Oligonucleotide for detection of monkeypox virus and varicella-zoster virus and use thereof

Abstract

To provide an effective method or the like for detecting monkeypox virus and / or varicella-zoster virus.SOLUTION: A probe for detecting monkeypox virus is provided, comprising: (A) a base sequence A1 of at least 15 bases including a specific base sequence, or a base sequence A2 in which 1 to 3 bases in the base sequence A1 are substituted, deleted, inserted, or added; and (B) a label attached exclusively to either the 5' or 3' end; and a method is provided for the simultaneous detection of monkeypox virus and varicella-zoster virus possibly present in a sample in a single reaction using the probe.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oligonucleotide for detecting monkeypox virus (MPV) and varicella zoster virus (VZV) that may be contained in a sample, etc. Furthermore, the present invention relates to a method for detecting monkeypox virus and varicella zoster virus that may be contained in a sample (particularly for simultaneously detecting them in one reaction) using the oligonucleotide, and a reagent and a kit for use in the method, etc. [Background technology]

[0002] The monkeypox virus is transmitted through contact with skin lesions, bodily fluids, or blood (including sexual contact) of infected people or animals. In the 2022 epidemic, mainly in Europe and the United States, more than 52,000 cases of infection have been reported, including deaths, and rapid response is required for monkeypox virus testing. Infections have also been confirmed in Japan, so it is necessary to keep a close eye on the future epidemic situation.

[0003] Furthermore, the varicella-zoster virus is an example of a virus whose clinical symptoms are very similar to those of the monkeypox virus. In Japan, following the outbreak of monkeypox virus infections, the National Institute of Infectious Diseases published a pathogen detection manual for the monkeypox virus in June 2022. The pathogen detection manual for the monkeypox virus published by the National Institute of Infectious Diseases also discloses a real-time PCR method using a double-labeled nucleic acid probe (also called a Taqman probe, hydrolysis probe, etc.) for the simultaneous detection of the monkeypox virus and the varicella-zoster virus. (Non-Patent Document 1) [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] National Institute of Infectious Diseases, Pathogen Detection Manual Monkeypox Virus, 1st Edition, released in June 2022 Summary of the Invention [Problem to be solved by the invention]

[0005] The real-time PCR method described in Non-Patent Document 1 allows for quantitative detection, but requires photometry for each PCR cycle, and therefore requires about one hour for detection even in the shortest case.

[0006] Melting curve analysis is known as a method for detecting nucleic acid amplification products. In melting curve analysis, nucleic acid amplification and detection can be performed in separate steps, and measurements can be performed relatively easily in as little as 30 minutes. Furthermore, melting curve analysis using fluorescently labeled nucleic acid probes has the advantage of being easily adapted to genetic testing using automated analyzers. However, there are no known nucleic acid probes that can simultaneously detect monkeypox virus and varicella-zoster virus in a single reaction with high sensitivity using melting curve analysis.

[0007] One object of the present invention is to provide a useful method for detecting monkeypox virus and / or varicella-zoster virus that may be contained in a sample. [Means for solving the problem]

[0008] As a result of intensive research conducted by the present inventors to achieve the above object, they discovered a method for detecting monkeypox virus and varicella-zoster virus (particularly simultaneously in one reaction) easily and with high sensitivity by using a specific nucleic acid probe. Based on this finding, they conducted further investigations and completed the present invention.

[0009] The present invention includes the following aspects. [Item 1] A probe for detecting monkeypox virus, having the following characteristics (A) and (B): (A) It has a base sequence A1 of at least 15 consecutive bases in the base sequence of positions 65 to 100 of SEQ ID NO: 1 or its complementary base sequence, or a base sequence A2 in which 1 to 3 bases have been substituted, deleted, inserted or added in base sequence A1. (B) Only either the 5' or 3' end is labeled. [Item 2] The probe according to Item 1, wherein the length of the base sequence of (A) is 15 to 20 bases. [Item 3] The probe according to Item 1 or 2, wherein the base sequence of (A) comprises a base sequence shown in any one of SEQ ID NOs: 2 to 7 or a complementary base sequence thereto. [Item 4] The probe according to any one of Items 1 to 3, wherein the label (B) is a fluorescent dye label. [Item 5] The probe according to any one of Items 1 to 4, wherein the label (B) is a label with a fluorescent quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe. [Item 6] The probe according to any one of Items 1 to 5, wherein the label (B) is a label with a fluorescence quenching dye that is quenched by interaction with guanine. [Item 7] The probe according to any one of Items 1 to 6, wherein the label (B) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof. [Item 8] The probe according to any one of Items 1 to 7, wherein the label (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 9] The probe according to any one of Items 1 to 8, wherein in (B), the labeled terminal base is cytosine. [Item 10] A probe for detecting varicella-zoster virus, having the following characteristics (C) and (D): (C) has a base sequence C1 of at least 14 consecutive bases in the base sequence from positions 95 to 125 of SEQ ID NO: 8 or its complementary base sequence, or a base sequence C2 in which 1 to 3 bases have been substituted, deleted, inserted or added in base sequence C1. (D) Only either the 5' or 3' end is labeled. [Item 11] The probe according to Item 10, wherein the length of the base sequence of (C) is 14 to 20 bases. [Item 12] The probe according to item 10 or 11, wherein the base sequence of (C) comprises a base sequence shown in any one of SEQ ID NOs: 9 to 13 or a complementary base sequence thereto. [Item 13] The probe according to any one of Items 10 to 12, wherein the label (D) is a fluorescent dye label. [Item 14] The probe according to any one of Items 10 to 13, wherein the label (D) is a label with a fluorescent quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe. [Item 15] The probe according to any one of Items 10 to 14, wherein the label (D) is a label with a fluorescence quenching dye that is quenched by interaction with guanine. [Item 16] The probe according to any one of Items 10 to 15, wherein the label (D) is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof. [Item 17] The probe according to any one of Items 10 to 16, wherein the label (D) 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 18] The probe according to any one of Items 10 to 17, wherein in (D), the labeled terminal base is cytosine. [Item 19] A method for simultaneously detecting monkeypox virus and varicella-zoster virus that may be contained in a sample in a single reaction, using the probe according to any one of items 1 to 9 and the probe according to any one of items 10 to 18. [Item 20] The following steps (1), (2), and (3): (1) providing a sample that may contain monkeypox virus and varicella-zoster virus; (2) carrying out a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more probes according to any one of items 1 to 9 and one or more probes according to any one of items 10 to 18; 20. The method according to claim 19, comprising: [Item 21] The method according to Item 20, wherein the step (2) is carried out by a PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to Family B. [Item 22] The method according to Item 21, wherein the DNA polymerase belonging to Family B is a KOD-derived DNA polymerase or a mutant thereof. [Item 23] The method according to any one of Items 20 to 22, wherein the primer set for detecting monkeypox virus used in the nucleic acid amplification reaction in the step (2) comprises a first primer having a base sequence S1 of at least 18 consecutive bases in the base sequence of positions 1 to 70 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S1, and a second primer having a base sequence S3 of at least 20 consecutive bases in the base sequence of positions 95 to 150 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S4 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S3, wherein the second primer is complementary to a DNA extension product of the first primer. [Item 24] The method according to Item 23, wherein the first primer has a base sequence shown in any one of SEQ ID NOs: 14 to 17, a complementary base sequence thereof, or these base sequences in which 1 to 3 bases have been substituted, deleted, inserted or added, and the second primer has a base sequence shown in any one of SEQ ID NOs: 18 to 20, a complementary base sequence thereof, or these base sequences in which 1 to 3 bases have been substituted, deleted, inserted or added. [Item 25] The method according to any one of Items 20 to 24, wherein the primer set for detecting Varicella-Zoster virus used in the nucleic acid amplification reaction in the step (2) comprises a first primer having a base sequence S5 of at least 19 consecutive bases in the base sequence of positions 1 to 100 of SEQ ID NO: 8 or a complementary base sequence thereto, or a base sequence S6 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S5, and a second primer having a base sequence S7 of at least 18 consecutive bases in the base sequence of positions 120 to 175 of SEQ ID NO: 8 or a complementary base sequence thereto, or a base sequence S8 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S7, wherein the second primer is complementary to a DNA extension product of the first primer. [Item 26] The method according to Item 25, wherein the first primer has a base sequence shown in any one of SEQ ID NOs: 21 to 23, a complementary base sequence thereof, or these base sequences in which 1 to 3 bases have been substituted, deleted, inserted or added, and the second primer has a base sequence shown in any one of SEQ ID NOs: 24 to 26, a complementary base sequence thereof, or these base sequences in which 1 to 3 bases have been substituted, deleted, inserted or added. [Item 27] ​​The method according to any one of Items 20 to 26, wherein the detection step in step (3) is carried out by melting curve analysis. [Item 28] A kit for simultaneously detecting monkeypox virus and varicella-zoster virus in one reaction, comprising the probe according to any one of items 1 to 9 and the probe according to any one of items 10 to 18. [Item 29] A kit for simultaneously detecting monkeypox virus and varicella-zoster virus in one reaction, comprising the primer set according to item 23 or 24 and the primer set according to item 25 or 26. Effect of the Invention

