects the change in the signal generated by the second oligonucleotide set at the first detection temperature and the signal generated at the second detection temperature in the presence of the second target nucleic acid.

[0434] The present inventors have developed an optimal method for determining the presence of each target nucleic acid based on signals measured at different temperatures when multiple target nucleic acids are detected simultaneously. Specifically, they have confirmed that the presence or absence of each target nucleic acid can be effectively determined while minimizing false positive / false negative errors when signals are extracted using the above three types of reference values ​​among various reference values.

[0435] In one embodiment, the extracted signal provided using the reference value and the measured signal can be used to determine the presence of at least one target nucleic acid.

[0436] In certain embodiments, the presence of a first target nucleic acid can be determined from an extracted signal for the first target nucleic acid at a first detection temperature, and the presence of a second target nucleic acid can be determined from an extracted signal for the second target nucleic acid at a second detection temperature.

[0437] As described above, the presence of the third target nucleic acid can be determined by confirming the significance of a signal measured at a third detection temperature where only a single signal from the third target nucleic acid can be detected, including when the absence of the third target nucleic acid includes when only a signal having an intensity similar to the background signal is detected at the third detection temperature.

[0438] In contrast, the signal for the first target nucleic acid may be mixed with the signal for the second target nucleic acid and the signal for the third target nucleic acid at the first detection temperature, and the signal for the second target nucleic acid may be mixed with the signal for the third target nucleic acid at the second detection temperature. Therefore, in order to confirm the presence of each target nucleic acid using the detection signal, it is necessary to use the above-mentioned difference and reference value to extract the signals for each of the first and second target nucleic acids.

[0439] Meanwhile, in the case of a first target nucleic acid, since the first oligonucleotide set provides a signal only at the first detection temperature, the signal for the first target nucleic acid may be extracted only from the signal measured at the first detection temperature. In contrast, in the case of a second target nucleic acid, since the second oligonucleotide set provides a signal at both the first detection temperature and the second detection temperature, the signal for the second target nucleic acid may be extracted from the signal measured at the first detection temperature or the signal measured at the second detection temperature. In this regard, the present inventors have confirmed that determining the presence of the second target nucleic acid at the second detection temperature rather than the first detection temperature minimizes errors in the reference values ​​and mathematical processing processes obtained by the various methods described above, thereby deriving more reliable results. Thus, a method for determining the presence of the first target nucleic acid at the first detection temperature, the second target nucleic acid at the second detection temperature, and the third target nucleic acid at the third detection temperature has been established.

[0440] In one embodiment, the extracted signal for the second target nucleic acid at the second detection temperature may be provided using a reference value that reflects the signal generated by the third oligonucleotide set at the second temperature in the presence of the third target nucleic acid and the change in the signal generated at the third detection temperature.

[0441] For example, the extracted signal for the second target nucleic acid at the second detection temperature can be provided by processing the signal measured at the third detection temperature and a reference value for the third target nucleic acid at the second and third detection temperatures, and subtracting the signal for the third target nucleic acid from the signal measured at the second detection temperature. In other words, the signal for the third target nucleic acid at the second detection temperature can be obtained by processing the signal measured at the third detection temperature with a reference value that reflects the change in the signal for the third target nucleic acid at the second and third detection temperatures.

[0442] In one embodiment, an extraction signal (eS T2 ) can be treated to provide the following formula I:

[0443] <Formula I> eS T2 =RFU D2 -RV T3(D2D3) *RFU D3

[0444] eS T2 : an extraction signal for a second target nucleic acid at a second detection temperature RFU D2 : Signal measured at the second temperature RV T3(D2D3) : a reference value reflecting the change in signal at the second detection temperature and the third detection temperature of the third target nucleic acid RFU D3 : Signal measured at the third temperature

[0445] The term "reference value processing" refers to mathematical processing of reference values ​​for measured signals. When confirming the presence of each target within the same experiment, various signal extraction methods using reference value application using various mathematical formulas, including multiplication or division, may be applied in the same way.

[0446] Additionally, the extracted signal for the first target nucleic acid at the first detection temperature may be provided by subtracting (i) the signal for the third target nucleic acid at the first detection temperature and (ii) the signal for the second target nucleic acid at the first detection temperature from the signal measured at the first detection temperature.

[0447] In one embodiment, an extraction signal (eS T1 ) can be treated to provide the following formula II:

[0448] <Formula II> eS T1 =RFU D1 -eS T2 -eS T3

[0449] eS T1 : an extraction signal for a first target nucleic acid at a first detection temperature RFU D1 : Signal measured at the first temperature eS T2 : an extraction signal for a second target nucleic acid at a first detection temperature eS T3 : Extraction signal for the third target nucleic acid at the first detection temperature

[0450] In one embodiment, the extracted signal for the first target nucleic acid at the first detection temperature may be provided using three reference values: a reference value reflecting the signal generated by the third oligonucleotide set at the second detection temperature in the presence of the third target nucleic acid and the change in the signal generated at the third detection temperature; a reference value reflecting the signal generated by the third oligonucleotide set at the first detection temperature in the presence of the third target nucleic acid and the change in the signal generated at the third detection temperature; and a reference value reflecting the signal generated by the second oligonucleotide set at the first detection temperature in the presence of the second target nucleic acid and the change in the signal generated at the second detection temperature.

[0451] In one embodiment, to remove the signal generated by the third target nucleic acid from the signal measured at the first detection temperature, a reference value reflecting the change in the signal generated by the third oligonucleotide set at the first detection temperature and the signal generated at the third detection temperature in the presence of the third target nucleic acid can be used to obtain the signal for the third target nucleic acid at the first detection temperature. For example, the signal for the third target nucleic acid at the first detection temperature can be obtained by multiplying the signal measured at the third detection temperature by the reference values ​​for the third target nucleic acid at the first and third detection temperatures.

[0452] Meanwhile, to remove the signal generated by the third target nucleic acid from the signal measured at the first detection temperature, the signal generated by the third target nucleic acid at the second detection temperature, but not at the third detection temperature, can be removed. For example, (i) the signal measured at the third detection temperature is multiplied by a reference value reflecting the signal change of the third target nucleic acid at the second and third detection temperatures, and the result is subtracted from the signal measured at the second detection temperature. (ii) The signal generated by the third target nucleic acid at the first detection temperature can be obtained by multiplying the signal generated by the third target nucleic acid at the second detection temperature obtained in (i) by a reference value reflecting the signal change of the third target nucleic acid at the first and second detection temperatures. This may result in errors during fine correction and plotting of the signal and reference value when the method is actually implemented by a program to realize a signal indicating the presence of a target nucleic acid. Therefore, the present invention determines the presence of a first target nucleic acid without using reference values ​​for the third target nucleic acid at the first and second detection temperatures.

