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WO2025053620A1 - Procédé de détection d'acide nucléique cible à l'aide de trois sondes - Google Patents

Procédé de détection d'acide nucléique cible à l'aide de trois sondes Download PDF

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Publication number
WO2025053620A1
WO2025053620A1 PCT/KR2024/013368 KR2024013368W WO2025053620A1 WO 2025053620 A1 WO2025053620 A1 WO 2025053620A1 KR 2024013368 W KR2024013368 W KR 2024013368W WO 2025053620 A1 WO2025053620 A1 WO 2025053620A1
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target nucleic
nucleic acid
signal
probe
quencher
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Korean (ko)
Inventor
성민선
김상균
민일재
이한빛
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Seegene Inc
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Seegene Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present disclosure relates to a composition for detecting a target nucleic acid comprising three probes and a method for detecting a target nucleic acid using the same.
  • Real-time detection methods For the detection of target nucleic acids, real-time detection methods are widely used, which can detect target nucleic acids while monitoring target amplification in real time. Real-time detection methods generally utilize labeled probes or primers that specifically hybridize with target nucleic acids.
  • Examples of methods utilizing hybridization between labeled probes and target nucleic acids include the molecular beacon method (Tyagi et al., Nature Biotechnology v. 14 MARCH 1996) utilizing dual-labeled probes having a hairpin structure, the HyBeacon method (French DJ et al., Mol. Cell Probes, 15(6):363-374(2001)), the hybridization probe method using two probes each labeled as a donor and an acceptor (Bernad et al., 147-148 Clin Chem 2000; 46) and the Lux method (U.S. Pat. No. 7,537,886) utilizing single-labeled oligonucleotides.
  • the TaqMan method U.S. Patent Nos. 5,210,015 and 5,538,848) utilizing dual-labeled probes and cleavage of the probes by the 5'-nuclease activity of DNA polymerase is widely used in the art.
  • Examples of methods utilizing labeled primers include the Sunrise primer method (Nazarenko et al., 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., 804-807, Nature Biotechnology v. 17 AUGUST 1999 and U.S. Pat. No. 6,326,145), and the TSG primer method (WO 2011-078441).
  • the number of target nucleic acids that can be detected simultaneously in one reaction is limited by the number of available labels (e.g., 5 or less).
  • the present inventors have made extensive research efforts to develop a novel method for detecting a target nucleic acid in real time with improved convenience and high efficiency. In particular, they have made efforts to develop a method capable of detecting a plurality of target nucleic acid sequences using a single type of label.
  • a novel protocol for detecting a target nucleic acid has been established using a reporter probe including a nucleotide sequence that hybridizes to a first region of a target nucleic acid and a reporter molecule linked thereto, a first quencher probe including a nucleotide sequence that hybridizes to the reporter probe and a first quencher molecule linked thereto, and a second quencher probe including a nucleotide sequence that hybridizes to a second region of a target nucleic acid and a second quencher molecule linked thereto.
  • the protocol according to the present disclosure can detect not only a single target nucleic acid but also a plurality of target nucleic acids.
  • an object of the present disclosure is to provide a composition for detecting a target nucleic acid comprising three probes.
  • Another object of the present disclosure is to provide a method for detecting a target nucleic acid in a sample using three probes.
  • Another object of the present disclosure is to provide a method for detecting n target nucleic acids in a sample.
  • composition for detecting a target nucleic acid comprising:
  • the reporter probe comprises a nucleotide sequence that hybridizes to a first region of the target nucleic acid
  • the above reporter probe has a reporter molecule linked thereto;
  • the first quencher probe comprises a nucleotide sequence that hybridizes to the reporter probe
  • the first quencher probe has a first quencher molecule linked thereto,
  • the first quencher molecule When a first hybrid is formed between the reporter probe and the first quencher probe, the first quencher molecule is at a position to quench a signal from the reporter probe;
  • the second quencher probe comprises a nucleotide sequence that hybridizes to a second region of the target nucleic acid
  • the second quencher probe has a second quencher molecule linked thereto,
  • the first region and the second region of the above target nucleic acid are adjacent to each other,
  • the second quencher molecule is at a position to quench a signal from the reporter molecule
  • the first hybrid compound has a melting temperature (Tm) that is different from the Tm of the second hybrid compound and the Tm of the third hybrid compound.
  • formation of the second hybrid is superior to formation of the first hybrid.
  • the length of the first quencher probe is 1 to 30 nucleotides shorter than the length of the reporter probe.
  • the second quencher molecule is positioned immediately adjacent to the reporter molecule, or is positioned within 1 to 4 nucleotides from the reporter molecule.
  • the reporter molecule when the first region of the target nucleic acid is located 5'-upstream of the second region of the target nucleic acid, the reporter molecule is located at the 5'-terminal portion of the reporter probe, and the second quencher molecule is located at the 3'-terminal portion of the second quencher probe.
  • the reporter molecule when the second region of the target nucleic acid is located 5'-upstream of the first region of the target nucleic acid, the reporter molecule is located at the 3'-terminal portion of the reporter probe, and the second quencher molecule is located at the 5'-terminal portion of the second quencher probe.
  • the first quencher molecule and the second quencher molecule are of the same type.
  • the composition for detecting a target nucleic acid provides a signal dependent on the presence of the target nucleic acid.
  • the signal is provided at a temperature at which one of the second hybrid and the third hybrid maintains its double-stranded state and the other dissociates into a single strand.
  • the temperature is dependent on the Tms of the first hybrid, the second hybrid and the third hybrid.
  • the composition for detecting a target nucleic acid has a signal-changing temperature range (SChTR) in which a signal changes depending on the presence of the target nucleic acid in an amplification reaction for the target nucleic acid, and two signal-constant temperature ranges (SCoTRs) in which the signal is constant even when the target nucleic acid is present.
  • SChTR signal-changing temperature range
  • SCoTRs signal-constant temperature ranges
  • the signal-change temperature range is higher than a first signal-constant temperature range of the two signal-constant temperature ranges and lower than a second signal-constant temperature range of the two signal-constant temperature ranges.
  • the second hybrid and the third hybrid in the presence of the target nucleic acid, maintain their double-stranded state at a temperature within the first signal-specific temperature range.
  • the second hybridization and the third hybridization are dissociated into single strands at a temperature within the second signal-specific temperature range.
  • one of the second hybridization and the third hybridization in the presence of the target nucleic acid, at a temperature within the signal-change temperature range, one of the second hybridization and the third hybridization maintains its double-stranded state, and the other dissociates into two single-stranded compounds.
  • a method for detecting a target nucleic acid in a sample comprising:
  • the above reporter probe has a reporter molecule linked to it
  • the first quencher probe comprises a nucleotide sequence that hybridizes to the reporter probe
  • the first quencher probe has a first quencher molecule linked thereto,
  • the first quencher molecule When a first hybrid is formed between the reporter probe and the first quencher probe, the first quencher molecule is at a position to quench a signal from the reporter probe,
  • the second quencher probe comprises a nucleotide sequence that hybridizes to a second region of the target nucleic acid
  • the second quencher probe has a second quencher molecule linked thereto,
  • the first region and the second region of the above target nucleic acid are adjacent to each other,
  • the second quencher molecule is at a position to quench a signal from the reporter molecule
  • the first hybrid has a melting temperature (Tm) different from the Tm of the second hybrid and the Tm of the third hybrid;
  • step (c) a step of determining the presence of the target nucleic acid from the signal measured in the step (b).
  • the incubation comprises a nucleic acid amplification reaction.
  • the signal is measured at a temperature at which one of the second hybrid and the third hybrid maintains its double-stranded state and the other dissociates into a single strand.
  • the temperature is dependent on the Tms of the first hybrid, the second hybrid and the third hybrid.
  • formation of the second hybrid is superior to formation of the first hybrid.
  • the first quencher molecule and the second quencher molecule are of the same type.
  • a method of detecting n target nucleic acids in a sample comprising:
  • n is an integer greater than or equal to 2
  • the above incubation comprises multiple reaction cycles, and the measurement of the signal is performed in one or more of the multiple reaction cycles,
  • Each of the above n target nucleic acid detection compositions provides a change in a signal indicating the presence of the corresponding target nucleic acid at a corresponding detection temperature among the n detection temperatures in the presence of the corresponding target nucleic acid,
  • the i target nucleic acid detection composition provides a change in a signal indicating the presence of the i target nucleic acid at the i detection temperature among the n detection temperatures in the presence of the i target nucleic acid, and provides a constant signal at other detection temperatures.
  • the above i represents an integer from 1 to n , and the i- th detection temperature is lower than the i +1-th detection temperature.
  • the composition for detecting the i- th target nucleic acid has a signal-changing temperature range (SChTR) in which a signal changes depending on the presence of the i- th target nucleic acid and one or two signal-constant temperature ranges (SCoTR) in which a signal is constant even when the i-th target nucleic acid is present.
  • SChTR signal-changing temperature range
  • SCoTR signal-constant temperature ranges
  • composition for detecting the above i target nucleic acid is,
  • At least one of the above n target nucleic acid detection compositions is a composition comprising the above-described reporter probe, the first quencher probe and the second quencher probe, and;
  • step (b) a step of determining the presence of n target nucleic acids from the signals measured in the step (a), wherein the step of determining the presence of the i- th target nucleic acid by the signal measured at the i- th detection temperature.
  • the first feature of the present disclosure is that one reporter probe and two quencher probes are used to detect a target nucleic acid.
  • the one reporter probe and two quencher probes can form three hybrids when a target nucleic acid is present, specifically, a first hybrid between the reporter probe and the first quencher probe, a second hybrid between the reporter probe and the target nucleic acid, and a third hybrid between the second quencher probe and the target nucleic acid.
  • the second feature of the present disclosure is that the Tm of the first hybrid is different from the Tm of the second hybrid and the third hybrid. Due to this feature, the composition for detecting a target nucleic acid according to the present disclosure has a signal-changing temperature range in which the signal changes depending on the presence of the target nucleic acid and two signal-constant temperature ranges in which the signal is constant even when the target nucleic acid is present. That is, the composition for detecting a target nucleic acid and the method for detecting a target nucleic acid according to the present disclosure can be used as an InterSC-type composition and an InterSC-type signal generating method.
  • the composition for detecting target nucleic acids according to the present disclosure not only enables detection of a single target nucleic acid, but also enables detection of a plurality of target nucleic acids using a single type of label.
  • the analysis time is drastically shortened compared to the conventional technology that requires melting analysis after target amplification in order to detect a plurality of target nucleic acids using a single type of label.
  • FIG. 1 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe in the absence of a target nucleic acid or before a reaction between a composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid, (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • Tm1 of a first hybridization between the reporter probe and the first quencher probe is lower than Tm2 of a second hybridization between the reporter probe and the first region of the target nucleic acid and Tm3 of a third hybridization between the second quencher probe and the second region of the target nucleic acid.
  • FIG. 2 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe after a reaction between a composition for detecting a target nucleic acid according to the present disclosure and a target nucleic acid in (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • Tm1 of a first hybridization between the reporter probe and the first quencher probe is lower than Tm2 of a second hybridization between the reporter probe and the first region of the target nucleic acid and Tm3 of a third hybridization between the second quencher probe and the second region of the target nucleic acid.
  • FIG. 3 shows the content ratio and melt curve of the reaction result between the composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid.
  • FIG. 3A shows the content ratio (or abundance ratio) of the first hybridization product and the second hybridization product (or the third hybridization product) and their melt curves in the initial cycle, the middle cycle, and the final cycle of the target nucleic acid amplification reaction using the composition for detecting a target nucleic acid according to the present disclosure.
  • FIG. 3B shows a plot in which the three melt curves of FIG. 3A are merged.
  • the Tm1 of the first hybridization between the reporter probe and the first quencher probe is lower than the Tm2 of the second hybridization between the reporter probe and the first region of the target nucleic acid and the Tm3 of the third hybridization between the second quencher probe and the second region of the target nucleic acid.
  • FIG. 4 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe in the absence of a target nucleic acid or before a reaction between a composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid, (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • Tm1 of a first hybridization between the reporter probe and the first quencher probe is lower than Tm2 of a second hybridization between the reporter probe and the first region of the target nucleic acid and higher than Tm3 of a third hybridization between the second quencher probe and the second region of the target nucleic acid.
  • FIG. 5 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe after a reaction between a composition for detecting a target nucleic acid according to the present disclosure and a target nucleic acid in (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • Tm1 of a first hybridization between the reporter probe and the first quencher probe is lower than Tm2 of a second hybridization between the reporter probe and the first region of the target nucleic acid and higher than Tm3 of a third hybridization between the second quencher probe and the second region of the target nucleic acid.
  • FIG. 6 shows the content ratio and melt curve of the reaction result between the composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid.
  • FIG. 6A shows the content ratio (or abundance ratio) of the first hybridization product and the second hybridization product (or the third hybridization product) and their melt curves in the initial cycle, the middle cycle, and the final cycle of the target nucleic acid amplification reaction using the composition for detecting a target nucleic acid according to the present disclosure.
  • FIG. 6B shows a plot in which the three melt curves of FIG. 6A are merged.
  • the Tm1 of the first hybridization between the reporter probe and the first quencher probe is lower than the Tm2 of the second hybridization between the reporter probe and the first region of the target nucleic acid, and is higher than the Tm3 of the third hybridization between the second quencher probe and the second region of the target nucleic acid.
  • Figure 7 shows the real-time PCR results in Example 1.
  • Figure 8 shows the real-time PCR results in Example 2.
  • Figure 9 shows the conformational changes of the reporter probe, the first quencher probe and the second quencher probe of each of the two compositions used in Example 3 depending on the presence or absence of the corresponding target nucleic acid and the temperature.
  • Figure 10 shows the multiplex real-time PCR results in Example 3.
  • the present inventors have made extensive research efforts to develop a novel method for detecting a target nucleic acid in real time with improved convenience and high efficiency. In particular, they have made efforts to develop a method capable of detecting a plurality of target nucleic acid sequences using a single type of label.
  • a novel protocol for detecting a target nucleic acid has been established using a reporter probe having a reporter molecule that includes a nucleotide sequence that hybridizes to a first region of a target nucleic acid and is linked thereto, a first quencher probe having a nucleotide sequence that hybridizes to the reporter probe and is linked thereto, and a second quencher probe having a second quencher molecule that includes a nucleotide sequence that hybridizes to a second region of a target nucleic acid and is linked thereto.
  • the protocol according to the present disclosure can detect not only a single target nucleic acid but also a plurality of target nucleic acids.
  • the present disclosure provides a composition for detecting a target nucleic acid, comprising:
  • the reporter probe comprises a nucleotide sequence that hybridizes to a first region of the target nucleic acid
  • the above reporter probe has a reporter molecule linked thereto;
  • the first quencher probe comprises a nucleotide sequence that hybridizes to the reporter probe
  • the first quencher probe has a first quencher molecule linked thereto,
  • the first quencher molecule When a first hybrid is formed between the reporter probe and the first quencher probe, the first quencher molecule is at a position to quench a signal from the reporter probe;
  • the second quencher probe comprises a nucleotide sequence that hybridizes to a second region of the target nucleic acid
  • the second quencher probe has a second quencher molecule linked thereto,
  • the first region and the second region of the above target nucleic acid are adjacent to each other,
  • the second quencher molecule is at a position to quench a signal from the reporter molecule
  • the first hybrid compound has a melting temperature (Tm) that is different from the Tm of the second hybrid compound and the Tm of the third hybrid compound.
  • a composition for detecting a target nucleic acid of the present disclosure comprises one reporter probe and two quencher probes.
  • the reporter probe comprises a nucleotide sequence that hybridizes to a first region of the target nucleic acid and has a reporter molecule linked thereto;
  • the first quencher probe comprises a nucleotide sequence that hybridizes to the reporter probe and has a first quencher molecule linked thereto;
  • the second quencher probe comprises a nucleotide sequence that hybridizes to a second region of the target nucleic acid and has a second quencher molecule linked thereto.
  • target nucleic acid means a nucleic acid sequence to be detected, which anneals or hybridizes with a probe or primer under hybridization, annealing, or amplification conditions.
  • the target nucleic acid sequence includes double-stranded as well as single-stranded.
  • the target nucleic acid sequence includes sequences that are initially present in the nucleic acid sample as well as sequences that are newly generated in the reaction.
  • the above target nucleic acid includes all DNA (gDNA and cDNA), RNA molecules and hybrids thereof (chimeric nucleic acids).
