Oligonucleotides for detecting influenza virus and uses thereof

Abstract

To provide a labeled probe, a detection method and a reagent kit for detecting an influenza virus.SOLUTION: A labeled probe having the following features (A) and (B) is used as a labeled probe used for detecting an influenza virus that can be included in a specimen sample: (A) including a base sequence showing 85% or more of identity with a base sequence shown by a specific sequence number or a base sequence of at least 10 bases or more continuous in a base sequence complementary with the sequence; and (B) only one of 5' terminal and 3' terminal is labeled. In a preferred embodiment, two or more of the labeled probes are used so as to coexist in reaction liquid.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to oligonucleotides for rapidly, reliably and simply detecting influenza virus contained in a specimen sample. Furthermore, the present invention relates to a method for detecting influenza virus contained in a specimen sample using the oligonucleotide, and reagents, kits and the like for use in the method.

Background Art

[0002] Respiratory infections caused by influenza virus are one of the most well-known diseases. Influenza viruses are classified into multiple types based on the antigenicity differences of nucleoprotein (NP) and matrix protein (M), among which types A and B are frequently seen in clinical practice. In addition, various subtypes exist for each type of influenza virus.

[0003] As methods for examining influenza virus, genetic tests, antigen tests, and antibody tests have been developed so far. Among these, antigen tests have the advantage of being able to be examined simply, but currently have difficulties in terms of sensitivity. Antibody tests can confirm whether one has suffered from the disease in the past, but are not suitable for testing whether one is currently infected. In contrast, genetic tests are characterized by being able to detect influenza virus specifically with excellent sensitivity.

[0004] In the influenza gene detection method, first, the genomic RNA of the influenza virus is reverse transcribed into cDNA by reverse transcriptase (RT: reverse transcription), and then nucleic acid amplification and detection are performed by methods such as the PCR method or the LAMP (Loop-mediated Isothermal Amplification) method. These are called the RT-PCR method and the RT-LAMP method, respectively. In the detection by the most commonly used RT-PCR method at present, a method of quantitative detection is mainly carried out by performing real-time PCR using a double-labeled nucleic acid probe (also called Taqman probe, hydrolysis probe, etc.) (Non-Patent Document 1). In this method, it is possible to quantify the virus amount based on the control RNA, but since it is necessary to measure the photometry for each cycle of PCR, it takes about 1 hour for detection even in the shortest case.

[0005] Furthermore, as a method for detecting nucleic acid amplification products, the melting curve analysis method is known. The melting curve analysis method can perform nucleic acid amplification and detection in separate steps, and can be measured relatively simply in about 30 minutes at the shortest. Furthermore, the melting curve analysis using a fluorescently labeled nucleic acid probe has the advantage that it is easy to adapt to gene detection using an automatic analyzer. However, no nucleic acid probe that can detect the influenza virus with high sensitivity by the melting curve analysis method is known yet.

[0006] Influenza viruses mainly exist as type A and type B. Furthermore, the novel influenza virus that spread globally in 2009 was a special type that was type A but could not be detected by existing nucleic acid amplification methods. This type of influenza virus was particularly designated as H1N1pdm09. Therefore, in order to detect each type of influenza virus at once, it is necessary to put multiple primer-probe sets into the same reaction system. On the other hand, putting multiple primer-probe sets into the same reaction system increases the possibility of interactions such as forming dimers between oligonucleotides, and more trial and error is required to find the optimal primer and probe sequences. Also, influenza viruses can mutate in the genomic RNA region. In fact, various subtype base sequences are published in the database.

[0007] When there is a mismatch between the base sequences of the primer and probe and the base sequence of the targeted influenza virus, the binding ability of the primer or probe to the viral RNA or its amplification product decreases. Especially in the real-time RT-PCR method, when there are many mismatches between the target nucleic acid and the probe, the probe cannot bind sufficiently to the target nucleic acid, so it is expected that the rise of the amplification curve will be delayed apparently or there will be no rise at all. Such a situation can induce false negatives. In the case of the melting curve analysis method, since the detection step is performed after RT-PCR is completed, even if there is a mismatch in the primer-probe, as long as the amplification product is finally obtained, detection is possible, so the influence of mismatches is relatively less than that of the real-time RT-PCR method.

[0008] In addition, in the inspection of influenza virus, usually, RNA is extracted and purified from specimens such as nasopharyngeal swab specimens, sputum, and saliva using an RNA extraction and purification kit, and the RNA extract is generally used for the next step RT-PCR method or the like. However, since RNA extraction and purification require a great deal of labor and cost, it is required to use a sample prepared only by simple pretreatment for the inspection. That is, instead of extracting and purifying the genomic RNA of influenza virus from a specimen containing influenza virus, a method that can detect using a sample (simple RNA extract) in which RNA is simply extracted is required.

[0009] As described above, in the inspection of influenza virus, there is a need for a measurement method that can have advantages such as (1) short measurement time, (2) high sensitivity, (3) strong resistance to amplification inhibition by specimen samples, (4) ability to cope with mutations of influenza virus, and (5) ability to incorporate an internal control that can detect amplification inhibition by specimens into the reaction system.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0011] The present invention has been made against the background of such problems of the prior art. That is, the object of the present invention is to have advantages such as being able to detect influenza virus contained in a specimen sample in a shorter time, with higher sensitivity, being strong against amplification inhibition by a simple extraction specimen, being able to cope with mutations of influenza virus, and being able to incorporate an internal control that can detect amplification inhibition by a specimen, and to provide a method for detecting influenza virus.

Means for Solving the Problem

[0012] As a result of intensive research, the present inventor has found a method for detecting influenza virus that can meet the above requirements by using a labeled probe having a specific sequence, and in particular, by combining a plurality of specific labeled probes in a reaction solution, and has completed the present invention. That is, the outline of the present invention is as follows.