[0010] The present invention can provide a useful method for detecting monkeypox virus and varicella-zoster virus that may be contained in a sample. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows a representative example of the results of Test Example 1 (a graph showing the detection results when melting curve analysis was carried out using the probe shown in SEQ ID NO: 7). [Diagram 2] FIG. 1 shows a representative example of the results of Test Example 2 (a graph showing the detection results when melting curve analysis was carried out using the probe shown in SEQ ID NO: 11). [Diagram 3] FIG. 1 shows a representative example of the results of Test Example 5 (a graph showing the detection results when melting curve analysis was performed using primers represented by the combinations of SEQ ID NOs: 15, 18, 21, and 24). [Figure 4] FIG. 1 shows a representative example of the results of Test Example 6 (a graph showing the detection results when melting curve analysis was performed using primers represented by the combinations of SEQ ID NOs: 15, 18, 21, and 24). [Diagram 5] 1 is a schematic diagram of primer and probe designs. [Figure 6] 1 is a schematic diagram of primer and probe designs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in further detail below with reference to embodiments of the present invention, but the present invention is not limited thereto. All non-patent and patent documents described in this specification are incorporated herein by reference in their entirety. In addition, "~" in this specification means "more than or equal to, less than or equal to", for example, if "X~Y" is described in the specification, it means "more than or equal to X, less than or equal to Y". In this specification, "and / or" means any one or any possible combination of two or more of the listed elements. In this specification, "comprising" includes the concepts of "consisting essentially of" and "consisting only of".

[0013] In addition, in this specification, a nucleic acid primer may be simply referred to as a primer, and a nucleic acid probe and a labeled probe may be simply referred to as a probe, and these are also collectively referred to as oligonucleotides.

[0014] In one embodiment, the present invention provides a method for detecting monkeypox virus and / or varicella-zoster virus using a labeled probe containing a specific base sequence. The method can simultaneously detect monkeypox virus and varicella-zoster virus in one reaction with high sensitivity in a short time. In one embodiment, the present invention can detect monkeypox virus and varicella-zoster virus with high sensitivity in, for example, melting curve analysis by designing a labeled probe targeting a specific base sequence region derived from monkeypox virus and varicella-zoster virus. In this specification, SEQ ID NO: 1 is a base sequence derived from monkeypox virus. Specifically, SEQ ID NO: 1 is a part of the base sequence of the F3L gene of monkeypox virus. SEQ ID NO: 8 is a base sequence derived from varicella-zoster virus. Specifically, SEQ ID NO: 8 is a part of the base sequence of the ORF38 gene of varicella-zoster virus. The present invention encompasses labeled probes (also referred to herein as "nucleic acid probes") that contain specific base sequences that target the regions of the base sequences shown in SEQ ID NO:1 or 8, respectively.

[0015] One embodiment of the present invention is a method for detecting monkeypox virus and varicella-zoster virus that may be contained in a sample (particularly, for simultaneous detection in one reaction). The method of the present invention uses a labeled probe having a specific base sequence described below. In a specific embodiment, by using one specific labeled probe in a reaction solution, monkeypox virus and varicella-zoster virus that may be contained in a sample can be simultaneously detected with high sensitivity in one reaction, for example, in PCR-melting curve analysis (including RT-PCR-melting curve analysis) or the like.

[0016] In a particular embodiment, the method of the present invention comprises at least the following steps (A) and (B): (A) performing a nucleic acid amplification reaction using a nucleic acid primer set consisting of a plurality of nucleic acid primers containing a specific base sequence, using a specific region present in a base sequence derived from monkeypox virus and / or varicella-zoster virus as a template, to generate one or more nucleic acid amplification products; and (B) detecting one or more amplification products obtained in step (A) using one or more labeled probes of the present invention consisting of specific base sequences; Preferably, the method is a PCR-melting curve analysis method in which a PCR reaction is carried out in step (A) and a melting curve analysis is carried out in step (B). Steps (A) and (B) may be carried out in the same reaction solution. Steps (A) and (B) may also be carried out consecutively or simultaneously.

[0017] In a specific embodiment, a method for simultaneously detecting monkeypox virus and varicella-zoster virus that may be contained in a sample in one reaction includes at least the following steps (1), (2), and (3): (1) providing a sample that may contain monkeypox virus and varicella-zoster virus; (2) carrying out a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products of step (2) using one or more labeled probes for detecting monkeypox virus of the present invention and one or more labeled probes for detecting varicella-zoster virus of the present invention; It is preferable that the method comprises carrying out step (2) by a PCR reaction and carrying out step (3) by melting curve analysis (PCR-melting curve analysis method). Steps (2) and (3) may be carried out in the same reaction solution. Moreover, steps (2) and (3) may be carried out consecutively or simultaneously.

[0018] [Process (1)] In one embodiment, in step (1), it is preferable to prepare a sample that may contain monkeypox virus and varicella-zoster virus (for example, biological samples such as animal and plant tissues, body fluids, excrement, cells, etc.; environmental samples such as wipes of facility walls, floors, equipment and fixtures, toilets, etc., or cleaning fluids used to clean them, etc.). Samples that can be used in the present invention are not particularly limited as long as they may contain monkeypox virus and varicella-zoster virus. For example, blood, blood culture fluid, cervical scrapings, urethral scrapings, male urethral scrapings, urine, pus, cerebrospinal fluid, pleural effusion, pharyngeal swabs, nasal swabs, sputum, saliva, oral scrapings, tissue sections, skin, vomit, feces, isolated culture colonies, catheter washings, etc. collected from subjects suspected of infection with monkeypox virus and / or varicella-zoster virus may be included, but are not limited to these. Considering that the main symptom of monkeypox virus and varicella-zoster virus is a systemic rash, the sample to be used is preferably, for example, a tissue section, skin, blood, isolated culture colony, blister fluid, pustule fluid, blister cap, pustule cap, or throat swab, and more preferably, a swab of a lesion. When a biological sample is used as the measurement target, although there are no particular limitations depending on each biological sample, pretreatment such as dilution or suspension, centrifugation, enzyme treatment, filtration, heat treatment, acid treatment, alkali treatment, organic solvent treatment, crushing treatment, grinding treatment, etc., or nucleic acid extraction may or may not be performed.