[0453] In one embodiment, to remove the signal generated by the second target nucleic acid from the signal measured at the first detection temperature, it is necessary to first extract the signal generated by the second target nucleic acid at the second detection temperature. This may be the same as the method for extracting a signal to determine the presence of the second target nucleic acid described above. For example, the extracted signal for the second target nucleic acid at the second detection temperature may be obtained by treating the signal measured at the third detection temperature with the second detection temperature and a reference value for the third target nucleic acid at the third detection temperature, and then removing the extracted signal for the third target nucleic acid from the signal measured at the second detection temperature. Then, the signal for the second target nucleic acid at the first detection temperature may be obtained using the extracted signal for the second target nucleic acid at the second detection temperature and the reference value for the second target nucleic acid at the first detection temperature and the second detection temperature.

[0454] In one embodiment, an extraction signal (eS T1 ) can be treated to provide the following formula III:

[0455] <Formula III> eS T1 =RFU‐ D1 -(RFU D2 -RV T3(D2D3) *RFU D3 )-RV T3(D1D3) *RFU D3

[0456] eS T1 : an extraction signal for a first target nucleic acid at a first detection temperature RFU D2 : Signal measured at the second temperature RV T3(D2D3) : a reference value reflecting the change in signal at the second detection temperature and the third detection temperature of the third target nucleic acid RFU D3 : Signal measured at the third temperature RV T3(D1D3) : a reference value reflecting the change in signal of the third target nucleic acid at the first detection temperature and the third detection temperature RFU D3 : Signal measured at the third temperature

[0457] In certain embodiments, if it is previously determined that the third target nucleic acid is not present, the presence of the second target nucleic acid may be determined from the signal measured at the second detection temperature. In the absence of the third target nucleic acid, only the second target nucleic acid can provide a signal at the second detection temperature, and therefore, the presence of the second target nucleic acid can be determined by determining the significance of the detection of the signal measured at the second detection temperature.

[0458] In certain embodiments, when it is previously determined that the second target nucleic acid is not present, the extracted signal for the first target nucleic acid at the first detection temperature may be obtained by processing the signal measured at the third detection temperature and the reference values ​​for the third target nucleic acid at the first and third detection temperatures, and subtracting the extracted signal for the third target nucleic acid at the first detection temperature from the signal measured at the first detection temperature.

[0459] II. Target Nucleic Acid Detection Kit

[0460] In yet another aspect of the invention, the invention provides a kit for detecting three target nucleic acids in a sample, comprising:

[0461] (a) a first oligonucleotide set for a first target nucleic acid, a second oligonucleotide set for a second target nucleic acid, and a third oligonucleotide set for a third target nucleic acid;

[0462] Each of the oligonucleotide sets comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that serves to generate a signal in the presence of the target nucleic acid and that is bound to a fluorescent label, wherein the amplification oligonucleotide and the signaling oligonucleotide are the same or different, and each oligonucleotide set commonly comprises a single type of fluorescent label;

[0463] the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid;

[0464] (b) Instructions describing one embodiment of the method.

[0465] Since the kits of the present invention are manufactured for carrying out the methods of the present invention, the common content between the two will be omitted to avoid excessive duplication that would otherwise complicate this specification.

[0466] Any of the kits of the present invention described above may optionally include reagents necessary to perform a target amplification PCR 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 to inhibit DNA polymerase activity. The kit may also include reagents necessary to perform positive and negative control reactions. The optimal amounts of reagents to be used in a particular reaction may be readily determined by one of skill in the art having the benefit of this disclosure. The components of the kit may be present in separate containers, or multiple components may be present in a single container.

[0467] The instructions for describing or carrying out the methods of the invention may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper and plastic. In other embodiments, the instructions may be present as an electronic storage data file present on a suitable computer-readable recording medium, such as a CD-ROM or diskette. In still other embodiments, no substantive instructions are included in the kit, but means for obtaining the instructions from a remote source, for example, via the internet, are provided. One such embodiment is a kit that includes a web address where the instructions can be read and / or from which the instructions can be downloaded.

[0468] III. Recording Media and Devices for Detection of Target Nucleic Acids

[0469] The recording medium, device, and computer program of the present invention described below are for executing the present invention on a computer, and common content between them will be omitted to avoid excessive duplication that would complicate this specification.

[0470] In another aspect of the invention, the invention provides a computer-readable medium comprising instructions embodying a processor for performing a method for detecting three target nucleic acids in a sample, the method comprising:

[0471] The method comprises the steps of:

[0472] (a) receiving signals measured at a first detection temperature, a second detection temperature, and a third detection temperature, wherein a first oligonucleotide set is directed to a first target nucleic acid, a second oligonucleotide set is directed to a second target nucleic acid, and a third oligonucleotide set is directed to a third target nucleic acid;

[0473] Each of the oligonucleotide sets comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that serves to generate a signal in the presence of the target nucleic acid and that is bound to a fluorescent label, wherein the amplification oligonucleotide and the signaling oligonucleotide are the same or different, and each oligonucleotide set commonly comprises a single type of fluorescent label;

[0474] the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid;

[0475] the first detected temperature, the second detected temperature, and the third detected temperature are different from each other;

[0476] (b) determining the presence of each target nucleic acid based on signals measured at the first detection temperature, the second detection temperature, and the third detection temperature;

[0477] The presence of the first target nucleic acid is determined by the difference between the signals measured at the first, second and third detection temperatures, the presence of the second target nucleic acid is determined by the difference between the signals measured at the second and third detection temperatures, and the presence of the third target nucleic acid is determined by the signal measured at the third detection temperature.

[0478] According to one embodiment, specific reference values ​​are used to determine the presence of the three target nucleic acids.

[0479] According to an embodiment of the present invention, the reference value is stored in a computer-readable recording medium. According to an embodiment of the present invention, the computer-readable recording medium includes instructions for inputting the reference value during execution of the method. According to an embodiment of the present invention, the computer-readable recording medium further includes instructions for causing a processor to execute a method for obtaining the reference value.

