Target RNA detection method and probe kit

Abstract

To provide a target RNA detection method capable of detecting even short-chain RNA such as miRNA with high sensitivity and ease.SOLUTION: The method for detecting a target RNA comprises: (I) a hybridization step for hybridizing a DNA probe and a chimera probe to a single stranded target RNA, where, with respect to a first region of a target RNA which is the 5' end side or the 3' end side of the single stranded target RNA and a second region which is the other end from the first region side and which does not overlap with the first region, a DNA probe comprising a base sequence D1 consisting of DNA complementary to the first region of the target RNA and a region SG capable of binding a labelling substance is hybridized to the target RNA and a chimera probe comprising a base sequence D2 consisting of DNA complementary to the second region of the target RNA, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA in this order, the base sequence SD and the base sequence SR consisting of base sequences that are complementary to each other, and the base sequence SD, linker, and the base sequence SR forming a stem loop structure, is hybridized with the target RNA; (II) a capturing step for capturing a complex of the target RNA, the DNA probe, and the chimera probe with a capture body containing a water-insoluble carrier and an anti-DNA / RNA chimera antibody immobilized on the water-insoluble carrier; and (III) a detection step of detecting the complex captured by the capturing body.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting a target RNA and a probe kit, and more particularly to a method for detecting a target RNA and a probe kit that can be suitably used for the method. [Background technology]

[0002] Cells contain a large amount of short-stranded RNAs that are not translated into proteins. While these short-stranded RNAs were once thought to have no function, research since the 1990s has revealed that they play important roles in living organisms. For example, among these short-stranded RNAs, single-stranded RNAs with a length of approximately 19 to 25 bases called miRNAs (microRNAs) have been shown to play an important role in cellular function by regulating gene expression.

[0003] The miRNA production process is typically as follows: First, miRNA genes on the genome are transcribed by RNA polymerase to produce pri-miRNA (primary miRNA) containing a hairpin structure. The pri-miRNA is then cleaved by the nuclear RNase Drosha to produce pre-miRNA (precursor miRNA) with a 60- to 70-base hairpin structure. The pre-miRNA is then transported from the nucleus to the cytoplasm, where it is cleaved by the cytoplasmic RNase Dicer to produce a 21- to 24-base double-stranded RNA (miRNA duplex). The miRNA duplex is then incorporated into Ago proteins, and only one of the RNA strands of the duplex ultimately becomes the mature miRNA.

[0004] Mature miRNAs bind to Ago proteins to form the RNA-induced silencing complex (RISC), which binds to target mRNAs that contain sequences partially complementary to the mature miRNAs contained in the complex, suppressing the translation of the target mRNAs and thereby regulating the expression of numerous genes.

[0005] It has also become clear that miRNAs are closely related to various diseases. In recent years, it has been reported that miRNAs exhibit abnormal expression in various diseases such as cancer, and therefore, there are expectations for the development of technologies to detect such miRNAs as diagnostic tools for these diseases.

[0006] As a method for detecting short-stranded RNA such as miRNA, a microarray method using a microarray pre-loaded with a probe that specifically binds to a target RNA is widely used (for example, Patent Document 1, etc.).

[0007] Nucleic acid amplification methods are also used as methods for detecting short-chain RNAs such as miRNAs. For example, a method has been developed in which the target miRNA is detected by polyadenylation of the target miRNA to add a polyA sequence, followed by synthesis of cDNA by reverse transcription using a primer containing a polyT sequence and a sequence complementary to the base sequence of the target miRNA, and then nucleic acid amplification is performed using this cDNA as a template (Patent Document 2).

[0008] Furthermore, a method for detecting a target miRNA has also been developed, for example, by hybridizing the target miRNA to a primer having a 3' region and a stem-loop that are complementary to the base sequence of the target miRNA, carrying out an extension reaction of the primer, and then carrying out nucleic acid amplification using the extended primer as a template (Patent Document 3).

[0009] Another method for detecting nucleic acids by nucleic acid amplification has been developed, in which, for example, a chimeric probe containing RNA in part is hybridized to target DNA, and the resulting DNA-RNA hybrid chain is cleaved with ribonuclease to release the chimeric probe fragment, which is then used as a primer for an extension reaction to detect the target DNA (Patent Document 4).