[0013] [Item 1] A labeled probe having the following characteristics (A) and (B) and used for detecting influenza virus that may be contained in a specimen sample: (A) It contains a base sequence that shows 85% or more identity with the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence that is complementary to them and includes at least 10 consecutive bases; and (B) Only one of the 5'-end or 3'-end is labeled. [Item 2] The labeled probe according to Item 1, wherein the labeled probe contains (A) a base sequence that shows 85% or more identity with the base sequence represented by SEQ ID NO: 1 or a base sequence that is complementary to them and includes at least 10 consecutive bases, and is used in coexistence in a reaction solution with a labeled probe that contains (A-2) a base sequence that shows 85% or more identity with the base sequence represented by SEQ ID NO: 2 or a base sequence that is complementary to them and includes at least 10 consecutive bases, and (B-2) only one of the 5'-end or 3'-end is labeled. [Item 3] The labeled probe according to Item 1, wherein the labeled probe contains (A) a base sequence that shows 85% or more identity with the base sequence represented by SEQ ID NO: 2 or a base sequence that is complementary to them and includes at least 10 consecutive bases, A labeled probe according to claim 1, comprising a base sequence of at least 10 consecutive bases in a base sequence having 85% or more identity with the base sequence represented by SEQ ID NO: 1 or a base sequence complementary thereto, and being used in coexistence in a reaction solution with a labeled probe having only one of the 5'-end or 3'-end labeled. [Claim 4] The labeled probe according to any one of claims 1 to 3, wherein the base sequence of at least 10 consecutive bases described in (A) above consists of a base sequence of at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto, or a base sequence in which one or several bases are substituted, deleted, or added in those base sequences. [Claim 5] The labeled probe according to any one of claims 1 to 4, wherein the base sequence of at least 10 consecutive bases described in (A) above consists of a base sequence represented by any one of SEQ ID NOs: 3 to 32 or a base sequence complementary thereto. [Claim 6] The labeled probe according to any one of claims 1 to 5, wherein the label is a fluorescent dye label. [Claim 7] The labeled probe according to any one of claims 1 to 6, wherein the labeled probe is labeled with a fluorescence quenching dye that quenches when bound to a nucleic acid containing a base sequence having 90% or more identity with a base sequence complementary to the base sequence of the labeled probe. [Claim 8] The labeled probe according to any one of claims 1 to 7, wherein the labeled probe is labeled with a fluorescence quenching dye that quenches by interaction with guanine. [Claim 9] The labeled probe according to any one of claims 1 to 8, wherein the label in (B) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives. [Item 10] The labeled probe according to any one of Items 1 to 9, wherein the label of (B) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 11] The labeled probe according to any one of Items 1 to 10, wherein the terminal base labeled in (B) is cytosine. [Item 12] The labeled probe according to any one of Items 1 to 11, which is used for detecting influenza virus that may be contained in a biological specimen sample that has not undergone a nucleic acid purification step. [Item 13] A labeled probe set used for detecting influenza virus that may be contained in a specimen sample, comprising at least the following first labeled probe and second labeled probe: (A-1) comprising a base sequence having 85% or more identity with the base sequence shown in SEQ ID NO: 1 or a base sequence complementary thereto and having at least 10 consecutive bases; and (B-1) A first labeled probe, characterized in that only one of the 5'-end or 3'-end is labeled; and (A-2) comprising a base sequence having 85% or more identity with the base sequence shown in SEQ ID NO: 2 or a base sequence complementary thereto and having at least 10 consecutive bases; and (B-2) A second labeled probe, characterized in that only one of the 5'-end or 3'-end is labeled. [Item 14] A method for detecting influenza virus that may be contained in a specimen sample, using the labeled probe according to any one of Items 1 to 12 or the labeled probe set according to Item 13. [Item 15] The method for detecting influenza virus according to Item 14, wherein the detection is performed by RT-PCR-melting curve analysis. [Item 16] The detection method according to item 15, wherein the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B. [Item 17] The detection method according to item 16, wherein the DNA polymerase belonging to family B is a DNA polymerase derived from KOD or a variant thereof. [Item 18] The detection method according to any one of items 14 to 17, wherein the enzyme having reverse transcription activity used in the RT reaction is a reverse transcriptase derived from M-MLV (Moloney Murine Leukemia Virus) or a variant thereof. [Item 19] The detection method according to any one of items 14 to 18, wherein the sample is a biological sample that has not undergone a nucleic acid purification step. [Item 20] A reagent kit for detecting influenza virus that may be contained in a sample, comprising the labeled probe according to any one of items 1 to 12 or the labeled probe set according to item 13. [Item 21] The detection method according to any one of items 14 to 19, which can distinguish between influenza A virus and influenza B virus. [Advantages of the Invention]

[0014] According to the present invention, influenza virus contained in a sample can be detected quickly and with high sensitivity. According to the method of the present invention, for example, even when using a sample in which nucleic acids are not highly isolated and purified, that is, a sample obtained by simple RNA extraction, it is less susceptible to amplification inhibition and can detect influenza virus with extremely high sensitivity. By using the detection method, reagent or kit of the oligonucleotide of the present invention, simple, rapid and highly sensitive detection of influenza virus becomes possible, which can greatly contribute to the field of clinical diagnosis. [Brief Description of the Drawings]

[0015]

Figure 1

Figure 2

[0016] Hereinafter, while showing embodiments of the present invention, the present invention will be described in more detail, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference. In this specification, "~" means "above and below". For example, if "X~Y" is described in the specification, it means "X or more and Y or less". Also, "and / or" in this specification means either one or both.

[0017] In this specification, a nucleic acid primer may be referred to as an oligonucleotide primer or simply a primer, and a nucleic acid probe may be referred to as an oligonucleotide probe or simply a probe, and these are collectively referred to as oligonucleotides.

[0018] [Method for Detecting Influenza Virus] One of the embodiments of the present invention is a method for highly sensitively detecting influenza virus that may be contained in a specimen sample. The present invention is characterized by using a labeled probe having a specific base sequence described later (also referred to as a "nucleic acid probe" in this specification). In a specific embodiment, by using two or more kinds of the labeled probes of the present invention corresponding to the genomic sequences of influenza A virus and influenza B virus in combination, for example, in RT-PCR-melting curve analysis method, etc., for the influenza virus that may be contained in a specimen sample, influenza A virus and influenza B virus can be detected quickly, simply, and highly sensitively while being discriminated. In a specific embodiment, the method of the present invention includes at least the following steps (1) to (3): (1) A step of converting target RNA into cDNA using an enzyme having reverse transcriptase activity in a specimen sample; (2) Using one or more nucleic acid primer sets consisting of specific base sequences, performing a nucleic acid amplification reaction to generate one or more nucleic acid amplification products using the cDNA obtained in the step (1) as a template; and (3) Detecting the one or more amplification products obtained in the step (2) by a melting curve analysis method using one or more labeled probes of the present invention consisting of specific base sequences, characterized by comprising the above. That is, in the detection of influenza virus, one feature of the present invention is to use one or more labeled probes of specific base sequences in the nucleic acid amplification detection reaction in order to obtain a highly sensitive determination result.

[0019] [Reverse transcription reaction] In one embodiment, the method of the present invention performs a reverse transcription (RT) reaction using a nucleic acid primer and a reverse transcriptase to generate cDNA from a target RNA. The reverse transcriptase is not particularly limited as long as it has reverse transcription activity. For example, reverse transcriptases (RNA-dependent DNA polymerases) derived from M-MLV (Moloney Murine Leukemia Virus) or AMV (avian myeloblastosis virus) and mutants thereof can be mentioned. Examples of mutants include mutants in which one or several amino acids are deleted, substituted, and / or added in the wild-type amino acid sequence. Specifically, mutants lacking RNase H activity can be mentioned in order to increase the synthesis efficiency of cDNA. Also, under specific conditions, Tth DNA polymerase and its mutants are known to have reverse transcription activity and may be used as reverse transcriptases in the present invention.