[0019] The method of collecting and preparing the sample is not particularly limited, and known methods can be used depending on the type of sample, purpose, etc. In a specific preferred embodiment, the sample does not need to be a sample from which DNA has been isolated or purified, and for example, a specimen collected from a living body may be subjected to proteolytic denaturation treatment using a protease (e.g., proteinase K) and / or heat treatment (e.g., at 60 to 100°C for 1 second to 10 minutes) to previously decompose and remove DNase (DNA decomposition enzyme) present in the specimen, and the resulting sample may be used as is.

[0020] The method for extracting nucleic acid is not particularly limited, and known methods can be used depending on the type of sample, purpose, etc. For nucleic acid extraction, for example, a kit sold by each manufacturer may be used, or an automatic nucleic acid extraction and purification device may be used.

[0021] [Process (2)] In one embodiment, step (2) is preferably a step of generating one or more nucleic acid amplification products by a nucleic acid amplification method (carrying out a nucleic acid amplification reaction using one or more nucleic acid primer sets). The nucleic acid amplification method is a technique for amplifying several copies of a target nucleic acid to a level at which it can be visualized, i.e., to hundreds of millions of copies or more, and is 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. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, TRC, and TMA. These techniques have already been established in the technical field, and a method can be selected according to the purpose. The nucleic acid amplification method is preferably, but is not limited to, the PCR method (including the RT-PCR method).

[0022] (PCR reaction) PCR reaction is a reaction catalyzed mainly by DNA polymerase. PCR reaction usually includes three steps, namely (i) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (ii) annealing of a primer to a template single-stranded DNA, and (iii) extension of the primer using DNA polymerase, as one cycle, and the cycle is repeated. Examples of DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof. In the present invention, it is preferable to use a DNA polymerase belonging to family B from the viewpoints of simplicity, speed, high sensitivity, and resistance to amplification inhibition by a sample. In addition, when step (3) is performed by melting curve analysis, it is preferable to use a DNA polymerase belonging to family B that does not have 5'→3' exonuclease activity from the viewpoint of using a fluorescence quenching probe.

[0023] The conditions of the PCR reaction are not particularly limited as long as the reaction proceeds. For example, the first step (i) may be performed at 80 to 100°C for about 0 to 300 seconds (for example, about 0.5 to 300 seconds), the second or subsequent (repeated) step (i) may be performed at 80 to 100°C for about 0.5 to 300 seconds, step (ii) may be performed at 35 to 80°C for about 1 to 300 seconds, and step (iii) may be performed at 35 to 85°C for about 1 to 300 seconds. It is preferable that the cycle from step (i) to (iii) is repeated 30 to 70 times. The temperature and time of the repeated cycle may be changed every 1 to 3 cycles.

[0024] (DNA polymerase) The DNA polymerase that can be used in step (2) is preferably, but is not limited to, a DNA polymerase belonging to family B. The DNA polymerase belonging to family B is not particularly limited, but is preferably a DNA polymerase derived from Archea, and more preferably, includes DNA polymerases derived from bacteria of the genus Pyrococcus and Thermococcus. In addition, suitable DNA polymerases also include mutants thereof that do not lose DNA polymerase activity derived from Archea belonging to family B. Examples of the mutants include mutants in which 1 to 3 amino acids (for example, 1 or 2, or 1) are deleted, substituted, inserted, and / or added in the wild-type amino acid sequence, or mutants that show 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more amino acid sequence identity with the wild-type amino acid sequence. Specifically, examples of DNA polymerase mutants include, but are not limited to, mutants aimed at enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, and the like.

[0025] Examples of DNA polymerases 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, and Pyrococcus horikoshii, as well as mutants derived therefrom that have not lost their DNA polymerase activity.

[0026] 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. 9 degrees North-7 (Thermococcus sp. 9°N-7), and Thermococcus siculi, and mutants thereof that have not lost their DNA polymerase activity. Preferably, DNA polymerases derived from Thermococcus kodakaraensis and mutants thereof (e.g., KOD-derived DNA polymerase lacking 3'→5' exonuclease activity) can be particularly preferably used in the present invention from the viewpoint of excellent extensibility and thermostability.

[0027] 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), and the like, any of which can be used in the present invention.

[0028] (DNA polymerase derived from KOD) In the present specification, KOD-derived DNA polymerase (also referred to as KOD DNA polymerase) refers to Thermococcus kodakaraensis-derived DNA polymerase and mutants thereof (e.g., KOD-derived DNA polymerase in which 3'→5' exonuclease activity has been deleted by substituting, deleting, inserting and / or adding 1 to 3 (e.g., 1 or 2, or 1) amino acids in the naturally occurring amino acid sequence). In one preferred embodiment, step (2) involves performing a nucleic acid amplification reaction using such a KOD-derived DNA polymerase. Compared to Taq DNA polymerase, which is a DNA polymerase belonging to family A, KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility and resistance to amplification inhibition by sample-derived inhibitors. In the present invention, the use of such a KOD DNA polymerase is more preferable in terms of simultaneously detecting monkeypox virus and varicella-zoster virus in a simple, rapid and highly sensitive manner, as shown in the Examples described later.

[0029] (Nucleic acid primer set) The nucleic acid primer set that can be used in step (2) is not particularly limited as long as it can amplify a nucleic acid fragment derived from monkeypox virus or varicella-zoster virus with which a probe described below can form a complex. From the viewpoint of easily obtaining a highly sensitive determination result, the nucleic acid primer set is preferably a nucleic acid primer set that can amplify at least the nucleotide sequence shown in SEQ ID NO: 1 for amplifying monkeypox virus, and is preferably a nucleic acid primer set that can amplify at least the nucleotide sequence shown in SEQ ID NO: 8 for amplifying varicella-zoster virus.

[0030] For example, the nucleic acid primer set for amplifying monkeypox virus preferably includes a first primer having a base sequence S1 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of positions 25 to 70 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S2 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added in the base sequence S1, and a second primer having a base sequence S3 of at least 20 consecutive bases (e.g., 20 to 35 bases) in the base sequence of positions 95 to 145 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S4 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added in the base sequence S3, wherein the second primer is complementary to a DNA extension product of the first primer.

[0031] More preferably, the nucleic acid primer set includes a first primer having a base sequence shown in any one of SEQ ID NOs: 14 to 17, a complementary base sequence thereof, or a base sequence in which one to three (e.g., one or two, or one) bases have been substituted, deleted, inserted or added, and a second primer having a base sequence shown in any one of SEQ ID NOs: 18 to 20, a complementary base sequence thereof, or a base sequence in which one to three (e.g., one or two, or one) bases have been substituted, deleted, inserted or added, wherein one primer is complementary to a DNA extension product of the other primer. From the viewpoint of facilitating highly sensitive detection with a high rate of fluorescence change even when detecting small amounts of monkeypox virus (e.g., about 1 to 20 copies), it is particularly preferred that the nucleic acid primer set that can be used in the present invention is a nucleic acid primer set that includes a first primer having a base sequence shown in either SEQ ID NO: 14 or 15, a complementary base sequence thereof, or a base sequence in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added, and a primer having a base sequence shown in SEQ ID NO: 18, a complementary base sequence thereof, or a base sequence in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added, wherein one primer is complementary to the DNA extension product of the other primer.

