Nucleic acid probe

Abstract

The present invention addresses the problem of making it possible, for a nucleic acid probe for use in the real-time detection of a nucleic acid based on RPA method, to be reduced in probe length and / or to avoid internal modification.　Provided is a nucleic acid probe for detecting a target nucleic acid sequence, the nucleic acid probe comprising: one or more abasic sites, oxidized bases or naturally occurring ribonucleotides; and one or more non-naturally occurring nucleotides. The nucleic acid probe includes, except for the abasic sites, a nucleotide sequence that is complementary to a probe-binding region in the target nucleic acid sequence or a sequence complementary to the target nucleic acid sequence. The nucleic acid probe also has a fluorescent substance and a quenching sequence both bonded to the nucleic acid probe.

Description

Nucleic acid probes

[0001] The present invention relates to a nucleic acid probe for detecting a target nucleic acid sequence, a composition or kit for detecting a target nucleic acid sequence, a method for detecting a target nucleic acid sequence, and the like.

[0002] Isothermal nucleic acid amplification is a technique for amplifying nucleic acids under isothermal conditions without requiring temperature changes. Unlike the polymerase chain reaction (PCR), which is a typical nucleic acid amplification method, isothermal nucleic acid amplification does not require complex temperature control for the amplification reaction, and therefore has the great advantage of being extremely simple to use.

[0003] Various isothermal nucleic acid amplification methods have been developed to date, including recombinase polymerase amplification (hereinafter sometimes abbreviated as "RPA") and loop-mediated isothermal amplification (LAMP). Among these, the RPA method is characterized by the use of nucleic acid recombination-related enzymes (recombinase and / or accessory proteins) in the amplification reaction. In PCR, the double-stranded nucleic acid template is thermally denatured and then cooled to anneal the nucleic acid primer to the template. In RPA, however, the nucleic acid recombination-related enzyme forms a complex with the nucleic acid primer, and the nucleic acid primer binds to the template through the action of the nucleic acid recombination-related enzyme (Figure 1). Because the RPA method completes the amplification reaction in approximately several tens of minutes, it has become increasingly popular in the medical field and is also used for rapid pathogen detection, etc.

[0004] In the PCR method, a method for detecting nucleic acids in real time has also been developed and is widely used as a real-time PCR method. Similar to the PCR method, a real-time detection method has also been developed for the RPA method, but as will be explained below, there is a difference between the PCR method and the RPA method in the structure of the nucleic acid probe used.

[0005] The TaqMan® probe method, one type of real-time PCR method, uses a nucleic acid probe with a fluorescent substance (fluorophore) and a quencher bound to the 5' and 3' ends of the nucleic acid strand, respectively. This nucleic acid probe binds to the amplified region of the template through cycles of thermal denaturation and cooling, similar to the annealing of a nucleic acid primer. Subsequently, DNA polymerase extends the nucleic acid strand in the 3' direction from the nucleic acid primer, and the nucleic acid probe bound to the end is degraded by the 5' exonuclease activity of the DNA polymerase moving in the 3' direction. This degradation separates the fluorescent substance bound to the nucleic acid probe from the quencher, causing it to emit fluorescence. In real-time PCR, the amount of nucleic acid amplification can be measured by measuring this change in fluorescence over time.

[0006] In contrast, the RPA method, which uses nucleic acid recombination-related enzymes, uses nucleic acid probes of approximately 30-60 mers that have a different structure from the nucleic acid probes used in real-time PCR. The reason for this is that a length of approximately 28 mers or more is required for a nucleic acid chain to form a complex with a nucleic acid recombination-related enzyme. However, when a nucleic acid recombination-related enzyme binds, the structure of the nucleic acid probe extends and the ends are spatially separated from each other. Therefore, if a fluorescent substance and a quencher are bound to the 5' end and 3' end of the nucleic acid probe, respectively, no quenching effect on the fluorescent substance can be obtained.

[0007] Therefore, in the real-time detection method based on the RPA method, the fluorescent substance and quencher are bound to the nucleic acid probe not at either end, but at positions close to each other within the probe. By locating the quencher in close proximity, the quenching effect on the fluorescent substance can be obtained even when the nucleic acid recombination-related enzyme binds to the nucleic acid probe.

[0008] In addition, the nucleic acid probe used in the RPA method has an abasic site mimic located between the binding sites of the fluorophore and the quencher. When the nucleic acid probe binds to the template, Escherichia coli exonuclease III cleaves the nucleic acid probe at this abasic site mimic. As a result, the fluorophore is separated from the quencher and begins to emit fluorescence (Figure 2).

[0009] Special Publication No. 2009-502161

[0010] In conventional real-time detection methods based on RPA, primers and nucleic acid probes bind to double-stranded target DNA via nucleic acid recombination-related enzymes, resulting in target amplification and detection. In other words, both the primers and nucleic acid probes must serve as substrates for the nucleic acid recombination-related enzymes. To serve as good substrates for the nucleic acid recombination-related enzymes, primers and nucleic acid probes must be approximately 30-60 mers in length. However, due to the length of these sequences, nonspecific amplification due to dimer formation often becomes a problem. This tendency is more pronounced in multiplex systems where numerous primers and probes are present in the system.

[0011] Another problem is that in the nucleic acid probes used in conventional RPA methods, as mentioned above, the fluorophore and quencher must be attached to positions close to each other within the nucleic acid probe, rather than to both ends, and the positions at which the fluorophore and quencher are introduced are limited to thymine residues, resulting in limited flexibility in the design of the nucleic acid probe. Furthermore, internal modification is more expensive than terminal modification, which is an obstacle to low-cost testing in the medical field, for example.

[0012] Therefore, an objective of the present invention is to enable either or both of reducing the probe length and avoiding internal modifications in nucleic acid probes used for real-time detection of nucleic acids based on the RPA method.

[0013] The present inventors have discovered that the above-mentioned problems of conventional RPA methods can be overcome by combining a primer that serves as a substrate for a nucleic acid recombination-related enzyme with a nucleic acid probe that does not bind to the nucleic acid recombination-related enzyme in real-time detection. Specifically, detection is achieved by binding a nucleic acid probe that does not bind to the nucleic acid recombination-related enzyme to a single-stranded region that is generated when a primer that serves as a substrate for the nucleic acid recombination-related enzyme invades and extends a double-stranded nucleic acid during nucleic acid amplification (Figure 3). According to the present invention, since binding of the nucleic acid probe to the target sequence is not required, it is possible to design a probe that is shorter than the nucleic acid probes used in conventional RPA methods, thereby reducing the risk of nonspecific amplification due to dimer formation, etc. In addition, since fluorescent substances and quenchers can be modified at both ends of the nucleic acid probe, the probe can be designed with greater flexibility and at lower costs.

[0014] In designing the nucleic acid probe of the present invention, the inventors first designed a probe of approximately 28 mer or less that would not intercalate into nucleic acid chains, but its fluorescence intensity increased in the presence of nucleic acid recombination-related enzymes, regardless of amplification. The reason for this is thought to be that although nucleic acid probes of approximately 28 mer or less do not efficiently invade nucleic acid chains, they do bind to nucleic acid recombination-related enzymes, which extends the structure of the nucleic acid probe and spatially separates the fluorescent substance and quencher modified at both ends. After extensive research, the inventors discovered a nucleic acid probe that suppresses binding to nucleic acid recombination-related enzymes by incorporating non-natural nucleotides into the nucleic acid probe, leading to the invention of a new real-time detection method for RPA.

[0015] Based on this idea, the present inventors have discovered a new nucleic acid probe configuration and a new real-time detection method based thereon, and have found that this method has superior specificity and sensitivity compared to conventional methods and can also achieve multiplex detection, which was difficult with conventional methods. The present invention is based on the above findings and provides the following.

[0016] <1> A nucleic acid probe for detecting a target nucleic acid sequence, comprising one or more abasic sites, oxidized bases, or natural ribonucleotides, and one or more non-natural nucleotides, comprising a base sequence complementary to a probe binding region in the target nucleic acid sequence or its complementary sequence excluding the abasic site, and comprising a fluorescent substance and a quencher bound to the nucleic acid probe. <2> The nucleic acid probe according to <1>, wherein the non-natural nucleotide is a bridged nucleotide and / or a 2'-modified nucleotide. <3> The nucleic acid probe according to <2>, wherein the bridged nucleotide comprises one or more AmNA nucleotides and / or GuNA nucleotides. <4> The nucleic acid probe according to <3>, comprising 2 to 8 AmNA nucleotides and / or GuNA nucleotides. <5> The nucleic acid probe according to <3> or <4>, further comprising one or more LNA nucleotides. <6> The nucleic acid probe according to <2>, wherein a variable L indicating the base length of the nucleic acid probe and a variable N indicating the number of the bridged nucleotides and / or 2'-modified nucleotides in the nucleic acid probe satisfy the following formulas I and II: N≧0.8×L−5.6 (Formula I), and L>N (Formula II). <7> The nucleic acid probe according to <6>, which further satisfies the following formula III: 10≦L≦20 (Formula III). <8> The nucleic acid probe according to <6> or <7>, wherein the bridged nucleotide is an LNA nucleotide. <9> The nucleic acid probe according to <8>, wherein the bridged nucleotide further comprises an AmNA nucleotide or a GuNA nucleotide. <10> The nucleic acid probe according to any one of <1> to <9>, which comprises a fluorescent substance bound to the 5' end or the 3' end of the nucleic acid probe and a quencher bound to the other end.<11> A composition or kit for detecting a target nucleic acid sequence, comprising: the nucleic acid probe according to any one of <1> to <10>; a first nucleic acid primer comprising a base sequence complementary to a first primer binding region located at the 3' end of the target nucleic acid sequence and serving as a substrate for a nucleic acid recombination-related enzyme; and a second nucleic acid primer comprising a base sequence complementary to a second primer binding region located at the 3' end of the complementary sequence of the target nucleic acid sequence and serving as a substrate for a nucleic acid recombination-related enzyme. <12> The composition or kit according to <11>, which is for use in an isothermal nucleic acid amplification method. <13> The composition or kit according to <12>, further comprising a strand-displacing DNA polymerase, exonuclease III or ribonuclease H, and / or a nucleic acid recombination-related enzyme. <14> A composition or kit for multiplex detection, comprising a plurality of probe / primer sets for detecting a plurality of different target nucleic acid sequences or their complementary sequences, wherein each of the probe / primer sets comprises the nucleic acid probe according to <6> or <7>, a first nucleic acid primer, and a second nucleic acid primer, and each of the nucleic acid probes in the plurality of probe / primer sets comprises a fluorescent substance detectable at a different fluorescent wavelength. <15> The composition or kit according to <14>, wherein the plurality of different target nucleic acid sequences are derived from the same or different viruses, viroids, and / or organisms. <16> A method for detecting a target nucleic acid sequence in a sample, comprising: an amplification step of amplifying the target nucleic acid sequence and / or its complementary sequence in the sample by a nucleic acid amplification method using the nucleic acid probe according to <11> or <12>, a first nucleic acid primer, and a second nucleic acid primer; and a detection step of irradiating the nucleic acid probe with excitation light during and / or after the amplification step, and detecting fluorescence emitted from the fluorescent substance. <17> The method according to <16>, wherein the nucleic acid amplification method is an isothermal nucleic acid amplification method. <18> The method according to <17>, wherein the amplification step is performed using a strand displacement DNA polymerase.<19> The method according to <18>, wherein the amplification step is performed in the presence of a nucleic acid recombination-related enzyme, and exonuclease III, ribonuclease H, DNA glycosylase, or endonuclease IV. <20> The method according to any one of <16> to <19>, wherein the target nucleic acid sequence is RNA, and further comprises a reverse transcription step of reverse transcribing the RNA before and / or during the amplification step. <21> The method according to any one of <16> to <20>, further comprises an extraction step of extracting nucleic acid containing the target nucleic acid sequence from the sample before the amplification step. <22> The method according to any one of <16> to <21>, wherein the sample is a cell, tissue, cell extract, tissue extract, tissue fluid, or body fluid. <23> The method according to any one of <16> to <22>, wherein the target nucleic acid sequence is derived from a virus, viroid, and / or organism. This specification includes the disclosure of Japanese Patent Application No. 2024-051209, from which the present application claims priority.