[0480] In yet another aspect of the present invention, there is provided a computer program storable on a computer readable recording medium embodying a processor for carrying out a method for detecting three target nucleic acids in a sample, said method comprising the steps of:

[0481] (a) receiving signals measured at a first detection temperature, a second detection temperature, and a third detection temperature, wherein a first oligonucleotide set is directed to a first target nucleic acid, a second oligonucleotide set is directed to a second target nucleic acid, and a third oligonucleotide set is directed to a third target nucleic acid;

[0482] Each of the oligonucleotide sets comprises (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that serves to generate a signal in the presence of the target nucleic acid and that is bound to a fluorescent label, wherein the amplification oligonucleotide and the signaling oligonucleotide are the same or different, and each oligonucleotide set commonly comprises a single type of fluorescent label;

[0483] the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second, and third detection temperatures in the presence of a third target nucleic acid;

[0484] the first detected temperature, the second detected temperature, and the third detected temperature are different from each other;

[0485] (b) determining the presence of each target nucleic acid based on signals measured at the first detection temperature, the second detection temperature, and the third detection temperature;

[0486] The presence of the first target nucleic acid is determined by the difference between the signals measured at the first, second and third detection temperatures, the presence of the second target nucleic acid is determined by the difference between the signals measured at the second and third detection temperatures, and the presence of the third target nucleic acid is determined by the signal measured at the third detection temperature.

[0487] The program instructions, when executed by the processor, operate to cause the processor to perform the method of the invention described above. The program instructions may include instructions for receiving a signal and for using the received signal to determine the presence of the three target nucleic acid sequences.

[0488] The methods of the present invention described above are implemented on a processor, for example, a stand-alone computer, a network attached computer, or a processor located in a data acquisition device such as a real-time PCR instrument.

[0489] The computer-readable recording medium includes various recording media such as CD-R, CD-ROM, DVD, flash memory, floppy disk, hard drive, portable HDD, USB, magnetic tape, MINIDISC, non-volatile memory card, EEPROM, optical disk, optical recording medium, RAM, ROM, system memory, and web server.

[0490] Data associated with the signal (e.g., intensity, amplification cycle number, and detection temperature) may be received by several devices. For example, the data may be collected by a processor in a PCR data collection device. The data may be provided in real time as the data is collected, or may be stored in a memory unit or buffer and provided to the processor after the experiment is completed. Similarly, the data set may be provided to another system, such as a desktop computer system, via a network connection (e.g., LAN, VPN, Internet, and Intranet) or direct connection (e.g., USB or other direct wired or wireless connection) with the collection device, or on portable media such as a CD, DVD, floppy disk, portable HDD, or stand-alone computer system. Similarly, the data set may be provided to a client, such as a laptop or desktop computer system, via a network connection (e.g., LAN, VPN, Internet, Intranet, and wireless communication network) to a server system. After the data is received or collected, if the signal is detected at the elevated detection temperature, the data analysis process provides a processed (extracted) signal derived from the difference between the signals to determine the presence of the target nucleic acid sequence. The processor processes the received data associated with the signals and provides a processed signal that reflects the difference between the signals at the three sensed temperatures.

[0491] Instructions for implementing a processor to carry out the present invention may be included in a logic system. The instructions may be provided on any software storage medium, such as a portable HDD, USB, floppy disk, CD, or DVD, or may be downloadable or stored in a memory module (e.g., a hard drive or other memory, such as local or attached RAM or ROM). Computer code for carrying out the present invention may be implemented in a variety of coding languages, such as C, C++, Java, Visual Basic, VBScript, JavaScript, Perl, and XML. Additionally, a variety of languages ​​and protocols may be used for external and internal storage and transmission of data and instructions according to the present invention.

[0492] In yet another aspect, the present invention provides an apparatus for detecting a target nucleic acid in a sample using different detection temperatures, comprising: (a) a computer processor; and (b) a computer-readable recording medium coupled to the computer processor.

[0493] According to one embodiment, the device further includes a reaction vessel capable of accommodating a sample and an oligonucleotide set, a temperature control means for controlling the temperature of the reaction vessel, and / or a detector including a single type of detection channel capable of detecting a signal.

[0494] According to one embodiment, the processor may be implemented by installing software in a conventional device for detecting target nucleic acids (e.g., a real-time PCR device). According to one embodiment, the device includes a processor that detects signals and extracts only signals corresponding to each target nucleic acid from the signals.

[0495] The present invention will be described in more detail using examples. It will be apparent to those skilled in the art that these examples are provided to more specifically explain the present invention, and that the scope of the present invention as defined in the appended claims is not limited by these examples.

[0496] Example

[0497] Example 1: Determining the presence of 14 HPV types and an internal control group using signals measured at three temperatures

[0498] The inventors conducted an experiment to determine whether 15 target nucleic acids (14 HPV types and an internal control group) could be detected in one reaction vessel using PTOCE real-time PCR, which includes five detection channels and signal detection at three different temperatures.

[0499] <1-1> Amplification and signal detection of 14 HPV types and one internal control group

[0500] Taq DNA polymerase, which has 5' nuclease activity, was used to extend the forward and reverse primers, cleave the PTO, and extend the PTO fragment.

[0501] Genomic DNA of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and an internal control group was used as target nucleic acid.

[0502] PTOCE real-time PCR was used to detect the target nucleic acid.

[0503] In this example, five detection channels were used to detect five fluorescent signals, and the fluorescent labels used to generate the fluorescent signals were FAM (Biosearch Technologies Inc.), CAL Fluor Orange 560 (Biosearch Technologies Inc.), CAL Fluor Red 610 (Biosearch Technologies Inc.), ATTO 647N (ATTO-TEC GmbH), and ATTO 680 (ATTO-TEC GmbH).

[0504] The signal detection temperatures for each channel were 60°C, 72°C, and 83°C, and the HPV types assigned to each detection temperature for each channel are shown in Table 1 below.

[0505] [Table 1]

[0506] As can be seen in Table 1, for a target nucleic acid assigned a detection temperature of 60°C, the CTO used in the PTOCE method was designed to form a duplex to provide a signal at the detection temperature of 60°C, but not to form a duplex and dissociate to provide a signal at the detection temperatures of 72°C and 83°C; for a target nucleic acid assigned a detection temperature of 72°C, the CTO used in the PTOCE method was designed to form a duplex to provide a signal at the detection temperatures of 60°C and 72°C, but not to form a duplex and dissociate to provide a signal at the detection temperature of 83°C; for a target nucleic acid assigned a detection temperature of 83°C, the CTO used in the PTOCE method was designed to form a duplex to provide a signal at all detection temperatures of 60°C, 72°C, and 83°C.