[0032] Furthermore, the nucleic acid primer set for amplifying varicella-zoster virus preferably comprises a first primer having a base sequence S5 of at least 19 consecutive bases (e.g., 19 to 35 bases) in the base sequence of positions 30 to 100 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added in the base sequence S5, and a second primer having a base sequence S7 of at least 18 consecutive bases (e.g., 18 to 35 bases) in the base sequence of positions 120 to 180 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added in the base sequence S7, wherein the second primer is complementary to a DNA extension product of the first primer.

[0033] More preferably, the nucleic acid primer set includes a first primer having a base sequence shown in any one of SEQ ID NOs: 21 to 23, a complementary base sequence thereof, or a base sequence in which one to three (e.g., one or two, or one) bases have been substituted, deleted, inserted or added, and a second primer having a base sequence shown in any one of SEQ ID NOs: 24 to 26, a complementary base sequence thereof, or a base sequence in which one to three (e.g., one or two, or one) bases have been substituted, deleted, inserted or added, wherein one primer is complementary to a DNA extension product of the other primer. From the viewpoint of facilitating highly sensitive detection with a high rate of fluorescence change even when detecting a small amount (e.g., about 1 to 20 copies) of Varicella-Zoster virus, it is particularly preferred that the nucleic acid primer set that can be used in the present invention is a nucleic acid primer set that includes a first primer having the nucleotide sequence shown in SEQ ID NO: 21 or 22, a complementary nucleotide sequence thereof, or said nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted or added, and a primer having the nucleotide sequence shown in SEQ ID NO: 24 or 26, a complementary nucleotide sequence thereof, or said nucleotide sequence in which 1 to 3 (e.g., 1 or 2, or 1) nucleotides have been substituted, deleted, inserted or added, wherein one primer is complementary to the DNA extension product of the other primer.

[0034] [Process (3)] The embodiment of step (3) is not particularly limited, and can be carried out by any method known in the art. The monkeypox virus and varicella-zoster virus to be detected can mutate like other infectious microorganisms. Here, if a mismatch occurs between the base sequence of the primer or probe and the base sequence of the target monkeypox virus or varicella-zoster virus mutant, the binding strength of the primer or probe to the target gene or the nucleic acid amplification product derived therefrom (target nucleic acid) can be reduced. In particular, when there are many mismatches between the target nucleic acid and the probe in the real-time PCR method, the probe cannot sufficiently bind to the target nucleic acid, so that the rise of the amplification curve may be delayed or may not rise at all, which may induce a false negative. In the case of the melting curve analysis method, since step (3) is performed after the completion of PCR, even if there is a mismatch in the primer or probe, detection is possible as long as the nucleic acid amplification product is finally obtained, so that the effect of mismatch can be suppressed more than in the real-time PCR method. Therefore, in step (3), it is particularly preferable to detect the nucleic acid amplification product by melting curve analysis. Furthermore, by detecting the nucleic acid amplification products using melting curve analysis, it is possible to detect them in a shorter time (for example, 45 minutes or less, preferably 40 minutes or less, and more preferably 35 minutes or less from the start of the nucleic acid amplification reaction to the completion of melting curve analysis).

[0035] In one embodiment, step (3) comprises the following steps (3-1) and (3-2): (3-1) a step of hybridizing one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of the step (2) with one or more nucleic acid probes of the present invention to form a complex; and (3-2) detecting the complex of step (3-1) In order to obtain highly sensitive determination results in the detection of monkeypox virus and varicella-zoster virus, it is preferable to use a nucleic acid probe described below that can specifically react with a nucleic acid amplification product generated in the nucleic acid amplification reaction in step (2) to form a complex. The hybridization in step (3-1) is preferably carried out under temperature conditions that allow sufficient hybridization of the nucleic acid amplification product with the nucleic acid probe, such as, but not limited to, a temperature at least 5°C lower than the Tm value of the nucleic acid probe, more preferably at least 10°C lower.

[0036] (Nucleic acid probe) The nucleic acid probe of the present invention is not particularly limited as long as it has the characteristics of (A) and (B), or (C) and (D). However, in the case of a nucleic acid probe labeled with a fluorescent quenching dye that is quenched by interaction with guanine, as described below, it is preferable that at least one terminal base labeled with the dye is cytosine.

[0037] In one embodiment, the nucleic acid probe of the present invention can detect monkeypox virus (including monkeypox virus mutants) having a nucleotide sequence that shows 85% or more identity to the nucleotide sequence of bases 65 to 100 of SEQ ID NO: 1, or varicella-zoster virus (including varicella-zoster virus mutants) having a nucleotide sequence that shows 85% or more identity to the nucleotide sequence of bases 65 to 100 of SEQ ID NO: 8, and is preferably a nucleic acid probe that can detect monkeypox virus (including monkeypox virus mutants) having a nucleotide sequence that shows 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity to the nucleotide sequence of bases 65 to 100 of SEQ ID NO: 1, or varicella-zoster virus (including varicella-zoster virus mutants) having a nucleotide sequence that shows 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity to the nucleotide sequence of bases 65 to 100 of SEQ ID NO: 8.

[0038] In a specific embodiment, the nucleic acid probe of the present invention may be a probe containing a base sequence in which 1 to 3 bases are substituted, deleted, inserted or added in the base sequence shown in (A) or (C). The number of bases that may be substituted, deleted, inserted or added may preferably be 1 or 2. When a base is substituted, deleted, inserted or added in this way, the probe is said to contain a mismatched base, and it has been confirmed in the examples described below that a probe containing such a mismatched base can also be suitably used in the present invention.

[0039] In this specification, containing a mismatched base (or simply referred to as "mismatch") means containing a base that is not complementary to the base sequence of the target nucleic acid (when the target nucleic acid becomes double-stranded after the nucleic acid amplification reaction, the base sequence of any single-stranded nucleic acid resulting from 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 probe is a base other than guanine (e.g., an adenine base, a cytosine base, a thymine base, and a universal base). For example, when detecting a region of the target nucleic acid that is prone to mutation, a mismatched base (e.g., a universal base) can be selected at the position of the probe that corresponds to the base that is prone to mutation.

[0040] When the nucleic acid probe of the present invention contains a mismatched base, the position of the mismatched base is not particularly limited as long as it does not inhibit the effect of the present invention. From the viewpoint of more reliable detection of the nucleic acid amplification product, it is preferable that the mismatched base is not the terminal base of each probe. For example, the position of the mismatched base is preferably within 5 mers around the center of the total length n of the base sequence constituting the probe ((n+1) / 2 when n is an odd number, n / 2 when n is an even number), more preferably within 4 mers around, even more preferably within 3 mers around, even more preferably within 2 mers around, and particularly preferably within 1 mer around.