[0017] According to the present invention, it is possible to reduce the probe length and / or avoid internal modifications of nucleic acid probes used for real-time detection of nucleic acids based on the RPA method.

[0018] FIG. 7 shows an overview of the RPA method. In the figure, "SSB" stands for single-stranded binding protein. FIG. 7 shows an overview of a conventional real-time detection method based on the RPA method. FIG. 7 shows an overview of a new real-time detection method based on the RPA method. FIG. 7 shows one embodiment of the nucleic acid probe of the present invention. The results of evaluating binding by nucleic acid recombination-related enzymes for various probes differing in the number of unnatural nucleotides contained in the probe and probe length are shown. Probes that were able to avoid binding by nucleic acid recombination-related enzymes are shown as "Yes," and probes that were not able to avoid binding are shown as "No." The conditions for probes that can avoid binding by nucleic acid recombination-related enzymes are shown. When the combination of probe length (X coordinate) and number of unnatural nucleotides (Y coordinate) is located inside or on the edge of a rectangle, the probe can avoid binding by nucleic acid recombination-related enzymes. The results of real-time detection using various probes differing in the number of unnatural nucleotides contained in the probe and probe length are shown. FIG. 7A shows the results of an amplification reaction using S-probes 17 to 19. FIG. 7B shows the results of an amplification reaction using S-probes 20 to 22. The results of comparing the specificity of the real-time detection method of the present invention with that of a conventional real-time detection method. FIG. 8A shows the results of analyzing the specificity of S-probe5 (probe length: 56 mer, no LNAs), a probe based on a conventional method. FIG. 8B shows the results of analyzing the specificity of S-probe17 (probe length: 12 mer, 4 LNAs), a probe of the present invention. The results of an amplification reaction performed in the presence of a template nucleic acid are shown as "template (+)," and the results of an amplification reaction performed in the absence of a template nucleic acid are shown as "template (-)." This figure shows the results of comparing the detection limits for template DNA of the real-time detection method of the present invention and a conventional real-time detection method. FIG. 9A shows the results of analyzing the detection limit for template DNA of S-probe5 (probe length: 56 mer, no LNAs), a probe based on a conventional method. FIG. 9B shows the results of analyzing the detection limit for template DNA of S-probe17 (probe length: 12 mer, 4 LNAs), a probe of the present invention. This figure shows the results of comparing the detection limits for template RNA of the probe of the present invention (novel probe) and a probe based on a conventional method.FIG. 10A shows the results of an analysis of the detection limit for template RNA of S-probe5 (56-mer probe length, no LNA), a probe based on a conventional method. FIG. 10B shows the results of an analysis of the detection limit for template RNA of S-probe17 (12-mer probe length, 4 LNAs), a probe of the present invention. These figures show the results of real-time detection based on the RPA method under conditions in which various contaminants are present. FIG. 11A shows the results using human genomic DNA as the contaminant. FIG. 12B shows the results using a saliva-derived DNA extract as the contaminant. The results of amplification reactions performed in the presence of template nucleic acid are shown as "template (+)" and amplification reactions performed in the absence of template nucleic acid are shown as "template (-)." The results of amplification reactions performed in the presence of each contaminant are shown as "contaminant (+)," and amplification reactions in the absence of contaminants are shown as "contaminant (-)." Figures 12A and 12B show the results of singleplex real-time detection using a plasmid DNA sequence derived from Chlamydia trachomatis and the porA gene derived from Neisseria gonorrhoeae as detection targets. Figure 12A shows the results of detection of the ORF4 sequence of plasmid DNA derived from Chlamydia trachomatis. Figure 12B shows the results of detection of the porA gene derived from Neisseria gonorrhoeae. Figures 12A and 12B show the results of detection of the porA gene derived from Neisseria gonorrhoeae as detection targets, and show the results of multiplex real-time detection of each target sequence in the same sample using the ORF4 sequence of plasmid DNA derived from Chlamydia trachomatis and the porA gene derived from Neisseria gonorrhoeae as detection targets. Figure 13A shows the results of detection of the porA gene derived from Neisseria gonorrhoeae. Figure 13B shows the results of detection of a plasmid DNA sequence derived from Chlamydia trachomatis. Figures 12A and 12B show the results of real-time detection using three different probes (S-probe 17, S-probe 25, and S-probe 26) as detection targets for the S gene of SARS-CoV-2. Figure 14A shows the detection results using a synthetic DNA fragment consisting of SEQ ID NO: 20 (200 copies / reaction solution) as template DNA. Figure 14B shows the detection results using a synthetic DNA fragment consisting of SEQ ID NO: 20 (20 copies / reaction solution) as template DNA. The results of real-time detection using three different probes with the ORF4 sequence of plasmid DNA derived from Chlamydia trachomatis as the detection target are shown. Figure 15A shows the detection results using CTP-probe1.Figure 15B shows the detection results using CTP-probe 3. Figure 15C shows the detection results using CTP-probe 4. Figures 15A and 15B show the detection results using CTP-probe 4. Figures 15A and 15B show the detection results using CTP-probe 3. Figure 15C shows ... 15C shows the detection results using CTP-probe 3. Figure 15C shows the detection results using CTP-probe 4. Figure 15C shows the detection results using CTP-probe 3.

[0019] 1. Nucleic Acid Probe for Detecting a Target Nucleic Acid Sequence 1-1. Overview A first aspect of the present invention is a nucleic acid probe for detecting a target nucleic acid sequence. The nucleic acid probe for detecting a target nucleic acid sequence is characterized by comprising one or more abasic sites, oxidized bases, or natural ribonucleotides, and one or more non-natural nucleotides, comprising a base sequence complementary to a probe binding region in the target nucleic acid sequence or its complementary sequence excluding the abasic sites, and comprising a fluorescent substance and a quencher bound to the nucleic acid probe.

[0020] 1-2. Definitions of Terms The following terms frequently used in this specification are defined below.

[0021] As used herein, the term "nucleic acid amplification method" refers to a method in which a specific region of a target nucleic acid is amplified by a nucleic acid polymerase using a nucleic acid primer. Specific examples of nucleic acid amplification methods include PCR based on a thermal denaturation reaction, and the RPA method and LAMP method, which are isothermal nucleic acid amplification methods described below.

[0022] As used herein, the term "isothermal nucleic acid amplification method" refers to a nucleic acid amplification method that can be carried out isothermally. Various methods have been developed as isothermal nucleic acid amplification methods. Examples of isothermal nucleic acid amplification methods include Recombinase Polymerase Amplification (RPA), Loop-mediated Isothermal Amplification (LAMP), Strand Displacement Amplification (SDA), EXPAR (Exponential Amplification Reaction), RCA (Rolling Circle Amplification), and ICAN (Isothermal and Chimeric Primer-initiated Amplification). Examples of isothermal nucleic acid amplification methods include the isothermal amplification of nucleic acids (Isothermal Amplification of Nucleic Acids) method. In these isothermal nucleic acid amplification methods, annealing of the nucleic acid primer to the template and the subsequent extension reaction of the nucleic acid primer occur simultaneously in a reaction solution kept at a constant temperature. For details of each isothermal nucleic acid amplification method, see Beatriz, BO, Bruno, V and Pedro, VB, (2021) Isothermal Amplification of Nucleic Acids: The Race for the Next "Gold Standard, Front. Sens. and other literature.

[0023] The "recombinase polymerase amplification (RPA) method" is a nucleic acid amplification method characterized by the use of recombinase. In PCR, the double-stranded nucleic acid template is thermally denatured and then cooled to anneal the nucleic acid primer to the template. In RPA, however, a nucleic acid recombination-related enzyme forms a complex with the nucleic acid primer, and the nucleic acid primer binds to the template through the action of the nucleic acid recombination-related enzyme (Figure 1). Furthermore, while PCR uses a thermostable DNA polymerase, RPA primarily uses a strand-displacing DNA polymerase. This is because synthesis is performed while dissociating the double-stranded DNA template. While PCR requires 1 to 2 hours or more to complete the amplification reaction, RPA has the advantage of completing the amplification reaction in approximately several tens of minutes.

[0024] As used herein, "real-time detection" refers to quantifying the degree of nucleic acid synthesis during nucleic acid amplification. For example, a real-time detection method based on PCR is called real-time PCR. Examples of real-time PCR include intercalator methods that use reagents that specifically bind to double-stranded DNA (e.g., SYBR Green, TB Green, Eva Green, etc.), and methods that use fluorescently labeled probes (e.g., TaqMan® probe method and cycling probe method). For example, the TaqMan® probe method uses a probe modified with a quencher substance at the 5' end and a fluorescent dye at the 3' end. Normally, the quencher substance at the 5' end inhibits the fluorescent dye at the 3' end, but during the extension reaction, the probe is degraded by the 5'→3' exonuclease activity of Taq polymerase, thereby releasing the inhibition by the quencher substance and allowing fluorescence to be emitted. The amount of fluorescence reflects the amount of amplification product. Since the cycle number (CT) or time at which the amplification product reaches the detection threshold is inversely correlated with the amount of initial template, the amount of initial template can be quantified by measuring the CT or time in real-time detection methods. By measuring the CT or time using several levels of known amounts of template and creating a calibration curve, the absolute value of the amount of initial template in an unknown sample can be calculated.

[0025] Real-time detection methods based on the RPA method are also known, and as described above, conventional methods use nucleic acid probes of approximately 30-60 mer length for detection, in which a fluorescent substance and a quencher are bound to a base other than the terminal base of the nucleic acid probe, typically a thymine base.

[0026] As used herein, the term "nucleic acid chain" refers to a polymer that, in principle, has nucleotides as its constituent units and is linked by internucleotide bonds.

[0027] As used herein, the term "target nucleic acid sequence" refers to the base sequence of a nucleic acid that is the target for detection by a nucleic acid probe. The target nucleic acid sequence may be, for example, a target DNA sequence or a target RNA sequence. If the target DNA sequence is a genomic DNA sequence, a cDNA sequence obtained by reverse transcription from mRNA, or a vector DNA sequence such as a plasmid DNA sequence. If the target RNA sequence is an mRNA sequence, it may be the sequence of a non-coding RNA such as miRNA, lncRNA, tRNA, or ribozyme.

[0028] As used herein, the term "nucleic acid probe" (sometimes simply abbreviated as "probe") refers to a nucleic acid strand used to specifically bind to and detect a nucleic acid having a target nucleic acid sequence. Generally, a nucleic acid probe composed mainly of DNA is called a DNA probe, and a nucleic acid probe composed mainly of RNA is called an RNA probe. Furthermore, a nucleic acid probe containing both DNA and RNA is called a DNA / RNA mixed probe.

[0029] As used herein, a "nucleic acid primer" (sometimes simply abbreviated as "primer") refers to a nucleic acid strand that specifically binds to a portion of a nucleic acid having a target nucleic acid sequence and provides a starting point for a nucleic acid synthesis reaction catalyzed by a polymerase enzyme. A nucleic acid primer is usually single-stranded, and preferably has a base sequence complementary to a portion of the target nucleic acid sequence that serves as a template for the polymerase reaction.

[0030] As used herein, the term "probe binding region" refers to the region to which a nucleic acid probe binds in a target nucleic acid sequence or its complementary sequence.

[0031] As used herein, the term "natural base" refers to adenine, cytosine, guanine, thymine, uracil, and any modified base thereof that occurs in nature.

[0032] As used herein, the term "modified base" refers to any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. Examples of modified bases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, N6-methyladenine, 8-bromoadenine, N2-methylguanine, or 8-bromoguanine.

[0033] As used herein, the term "abasic site" refers to a site in a nucleotide where a nucleobase has been removed. More specifically, it refers to a site in a nucleotide where no nucleobase is bound to the 1'-position of a sugar moiety such as ribose or deoxyribose. An abasic site is also called an apurinic / apyrimidinic site or AP site. An abasic site may be an abasic site mimic (also referred to as an abasic site mimic). Preferred embodiments of an abasic site mimic include a tetrahydrofuran residue, a d-spacer, and the like.

[0034] As used herein, the term "oxidized base" refers to an oxidized natural base or modified base. Examples of oxidized natural bases include 8-oxoguanine, 2-hydroxyadenine, 8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxycytosine and 5-hydroxy-uracil, 5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydantoin, uracil glycol, and 6-hydroxy-5,6-dihydrothymine. The oxidized base can be removed by DNA glycosylase to become an abasic site. DNA glycosylases include those that have only DNA glycosylase activity and those that have both DNA glycosylase activity and AP lyase activity. DNA glycosylases with AP lyase activity also cleave abasic sites.