  • the sequence may be in double-stranded or single-stranded form.
  • the target nucleic acid includes any naturally occurring prokaryotic nucleic acid, eukaryotic (e.g., protozoa and parasites, fungi, yeast, higher plants, lower animals, and higher animals including mammals and humans) nucleic acid, viral (e.g., herpes virus, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, poliovirus, etc.) nucleic acid, or viroid nucleic acid.
  • the target nucleic acid can be any nucleic acid molecule that is or can be produced recombinantly or any nucleic acid molecule that is or can be synthesized chemically. Accordingly, the target nucleic acid may or may not be found in nature.
  • the target nucleic acid can include a known or unknown sequence.
  • the target nucleic acid may comprise a nucleotide variation.
  • nucleotide variation may mean a substitution, deletion or insertion of any single or multiple nucleotides in a DNA sequence at a specific position in a continuous DNA segment. Such continuous DNA segments may include a gene or any other portion of a chromosome. Such nucleotide variations may be mutations or polymorphic allele variations.
  • the nucleotide variations detected in the present disclosure include single nucleotide polymorphisms (SNPs), mutations, deletions, insertions, substitutions and translocations.
  • nucleotide variations include various variations in the human genome (e.g., variations in the methylenetetrahydrofolate reductase (MTHFR) gene), variations associated with drug resistance in pathogens, and tumorigenic variations.
  • MTHFR methylenetetrahydrofolate reductase
  • nucleotide variation as used herein includes all variations at a specific position of a nucleic acid sequence. That is, the term nucleotide variation includes the wild type and all mutant forms thereof at a specific position of a nucleic acid sequence.
  • sample means a cell, tissue or fluid from a biological source, or any other medium that can be beneficially evaluated according to the present disclosure, and includes 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).
  • the sample also includes natural-occurring and synthetic nucleic acid molecules isolated from a biological source.
  • primer refers to an oligonucleotide that can act as an initiation point of synthesis when the conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand (template) are present, i.e., in the presence of nucleotides and a polymerization agent, such as DNA polymerase, and at suitable temperature and pH.
  • the primer must be sufficiently long to prime the synthesis of an extension product in the presence of the polymerization agent. The exact length of the primer will depend on many factors, including temperature, application, and source of the primer.
  • probe refers to a single-stranded nucleic acid molecule comprising hybridization site(s) for a target nucleic acid sequence.
  • the probe and primer are single-stranded deoxyribonucleotide molecules.
  • the probe or primer utilized in the present disclosure may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. Additionally, the probe or primer may include ribonucleotides.
  • annealing or “priming” as used herein refers to the apposition of an oligodeoxynucleotide or nucleic acid to a template nucleic acid, which causes a polymerase to polymerize the nucleotides to produce a nucleic acid molecule complementary to the template nucleic acid or a portion thereof.
  • hybridize refers to the formation of a double strand by non-covalent association between two complementary single-stranded polynucleotides under specific hybridization conditions or stringent conditions.
  • Hybridization can occur when the complementarity between the two nucleic acid strands is perfect (perfect match) or can occur even if some mismatched bases are present.
  • the degree of complementarity required for hybridization can vary depending on the hybridization conditions and can be controlled, in particular, by temperature.
  • Hybridization herein can be performed under suitable hybridization conditions, which are generally determined by optimization procedures. Conditions such as temperature, concentration of components, hybridization and wash times, buffer components, and their pH and ionic strength can vary depending on various factors, including the length and GC content of the oligonucleotides (primers and probes) and the target nucleotide sequence. For example, when using relatively short oligonucleotides, it is preferable to select low stringency conditions. Detailed conditions for hybridization can be found in Joseph Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999).
  • an oligonucleotide e.g., a reporter probe or a second quencher probe
  • a nucleotide sequence that hybridizes to another oligonucleotide (e.g., a target nucleic acid) means that all or a portion of the sequence of said one oligonucleotide comprises a complementary nucleotide sequence necessary for hybridization with all or a portion of the sequence of the other oligonucleotide.
  • the portion when referring to hybridization between a portion within an oligonucleotide and another oligonucleotide herein, the portion may be regarded as a separate oligonucleotide and hybridization between the other oligonucleotides may be expressed.
  • complementary means sufficiently complementary to allow a primer or probe to selectively hybridize to a target nucleic acid under given annealing conditions or stringent conditions, and includes both “substantially complementary” and “perfectly complementary,” specifically, perfectly complementary.
  • non-complementary means sufficiently non-complementary such that a primer or probe will not selectively hybridize to a particular sequence under given annealing or stringent conditions, and encompasses both “substantially non-complementary” and “perfectly non-complementary,” specifically, completely non-complementary.
  • a reporter probe according to the present disclosure has a reporter molecule linked thereto and comprises a nucleotide sequence that hybridizes to a first region of the target nucleic acid.
  • a first quencher probe according to the present disclosure has a first quencher molecule linked thereto and comprises a nucleotide sequence that hybridizes to the reporter probe.
  • the first quencher probe can hybridize to the reporter probe to form a first hybrid product.
  • the first quencher molecule of the first quencher probe when the first quencher probe hybridizes to the reporter probe, the first quencher molecule of the first quencher probe is positioned close to the reporter molecule of the reporter probe such that the first quencher molecule quenches a signal from the reporter molecule. Accordingly, when the first quencher probe hybridizes with the reporter probe to form a first hybrid, the first quencher molecule quenches a signal from the reporter molecule, and when the first hybrid dissociates into two single strands, the first quencher molecule unquenchs a signal from the reporter.
  • the reporter probe and the first quencher probe have different lengths.
  • the length of the first quencher probe is shorter than the length of the reporter probe.
  • the length of the first quencher probe is 1 to 30 nucleotides, 1 to 25 nucleotides, 1 to 20 nucleotides, 1 to 15 nucleotides, 1 to 10 nucleotides, 2 to 30 nucleotides, 2 to 25 nucleotides, 2 to 20 nucleotides, 2 to 15 nucleotides, 2 to 10 nucleotides, 3 to 30 nucleotides, 3 to 25 nucleotides, 3 to 20 nucleotides, 3 to 15 nucleotides, 3 to 10 nucleotides, 2 to 7 nucleotides or 1 to 5 nucleotides shorter than the length of the reporter probe.
  • the first quencher probe has a non-complementary base or a universal base with a base on the reporter probe. In this way, including the non-complementary base or the universal base on the first quencher probe can cause the Tm of the first hybrid to be lower than the Tm of the second hybrid even when the first quencher probe has the same or a longer length than the reporter probe.
  • the first quencher probe can have 1 to 5 non-complementary bases or universal bases.
  • the universal base is deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2'-OMe 3-nitropyrrole, 2'-F 3-nitropyrrole, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5-nitropyrrole, 5-nitroindole, 2'-OMe 5-nitroindole, 2'-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole, 4-aminobenzimidazole, deoxy Nebularine, 2'-F nebularine, 2'-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebularine, PNA-5-
  • a double-stranded oligonucleotide composed of two strands with different lengths spontaneously reacts with a single-stranded oligonucleotide, and the relatively shorter strand of the double-stranded oligonucleotide is displaced by the single-stranded oligonucleotide to form a new double-stranded oligonucleotide that is thermodynamically more stable.
  • a first hybrid formed between a reporter probe, which is a double-stranded oligonucleotide, and a first quencher probe can spontaneously form a second hybrid, which is thermodynamically more stable, by displacing the first quencher probe, which is a single-stranded oligonucleotide, from the target nucleic acid.
  • the formation of the second hybrid is superior to the formation of the first hybrid.
  • the reporter probe and the first quencher probe hybridize with each other to form the first hybrid.
  • the first quencher probe of the first hybrid is replaced by the target nucleic acid, forming a second hybrid between the reporter probe and the target nucleic acid.
  • a second quencher probe according to the present disclosure has a second quencher molecule linked thereto and comprises a nucleotide sequence that hybridizes to a second region of the target nucleic acid.
  • the reporter probe hybridizes to a first region of the target nucleic acid to form a second hybrid
  • the second quencher probe hybridizes to a second region of the target nucleic acid to form a third hybrid.
  • the reporter probe, the first quencher probe and/or the second quencher probe are "blocked" at their 3'-ends to prevent extension.
  • Blocking can be accomplished by any conventional method. For example, blocking can be accomplished 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 moiety, to the 3'-hydroxyl group of the last nucleotide. Alternatively, blocking can be accomplished by removing the 3'-hydroxyl group of the last nucleotide or by using a nucleotide lacking a 3'-hydroxyl group, such as a dideoxynucleotide.
  • a chemical moiety such as biotin, a label, a phosphate group, an alkyl group, a non-nucleotide linker, a phosphorothioate or an alkane-
  • the first region and the second region of the target nucleic acid are adjacent to each other.
  • adjacent as used herein when referring to a first region and a second region of a target nucleic acid means that a reporter molecule of a reporter probe hybridized to the first region of the target nucleic acid and a second quencher molecule of a second quencher probe hybridized to the second region of the target nucleic acid are sufficiently adjacent to each other to interact with each other.
  • the first region and the second region of the target nucleic acid can be positioned immediately adjacent to each other or in close proximity with some separation, as long as the second quencher molecule of the second quencher probe hybridized to the second region of the target nucleic acid can quench a signal from the reporter molecule of the reporter probe hybridized to the first region of the target nucleic acid.
  • the first region and the second region of the target nucleic acid can be positioned directly adjacent to each other.
  • the first region and the second region of the target nucleic acid can be positioned directly adjacent to each other to form a nick.
  • the first region and the second region of the target nucleic acid can be positioned directly adjacent to each other.
  • the first region of the target nucleic acid can be positioned 1-30 nucleotides, 1-20 nucleotides, 1-15 nucleotides, 1-10 nucleotides, 1-5 nucleotides, or 1-4 nucleotides apart from the second region of the target nucleic acid.
  • the first region and the second region of the target nucleic acid can overlap each other.
  • 1-10 nucleotides, 1-7 nucleotides, 1-5 nucleotides, 1-3 nucleotides or 1-2 nucleotides may overlap.
  • the second quencher molecule linked to the second quencher probe when the reporter probe and the second quencher probe are hybridized to the target nucleic acid, the second quencher molecule linked to the second quencher probe is positioned in proximity to the reporter molecule linked to the reporter probe hybridized to the target nucleic acid such that the second quencher molecule can quench a signal from the reporter molecule.
  • the second quencher molecule linked to the second quencher probe is positioned immediately adjacent to the reporter molecule linked to the reporter probe or within 1 to 4 nucleotides from the reporter molecule.
  • the reporter molecule of the reporter probe, the first quencher molecule of the first quencher probe and the second quencher molecule of the second quencher probe are each linked to a position where quenching and unquenching can be induced under specific conditions, taking into account the forms of the first hybrid, the second hybrid and the third hybrid.
  • the reporter molecule, the first quencher molecule and the second quencher molecule are linked to positions that satisfy both of the following conditions.
  • the first quencher molecule when the first hybrid is formed, the first quencher molecule quenches the signal from the reporter molecule, and when the first hybrid is dissociated, the first quencher molecule unquenchs the signal from the reporter molecule;
  • the reporter probe and the second quencher probe each hybridize to the first region and the second region of the target nucleic acid, respectively, the second quencher molecule linked to the second quencher probe approaches the reporter molecule of the reporter probe hybridized to the target nucleic acid, such that the second quencher molecule linked to the second quencher probe quenches a signal from the reporter molecule linked to the reporter probe.
  • the first region of the target nucleic acid can be located upstream from the 5'-terminus of the second region of the target nucleic acid. In another embodiment, the first region of the target nucleic acid can be located downstream from the 3'-terminus of the second region.
  • the reporter molecule is linked to the 3'-terminal portion or the 5'-terminal portion of the reporter probe.
  • the first quencher molecule is linked to the 5'-terminal portion or the 3'-terminal portion of the first probe.
  • the second quencher molecule is linked to the 5'-terminal portion or the 3'-terminal portion of the second quencher probe.
  • the reporter molecule when the first region of the target nucleic acid is located 5'-upstream of the second region of the target nucleic acid, the reporter molecule is located at the 5'-terminal portion of the reporter probe, and the second quencher molecule is located at the 3'-terminal portion of the second quencher probe.
  • the reporter molecule when the second region of the target nucleic acid is located 5'-upstream of the first region of the target nucleic acid, the reporter molecule is located at the 3'-terminal portion of the reporter probe, and the second quencher molecule is located at the 5'-terminal portion of the second quencher probe.
  • the reporter molecules and quencher molecules used in the present disclosure are interactive labels.
  • a fluorescence resonance energy transfer (FRET) labeling system includes a fluorescent reporter molecule (donor molecule) and a quencher molecule (acceptor molecule).
  • FRET fluorescence resonance energy transfer
  • the energy donor is fluorescent, but the energy acceptor can be fluorescent or non-fluorescent.
  • the energy donor is non-fluorescent, such as a chromophore, and the energy acceptor is fluorescent.
  • the energy donor is luminescent, such as bioluminescent, chemiluminescent, or electrochemiluminescent, and the acceptor is fluorescent.
  • the donor molecule and the acceptor molecule may be respectively described as a reporter molecule and a quencher molecule in the present disclosure.
  • Interactive labels comprise a pair of labels that provide a detectable signal 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 label system includes all instances where a signal change is induced by an interaction between at least two molecules (e.g., dyes).
  • Reporter molecules and quencher molecules useful as interactive labels may include any molecules known in the art. Examples include: Cy2TM (506), YO-PROTM-1 (509), YOYOTM-1 (509), Calcein (517), FITC (518), FluorXTM (519), AlexaTM (520), Rhodamine 110 (520), Oregon GreenTM 500 (522), Oregon GreenTM 488 (524), RiboGreenTM (525), Rhodamine GreenTM (527), Rhodamine 123 (529), Magnesium GreenTM (531), Calcium GreenTM (533), TO-PROTM-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3TM (570), AlexaTM 546 (570), TRITC (572), Magnesium OrangeTM (575), Phycoerythrin R&B (575), Rhodamine Phalloidin
  • Suitable reporter-quencher pairs are described in various references, including: Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color AND Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, 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, R. P., Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996); U.S. Patent Nos. 3,996,345 and
  • non-fluorescent black quencher molecules capable of quenching fluorescence of a broad wavelength or a specific wavelength can be utilized.
  • non-fluorescent black quencher molecules are BHQ and DABCYL.
  • the reporter comprises a donor of FRET and the quencher comprises the remaining partner (acceptor) of FRET.
  • fluorescein dye is used as a reporter and rhodamine dye is used as a quencher.
  • the first quencher molecule and the second quencher molecule are quencher molecules of the same type.
  • the first quencher molecule is a non-fluorescent black quencher molecule (e.g., BHQ)
  • the second quencher molecule can use the same type of non-fluorescent black quencher molecule (e.g., BHQ)
  • the first quencher molecule is a fluorescent acceptor molecule (e.g., rhodamine dye)
  • the second quencher molecule can also use the same type of fluorescent acceptor molecule (e.g., rhodamine dye).
  • a composition for detecting a target nucleic acid comprising a reporter probe, a first quencher probe and a second quencher probe according to the present disclosure described above reacts with a target nucleic acid to provide a signal dependent on the presence of the target nucleic acid.
  • a composition for detecting a target nucleic acid comprising three probes according to the present disclosure provides a signal change indicating the presence of the target nucleic acid as the target nucleic acid is amplified.
  • the provision of the signal includes “signal generation or extinguishment” and “signal increase or decrease.”
  • the provision of the signal means provision of a significant signal, i.e., a signal indicating the presence of the target nucleic acid.
  • the significant signal i.e., a signal indicating the presence of the target nucleic acid
  • the significant signal i.e., a signal indicating the presence of the target nucleic acid
  • the significant signal means a signal having an intensity after subtracting the intensity of the background signal or the intensity of a signal that can be provided in the absence of the target nucleic acid from the intensity of the provided signal.
  • the provision of a signal in this invention is interpreted as the provision of a change in signal, and the provision of said change in signal means providing a change in the signal in the presence of a target compared to the signal in the absence of the target.
  • the change in signal is provided by a change in the sum of the signal provided from the first hybrid and the signal provided from the second or third hybrid.
  • the reaction with the target nucleic acid may include an amplification reaction, such as a signal amplification reaction and/or a nucleic acid amplification reaction.