[0041] From the viewpoint of enabling more sensitive detection, the nucleic acid probe of the present invention is preferably a fluorescently labeled probe having a base sequence shown in any one of SEQ ID NOs: 2 to 7 or a complementary base sequence thereto, and / or a fluorescently labeled probe having a base sequence shown in any one of SEQ ID NOs: 9 to 13 or a complementary base sequence thereto. Furthermore, from the viewpoint of enabling higher detection in a synergistic manner together with a fluorescently labeled probe having a base sequence shown in any one of SEQ ID NOs: 11 to 13 or a complementary base sequence thereto, a fluorescently labeled probe having a base sequence shown in any one of SEQ ID NOs: 3, 4, 6, and 7 or a complementary base sequence thereto is preferred, from the viewpoint of enabling higher detection in a synergistic manner together with a fluorescently labeled probe having a base sequence shown in SEQ ID NO: 11 or 12 or a complementary base sequence thereto, a fluorescently labeled probe having a base sequence shown in SEQ ID NO: 6 or 7 or a complementary base sequence thereto is more preferred, and from the viewpoint of enabling higher detection in a synergistic manner together with a fluorescently labeled probe having a base sequence shown in SEQ ID NO: 11 or a complementary base sequence thereto, a fluorescently labeled probe having a base sequence shown in SEQ ID NO: 7 or a complementary base sequence thereto is preferred.

[0042] The length of the nucleic acid probe of the present invention is not particularly limited as long as it is 14 bases or more, and is, for example, 15 bases or more, preferably 16 bases or more, and usually 25 bases or less, preferably 20 bases or less, and more preferably 19 bases or less. The length of the probe may be more preferably 14 to 25 bases, and more preferably 14 to 20 bases. In a specific embodiment, the length of the nucleic acid probe of the present invention may be 14 to 17 bases, 14 to 16 bases, or 15 to 20 bases. By using a probe of such a length, monkeypox virus and / or varicella-zoster virus can be detected with higher sensitivity.

[0043] In a specific preferred embodiment, specific examples of the nucleic acid probe of the present invention include a probe containing a base sequence shown in any one of SEQ ID NOs: 4 to 7, a complementary base sequence thereto, or a base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in said base sequence. By using a probe having such a specific base sequence, monkeypox virus and / or varicella-zoster virus can be detected with even higher sensitivity.

[0044] The above-mentioned probe is preferably labeled at only one of the 5'-end or 3'-end. In one embodiment, the nucleic acid probe of the present invention is preferably labeled to generate quenching or fluorescence when bound to a nucleic acid containing a base sequence that shows 85% or more, preferably 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity with a base sequence complementary to the base sequence of the nucleic acid probe, and more preferably labeled to generate quenching. The labeling substance is not particularly limited, but is more preferably a fluorescent dye.

[0045] The fluorescent dye may be either a fluorescent substance that generates fluorescence or a fluorescent substance that quenches fluorescence by hybridizing with a target nucleic acid amplification product to form a complex, but is preferably a fluorescent substance that quenches fluorescence when hybridized with a target nucleic acid amplification product, and is particularly preferably a fluorescence quenching dye that is quenched by interaction with guanine when hybridized with a target nucleic acid amplification product (for example, a fluorescence quenching dye that is quenched by interaction with guanine). Specific examples of the fluorescent quenching dye include, but are not limited to, at least one fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives (e.g., fluorescein isothiocyanate (FITC)), rhodamine and its derivatives (e.g., 5-carboxyrhodamine 6G (GR6G), tetramethylrhodamine (TAMRA), carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), and BODIPY and its derivatives (e.g., 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).

[0046] More specifically, examples of the fluorescence quenching dye that is quenched by interaction with guanine include 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 (CR6G), TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue), and these fluorescence quenching dyes can be suitably used in the present invention.

[0047] In a particular preferred embodiment, a probe in which the terminal base labeled with a fluorescence quenching dye is cytosine is more preferred. When such a probe hybridizes to a nucleic acid amplification product, it can form a base pair with a guanine base in the nucleic acid amplification product and interact with it to quench the fluorescence, making it very easy to measure the change in the fluorescence intensity of the reaction solution.

[0048] In addition, when the probe is hybridized, even if the cytosine base of the probe and the guanine base in the nucleic acid amplification product do not form a base pair, the fluorescence can be quenched as long as the distance between these bases is close. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. That is, when the probe is hybridized, the fluorescence can be quenched if the guanine base in the nucleic acid amplification product is present within a range of, for example, 1 to 3 bases relative to the cytosine base of the probe (a base that forms a base pair with the cytosine base is considered to be 1).

[0049] Therefore, even if at least one terminal base labeled with a fluorescence quenching dye is not cytosine, the change in the fluorescence intensity of the reaction solution can be measured. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. For example, when the probe is hybridized, quenching can be achieved if a guanine base in the nucleic acid amplification product is present within a range of, for example, 1 to 3 bases relative to the terminal base labeled with the fluorescence quenching dye (the base that forms a base pair with the terminal base is counted as 1).

[0050] In a specific preferred embodiment, the nucleic acid probe of the present invention is used in step (3). In this manner, the monkeypox virus and the varicella-zoster virus can be detected by using the nucleic acid probe of the present invention. In the method of the present invention, one type of the probe of the present invention may be used, or two or more types of the probe may be used in combination.

[0051] In one embodiment, the step (3) may include at least one of the following steps (3-a), (3-b), and (3-c): (3-a) Simultaneously with step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution, thereby monitoring the progress of the reaction (nucleic acid amplification reaction) of step (2) in real time. (3-b) After completion of step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution, thereby monitoring the progress of the reaction (nucleic acid amplification reaction) of step (2) at an endpoint. (3-c) after completion of the step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the temperature dependence of the fluorescence intensity of the reaction solution. By the step (3-a), (3-b), or (3-c), the formation of a complex formed between a nucleic acid amplification product and a nucleic acid probe can be detected simply, quickly, and with high sensitivity. The steps (3-a), (3-b), and (3-c) may be performed in combination, for example, both steps (3-a) and (3-b), or both steps (3-a) and (3-c) can be performed. In one embodiment, from the viewpoint of more rapid detection of a nucleic acid amplification product, the step (3-b) or (3-c) is preferred. In particular, the step (3-c), i.e., the melting curve analysis method, is preferred.

[0052] (Step (3-a)) Step (3-a) is a method for monitoring the progress of a nucleic acid amplification reaction in real time (so-called real-time PCR method), and quantitative analysis is possible by comparing with a control substance of known concentration.

[0053] (Step (3-b)) In step (3-b), the progress of the nucleic acid amplification reaction is monitored at the end point, thereby enabling rapid detection of the target nucleic acid contained in the sample. Furthermore, the approximate amount of the target nucleic acid can be estimated by comparing the fluorescence intensity at the end point. For example, the progress of the nucleic acid amplification reaction is monitored at an end point by measuring the fluorescence intensity of a reaction solution containing a nucleic acid probe labeled with a fluorescent quenching dye. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured, and the fluorescence intensity of the reaction solution after the reaction is compared with the fluorescence intensity of the reaction solution before the reaction, thereby confirming the presence or absence of amplification of the target nucleic acid. Alternatively, the presence or absence of the target nucleic acid contained in the sample can also be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of a control reaction solution. The control reaction solution is a reaction solution to which a sample known to be negative or positive is added instead of the sample to be measured. The progress of the nucleic acid amplification reaction generally needs to be monitored in real time, but for the purpose of more rapid and convenient detection, it is preferable to measure it at an end point.