[0035] As used herein, the term "natural nucleotide" refers to a nucleotide that exists in nature, such as a ribonucleotide consisting of ribose and a base such as adenine, cytosine, guanine, or uracil, or a deoxyribonucleotide consisting of deoxyribose and a base such as adenine, cytosine, guanine, or thymine.

[0036] As used herein, the term "natural ribonucleotide" refers to a ribonucleotide that exists in nature. Examples include ribonucleotides consisting of ribose and a base such as adenine, cytosine, guanine, or uracil. Note that, in this specification, nucleotides consisting of ribose and a thymine base are also included in natural ribonucleotides.

[0037] As used herein, the term "non-naturally occurring nucleotide" refers to any nucleotide other than a naturally occurring nucleotide, including modified nucleotides and nucleotide mimetics. Note that, as used herein, non-naturally occurring nucleotides do not include abasic sites or oxidized bases.

[0038] As used herein, the term "modified nucleotide" refers to a nucleotide having a modified moiety, such as a modified base and / or a modified sugar moiety. Examples of modified nucleotides include bridged nucleotides and 2'-modified nucleotides.

[0039] As used herein, the term "bridged nucleotide" refers to a nucleotide containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are commonly referred to as bridged nucleic acids. A bicyclic sugar may be a sugar in which the 2'- and 4'-carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those skilled in the art. Examples of nucleic acids containing bicyclic sugars (BNAs) include, but are not limited to, methyleneoxy (4'-CH ２ -O-2')BNA (known as "LNA"), ethyleneoxy (4'-(CH ２ ) ２ -O-2') BNA (also known as "ENA"), cEt BNA, scpBNA, cMOE BNA, AmNA, GuNA, and the like.

[0040] As used herein, "AmNA" is also called amide-bridged nucleic acid or amide BNA, and includes structures of (4'-C(O)-N(R)-2')BNA where R=H, R=Me, R=Et, R=nPr, R=iPr, R=Bn, and R=Phen. In the following formula (IV), the structure where R=H is called AmNA[N-H], the structure where R=Me is called AmNA[N-Me], the structure where R=Et is called AmNA[N-Et], the structure where R=nPr is called AmNA[N-nPr], the structure where R=iPr is called AmNA[N-iPr], the structure where R=Bn is called AmNA[N-Bn], and the structure where R=Phen is called AmNA[N-Phen]. As used herein, AmNA may be any of AmNA[N-H], AmNA[N-Me], AmNA[N-Et], AmNA[N-nPr], AmNA[N-iPr], AmNA[N-Bn], and AmNA[N-Phen], but is preferably AmNA[N-Me]. [wherein R represents a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an i-propyl group, a benzyl group, or a phenyl group]

[0041] As used herein, "GuNA" refers to a bridged nucleic acid, also known as a guanidine-bridged nucleic acid or guanidine-BNA. In the following formula (V), the structure where R=H is called GuNA[N-H], the structure where R=Me is called GuNA[N-Me], and the structure where R=t-Bu is called GuNA[N-t-Bu]. As used herein, GuNA may be any of GuNA[N-H], GuNA[N-Me], or GuNA[N-t-Bu], but is preferably GuNA[N-H]. [wherein R represents a hydrogen atom, a methyl group, or a tert-butyl group]

[0042] As used herein, the term "2'-modified nucleotide" refers to a nucleotide containing a 2'-modified sugar. Examples of the substituent at the 2' position of a 2'-modified nucleotide include 2'-F (2'-fluoro group), -OCH ３ (methoxy), -OCH ２ CH ３ (ethoxy), —OCH ２ NH ２ (aminomethoxy), —OCH２ CH ２ NH ２ (aminoethoxy), —OCH ２ CH ２ F (fluoromethylmethoxy), —OCH ２ CH ２ CH ２ F (methylethoxy fluoride), -CH ３ (methyl), —CH ２ CH ３ (ethyl), —CH ２ CH ２ CH ３ (propyl), —CH ２ OCH ３ (methoxymethyl), —CH ２ CH ２ OCH ３ (Methoxyethyl; MOE), —OCH ２ OCH ３ , -OCH ２ CH ２ OCH ３ , -CH ２ OCH ２ OCH ３ , and -CH ２ OCH ２ CH ２ OCH ３ (methoxyethoxymethyl; MEM).

[0043] As used herein, the term "fluorescent substance (fluorophore)" refers to a substance that has the property of becoming excited by absorbing excitation light of a specific wavelength and emitting fluorescence when returning to its original ground state. For example, fluorescein isothiocyanate (FITC), Texas Red, Texas Red, Cyanine 3, Cyanine 5, Cyanine 5.5, Cyanine 7, Cyanine 7.5, carboxyfluorescein (FAM), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), VIC, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), carboxy-X-rhodamine (ROX), tetrachlorofluorescein (TET), Yakima Yellow (registered trademark), Bodipy 493, NBD, 6-carboxytetramethylrhodamine (TAMRA), Quasar 670, Quasar 705, CAL Fluor Examples of the dye include Red 610, SYBR Green, TB Green, Eva Green, SYTOX Green, fluorescamine or a derivative thereof, fluorescein or a derivative thereof, azo compounds, rhodamine or a derivative thereof, coumarin or a derivative thereof, pyrene or a derivative thereof, cyanine or a derivative thereof, etc. These dyes may be used alone or in combination of two or more.

[0044] As used herein, the term "quencher" refers to a substance that has the property of absorbing the excitation energy of the above-mentioned fluorescent substance and suppressing fluorescence. Examples of such a substance include TAMRA, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), IBRQ, IBFQ, BHQ (registered trademark) 1, BHQ2, and BHQ3. These may be used alone or in combination of two or more.

[0045] As used herein, a "nucleic acid recombination-related enzyme" refers to an enzyme that acts on two nucleic acid strands and directly or indirectly promotes recombination between the two nucleic acid strands. More specifically, a "nucleic acid recombination-related enzyme" refers to a "recombinase" and / or an "accessory protein." As used herein, a "recombinase" refers to an enzyme that can directly promote three reaction processes involved in the "recombination reaction" described below. As used herein, an "accessory protein" refers to an enzyme that can directly or indirectly promote one or two reaction processes selected from the three reaction processes involved in the "recombination reaction" described below. A nucleic acid recombination-related enzyme may be derived from any organism, virus, or bacteriophage, for example, from bacteria, archaea, eukaryotes such as mammals or yeast, or from bacteriophages. Furthermore, a nucleic acid recombination-related enzyme may be a mesophilic or thermophilic enzyme. Known recombination reactions that can be catalyzed by a nucleic acid recombination-related enzyme include homologous recombination and site-specific recombination. Homologous recombination is a recombination reaction between two nucleic acid strands having homologous base sequences. Site-specific recombination is a recombination reaction between two nucleic acid strands having homologous base sequences with specific sequences. In the present invention, the nucleic acid recombination-related enzyme is preferably an enzyme that catalyzes homologous recombination. Examples of recombinases that catalyze homologous recombination include RecA protein, T4 uvsX protein, Rad51 protein, and RadA protein. Examples of accessory proteins include single-strand-binding protein (SSB), T4 uvsY protein, RecO protein, RecR protein, RecF protein, and RuvA / RuvB complex protein.

[0046] As used herein, the term "recombination reaction" refers to a reaction that includes a reaction process in which a nucleic acid recombination-related enzyme binds to a nucleic acid strand (e.g., single-stranded DNA) to form a complex, a reaction process in which the nucleic acid recombination-related enzyme searches for a base sequence on another nucleic acid strand that is homologous to the base sequence of the nucleic acid strand that formed the complex, and a reaction process in which the nucleic acid recombination-related enzyme invades the nucleic acid strand.

[0047] As used herein, the phrase "a nucleic acid strand such as a nucleic acid primer or a nucleic acid probe serves as a substrate for a nucleic acid recombination-related enzyme" means that, in a recombination reaction catalyzed by a nucleic acid recombination-related enzyme, the nucleic acid recombination-related enzyme binds to the nucleic acid strand to form a complex and can directly or indirectly promote invasion between nucleic acid strands, or that this occurs with sufficiently high efficiency. Furthermore, as used herein, the phrase "a nucleic acid strand such as a nucleic acid primer or a nucleic acid probe does not serve as a substrate for a nucleic acid recombination-related enzyme" means that, in a recombination reaction catalyzed by a nucleic acid recombination-related enzyme, the nucleic acid recombination-related enzyme does not bind to the nucleic acid strand to form a complex and / or does not invade between nucleic acid strands, or that this occurs with sufficiently low efficiency. The nucleic acid probes of the present invention do not bind to a nucleic acid recombination-related enzyme to form a complex, or do not substantially form a complex, or that this occurs with sufficiently low efficiency. Whether a particular nucleic acid strand serves as a substrate for a nucleic acid recombination-related enzyme, or whether this efficiency is high or low, can be determined by measuring the recombination activity of the nucleic acid strand or by evaluating binding by a nucleic acid recombination-related enzyme, as described in the Examples below. For example, a nucleic acid strand having a fluorescent substance and a quencher bound to the 5'-end and 3'-end is incubated in the presence or absence of a nucleic acid recombination-related enzyme, and if the relative fluorescence intensity in the presence of the nucleic acid recombination-related enzyme to the fluorescence intensity in the absence of the nucleic acid recombination-related enzyme is a specific value or greater (e.g., 2 or greater), it can be determined that the nucleic acid strand serves as a substrate for the nucleic acid recombination-related enzyme.

[0048] As used herein, "single strand-binding protein (SSB)" refers to any protein capable of binding to single-stranded nucleic acids. By binding to single-stranded nucleic acids, SSB can prevent secondary structure formation in nucleic acids, protect them from nuclease digestion, and / or promote nucleic acid replication. Specific examples of SSB include SSB derived from Escherichia coli, T4 gp32, SSB derived from phage phi29, SSB derived from Thermus aquaticus, and SSB derived from hyperthermophilic microorganisms.

[0049] As used herein, the term "cleavage enzyme" refers to an enzyme that cleaves a nucleic acid probe at the cleavage site described below, depending on the binding between the nucleic acid probe of the present invention and a probe binding region in a target nucleic acid sequence or its complementary sequence. The type of cleavage enzyme can be appropriately selected depending on the type of cleavage site. When the cleavage site is an abasic site, the cleavage enzyme may be exonuclease III or endonuclease IV. Specific examples of exonuclease III include Escherichia coli exonuclease III and APE1 protein. Specific examples of endonuclease IV include Escherichia coli endonuclease IV (Nfo) and Tth endonuclease IV. When the cleavage site is a natural ribonucleotide, the cleavage enzyme may be ribonuclease H. Specific examples of ribonuclease H include Escherichia coli ribonuclease H and Tli RNase H. When the cleavage site is an oxidized base, the cleavage enzyme may be DNA glycosylase, exonuclease III, and / or endonuclease IV. The oxidized base is removed by DNA glycosylase to form an abasic site, which can be cleaved by DNA glycosylase, exonuclease III, or endonuclease IV, which have AP lyase activity. Specific examples of DNA glycosylases include Fpg protein, Nth protein, hOGG1 protein, and NTHL1 protein.

[0050] As used herein, the term "cleavage site" refers to a site at which a nucleic acid probe can be cleaved depending on binding to a probe binding region within a target nucleic acid sequence or its complementary sequence.

[0051] As used herein, the term "strand displacement DNA polymerase" refers to a DNA polymerase that, when a template DNA forms a double strand by base pairing with its complementary strand in the extension direction during the process of synthesizing a DNA strand complementary to the template DNA, can continue synthesis of a complementary strand while dissociating the double strand. Furthermore, synthesis of a complementary strand while dissociating the double strand is referred to as a "strand displacement reaction."

[0052] As used herein, the term "complementary" refers to a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) via hydrogen bonds. In the present invention, a base sequence is acceptable if it has at least 80%, preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. The complementarity of a base sequence can be determined using a BLAST program or the like.

[0053] The term "detection" as used herein may be replaced with the terms "test," "measurement," "determination," or "determination support." Furthermore, the term "evaluation" as used herein is used to mean supporting a diagnosis or evaluation based on test results or measurement results.