  • an amplification reaction such as a signal amplification reaction and/or a nucleic acid amplification reaction.
  • the composition for detecting a target nucleic acid according to the present disclosure can form a first hybrid, and when a target nucleic acid is present, can form not only the first hybrid, but also a second hybrid and a third hybrid.
  • the first hybrid formed by the composition for detecting a target nucleic acid according to the present disclosure is characterized in that it has a melting temperature (Tm) that is different from the Tms of the second hybrid and the third hybrid. That is, the Tm of the first hybrid (referred to as Tm1 herein) is different from the Tm of the second hybrid (referred to as Tm2 herein) and the Tm of the third hybrid (referred to as Tm3 herein).
  • melting temperature means the melting temperature at which half of the population of double-stranded nucleic acid molecules dissociates into single-stranded molecules. Tm is determined by the length and G/C content of the hybridizing nucleotides. Tm can be calculated by conventional methods such as the Wallace rule (R.B. Wallace et al., Nucleic Acids Research, 6:3543-3547(1979)) and the nearest-neighbor method (SantaLucia J. Jr. et al., Biochemistry, 35:3555-3562(1996)); Sugimoto N. et al., Nucleic Acids Res., 24:4501-4505(1996)).
  • the signal dependent on the presence of the target nucleic acid is provided at a temperature at which one of the second hybrid and the third hybrid maintains its double-stranded state and the other dissociates into a single strand. That is, the temperature at which the signal is provided is dependent on the Tms of the first hybrid, the second hybrid and the third hybrid.
  • Tm1 and Tm2 are adjusted such that formation of the second hybrid is favored over formation of the first hybrid.
  • Tm1 is lower than Tm2, and specifically, Tm1 is at least 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C or 15°C lower than Tm2.
  • Tm3 can be higher or lower than Tm1.
  • Tm1 is controllable by the sequence and/or length of the first quencher probe.
  • Tm2 is controllable by the sequence and/or length of the reporter probe.
  • Tm3 is controllable by the sequence and/or length of the second quencher probe.
  • Tm1 is different from both Tm2 and Tm3, Tm2 and Tm3 may be the same or different from each other.
  • the second hybrid and the third hybrid have different Tm's. That is, Tm2 and Tm3 are different from each other, for example, Tm2 and Tm3 are different from each other by at least 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C or 20°C.
  • the second hybrid and the third hybrid have the same Tm, i.e., Tm2 and Tm3 are the same.
  • the composition for detecting a target nucleic acid has a temperature range in which a signal changes depending on the presence of the target nucleic acid in a reaction with the target nucleic acid (e.g., an amplification reaction), i.e., a signal-changing temperature range (SChTR), and two temperature ranges in which the signal does not change even in the presence of the target nucleic acid, i.e., two signal-constant temperature ranges (SCoTRs).
  • SChTR signal-changing temperature range
  • SCoTRs two temperature ranges in which the signal does not change even in the presence of the target nucleic acid
  • the signal-change temperature range of the composition for detecting a target nucleic acid according to the present disclosure is higher than the first signal-constant temperature range among the two signal-constant temperature ranges and lower than the second signal-constant temperature range among the two signal-constant temperature ranges.
  • Figures 1 and 2 show the predominant conformations of the reporter probe, the first quencher probe and the second quencher probe depending on the presence of the target nucleic acid and the temperature when Tm1 is lower than both Tm2 and Tm3.
  • FIG. 1 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe in the absence of a target nucleic acid or before a reaction between a composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid, in (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • FIG. 1 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe in the absence of a target nucleic acid or before a reaction between a composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid, in (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • FIG. 1 shows conformational changes of a reporter probe, a first quencher probe and a
  • FIG. 2 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe after a reaction between a composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid, in (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • the reporter probe and the first quencher probe hybridize with each other to form a first hybrid (see (i) of FIG. 1). That is, at the temperature, the reporter molecule linked to the reporter probe and the first quencher molecule linked to the first quencher probe come into close proximity to each other, such that the first quencher molecule quenches a signal from the reporter molecule.
  • the first hybridization between the reporter probe and the first quencher probe dissociates into two single strands (see (ii) and (iii) of FIG. 1). That is, at the temperature, the reporter molecule linked to the reporter probe and the first quencher molecule linked to the first quencher probe are separated from each other, so that the first quencher molecule unquenches a signal from the reporter molecule.
  • the second quencher probe exists as a single strand at a temperature within the entire temperature range.
  • the reporter probe and the second quencher probe hybridize to the target nucleic acid to form a second hybrid and a third hybrid, respectively (see (i) of FIG. 2). That is, at the temperature, the reporter molecule linked to the reporter probe and the second quencher molecule linked to the second quencher probe are close to each other, such that the second quencher molecule quenches a signal from the reporter molecule. Therefore, as shown in (i) of FIG. 1 and (i) of FIG.
  • the reporter molecule linked to the reporter probe is quenched by the first quencher molecule or the second quencher molecule regardless of the presence of the target nucleic acid. Therefore, in the first signal-constant temperature range, the signal is constant without a signal change even when the target nucleic acid is present.
  • the reporter probe and the second quencher probe dissociate from the target nucleic acid and each exist as a single strand (see (iii) of FIG. 2). That is, at the temperature, the reporter molecule linked to the reporter probe and the second quencher molecule linked to the second quencher probe are spaced apart from each other, so that the second quencher molecule unquenches a signal from the reporter molecule. Therefore, as shown in (iii) of FIG. 1 and (iii) of FIG.
  • the reporter molecule linked to the reporter probe is separated from both the first quencher molecule and the second quencher molecule and unquenched regardless of the presence of the target nucleic acid. Therefore, in the second signal-constant temperature range, the signal is constant without signal change even when the target nucleic acid is present.
  • the following forms may coexist: (ii-1) a reporter probe and a second quencher probe hybridize to the target nucleic acid to form a second hybrid product and a third hybrid product at a temperature within a signal-change temperature range; and (ii-2) a form in which the reporter probe hybridizes to the target nucleic acid to form a second hybrid product and the second quencher probe dissociates from the target nucleic acid and exists as a single strand (see (ii) of FIG. 2).
  • the reporter molecule linked to the reporter probe and the second quencher molecule linked to the second quencher probe are close to each other so that the second quencher molecule quenches a signal from the reporter molecule
  • the reporter molecule linked to the reporter probe and the second quencher molecule linked to the second quencher probe are spaced apart from each other so that the second quencher molecule unquenchs a signal from the reporter molecule.
  • a reporter probe, a first quencher probe, and a second quencher probe that have not yet participated in the reaction may exist together, and at this time, the three probes exist in the forms (i) to (iii) of FIG. 1 depending on the temperature.
  • the contents of the reporter probe, the first quencher probe and the second quencher probe are constant at the concentrations included in the initial target nucleic acid detection composition.
  • the reporter probe, the first quencher probe and the second quencher probe included in the composition for detecting a target nucleic acid can exist in the forms of FIGS. 1 and 2 depending on the presence or absence of the target nucleic acid and/or a change in temperature, as described above, and the contents of the reporter probe, the first quencher probe and the second quencher probe existing in the forms of FIGS. 1 and 2 change depending on the degree of reaction with the target nucleic acid (e.g., the degree of amplification of the target nucleic acid).
  • the reporter probe forms a first hybrid with the first quencher probe, and when it reacts with the target nucleic acid, the relatively short first quencher probe is displaced by the target nucleic acid, so that the reporter probe spontaneously forms a second hybrid that is thermodynamically more stable with the target nucleic acid.
  • the reporter probe that formed the first hybrid can be considered to be consumed as it forms the second hybrid, that is, the content of the first hybrid can be considered to decrease and the content of the second hybrid can be considered to increase.
  • FIG. 3 shows the content ratio and melt curve of the composition for detecting a target nucleic acid according to the present disclosure and the reaction result with the target nucleic acid.
  • FIG. 3A shows the content ratio (or abundance ratio) of the first hybridization product and the second hybridization product (or the third hybridization product) and their melt curves in the initial cycle, middle cycle, and final cycle of the target nucleic acid amplification reaction using the composition for detecting a target nucleic acid according to the present disclosure.
  • FIG. 3B shows a plot in which the three melt curves of FIG. 3A are merged.
  • the number in the (i) row in FIG. 3A represents the total content ratio of the first hybridization product existing in the form of (i) to (iii) of FIG. 1
  • the number in the (ii) row represents the total content ratio of the second hybridization product (or third hybridization product) existing in the form of (i) to (iii) of FIG. 2.
  • the content ratios in the (i) and (ii) rows change as the cycle increases, that is, as the target nucleic acid is amplified.
  • a graph such as FIG. 3B can be obtained.
  • the composition for detecting a target nucleic acid according to the present disclosure has two temperature ranges in which the signal is constant even when the target nucleic acid is present, and a temperature range in which the signal changes as the target nucleic acid is amplified.
  • the content of the second hybridization product (and/or the third hybridization product) increases as the target nucleic acid is amplified.
  • the content of the first hybridization product decreases as the target nucleic acid is amplified.
  • the composition for detecting a target nucleic acid according to the present disclosure can provide a signal dependent on the presence of the target nucleic acid.
  • the signal does not change and is constant within two signal constant-temperature ranges. Specifically, at a temperature within the first signal-constant temperature range, the reporter probe is quenched by the first quencher probe or the second quencher probe, so that the signal is constant, and at a temperature within the second signal-constant temperature range, the reporter probe is separated from both the first quencher probe and the second quencher probe and is unquenched, so that the signal is constant.
  • Figures 4 and 5 show the predominant conformations of the reporter probe, the first quencher probe and the second quencher probe depending on the presence of the target nucleic acid and the temperature when Tm1 is lower than Tm2 and higher than Tm3.
  • FIG. 4 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe in the absence of a target nucleic acid or before a reaction between a composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid, in (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • FIG. 4 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe in the absence of a target nucleic acid or before a reaction between a composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid, in (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • FIG. 5 shows conformational changes of a reporter probe, a first quencher probe and a second quencher probe after a reaction between a composition for detecting a target nucleic acid according to the present disclosure and the target nucleic acid, in (i) a first signal-constant temperature range, (ii) a signal-change temperature range and (iii) a second signal-constant temperature range.
  • the reporter probe and the first quencher probe hybridize with each other to form a first hybrid (see (i) of FIG. 4). That is, at the temperature, the reporter molecule linked to the reporter probe and the first quencher molecule linked to the first quencher probe come into close proximity to each other such that the first quencher molecule quenches a signal from the reporter molecule.
  • the first hybrid between the reporter probe and the first quencher probe dissociates and exists as two single strands (see (iii) of FIG. 4). That is, at the temperature, the reporter molecule linked to the reporter probe and the first quencher molecule linked to the first quencher probe are separated from each other, so that the first quencher molecule unquenches a signal from the reporter molecule.
  • composition for detecting a target nucleic acid reacts with the target nucleic acid or in the absence of the target nucleic acid, at a temperature within the signal-change temperature range, (ii-1) a form in which the reporter probe and the first quencher probe hybridize with each other to form a first hybrid product and (ii-2) a form in which the first hybrid product between the reporter probe and the first quencher probe dissociates and exists as two single strands can coexist (see (ii) of FIG. 4).
  • the reporter molecule linked to the reporter probe and the first quencher molecule linked to the first quencher probe are close to each other so that the first quencher molecule quenches a signal from the reporter molecule
  • the reporter molecule linked to the reporter probe and the first quencher molecule linked to the first quencher probe are spaced apart from each other so that the first quencher molecule unquenchs a signal from the reporter molecule.
  • the second quencher probe exists as a single strand at a temperature within the entire temperature range.
  • the reporter probe and the second quencher probe hybridize to the target nucleic acid to form a second hybrid and a third hybrid, respectively (see (i) of FIG. 5). That is, at the temperature, the reporter molecule linked to the reporter probe and the second quencher molecule linked to the second quencher probe are close to each other so that the second quencher molecule quenches a signal from the reporter molecule. Therefore, as shown in (i) of FIG. 4 and (i) of FIG.
  • the reporter molecule linked to the reporter probe is quenched by the first quencher molecule or the second quencher molecule regardless of the presence of the target nucleic acid. Therefore, in the first signal-constant temperature range, the signal is constant without a signal change even when the target nucleic acid is present.
  • the reporter probe and the second quencher probe dissociate from the target nucleic acid and each exist as a single strand (see (iii) of FIG. 5). That is, at the temperature, the reporter molecule linked to the reporter probe and the second quencher molecule linked to the second quencher probe are spaced apart from each other, so that the second quencher molecule unquenches a signal from the reporter molecule. Therefore, as shown in (iii) of FIG. 4 and (iii) of FIG.
  • the reporter molecule linked to the reporter probe is separated from both the first quencher molecule and the second quencher molecule and unquenched regardless of the presence of the target nucleic acid. Therefore, in the second signal-constant temperature range, the signal is constant without signal change even when the target nucleic acid is present.
  • the reporter probe when a target nucleic acid is present, at a temperature within a signal-change temperature range, the reporter probe hybridizes to the target nucleic acid to form a second hybrid and the second quencher probe dissociates from the target nucleic acid and exists as a single strand (see (ii) of FIG. 5). That is, at the temperature, the reporter molecule linked to the reporter probe and the second quencher molecule linked to the second quencher probe are spaced apart from each other so that the second quencher molecule unquenches a signal from the reporter molecule. That is, as shown in (ii) of FIG. 4 and (ii) of FIG.
  • the first quencher molecule linked to (some) of the first quencher probes quenches a signal from the reporter molecule linked to the reporter probe (i.e., in the form of (ii-1) of FIG. 4), whereas when the target nucleic acid is present, the first quencher molecule linked to the reporter probe unquenches a signal. Due to this, the signal changes dependently on the presence of the target nucleic acid in the signal-change temperature range.
  • a reporter probe, a first quencher probe, and a second quencher probe that have not yet participated in the reaction may exist together, and at this time, the three probes exist in the forms (i) to (iii) of FIG. 4 depending on the temperature.
  • the contents of the reporter probe, the first quencher probe and the second quencher probe are constant at the concentrations included in the initial target nucleic acid detection composition.
  • the reporter probe, the first quencher probe and the second quencher probe included in the composition for detecting a target nucleic acid can exist in the forms of FIGS. 4 and 5 depending on the presence or absence of the target nucleic acid and/or a change in temperature, as described above, and the contents of the reporter probe, the first quencher probe and the second quencher probe existing in the forms of FIGS. 4 and 5 change depending on the degree of reaction with the target nucleic acid (e.g., the degree of amplification of the target nucleic acid).
  • the reporter probe forms a first hybrid with the first quencher probe, and when it reacts with the target nucleic acid, the relatively short first quencher probe is displaced by the target nucleic acid, so that the reporter probe spontaneously forms a second hybrid that is thermodynamically more stable with the target nucleic acid.
  • the reporter probe that formed the first hybrid can be considered to be consumed as it forms the second hybrid, that is, the content of the first hybrid can be considered to decrease and the content of the second hybrid can be considered to increase.
  • FIG. 6 shows the content ratio and melt curve of the composition for detecting a target nucleic acid according to the present disclosure and the reaction result with the target nucleic acid.
  • FIG. 6A shows the content ratio (or abundance ratio) of the first hybridization product and the second hybridization product (or the third hybridization product) and their melt curves in the initial cycle, middle cycle, and final cycle of the target nucleic acid amplification reaction using the composition for detecting a target nucleic acid according to the present disclosure.
  • FIG. 6B shows a plot in which the three melt curves of FIG. 6A are merged.
  • the number in the (i) row in FIG. 6A represents the total content ratio of the first hybridization product existing in the form of (i) to (iii) of FIG. 4, and the number in the (ii) row represents the total content ratio of the second hybridization product (or third hybridization product) existing in the form of (i) to (iii) of FIG. 5.
  • the content ratios in the (i) and (ii) rows change as the cycle increases, that is, as the target nucleic acid is amplified.
  • a graph such as FIG. 6B can be obtained.
  • the composition for detecting a target nucleic acid according to the present disclosure has two temperature ranges in which the signal is constant even when the target nucleic acid is present, and a temperature range in which the signal changes as the target nucleic acid is amplified.
  • the content of the second hybridization product (and/or the third hybridization product) increases as the target nucleic acid is amplified.
  • the content of the first hybridization product decreases as the target nucleic acid is amplified.
  • the composition for detecting a target nucleic acid according to the present disclosure can provide a signal dependent on the presence of the target nucleic acid.