[0054] (Step (3-c)) In step (3-c), measuring the temperature dependency of the fluorescence intensity may specifically mean measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high. The melting temperature (Tm value) specific to the nucleic acid probe used can be obtained by first differentiating the obtained fluorescence intensity with respect to temperature. The fluorescence intensity may also be converted into a fluorescence quenching rate or the like according to the purpose. The detection and analysis of target nucleic acids using the Tm value is called melting curve analysis. In general, the Tm value refers to the temperature at which the proportion of an oligonucleotide that forms a double strand with its complementary strand is equal to the proportion that does not form a double strand and is single-stranded. Since the Tm value is a value specific to a base sequence, melting curve analysis can be used as a method for analyzing base sequence polymorphisms of target nucleic acids. Base sequence polymorphisms referred to here include single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

[0055] As an example, melting curve analysis is also applied to SNP analysis. When there is a mutation in the base sequence of the target nucleic acid relative to the probe, the bases mismatch when the probe hybridizes, and the Tm value is generally low. Therefore, single nucleotide polymorphism analysis (SNP analysis) can also be performed by comparing the magnitude of the Tm value.

[0056] [Reagents for detecting monkeypox and / or varicella-zoster viruses] In another embodiment, the present invention provides a reagent for detecting monkeypox virus and / or varicella-zoster virus. The reagent preferably contains at least components necessary for nucleic acid amplification reaction and detection in addition to the nucleic acid probe of the present invention described above (monkeypox virus detection probe and / or varicella-zoster virus detection probe). The necessary components can be known. For example, the reagent of the present invention preferably contains at least a nucleic acid primer set for PCR, DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and inorganic salts such as magnesium salts. The nucleic acid primer set for PCR and the nucleic acid probe for detection can contain multiple sets in order to amplify multiple regions of monkeypox virus and / or varicella-zoster virus. The concentration of each component can be appropriately adjusted, but 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 contained in the probe set is preferably within the above-mentioned concentration range. The nucleic acid primer is preferably at 0.01 to 10 μM. The DNA polymerase is preferably at 0.01 to 1 U / μL, more preferably at 0.02 to 0.5 U / μL. The deoxyribonucleoside triphosphates (dNTPs) are preferably at 0.02 to 1 mM, more preferably at 0.1 to 0.5 mM. The inorganic salts such as magnesium salts are preferably at 0.1 to 10 mM, more preferably at 1 to 5 mM.

[0057] Furthermore, the reagent of the present invention may contain additives known in the art for the purpose of suppressing non-specific amplification and promoting the reaction. Examples of additives for suppressing non-specific amplification include known anti-DNA polymerase antibodies and phosphoric acid. Examples of additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, 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 (registered trademark) P40, and the like. In order to facilitate the determination of false negatives, the reagent of the present invention preferably contains an internal control known in the art. In the present invention, these additives may be used alone or in combination of two or more.

[0058] In a specific embodiment, the reagent of the present invention preferably includes an internal control. By using the internal control in combination, it is possible to easily confirm that the nucleic acid amplification reaction is performed normally, reduce the risk of false negatives, and enable more accurate monkeypox virus and / or varicella-zoster virus testing. When using an internal control in combination in the melting curve analysis method, it is preferable that the detection temperature of the internal control and the detection temperature of the monkeypox virus or varicella-zoster virus detection probe of the present invention are different to a certain extent (for example, 5°C or more). For example, when using an internal control and / or a monkeypox virus or varicella-zoster virus detection probe of the present invention in the melting curve analysis method, the detection temperature can be determined by a method known in the art, and a person skilled in the art can appropriately select and use one whose detection temperatures differ from each other by, for example, 5°C or more.

[0059] [Kit for detecting monkeypox virus and / or varicella-zoster virus] In another embodiment, the present invention provides a kit for detecting monkeypox virus and / or varicella-zoster virus. The kit of the present invention is not particularly limited as long as it contains the nucleic acid probe of the present invention (monkeypox virus detection probe and / or varicella-zoster virus detection probe) described above or the reagent of the present invention and is configured to detect (including differentiation) monkeypox virus and / or varicella-zoster virus. For example, the kit of the present invention can optionally contain a reagent capable of detecting (including quantification) the presence of a target substance and / or an instruction manual for use that explains the method of use, etc. For example, the kit of the present invention can be provided in a form in which the nucleic acid probe, components necessary for nucleic acid amplification reaction, and components necessary for detection of the amplification product are sealed in the same container or in separate containers, and packaged in, for example, a single package, and includes information on the method of use of the kit. The kit of the present invention can also contain a positive control solution and / or a negative control solution. EXAMPLES

[0060] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0061] Test Example 1: Confirmatory test for detection of monkeypox virus alone (1-1) Method In order to search for a nucleic acid probe capable of specifically detecting monkeypox virus, 100 copies of monkeypox virus DNA were used per reaction, and the presence or absence of non-specific amplification and detection of monkeypox virus were confirmed using a total of six types of nucleic acid probes designed based on the base sequence shown in SEQ ID NO: 1. Purified water was used as a negative sample for the measurement of the presence or absence of non-specific amplification. The nucleic acid probes used were oligonucleotides consisting of the base sequences shown in SEQ ID NO: 2 to 7, which were synthesized according to a conventional method (only either the 5' or 3' end was labeled with BODIPY-FL. See FIG. 5 for the labeling position in each probe). In addition, a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 14 and a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 18 were used as the nucleic acid primer set.

[0062] (1-2) Reaction solution The following solutions were prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) was used by adjusting the amount used as described in the respective instruction manuals. An internal control (IC) of a known sequence was also added to the reagent to confirm whether the nucleic acid amplification was performed normally. 3.0 μM primer shown in SEQ ID NO:14 0.5 μM primer shown in SEQ ID NO: 18 0.5 μM of a probe represented by any one of SEQ ID NOs: 2 to 7

[0063] (1-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. (Nucleic acid amplification and melting curve analysis) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0064] (1-4) Results 1 is a detection graph obtained when 100 copies of monkeypox virus were detected by nucleic acid amplification and melting curve analysis using the probe shown in SEQ ID NO: 7 as a representative example. Similarly, the measurement results using the probe used in this test example are summarized in Table 1. The rate of change in fluorescence (representing the quenching rate in this example) was calculated by the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0065] As shown in the results in Table 1, even when probes were designed in the same region, it was shown that high fluorescence intensity and stable detection were possible depending on the probe sequence. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed regardless of which probe was used.

[0066] [Table 1]

[0067] Test Example 2: Confirmatory test for detection of varicella-zoster virus alone (2-1) Method In order to search for a nucleic acid probe capable of specifically detecting the varicella-zoster virus, 100 copies of the DNA of the varicella-zoster virus were used per reaction, and the presence or absence of non-specific amplification and the detection of the varicella-zoster virus were confirmed using a total of five nucleic acid probes designed based on the base sequence shown in SEQ ID NO:8. Purified water was used as a negative sample for the presence or absence of non-specific amplification. The nucleic acid probes used were oligonucleotides consisting of the base sequences shown in SEQ ID NO:9 to 13, which were synthesized according to a conventional method (only either the 5' or 3' end was labeled with CR6G. See FIG. 6 for the labeling position in each probe). In addition, a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO:21 and the base sequence shown in SEQ ID NO:24 were used as the nucleic acid primer set.