[0054] As used herein, a "subject" refers to any living organism, including animals and plants. The animal may be a mammal, such as a human, a primate, including chimpanzees, a pet animal, such as a dog or cat, a livestock animal, such as a cow, horse, sheep, or goat, a rodent, such as a mouse or rat, or an animal kept in a zoo. A preferred subject is a human.

[0055] As used herein, the term "sample" to be detected or evaluated refers to a sample collected or isolated from a subject, a healthy subject, or a group of healthy subjects, and subjected to the detection method of the present invention. Examples of such samples include cells, tissues, cell extracts, tissue extracts, tissue fluids, or other body fluids, including feces and hair. Examples of body fluids include cerebrospinal fluid, interstitial fluid, blood (including serum, plasma, and interstitial fluid), lymph, tissue or cell extracts, pleural effusion, sputum, tears, nasal discharge, saliva, and urine. Blood may be serum or plasma prepared from blood. Tissues and cells include, for example, tissues and cells of a subject that are or may be affected by a disease (e.g., an infectious disease), as well as corresponding tissues and cells in a healthy subject. In addition to the above, samples may also be biological samples extracted from these.

[0056] As used herein, the term "healthy individual" refers to an individual not suffering from a specific disease, preferably an individual not suffering from any disease. However, as used herein, healthy cells are also included in the broad definition of healthy individuals. Therefore, a healthy individual is defined as an individual that is in a healthy state not only at the individual level but also at the cellular level.

[0057] In this specification, the term "plurality" refers to an integer of 2 or more, for example, an integer of 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

[0058] As used herein, the term "identity (base identity)" of a base sequence refers to the percentage (%) of the number of matching bases in the total number of bases when two base sequences are compared and aligned by inserting appropriate gaps into one or both of them as needed to maximize the number of matching bases. Identity can be determined using methods well known to those skilled in the art, sequence analysis software, etc.

[0059] 1-3. Configuration The nucleic acid probe for detecting a target nucleic acid sequence of the present invention (hereinafter often referred to as the "nucleic acid probe of the present invention") contains a base sequence complementary to the target nucleic acid sequence or its complementary sequence. When the nucleic acid probe contains one or more abasic sites as described below, the nucleic acid probe can contain a base sequence complementary to a sequence excluding bases corresponding to the abasic sites of the nucleic acid probe in the probe binding region of the target nucleic acid sequence or its complementary sequence.

[0060] The nucleic acid probe of the present invention contains one or more cleavage sites. Specific cleavage sites are selected from abasic sites, oxidized bases, and natural ribonucleotides. Abasic sites are cleaved by exonuclease III or endonuclease IV. Natural ribonucleotides are cleaved by ribonuclease H. Oxidized bases are cleaved by DNA glycosylase, exonuclease III, and / or endonuclease IV. The number of cleavage sites in a nucleic acid probe is not limited as long as it is one or more. For example, it may be one or more, two or more, three or more, four or more, and / or ten or fewer, nine or fewer, eight or fewer, or seven or fewer, more specifically, one, two, or three. Furthermore, the location of the cleavage site in a nucleic acid probe is not limited, and the cleavage site may be located at a nucleotide located within the nucleic acid probe (a nucleotide located other than the 5' end and 3' end), and / or at a nucleotide located at the 5' end and / or 3' end. In principle, the cleavage site is composed of one of an abasic site, an oxidized base, or a natural ribonucleotide, but it is also possible to combine any two or three types selected from an abasic site, an oxidized base, and a natural ribonucleotide.

[0061] The nucleic acid probe of the present invention comprises a fluorescent substance and a quencher bound to the nucleic acid probe. The combination of fluorescent substance and quencher is not limited as long as the quencher can absorb the excitation energy of the fluorescent substance and suppress its fluorescence. The number of fluorescent substances may be, for example, 1 or more, 2 or more, 3 or more, and / or 6 or less, 5 or less, or 4 or less, and the number of quenchers may be, for example, 1 or more, 2 or more, 3 or more, and / or 6 or less, 5 or less, or 4 or less. The number of fluorescent substances and quenchers bound to the nucleic acid probe may be the same or different. For example, the number of fluorescent substances and quenchers may be, for example, one each, one and two, or two and one. Furthermore, the positions at which the fluorescent substance and quencher are bound may be within the nucleic acid probe (any position other than the 5' end and the 3' end), and / or the 5' end and / or the 3' end. The positional relationship between the fluorescent substance and the quencher is not particularly limited, as long as one or more cleavage sites are located between the fluorescent substance and the quencher so that the fluorescent substance is separated from the quencher after the nucleic acid probe is cleaved at the cleavage site. In one embodiment, if the fluorescent substance is located at the 5'-end or 3'-end of the nucleic acid probe, the quencher is located at the other end, and the cleavage site is located at any position other than the 5'-end or 3'-end, this is preferable in terms of synthesis cost because it can avoid introducing the fluorescent substance and the quencher into the nucleic acid probe.

[0062] The nucleic acid probe of the present invention contains one or more non-natural nucleotides. The type of non-natural nucleotide is not limited as long as it is effective in binding to the substrate of the nucleic acid recombination-related enzyme without forming a complex.

[0063] In one embodiment, the non-natural nucleotide is a bridged nucleotide or a 2'-modified nucleotide. For example, the bridged nucleotide may be ENA, cEt BNA, cMOE BNA, AmNA, or GuNA. A preferred bridged nucleotide is LNA, AmNA, or GuNA. The bridged nucleotide may also be a combination of two or three of LNA, AmNA, and GuNA, for example, a combination of LNA and AmNA or a combination of LNA and GuNA. The 2'-modified nucleotide may be, for example, a 2'-methyl-modified nucleotide, a 2'-MOE-modified nucleotide, or a 2'-fluoro-modified nucleotide. Whether a nucleic acid probe serves as a substrate for a nucleic acid recombination-related enzyme can be determined by measuring recombination activity or evaluating binding by a nucleic acid recombination-related enzyme, as described above.

[0064] In one embodiment, the bridged nucleotide comprises one or more AmNA and / or GuNA nucleotides. In this embodiment, the bridged nucleotide may further comprise an LNA nucleotide in addition to one or more AmNA and / or GuNA nucleotides.

[0065] In the nucleic acid probes of the present invention, the number of unnatural nucleotides is not limited as long as it is one or more. The lower limit of the number of unnatural nucleotides may be, for example, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more. The upper limit of the number of unnatural nucleotides is not particularly limited, and may be, for example, 25 or less, 20 or less, 15 or less, 12 or less, 11 or less, or 10 or less. Exemplary ranges include 2 to 8, 3 to 6, or 4 to 5. For example, the nucleic acid probes of the present invention may contain 2 to 8 AmNA nucleotides and / or GuNA nucleotides, and may optionally further contain 1 to 8, 2 to 6, or 3 to 5 LNA nucleotides.

[0066] The position of the unnatural nucleotide in the nucleic acid probe of the present invention is not limited. For example, in the nucleic acid probe of the present invention, segments composed of unnatural nucleotides having a randomly selected length or a periodic length (e.g., 1 to 10 bases long, 2 to 9 bases long, 3 to 8 bases long, or 4 to 7 bases long) are alternated with segments composed of natural nucleotides.

[0067] The nucleic acid probe of the present invention may contain natural nucleotides in addition to unnatural nucleotides, and the natural nucleotides may be deoxyribonucleotides or ribonucleotides. For example, the nucleic acid probe of the present invention is composed of unnatural nucleotides and natural deoxyribonucleotides, and does not contain 10 or more, 9 or more, 8 or more, 7 or more, 6 or more, 5 or more, 4 or more, 3 or more, or 2 or more consecutive natural deoxyribonucleotides.

[0068] The length of the nucleic acid probe of the present invention may be, for example, 8 bases or more, 9 bases or more, 10 bases or more, 11 bases or more, 12 bases or more, 13 bases or more, 14 bases or more, or 15 bases or more, and / or 30 bases or less, 29 bases or less, 28 bases or less, 27 bases or less, 26 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. Exemplary ranges include 8 to 25 bases, 10 to 20 bases, and 12 to 18 bases.

[0069] In one embodiment of the nucleic acid probe of the present invention, a variable L indicating the base length of the nucleic acid probe and a variable N indicating the number of unnatural nucleotides in the nucleic acid probe satisfy the following formulas I and II: N≧0.8×L−5.6 (formula I), and L>N (formula II).

[0070] In one embodiment, the nucleic acid probe of the present invention further satisfies the following formula III: 10≦L≦20 (Formula III).

[0071] The non-natural nucleotide defined by the variable N satisfying the above formula I and formula II may be a bridged nucleotide or a 2'-modified nucleotide. The bridged nucleotide may be any one of LNA, AmNA, or GuNA.

[0072] In one embodiment, the non-natural nucleotide defined by the variable N satisfying Formula I and II above is a bridged nucleotide, and the bridged nucleotide is an LNA. In this embodiment, the number of LNA nucleotides is equal to or less than the variable N satisfying Formula I and II above.

[0073] In another embodiment, the non-natural nucleotides defined by the variable N satisfying the above formula I and formula II are bridged nucleotides, and the bridged nucleotides include a combination of LNA and AmNA or a combination of LNA and GuNA. In this embodiment, the total number of LNAs and AmNAs or the total number of LNAs and GuNAs is equal to or less than the variable N satisfying the above formula I and formula II.

[0074] 1-4. Effects The nucleic acid probe of the present invention does not bind to nucleic acid recombination-related enzymes to form complexes, or does so with low efficiency. Therefore, unlike nucleic acid probes used in conventional real-time detection methods based on RPA, it is not necessary to introduce a fluorescent substance and a quencher into the nucleic acid probe. For example, even if a fluorescent substance and / or a quencher is introduced into the 5' end and / or 3' end of the nucleic acid probe, the quenching effect of the quencher is not lost by the nucleic acid recombination-related enzyme.

[0075] In nucleic acid probes used in conventional real-time detection methods based on the RPA method, fluorescent substances and quenchers are generally introduced into thymidine bases within the nucleic acid probe, but this has the problem of limited freedom in selecting the base sequence that constitutes the nucleic acid probe, as well as the high cost of internal modification. In contrast, the nucleic acid probe of the present invention, based on the above-mentioned characteristics, allows for the introduction of fluorescent substances and / or quenchers into the terminal end of the nucleic acid probe, allowing for a high degree of design freedom and making it possible to avoid the high costs associated with internal modification.

[0076] Furthermore, nucleic acid probes used in conventional real-time detection methods based on RPA require a length of approximately 30-60 mers in order to serve as substrates for nucleic acid recombination-related enzymes. This has led to problems such as the tendency for dimers to form, leading to nonspecific amplification, and the tendency for primers and probes to bind to each other in multiplex systems, leading to nonspecific amplification. In contrast, the nucleic acid probes of the present invention can be made shorter than 30 mers, thereby avoiding the problem of nonspecific amplification and enabling low-cost, high-throughput multiplex analysis.

[0077] According to the present invention, there is provided a nucleic acid probe for detecting the target nucleic acid sequence according to this aspect, and further, there is provided a detection agent for the target nucleic acid sequence comprising the nucleic acid probe of the present invention.

[0078] 2. Composition or kit for detecting a target nucleic acid sequence 2-1. Overview A second aspect of the present invention is a composition or kit for detecting a target nucleic acid sequence. The composition or kit of this aspect comprises the nucleic acid probe and nucleic acid primer described in the first aspect.

[0079] 2-2. Configuration The composition or kit for detecting a target nucleic acid sequence of the present invention (hereinafter often referred to as the "composition or kit of the present invention") comprises, as essential components, the nucleic acid probe according to the first aspect and nucleic acid primers for detecting a target nucleic acid sequence or a sequence complementary thereto.

[0080] The number of nucleic acid primers contained in the composition or kit of the present invention is not limited as long as it is capable of amplifying a target nucleic acid sequence and / or its complementary sequence. However, since a pair of nucleic acid primers is generally required to amplify a target nucleic acid sequence and / or its complementary sequence, the number of nucleic acid primers is generally two or more, and may be, for example, three or more or four or more. In the following description, a pair of nucleic acid primers among the two or more primers contained in the composition or kit of the present invention will be referred to as a first nucleic acid primer and a second nucleic acid primer.