[0020] The nucleic acid primer used in the reverse transcription reaction may also serve as one of the PCR primers used in the subsequent nucleic acid amplification reaction. Also, the conditions of the reverse transcription reaction are not particularly limited as long as the effects of the present invention are achieved. For example, 37 to 55 °C for 0 seconds to 60 minutes is preferable. Particularly, 42 to 50 °C for 2 to 15 minutes is preferable.

[0021] [Nucleic acid amplification reaction] In one embodiment, the method of the present invention performs a nucleic acid amplification reaction using a nucleic acid primer set to generate a nucleic acid amplification product. Nucleic acid amplification methods are techniques for amplifying several copies of a target nucleic acid to a visible level, i.e., hundreds of millions of copies or more, and are widely used not only in the field of life science research but also in fields such as clinical diagnosis, food hygiene inspection, and environmental inspection. Such nucleic acid amplification methods include the PCR method, LAMP method, LCR method, TMA method, SDA method, RT-PCR method, RT-LAMP method, NASBA method, TRC method, TMA method, etc. These techniques have already been established in the technical field, and the method can be selected according to the purpose. The nucleic acid amplification method performed in the present invention is preferably the PCR method (including the RT-PCR method), but is not limited thereto.

[0022] [PCR reaction] The PCR reaction is mainly a reaction catalyzed by DNA polymerase, and one cycle consists of three steps: (1) DNA denaturation by heat treatment (separation from double-stranded DNA to single-stranded DNA), (2) annealing of the primer to the template single-stranded DNA, and (3) extension of the primer using DNA polymerase. By repeating this cycle, the target nucleic acid is amplified. Examples of DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and their variants. In the present invention, from the viewpoints of rapidity, high sensitivity, and resistance to amplification inhibition by the sample, it is preferable to use a DNA polymerase belonging to family B. In addition, when performing melting curve analysis, from the viewpoint of using a fluorescence quenching probe, it is also preferable to use a DNA polymerase belonging to family B that does not have 5'→3' exonuclease activity.

[0023] The conditions of the PCR reaction are not particularly limited as long as the effects of the present invention are achieved. For example, the first heat denaturation step is carried out at 80 to 100°C for 0 seconds to 5 minutes, the repeated heat denaturation step is carried out at 80 to 100°C for 0.5 to 300 seconds, the annealing is carried out at 35 to 80°C for 1 to 300 seconds, and the extension reaction step is carried out at 35 to 85°C for about 1 to 300 seconds, and this repetition is preferably repeated 30 to 70 times. Here, the temperature and time of the cycle repeated may be changed every 1 to several cycles.

[0024] [DNA polymerase belonging to Family B] The DNA polymerase used in the present invention is preferably a DNA polymerase belonging to Family B, but is not limited thereto. The DNA polymerase belonging to Family B is not particularly limited, but is preferably a DNA polymerase derived from archaea, and more preferably a DNA polymerase derived from bacteria of the genus Pyrococcus and the genus Thermococcus. In addition, the present invention also includes mutants thereof that have not lost the DNA polymerase activity derived from archaea belonging to Family B. Mutants of DNA polymerase include, but are not limited to, mutants for the purpose of enhancing polymerase activity, deleting exonuclease activity, and adjusting substrate specificity. Examples of the DNA polymerase derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, Pyrococcus horikoshii, and mutants thereof that have not lost the DNA polymerase activity derived therefrom. Examples of DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9degrees North-7 (Thermococcus sp. 9°N-7), Thermococcus siculi, and mutants thereof that have not lost the DNA polymerase activity derived therefrom. Preferably, DNA polymerases derived from Thermococcus kodakaraensis and mutants thereof (for example, KOD-derived DNA polymerase lacking 3'→5' exonuclease activity, etc.) can be particularly preferably used in the present invention from the viewpoint of excellent elongation property and thermal stability. PCR enzymes using these DNA polymerases are commercially available, and examples include Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), Pwo (Roche), etc., and any of them can be used in the present invention.

[0025] [DNA polymerase derived from KOD] As used herein, the DNA polymerase derived from KOD (also referred to as KOD DNA polymerase) refers to the DNA polymerase derived from Thermococcus kodakaraensis and its variants (for example, the DNA polymerase derived from KOD in which the 3'→5' exonuclease activity is deleted by substituting, deleting, and / or adding one or several amino acids in the naturally derived amino acid sequence, etc.). In one preferred embodiment, the present invention performs a nucleic acid amplification reaction using such a DNA polymerase derived from KOD. KOD DNA polymerase is superior to Taq DNA polymerase, which is a DNA polymerase belonging to family A, in terms of accuracy, amplification efficiency, elongation property, and resistance to amplification inhibition by inhibitors derived from samples. In the present invention, by using such KOD DNA polymerase, as shown in the examples described below, it becomes possible to detect influenza virus simply, rapidly, and with high sensitivity.

[0026] [Nucleic acid primer set] The nucleic acid primer set described in the above (2) and used for detecting influenza virus in the present invention is not particularly limited as long as it can amplify a nucleic acid fragment derived from influenza virus with which a labeling probe described later can form a complex. From the viewpoint that a more sensitive determination result is easily obtained, the nucleic acid primer set is a nucleic acid primer set capable of amplifying a base sequence containing part or all of the base sequence of the influenza virus genomic RNA represented by SEQ ID NO: 1 or 2. For example, the nucleic acid primer set may be a nucleic acid primer set composed of at least one nucleic acid primer consisting of the base sequence represented by SEQ ID NO: 33 or 34 and at least one nucleic acid primer consisting of the base sequence represented by SEQ ID NO: 35 or 36; a nucleic acid primer set composed of at least one nucleic acid primer consisting of the base sequence represented by SEQ ID NO: 37 or 38 and at least one nucleic acid primer consisting of the base sequence represented by SEQ ID NO: 39 or 40; a nucleic acid primer set composed of at least one nucleic acid primer consisting of the base sequence represented by SEQ ID NO: 41 or 42 and at least one nucleic acid primer consisting of the base sequence represented by SEQ ID NO: 43 or 44, but is not limited thereto.

[0027] In this specification, SEQ ID NO: 1 and SEQ ID NO: 2 are nucleotide sequences corresponding to those obtained by reverse transcribing a part of the RNA sequence of influenza virus into DNA. SEQ ID NO: 1 corresponds to the nucleotide sequence of influenza A virus, and SEQ ID NO: 2 corresponds to the nucleotide sequence of influenza B virus. The present invention can detect influenza virus with high sensitivity in melting curve analysis and the like by targeting the influenza virus genomic RNA region corresponding to the nucleotide sequences shown in these SEQ ID NO: 1 or 2. In particular, when simultaneously detecting both the genomic region of influenza virus shown by SEQ ID NO: 1 and the genomic region of influenza virus shown by SEQ ID NO: 2 as target sequences, a new finding has been made that it is also possible to discriminate between influenza A virus and influenza B virus in melting curve analysis and the like. Therefore, the nucleic acid primer set used in the present invention preferably can amplify a nucleotide sequence containing a part or all of each of the two regions shown by SEQ ID NO: 1 and 2.