  • the signal does not change and is constant within two signal constant-temperature ranges. Specifically, at a temperature within the first signal-constant temperature range, the reporter probe is quenched by the first quencher probe or the second quencher probe, so that the signal is constant, and at a temperature within the second signal-constant temperature range, the reporter probe is separated from both the first quencher probe and the second quencher probe and is unquenched, so that the signal is constant.
  • the composition for detecting a target nucleic acid has a signal-changing temperature range (SChTR) in which a signal changes depending on the presence of a target nucleic acid in an amplification reaction for the target nucleic acid, and two signal-constant temperature ranges (SCoTRs) in which the signal is constant even when the target nucleic acid is present.
  • SChTR signal-changing temperature range
  • SCoTRs two signal-constant temperature ranges
  • the term "signal-constant temperature range" refers to a temperature range over which a composition for detecting a target nucleic acid provides a constant signal despite the presence of a target nucleic acid.
  • the signal-constant temperature range is a temperature range over which a composition for detecting a target nucleic acid provides a constant signal over the course of a reaction time, which may also be referred to herein as a temperature range over which a composition for detecting a target nucleic acid does not provide a significant signal, or a temperature range over which a signal indicating the presence of a target nucleic acid is not provided.
  • the term “constant signal” means that the signal does not substantially change during the reaction between the target nucleic acid and the composition for detecting the target nucleic acid (e.g., the target nucleic acid amplification reaction). That is, the term “constant signal” means all or any signal pattern excluding a significant signal change that appears due to the amplification of the existing target nucleic acid. In particular, the constant signal means no signal change.
  • the signal when the signal does not exceed the intensity of the background signal or the intensity of the signal that can occur in the absence of the target nucleic acid during the amplification reaction, it can be expressed as "the signal is constant.”
  • the constant signal can be used interchangeably with a signal that does not change or a signal that does not show a change.
  • the term "signal-change temperature range” refers to a temperature range over which a composition for detecting a target nucleic acid provides a signal that changes dependently on the presence of a target nucleic acid.
  • the signal-change temperature range is a temperature range over which a composition for detecting a target nucleic acid provides a signal that changes over the course of a reaction time, which may also be referred to herein as a temperature range over which a composition for detecting a target nucleic acid provides a significant signal, or a temperature range over which a signal indicative of the presence of a target nucleic acid is provided.
  • the expression “one temperature range is lower than the other temperature range” used in relation to the signal-change temperature range and the signal-constant temperature range of the target nucleic acid detection composition means that the highest temperature within one temperature range is lower than the lowest temperature within the other temperature range.
  • the expression “one temperature range is higher than the other temperature range” means that the lowest temperature within one temperature range is higher than the highest temperature within the other temperature range.
  • the expression that the signal-constant temperature range is higher than the signal-change temperature range means that the lowest temperature within the signal-constant temperature range is higher than the highest temperature within the signal-change temperature range.
  • WO2022-265463 discloses that various signal generation methods known in the art for detecting a target nucleic acid have a signal-changing temperature range (SChTR) in which the signal changes depending on the presence of the target nucleic acid and one or two signal-constant temperature ranges (SCoTR) in which the signal does not change even in the presence of the target nucleic acid.
  • SChTR signal-changing temperature range
  • SCoTR signal-constant temperature ranges
  • WO2022-265463 discloses that various conventional signal generation methods can be classified into the following three types according to the number and order of signal-changing temperature ranges and signal-constant temperature ranges:
  • compositions for implementing the above three types of signal generation methods can be classified into UnderSC-type compositions, InterSC-type compositions, and OverSC-type compositions, respectively.
  • composition for detecting target nucleic acid according to the present disclosure can also be classified as an InterSC-type composition among the three signal generation methods (UnderSC-type, InterSC-type, and OverSC-type) described in WO2022-265463.
  • the signal-change temperature range is determined dependently on (i) the Tm value of the first hybrid compound and (ii) the Tm values of the second hybrid compound and/or the third hybrid compound. Accordingly, the signal-change temperature range and the two signal-constant temperature ranges can be controlled by controlling the Tm of the first hybrid compound and the Tm of the second hybrid compound or the third hybrid compound.
  • the second hybrid and the third hybrid in the presence of the target nucleic acid, maintain their double-stranded state at a temperature within the first signal-constant temperature range. Specifically, most of the second hybrid and most of the third hybrid maintain their double-stranded state.
  • the first hybrid largely maintains its double-stranded state.
  • the second hybrid and the third hybrid dissociate from the target nucleic acid and exist as single strands. Specifically, most of the second hybrid and most of the third hybrid dissociate from the target nucleic acid and exist as single strands.
  • the first hybrid dissociates predominantly into two single strands.
  • At least one of the second hybrid and the third hybrid maintains its double-stranded state over the signal-change temperature range.
  • the second hybrid and the third hybrid when Tm1 is lower than Tm2 and Tm3, in the presence of the target nucleic acid, the second hybrid and the third hybrid maintain their double-stranded state in the signal-change temperature range. Specifically, most of the second hybrid maintains its double-stranded state, and some of the third hybrid may maintain its double-stranded state (e.g., form (ii-1) of FIG. 2) and some may dissociate from the target nucleic acid and exist as a single strand (e.g., form (ii-2) of FIG. 2). Here, most of the first hybrid dissociates into two single strands.
  • the second hybridization when Tm1 is lower than Tm2 and higher than Tm3, in the presence of the target nucleic acid, the second hybridization maintains its double-stranded state and the third hybridization dissociates into single strands in the signal-change temperature range. Specifically, most of the second hybridization maintains its double-stranded state and most of the third hybridization dissociates from the target nucleic acid and exists as single strands.
  • some of the first hybridization maintains its double-stranded state and some dissociates into two single strands.
  • a portion when used herein to refer to a form of the first hybrid, the second hybrid and/or the third hybrid at a particular temperature range (or at a particular temperature) means a portion of the total content of the hybrid at the particular temperature range, for example, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% of the total content.
  • the term "most" when used herein to refer to a form of the first hybrid, the second hybrid and/or the third hybrid at a particular temperature range (or a particular temperature) means a majority of the total amount of the hybrid at the particular temperature range, for example, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the total amount, and is meant to encompass substantially all of the hybrid.
  • the reporter probe, the first quencher probe and the second quencher probe are (i) contained in a single vessel or (ii) contained respectively in separate vessels.
  • the composition for detecting a target nucleic acid may additionally include a primer capable of amplifying a nucleic acid sequence including the first region and the second region of the target nucleic acid.
  • the primer may comprise a forward primer (also referred to as an upstream primer or an upstream oligonucleotide), a reverse primer (also referred to as a downstream primer or a downstream oligonucleotide), or both.
  • the primer may be an oligonucleotide having a structure known in the art and may be synthesized by methods known in the art.
  • the composition for detecting a target nucleic acid may additionally include a polymerase for amplifying the target nucleic acid.
  • the polymerase is a polymerase lacking nuclease activity (e.g., 5' -> 3' exonuclease activity).
  • the polymerase is a polymerase having nuclease activity (e.g., 5'->3' exonuclease activity).
  • the reporter probe, the first quencher probe and/or the second quencher probe are resistant to nuclease activity.
  • the composition for detecting a target nucleic acid according to the present disclosure is characterized by having a signal-change temperature range and two signal-constant temperature ranges. Due to these features, by controlling the signal-change temperature range, not only the detection of a single target nucleic acid but also the detection of a plurality of target nucleic acids using a single type of label is possible. However, when a polymerase having nuclease activity is used, the reporter probe, the first quencher probe and/or the second quencher probe can be cleaved by the nuclease activity.
  • the cleavage of the probe induces the release of the reporter molecule, the first quencher molecule and/or the second quencher molecule linked to the probe, and the reporter molecule is unquenched from the first quencher molecule and/or the second quencher molecule over the entire temperature range. That is, the composition for detecting a target nucleic acid according to the present disclosure causes a signal to change dependently on the presence of a target nucleic acid over the entire temperature range without a signal-constant temperature range.
  • the resistance to activity of a 5' -> 3' exonuclease is conferred by a nucleotide having a backbone having resistance to activity of a 5' -> 3' exonuclease, the nucleotide comprising a variety of phosphorothioate linkages, phosphonate linkages, phosphoroamidate linkages and 2'-carbohydrate modifications, more preferably phosphorothioate linkages, alkyl phosphotriester linkages, aryl phosphotriester linkages, alkyl phosphonate linkages, aryl phosphonate linkages, hydrogen phosphonate linkages, alkyl phosphoroamidate linkages, aryl phosphoroamidate linkages, phosphoroselenate linkages, 2'-O-aminopropyl modifications, 2'-O-alkyl modifications, 2'-O-allyl modifications, 2'-O-butyl modifications, ⁇ -anomeric oligodeoxy
  • the composition for detecting a target nucleic acid may optionally include reagents necessary for performing a target nucleic acid amplification reaction (e.g., a PCR reaction), such as a buffer, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate.
  • a target nucleic acid amplification reaction e.g., a PCR reaction
  • the composition may also include various polynucleotide molecules, a reverse transcriptase enzyme, various buffers and reagents, and an antibody that inhibits DNA polymerase activity.
  • the composition may also include reagents necessary for performing positive control and negative control reactions. The optimal amount of reagents to be used in a particular reaction can be readily determined by one of ordinary skill in the art having the benefit of the present disclosure.
  • the components of the composition may be present in separate containers, or a plurality of components may be present in a single container.
  • the present disclosure provides a method for detecting a target nucleic acid in a sample, comprising:
  • the reporter probe comprises a nucleotide sequence that hybridizes to a first region of the target nucleic acid
  • the above reporter probe has a reporter molecule linked to it
  • the first quencher probe comprises a nucleotide sequence that hybridizes to the reporter probe
  • the first quencher probe has a first quencher molecule linked thereto,
  • the first quencher molecule When a first hybrid is formed between the reporter probe and the first quencher probe, the first quencher molecule is at a position to quench a signal from the reporter probe,
  • the second quencher probe comprises a nucleotide sequence that hybridizes to a second region of the target nucleic acid
  • the second quencher probe has a second quencher molecule linked thereto,
  • the first region and the second region of the above target nucleic acid are adjacent to each other,
  • the second quencher molecule is at a position to quench a signal from the reporter molecule
  • the first hybrid has a melting temperature (Tm) different from the Tm of the second hybrid and the Tm of the third hybrid;
  • step (c) a step of determining the presence of the target nucleic acid from the signal measured in the step (b).
  • a sample suspected of containing a target nucleic acid is mixed and incubated with a composition for detecting a target nucleic acid, which comprises a reporter probe, a first quencher probe, and a second quencher probe.
  • the reporter probe comprises a nucleotide sequence that hybridizes to a first region of the target nucleic acid, and the reporter probe has a reporter molecule linked thereto.
  • the second quencher probe comprises a nucleotide sequence that hybridizes to a second region of the target nucleic acid, and the second quencher probe has a second quencher molecule linked thereto.
  • the first region and the second region of the above target nucleic acid are adjacent to each other.
  • the second quencher molecule is at a position to quench a signal from the reporter molecule.
  • the first hybrid compound has a melting temperature (Tm) that is different from the Tm of the second hybrid compound and the Tm of the third hybrid compound.
  • the first quencher molecule and the second quencher molecule are of the same type.
  • the incubation reaction means a reaction in which a target nucleic acid reacts with the target nucleic acid detection composition to provide a signal dependent on the presence of the target nucleic acid at a temperature selected within the signal-change temperature range of the target nucleic acid detection composition.
  • the step (a) incubation comprises a nucleic acid amplification reaction, specifically, an amplification reaction of a target nucleic acid.
  • the amplification reaction comprises multiple cycles.
  • the amplification of the target nucleic acid can be performed by polymerase chain reaction (PCR), more specifically, real-time polymerase chain reaction (real-time PCR).
  • PCR polymerase chain reaction
  • Real-time PCR real-time polymerase chain reaction
  • Polymerase chain reaction is widely used in the art to amplify target nucleic acid, and includes repeated cycles of denaturation of target nucleic acid, annealing (hybridization) between target nucleic acid and primer, and primer extension (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354).
  • Methods for separating the double-strands include, but are not limited to, heating, alkali, formamide, urea and glycoxal treatment, enzymatic methods (e.g., helicase action), and binding proteins.
  • separation of the strands can be accomplished by heating at a temperature in the range of 80° C. to 105° C.
  • a general method for accomplishing such treatments is provided by Joseph Sambrook, et. al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
  • Annealing of the primer and target nucleic acid can be carried out under suitable hybridization conditions, which are generally determined by optimization procedures. Conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength can vary depending on various factors including the length and GC content of the oligonucleotide (primer) and the target nucleic acid. Detailed conditions for hybridization can be found in Joseph Sambrook et. al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999).
  • the primer annealed to the target nucleic acid is extended by a template-dependent polymerase, which includes the "Klenow" fragment of E. coli DNA polymerase I, a thermostable DNA polymerase, and bacteriophage T7 DNA polymerase.
  • a template-dependent polymerase which includes the "Klenow" fragment of E. coli DNA polymerase I, a thermostable DNA polymerase, and bacteriophage T7 DNA polymerase.
  • the template-dependent polymerase is a thermostable DNA polymerase obtained from a variety of bacterial species.
  • the components required for the reaction may be provided in excess to the reaction vessel.
  • excess means an amount of each component such that the ability to achieve the desired extension is not substantially limited by the concentration of said components. It is desirable to provide the necessary cofactors such as Mg 2+ , dATP, dCTP, dGTP and dTTP to the reaction mixture in sufficient amounts to cause the desired reaction to occur.
  • a reverse transcription step is essential prior to the annealing step, and the details thereof are disclosed in the literature [Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and Noonan, K. F. et al., Nucleic Acids Res. 16:10366 (1988)].
  • an oligonucleotide dT primer, a random primer or a target-specific primer capable of hybridizing to the poly A tail of mRNA can be used.
  • ligase chain reaction 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 WO 90/01069, European Patent No. 439182 and WO 93/00447
  • Q-beta replicase amplification see Cahill P, et al., Clin Chem., 37(9):1482-5(1991), U.S. Patent No.
  • amplification-mediated amplification e.g., European Patent No. 497272
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription-mediated amplification
  • RPA recombinase polymerase amplification
  • LAMP loop-mediated isothermal amplification
  • the amplification method described above can amplify target nucleic acids through repetition of a series of reactions with or without changing the temperature.
  • the unit of amplification including repetition of the series of reactions is expressed as a "cycle.”
  • the unit of the cycle can be expressed as the number of repetitions or time depending on the amplification method.
  • the above series of reactions can be performed sequentially.
  • the annealing reaction of the primer can be performed, and then the extension reaction of the primer can be performed sequentially.
  • the cycle can be expressed as the number of repetitions.
  • the above series of reactions may be performed simultaneously.
  • annealing of the primer may be performed in some of the multiple templates at the same time, and in other templates, the primer may already be annealed and the primer extension reaction may be performed.
  • the cycle may be expressed in time. Specifically, one cycle may be 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, or 2 hours.
  • the incubation can be performed for a number of cycles sufficient to detect a change in signal dependent on the presence of the target nucleic acid.
  • the number of the plurality of cycles is 2 to 100 cycles, 2 to 90 cycles, 2 to 80 cycles, 2 to 70 cycles, 2 to 60 cycles, 2 to 50 cycles, 2 to 40 cycles, 2 to 30 cycles, 2 to 20 cycles, 2 to 10 cycles, 5 to 100 cycles, 5 to 90 cycles, 5 to 80 cycles, 5 to 70 cycles, 5 to 60 cycles, 5 to 50 cycles, 5 to 40 cycles, 5 to 30 cycles, 5 to 20 cycles, 5 to 10 cycles, 10 to 100 cycles, 10 to 90 cycles, 10 to 80 cycles, 10 to 70 cycles, 10 to 60 cycles, 10 to 50 cycles, 10 to 40 cycles, 10 to 30 cycles, It can be 10 to 20 cycles, 20 to 100 cycles, 20 to 90 cycles, 20 to 80 cycles, 20 to 70 cycles, 20 to 60 cycles, 20 to 50 cycles, 10 to 40 cycles, 10 to 30 cycles, It can be 10 to 20 cycles, 20 to 100 cycles, 20 to 90 cycles, 20 to 80 cycles
  • a signal provided by incubation (specifically, an amplification reaction) of a target nucleic acid and a composition for detecting the target nucleic acid is measured.