[0507] Therefore, at a detection temperature of 83°C, only the signal for the target nucleic acid assigned to the detection temperature of 83°C will be generated and detected; at a detection temperature of 72°C, not only the signal for the target nucleic acid assigned to the detection temperature of 72°C but also the signal for the target nucleic acid assigned to the detection temperature of 83°C will be generated and detected in an undifferentiated form, i.e., a mixed form; at a detection temperature of 60°C, the signal for the target nucleic acid assigned to the detection temperature of 83°C, the signal for the target nucleic acid assigned to the detection temperature of 72°C, and the signal for the target nucleic acid assigned to the detection temperature of 60°C will be generated and detected in an undifferentiated form, i.e., a mixed form.

[0508] The 3' ends of the PTO and CTO are blocked with a carbon spacer to prevent their extension, and the CTO is labeled with a quencher molecule (BHQ-2) at its 5' end and a fluorescent reporter molecule (FAM, CAL Fluor Orange 560, CAL Fluor Red 610, ATTO 647N, or ATTO 680) at its templating site.

[0509] One reaction tube containing all HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and an internal control was prepared.

[0510] The sequences of the forward primer, reverse primer, PTO and CTO used in this example are shown in Table 2 below.

[0511] [Table 2] JPEG2025513004000014.jpg90169

[0512] Real-time PCR was performed in a final volume of 20 μl containing target nucleic acids (10 pg each of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and the internal control), 1 to 3.2 pmoles of forward primers for amplification and detection of each target nucleic acid, 1 to 3.2 pmoles of reverse primers, 0.8 to 2.8 pmoles of PTO, 0.2 to 0.6 pmoles of CTO, and 10 μl of 2X master mix (final 200 μM dNTPs, 2 mM MgCl2, and 2 U Taq DNA polymerase).

[0513] The tube containing the reaction mixture was placed in a real-time thermocycler (CFX96, Bio-Rad) and denatured at 95°C for 15 minutes, followed by 45 cycles of 95°C for 3 seconds, 60°C for 10 seconds, 72°C for 10 seconds, and 83°C for 5 seconds. Signal detection was performed at 60°C, 72°C, and 83°C for each cycle.

[0514] The signals measured for each detection channel at 60°C, 72°C and 83°C for each cycle of the PCR reaction are shown in Figures 1, 3, 5, 7 and 9.

[0515] As shown in Figures 1, 3, 5, 7, and 9, the signals measured at a detection temperature of 60°C for each detection channel contained signals for three HPV types, and the signals measured at a detection temperature of 72°C contained signals for two HPV types. Therefore, it was not possible to immediately determine the presence of each HPV type from these measured signals.

[0516] <1-2> Identification of 14 HPV types and one internal control group

[0517] In this example, a method using a reference value for each target and signals from three detection temperatures (83°C, 72°C, and 60°C) provides an amplification curve showing an extracted signal that can confirm the presence of each target nucleic acid. Specifically, the present invention determines the presence or absence of the first and second targets based on the extracted signal for each target extracted using the reference value. In the case of the third target, the signal at 83°C contains only the signal generated by the third oligonucleotide set, so the presence or absence of the third target nucleic acid is determined from the signal measured at the third detection temperature (83°C).

[0518] The reference values ​​for each target were calculated using the signals from single samples of HPV 16, 18, 31, 35, 39, 45, 52, 58, 59, and 68, and are shown in Tables 3 to 5.

[0519] [Table 3]

[0520] [Table 4]

[0521] [Table 5]

[0522] To distinguish the presence of the three target nucleic acids assigned to each channel, the signals measured at each detection temperature and the target-specific extracted signals obtained from these were used. Specifically, for HPV 58, HPV 16, HPV 52, HPV 18, and HPV 31, which generate signals at 60°C, 72°C, and 83°C, the presence or absence of the target was confirmed based on the signal measured at 83°C without a separate signal extraction process.

[0523] On the other hand, for HPV 45, HPV 59, HPV 39, HPV 35, and HPV 68, which generate signals only at 60°C and 72°C, the presence or absence of targets was confirmed based on the signals extracted for each channel. The extracted signals were obtained by multiplying the signal measured at 83°C by a reference value reflecting the signal change at 72°C and 83°C listed in Table 5 (targets that generate signals at 60°C, 72°C, and 83°C), and subtracting this result from the signal measured at 72°C. The reference value was obtained by calculating the ratio of the 72°C signal to the 83°C signal for a standard sample containing only one target nucleic acid that generates signals at 60°C, 72°C, and 83°C.

[0524] Specifically, the extracted signal for HPV 45 at 72°C was obtained by multiplying the signal measured at 83°C in the FAM channel by the reference value of HPV 58, 1.16, and then subtracting the result from the signal measured at 72°C. The extracted signal for HPV 59 at 72°C was obtained by multiplying the signal measured at 83°C in the CAL Fluor Orange 560 channel by the reference value of HPV 16, 1.02, and then subtracting the result from the signal measured at 72°C. The extracted signal for HPV 39 at 72°C was obtained by multiplying the signal measured at 83°C in the CAL Fluor Red 610 channel by the reference value of HPV 52, 1.05, and then subtracting the result from the signal measured at 72°C. The extracted signal for HPV 35 at 72°C was obtained by multiplying the signal measured at 83°C in the ATTO 647N channel by the reference value of HPV 18, 1.05, and then subtracting this from the signal measured at 72°C. The extracted signal for HPV 68 at 72°C was obtained by multiplying the signal measured at 83°C in the ATTO 680 channel by the reference value of HPV 31, 1.11, and then subtracting this from the signal measured at 72°C.