[0028] [Step of detecting amplification product] In the method of the present invention, as step (3), it includes the step of detecting the amplification product obtained in the above steps (1) to (2). The mode of this detection step is not particularly limited and can be carried out by any method known in the art. In the present invention, the melting curve analysis method is preferred.

[0029] In one embodiment, the above step (3) includes the following steps (3-1) to (3-2): (3-1) A step of hybridizing the nucleic acid amplification product obtained in the above step (2) with a nucleic acid probe to form a complex; and (3-2) A step of detecting the complex obtained in the above step (3-1). It is characterized by including. In a preferred embodiment of the present invention, in the detection of influenza virus, in order to obtain a highly sensitive determination result, one feature is to use a labeled probe that can specifically react with the nucleic acid amplification product derived from influenza virus obtained by the above steps (1) and (2) to form a complex. From the perspective of enabling highly sensitive detection even when only simple RNA extraction is performed, it is preferable to use one or more nucleic acid labeled probes that can specifically react with one or more nucleic acid amplification products derived from influenza virus obtained by the above steps (1) and (2) to form a complex.

[0030] [Nucleic acid probe] In one embodiment, the present invention is a nucleic acid probe that can specifically react with a nucleic acid amplification product derived from influenza virus amplified with a nucleic acid primer set containing the above nucleic acid primers to form a complex. The labeled probe of the present invention that specifically reacts with a nucleic acid amplification product derived from influenza virus can be a nucleic acid probe for detecting influenza virus having the following characteristics: (A) It contains a base sequence having 85% or more identity with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto and having at least 10 consecutive bases; and (B) Only one of the 5'-end or 3'-end is labeled. By using a nucleic acid probe having such characteristics, for example, it becomes possible to detect influenza virus with high sensitivity even in melting curve analysis.

[0031] The nucleic acid probe of the present invention is not particularly limited as long as it has a sequence highly homologous to the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto. However, when it is a nucleic acid probe labeled with a fluorescence quenching dye that quenches by interaction with guanine described later, it is preferable that at least one terminal base labeled with the dye is cytosine.

[0032] In one embodiment, the labeled probe used in the present invention may consist of a nucleotide sequence having at least 10 consecutive nucleotides with 85% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence complementary thereto. Preferably, it may be a nucleic acid probe consisting of a nucleotide sequence having at least 10 consecutive nucleotides with 90% or more, more preferably 93% or more, still more preferably 95% or more, and even more preferably 98% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence complementary thereto. Particularly preferably, it is a nucleic acid probe consisting of a nucleotide sequence having 100% identity to the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence complementary thereto and having at least 10 consecutive nucleotides.

[0033] In a specific embodiment, the labeled probe of the present invention may be, for example, a labeled probe consisting of a nucleotide sequence in which one or several nucleotides are substituted, deleted, or added in the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. Here, one or several means, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and still more preferably 1 to 2. By using a labeled probe having a nucleotide sequence highly homologous to the nucleotide sequence represented by SEQ ID NO: 1 or 2 in this way, it becomes possible to detect influenza virus with high sensitivity.

[0034] When having one or several nucleotide substitutions, deletions, or additions as described above, the probe is said to contain mismatched nucleotides, and in the present invention, a labeled probe containing such mismatched nucleotides can also be preferably used.

[0035] As used herein, "comprising a mismatched base" (or simply "mismatch") means containing a base that is not complementary to the base sequence of the target nucleic acid (when the target nucleic acid is double-stranded, the base sequence of either single-stranded nucleic acid after dissociation of the double strand). For example, when a cytosine base is present in the base sequence of the target nucleic acid, the base at the position corresponding to the cytosine base in the target probe is a base other than guanine (e.g., adenine base, cytosine base, guanine base, thymine base, and universal base). The mismatched bases in the first target probe and / or the second target probe may be any of adenine base, cytosine base, guanine base, thymine base, universal base (e.g., hypoxanthine, nebularine, 5-nitroindole, etc.), and those skilled in the art can appropriately select non-complementary bases to design probes. For example, when detecting a target nucleic acid that is prone to mutation, a universal base can be selected at the position of the target probe corresponding to the base that is prone to mutation.

[0036] When the labeled probe of the present invention contains a mismatched base, the position where the mismatch is introduced is not particularly limited as long as the effects of the present invention are not inhibited. From the perspective of more reliably facilitating the detection of the target nucleic acid, it is preferably not the terminal base of each probe. For example, when the target probe contains a mismatched base, the position of the mismatched base is preferably within 8 mers before and after the center of the entire base sequence constituting the target probe, and more preferably within 5 mers before and after the center of the entire length.

[0037] In one preferred embodiment, the labeled probe of the present invention is a labeled probe consisting of a base sequence of at least 10 consecutive bases or more in the base sequence shown from the 15th to the 49th of the base sequence shown in SEQ ID NO: 1 or a base sequence complementary to the base sequence, or a base sequence in which one or several bases are substituted, deleted, or added in those base sequences. By reacting such a labeled probe with a nucleic acid amplification product, it becomes possible to more favorably detect influenza A virus.

[0038] In another preferred embodiment, the labeled probe of the present invention is a labeled probe consisting of a base sequence of at least 10 consecutive bases or more in the base sequence shown at positions 22 to 40 of the base sequence shown in SEQ ID NO: 2 or a base sequence complementary thereto, or a base sequence in which one or several bases are substituted, deleted, or added in these base sequences. By reacting such a labeled probe with a nucleic acid amplification product, it becomes possible to detect influenza B virus better.

[0039] The length of the labeled probe is not particularly limited. For example, it can be 10 bases or more, preferably 14 bases or more, more preferably 14 to 26 bases, and even more preferably 14 to 20 bases. By using a labeled probe of such a length, it is possible to detect influenza virus with higher sensitivity.

[0040] In a specific preferred embodiment, specific examples of the labeled probe used in the present invention include a base sequence represented by any of SEQ ID NOs: 3 to 32 or a base sequence complementary thereto, or a labeled probe consisting of a base sequence in which one or several bases are substituted, deleted, or added in these base sequences. The labeled probe corresponding to the base sequence shown in SEQ ID NOs: 3 to 25 is useful for the detection of influenza A virus, and the labeled probe corresponding to the base sequence shown in SEQ ID NOs: 26 to 32 is useful for the detection of influenza B virus. By using a labeled probe having such a specific base sequence, it becomes possible to detect influenza virus with even higher sensitivity.