[0068] (2-2) Reaction solution The following solutions were prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) was used by adjusting the amount used as described in the respective instruction manuals. An internal control (IC) of a known sequence was also added to the reagent to confirm whether the nucleic acid amplification was performed normally. 3.0 μM primer shown in SEQ ID NO:21 0.5 μM primer shown in SEQ ID NO:24 0.4 μM of a probe represented by any one of SEQ ID NOs: 9 to 13

[0069] (2-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. (Nucleic acid amplification and melting curve analysis) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0070] (2-4) Results 2 is a detection graph obtained when 100 copies of Varicella-Zoster virus were detected by nucleic acid amplification and melting curve analysis using the probe shown in SEQ ID NO: 11 as a representative example. Similarly, the measurement results using the probe used in this test example are summarized in Table 2. The rate of change in fluorescence (representing the quenching rate in this example) was calculated by the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0071] As shown in the results in Table 2, even when probes were designed in the same region, it was shown that high fluorescence intensity and stable detection were possible depending on the probe sequence. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed regardless of which probe was used.

[0072] [Table 2]

[0073] Test Example 3: Confirmation test for detection of monkeypox virus using nucleic acid primer combinations (3-1) Method Based on the results of Test Example 1, the combination of nucleic acid primers for detecting monkeypox virus was verified. 100 copies of monkeypox virus DNA were used per reaction, and the presence or absence of non-specific amplification and detection of monkeypox virus were confirmed using a total of eight combinations of nucleic acid primers designed based on the base sequence shown in SEQ ID NO: 1. Purified water was used as a negative sample for the measurement of the presence or absence of non-specific amplification. The nucleic acid probe used was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7, synthesized according to a conventional method (only either the 5' end or the 3' end was labeled with BODIPY-FL. See FIG. 5 for the labeling position in each probe). In addition, a set of nucleic acid primers consisting of a base sequence shown in any one of SEQ ID NOs: 14 to 17 and a set of nucleic acid primers consisting of a base sequence shown in any one of SEQ ID NOs: 18 to 20 were used as the nucleic acid primer set.

[0074] (3-2) Reaction solution The following solutions were prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) was used by adjusting the amount used as described in the respective instruction manuals. An internal control (IC) of known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.5 μM of a primer represented by any one of SEQ ID NOs: 14 to 17 3.0 μM of a primer represented by any one of SEQ ID NOs: 18 to 20 0.5 μM probe shown in SEQ ID NO:4

[0075] (3-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. (Nucleic acid amplification and melting curve analysis) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0076] (3-4) Results The measurement results for the combination of nucleic acid primers used in this test example are summarized in Table 3. The fluorescence change rate (representing the quenching rate in this example) was calculated by the formula (fluorescence intensity before amplification-fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0077] As shown in the results in Table 3, even when nucleic acid primers were designed in the same region, it was shown that high fluorescence intensity and stable detection were possible depending on the nucleic acid primer sequence. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed regardless of which primer was used.

[0078] [Table 3]

[0079] Test Example 4: Detection confirmation test of varicella-zoster virus using a combination of nucleic acid primers (4-1) Method From the results of Test Example 2, the combination of nucleic acid primers for detecting the varicella-zoster virus was verified. 100 copies of the DNA of the varicella-zoster virus were used per reaction, and the presence or absence of non-specific amplification and the detection of the varicella-zoster virus were confirmed with a combination of a total of six nucleic acid primers designed based on the base sequence shown in SEQ ID NO: 8. Purified water was used as a negative sample for the measurement of the presence or absence of non-specific amplification. The nucleic acid probe used was an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10, synthesized according to a conventional method (only either the 5' end or the 3' end was labeled with CR6G. See FIG. 6 for the labeling position in each probe). In addition, a set of nucleic acid primers consisting of a base sequence shown in any one of SEQ ID NOs: 21 to 23 and a set of nucleic acid primers consisting of a base sequence shown in any one of SEQ ID NOs: 24 to 26 were used as the nucleic acid primer set.

[0080] (4-2) Reaction solution The following solutions were prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark). GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) and ReverTra Ace (registered trademark) were used in the amounts adjusted as described in their respective instruction manuals (ReverTra Ace (registered trademark) was used at 0.1 U / μL). An internal control (IC) of a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 3.0 μM of a primer represented by any one of SEQ ID NOs: 21 to 23 0.5 μM of a primer represented by any one of SEQ ID NOs: 24 to 26 0.5 μM probe shown in SEQ ID NO:8

[0081] (4-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. (Nucleic acid amplification and melting curve analysis) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0082] (4-4) Results The measurement results for the combination of nucleic acid primers used in this test example are summarized in Table 4. The fluorescence change rate (representing the quenching rate in this example) was calculated by the formula (fluorescence intensity before amplification-fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0083] As shown in the results in Table 4, even when nucleic acid primers were designed in the same region, it was shown that high fluorescence intensity and stable detection were possible depending on the nucleic acid primer sequence. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed regardless of which probe was used.

[0084] [Table 4]

[0085] Test Example 5: Simultaneous detection and confirmation test for monkeypox virus and varicella-zoster virus (5-1) Method Based on the results obtained so far, we verified the simultaneous detection of monkeypox virus and varicella-zoster virus. When 50 copies and 10 copies of DNA of monkeypox virus and varicella-zoster virus were used per reaction, the presence or absence of non-specific amplification and the detection of monkeypox virus and varicella-zoster virus were confirmed by combining the primer probe for detecting monkeypox virus and the primer probe for detecting varicella-zoster virus. Purified water was used as a negative sample for the presence or absence of non-specific amplification. The nucleic acid probes used were oligonucleotides consisting of the base sequence shown in SEQ ID NO: 7 for detecting monkeypox virus and SEQ ID NO: 11 for detecting varicella-zoster virus, which were synthesized according to a conventional method (only either the 5' or 3' end was labeled with BODIPY-FL or CR6G. See Figures 5 and 6 for the labeling positions in each probe).

[0086] (5-2) Reaction solution The following solutions were prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) was used by adjusting the amount used as described in the respective instruction manuals. An internal control (IC) of a known sequence was also added to the reagent to confirm whether the nucleic acid amplification was performed normally. For monkeypox virus detection 3.0 μM of a primer represented by any one of SEQ ID NOs: 14 to 17 0.5 μM primer shown in SEQ ID NO: 18 0.5 μM probe shown in SEQ ID NO:7 Varicella-zoster virus detection 3.0 μM of a primer represented by any one of SEQ ID NOs: 21 to 23 0.5 μM primer shown in SEQ ID NO:24 0.5 μM probe shown in SEQ ID NO:11

[0087] (5-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. (Nucleic acid amplification and melting curve analysis) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0088] (5-4) Results The measurement results for the combination of monkeypox virus detection and varicella-zoster virus detection used in this test example are summarized in Table 5. The rate of change in fluorescence (representing the quenching rate in this example) was calculated by the formula: (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0089] Figure 3 shows a detection graph obtained when 10 copies of monkeypox virus and varicella-zoster virus were detected by nucleic acid amplification and melting curve analysis using combinations of SEQ ID NOs: 15, 18, 21, and 24 as representative examples. As shown in the results of Table 5, it was confirmed that 50 copies of monkeypox virus and varicella-zoster virus could be simultaneously detected for each combination. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed for any of the probes used.

[0090] [Table 5]

[0091] Test Example 6: Simultaneous detection and confirmation test for monkeypox virus and varicella-zoster virus using blister swab specimens (6-1) Method Based on the results obtained so far, we will confirm whether it is possible to detect monkeypox virus and varicella-zoster virus by simple processing of blister swab specimens. Negative blister swab specimens were centrifuged at 13,000 rpm, and 50 copies of monkeypox virus and varicella-zoster virus DNA were used per reaction for the supernatant obtained, and the detection of monkeypox virus and varicella-zoster virus was confirmed. The nucleic acid probes used were the oligonucleotides consisting of the base sequence shown in SEQ ID NO: 7 for monkeypox virus detection and SEQ ID NO: 11 for varicella-zoster virus detection, which were synthesized according to a conventional method (only either the 5' or 3' end was labeled with BODIPY-FL or CR6G. See Figures 5 and 6 for the labeling positions in each probe).