[0525] In addition, for HPV 66, HPV 51, HPV 33, the internal control, and HPV 56, which only generate signals at 60°C, the signals of other targets were removed using the signals measured at 83°C, the signals measured at 72°C, and the respective reference values ​​from the signal measured at 60°C in the corresponding channel, and then the presence or absence of HPV 66, HPV 51, HPV 33, the internal control, and HPV 56 was confirmed. This is explained in detail as follows:

[0526] First, the extracted signal was obtained by multiplying the signal measured at 83°C by a reference value reflecting the change in signal at 72°C and 83°C listed in Table 5 (targets that generate signals at 60°C, 72°C, and 83°C), and subtracting this result from the signal measured at 72°C. The reference value was obtained by calculating the ratio of the 72°C signal to the 83°C signal for a standard sample containing only one target nucleic acid that generates signals at 60°C, 72°C, and 83°C.

[0527] Next, the extracted signal for the target that generates a signal only at 60°C and 72°C was multiplied by a reference value reflecting the signal change at 60°C and 72°C listed in Table 3 (targets that generate a signal at 60°C and 72°C), and this result was subtracted from the signal measured at 60°C. Subsequently, the signal measured at 83°C was multiplied by a reference value reflecting the signal change at 60°C and 83°C listed in Table 4 (targets that generate a signal at 60°C, 72°C, and 83°C), and this result was further subtracted, and the presence or absence of the target was confirmed based on the extracted signal.

[0528] Specifically, at 60°C, the extracted signal for HPV 66 can be obtained in the FAM channel as follows:

[0529] First, the extracted signal for HPV 45 at 72°C was obtained by multiplying the signal measured at 83°C by the reference value of HPV 58, 1.16, and then subtracting it from the signal measured at 72°C. Second, the extracted signal for HPV 45 at 60°C was obtained by multiplying the extracted signal for HPV 45 at 72°C by the reference value of HPV 45, 1.28. Third, the extracted signal for HPV 58 at 60°C was obtained by multiplying the signal measured at 83°C by the reference value of HPV 58, 1.21. Fourth, the extracted signal for HPV 66 at 60°C was obtained by subtracting the extracted signal for HPV 45 at 60°C and the extracted signal for HPV 58 at 60°C from the signal measured at 60°C.

[0530] Also, the extracted signal for HPV 51 at 60°C can be obtained in the CAL Fluor Orange 560 channel as follows:

[0531] First, the extracted signal for HPV 59 at 72°C was obtained by multiplying the signal measured at 83°C by the HPV 16 reference value of 1.02 and then subtracting it from the signal measured at 72°C. Second, the extracted signal for HPV 59 at 60°C was obtained by multiplying the extracted signal for HPV 59 at 72°C by the HPV 59 reference value of 1.21. Third, the extracted signal for HPV 16 at 60°C was obtained by multiplying the signal measured at 83°C by the HPV 16 reference value of 1.00. Fourth, the extracted signal for HPV 51 at 60°C was obtained by subtracting the extracted signal for HPV 59 at 60°C and the extracted signal for HPV 16 at 60°C from the signal measured at 60°C.

[0532] Also, the extracted signal for HPV33 at 60°C can be obtained in the CAL Fluor Red 610 channel as follows:

[0533] First, the extracted signal for HPV 39 at 72°C was obtained by multiplying the signal measured at 83°C by the HPV 52 reference value of 1.05 and then subtracting it from the signal measured at 72°C. Second, the extracted signal for HPV 39 at 60°C was obtained by multiplying the extracted signal for HPV 39 at 72°C by the HPV 39 reference value of 1.09. Third, the extracted signal for HPV 52 at 60°C was obtained by multiplying the signal measured at 83°C by the HPV 52 reference value of 0.94. Fourth, the extracted signal for HPV 33 at 60°C was obtained by subtracting the extracted signal for HPV 39 at 60°C and the extracted signal for HPV 52 at 60°C from the signal measured at 60°C.

[0534] Also, the extracted signal for the internal control at 60°C can be obtained in the ATTO 647N channel as follows:

[0535] First, the extracted signal for HPV 35 at 72°C was obtained by multiplying the signal measured at 83°C by the HPV 18 reference value of 1.05 and then subtracting it from the signal measured at 72°C. Second, the extracted signal for HPV 35 at 60°C was obtained by multiplying the extracted signal for HPV 35 at 72°C by the HPV 35 reference value of 1.03. Third, the extracted signal for HPV 18 at 60°C was obtained by multiplying the signal measured at 83°C by the HPV 18 reference value of 1.00. Fourth, the extracted signal for the internal control at 60°C was obtained by subtracting the extracted signal for HPV 35 at 60°C and the extracted signal for HPV 18 at 60°C from the signal measured at 60°C.

[0536] Also, the extracted signal for HPV 56 at 60°C can be obtained in the ATTO 680 channel as follows:

[0537] First, the extracted signal for HPV 68 at 72°C was obtained by multiplying the signal measured at 83°C by the HPV 31 reference value of 1.11 and then subtracting it from the signal measured at 72°C. Second, the extracted signal for HPV 68 at 60°C was obtained by multiplying the extracted signal for HPV 68 at 72°C by the HPV 68 reference value of 1.01. Third, the extracted signal for HPV 31 at 60°C was obtained by multiplying the signal measured at 83°C by the HPV 31 reference value of 1.00. Fourth, the extracted signal for HPV 56 at 60°C was obtained by subtracting the extracted signal for HPV 68 at 60°C and the extracted signal for HPV 31 at 60°C from the signal measured at 60°C.

[0538] The signals extracted for each target in each detection channel using the above process are shown in Figures 2, 4, 6, 8, and 10. All RFU values ​​were derived from the "baseline subtracted curve fit" analysis data in the instrument software. Therefore, the signals for HPV 58, HPV 16, HPV 52, HPV 18, and HPV 31 were substantially identical to the signals measured at 83°C in Figures 1, 3, 5, 7, and 9, although there were slight differences due to fit analysis. To confirm the significance of the extracted signals, an appropriate threshold of 110 was selected based on the results of each individual sample.

[0539] As shown in Figure 2, the amplification curve extracted at 60°C confirmed the presence or absence of HPV 66, which generates a signal only at 60°C, and the amplification curve extracted at 72°C confirmed the presence or absence of HPV 45, which generates signals at both 60°C and 72°C. Furthermore, for HPV 58, which is designed to generate signals at all temperatures, 60°C, 72°C, and 83°C, the presence or absence of the target could be confirmed substantially based on the signal measured at 83°C.