  • the measurement of the signal is performed at a temperature selected from the signal-change temperature range (i.e., a detection temperature). Specifically, the measurement of the signal is performed at a temperature at which one of the second hybrid and the third hybrid maintains its double-stranded state and the other dissociates into a single strand.
  • the detection temperature is dependent on the Tms of the first hybrid, the second hybrid and the third hybrid.
  • the formation of the second hybrid is superior to the formation of the first hybrid.
  • the measurement of the signal is performed during and/or after the incubation reaction.
  • the measurement of the signal can be performed at each cycle of the incubation reaction comprising a plurality of cycles, at a selected portion of the cycles, or at the end-point of the reaction.
  • the measurement of the signal is performed one or more times.
  • the measurement of the signal can be performed in at least two cycles.
  • the first cycle and the last cycle in which the signal is measured may be selected to be separated from each other by at least 1 cycle to 20 cycles.
  • the first cycle and the last cycle in which the signal is measured may be selected to be separated from each other by 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 11 cycles, 12 cycles, 13 cycles, 14 cycles, 15 cycles, 16 cycles, 17 cycles, 18 cycles, 19 cycles or 20 cycles, and more specifically, may be selected to be separated from each other by 5 cycles, 10 cycles, 15 cycles, 20 cycles or 30 cycles or more.
  • At least one of the cycles in which the signal is measured includes either an intermediate cycle including an exponential phase region or a late cycle including a plateau region.
  • an intermediate cycle including an exponential phase region For example, when measuring signals in two cycles, one may be performed in either an early cycle including a baseline region and the other may be performed in either an intermediate or late cycle, or one may be performed in either an intermediate cycle and the other may be performed in either an intermediate or late cycle.
  • the initial cycle includes a cycle up to an adjacent cycle and a value obtained by dividing the end cycle by 3. For example, if the end cycle is 45, since 45 divided by 3 is 15, the initial cycle may be determined as 1 to 20 cycles, 1 to 15 cycles, 1 to 10 cycles, or 1 to 5 cycles.
  • the middle cycle may be determined as an adjacent cycle and a value obtained by dividing the end cycle by 2. For example, if the end cycle is 45, since 45 divided by 2 is 22.5, the middle cycle may be determined as 16 to 30 cycles, 18 to 30 cycles, 20 to 30 cycles, 16 to 27 cycles, 18 to 27 cycles, 20 to 27 cycles, 16 to 25 cycles, 18 to 25 cycles, or 20 to 25 cycles.
  • the measurement of the signal can be performed in one cycle.
  • the one cycle in which the signal is measured is any one of an intermediate cycle including an exponential phase region excluding an early cycle including a baseline region, or a late cycle including a (plateau) region.
  • the presence of the target nucleic acid is determined from the signal measured in step (b).
  • the step (c) detects a change in the signal using the signal measured in at least two cycles in the step (b) or the signal measured in at least one cycle and a reference signal value, and if the signal changes, it is determined that the target nucleic acid is present. On the other hand, if the signal is constant, it is determined that the target nucleic acid is not present.
  • the signal change can be detected using the signal measured in the at least two cycles.
  • the incubation reaction e.g., PCR
  • the signal can be measured at the detection temperature for each cycle.
  • the signal values measured in each cycle can be plotted as an amplification curve (a set of data points consisting of RFU and cycles).
  • amplification curve refers to a curve obtained through a signal generation reaction of a target analyte (specifically, a target nucleic acid), particularly an amplification reaction.
  • the amplification curve includes a curve obtained when a target nucleic acid is present in a sample during an amplification reaction and a curve or line obtained when a target nucleic acid is not present in a sample during an amplification reaction.
  • a signal change or a constant signal can be detected as an indicator of amplification of a target nucleic acid.
  • the term "indicator of amplification” as used herein means an indicator that is closely related to the occurrence of amplification of a target nucleic acid, which can be obtained from the signal provided in step (a).
  • the indicator may mean a value that is generated dependently on the amplification of the target nucleic acid.
  • the indicator may be an indicator that provides a larger value as the amplification of the target nucleic acid increases (i.e., the amount of the target nucleic acid increases), or an indicator that provides a smaller value as the amplification increases.
  • the indicator may be any indicator as long as it indicates amplification.
  • the indicator may include something obtained from an amplification curve or a melting curve.
  • the indicator may include a value of a signal at a specific cycle in an amplification curve (e.g., RFU), a value of a signal at each cycle, a difference in a value of a signal between specific cycles, or a difference in a value of a signal between a reference signal value and specific cycles, or a height, width or area of a maximum melting peak in a melting curve.
  • the indicator may be, but is not limited to, a Ct (cycle threshold) value, ⁇ RFU (e.g., a difference in RFU between two cycles or a difference in RFU between a reference RFU and a specific cycle, etc.), an RFU ratio (e.g., a ratio of RFU between two cycles or a ratio of RFU between a reference RFU and a specific cycle, etc.), and a melting peak height/area/width (e.g., a height/area/width of a maximum peak in a melting curve).
  • ⁇ RFU e.g., a difference in RFU between two cycles or a difference in RFU between a reference RFU and a specific cycle, etc.
  • RFU ratio e.g., a ratio of RFU between two cycles or a ratio of RFU between a reference RFU and a specific cycle, etc.
  • a melting peak height/area/width e.g., a height/area/width of a maximum peak in
  • the indicator representing amplification is a Ct value or a Cq value.
  • the concepts of Ct value and Cq value are widely known in the art.
  • the indicator representing the amplification is ⁇ RFU or RFU ratio of the RFU values obtained in the amplification reaction.
  • the indicator is the difference (subtraction) or ratio between the RFUs of two cycles, or the difference (subtraction) or ratio between the RFU of a reference RFU and the RFU of a specific cycle.
  • the signal change can be detected using the signal measured in the one cycle and the reference signal value.
  • the above “reference signal value” may refer to a value that can confirm a signal change dependent on the presence of a target nucleic acid through a separate reaction.
  • the reference signal value may be obtained from a reaction in which the target nucleic acid is absent at the detection temperature (e.g., a negative control reaction).
  • the reference signal value may be a "signal value at the detection temperature" in the case in which the target nucleic acid is absent.
  • the reference signal value may be a threshold value determined in advance from a negative control reaction, considering the background signal of the detector, sensitivity, or characteristics of the label used.
  • the significance of the signal change can be determined using the threshold value.
  • the threshold value can be determined according to a conventional threshold value setting method. For example, the threshold value can be determined by considering the background signal, sensitivity, label characteristics, signal variation of the detector, or error range.
  • a threshold value when used as the reference signal value, it can be determined that the signal has changed if the signal value measured in step (b) is equal to or greater than the threshold value.
  • the present disclosure provides a method of detecting n target nucleic acids in a sample, comprising:
  • n is an integer greater than or equal to 2
  • the above incubation comprises multiple reaction cycles, and the measurement of the signal is performed in one or more of the multiple reaction cycles,
  • Each of the above n target nucleic acid detection compositions provides a change in a signal indicating the presence of the corresponding target nucleic acid at a corresponding detection temperature among the n detection temperatures in the presence of the corresponding target nucleic acid,
  • the i target nucleic acid detection composition provides a change in a signal indicating the presence of the i target nucleic acid at the i detection temperature among the n detection temperatures in the presence of the i target nucleic acid, and provides a constant signal at other detection temperatures.
  • the above i represents an integer from 1 to n , and the i- th detection temperature is lower than the i +1-th detection temperature.
  • the composition for detecting the i- th target nucleic acid has a signal-changing temperature range (SChTR) in which a signal changes depending on the presence of the i- th target nucleic acid and one or two signal-constant temperature ranges (SCoTR) in which a signal is constant even when the i-th target nucleic acid is present.
  • SChTR signal-changing temperature range
  • SCoTR signal-constant temperature ranges
  • composition for detecting the above i target nucleic acid is,
  • At least one of the above n target nucleic acid detection compositions is a composition comprising the above-described reporter probe, the first quencher probe and the second quencher probe, and;
  • step (b) a step of determining the presence of n target nucleic acids from the signals measured in the step (a), wherein the step of determining the presence of the i- th target nucleic acid by the signal measured at the i- th detection temperature.
  • the third aspect of the present disclosure utilizes the composition of the first aspect and the method of the second aspect described above, common content therebetween is omitted to avoid excessive duplication which would complicate the present specification.
  • a method for detecting n target nucleic acids according to the present disclosure uses n compositions for detecting n target nucleic acids, wherein the n compositions include a composition for detecting target nucleic acids including at least three probes according to the present disclosure, and can be variously combined with UnderSC-type, InterSC-type and OverSC-type compositions that adopt various signal generating methods known in the art for detecting target nucleic acids.
  • the method for detecting n target nucleic acids adjusts the signal-change temperature ranges of n compositions for detecting n target nucleic acids so that only a signal (specifically, a signal change) indicating the presence of the corresponding target nucleic acid is provided at each detection temperature, thereby enabling the presence of a specific target nucleic acid to be confirmed by a signal change detected only at a specific detection temperature.
  • a signal specifically, a signal change
  • a sample suspected of containing one or more of the n target nucleic acids is mixed with the n target nucleic acid detection compositions in one reaction vessel and incubated.
  • the n target nucleic acids can include nucleotide variations.
  • one of the n target nucleic acids can include one type of nucleotide variation and another can include another type of nucleotide variation.
  • n target nucleic acids herein may be genes from n different organisms, n different genes from the same organism, or a combination thereof.
  • the incubation reaction means any reaction that induces a change in signal dependent on the presence of the corresponding target nucleic acid at the corresponding detection temperature, as each target nucleic acid reacts with the corresponding target nucleic acid detection composition.
  • incubation comprises multiple cycles.
  • the incubation of step (a) may include an amplification reaction, such as a signal amplification reaction and/or a nucleic acid amplification reaction.
  • an amplification reaction such as a signal amplification reaction and/or a nucleic acid amplification reaction.
  • the amplification reaction comprises multiple cycles.
  • the incubation of step (a) is performed under conditions that allow target amplification together with signal changes by the composition for detecting target nucleic acids.
  • conditions include temperature, salt concentration, and pH of the solution.
  • step (a) the incubation of step (a) is performed in a signal amplification process without nucleic acid amplification.
  • the signal can be amplified simultaneously with amplification of the target nucleic acid.
  • the signal can be amplified without amplification of the target nucleic acid.
  • the signal change occurs in a process that includes signal amplification and amplification of the target nucleic acid.
  • Amplification of the target nucleic acid can be described in detail with reference to the description of the second aspect described above.
  • the amplification reaction of the target nucleic acid may be a multiple target nucleic acid sequence amplification reaction.
  • multiple target nucleic acid amplification reaction refers to a reaction that targets two or more nucleic acids and amplifies them in a single reaction vessel.
  • a multiple target nucleic acid amplification reaction refers to a reaction that amplifies two or more nucleic acids together.
  • a multiple target nucleic acid amplification reaction can amplify 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more target nucleic acids together in a single reaction.
  • the method according to the present disclosure comprises using a single type of label in one reaction vessel to produce 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 12, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 15, 3 to 12, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 12, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6 or 4 to 5 target nucleic acids can be detected.
  • a method according to the present disclosure is used to determine whether at least one of n target nucleic acids is present in a sample. For example, when n is 2, the present disclosure can be used to determine whether at least one of a first target nucleic acid and a second target nucleic acid is present in the sample. As another example, when n is 3, the present disclosure can be used to determine whether at least one of a first target nucleic acid, a second target nucleic acid and a third target nucleic acid is present in the sample.
  • n is an integer greater than or equal to 2.
  • n may be, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50.
  • the present disclosure uses a combination of n target nucleic acid detection compositions to detect n target nucleic acids ( n is an integer greater than or equal to 2). That is, a combination of first to n target nucleic acid detection compositions is used to detect first to nth target nucleic acids.
  • a combination of the first through nth target nucleic acid detection compositions refers to a compilation or mixture of compositions that are specific for each of the first through nth target nucleic acids.
  • a target nucleic acid detection composition is specific for a corresponding target nucleic acid.
  • the expression "a target nucleic acid detection composition is specific for a corresponding target nucleic acid” means that the target nucleic acid detection composition is involved in the detection of the corresponding target nucleic acid but is not involved in the detection of other target nucleic acids. In other words, the expression means that the target nucleic acid detection composition interacts with the corresponding target nucleic acid but does not interact with other target nucleic acids.
  • the composition for detecting the nth target nucleic acid is specific for the nth target nucleic acid, for example, the composition for detecting the first target nucleic acid is specific for the first target nucleic acid, the composition for detecting the second target nucleic acid is specific for the second target nucleic acid, and the third The composition for detecting a target nucleic acid is specific for a third target nucleic acid.
  • the combination of the first to nth target nucleic acid detection compositions used in the present invention is used together in one reaction. That is, the first to nth target nucleic acid detection compositions exist together in one reaction solution or reaction vessel.
  • composition for detecting a target nucleic acid means a composition containing components used for detecting a target nucleic acid.
  • each of the first to nth target nucleic acid detection compositions includes a label that provides a signal dependent on the presence of a corresponding target nucleic acid among the first to nth target nucleic acids, and the signals provided from each of the first to nth target nucleic acid detection compositions are not distinguished from each other by a single detection channel.
  • a composition for detecting a target nucleic acid may include various oligonucleotides involved in the amplification and/or detection of the target nucleic acid.
  • the label may be linked to the oligonucleotide or may be present in a free form. Alternatively, the label may be incorporated into the oligonucleotide during the incubation.
  • the above-mentioned labels and oligonucleotides have been mentioned as core elements in the composition for detecting the first to nth target nucleic acids, and that various components may be additionally included in addition to the labels and oligonucleotides.
  • components included in the composition for detecting the target nucleic acid include, but are not limited to, an oligonucleotide set used to amplify or detect the target nucleic acid, a label, a nucleic acid polymerase, a buffer, a polymerase cofactor, and deoxyribonucleotide-5-triphosphate.
  • the composition may also include various polynucleotide molecules, a reverse transcriptase, various buffers and reagents, and an antibody that inhibits DNA polymerase activity.
  • the composition may also include reagents necessary to perform positive control and negative control reactions. The optimal amount of reagents to be used in a particular reaction can be readily determined by one of ordinary skill in the art having the benefit of the present disclosure.
  • the components of the composition may be present in separate containers, or multiple components may be present in a single container.
  • the first target nucleic acid detection composition is an UnderSC-type or InterSC-type composition
  • the second target nucleic acid detection composition is an InterSC-type or OverSC-type composition
  • the first target nucleic acid detection composition is an UnderSC-type or InterSC-type composition
  • the n-th target nucleic acid detection composition is an InterSC-type or OverSC-type composition
  • the remaining compositions except the first and n- th target nucleic acid detection compositions are InterSC-type compositions.
  • the n target nucleic acid detection compositions used in the present disclosure are each one of (i) an UnderSC-type composition, (ii) an InterSC-type composition, and (iii) an OverSC-type composition, wherein at least one of the n target nucleic acid detection compositions is an InterSC-type composition.
  • the target nucleic acid detection composition according to the present disclosure comprises three probes.
  • n 2
  • at least one of the first target nucleic acid detection composition and the second target nucleic acid detection composition is a target nucleic acid detection composition comprising three probes.
  • exemplary combinations of the first target nucleic acid detection composition and the second target nucleic acid detection composition are as shown in Table 1 below.
  • exemplary combinations of the first to third target nucleic acid detection compositions are as shown in Table 2.
  • n 2 Target 1 Second target 1 UnderSC Composition according to the present disclosure 2 Composition according to the present disclosure Composition according to the present disclosure 3 Composition according to the present disclosure InterSC 4 InterSC Composition according to the present disclosure 5 Composition according to the present disclosure OverSC
  • each of the n target nucleic acid detection compositions provides a change in a signal indicating the presence of a corresponding target nucleic acid at a corresponding detection temperature among the n detection temperatures.
  • a composition for detecting an i - th target nucleic acid among the n target nucleic acids provides a signal change at an i - th detection temperature among the n detection temperatures in the presence of the i -th target nucleic acid, and provides a constant signal at other detection temperatures.
  • the composition for detecting the i- th target nucleic acid provides a signal change (i.e., a change in the i - th signal) at the i -th detection temperature in response to amplification of the target nucleic acid when the i -th target nucleic acid is present, whereas at detection temperatures other than the i -th detection temperature, the composition does not provide a signal change even if the target nucleic acid is amplified (i.e., the signal is constant).