[0081] The first nucleic acid primer included in the composition or kit of the present invention contains a base sequence complementary to the base sequence located at the 3' end of the target nucleic acid sequence so as to bind thereto. Herein, the region to which the first nucleic acid primer binds in the target nucleic acid sequence is referred to as the "first primer binding region." Furthermore, the second nucleic acid primer included in the composition or kit of the present invention contains a base sequence complementary to the base sequence located at the 3' end of the sequence complementary to the target nucleic acid sequence so as to bind thereto. Herein, the region to which the second nucleic acid primer binds in the complementary sequence of the target nucleic acid sequence is referred to as the "second primer binding region." The first nucleic acid primer and the second nucleic acid primer amplify a nucleic acid strand consisting of the target nucleic acid sequence and its complementary strand.

[0082] The first nucleic acid primer and the second nucleic acid primer contained in the composition or kit of the present invention serve as substrates for nucleic acid recombination-related enzymes.

[0083] The length of the nucleic acid primer that serves as a substrate for the nucleic acid recombination-related enzyme may be, for example, 12 or more bases, 15 or more bases, 16 or more bases, 17 or more bases, 18 or more bases, 19 or more bases, 20 or more bases, 21 or more bases, 22 or more bases, 23 or more bases, 24 or more bases, 25 or more bases, 27 or more bases, or 30 or more bases. There is no particular upper limit on the length, but the nucleic acid primer may be, for example, 100 or less bases, 90 or less bases, 80 or less bases, 70 or less bases, 60 or less bases, 55 or less bases, 50 or less bases, 45 or less bases, or 40 or less bases. Exemplary ranges include 25 to 65 bases, or 35 to 55 bases.

[0084] Furthermore, the types of nucleotides constituting the nucleic acid primers that serve as substrates for nucleic acid recombination-related enzymes are not particularly limited, and the number of non-natural nucleotides contained in the nucleic acid primers is, for example, 10 or less, 8 or less, 6 or less, or 5 or less, and preferably 4 or less, 3 or less, 2 or less, 1 or less, or 0. The first nucleic acid primer and the second nucleic acid primer may be composed only of natural nucleotides, for example, only of DNA nucleotides.

[0085] The number of nucleic acid probes according to the first aspect contained in the composition or kit of the present invention is not particularly limited as long as it is 1 or more, and may be, for example, 1 or 2, or 2 or more, 3 or more, 4 or more, or 5 or more. There is no particular upper limit, but the number of nucleic acid probes may be, for example, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less. For example, the composition or kit of the present invention may contain a nucleic acid probe for detecting a target nucleic acid sequence and / or a nucleic acid probe for detecting a complementary sequence of the target nucleic acid sequence.

[0086] One embodiment of the composition or kit for detecting a target nucleic acid sequence of the present invention is a composition for detecting a target nucleic acid sequence (hereinafter, sometimes referred to as the "composition of the present invention"). The composition of the present invention comprises the nucleic acid probe described in the first aspect, a first nucleic acid primer, and a second nucleic acid primer as essential components, and may optionally comprise a solvent and / or additives. The solvent may be, for example, water, an aqueous solution, or an organic solvent. The aqueous solution may be, for example, physiological saline, phosphate buffer, sodium acetate buffer, Tris buffer, etc. In addition, examples of additives include chelating agents, pH adjusters, suspending agents, surfactants, stabilizers, excipients, preservatives, diluents, isotonicity agents, buffers, solubilizing agents, etc. In particular, chelating agents such as EDTA and EGTA are preferred for stabilizing nucleic acid probes and nucleic acid primers because they can inactivate trace amounts of nucleases when they are present.

[0087] One embodiment of the composition or kit for detecting a target nucleic acid sequence of the present invention is a kit for detecting a target nucleic acid sequence (hereinafter, sometimes referred to as the "kit of the present invention"). The kit of the present invention comprises the nucleic acid probe described in the first aspect, a first nucleic acid primer, and a second nucleic acid primer as essential components. In the kit of the present invention, the nucleic acid probe, the first nucleic acid primer, and the second nucleic acid primer may be contained in separate containers, or may be contained in the same container as in the composition of the present invention described above. Each container may contain a solvent and / or additive as an optional component in addition to the nucleic acid probe, the first nucleic acid primer, and / or the second nucleic acid primer. The kit of the present invention may also include, as optional components, other reagents necessary for nucleic acid amplification or target nucleic acid detection, such as strand-displacing DNA polymerase, exonuclease III, ribonuclease H, DNA glycosylase, endonuclease IV, nucleic acid recombination-related enzymes, creatine kinase and phosphocreatine, or pyruvate kinase and phosphophenyl-pyruvate, buffer, dNTP, ATP, DTT, additives, sample collection equipment, nucleic acid extraction reagents, nucleic acid purification reagents, and / or instructions describing methods for nucleic acid amplification or target nucleic acid detection. The kit of the present invention can also be provided as a reagent, such as a reagent kit.

[0088] In one embodiment, the composition or kit for detecting a target nucleic acid sequence of the present invention can be used in a nucleic acid amplification method such as an isothermal nucleic acid amplification method. The nucleic acid amplification method such as an isothermal nucleic acid amplification method may be a quantitative nucleic acid amplification method, for example, real-time detection.

[0089] In one embodiment, the composition or kit of the present invention for detecting a target nucleic acid sequence is a composition or kit for singleplex detection. As used herein, "singleplex detection" refers to the detection of a single target nucleic acid sequence by the composition or kit for detecting a target nucleic acid sequence. In this embodiment, the composition or kit of the present invention includes the above-described first and second nucleic acid primers as a pair of nucleic acid primers for amplifying a single target nucleic acid sequence, and may include one nucleic acid probe according to the first aspect for detecting the target nucleic acid sequence or its complementary sequence, or two or more nucleic acid probes according to the first aspect bound to fluorescent substances having fluorescence wavelengths in the same or overlapping wavelength ranges for detecting the target nucleic acid sequence and / or its complementary sequence.

[0090] In another embodiment, the composition or kit of the present invention for detecting a target nucleic acid sequence is a composition or kit for multiplex detection. As used herein, "multiplex detection" refers to the detection of two or more target nucleic acid sequences by a composition or kit for detecting a target nucleic acid sequence. In this embodiment, the composition or kit of the present invention includes multiple probe / primer sets for detecting multiple different target nucleic acid sequences. Here, each probe / primer set includes the nucleic acid probe described in the first aspect, a first nucleic acid primer, and a second nucleic acid primer. Furthermore, each of the nucleic acid probes described in the first aspect in the multiple probe / primer sets includes a fluorescent substance detectable at a different fluorescent wavelength. Because the fluorescent substances detectable at different fluorescent wavelengths are bound to the multiple nucleic acid probes, each of the different target nucleic acid sequences can be detected at a different fluorescent wavelength. The multiple different target nucleic acid sequences may be multiple different sequences from the same virus, viroid, and / or organism, or may be sequences derived from different viruses, viroids, and / or organisms.

[0091] 2-4. Effects The composition or kit for detecting a target nucleic acid sequence of the present invention allows for high flexibility in the design of the nucleic acid probe contained in the composition or kit of the present invention, allowing for low-cost detection of the target nucleic acid sequence. Furthermore, the composition or kit of the present invention causes little nonspecific amplification. Furthermore, the composition or kit of the present invention can also be used for low-cost, high-throughput multiplex analysis.

[0092] 3. Method for detecting a target nucleic acid sequence 3-1. Overview A third aspect of the present invention is a method for detecting a target nucleic acid sequence (hereinafter, sometimes simply referred to as a "detection method"). The detection method of this aspect uses the nucleic acid probe described in the first aspect and the first and second nucleic acid primers described in the second aspect to amplify a target nucleic acid sequence in a sample, induces cleavage of the nucleic acid probe based on the amplified target nucleic acid sequence, and detects fluorescence emitted from a fluorescent substance.

[0093] The detection method of the present invention includes an amplification step and a detection step as essential steps, and includes a reverse transcription step and an extraction step as selective steps. Each step in the method of this embodiment will be specifically described below.

[0094] (Amplification step) In the method of this embodiment, the "amplification step" is a step of amplifying a target nucleic acid sequence in a sample by a nucleic acid amplification method using any of the nucleic acid probes described in the first embodiment and the first and second nucleic acid primers described in the second embodiment.

[0095] Nucleic acid amplification methods that can be used in this step are known in the art, and can be performed by referring to the conditions described in various protocols. Examples of protocol collections include the aforementioned Green, MR and Sambrook, J, (2012), Domingues L. (2017) PCR: Methods and Protocols, Methods in Molecular Biology, Humana Press, or Park DJ, (2010) PCR Protocols, Methods in Molecular Biology, Third Edition, Humana Press. Nucleic acid amplification kits are also commercially available from life science manufacturers, and these can also be used. In this case, the conditions of the nucleic acid amplification method may be determined in accordance with the attached instructions or the protocol recommended by each manufacturer. Furthermore, to accelerate the nucleic acid amplification reaction, the reaction solution may be stirred, shaken, and / or mixed before and / or during the reaction. The specific method of stirring, shaking, and / or mixing is not limited, and examples include methods using a stirrer or vortex, mixing by inversion, pipetting, and tapping. The number and frequency of stirring, shaking, and / or mixing are also not limited, and may be performed, for example, several to several tens of times during the reaction.

[0096] In one embodiment, the nucleic acid amplification method used in this step is an isothermal nucleic acid amplification method, which may be, for example, the RPA method, the EXPAR method, or the RCA method.

[0097] The reaction conditions for amplifying nucleic acids by isothermal nucleic acid amplification may be selected so that the polymerase used for amplification and the cleavage enzyme used for cleaving the cleavage site can function sufficiently. For example, the reaction time may be 30 seconds to 3 hours, 1 minute to 2 hours, 5 minutes to 60 minutes, 10 minutes to 50 minutes, 15 minutes to 45 minutes, 20 minutes to 40 minutes, or 25 minutes to 35 minutes under isothermal conditions of 20°C or higher, 25°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, or 36°C or higher, and / or 65°C or lower, 60°C or lower, 55°C or lower, 50°C or lower, 47°C or lower, 45°C or lower, 43°C or lower, 42°C or lower, 41°C or lower, 40°C or lower, 39°C or lower, 38°C or lower, or 37°C or lower.

[0098] In one embodiment, amplification in this step is performed using a strand-displacing DNA polymerase. The strand-displacing DNA polymerase may be, for example, Escherichia coli DNA polymerase I large fragment, Tli DNA polymerase, T7 DNA polymerase, T5 DNA polymerase, Phi29 DNA polymerase, Bsu DNA polymerase, Bsu DNA polymerase large fragment, Bst DNA polymerase, or Bst DNA polymerase large fragment. Commercially available strand-displacing DNA polymerases may be used. Examples of commercially available products include Bsu DNA Polymerase, Large Fragment (manufactured by New England BioLabs), Bst DNA Polymerase (manufactured by Nippon Gene Co., Ltd.), Bst 2.0 DNA Polymerase (manufactured by New England BioLabs), Bst 2.0 WarmStart DNA Polymerase (manufactured by New England BioLabs), and Bst 3.0 DNA Polymerase (manufactured by New England BioLabs).

[0099] In one embodiment, this step involves amplification in the presence of a nucleic acid recombination-related enzyme and a cleavage enzyme (e.g., exonuclease III, ribonuclease H, DNA glycosylase, or endonuclease IV). The specific type of cleavage enzyme can be appropriately selected depending on the type of cleavage site contained in the nucleic acid probe, as described above. For example, when the cleavage site is an abasic site, the cleavage enzyme is exonuclease III or endonuclease IV, and when the cleavage site is a natural ribonucleotide, the cleavage enzyme is ribonuclease H. On the other hand, when the cleavage site is an oxidized base, the cleavage enzyme may be DNA glycosylase, exonuclease III, and / or endonuclease IV. The oxidized base can be removed by DNA glycosylase to become an abasic site. This is because this abasic site can be cleaved by DNA glycosylase, exonuclease III, or endonuclease IV. Commercially available cleavage enzymes may be used. Examples of commercially available products include Exonuclease III (manufactured by Takara Bio Inc.), Endonuclease IV (manufactured by New England Biolabs), and Fpg (manufactured by New England Biolabs).