[0540] 4, the amplification curve extracted at 60°C confirmed the presence or absence of HPV 51, which generates a signal only at 60°C, while the amplification curve extracted at 72°C confirmed the presence or absence of HPV 59, which generates signals at both 60°C and 72°C. Furthermore, with HPV 16, which is designed to generate signals at all temperatures, 60°C, 72°C, and 83°C, the presence or absence of the target could be confirmed substantially based on the signal measured at 83°C.

[0541] As shown in Figure 6, the amplification curve extracted at 60°C confirmed the presence or absence of HPV33, which generates a signal only at 60°C, and the amplification curve extracted at 72°C confirmed the presence or absence of HPV39, which generates signals at both 60°C and 72°C. Furthermore, with HPV52, which is designed to generate signals at all temperatures, 60°C, 72°C, and 83°C, the presence or absence of the target could be confirmed substantially based on the signal measured at 83°C.

[0542] As shown in Figure 8, the amplification curve extracted at 60°C confirmed the presence or absence of an internal control group that generates a signal only at 60°C, while the amplification curve extracted at 72°C confirmed the presence or absence of HPV 35, which generates signals at both 60°C and 72°C. Furthermore, for HPV 18, which is designed to generate signals at all temperatures, 60°C, 72°C, and 83°C, the presence or absence of the target could be confirmed essentially based on the signal measured at 83°C.

[0543] 10 , the amplification curve extracted at 60° C. confirmed the presence or absence of HPV 56, which generates a signal only at 60° C., and the amplification curve extracted at 72° C. confirmed the presence or absence of HPV 68, which generates signals at both 60° C. and 72° C. Furthermore, for HPV 31, which is designed to generate signals at all temperatures, 60° C., 72° C., and 83° C., the presence or absence of the target could be confirmed substantially based on the signal measured at 83° C.

[0544] The above results demonstrate that the signals measured at each detection temperature and the reference values ​​can be used to extract or distinguish effective signals for each target nucleic acid. Therefore, using real-time PCR involving signal detection at different temperatures and a single detection channel, three target nucleic acids can be detected in one reaction vessel, and by simultaneously analyzing five channels, the presence of 15 targets (including IC) can be determined in a more convenient and reliable manner.

[0545] Example 2: Confirmation of signal quality depending on the type of fluorescent label and temperature change

[0546] The present inventors conducted an experiment to determine whether the fluorescent labeling would enable the long wavelength detection channel to detect an appropriate signal at three different temperatures, including temperatures above 75°C.

[0547] Except for the change in fluorescent label, the target nucleic acid was amplified in the same manner as in Example <1-1>, and signals were detected at 60°C, 72°C, and 83°C. For HPV 18, an oligonucleotide set containing Quasar 670 fluorescent label and an oligonucleotide set containing ATTO 647N fluorescent label were used, respectively. All experiments were performed in duplicate.

[0548] Due to the characteristics of the oligonucleotide sets used in this example, as the detection temperature increases, the intensity of the signal generated by each oligonucleotide set may decrease somewhat due to dissociation of some of the dimers.

[0549] The results are shown in Figure 11.

[0550] Referring to Figure 11, the Quasar 670 fluorescently labeled oligonucleotide set exhibited significantly lower signal intensities across all detection temperatures compared to the ATTO 647N fluorescently labeled oligonucleotide set. Furthermore, the Quasar 670 fluorescently labeled oligonucleotide set exhibited relatively low average fluorescence intensity, confirming that the dye itself exhibited significant signal degradation due to temperature. In particular, the Quasar 670 fluorescently labeled oligonucleotide set generated a signal close to the threshold value even at 83°C, despite the presence of target nucleic acid. In contrast, the ATTO 647N fluorescently labeled oligonucleotide set exhibited only a slight decrease in fluorescence intensity with increasing temperature, and even at 83°C, the average fluorescence intensity exceeded 1700 RFU, demonstrating excellent long-wavelength fluorescence signal even at high temperatures.

[0551] Therefore, it was demonstrated that ATTO 647N, a carbopyronine dye, is more suitable for the present invention, which requires detecting signals at three temperatures and performing comparative analysis.

[0552] Although preferred embodiments of the present invention have been described in detail, it will be apparent to those skilled in the art that variations and modifications can be made in accordance with the principles of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting a target nucleic acid in a sample, comprising the steps of: (a) incubating a sample in a reaction vessel with a first set of oligonucleotides directed to a first target nucleic acid, a second set of oligonucleotides directed to a second target nucleic acid, and a third set of oligonucleotides directed to a third target nucleic acid; Each of the oligonucleotide sets includes (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that is bound to a fluorescent label and serves to generate a signal in the presence of the target nucleic acid, the amplification oligonucleotide and the signaling oligonucleotide being the same or different, and each oligonucleotide set includes a single type of fluorescent label in common; the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second and third detection temperatures in the presence of a third target nucleic acid; (b) measuring signals at a first detection temperature, a second detection temperature, and a third detection temperature; the first detection temperature, the second detection temperature, and the third detection temperature are different from each other; (c) determining the presence of each target nucleic acid based on signals measured at the first detection temperature, the second detection temperature, and the third detection temperature; The presence of the first target nucleic acid is determined by the difference between the signals measured at the first detection temperature, the second detection temperature, and the third detection temperature, the presence of the second target nucleic acid is determined by the difference between the signals measured at the second detection temperature and the third detection temperature, and the presence of the third target nucleic acid is determined by the signal measured at the third detection temperature.

2. The method of claim 1 , wherein the difference includes a difference obtained by mathematically processing the signals measured at each temperature.

3. 2. The method of claim 1, wherein the presence of the first target nucleic acid and the second target nucleic acid is determined using the difference and a reference value, the reference value reflecting a change in a signal provided by each oligonucleotide set in the presence of each target nucleic acid at two temperatures selected from a first detection temperature, a second detection temperature, and a third detection temperature.

4. 2. The method of claim 1, wherein the reference value is obtained by (i) incubating at least one of the first oligonucleotide set, the second oligonucleotide set, and the third oligonucleotide set with at least one of the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid in a reaction vessel different from the reaction vessel in step (a), (ii) measuring signals at at least two temperatures selected from the first detection temperature, the second detection temperature, and the third detection temperature, and then (iii) calculating the difference between the signals measured at the selected two temperatures.