  • a signal change i.e., a change in the i - th signal
  • the composition for detecting the i- th target nucleic acid has a signal-change temperature range in which a signal changes as the i- th target nucleic acid is amplified and a signal-constant temperature range in which a signal is constant even if the i -th target nucleic acid is amplified.
  • i - th signal means a signal provided by the i - th target nucleic acid detection composition at the i -th detection temperature, and is used interchangeably with “signal at the i -th detection temperature.”
  • the composition for detecting the i- th target nucleic acid provides a constant signal without signal change at the i - th detection temperature during an incubation reaction (e.g., a target nucleic acid amplification reaction) when the i- th target nucleic acid is not present.
  • an incubation reaction e.g., a target nucleic acid amplification reaction
  • the signal-change temperature range is a temperature range in which the signal value changes depending on the degree of amplification of the target nucleic acid (e.g., the amount of amplified target nucleic acid).
  • the signal-constant temperature range is a temperature range in which the value of the signal does not change regardless of the presence of the target nucleic acid.
  • the signal-constant temperature range is a temperature range in which there is no difference between the signal value when the target nucleic acid is present and the signal value when the target nucleic acid is not present.
  • the i detection temperature may be selected within the signal-change temperature range of the i target nucleic acid detection composition.
  • the i target nucleic acid detection composition herein is referred to as having the i detection temperature.
  • the i target nucleic acid corresponding to the i target nucleic acid detection composition may be referred to as a target nucleic acid having the i detection temperature.
  • one detection temperature determined by the corresponding target nucleic acid detection composition is assigned to one target nucleic acid.
  • the first target nucleic acid detection composition when n is 2, provides a change in signal at a first detection temperature in the presence of the first target nucleic acid, and a constant signal at another detection temperature, i.e., the second detection temperature; and the second target nucleic acid detection composition provides a change in signal at the second detection temperature in the presence of the second target nucleic acid, and a constant signal at another detection temperature, i.e., the first detection temperature.
  • the first target nucleic acid detection composition when n is 3, provides a change in signal at a first detection temperature in the presence of the first target nucleic acid, and a constant signal at other detection temperatures, i.e., the second detection temperature and the third detection temperature; the second target nucleic acid detection composition provides a change in signal at the second detection temperature in the presence of the second target nucleic acid, and a constant signal at other detection temperatures, i.e., the first detection temperature and the third detection temperature; and the third target nucleic acid detection composition provides a change in signal at the third detection temperature in the presence of the third target nucleic acid, and a constant signal at other detection temperatures, i.e., the first detection temperature and the second detection temperature.
  • the i detection temperature is selected within a signal-change temperature range of the i target nucleic acid detection composition, and the i detection temperature is not included in a signal-change temperature range of another target nucleic acid detection composition.
  • a signal-change temperature range of any one of the target nucleic acid detection compositions may overlap with a signal-change temperature range of a target nucleic acid detection composition having an adjacent detection temperature, but may not overlap with a signal-change temperature range of a target nucleic acid detection composition having a non-adjacent detection temperature.
  • the detection temperature of the target nucleic acid detection composition having a signal-change temperature range overlapping with a signal-change temperature range of another target nucleic acid detection composition is selected from a temperature range that does not overlap with the signal-change temperature range of the other target nucleic acid detection composition among the signal-change temperature ranges.
  • the signal-change temperature range of one of the target nucleic acid detection compositions may overlap with the signal-change temperature range of a target nucleic acid detection composition having an adjacent detection temperature, but neither of the two signal-change temperature ranges is completely included in the other signal-change temperature range.
  • adjacent detection temperatures is used to refer to consecutive detection temperatures among n detection temperatures, for example, the adjacent detection temperature of an i -th detection temperature is an i- 1-th detection temperature or an i +1-th detection temperature.
  • the signal-change temperature range of the i target nucleic acid detection composition may partially overlap with the signal-change temperature range of the target nucleic acid detection composition having an adjacent detection temperature, but does not overlap with the signal-change temperature range of the target nucleic acid detection composition having a non-adjacent detection temperature.
  • the composition for detecting the i target nucleic acid comprises a label that provides a signal dependent on the presence of the i target nucleic acid.
  • the label may be linked to the oligonucleotide or may be present in a free form.
  • the label may be incorporated into the oligonucleotide during the incubation (e.g., a nucleic acid amplification reaction). That is, the composition for detecting a target nucleic acid may initially include an oligonucleotide to which the label is linked, or the label may be incorporated into an oligonucleotide (e.g., an extended strand) newly formed during the incubation reaction to provide an oligonucleotide to which the label is linked.
  • the composition for detecting the i target nucleic acid comprises an incorporating label that is incorporated into the oligonucleotide during incubation and provides a signal dependent on the presence of the i target nucleic acid.
  • the composition for detecting the i target nucleic acid provides a label-linked oligonucleotide that serves to provide a signal dependent on the presence of the i target nucleic acid.
  • the composition for detecting the i target nucleic acid comprises from the beginning a labeled oligonucleotide linked thereto, which serves to provide a signal dependent on the presence of the i target nucleic acid.
  • the reporter probe, the first quencher probe and the second quencher probe are examples of labeled oligonucleotides herein.
  • the composition for detecting the i- th target nucleic acid may include a label and an oligonucleotide that provides a signal dependent on the presence of the i -th target nucleic acid, and the label may be incorporated into the oligonucleotide during an incubation reaction (e.g., a nucleic acid amplification reaction) to provide an oligonucleotide linked to a label that serves to provide a signal dependent on the presence of the i-th target nucleic acid.
  • an incubation reaction e.g., a nucleic acid amplification reaction
  • label-linked oligonucleotide as used herein is an oligonucleotide that is involved in the generation of a detectable signal.
  • the label-linked oligonucleotide can include an oligonucleotide (e.g., a probe or a primer) that specifically hybridizes to a target nucleic acid; when the probe or primer hybridized to the target nucleic acid is cleaved to release a fragment, the label-linked oligonucleotide can include a capture oligonucleotide that specifically hybridizes to the fragment; when the fragment hybridized to the capture oligonucleotide is extended to form an extended strand, the label-linked oligonucleotide can include an oligonucleotide that specifically hybridizes to the extended strand, an oligonucleotide generated by inserting a label during the extension of the fragment, an oligonucleotide that specifically hybridizes to the capture oligonucleotide, and combinations thereof.
  • an oligonucleotide e.g., a probe or a primer
  • the label-linked oligonucleotide comprises an oligonucleotide that is involved in actual signal generation. For example, hybridization or non-hybridization of the label-linked oligonucleotide with another oligonucleotide (e.g., the label-linked oligonucleotide or an oligonucleotide comprising a nucleotide sequence complementary to a target nucleic acid) determines signal generation.
  • another oligonucleotide e.g., the label-linked oligonucleotide or an oligonucleotide comprising a nucleotide sequence complementary to a target nucleic acid
  • the oligonucleotide to which the label is linked may be a 'probe' known in the art.
  • the 3'-end of the probe is "blocked" to prevent its extension. Blocking may be accomplished by conventional methods.
  • the label-linked oligonucleotide can be comprised of at least one oligonucleotide. In one embodiment, when the label-linked oligonucleotide is comprised of a plurality of oligonucleotides, the label-linked oligonucleotide can have labels in various ways. For example, all or some of the plurality of oligonucleotides can have at least one label.
  • the label may be a single label or an interactive label.
  • the single label includes a fluorescent label, a luminescent label, a chemiluminescent label, an electrochemical label, and a metal label.
  • the single label provides different signals (e.g., different signal intensities) depending on whether it is present in the double strand or the single strand.
  • the single label is a fluorescent label. Preferred types and binding sites of the single fluorescent labels used in the present disclosure are disclosed in U.S. Patent Nos. 7,537,886 and 7,348,141, the teachings of which are incorporated herein by reference in their entirety.
  • the single fluorescent labels include JOE, FAM, TAMRA, ROX, and fluorescein-based labels.
  • the single labels can be linked to the oligonucleotide by a variety of methods.
  • the labels are linked to the probe via a spacer comprising carbon atoms (e.g., a 3-carbon spacer, a 6-carbon spacer, or a 12-carbon spacer).
  • the interaction label can be an interaction label comprising at least one reporter molecule and at least one quencher molecule.
  • the interaction label can be an interaction dual label comprising one reporter molecule and one quencher molecule.
  • the interaction label can be an interaction label comprising one reporter molecule and two quencher molecules.
  • Reporter molecules and quencher molecules useful in the present disclosure may include any molecules known in the art, for detailed descriptions of which see the section describing reporter molecules and quencher molecules in the first aspect described above.
  • the interaction label when the label is an interaction label, the interaction label may be an interaction label comprising at least one reporter molecule and at least one quencher molecule, and the interaction labels may be linked all to one oligonucleotide, or may be linked individually to a plurality of oligonucleotides.
  • the insertion label can be used in a process of generating a signal by inserting the label during primer extension (e.g., the Plexor method, Sherrill CB, et al., Journal of the American Chemical Society , 126:4550-45569 (2004)).
  • the insertion label can also be used in signal generation by a duplex formed in a manner dependent on the cleavage of an intermediate oligonucleotide hybridized to the target nucleic acid sequence.
  • the insertion tag may generally be linked to a nucleotide. Additionally, nucleotides having non-natural bases may also be utilized.
  • non-natural base refers to derivatives of natural bases, such as adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U), which can form hydrogen-bonded base pairs.
  • non-natural base includes bases that have base pairing patterns that are different from the natural bases as the mother compounds, and are described, for example, in U.S. Patent Nos. 5,432,272, 5,965,364, 6,001,983, and 6,037,120.
  • Base pairing between non-natural bases involves two or three hydrogen bonds, like natural bases. Base pairing between non-natural bases also occurs in specific ways.
  • unnatural bases include the following base pairing combinations: iso-C/iso-G, iso-dC/iso-dG, Z/P, V/J, K/X, H/J, Pa/Ds, Pa/Q, Pn/Ds, Pn/Dss, Px/Ds, NaM/5SICS, 5FM/5SICS, and M/N (see US Pat. Nos. 5,432,272; 5,965,364; 6,001,983; 6,037,120; 6,140,496; 6,627,456; 6,617,106; and 7,422,850; and Filip Wojciechowsk i et al., Chem. Soc. Rev. , 2011, 40, 5669-5679).
  • the method according to the present disclosure can detect multiple target nucleic acids in real time without additional analysis, such as melting curve analysis, even while using a single type of label (e.g., one fluorescent label) by using a composition for detecting target nucleic acids that provides a dimer.
  • each of the n target nucleic acid detection compositions provides at least one dimer.
  • duplex refers to a double-stranded nucleic acid molecule formed by hybridization under hybridization conditions of two single-stranded nucleic acid molecules having partially or fully complementary sequences.
  • the two single-strands forming the duplex may exist in an associated form (i.e., a double-stranded molecule) or in a dissociated form (i.e., two single-stranded molecules) depending on the temperature (particularly, the detection temperature).
  • the term “duplex” may be used to encompass a duplex in associated form and a duplex in dissociated form.
  • the dimer may be referred to as a “hybrid.”
  • the expression "the composition for detecting a target nucleic acid provides a dimer” as used herein can mean providing a dimer in an associated form and/or a dissociated form.
  • the expression "the composition for detecting a target nucleic acid generates a dimer during incubation” as used herein can mean generating a dimer in an associated form and/or a dissociated form during the incubation reaction.
  • At least one of the dimers provided by the composition for detecting the target nucleic acid is a dimer that provides a signal.
  • the dimer is a dimer that provides a signal change. That is, the composition for detecting the i- th target nucleic acid provides a dimer that provides a signal, and specifically, the composition for detecting the i- th target nucleic acid provides a dimer that provides a signal change dependent on the presence of the i- th target nucleic acid.
  • signal-providing dimer means a dimer capable of providing a signal that can be distinguished depending on whether the dimer is in an associated or dissociated state. For example, this means that the dimer in an associated form generates (or extinguishes) a signal, and the dimer in a dissociated form extinguishes (or generates) a signal.
  • the signal providing dimer may comprise at least one label.
  • dimer providing a signal change means a dimer whose content changes depending on the presence of the target nucleic acid, thereby providing a signal change indicating the presence of the target nucleic acid.
  • the first hybrid, the second hybrid, and the third hybrid described above are examples of dimers providing a signal change described herein.
  • the duplexer providing the signal change comprises a label. Specifically, at least one label is linked to at least one of the two single strands constituting the duplexer.
  • the duplexer providing the signal change comprises a single label, in which case the single label is linked to either one of the two single strands constituting the duplexer.
  • the duplexer providing the signal change comprises an interaction label, in which case the interaction label is linked to both one of the two strands constituting the duplexer providing the signal change, or one of the interaction labels is linked to either one of the two single strands and the other of the interaction labels is linked to the other one of the two single strands.
  • the association and dissociation of the dimer can be caused by temperature.
  • the dimer providing the signal change may be a dimer that is included in the composition for detecting a target nucleic acid from the beginning.
  • the duplexer when the duplexer providing the signal change is initially included in the composition for detecting the target nucleic acid, the duplexer can be generated by hybridization between an oligonucleotide to which a label is linked and an oligonucleotide capable of hybridizing with the oligonucleotide to which the label is linked.
  • An example of this is the first hybridization between the reporter probe and the first quencher probe in the present disclosure.
  • the content of the dimer providing the signal change varies depending on the presence of the target nucleic acid, specifically, the content decreases, thereby providing the signal change.
  • the content of the first hybrid decreases while generating a second hybrid between the reporter probe and the target nucleic acid depending on the presence of the target nucleic acid, thereby providing a signal change depending on the presence of the target nucleic acid.
  • the duplex providing the signal change generated during the incubation reaction can be provided by hybridization between the label-linked oligonucleotide and the target nucleic acid.
  • the second hybrid and the third hybrid in the present disclosure are examples.
  • the signal by the formation of a dimer between the label-linked oligonucleotide and the target nucleic acid can be detected by the Scorpion method (Whitcombe et al., Nature Biotechnology 17:804-807 (1999)), the Sunrise (or Amplifluor) method (Nazarenko et al., Nucleic Acids Research , 25(12):2516-2521 (1997), and U.S. Pat. No. 6,117,635 ), the Lux method (U.S. Pat. No.
  • the signal change is caused by a duplex formed in a manner dependent on cleavage of an intermediate oligonucleotide that hybridizes specifically to the target nucleic acid.
  • intermediate oligonucleotide is an oligonucleotide that mediates the formation of a duplexer that does not include a target nucleic acid.
  • cleavage of the intermediate oligonucleotide itself does not generate a signal, but rather, after hybridization and cleavage of the intermediate oligonucleotide, a fragment (cleavage product) generated by the cleavage participates in a continuous reaction for signal generation.
  • hybridization or cleavage of the intermediate oligonucleotide itself does not generate a signal.
  • the mediating oligonucleotide comprises an oligonucleotide that mediates formation of a duplex by hybridizing to a target nucleic acid and cleaving it to release a fragment.
  • the fragment mediates the formation of a duplex by extension of the fragment onto a capture oligonucleotide.
  • the intermediate oligonucleotide comprises (i) a targeting moiety comprising a nucleotide sequence that hybridizes to a target nucleic acid and (ii) a tagging moiety comprising a nucleotide sequence that non-hybridizes to the target nucleic acid.
  • the composition for detecting the target nucleic acid comprises a tagging oligonucleotide that hybridizes to the target nucleic acid, and the cleavage reaction dependent on the presence of the target nucleic acid can comprise cleavage of the tagging oligonucleotide.
  • the tagging oligonucleotide is an example of an intermediary oligonucleotide as described above.
  • cleavage of the intermediate oligonucleotide releases a fragment, which fragment specifically hybridizes to the capture oligonucleotide and extends onto the capture oligonucleotide.
  • the capture oligonucleotide comprises a label
  • the capture oligonucleotide is an example of an oligonucleotide to which a label is linked herein.
  • an intermediary oligonucleotide hybridized to a target nucleic acid is cleaved to release a fragment, which specifically hybridizes to a capture oligonucleotide, which is extended to produce an extended strand, which causes formation of an extended duplex between the extended strand and the capture oligonucleotide, thereby providing a signal indicating the presence of the target nucleic acid.