[0041] The above-mentioned labeled probe is characterized in that only one of the 5'-end or 3'-end is labeled. In one embodiment, the labeled probe of the present invention is preferably labeled so as to produce quenching or fluorescence when bound to a nucleic acid containing a base sequence having 90% or more identity with the base sequence complementary to the base sequence of the labeled probe, and more preferably labeled so as to produce quenching. There is no particular limitation on the labeling substance, but a fluorescent dye label using a fluorescent substance is more preferable.

[0042] As the fluorescent label, it may be either a fluorescent substance that generates fluorescence or a fluorescent substance that generates quenching by forming a complex by hybridizing with the target nucleic acid amplification product. Preferably, it is a fluorescent substance that generates quenching when hybridized with the target nucleic acid amplification product. Particularly preferably, it is a fluorescence quenching dye that quenches by interaction with guanine in hybridization with the target nucleic acid amplification product (for example, a fluorescence quenching dye that quenches by interaction with guanine). Specifically, at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives (for example, fluorescein isothiocyanate (FITC)), rhodamine and its derivatives (for example, 5-carboxyrhodamine 6G (GR6G), tetramethylrhodamine (TAMRA), carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), and BODIPY and its derivatives (for example, BODIPY-FL, BODIPY-FL / C3, BODIPY-FL / C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, BODIPY-665) is included, but not limited thereto.

[0043] More specifically, as the fluorescence quenching dye that quenches by interaction with guanine, for example, at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue) can be mentioned, and these fluorescence quenching dyes can be preferably used in the present invention.

[0044] In a specific preferred embodiment, a labeled probe in which the terminal base labeled with a fluorescence quenching dye is cytosine is more preferred. When such an oligonucleotide probe hybridizes to an amplification product, it can be quenched by forming a base pair and interacting with the guanine base in the amplification product. Therefore, the change in the fluorescence intensity of the reaction solution can be measured very simply.

[0045] In addition, when the probe hybridizes, even if the cytosine base of the probe and the guanine base in the amplification product do not form a base pair, fluorescence can be quenched if the distance between these bases is close. For example, the details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. That is, when the probe hybridizes, if the guanine base in the amplification product is present within a range of, for example, 1 to 3 bases with respect to the cytosine base of the probe (taking the base forming a base pair with the cytosine base as 1), quenching can occur.

[0046] Furthermore, in a specific embodiment, even a nucleic acid probe in which at least one terminal base labeled with a fluorescence quenching dye (that is, when both ends are labeled, at least one of the terminal bases) is not cytosine can measure the change in the fluorescence intensity of the reaction solution. For example, the details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. For example, when the probe hybridizes, if the guanine base in the amplification product is present within a range of, for example, 1 to 3 bases with respect to at least one terminal base labeled with a fluorescence quenching dye (taking the base forming a base pair with the terminal base as 1), quenching can occur.

[0047] In a specific preferred embodiment, in the method for detecting influenza virus of the present invention, two or more of the labeled probes of the present invention are used in combination in a detection reaction solution. Even when a plurality of the labeled probes of the present invention are used so as to coexist in the reaction solution in this way, problems such as dimer formation between the probes are less likely to occur, and not only can influenza virus be detected, but it is also possible to distinguish between influenza A virus and influenza B virus. For example, by combining two or more of the labeled probes of the present invention in this way and allowing them to coexist in the detection reaction solution, it may be possible to distinguish and detect influenza A virus and influenza B virus with sufficient sensitivity even from a biological sample that has not undergone a step of purifying nucleic acid in advance.

[0048] In one preferred embodiment, a labeled probe containing a base sequence having 85% or more identity with the base sequence represented by SEQ ID NO: 1 or a base sequence of at least 10 consecutive bases in a base sequence complementary thereto is used so as to coexist in a reaction solution with a labeled probe having the following characteristics (A-2) and (B-2): (A-2) containing a base sequence having 85% or more identity with the base sequence represented by SEQ ID NO: 2 or a base sequence of at least 10 consecutive bases in a base sequence complementary thereto; and (B-2) Only one of the 5'-end or 3'-end is labeled.

[0049] In another preferred embodiment, a labeled probe containing a base sequence having 85% or more identity with the base sequence represented by SEQ ID NO: 2 or a base sequence of at least 10 consecutive bases in a base sequence complementary thereto is used so as to coexist in a reaction solution with a labeled probe having the following characteristics (A-1) and (B-1): (A-1) containing a base sequence having 85% or more identity with the base sequence represented by SEQ ID NO: 1 or a base sequence of at least 10 consecutive bases in a base sequence complementary thereto; and (B-1) Only one of the 5'-end or 3'-end is labeled.

[0050] Accordingly, the present invention further provides an embodiment of a labeled probe set including at least two of the above-described labeled probes. The labeled probe set of the present invention can be used, for example, to coexist a plurality of labeled probes included in the set in a reaction solution for performing melting curve analysis. In one embodiment, the labeled probe set of the present invention is a labeled probe set used for detecting influenza virus that may be included in a sample, including at least the following first labeled probe and second labeled probe: (A-1) comprising a base sequence having 85% or more identity with the base sequence shown in SEQ ID NO: 1 or a base sequence of at least 10 consecutive bases in a base sequence complementary thereto; and (B-1) a first labeled probe characterized in that only one of the 5'-end or 3'-end is labeled; and (A-2) comprising a base sequence having 85% or more identity with the base sequence shown in SEQ ID NO: 2 or a base sequence of at least 10 consecutive bases in a base sequence complementary thereto; and (B-2) a second labeled probe characterized in that only one of the 5'-end or 3'-end is labeled. The base sequence, base length, fluorescent label, etc. of the labeled probe in the above-described labeled probe set (also referred to as "nucleic acid probe set" in this specification) are the same as those detailed in the above-described labeled probe. The labeled probe set of the present invention can be used in the same manner as the above-described labeled probe of the present invention.

[0051] In one preferred embodiment, in the above nucleic acid probe or nucleic acid probe set, the base sequence of at least 10 bases or more of (A-1) can be the base sequence shown in any of SEQ ID NOs: 3 to 25 or a base sequence complementary thereto. In another preferred embodiment, in the above nucleic acid probe or nucleic acid probe set, the base sequence of at least 10 bases or more of (A-2) can be the base sequence shown in any of SEQ ID NOs: 26 to 32 or a base sequence complementary thereto. In a particularly preferred embodiment, a nucleic acid probe or nucleic acid probe set having the base sequences described in the above (A-1) and (A-2) is used in combination.

[0052] [Step of detecting the complex] In the step of detecting the amplification product of the present invention, the mode of the step (3-2) of detecting the complex formed by hybridizing the nucleic acid amplification product and the labeled probe is not particularly limited, and it can be carried out by any method known in the art.