[0100] In one embodiment, in this step, components necessary for the amplification reaction and cleavage reaction, such as dNTP, ATP, DTT, crowding agent, buffer, reverse transcriptase, additives, etc., may be added to the reaction solution. Creatine kinase and phosphocreatine, or pyruvate kinase and phosphophenylpyruvate may also be added as an ATP regeneration system. Uracil DNA glycosylase (UNG) may also be added to the reaction solution to prevent carryover contamination of the amplification product. In this case, dUTP is used instead of dTTP as the dNTP.

[0101] The sample subjected to this step may be any sample containing a target nucleic acid sequence, and may be either a purified sample containing purified nucleic acid or an unpurified sample containing unpurified nucleic acid. The purified sample may be a crude extract obtained by crude extraction using heat, alkali, a surfactant, or the like.

[0102] (Detection Step) In the method of this embodiment, the "detection step" is a step of irradiating the nucleic acid probe with excitation light and detecting the fluorescence emitted from the fluorescent substance.

[0103] The wavelength of the excitation light used in this step may be appropriately selected depending on the type of fluorescent substance bound to the nucleic acid probe, and the fluorescence wavelength for detecting the fluorescence in this step may also be appropriately selected depending on the type of fluorescent substance.

[0104] This step may be carried out simultaneously with or after the amplification step. When the detection step is carried out during the amplification step, the amplification process can be measured over time by carrying out this step at regular time intervals, for example, every 5 seconds, every 10 seconds, every 20 seconds, every 30 seconds, every minute, every 2 minutes, every 5 minutes, or every 10 minutes.

[0105] (Reverse transcription step) In the method of this embodiment, the "reverse transcription step" refers to a step of reverse transcribing RNA before and / or during the above-described amplification step when the target nucleic acid sequence is RNA. The reverse transcription step is a step of generating a reverse transcription product (cDNA) using sample-derived RNA as a template, a reverse transcription primer such as an oligo-dT primer, and an enzyme such as a reverse transcription polymerase.

[0106] The reverse transcription method used in this step can be any method known in the art. For example, the reverse transcription method may be performed in accordance with the reverse transcription method described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press. Furthermore, to accelerate the reverse transcription reaction, the reaction solution may be stirred, shaken, and / or mixed before and / or during the reaction. Specific methods for stirring, shaking, and / or mixing are not limited, and examples include methods using a stirrer or vortex, mixing by inversion, pipetting, and tapping. The number and frequency of stirring, shaking, and / or mixing are also not limited, and may be performed, for example, several to several tens of times during the reaction.

[0107] (Extraction Step) In the method of this embodiment, the "extraction step" refers to a step of extracting nucleic acid containing a target nucleic acid sequence from a sample before the above-mentioned amplification step.

[0108] In this step, nucleic acids such as DNA may be extracted using conventional methods known in the art. For example, the method described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York may be used. Alternatively, the DNA may be prepared using a commercially available DNA extraction kit. Examples of commercially available DNA extraction kits include Kaneka Easy DNA Extraction Kit Version 2 (manufactured by Kaneka Corporation), QIA prep Spin Miniprep Kit (manufactured by QIAGEN), and ISOSPIN Tissue DNA (manufactured by Nippon Gene Co., Ltd.).

[0109] When the method of this embodiment includes the reverse transcription step described above, this extraction step extracts RNA from the sample. The method for extracting RNA from the sample is not particularly limited. For example, a common acid phenol method (Acid Guanidinium-Phenol-Chloroform (AGPC) method) may be used, or an RNA extraction reagent containing acid phenol may be used. Examples of RNA extraction reagents that can be used in this step include Trizol (Life Technologies) and Isogen (Nippon Gene Co., Ltd.). RNA extraction kits are commercially available from life science manufacturers such as Qiagen, Takara Bio, Toyobo, Thermo Fisher Scientific, and Promega, and these kits can also be used.

[0110] In this or the first aspect, the target nucleic acid sequence may be derived from a virus, a viroid, and / or an organism.

[0111] As used herein, the term "virus" is not limited and may be either a DNA virus or an RNA virus. Examples of DNA viruses include Poxviridae (e.g., smallpox virus), Herpesviridae (e.g., herpes simplex virus, Epstein-Barr virus, and varicella-zoster virus), Baculoviridae, Parvoviridae, Adenoviridae, Papillomaviridae (e.g., papillomavirus), and Hepadnaviridae (e.g., hepatitis B virus). Examples of RNA viruses include Retroviridae (e.g., human T-cell leukemia virus type 1 and human immunodeficiency virus), Togaviridae, Coronaviridae (e.g., SARS-CoV and SARS-CoV-2), Flaviviridae (e.g., dengue virus, yellow fever virus, and hepatitis C virus), Paramyxoviridae (e.g., measles virus and mumps virus), Orthomyxoviridae (e.g., influenza virus), Bunyaviridae, Caliciviridae (e.g., norovirus), Filoviridae (e.g., Ebola virus), Matonaviridae (e.g., rubella virus), Picornaviridae (e.g., poliovirus), and Rhabdoviridae (e.g., rabies virus).

[0112] In this specification, the term "viroid" is not limited, and examples of viroids include the genus Absinthe viroid (e.g., Avocado sunblotch viroid), the genus Paremo viroid (e.g., Chrysanthemum chlorotic mottle viroid), the genus Pospiviroid (e.g., Potato spindle viroid), and the genus Apcus viroid (e.g., Apple pitted fruit viroid).

[0113] As used herein, the term "organism" is not limited, and may be, for example, a unicellular organism such as a bacterium, fungus, or archaea, or a multicellular organism such as a protist, plant, or animal. The organism may also be a pathogen such as an infectious disease. Examples of bacteria that fall under the category of pathogenic bacteria include Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Neisseria meningitidis, Staphylococcus aureus, α-group hemolytic streptococcus, Streptococcus pneumoniae, Shigella dysenteriae, and Salmonella. enterica), Vibrio cholerae, Pseudomonas aeruginosa, Bordetella pertussis, Mycobacterium tuberculosis, Corynebacterium diphtheriae, Mycoplasma pneumoniae, Rickettsia japonica, Salmonella enterica serovar Typhi, and the like.

[0114] In one embodiment, the method of this aspect may be singleplex detection or multiplex detection. For example, in multiplex detection, multiple different target nucleic acid sequences can be amplified and detected using multiple probe / primer sets in the amplification and detection steps. Each of the nucleic acid probes in the multiple probe / primer sets for multiplex detection contains a fluorescent substance that can be detected at a different fluorescent wavelength, so that each of the different target nucleic acid sequences can be detected at a different fluorescent wavelength.

[0115] 3-3. Effects The detection method of the present invention allows for a high degree of freedom in the nucleic acid probes that can be used, enabling target nucleic acid sequences to be detected at low cost. Furthermore, the detection method of the present invention reduces nonspecific amplification. Furthermore, the detection method of the present invention also enables low-cost, high-throughput multiplex analysis.

[0116] Furthermore, by applying the detection method of the present invention to a sample obtained from a subject, it becomes possible to diagnose or assist in the diagnosis of a disease associated with the target nucleic acid sequence (e.g., an infectious disease such as an infection with a pathogen from which the target nucleic acid sequence is derived). When a target nucleic acid sequence is detected by the detection step of this embodiment, it indicates that the subject from whom the sample was derived is suffering from a disease associated with the target nucleic acid sequence. For example, when a target nucleic acid sequence derived from the SARS-CoV virus or SARS-CoV-2 virus is detected by the detection method of the present invention, it indicates that the subject is infected with SARS-CoV and SARS-CoV-2, respectively. Furthermore, when a target nucleic acid sequence derived from Neisseria gonorrhoeae or Chlamydia trachomatis is detected by the detection method of the present invention, it indicates that the subject is infected with gonorrhea or chlamydia, respectively.

[0117] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0118] Example 1: Investigation of probe structure (Objective) To investigate probe structures that do not bind to nucleic acid recombination-related enzymes to form complexes. Specifically, to investigate the number of unnatural nucleotides contained in the probe and the probe length.

[0119] (Methods and Results) (1) Evaluation of Binding by Nucleic Acid Recombination-Related Enzymes The probes shown in Table 1 below were prepared. S-probe 6 to S-probe 16 contain various numbers of LNA nucleotides and have various probe lengths.

[0120]

[0121] The ability of each probe, S-probe6 to S-probe16, to avoid binding by nucleic acid recombination-related enzymes was examined using the following method. 200 nM of each probe was added to a solution containing 160 ng / μL UvsX recombinase, 40 ng / μL UvsY accessory protein, and 700 ng / μL gp32 protein (SSB), supplemented with ATP, phosphocreatine, creatine kinase, etc., and incubated at 45°C. Fluorescence intensity was measured after 10 minutes. The same experiment was also performed for each probe in a solution free of UvsX recombinase and gp32 protein (SSB). The fluorescence intensity in the solution free of UvsX recombinase and gp32 protein (SSB) was set to 1, and the relative fluorescence intensity was calculated from the fluorescence intensity in the solution containing UvsX recombinase and gp32 protein (SSB). When the relative fluorescence intensity was 2 or more, it was determined that the probe was unable to avoid binding by the recombinase, and when the relative fluorescence intensity was less than 2, it was determined that the probe was able to avoid binding by the nucleic acid recombination-related enzyme.

[0122] The results are shown in Figure 5. Of S-probes 6 to 16, S-probes 7, 9 to 12, and 14 to 16 were able to avoid binding by nucleic acid recombination-related enzymes. On the other hand, S-probes 6, 8, and 13 were unable to avoid binding by nucleic acid recombination-related enzymes.

[0123] The conditions for a probe that can avoid binding by nucleic acid recombination-related enzymes are shown in Figure 6. In Figure 6, the horizontal axis represents probe length, and the vertical axis represents the number of unnatural nucleotides (LNAs). Figure 6 shows that binding by nucleic acid recombination-related enzymes can be avoided when the probe length and the number of unnatural nucleotides are located inside or on the sides of a rectangle (this corresponds to a case where the probe length is X, the number of unnatural nucleotides is Y, and the following three conditions are satisfied: Y≧0.8X−5.6, 10≦X≦20, and Y≦X).

[0124] (2) Real-time Detection To perform real-time detection based on the RPA method using probes having various LNA numbers and various probe lengths, the probes shown in Table 2 below were prepared.

[0125]

[0126] A synthetic DNA fragment (20,000 copies / reaction solution) consisting of a base sequence (SEQ ID NO: 20) derived from the S gene in the SARS-CoV-2 genome was added as template DNA to the amplification reaction solution, and 420 nM of a forward primer (SEQ ID NO: 18) and a reverse primer (SEQ ID NO: 19) were added as amplification primers, and 120 nM of any of the probes listed in Table 2 above was added as a detection probe. Furthermore, 5 U of Bsu DNA polymerase large fragment (manufactured by New England BioLabs, Inc., model number M0330S), 100 U of Exonuclease III (manufactured by Takara Bio Inc., model number 2170A), 160 ng / μL of UvsX recombinase (manufactured by Intact Genomics, Inc., model number 3562), 40 ng / μL of UvsY accessory protein (manufactured by Intact Genomics, Inc., model number 3572), and 700 ng / μL of gp32 protein (single-stranded binding protein; SSB) (manufactured by Nippon Gene Co., Ltd., model number 312-03251) were added to the reaction solution along with necessary components such as dNTPs, ATP, phosphocreatine, and creatine kinase. Real-time detection was performed using an apparatus such as the LightCycler® 96 System, with the reaction carried out for 40 minutes at 45° C. Fluorescence was detected every 30 seconds.

[0127] The results of real-time detection using each probe listed in Table 2 are shown in Figure 7. When S-probe 17 to S-probe 19, which had a probe length of 12 to 18 mer and contained 4 to 8 LNAs, were used, the target gene sequence was detected. In contrast, when S-probe 20 to S-probe 22, which had a probe length of 12 to 25 mer and contained no LNAs, were used, the fluorescent signal already increased from the start of the reaction, and amplification of the target gene sequence could not be detected.

[0128] Example 2: Comparison with conventional real-time detection methods (Purpose) As described above, conventional real-time detection methods based on RPA use probes of 30 mer or longer that do not contain unnatural nucleotides and in which fluorescent substances and quenchers are bound to the interior of the nucleic acid chain rather than to both ends. In this example, the probes of the present invention are compared with probes designed based on conventional methods in terms of probe specificity, detection limit, etc.