  • a third oligonucleotide comprising a nucleotide sequence that hybridizes to the extended strand may be additionally used.
  • hybridization of the third oligonucleotide and the extended strand forms a different type of duplexer to provide a signal indicating the presence of the target nucleic acid (e.g., PCE-SH).
  • the different type of duplexer is the duplexer that provides the signal change.
  • the signal by the dimer generated in a manner dependent on the cleavage of the above-mentioned intermediate oligonucleotide can be generated by various methods including the PTO cleavage and extension (PTOCE) method (WO 2012/096523), the PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization (PCE-SH) method (WO 2013/115442) and the PTO Cleavage and Extension-Dependent Non-Hybridization (PCE-NH) method (WO 2014/104818).
  • PTOCE PTO cleavage and extension
  • PCE-SH PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization
  • PCE-NH PTO Cleavage and Extension-Dependent Non-Hybridization
  • oligonucleotides are as follows: the intermediate 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 Signaling Oligonucleotide
  • HO Hybridization Oligonucleotide
  • the SO, HO, CTO, the extender strand or a combination thereof can serve as the oligonucleotide to which the label is linked.
  • the dimer providing the signal change may be a single-type dimer or a multi-type dimer.
  • the number of the dimers may be 1, and when the dimer providing the signal change is a multi-type dimer, the number of the dimers may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20, and more specifically, may be 2, 3 or 4, and even more specifically, may be 2 or 3.
  • the dimer providing the signal change is a single-type dimer
  • the content of the single-type dimer changes depending on the presence of the target nucleic acid, thereby changing the signal.
  • the dimer when the dimer is a multi-type dimer, the content ratio between the multi-type dimers changes depending on the presence of the target nucleic acid, thereby changing the signal.
  • the Tm values of the multiple-type dimers are different from each other.
  • the Tm values between the dimers are different from each other by at least 2°C, 3°C, 4°C, 5°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C or 20°C.
  • the content of the dimer means the sum of the amount of the dimer in a state where the two nucleic acid strands constituting the dimer are dissociated (i.e., the dimer in a dissociated form) and the amount of the dimer in a state where the two nucleic acid strands are hybridized (i.e., the dimer in a bound form).
  • At least two of the multiple-type duplexers comprise the same single-strand.
  • the multiple-type duplexers comprise the same single-strand
  • the same single-strand is included in the first duplex that is initially included in the composition for detecting a target nucleic acid, and a new second duplexer comprising the same single-strand is generated during the incubation reaction.
  • the same single-strand that was included in the first duplexer may be considered to be consumed during the incubation reaction to generate the second duplexer, and thus, the content of the first duplexer comprising the same single-strand may be considered to decrease, and the content of the second duplexer comprising the same single-strand may be considered to increase.
  • the signal-change temperature range of the composition for detecting the i target nucleic acid can be determined dependently on the length and/or sequence of the duplexer providing the signal change.
  • the composition for detecting the i- th target nucleic acid when the composition for detecting the i- th target nucleic acid provides a multi-type dimer, specifically, two types of dimers, the composition for detecting the i- th target nucleic acid may have one signal-change temperature range and two signal-constant temperature ranges.
  • the signal-change range and the signal-constant temperature range may be determined depending on the length and/or sequence of the two types of dimers.
  • any one of the n target nucleic acid detection compositions may comprise an amplification oligonucleotide that serves to amplify the corresponding target nucleic acid.
  • the amplification oligonucleotide may be identical to the oligonucleotide to which the label is linked.
  • amplification oligonucleotide collectively refers to oligonucleotides that serve to amplify target nucleic acids.
  • the amplification oligonucleotide may be a 'primer' known in the art.
  • the amplifying oligonucleotide and the label-linked oligonucleotide are identical means that one oligonucleotide functions both as an amplifying oligonucleotide that amplifies the target nucleic acid and as a label-linked oligonucleotide that generates a signal in the presence of the target nucleic acid.
  • the label-linked oligonucleotide can hybridize with the target nucleic acid to be extended and generate a signal.
  • composition for detecting a target nucleic acid used in the present disclosure does not provide a signal at all temperatures in the presence of the target nucleic acid.
  • the target nucleic acid detection compositions containing oligonucleotides of different sequences can be considered to be different from each other.
  • the different target nucleic acid detection compositions have different detection temperatures.
  • the detection temperature according to the present disclosure can be determined in advance by considering the signal-change temperature ranges of each of the n target nucleic acid detection compositions.
  • the signal-change temperature range of any one of the n target nucleic acid detection compositions can be determined dependently on the length and/or sequence of the dimer. That is, by controlling the Tm value of the dimer, the signal-change temperature range can be determined in advance.
  • the measurement of the signal can be successfully achieved at a predetermined detection temperature by controlling the Tm value of the label-linked oligonucleotide.
  • a label-linked oligonucleotide e.g., a molecular beacon
  • the signal when the signal is generated by a dimer generated in response to the presence of the target nucleic acid, measurement of the signal is successfully accomplished at a predetermined temperature by controlling the Tm of the dimer.
  • the detection temperature is determined by considering the signal-change temperature range that depends on the dimer provided by the composition for detecting target nucleic acid.
  • the detection temperature of any one of the n compositions for detecting the n target nucleic acids can be predetermined within a signal-change temperature range that does not overlap with the signal-change temperature ranges of the other compositions.
  • the detection temperatures assigned to the compositions for detecting target nucleic acids differ from each other by at least 2°C, 3°C, 4°C, 5°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 15°C or 20°C.
  • the n detection temperatures are 45°C to 97°C, 45°C to 96°C, 45°C to 95°C, 45°C to 94°C, 45°C to 93°C, 45°C to 92°C, 45°C to 91°C, 45°C to 90°C, 46°C to 97°C, 46°C to 96°C, 46°C to 95°C, 46°C to 94°C, 46°C to 93°C, 46°C to 92°C, 46°C to 91°C, 46°C to 90°C, 47°C to 97°C, 47°C to 96°C, 47°C to 95°C, 47°C to 94°C, 47°C to 93°C, 47°C to 92°C, 47°C to 91°C, 47°C to 90°C, 48°C to 97°C, 48°C to 96°C, 48°C to 95°C, 48°C to 94°C, 48°C
  • the highest detection temperature among the above detection temperatures is 70°C to 97°C, 70°C to 95°C, 70°C to 93°C, 70°C to 90°C, 73°C to 97°C, 73°C to 95°C, 73°C to 93°C, 73°C to 90°C, 75°C to 97°C, 75°C to 95°C, 75°C to 93°C, 75°C to 90°C, 78°C to 97°C, 78°C to 95°C, 78°C to 93°C, 78°C to 90°C, 80°C to 97°C, 80°C to 95°C, 80°C to 93°C, 80°C to 90°C, 83°C to 97°C, 83°C to 95°C, 83°C
  • the temperature range may be selected from 83°C to 90°C, 85°C to 97°C, 85°C to 97°C, 85°C
  • the lowest detection temperature (i.e., the first detection temperature) can be selected from a temperature range of 45 to 70°C, 45°C to 68°C, 45°C to 65°C, 45°C to 63°C, 45°C to 60°C, 45°C to 58°C, 45°C to 55°C, 48°C to 70°C, 48°C to 68°C, 48°C to 65°C, 48°C to 63°C, 48°C to 60°C, 48°C to 58°C, 48°C to 55°C, 50°C to 70°C, 50°C to 68°C, 50°C to 65°C, 50°C to 63°C, 50°C to 60°C, 50°C to 58°C, 50°C to 55°C.
  • the intermediate detection temperatures are 55°C to 85°C, 55°C to 83°C, 55°C to 80°C, 55°C to 78°C, 55°C to 75°C, 55°C to 73°C, 55°C to 70°C, 55°C to 68°C, 55°C to 65°C, 55°C to 63°C, 55°C to 60°C, 58°C to 85°C, 58°C to 83°C, 58°C to 80°C, 58°C to 78°C, 58°C to 75°C, 58°C to 73°C, 58°C to 70°C, 58°C to 68°C, 58°C to 65°C, 58°C to 63°C, 58°C to 60°C, 60°C to 85°C, 60°C to 83°C, 58°C to 80°C, 58°C to 78°C, 58°C to 75°C, 58°C to 73
  • n target nucleic acids are each assigned to n detection temperatures, and then n compositions for detecting n target nucleic acids suitable for the detection temperatures are prepared, and then step (a) can be performed.
  • the first detection temperature when n is 3, the first detection temperature may be selected from a temperature range of 50°C to 60°C, the second detection temperature may be selected from a temperature range of 65°C to 75°C, and the third detection temperature may be selected from a temperature range of 80°C to 95°C.
  • step (a) the signal is measured at n detection temperatures during incubation.
  • measurement of the signal can be performed at each cycle, at selected cycles, or at the end-point of the reaction.
  • the measurement of the signal can be performed in at least one cycle.
  • the signal can be measured at n detection temperatures in one selected cycle, or can be measured at n detection temperatures in two selected cycles, respectively.
  • the signals i.e., the first signal, the second signal, and the third signal
  • the signals are measured at the first detection temperature, the second detection temperature, and the third detection temperature in 1 cycle
  • the signals are measured at the first detection temperature, the second detection temperature, and the third detection temperature in 30 cycles.
  • the measurement of the signal can be performed in at least two cycles.
  • the signal change can be detected using the signals measured in the at least two cycles.
  • the nucleic acid amplification can be performed for 30 cycles, 40 cycles, 45 cycles, or 50 cycles of PCR, and the signals can be measured at n detection temperatures for each cycle. Then, the signal values measured at each detection temperature in the plurality of cycles can be plotted as an amplification curve (a set of data points consisting of RFU and cycle) at each detection temperature.
  • an amplification curve at a first detection temperature, an amplification curve at a second detection temperature, and an amplification curve at a third detection temperature can be obtained, and the change in the signal can be identified from the amplification curves.
  • the method according to the present disclosure utilizes the fact that the composition for detecting a target nucleic acid provides a signal change dependent on the presence of the target nucleic acid only at a corresponding detection temperature.
  • the method according to the present disclosure can detect the signal change using signal values measured at the detection temperature in at least two cycles.
  • the method according to the present disclosure can detect the signal change using a signal value at the detection temperature measured in one cycle (i.e., the signal value measured in step (a)) and a reference signal value (e.g., a signal value at the detection temperature measured when the target nucleic acid is not present).
  • the "signal value at the detection temperature (e.g., the i -th detection temperature)" measured in the absence of the target nucleic acid (e.g., the i -th target nucleic acid) can be obtained through a separate negative control reaction.
  • the reference signal value can be obtained by conducting a negative control reaction simultaneously or separately from the method according to the present disclosure.
  • the reference signal value can be obtained through a negative control reaction.
  • the reference signal value at the i detection temperature can be obtained by mixing a sample (e.g., distilled water) that does not contain the i target nucleic acid with n target nucleic acid detection compositions, amplifying the nucleic acid, and measuring a signal at the i detection temperature.
  • the signal measurement can be performed in any cycle.
  • a signal value measured in any one of the early cycles of the negative control reaction can be used as the reference signal value, or a signal value measured in any one of the late cycles of the negative control reaction can be used as the reference signal value.
  • a signal value measured in the same cycle as the cycle in which the signal is measured in step (a) can be used as the reference signal value.
  • the reference signal value can be obtained through a positive control reaction.
  • a sample containing the i- th target nucleic acid is mixed with a composition for detecting the i - th target nucleic acid (specifically, n compositions for detecting n target nucleic acids) to amplify the nucleic acid, and a signal is measured at the i -th detection temperature.
  • the cycle in which the signal value is detected may be a cycle in the baseline region of the positive control reaction.
  • the baseline region refers to a region in which a signal (e.g., a fluorescent signal) remains substantially constant during an initial cycle of an amplification reaction (e.g., PCR). Since the level of the amplification product in this region is not sufficient to be detected, most of the fluorescent signal in this region is due to a background signal including a fluorescent signal of the reaction sample itself and a fluorescent signal of the measurement system itself. That is, a signal value measured in a cycle in the baseline region of the positive control reaction will be substantially the same as a reference signal value obtained from a reaction in which a target nucleic acid is absent (e.g., a negative control reaction).
  • a signal change can be detected through the difference between the reference signal value and the signal value measured in step (a).
  • the reference signal value may be a threshold value determined in advance from a negative control reaction, taking into account the background signal and sensitivity of the detector, or the characteristics of the label used, etc.
  • the significance of the signal change can be determined using the threshold value.
  • the threshold value can be determined by a known threshold value setting method. For example, the threshold value can be determined by taking into account the background signal, sensitivity, label characteristics, signal deviation or error range of the detector, etc.
  • a threshold value when a threshold value is used as the reference signal value, it can be determined that the signal has changed if the signal value measured in step (a) is equal to or greater than the threshold value.
  • signal measurements at each of the n detection temperatures can be performed using a single type of detector.
  • the single type of detector is one detector.
  • the signals generated from the labels of the oligonucleotides linked to each label included in the composition for detecting n target nucleic acids are not distinguished from each other for each target nucleic acid by the single type of detector.
  • a single or one type of fluorescent label means a fluorescent label having identical or substantially identical signal characteristics (e.g., optical characteristics, emission wavelength, and electrical signal).
  • signal characteristics e.g., optical characteristics, emission wavelength, and electrical signal.
  • FAM and CAL Fluor 610 provide different types of signals.
  • a single or one type of fluorescent label means that the signals from the fluorescent labels are indistinguishable from each other using a detection channel.
  • This single or one type of fluorescent label is not dependent on the chemical structure of the fluorescent label, and thus even if two fluorescent labels have different chemical structures, they are considered as one type if they are indistinguishable from each other using a detection channel.
  • signals generated from n target nucleic acid detection compositions that commonly include one type of fluorescent label are not distinguished by one detection channel.
  • detection channel means a means for detecting a signal 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), LightCycler (Roche), etc., contain several channels (e.g., photodiodes) for detecting signals from several different types of fluorescent labels, and such channels correspond to detection channels herein.
  • a detection channel used in the present disclosure includes a means for measuring a signal.
  • the detection channel may be a photodiode capable of detecting a fluorescent signal of a specific wavelength.
  • the signals measured at each of the n detection temperatures are indistinguishable from one another by the single type of detector.
  • step (a) signal changes are detected from the signals measured at n detection temperatures and the presence of n target nucleic acids is determined.
  • the presence of the i- th target nucleic acid is determined by a signal change detected at the i-th detection temperature. For example, a signal change is detected from a signal measured at the i- th detection temperature, and the presence of the i- th target nucleic acid is determined.
  • the signal is constant at the i detection temperature, it can be determined that the i target nucleic acid is absent.
  • the signal change can be detected using a signal measured in at least two cycles, or using a signal measured in at least one cycle and a reference signal value.
  • Determining the presence of a target nucleic acid from the signal measured at each detection temperature can be performed by various methods known in the art, including the method of detecting the signal change described above.
  • n 2
  • the presence of the first target nucleic acid can be determined from signals measured at a first detection temperature (the first signal at 10 cycles and the first signal at 30 cycles)
  • the presence of the second target nucleic acid can be determined from signals measured at a second detection temperature (the second signal at 10 cycles and the second signal at 30 cycles).
  • the method according to the present disclosure can be performed together with a negative control reaction.
  • a signal value measured in the negative control reaction can be used as a reference signal value.
  • a signal measured in one cycle e.g., the last cycle
  • a signal measured in the same cycle e.g., the last cycle
  • the same detection temperature i.e., the i- th detection temperature
  • n 3
  • the presence of the first target nucleic acid can be determined from a signal measured at a first detection temperature (i.e., a first signal at 30 cycles) and a first reference signal value (e.g., a signal at the first detection temperature measured at 30 cycles of the negative control reaction)
  • the presence of the second target nucleic acid can be determined from a signal measured at a second detection temperature (i.e., a second signal at 30 cycles) and a second reference signal value (e.g., a signal at the second detection temperature measured at 30 cycles of the negative control reaction)
  • the presence of the third target nucleic acid can be determined from a signal measured at a third detection temperature (i.e., a third signal at 30 cycles) and a third reference signal value (e.g., a signal at the third detection temperature measured at 30 cycles of the negative control reaction).
  • the method according to the present disclosure can be performed together with a positive control reaction.
  • a signal value measured in the positive control reaction can be used as a reference signal value.