[0053] In one embodiment, the step (3) can be an embodiment including at least one of the following steps (3-a) to (3-c): (3-a) A step of hybridizing the nucleic acid probe to the amplification product that can be obtained and measuring the fluorescence intensity of the reaction solution to monitor the progress of the nucleic acid amplification reaction in real time. (3-b) After the completion of the step (2), a step of hybridizing the nucleic acid probe to the amplification product that can be obtained and measuring the fluorescence intensity of the reaction solution to monitor the progress of the nucleic acid amplification reaction at the endpoint. (3-c) After the completion of the step (2), a step of hybridizing the nucleic acid probe to the amplification product that can be obtained and measuring the temperature dependence of the fluorescence intensity of the reaction solution. According to the steps such as (3-a) to (3-c) above, it may be possible to simply and highly sensitively detect the formation of a complex formed by a nucleic acid amplification product and a nucleic acid probe. From the viewpoint of detecting a nucleic acid amplification product more rapidly, the detection step by (3-b) or (3-c) is preferred in the embodiments of the present invention. In particular, the method by (3-c), that is, the melting curve analysis method, is preferred.

[0054] [(3-a) Monitoring the progress of nucleic acid amplification reaction in real time] This is a method for monitoring the progress of a nucleic acid amplification reaction in real time. The so-called real-time PCR method enables quantitative analysis by comparing with a control substance of known concentration. On the other hand, since it is necessary to measure photometry for each cycle of PCR, the time required for the nucleic acid amplification reaction tends to be long.

[0055] [(3-b) Monitoring the progress of nucleic acid amplification reaction at the endpoint] Although there is also a method for monitoring the progress of a nucleic acid amplification reaction in real time, in the present invention, by monitoring the progress of the nucleic acid amplification reaction at the endpoint, the target nucleic acid contained in the sample can be rapidly detected. Furthermore, by comparing the fluorescence intensity or the like at the endpoint, an approximate estimation of the amount of the target nucleic acid is also possible. For example, by measuring the fluorescence intensity of a reaction solution containing an oligonucleotide probe labeled with a fluorescence quenching dye, the progress of the nucleic acid amplification reaction is monitored at the endpoint. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured. By comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of the reaction solution before the reaction, the presence or absence of amplification of the target nucleic acid can be confirmed. Alternatively, by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of the control reaction solution, the presence or absence of the target nucleic acid contained in the sample can also be confirmed. The control reaction solution is a reaction solution to which a sample determined to be negative or a sample determined to be positive is added instead of the sample to be measured. Generally, it is necessary to monitor the progress of the nucleic acid amplification reaction in real time, but for the purpose of making the amplification detection step more rapid and simple, it is preferable to measure at the endpoint.

[0056] [Measurement of Temperature Dependence of Fluorescence Intensity (3-c)] Measuring the temperature dependence of fluorescence intensity specifically means measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high. By performing a first-order differentiation of the obtained fluorescence intensity with respect to temperature, the melting temperature (Tm value) specific to the oligonucleotide probe used can be determined. Also, the fluorescence intensity may be converted into a fluorescence quenching rate or the like according to the purpose. Detection, analysis, etc. of a target nucleic acid using the Tm value is called melting curve analysis. Generally, the Tm value refers to the temperature at which the ratio of the oligonucleotide forming a double strand with its complementary strand and the ratio of being single-stranded without forming a double strand are equal. Since the Tm value is a value specific to the base sequence, melting curve analysis can be used as a method for analyzing the base sequence polymorphism of a target nucleic acid. The base sequence polymorphism referred to here includes single nucleotide polymorphism, base substitution, base deletion, base insertion, etc.

[0057] As an example, melting curve analysis is also applied to SNP analysis and the like. When there is a mutation in the base sequence of a target nucleic acid with respect to an oligonucleotide probe, since the bases are mismatched when the probe hybridizes, generally the Tm value becomes lower. Therefore, single nucleotide polymorphism analysis (SNP analysis) can also be performed by comparing the magnitudes of the Tm values.

[0058] [Specimen Sample] The specimen sample that can be used in the present invention is not particularly limited as long as it may contain influenza virus. For example, oral swabs, throat swab specimens, nasopharyngeal swab specimens, nasal swab specimens, nasal aspirates, sputum, bronchial washings, alveolar washings, saliva, etc. collected from a subject suspected of being infected with influenza virus can be mentioned, but are not limited thereto. When using a biological sample as the specimen sample to be measured, depending on each biological sample, although there is no particular limitation, pretreatment such as dilution, suspension, centrifugation, enzyme treatment, or nucleic acid extraction may be performed.

[0059] The methods for collecting and preparing a sample specimen are not particularly limited, and known methods can be used according to the type and purpose of the sample. In a specific preferred embodiment, the sample specimen used in the present invention does not have to be a sample from which RNA has been isolated and purified. For example, a specimen collected from a living body may be subjected to proteolytic enzyme (e.g., proteinase K) treatment and / or heat treatment (e.g., at 60 to 100 °C for 1 second to 10 minutes) for proteolytic denaturation treatment, and the sample in which RNase (ribonuclease) and DNase (DNA-degrading enzyme) present in the specimen have been decomposed and removed in advance may be used as it is.

[0060] The method for nucleic acid extraction is not particularly limited, and known methods can be used according to the type and purpose of the specimen. As for the nucleic acid, it may mainly extract and purify RNA, or may extract and purify nucleic acid without distinguishing between RNA and DNA. For nucleic acid extraction, for example, kits sold by each manufacturer may be used. For example, the QIAamp Viral RNA Mini Kit (QIAGEN) can be mentioned. Also, a manual method or an automatic extraction and purification device may be used.

[0061] In a specific preferred embodiment, the method of the present invention can omit the nucleic acid purification step that is usually considered essential in conventional nucleic acid amplification reactions. As shown in the results of the test examples described later, according to the method of the present invention, even in a sample specimen containing contaminants such as biological components other than the target nucleic acid (which can be inhibitors of the nucleic acid amplification reaction), influenza virus can be detected with high sensitivity. When performing nucleic acid purification, in addition to the need for dedicated reagents, there are problems such as complicated operations that are time-consuming and laborious. However, according to the gene amplification reaction of the present invention, it is not necessary to prepare dedicated equipment for such nucleic acid purification, and it is possible to omit complicated operations. Furthermore, since the time required for the centrifugation method can also be shortened, the time from the collection of a biological sample from a subject to obtaining a gene test result can be shortened. For example, the time from sample collection to obtaining a gene test result can be within 1 day, preferably within half a day, more preferably within 6 hours, still more preferably within 3 hours, and most preferably within 2 hours (for example, within 1 hour). Thus, the labeled probe and method of the present invention that can detect using a biological sample that has not undergone the step of purifying nucleic acids can save the trouble of nucleic acid purification and enable simple and rapid detection of influenza virus.