5. 4. The method of claim 3, wherein at least one of three reference values ​​is used in step (c), the three reference values ​​being a reference value reflecting a change in a signal generated by the third oligonucleotide set at the second detection temperature and a change in a signal generated at the third detection temperature in the presence of a third target nucleic acid, a reference value reflecting a change in a signal generated by the third oligonucleotide set at the first detection temperature and a change in a signal generated at the third detection temperature in the presence of a third target nucleic acid, and a reference value reflecting a change in a signal generated by the second oligonucleotide set at the first detection temperature and a change in a signal generated at the second detection temperature in the presence of a second target nucleic acid.

6. 4. The method according to claim 3, characterized in that it utilizes an extracted signal provided by means of a reference value and a measured signal to determine the presence of at least one target nucleic acid.

7. 7. The method of claim 6, wherein the presence of the first target nucleic acid is determined from an extraction signal for the first target nucleic acid at a first detection temperature, and the presence of the second target nucleic acid is determined from an extraction signal for the second target nucleic acid at a second detection temperature.

8. The extracted signal for the second target nucleic acid at the second detection temperature is The method of claim 7, characterized in that a third oligonucleotide set is provided with a reference value reflecting a change in signal generated at a second temperature in the presence of a third target nucleic acid and a change in signal generated at a third detection temperature.

9. The method of claim 8, characterized in that the extracted signal for the second target nucleic acid at the second detection temperature is provided by removing the signal for the third target nucleic acid at the second detection temperature from the signal measured at the second detection temperature, and the signal for the third target nucleic acid at the second detection temperature is obtained by processing the signal measured at the third detection temperature with reference values ​​for the third target nucleic acid at the second detection temperature and the third detection temperature.

10. 8. The method of claim 7, wherein the extracted signal for the first target nucleic acid at the first detection temperature is provided by subtracting (i) the signal for the third target nucleic acid at the first detection temperature and (ii) the signal for the second target nucleic acid at the first detection temperature from the signal measured at the first detection temperature.

11. 11. The method of claim 10, wherein the extracted signal for the first target nucleic acid at the first detection temperature is provided using at least one of three reference values, the three reference values ​​being a reference value reflecting a change in the signal generated by the third oligonucleotide set at the second detection temperature and the signal generated at the third detection temperature in the presence of the third target nucleic acid, a reference value reflecting a change in the signal generated by the third oligonucleotide set at the first detection temperature and the signal generated at the third detection temperature in the presence of the third target nucleic acid, and a reference value reflecting a change in the signal generated by the second oligonucleotide set at the first detection temperature and the signal generated at the second detection temperature in the presence of the second target nucleic acid.

12. The method of claim 11, characterized in that the signal for the third target nucleic acid at the first detection temperature is obtained using a signal measured at the third detection temperature and a reference value for the third target nucleic acid at the first detection temperature and the third detection temperature, and the signal for the second target nucleic acid at the first detection temperature is obtained using an extraction signal for the second target nucleic acid at the second detection temperature and a reference value for the second target nucleic acid at the first detection temperature and the second detection temperature.

13. If the second target nucleic acid is determined to be absent, the extracted signal for the first target nucleic acid at the first detection temperature is The method of claim 7, characterized in that the signal for the third target nucleic acid at the first detection temperature is provided by removing the signal for the third target nucleic acid at the first detection temperature from the signal measured at the first detection temperature, and the signal for the third target nucleic acid at the first detection temperature is obtained by processing the signal measured at the third detection temperature with reference values ​​for the third target nucleic acid at the first detection temperature and the third detection temperature.

14. 2. The method of claim 1, wherein: (i) the first detected temperature is lower than the second detected temperature and the second detected temperature is lower than a third detected temperature; or (ii) the first detected temperature is higher than the second detected temperature and the second detected temperature is higher than the third detected temperature.

15. 2. The method according to claim 1, wherein the first, second and third detected temperatures are spaced from each other by 5° C. to 25° C. and are equal to or less than 90° C.

16. 15. The method of claim 14, wherein the first detection temperature is selected from 55°C to 70°C, the second detection temperature is selected from 65°C to 80°C, and the third detection temperature is selected from 75°C to 90°C.

17. 15. The method of claim 14, wherein the first detection temperature is selected from 75°C to 90°C, the second detection temperature is selected from 65°C to 80°C, and the third detection temperature is selected from 55°C to 70°C.

18. 2. The method of claim 1, wherein the fluorescent label contained in the oligonucleotide set is selected from the group consisting of FAM or a substitute thereof, CAL Fluor Orange 560 or a substitute thereof, CAL Fluor Red 610 or a substitute thereof, Quasar 670 or a substitute thereof, and Quasar 705 or a substitute thereof.

19. The method according to claim 1, wherein the highest detection temperature among the first detection temperature, the second detection temperature, and the third detection temperature is selected from the range of 75°C to 90°C.

20. The method according to claim 1, wherein the highest detection temperature among the first detection temperature, the second detection temperature and the third detection temperature is selected from the range of 80°C to 86°C.

21. The method according to claim 1, wherein the fluorescent label has an excitation wavelength in the range of 620 nm to 710 nm.

22. 22. The method of claim 21, wherein the fluorescent label is a carbopyronin-based compound.

23. The method according to claim 21 , wherein the compound is represented by the following formula 1: 【Chemistry 1】 In Formula 1, R １ , R ２ , R ３ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ and R １１ are each independently hydrogen, C １ ~C １０ Alkyl, C ３ ~C ７ Cycloalkyl, C ２ ~C １０ Alkenyl, C ６ ~C １０ monocyclic or bicyclic aryl or heteroaryl containing heteroatoms selected from N, O and S, phenyl, benzyl, tolyl, halogen, hydroxyl, thiol, amino, sulfonyl, sulfoxide, sulfonic acid, nitro, carbonyl, carboxyl, carboxamide, aldehyde, phospho, nitrile, isonitrile, isocyanate, thiocyanate or isothiocyanate, or alternatively R １ and R ８ , R ２ and R ９ , and / or R ６ and R １０ taken together with the carbon or nitrogen atom to which they are attached may be substituted or unsubstituted, optionally forming a ring containing multiple bonds; In this case, the alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, R １ and R ８ , R ２ and R ９ , and / or R ６ and R １０ the ring formed by may be unsubstituted or substituted with halogen, hydroxyl, thiol, amino, sulfo, nitro, carbonyl, carboxyl, carboxamide, aldehyde, phospho, nitrile, isonitrile, isocyanate, thiocyanate or isothiocyanate; R ４ is represented by the following chemical formula 2: 【Chemistry 2】 In the formula 2, X is hydrogen, C １ ~C １０ Saturated or unsaturated, substituted or unsubstituted, linear, branched or cyclic alkyl of C ２ ~C １０ substituted or unsubstituted, linear, branched or cyclic alkenyl of C(O)NR ａ R ｂ , C.H. ２ OR ａ , (CH ２ ) ｎ N.R. ａ R ｂ , (CH ２ ) ｎ N.R. ａ R ｂ , OR ａ , N.R. ａ R ｂ , S.O. ２ Ra, S.O. ２ N.H. ２ , S.O. ２ N.R. ａ２ is hydroxy or halogen; The R ａ is C １ ~C ６ Alkyl, C ３ ~C ７ cycloalkyl, or heterocyclyl containing a heteroatom selected from N, O and S, where the alkyl, cycloalkyl is unsubstituted or substituted with halogen; The R ｂ is hydrogen, halogen, hydroxyl, thiol, amino, sulfo, nitro, carbonyl, carboxyl, carboxamide, aldehyde, phospho, nitrile, isonitrile, isocyanate, thiocyanate or isothiocyanate, n is 1 or 2; m is an integer from 1 to 3.