  • a first signal measured in cycle 30, which is a signal measured in one cycle, for example, 30 cycles, at the i -th detection temperature can be compared with a signal measured in a cycle prior to cycle 30, for example, 1 cycle, at the i-th detection temperature of the positive control reaction to detect a change in the signal.
  • the measurement of the signal in the positive control reaction may be performed in a cycle at least 30 cycles prior to, 20 cycles prior to, 10 cycles prior to, or 5 cycles prior to the cycle in which the signal is measured in step (a).
  • n 3
  • the presence of the first target nucleic acid can be determined from a signal measured at a first detection temperature (i.e., a first signal at 30 cycles) and a first reference signal value (e.g., a signal at the first detection temperature measured at 1 cycle of the first target nucleic acid positive control reaction)
  • the presence of the second target nucleic acid can be determined from a signal measured at a second detection temperature (i.e., a second signal at 30 cycles) and a second reference signal value (e.g., a signal at the second detection temperature measured at 1 cycle of the second target nucleic acid positive control reaction)
  • the presence of the third target nucleic acid can be determined from a signal measured at a third detection temperature (i.e., a third signal at 30 cycles) and a third reference signal value (e.g., a signal at the third detection temperature measured at 1 cycle of the third target nucleic acid positive control reaction).
  • target nucleic acid detection is possible using a composition for detecting target nucleic acids including three probes according to the present disclosure.
  • the Tms of three hybrids i.e., a first hybrid, a second hybrid, and a third hybrid
  • the target nucleic acids can be arbitrarily controlled and used in various implementation examples.
  • Example 1 Detection of a single target nucleic acid 1 (when Tm1 is lower than Tm2 and Tm3)
  • Example 1-1 Preparation of target nucleic acid and oligonucleotide
  • the genomic DNA of Chlamydia trachomatis (CT) (Accession Number: ATCC VR-1500, Coram Deo Lab Co., Ltd.) was used as a template of the target nucleic acid.
  • the detection temperature of the CT target nucleic acid was set to 60°C, and the first region and the second region were selected in the 3' to 5' direction on the target nucleic acid template. Then, the sequences and lengths of the reporter probe, the first quencher probe, and the second quencher probe were designed so that Tm1 was 54.8°C, Tm2 was 72.5°C, and Tm3 was 67.3°C.
  • the reporter probe includes a sequence complementary to the first region of the target nucleic acid, and a reporter molecule (Cal Fluor Red 610) was linked to the 3'-terminus thereof.
  • the first quencher probe includes a sequence complementary to the 5'-terminal portion of the reporter probe, and a first quencher molecule (BHQ-2) was linked to the 5'-terminus thereof.
  • the second quencher probe contained a sequence complementary to the second region of the target nucleic acid and was linked to a second quencher molecule (BHQ-2) at its 5'-end. The 3'-ends of the first quencher probe and the second quencher probe were blocked with Spacer C3 to prevent elongation by DNA polymerase.
  • Primer pairs for amplifying a region including the first region and the second region of the CT target nucleic acid and three probes for detecting the CT target nucleic acid were prepared as shown in Table 3.
  • Example 1-2 Real-time polymerase chain reaction
  • tubes 1 and 2 containing the reaction mixtures were placed in a real-time thermocycler (CFX96 Real-time Cycler, Bio-Rad) respectively and denatured at 95°C for 15 minutes, followed by 50 cycles of 60°C for 60 seconds, 50°C for 5 seconds, 72°C for 30 seconds, and 95°C for 10 seconds.
  • CFX96 Real-time Cycler Bio-Rad
  • the signal measurements were performed at (i) 50°C, a temperature within the first signal-constant temperature range, (ii) 60°C, a temperature within the signal-changing temperature range, and (iii) 95°C, a temperature within the second signal-constant temperature range, for each cycle.
  • tube 1 For tube 1, a signal change was detected only at 60°C, which is within the signal-change temperature range, and no signal change was detected at 50°C and 95°C, which are within the first and second signal-constant temperature ranges. Therefore, tube 1 was determined to contain CT.
  • tube 2 the negative control, showed no signal change at any of the three temperatures. Therefore, tube 2 was determined not to contain CT.
  • composition for detecting target nucleic acids according to the present disclosure is an InterSC-type composition for detecting target nucleic acids, as described above.
  • the target nucleic acid detection method can detect a signal (i.e., a change in signal) indicating the presence of a target nucleic acid using a reference signal value obtained from a negative control reaction. If the signal values at 50°C, 60°C, and 95°C are less than or equal to RFU - 200, which is a threshold based on the signal value of the negative control reaction (i.e., RFU: 0), the signal was considered to have changed.
  • a signal i.e., a change in signal
  • tube Ct (Cycle threshold) 50°C 60°C 95°C 1 N/A 26.85 N/A 2 N/A N/A N/A
  • Tube 1 10 pg of CT genomic DNA
  • Tube 2 Negative control
  • Example 2 Detection of a single target nucleic acid 2 (when Tm1 is lower than Tm2 and higher than Tm3)
  • Example 2-1 Preparation of target nucleic acid and oligonucleotide
  • the genomic DNA of Neisseria gonorrhoeae (Accession Number: ATCC 700825, Coram Deo Lab Co., Ltd.) was used as a template of the target nucleic acid.
  • the detection temperature of the NG target nucleic acid was set to 74°C, and the first region and the second region were selected in the 3' to 5' direction on the target nucleic acid template. Then, the sequences and lengths of the reporter probe, the first quencher probe, and the second quencher probe were designed so that Tm1 was 76.4°C, Tm2 was 80.8°C, and Tm3 was 71.1°C.
  • the reporter probe included a sequence complementary to the first region of the target nucleic acid, and a reporter molecule (Cal Fluor Red 610) was linked to the 3'-end thereof.
  • the first quencher probe contains a sequence complementary to the 5'-terminal portion of the reporter probe and has a first quencher molecule (BHQ-2) linked to its 5'-terminus.
  • the second quencher probe contains a sequence complementary to the second region of the target nucleic acid and has a second quencher molecule (BHQ-2) linked to its 5'-terminus.
  • the 3'-terminus of the first quencher probe and the second quencher probe were blocked with Spacer C3 to prevent elongation by DNA polymerase.
  • Primer pairs for amplifying a region including the first region and the second region of the NG target nucleic acid and three probes for detecting the NG target nucleic acid were prepared as shown in Table 5.
  • tubes 1 and 2 containing the reaction mixtures were placed in a real-time thermocycler (CFX96 Real-time Cycler, Bio-Rad) respectively and denatured at 95°C for 15 minutes, followed by 50 cycles of 60°C for 60 seconds, 72°C for 30 seconds, 74°C for 5 seconds, and 95°C for 10 seconds.
  • CFX96 Real-time Cycler Bio-Rad
  • the signal measurements were performed at (i) 60°C, a temperature within the first signal-constant temperature range, (ii) 74°C, a temperature within the signal-changing temperature range, and (iii) 95°C, a temperature within the second signal-constant temperature range, for each cycle.
  • tube 1 For tube 1, a signal change was detected only at 74°C within the signal-change temperature range, and no signal change was detected at temperatures of 60°C and 95°C within the first and second signal-constant temperature ranges. Therefore, tube 1 was determined to contain NG.
  • tube 2 the negative control, showed no signal change at any of the three temperatures. Therefore, tube 2 was determined to not contain NG.
  • the composition for detecting target nucleic acids according to the present disclosure is an InterSC-type target nucleic acid detection composition, as described above.
  • the results of Example 2 together with the results of Example 1 indicate that the Tms of three hybrids (i.e., the first hybrid, the second hybrid, and the third hybrid) formed by three probes and target nucleic acids can be arbitrarily controlled and used in various implementation examples.
  • the target nucleic acid detection method can detect a signal (i.e., a change in signal) indicating the presence of a target nucleic acid using a reference signal value obtained from a negative control reaction. If the signal values at 60°C, 74°C, and 95°C are RFU 200 or higher, which is a threshold based on the signal value of the negative control reaction (i.e., RFU: 0), it was considered that the signal had changed.
  • a signal i.e., a change in signal
  • Tube 1 7 pg of NG genomic DNA
  • a plurality of target nucleic acids can be detected using a single type of label in a single reaction vessel.
  • Example 3-1 Preparation of target nucleic acid and oligonucleotide
  • the genomic DNA of Chlamydia trachomatis (CT) (Accession No.: ATCC VR-1500, Coram Deo Lab Co., Ltd.) used in Example 1 was used as a template for the first target nucleic acid
  • CT Chlamydia trachomatis
  • NG Neisseria gonorrhoeae
  • the sequences and lengths of the CT-reporter probe, the CT-first quencher probe, and the CT-second quencher probe were designed so that Tm1 was 54.8°C, Tm2 was 72.5°C, and Tm3 was 67.3°C, and these are the same oligonucleotides as in Table 3 of Example 1-1.
  • the sequences and lengths of the NG-reporter probe, the NG-first quencher probe and the NG-second quencher probe were designed so that Tm1 was 76.7°C, Tm2 was 81.0°C and Tm3 was 71.3°C, and these are the same oligonucleotides as in Table 5 of Example 2-1.
  • FIG. 9 shows the predominant form of the reporter probe, the first quencher probe and the second quencher probe of each of the two compositions used in the present embodiment, depending on the presence or absence of the corresponding target nucleic acid and the temperature.
  • 50°C is a temperature within the first signal-constant temperature range of both target nucleic acid detecting compositions.
  • 60°C is a first detection temperature, which is a temperature within the signal-change temperature range of the first target nucleic acid detecting composition and a temperature within the first signal-constant temperature range of the second target nucleic acid detecting composition.
  • 74°C is a second detection temperature, which is a temperature within the second signal-constant temperature range of the first target nucleic acid detecting composition and a temperature within the signal-change temperature range of the second target nucleic acid detecting composition.
  • 95°C is a temperature within the second signal-constant temperature range of both target nucleic acid detecting compositions.
  • the reporter molecule linked to the reporter probe included in the first target nucleic acid detection composition is (i) quenched by the first quencher molecule and/or the second quencher molecule at 50°C regardless of the presence of the first target nucleic acid, (ii) unquenched at 60°C when the first target nucleic acid is absent and quenched by the second quencher molecule when the first target nucleic acid is present, and (iii) unquenched at 74°C and 95°C regardless of the presence of the first target nucleic acid. That is, the first target nucleic acid detection composition provides a signal change dependent on the presence of the first target nucleic acid only at the first detection temperature of 60°C, and provides a constant signal at other temperatures even when the first target nucleic acid is present.
  • the reporter molecule linked to the reporter probe included in the composition for detecting the second target nucleic acid is (i) quenched by the first quencher molecule and/or the second quencher molecule regardless of the presence of the second target nucleic acid at 50°C and 60°C, (ii) quenched by the first quencher molecule when the second target nucleic acid is absent at 74°C and unquenched when the second target nucleic acid is present, and (iii) unquenched at 95°C regardless of the presence of the second target nucleic acid.
  • the composition for detecting the second target nucleic acid provides a signal change dependent on the presence of the second target nucleic acid only at the second detection temperature of 74°C, and provides a constant signal at other temperatures even when the second target nucleic acid is present.
  • Example 3-2 Multiplex real-time polymerase chain reaction
  • Real-time polymerase chain reaction was performed using the above oligonucleotides in a single reaction vessel.
  • Klentaq1 polymerase was used for extension of the forward and reverse primers.
  • Tube 1 10 pg of CT genomic DNA; Tube 2: 7 pg of NG genomic DNA; Tube 3: 10 pg of CT genomic DNA and 7 pg of NG genomic DNA) and distilled water (Tube 4: negative control) each contained 4 pmole of CT-forward primer (SEQ ID NO: 2), 20 pmole of CT-reverse primer (SEQ ID NO: 3), 1 pmole of CT-reporter probe (SEQ ID NO: 4), 5 pmole of CT-first quencher probe (SEQ ID NO: 5), 5 pmole of CT-second quencher probe (SEQ ID NO: 6), 2 pmole of NG-forward primer (SEQ ID NO: 8), 20 pmole of NG-reverse primer (SEQ ID NO: 9), 1 pmole of NG-reporter probe (SEQ ID NO: 10), 5 pmole of NG-first quencher probe (SEQ ID NO: 11), 5 pmole of NG-second quencher probe (SEQ ID NO: 2
  • tubes 1 to 4 containing the reaction mixture were each placed in a real-time thermocycler (CFX96 Real-time Cycler, Bio-Rad) and denatured at 95°C for 15 minutes, followed by 50 cycles of 60°C for 60 seconds, 72°C for 30 seconds, 74°C for 5 seconds, and 95°C for 10 seconds.
  • CFX96 Real-time Cycler Bio-Rad
  • Signal measurements were performed at (i) the first detection temperature of 60°C and (ii) the second detection temperature of 74°C for each cycle.
  • tube 1 For tube 1, a change in signal was detected only at the first detection temperature, and no change in signal was detected at the second detection temperature. Therefore, tube 1 was determined to contain only CT.
  • tube 2 For tube 2, a change in signal was detected only at the second detection temperature, and no change in signal was detected at the first detection temperature. Therefore, tube 2 was determined to contain only NG.
  • tube 3 For tube 3, a change in signal was detected at both the first and second detection temperatures. Therefore, tube 3 was determined to contain both CT and NG.
  • tube 4 the negative control, showed no signal change at both the first and second detection temperatures. Therefore, tube 4 was determined to not contain CT and NG.
  • composition for detecting target nucleic acids according to the present disclosure which is an InterSC-type composition
  • the composition for detecting target nucleic acids according to the present disclosure can be used in various combinations with UnderSC-type, InterSC-type, and OverSC-type compositions that adopt various signal generating methods known in the art for detecting target nucleic acids, thereby indicating that the composition can detect a plurality of target nucleic acids in real time using a single type of label in a single reaction vessel.
  • the target nucleic acid detection method can detect a signal (i.e., a change in the signal) indicating the presence of a target nucleic acid by using a reference signal value obtained from a negative control reaction. If the signal value at the first detection temperature is equal to or lower than RFU -200, which is a threshold based on the signal value of the negative control reaction (i.e., RFU: 0), the signal is considered to have changed, and if the signal value at the second detection temperature is equal to or higher than RFU 200, which is a threshold based on the signal value of the negative control reaction (i.e., RFU: 0), the signal is considered to have changed.
  • RFU -200 which is a threshold based on the signal value of the negative control reaction
  • RFU 200 which is a threshold based on the signal value of the negative control reaction
  • Tube Ct(Cycle Threshold) 1st detection temperature (60°C) Second detection temperature (74°C) 1 26.88 N/A 2 N/A 28.74 3 26.40 28.44 4 N/A N/A
  • Tube 1 10 pg of CT genomic DNA
  • Tube 2 7 pg of NG genomic DNA
  • Tube 3 10 pg of CT genomic DNA and 7 pg of NG genomic DNA
  • the composition for detecting a target nucleic acid comprising three probes according to the present disclosure can not only detect a target nucleic acid, but also, by using the target nucleic acid detection method according to the present disclosure alone or in combination with other signal generation methods (e.g., UnerSC, InterSC and/or OverSC signal generation methods), a plurality of target nucleic acids can be detected with only a single detector using the same type of label in one reaction vessel.
  • signal generation methods e.g., UnerSC, InterSC and/or OverSC signal generation methods

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Abstract

La présente divulgation concerne une composition pour détecter un acide nucléique cible, la composition contenant trois sondes, et un procédé de détection d'un acide nucléique cible l'utilisant. Selon la présente divulgation, lorsqu'un acide nucléique cible est présent, les trois sondes forment une pluralité d'hybrides ayant différentes valeurs Tm, et ainsi, la composition pour détecter un acide nucléique cible selon la présente divulgation permet la détection d'un seul acide nucléique cible ainsi que l'activation de la détection d'une pluralité d'acides nucléiques cibles à l'aide d'un seul type d'étiquette.
PCT/KR2024/013368 2023-09-08 2024-09-05 Procédé de détection d'acide nucléique cible à l'aide de trois sondes Pending WO2025053620A1 (fr)

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JP2021509821A (ja) * 2017-11-29 2021-04-08 パナジーン・インコーポレイテッド 標的核酸増幅方法及び標的核酸増幅用組成物
KR20220031544A (ko) * 2019-05-13 2022-03-11 펜타베이스 에이피에스 변이체 핵산 검출을 위한 용융 온도 방법, 키트 및 리포터 올리고

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