[0129] (Methods and Results) (1) Preparation of Probes Based on Conventional Methods Probes based on conventional methods were prepared for comparison as control probes and the probes of the present invention. These are shown in Table 3 below.

[0130]

[0131] S-Probe 5 is a probe that targets the S gene of SARS-CoV-2 for detection, and contains two deoxyribonucleotides containing thymidine bases bound to a fluorescent substance or a quencher, an abasic site between the thymidine bases, and a length of 56 bases including the abasic site. It does not contain any unnatural nucleotides.

[0132] (2) Comparison of Specificity To compare the specificity of S-probe17, a probe of the present invention, and S-probe5, a probe based on a conventional method, amplification was performed by the RPA method as follows. When using S-probe17, a probe of the present invention, a primer pair consisting of a forward primer with the nucleotide sequence shown in SEQ ID NO: 18 and a reverse primer with the nucleotide sequence shown in SEQ ID NO: 21 was used. When using S-probe5, a probe based on a conventional method, a primer pair consisting of a forward primer with the nucleotide sequence shown in SEQ ID NO: 18 and a reverse primer with the nucleotide sequence shown in SEQ ID NO: 23 was used. For each probe, reactions were performed under two conditions: one in which a synthetic DNA fragment (20,000 copies / reaction solution) consisting of a nucleotide sequence derived from the S gene in the SARS-CoV-2 genome (SEQ ID NO: 20) was added as template DNA, and one in which no template DNA was added. The reaction was performed at 25°C, 37°C, 39.5°C, 40°C, 43°C, or 45°C for 40 minutes. Other reaction conditions were the same as those in Example 1.

[0133] The results of the comparison of specificity are shown in Figure 8. When the conventional real-time detection method was used, the fluorescent signal increased not only under the condition in which template DNA was added ("template (+)" in the figure) but also under the condition in which no template DNA was added ("template (-)" in the figure) under all temperature conditions (Figure 8A). In contrast, when the real-time detection method of the present invention was used, an increase in the fluorescent signal was observed only under the condition in which template DNA was added ("template (+)" in the figure) under all temperature conditions, but no increase in the fluorescent signal was observed under the condition in which no template DNA was added ("template (-)" in the figure) (Figure 8B). These results demonstrate that the probe of the present invention, which has a 12-mer probe length and four LNAs, has higher specificity than the conventional probe, which has a 56-mer probe length and no LNAs.

[0134] (3) Comparison of Detection Limits for Template DNA To compare the detection limits for template DNA of S-probe17, a probe of the present invention, and S-probe5, a probe based on a conventional method, amplification was performed using the RPA method as follows. For each probe, a synthetic DNA fragment consisting of a base sequence (SEQ ID NO: 20) derived from the S gene in the SARS-CoV-2 genome was used as template DNA and added at 2 copies / reaction solution, 20 copies / reaction solution, 200 copies / reaction solution, 2,000 copies / reaction solution, 20,000 copies / reaction solution, or 200,000 copies / reaction solution, or under conditions where no template DNA was added. The same primer pair as in (2) above was used for each probe in the amplification reaction, and the reaction temperature was 37°C for reactions using probes based on a conventional method and 45°C for reactions using the probes of the present invention. The other reaction conditions were the same as those in Example 1.

[0135] The results of the comparison of detection limits are shown in Figure 9. While the detection limit of the probe based on the conventional method (control probe) was 20 copies / reaction solution to 200 copies / reaction solution, the detection limit of the probe of the present invention was below 20 copies / reaction solution, and 20 copies of the template could be detected in 15 minutes. These results revealed that the probe of the present invention, which has a probe length of 12 mers and four LNAs, has a detection limit for template DNA that is more than 10 times lower than the conventional probe based on the conventional method, which has a probe length of 56 mers and no LNAs.

[0136] (4) Comparison of Detection Limits for Template RNA To compare the detection limits for template RNA of the probe of the present invention, S-probe17, and the conventional probe, S-probe5, amplification was performed using the RPA method as follows. For each probe, a synthetic RNA molecule consisting of a base sequence derived from the S gene in the SARS-CoV-2 genome (SEQ ID NO: 24; in the sequence, "T" represents "U") was added as template RNA at 2 copies / reaction solution, 20 copies / reaction solution, 200 copies / reaction solution, 2,000 copies / reaction solution, or 20,000 copies / reaction solution, or under conditions where no template DNA was added. 420 nM of forward primer and reverse primer were used as amplification primers, and 120 nM of S-probe17 or S-probe5 was used as detection probes. Bsu DNA polymerase large fragment, Exonuclease III, UvsX recombinase, UvsY accessory protein, and gp32 protein at concentrations similar to those in Example 1, as well as 200 U of M-MLV reverse transcriptase (manufactured by Nippon Gene Co., Ltd., model number 313-08161), were added to the reaction solution along with necessary components such as dNTPs, ATP, phosphocreatine, and creatine kinase. The same primer pairs as those in (2) above were used to detect each probe, and the amplification reaction was carried out at 45°C for 40 minutes.

[0137] The amplification results are shown in Figure 10. The detection limit of the probes based on conventional methods was 20 copies / reaction solution to 200 copies / reaction solution, whereas the detection limit of the probes of the present invention was 2 copies / reaction solution to 20 copies / reaction solution, and the probes of the present invention were able to detect 200 copies of template RNA in 20 minutes. These results demonstrate that the probes of the present invention, which have a 12-mer probe length and four LNAs, have a detection limit for template RNA that is approximately 10 times lower than the conventional probes, which have a 56-mer probe length and no LNAs.

[0138] Example 3: Real-time detection in a contaminated system (Objective) Real-time detection based on the RPA method is carried out under conditions in which various contaminants are present.

[0139] (Method and Results) Real-time detection based on RPA was performed using the S-probe17 probe of the present invention as follows. Amplification reactions were performed under the following conditions: a synthetic DNA fragment consisting of a base sequence (SEQ ID NO: 20) derived from the S gene of the SARS-CoV-2 genome was added as template DNA at 2,000 copies per reaction solution, and no template DNA was added; and also under the conditions with and without the addition of contaminants. Human genomic DNA (20 ng / reaction solution) or saliva-derived DNA extract (5 μL / reaction solution) was used as the contaminant. Saliva-derived DNA extract was prepared by adding 100 μL of Reagent A from the Kaneka Simple DNA Extraction Kit version 2 to 20 μL of human saliva, heating the mixture at 98°C for 10 minutes, and then suspending the mixture in 14 μL of Reagent B. The amplification primers used were a primer pair consisting of a forward primer with the base sequence shown in SEQ ID NO: 18 and a reverse primer with the base sequence shown in SEQ ID NO: 21, and the reaction temperature was 45° C. The other reaction conditions were the same as in Example 1.

[0140] The results of real-time detection are shown in Figure 11. It was shown that even in the presence of any of the contaminants, 2,000 copies of template DNA could be sufficiently detected using S-probe 17, a probe of the present invention.

[0141] Example 4: Detection of Chlamydia trachomatis and Neisseria gonorrhoeae (Objective) Real-time detection is carried out using DNA derived from Chlamydia trachomatis and Neisseria gonorrhoeae as detection targets.

[0142] (Methods and Results) (1) Preparation of Probes The probes shown in Table 4 below were prepared using the ORF4 sequence of plasmid DNA derived from Chlamydia trachomatis and the porA gene sequence derived from Neisseria gonorrhoeae as detection targets.

[0143]

[0144] CTP-probe1 targets the ORF4 sequence of plasmid DNA derived from Chlamydia trachomatis and is composed of eight LNA nucleotides, one abasic site, and six DNA nucleotides, with a fluorophore (FAM) and a quencher (BHQ1) attached to the 5' and 3' ends, respectively. CTP-probe2 differs from CTP-probe1 in that ROX is attached instead of FAM as the fluorophore. NG-probe1 targets the porA gene sequence derived from Neisseria gonorrhoeae and is composed of eight LNA nucleotides, one abasic site, and six DNA nucleotides, with a fluorophore (FAM) and a quencher (BHQ1) attached to the 5' and 3' ends, respectively.

[0145] (2) Singleplex Detection Amplification was performed by RPA using a plasmid DNA sequence derived from Chlamydia trachomatis and a porA gene derived from Neisseria gonorrhoeae as the detection targets, as follows: When a plasmid DNA sequence derived from Chlamydia trachomatis was the detection target, CTP-probe 1 shown in Table 4 above was used, and an amplification reaction was carried out using a primer pair consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 28 and a reverse primer shown in SEQ ID NO: 29, under the conditions of adding a synthetic DNA fragment (20,000 copies / reaction solution) consisting of the base sequence of the plasmid DNA derived from Chlamydia trachomatis (SEQ ID NO: 30) as template DNA, and under the conditions of not adding template DNA. When the porA gene derived from Neisseria gonorrhoeae was to be detected, amplification reactions were carried out using NG-probe 1 shown in Table 4 above, and a primer pair consisting of a forward primer with the nucleotide sequence shown in SEQ ID NO: 31 and a reverse primer with the nucleotide sequence shown in SEQ ID NO: 32, under conditions in which a synthetic DNA fragment (20,000 copies / reaction solution) with the nucleotide sequence of the porA gene derived from Neisseria gonorrhoeae (SEQ ID NO: 33) was added as template DNA, and under conditions in which no template DNA was added. The reaction was carried out at 45°C for 40 minutes. Reaction conditions other than those described above were the same as those in Example 1.

[0146] The results of real-time detection are shown in Figure 12. It was demonstrated that all of the targets could be detected using the probe of the present invention.

[0147] (3) Multiplex Detection The presence of each sequence is detected in real time using different fluorescent wavelengths from the same sample containing a nucleic acid having a plasmid DNA sequence derived from Chlamydia trachomatis and a nucleic acid containing a porA gene sequence derived from Neisseria gonorrhoeae. Specifically, the presence of each sequence is detected in real time using different fluorescent wavelengths. Specifically, the presence of only Chlamydia trachomatis total DNA control (AMPLIRUN® CHLAMYDIA TRACHOMATIS DNA CONTROL) (2,000 copies / reaction solution) manufactured by Vircell is added as template DNA, and the presence of Neisseria gonorrhoeae total DNA control (AMPLIRUN® NEISSERIA GONORRHOEAE DNA CONTROL) manufactured by Vircell is added as template DNA. Using CTP-probe 2 and NG-probe 1 listed in Table 4 above, a primer pair (SEQ ID NO: 28 and SEQ ID NO: 29) for amplifying a plasmid DNA sequence derived from Chlamydia trachomatis and a primer pair (SEQ ID NO: 31 and SEQ ID NO: 32) for amplifying the porA gene sequence derived from Neisseria gonorrhoeae were used to carry out reactions at 45°C for 40 minutes under the following conditions: adding only the Chlamydia trachomatis total DNA control (2,000 copies / reaction solution) as template DNA; adding both the Chlamydia trachomatis total DNA control and the Neisseria gonorrhoeae total DNA control as template DNA; and adding neither template DNA. The Chlamydia trachomatis plasmid DNA sequence was detected by fluorescence at 599 nm derived from ROX, and the Neisseria gonorrhoeae porA gene sequence was detected by fluorescence at 520 nm derived from FAM. The other reaction conditions were the same as those in Example 1.

[0148] The results of multiplex real-time detection are shown in Figure 13. The results shown in Figures 13A and 13B demonstrate that target nucleic acid sequences can be simultaneously detected in real time from a sample containing only nucleic acid having the ORF4 sequence of plasmid DNA derived from Chlamydia trachomatis, a sample containing only nucleic acid having the porA gene sequence derived from Neisseria gonorrhoeae, and a sample containing both nucleic acid having the ORF4 sequence of plasmid DNA derived from Chlamydia trachomatis and nucleic acid containing the porA gene sequence derived from Neisseria gonorrhoeae.

[0149] Example 5: Detection of SARS-CoV-2 S gene using AmNA-containing probe (Purpose) A probe is prepared by substituting all or some of the LNA nucleotides in S-probe 17 prepared in Example 1 with AmNA nucleotides, and real-time detection is performed using the S gene of SARS-CoV-2 as the detection target.

[0150] (Methods and Results) (1) Preparation of Probes All or part of the LNA nucleotides in S-probe 17 prepared in Example 1 were replaced with AmNA nucleotides to prepare the probes shown in Table 5 below.