[0062] [Reagents for Detecting Influenza Virus] As another embodiment of the present invention, there is provided a reagent for detecting influenza virus that may be contained in a sample. The reagent includes at least components necessary for reverse transcription reaction (RT), nucleic acid amplification reaction, and detection, in addition to the nucleic acid probe or nucleic acid probe set of the present invention described above. As the necessary components, known ones can be used respectively. For example, it preferably includes at least an oligonucleotide primer for reverse transcription, a set of nucleic acid primers for PCR, reverse transcriptase, DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and inorganic salts such as magnesium salts. A set of nucleic acid primers for PCR that also serves as an oligonucleotide primer for reverse transcription and a nucleic acid probe for detection can include multiple sets for amplifying multiple regions of the influenza virus. The concentration of each component can be adjusted as appropriate. For example, the nucleic acid probe is preferably 0.01 to 1 μM, more preferably 0.02 to 0.5 μM. When used as a nucleic acid probe set, each nucleic acid probe included in the probe set is preferably within the above concentration range. The nucleic acid primer is preferably 0.01 to 10 μM. The reverse transcriptase is preferably 0.01 to 10 U / μL, more preferably 0.02 to 2 U / μL. The DNA polymerase is preferably 0.01 to 1 U / μL, more preferably 0.02 to 0.5 U / μL. The deoxyribonucleoside triphosphates (dNTPs) are preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. The inorganic salts such as magnesium salts are preferably 0.1 to 10 mM, more preferably 1 to 5 mM.

[0063] Furthermore, additives and the like known in the art may be added for the purpose of suppressing non-specific amplification or promoting the reaction. As additives for suppressing non-specific amplification, known anti-DNA polymerase antibodies, phosphoric acid, etc. may be used. As additives for promoting the reaction, bovine serum albumin (BSA), protease inhibitor, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, carnitine, Triton, Tween 20, Nonidet P40, etc. may be mentioned. In the present invention, an inhibitor or suppressant of ribonuclease may be added in order to reduce the degradation of the genomic RNA of the target influenza virus. For example, RNase inhibitor may be mentioned. In the present invention, these additives may be used in combination of one or more kinds, but are not limited thereto.

[0064] [Kit for Detecting Influenza Virus] As another embodiment of the present invention, there is provided a kit for detecting influenza virus that may be contained in a sample. The kit of the present invention includes the nucleic acid probe or nucleic acid probe set of the present invention described above, and / or a reagent containing the same, and is not particularly limited as long as it is configured to be able to detect and / or discriminate influenza virus. For example, the kit of the present invention may optionally include a gene test reagent capable of detecting or quantifying the presence of a subject to be detected and / or an instruction manual for explaining the method of use and the like. For example, the nucleic acid probe enclosed in the same container or in separate containers may be packaged in, for example, a single package and provided in a manner including information on the method of using the kit. Also, a positive control solution and a negative control solution may be included.

Example

[0065] Hereinafter, the present invention will be specifically described based on examples of the present invention. The present invention is not limited to the following examples.

[0066] 〔Example 1: Evaluation 1 of Nucleic Acid Probe〕 (1) Preparation of Sample RNA extracted from influenza A virus subtype H1 was serially diluted with sterilized water and prepared to be 100 copies / test to obtain a sample. (2) Reverse Transcription, Nucleic Acid Amplification, Melting Curve Analysis, and Determination Each of the above samples was added to the following reagents, and influenza A virus was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for reverse transcription, nucleic acid amplification, and melting curve analysis. Determination was made positive when a peak was obtained by melting curve analysis.

[0067] Reagent To evaluate the performance of each probe represented by SEQ ID NOs: 7-8, 11-20, and 23, the following solution was prepared using Gene Cube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). 0.5 μM primer represented by SEQ ID NO: 33 3.0 μM primer shown in SEQ ID NO: 35 0.25 μM each probe shown in SEQ ID NOs: 7 - 8, 11 - 20, 23 (SEQ ID NOs: 7 - 8, 16 - 20 are labeled with BODIPY - FL at the 3' end; SEQ ID NOs: 11 - 15, 23 are labeled with BODIPY - FL at the 5' end and phosphorylated at the 3' end)

[0068] Reverse transcription, nucleic acid amplification, and melting curve analysis 42 °C for 2 minutes 97 °C for 15 seconds (The above is 1 cycle) 97 °C for 1 second 58 °C for 3 seconds 63 °C for 5 seconds (The above is 50 cycles) 94 °C for 30 seconds 39 °C for 30 seconds 40 °C to 75 °C (temperature increase at 0.09 °C / second)

[0069] (3) Results Figure 1 is a detection graph obtained when detecting A - type influenza virus RNA by nucleic acid amplification and melting curve analysis using the nucleic acid probe shown in SEQ ID NO: 7. As shown in Figure 1, a detection peak is obtained at 58 °C with this probe. Similarly, the measurement results using the nucleic acid probes used in this example are summarized in Table 1. This study revealed that all of the above - mentioned probes are capable of detecting A - type influenza virus RNA.

[0070]

Table 1

[0071] 〔Example 2: Evaluation of Nucleic Acid Probe 2〕 (1) Preparation of sample RNA extracted from B - type influenza virus was serially diluted with sterile water and prepared to be 100 copies / test as a sample. (2) Reverse transcription, nucleic acid amplification, melting curve analysis, and determination In the same manner as in Example 1, B - type influenza virus was detected.

[0072] Reagent To evaluate the performance of each probe represented by SEQ ID NOs: 27, 29 to 32, the following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). 0.5 μM primer represented by SEQ ID NO: 41 3.0 μM primer represented by SEQ ID NO: 44 0.25 μM each probe represented by SEQ ID NOs: 27, 29 to 32 (SEQ ID NOs: 27, 29, 31, 32 are labeled with BODIPY-FL at the 3'-end; SEQ ID NO: 30 is labeled with BODIPY-FL at the 5'-end and phosphorylated at the 3'-end)

[0073] Reverse transcription, nucleic acid amplification, and melting curve analysis Performed under the same conditions as in Example 1.

[0074] (3) Results Figure 2 is a detection graph obtained when detecting influenza B virus RNA by nucleic acid amplification and melting curve analysis using the nucleic acid probe represented by SEQ ID NO: 27. As shown in Figure 2, a detection peak was obtained at 51.3 °C with this probe. Similarly, the measurement results using the nucleic acid probes used in this example are summarized in Table 2. This study showed that all of the probes represented by the above SEQ ID NOs can detect influenza B virus RNA.

[0075] [Table 2]

[0076] [Example 3: Examination of Nucleic Acid Probe Combinations 1] (1) Preparation of Samples RNA extracted from influenza A H1N1 virus was prepared to be 50 copies / test, and RNA extracted from influenza B virus was prepared to be 20 copies / test, respectively, and used as samples. (2) Reverse Transcription, Nucleic Acid Amplification, Melting Curve Analysis, and Determination In the same manner as in Examples 1 and 2, detection of influenza A and B viruses was performed.