24. The method according to claim 23, wherein the compound is any one selected from the group consisting of compounds represented by the following formulas 3 to 5 and combinations thereof: 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】

25. The method according to claim 21, wherein the fluorescent label exhibits stable fluorescent properties at a pH range of 2 to 11.

26. The method according to claim 21, wherein the fluorescence intensity of the fluorescent label at temperatures of 75°C to 95°C is 1.1 to 10 times that of Quasar 670.

27. 2. The method of claim 1, wherein the incubation is by PCR, real-time PCR or isothermal amplification.

28. 2. The method of claim 1, wherein at least one of the oligonucleotide sets generates a signal by duplex formation or dissociation from a signaling oligonucleotide that occurs depending on the presence of a target nucleic acid.

29. The method according to claim 1, characterized in that at least one of the oligonucleotide sets generates a signal by cleavage of a signaling oligonucleotide after hybridization with a target nucleic acid, the cleavage occurring depending on the presence of the target nucleic acid.

30. The method according to claim 1, wherein at least one of the oligonucleotide sets generates a signal by forming or dissociating a complex between a cleavage product and a signaling oligonucleotide after cleavage of a mediation oligonucleotide hybridized to the target nucleic acid, which occurs depending on the presence of the target nucleic acid.

31. The method of claim 1, wherein at least one of the oligonucleotide sets generates a signal by dissociation of a post-cleavage duplex of a signaling oligonucleotide hybridized to a target nucleic acid, the dissociation occurring depending on the presence of the target nucleic acid.

32. 1. A kit for detecting a target nucleic acid in a sample, comprising: (a) a first oligonucleotide set for a first target nucleic acid, a second oligonucleotide set for a second target nucleic acid, and a third oligonucleotide set for a third target nucleic acid; Each of the oligonucleotide sets includes (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that is bound to a fluorescent label and serves to generate a signal in the presence of the target nucleic acid, the amplification oligonucleotide and the signaling oligonucleotide being the same or different, and each oligonucleotide set includes a single type of fluorescent label in common; the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second and third detection temperatures in the presence of a third target nucleic acid; (b) instructions describing the method according to any one of claims 1 to 31.

33. A computer readable recording medium comprising instructions embodying a processor for carrying out a method of detecting a sample target nucleic acid comprising the steps of: (a) receiving all of signals measured at a first detection temperature, a second detection temperature, and a third detection temperature, wherein a first oligonucleotide set is for a first target nucleic acid, a second oligonucleotide set is for a second target nucleic acid, and a third oligonucleotide set is for a third target nucleic acid; Each of the oligonucleotide sets includes (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that is bound to a fluorescent label and serves to generate a signal in the presence of the target nucleic acid, the amplification oligonucleotide and the signaling oligonucleotide being the same or different, and each oligonucleotide set includes a single type of fluorescent label in common; the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second and third detection temperatures in the presence of a third target nucleic acid; the first detection temperature, the second detection temperature, and the third detection temperature are different from each other; (b) determining the presence of each target nucleic acid based on signals measured at the first detection temperature, the second detection temperature, and the third detection temperature, the presence of the first target nucleic acid is determined by a difference between signals measured at the first detection temperature, the second detection temperature, and the third detection temperature, the presence of the second target nucleic acid is determined by a difference between signals measured at the second detection temperature and the third detection temperature, and the presence of the third target nucleic acid is determined by a signal measured at the third detection temperature;

34. 34. An apparatus for detecting a target nucleic acid in a sample, comprising: (a) a computer processor; and (b) the computer readable recording medium of claim 33 coupled to the computer processor.

35. A computer program executable on a computer-readable recording medium to implement a processor for carrying out a method for detecting a target nucleic acid in a sample, the method comprising the steps of: (a) receiving all of signals measured at a first detection temperature, a second detection temperature, and a third detection temperature, wherein a first oligonucleotide set is for a first target nucleic acid, a second oligonucleotide set is for a second target nucleic acid, and a third oligonucleotide set is for a third target nucleic acid; Each of the oligonucleotide sets includes (i) an amplification oligonucleotide that serves to amplify a target nucleic acid, and (ii) a signaling oligonucleotide that is bound to a fluorescent label and serves to generate a signal in the presence of the target nucleic acid, the amplification oligonucleotide and the signaling oligonucleotide being the same or different, and each oligonucleotide set includes a single type of fluorescent label in common; the first oligonucleotide set is designed to generate a signal at a first detection temperature in the presence of a first target nucleic acid, the second oligonucleotide set is designed to generate a signal at the first and second detection temperatures in the presence of a second target nucleic acid, and the third oligonucleotide set is designed to generate a signal at the first, second and third detection temperatures in the presence of a third target nucleic acid; the first detection temperature, the second detection temperature, and the third detection temperature are different from each other; (b) determining the presence of each target nucleic acid based on signals measured at the first detection temperature, the second detection temperature, and the third detection temperature, the presence of the first target nucleic acid is determined by a difference between signals measured at the first detection temperature, the second detection temperature, and the third detection temperature, the presence of the second target nucleic acid is determined by a difference between signals measured at the second detection temperature and the third detection temperature, and the presence of the third target nucleic acid is determined by a signal measured at the third detection temperature;

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