[0151]

[0152] S-Probe17, S-Probe25, and S-Probe26 target the S gene of SARS-CoV-2 for real-time detection. They are probes consisting of four unnatural nucleotides, one abasic site, and seven DNA nucleotides (total length 12 bases), with a fluorophore (FAM) and a quencher (BHQ1) attached to the 5' and 3' ends, respectively. The three probes differ in the composition of the unnatural nucleotides. S-Probe17 contains four LNA nucleotides. S-Probe25 contains four AmNA nucleotides. S-Probe26 contains two LNA nucleotides and two AmNA nucleotides.

[0153] (2) Evaluation of probe cleavage efficiency A DNA oligonucleotide (SEQ ID NO: 40) having a sequence complementary to S-Probe 17, S-Probe 25, and S-Probe 26 was added to each probe to prepare double-stranded DNA. 2 U of Exonuclease III was added to 120 nM of double-stranded DNA and the mixture was incubated at 37°C for 5 minutes, and the fluorescence was measured before and after the reaction. The fluorescence intensity in a solution without Exonuclease III was set to 1, and the relative fluorescence intensity was calculated from the fluorescence intensity in a solution containing Exonuclease III.

[0154] The relative fluorescence intensity of the solution containing Exonuclease III to that of the solution not containing Exonuclease III was calculated to be 3.1 for S-Probe 17, 3.7 for S-Probe 25, and 3.8 for S-Probe 26. The increase in fluorescence intensity upon addition of Exonuclease III indicated that all probes were cleaved by Exonuclease III. Furthermore, the cleavage efficiency of the probe containing a combination of AmNA nucleotides and LNA nucleotides was shown to be 20% or more higher than that of a probe containing only LNA nucleotides as unnatural nucleotides.

[0155] (3) Real-time detection In real-time detection, in addition to the probe prepared in (1) above, a forward primer consisting of the base sequence shown in SEQ ID NO: 18 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 19 were used as primers for nucleic acid amplification. A synthetic DNA fragment (20 or 200 copies / reaction solution) consisting of a base sequence (SEQ ID NO: 20) derived from the S gene in the SARS-CoV-2 genome was added as template DNA to the amplification reaction solution. 420 nM of the forward primer and reverse primer were added as amplification primers, and 120 nM of S-probe17, S-probe25, or S-probe26 was added as a detection probe. The reaction solution was amplified at 45°C for 15 minutes while stirring. Other reaction conditions were the same as in Example 2. Furthermore, from the obtained amplification curve, the time (minutes) at which the amplification curve intersected with the threshold (Tt) value was calculated.

[0156] The results of real-time detection performed by adding 200 copies of template DNA per reaction solution to the reaction solution are shown in Figure 14A. The Tt values ​​calculated for the different probes were 8.2 for S-probe17, 5.4 for S-probe25, and 4.7 for S-probe26.

[0157] The results of real-time detection performed by adding 20 copies of template DNA per reaction solution to the reaction solution are shown in Figure 14B. The Tt values ​​were calculated to be 9.4 for S-probe 17, 6.4 for S-probe 25, and 6.0 for S-probe 26.

[0158] The results shown in Figure 14 demonstrate that probes containing only AmNA nucleotides as unnatural nucleotides and probes containing a combination of AmNA and LNA nucleotides as unnatural nucleotides can detect the S gene of the SARS-CoV-2 genome more quickly than probes containing only LNA nucleotides as unnatural nucleotides. Specifically, a 30% to 40% reduction in Tt value was achieved.

[0159] Example 6: Detection of Chlamydia trachomatis using an AmNA-containing probe (Purpose) A probe is prepared by substituting all or some of the LNA nucleotides in the CTP-probe 1 prepared in Example 4 with AmNA nucleotides, and real-time detection is performed using DNA derived from Chlamydia trachomatis as the detection target.

[0160] (Methods and Results) (1) Preparation of Probes All or part of the LNA nucleotides in the CTP-probe 1 prepared in Example 4 were replaced with AmNA nucleotides to prepare the probes shown in Table 6 below.

[0161]

[0162] CTP-probe1, CTP-probe3, and CTP-probe4 are probes targeted for real-time detection of Chlamydia trachomatis-derived DNA, and are composed of eight unnatural nucleotides, one abasic site, and six to eight DNA nucleotides (total length 15 or 17 bases), with a fluorophore (FAM) and a quencher (BHQ1) bound to the 5' and 3' ends, respectively. The three probes differ in the composition of the unnatural nucleotides. CTP-probe1 contains eight LNA nucleotides. CTP-probe3 contains eight AmNA nucleotides. CTP-probe4 contains three LNA nucleotides and five AmNA nucleotides.

[0163] (2) Real-time detection In real-time detection, in addition to the probe prepared in (1) above, a forward primer consisting of the base sequence shown in SEQ ID NO: 28 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 29 were used as primers for nucleic acid amplification. A synthetic DNA fragment (20, 200, or 2000 copies / reaction solution) consisting of the ORF4 base sequence (SEQ ID NO: 30) of plasmid DNA derived from Chlamydia trachomatis was added as template DNA to the reaction solution for amplification, and 420 nM of the forward primer and reverse primer were added as amplification primers, and 120 nM of CTP-probe1, CTP-probe3, or CTP-probe4 was added as a detection probe. The reaction solution was amplified at 45°C for 15 minutes. The other reaction conditions were the same as those in Example 2.

[0164] The results of real-time detection are shown in Figure 15. The results shown in Figure 15 demonstrate that the probe containing only AmNA nucleotides as unnatural nucleotides and the probe containing a combination of AmNA nucleotides and LNA nucleotides as unnatural nucleotides can detect DNA derived from Chlamydia trachomatis to an equal extent as the probe containing only LNA nucleotides as unnatural nucleotides.

[0165] Example 7: Detection of Neisseria gonorrhoeae using AmNA-containing probes (Objective) A probe is prepared by substituting all or some of the LNA nucleotides in NG-probe 1 prepared in Example 4 with AmNA nucleotides, and real-time detection is performed using DNA derived from Neisseria gonorrhoeae as the detection target.

[0166] (Methods and Results) (1) Preparation of Probes All or some of the LNA nucleotides in NG-probe 1 prepared in Example 4 were replaced with AmNA nucleotides to prepare the probes shown in Table 7 below.

[0167]

[0168] NG-probe1, NG-probe2, and NG-probe3 target DNA derived from Neisseria gonorrhoeae for real-time detection. They are probes composed of eight unnatural nucleotides, one abasic site, and six DNA nucleotides (total length 15 bases), with a fluorescent substance (FAM) and a quencher (BHQ1) attached to the 5' and 3' ends, respectively. The three probes differ in the composition of the unnatural nucleotides. NG-probe1 contains eight LNA nucleotides. NG-probe2 contains eight AmNA nucleotides. NG-probe3 contains two LNA nucleotides and six AmNA nucleotides.

[0169] (2) Real-time detection In real-time detection, in addition to the probe prepared in (1) above, a forward primer consisting of the base sequence shown in SEQ ID NO: 31 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 32 were used as primers for nucleic acid amplification. A synthetic DNA fragment (20, 200, or 2000 copies / reaction solution) consisting of the base sequence of the porA gene derived from Neisseria gonorrhoeae (SEQ ID NO: 33) was added as template DNA to the reaction solution for amplification, and 420 nM of the forward primer and reverse primer were added as amplification primers, and 120 nM of NG-probe1, NG-probe2, or NG-probe3 was added as a detection probe. The reaction solution was amplified at 45°C for 15 minutes. The other reaction conditions were the same as those in Example 2.

[0170] The results of real-time detection are shown in Figure 16. The results shown in Figure 16 indicate that a probe containing only AmNA nucleotides as unnatural nucleotides, and a probe containing a combination of AmNA nucleotides and LNA nucleotides as unnatural nucleotides, can detect DNA derived from Neisseria gonorrhoeae to the same extent as a probe containing only LNA nucleotides as unnatural nucleotides. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

1. A nucleic acid probe for detecting a target nucleic acid sequence, comprising one or more abasic sites, oxidized bases, or natural ribonucleotides, and one or more non-natural nucleotides, comprising a base sequence complementary to a probe binding region in the target nucleic acid sequence or its complementary sequence except for the abasic sites, and comprising a fluorescent substance and a quencher bound to the nucleic acid probe.

2. The nucleic acid probe according to claim 1, wherein the non-natural nucleotide is a bridged nucleotide and / or a 2'-modified nucleotide.

3. The nucleic acid probe of claim 2, wherein the bridged nucleotide comprises one or more AmNA nucleotides and / or GuNA nucleotides.

4. The nucleic acid probe according to claim 3, which comprises 2 to 8 AmNA nucleotides and / or GuNA nucleotides.

5. The nucleic acid probe of claim 3, further comprising one or more LNA nucleotides.

6. The nucleic acid probe according to claim 2, wherein a variable L indicating the base length of the nucleic acid probe and a variable N indicating the number of bridged nucleotides and / or 2'-modified nucleotides in the nucleic acid probe satisfy the following formulas I and II: N≧0.8×L−5.6 (formula I), and L>N (formula II).

7. The nucleic acid probe according to claim 6, further satisfying the following formula III: 10≦L≦20 (Formula III).

8. The nucleic acid probe of claim 6, wherein the bridged nucleotide is an LNA nucleotide.

9. The nucleic acid probe of claim 8, wherein the bridged nucleotides further comprise AmNA nucleotides or GuNA nucleotides.

10. The nucleic acid probe of claim 1, comprising a fluorescent substance attached to the 5' or 3' end of the nucleic acid probe and a quencher attached to the other end.

11. A composition or kit for detecting a target nucleic acid sequence, comprising: the nucleic acid probe according to claim 1; a first nucleic acid primer which comprises a base sequence complementary to a first primer binding region located at the 3' end of the target nucleic acid sequence and serves as a substrate for a nucleic acid recombination-related enzyme; and a second nucleic acid primer which comprises a base sequence complementary to a second primer binding region located at the 3' end of the complementary sequence of the target nucleic acid sequence and serves as a substrate for a nucleic acid recombination-related enzyme.

12. The composition or kit according to claim 11 for use in an isothermal nucleic acid amplification method.

13. The composition or kit according to claim 12, further comprising a strand-displacing DNA polymerase, exonuclease III or ribonuclease H, and / or a nucleic acid recombination-related enzyme.

14. A composition or kit for multiplex detection, comprising a plurality of probe / primer sets for detecting a plurality of different target nucleic acid sequences or their complementary sequences, each of the probe / primer sets comprising the nucleic acid probe according to claim 11, a first nucleic acid primer, and a second nucleic acid primer, and each of the nucleic acid probes in the plurality of probe / primer sets comprising a fluorescent substance that can be detected at a different fluorescent wavelength.

15. The composition or kit of claim 14, wherein the different target nucleic acid sequences are derived from the same or different viruses, viroids, and / or organisms.

16. A method for detecting a target nucleic acid sequence in a sample, comprising: an amplification step of amplifying the target nucleic acid sequence and / or its complementary sequence in the sample by a nucleic acid amplification method using the nucleic acid probe, a first nucleic acid primer, and a second nucleic acid primer described in claim 11; and a detection step of irradiating the nucleic acid probe with excitation light during and / or after the amplification step and detecting fluorescence emitted from the fluorescent substance.

17. The method of claim 16, wherein the nucleic acid amplification method is an isothermal nucleic acid amplification method.

18. The method according to claim 17, wherein the amplification step uses a strand-displacing DNA polymerase.

19. The method according to claim 18, wherein the amplification step is carried out in the presence of a nucleic acid recombination-related enzyme, and exonuclease III, ribonuclease H, DNA glycosylase, or endonuclease IV.

20. The method of claim 16, wherein the target nucleic acid sequence comprises RNA, and further comprising a reverse transcription step of reverse transcribing the RNA prior to and / or during the amplification step.

21. The method of claim 16, further comprising an extraction step of extracting nucleic acid containing said target nucleic acid sequence from said sample prior to said amplification step.

22. The method of claim 16, wherein the sample is a cell, tissue, cell extract, tissue extract, tissue fluid, or body fluid.

23. The method of claim 16, wherein the target nucleic acid sequence is derived from a virus, viroid, and / or organism.

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