[0092] (6-2) Reaction solution The following solutions were prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). The Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) was used by adjusting the amount used as described in the respective instruction manuals. An internal control (IC) of a known sequence was also added to the reagent to confirm whether the nucleic acid amplification was performed normally. For monkeypox virus detection 3.0 μM of a primer represented by any one of SEQ ID NOs: 15 0.5 μM primer shown in SEQ ID NO: 18 0.5 μM probe shown in SEQ ID NO:7 Varicella-zoster virus detection 3.0 μM of a primer represented by any one of SEQ ID NOs: 21 0.5 μM primer shown in SEQ ID NO:24 0.5 μM probe shown in SEQ ID NO:11

[0093] (6-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. (Nucleic acid amplification and melting curve analysis) 97℃・1 second 58℃・3 seconds 63℃・5 seconds (more than 50 cycles) 94℃・30 seconds 39℃・30 seconds 40℃~75℃ (Temperature rises at 0.09℃ / sec) The time required from the start of the reverse transcription reaction to the completion of the melting curve analysis was 35 minutes.

[0094] (6-4)Result Fig. 4 shows a detection graph obtained when a negative blister swab specimen was centrifuged at 13,000 rpm, and DNA of monkeypox virus and varicella-zoster virus was added to the supernatant so that the DNA was 50 copies / test. The centrifugal supernatant of the negative blister swab specimen used in this test example did not undergo a nucleic acid purification process, and therefore contains biological impurities such as various proteins that inhibit nucleic acid amplification reactions. From the results of this test example, it was confirmed that according to the present invention, a highly sensitive measurement can be performed without problems even on a sample obtained by simply processing a blister swab specimen like this. [Industrial Applicability]

[0095] By using a method, reagent, or kit using the nucleic acid probe of the present invention, monkeypox virus and / or varicella-zoster virus that may be contained in a sample can be detected easily and with high sensitivity. Therefore, the present invention is not only useful for research applications, but also realizes early detection and early treatment of monkeypox virus and / or varicella-zoster virus without being affected by the skills of an operator or the performance of equipment, which leads to the prevention of the spread of sexually transmitted diseases and can greatly contribute to clinical diagnosis, environmental testing, etc.

Claims

1. A probe for detecting monkeypox virus, having the following characteristics (A) and (B): (A) It has a base sequence A1 of at least 15 consecutive bases in the base sequence from base 65 to 100 of SEQ ID NO: 1 or its complementary base sequence, or a base sequence A2 in which 1 to 3 bases have been substituted, deleted, inserted or added in base sequence A1. (B) Only either the 5' or 3' end is labeled.

2. The probe according to claim 1, wherein the length of the base sequence of (A) is 15 to 20 bases.

3. The probe according to claim 1, wherein the base sequence of (A) comprises a base sequence shown in any one of SEQ ID NOs: 2 to 7 or a complementary base sequence thereto.

4. The probe according to claim 1 , wherein the label (B) is a fluorescent dye label.

5. The probe according to claim 1, wherein the label (B) is a label with a fluorescent quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe.

6. The probe according to claim 1 , wherein the label (B) is a label with a fluorescent quenching dye that is quenched by interaction with guanine.

7. The probe according to claim 1, wherein the label (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.

8. The probe according to claim 1, 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, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

9. The probe according to claim 1 , wherein in (B), the labeled terminal base is cytosine.

10. A probe for detecting varicella-zoster virus, having the following characteristics (C) and (D): (C) A base sequence C1 of at least 14 consecutive bases in the base sequence from base 95 to 125 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence C2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence C1. (D) Only either the 5' or 3' end is labeled.

11. The probe according to claim 10, wherein the length of the base sequence of (C) is 14 to 20 bases.

12. The probe according to claim 10, wherein the base sequence of (C) comprises a base sequence shown in any one of SEQ ID NOs: 9 to 13 or a complementary base sequence thereto.

13. The probe according to claim 10 , wherein the label in (D) is a fluorescent dye label.

14. The probe according to claim 10, wherein the label (D) is a label with a fluorescent quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe.

15. The probe according to claim 10 , wherein the label (D) is a label with a fluorescent quenching dye that is quenched by interaction with guanine.

16. The probe according to claim 10, wherein the label (D) 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 carboxyrhodamine 6G and its derivatives.

17. The probe according to claim 10, wherein the label of (D) 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, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

18. The probe according to claim 10, wherein in (D), the labeled terminal base is cytosine.

19. A method for simultaneously detecting monkeypox virus and varicella zoster virus that may be contained in a sample in one reaction, using the probe according to any one of claims 1 to 9 and the probe according to any one of claims 10 to 18.

20. The following steps (1), (2), and (3): (1) providing a sample that may contain monkeypox virus and varicella-zoster virus; (2) performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more of the probes; 20. The method of claim 19, comprising:

21. The method according to claim 20, wherein the step (2) is carried out by a PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B.

22. The method according to claim 21 , wherein the DNA polymerase belonging to family B is a KOD-derived DNA polymerase or a mutant thereof.

23. The method according to claim 20, wherein the primer set for detecting monkeypox virus used in the nucleic acid amplification reaction of the step (2) comprises a first primer having a base sequence S1 of at least 18 consecutive bases in the base sequence of positions 1 to 70 of SEQ ID NO: 1 or a complementary base sequence thereto, or a base sequence S2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S1, and a second primer having a base sequence S3 of at least 20 consecutive bases in the base sequence of positions 95 to 150 of SEQ ID NO: 1 or a complementary base sequence thereto, or a base sequence S4 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S3, wherein the second primer is complementary to a DNA extension product of the first primer.

24. The method according to claim 23, wherein the first primer has a base sequence shown in any one of SEQ ID NOs: 14 to 17, a complementary base sequence thereof, or a base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in said base sequence, and the second primer has a base sequence shown in any one of SEQ ID NOs: 18 to 20, a complementary base sequence thereof, or a base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in said base sequence.

25. The method according to claim 20, wherein the primer set for detecting Varicella-Zoster virus used in the nucleic acid amplification reaction of the step (2) comprises a first primer having a base sequence S5 of at least 19 consecutive bases in the base sequence of positions 1 to 100 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S5, and a second primer having a base sequence S7 of at least 18 consecutive bases in the base sequence of positions 120 to 175 of SEQ ID NO: 8 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S7, wherein the second primer is complementary to a DNA extension product of the first primer.

26. The method according to claim 25, wherein the first primer has a base sequence shown in any one of SEQ ID NOs: 21 to 23, a complementary base sequence thereof, or a base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in said base sequence, and the second primer has a base sequence shown in any one of SEQ ID NOs: 24 to 26, a complementary base sequence thereof, or a base sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in said base sequence.

27. 21. The method of claim 20, wherein the detecting step in step (3) is carried out by melting curve analysis.

28. A kit for simultaneously detecting monkeypox virus and varicella zoster virus in one reaction, comprising the probe according to any one of claims 1 to 9 and the probe according to any one of claims 10 to 18.

29. A kit for simultaneously detecting monkeypox virus and varicella zoster virus in one reaction, comprising the primer according to claim 23 or 24 and the primer according to claim 25 or 26.

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