[0077] Reagent To examine the discrimination between influenza A virus and influenza B virus by the combination of probes evaluated in Example 1 and Example 2, a solution combining the primers and probes described in Table 3 was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). In the following solution, the primer concentration was 0.5 μM for the AF primer and 3.0 μM for the AR primer. However, when two AF primers or two AR primers were used, the total concentration of each primer was set to the above concentration. Also, the BF primer concentration was 0.5 μM and the BR primer concentration was 3.0 μM. The probe concentration was 0.25 μM for all. The labeling of the probe was such that only one of the 3'-end or 5'-end was labeled in the same manner as in Example 1 and Example 2. [Table 3]

[0078] Reverse transcription, nucleic acid amplification, melting curve analysis, and determination The conditions for reverse transcription, nucleic acid amplification, and melting curve analysis were the same as those in Example 1. Melting curve analysis was performed, and based on the Tm value calculated by the nearest-neighbor base pair method from the base sequence of each labeled probe, when detection was observed at 58°C or higher, it was regarded as positive for influenza A virus, and when detection was observed at 54°C or lower, it was regarded as positive for influenza B virus.

[0079] (3) Results The following Table 4 is a summary of the results of this example. Nucleic acid detection of influenza A H1N1 virus RNA and influenza B virus RNA was observed using any of the solutions, and the results of the two were distinguishable. From this example, it was shown that it is possible to discriminate between influenza A virus and influenza B virus by using the set combining the probes of the present invention.

[0080] [Table 4]

[0081] [Example 4: Examination of Nucleic Acid Probe Combinations 2] (1) Preparation of Sample A sample is prepared by spiking nasopharyngeal swab fluid with RNA extracted from influenza A H1N1 virus at a concentration of 50 copies / test and RNA extracted from influenza B virus at a concentration of 20 copies / test. Since this sample does not undergo further nucleic acid isolation and purification treatment, it corresponds to a sample containing biological contaminants. (2) Reverse Transcription, Nucleic Acid Amplification, Melting Curve Analysis, and Determination In the same manner as in Example 3, influenza A and B viruses are detected.

[0082] Reagents To examine the discrimination between influenza A virus and influenza B virus using the probe combinations evaluated in Examples 1 and 2, a solution is prepared by combining the primers and probes described in Table 5 using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). In the following solution, the primer concentration of AF primer is 0.5 μM and that of AR primer is 3.0 μM. However, when two AF primers or two AR primers are used, the total concentration of each primer is the above concentration. Also, the BF primer concentration is 0.5 μM and the BR primer concentration is 3.0 μM. The probe concentration is 0.25 μM for all. The labeling of the probes is the same as in Examples 1 and 2. [Table 5]

[0083] Reverse Transcription, Nucleic Acid Amplification, Melting Curve Analysis, and Determination The conditions for reverse transcription, nucleic acid amplification, and melting curve analysis are the same as those in Example 1. Based on the Tm value calculated by the nearest-neighbor base pair method from the base sequences of each labeled probe, if detection is observed at 58°C or higher, it is determined as positive for influenza A virus, and if detection is observed at 54°C or lower, it is determined as positive for influenza B virus. As shown in the results of Examples 1 and 2, the labeled probes of the present invention can detect influenza A virus and influenza B virus with high sensitivity. From this example, it can be seen that influenza A virus and influenza B virus can be discriminated and detected even in a sample containing contaminants such as components derived from a living body.

Industrial Applicability

[0084] By using the nucleic acid probe of the present invention, influenza virus that may be contained in a sample can be detected simply and with high sensitivity. Therefore, for example, even for a sample obtained by simple RNA extraction in which contaminants have not been completely removed, it is possible to detect it quickly and reliably, and it is expected to make a great contribution to clinical diagnosis and the like. In addition, by combining the nucleic acid probes of the present invention, it is also possible to discriminate between influenza A virus and influenza B virus.

Claims

1. A labeled probe having the following features (A) and (B) and used for detecting influenza A virus or influenza B virus that may be contained in a specimen sample: (A) consisting of a base sequence represented by any of SEQ ID NOs: 7, 8, 11 to 19, 23, 27, 29 to 32; and (B) only one of the 5'-end or 3'-end is labeled.

2. The labeled probe according to Claim 1, wherein the label is a fluorescent dye label.

3. The labeled probe according to Claim 1 or 2, wherein the labeled probe is labeled with a fluorescence quenching dye that quenches when binding to a nucleic acid containing the same base sequence as the base sequence complementary to the base sequence of the labeled probe.

4. The labeled probe according to any one of Claims 1 to 3, which is labeled with a fluorescence quenching dye that quenches by interaction with guanine.

5. The labeled probe according to any one of Claims 1 to 4, wherein the label in (B) is a label with at least one fluorescence quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, carboxyrhodamine 6G, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid.

6. The labeled probe according to any one of Claims 1 to 5, wherein the terminal base that is labeled in (B) is cytosine.

7. The labeled probe according to any one of Claims 1 to 6 and used for detecting influenza virus that may be contained in a biological specimen sample that has not undergone a nucleic acid purification step.

8. A labeled probe set used for detecting influenza A virus and influenza B virus that may be contained in a specimen sample, comprising at least the following first labeled probe and second labeled probe: (A-1) consisting of a base sequence represented by any of SEQ ID NOs: 7, 8, 11 to 19, 23; and (B-1) a first labeled probe, characterized in that only one of the 5'-end or 3'-end is labeled; and (A-2) consisting of a base sequence represented by any of SEQ ID NOs: 27, 29 to 32; and A second labeled probe, characterized in that only one of the 5'-end and 3'-end of (B-2) is labeled.

9. A method for detecting influenza virus that may be contained in a sample using the labeled probe according to any one of Claims 1 to 7 or the labeled probe set according to Claim 8.

10. The method for detecting influenza virus according to Claim 9, wherein the detection is performed by RT-PCR-melting curve analysis.

11. The method according to Claim 10, wherein the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to Family B.

12. The method according to Claim 11, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD DNA polymerase or a mutant in which 1 to 2 amino acids thereof are deleted, substituted and / or added.

13. The method according to any one of Claims 10 to 12, wherein the enzyme having reverse transcription activity used in the RT reaction is a reverse transcriptase derived from M-MLV (Moloney Murine Leukemia Virus) or a mutant in which 1 to 2 amino acids thereof are deleted, substituted and / or added.

14. The method according to any one of Claims 10 to 13, wherein the sample is a biological sample that has not undergone a step of purifying nucleic acid.

15. A reagent kit for detecting influenza virus that may be contained in a sample, comprising the labeled probe according to any one of Claims 1 to 7 or the labeled probe set according to Claim 8.

16. The method according to any one of Claims 10 to 14, wherein it is possible to distinguish between influenza A virus and influenza B virus.

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