[0010] Furthermore, methods for detecting specific polynucleotides have been developed, for example, by hybridizing a non-detectable polynucleotide sequence with a labeled nucleic acid probe containing a base sequence complementary to the non-detectable polynucleotide sequence and detecting the resulting DNA-RNA hybrid chain using an anti-DNA / RNA antibody (Patent Document 5). BioVendor has also developed a kit for hybridizing a biotinylated DNA probe containing a base sequence complementary to the target miRNA and detecting the resulting DNA-RNA-biotin complex by ELISA using an anti-DNA / RNA antibody (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-075095 [Patent Document 2] US Patent Application Publication No. 2009 / 0220969 [Patent Document 3] U.S. Patent No. 7,575,863 [Patent Document 4] Japanese Patent Application Laid-Open No. 2008-307029 [Patent Document 5] Japanese Patent Application Publication No. 179657 / 1983 [Non-patent literature]

[0012] [Non-Patent Document 1] DENIS Pharma Co., Ltd., "miREIA-miRNA (micro RNA) Enzyme Immunoassay Kit," [online], [Retrieved August 5, 2021], Internet<URL:https: / / research.sceti.co.jp / images / upload / flyer / 16 / 16.pdf> Summary of the Invention [Problem to be solved by the invention]

[0013] However, microarray methods have drawbacks, such as complex procedures and high costs. Furthermore, commonly used microarray and nucleic acid amplification methods involve first amplifying the target nucleic acid before detecting it, so results are often dependent on the accuracy of the amplification reaction. Furthermore, reverse transcription reactions can cause variations in detection accuracy, resulting in a lack of accurate reflection of the amount of target nucleic acid in the sample. Furthermore, the present inventors have newly discovered that conventional methods for detecting DNA-RNA complexes using anti-DNA / RNA antibodies with ELISA have insufficient sensitivity for some types of target RNA, and are particularly limited in the detection of short-stranded RNAs such as miRNAs.

[0014] The present invention has been made in consideration of the problems associated with the above-mentioned conventional techniques, and aims to provide a target RNA detection method that can detect even short-chain RNAs such as miRNAs with high sensitivity and in a simple manner, as well as a probe kit that can be suitably used for the method. [Means for solving the problem]

[0015] To achieve the above-mentioned object, the inventors of the present invention have conducted extensive research and have developed a method for capturing and detecting a hybridized complex between a target RNA and a DNA probe using an anti-DNA / RNA antibody. The DNA probes are designed to be divided into two types: a DNA probe to which a labeling substance is attached, and a chimeric probe whose stem region forms a DNA / RNA hybrid. The inventors have found that the combined use of these probes significantly improves detection sensitivity, even when the target RNA is a short-stranded RNA such as miRNA. Furthermore, because this method does not require a nucleic acid amplification reaction, highly accurate quantification that reflects the amount of target RNA in a sample is possible, regardless of the accuracy of the amplification reaction.

[0016] Furthermore, with this method, the steps from hybridization to detection can be carried out isothermally without temperature control such as heating, making it possible to detect the amount of target RNA with high accuracy. In addition to the fact that the above-mentioned nucleic acid amplification reaction is unnecessary, the inventors have also discovered that target RNA can be detected much more easily than conventional methods, and have thus completed the present invention.

[0017] The aspects of the present invention obtained based on these findings are as follows. [1] A method for detecting a target RNA, (I) a first region on the 5'-end or 3'-end side of a single-stranded target RNA, and a second region including the other end of the first region and not overlapping with the first region; a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region of the target RNA and a region SG to which a labeling substance can be bound; a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to the second region of the target RNA, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure. a hybridization step in which the (II) a capture step of capturing a complex of the target RNA, the DNA probe, and the chimeric probe with a capture body comprising a water-insoluble carrier and an anti-DNA / RNA chimeric antibody immobilized on the water-insoluble carrier; (III) a detection step of detecting the complex captured by the capturer; A method for detecting a target RNA, comprising: [2] The method for detecting a target RNA according to [1], wherein the detection step is a step of detecting the complex using a signal derived from a labeling substance bound to the region SG as an indicator. [3] The method for detecting a target RNA according to [1] or [2], wherein the target RNA is a single-stranded RNA molecule having a length of 19 to 25 bases. [4] The method for detecting a target RNA according to any one of [1] to [3], wherein the linker is a base sequence consisting of DNA and / or RNA. [5] The method for detecting a target RNA according to [4], wherein the linker has a length of 3 to 50 bases. [6] The method for detecting a target RNA according to any one of [1] to [5], wherein the length of the base sequence SD and the length of the base sequence SR are each 3 to 30 bases long. [7] The method for detecting a target RNA according to any one of [1] to [6], wherein the temperature in the hybridization step is 15 to 60°C. [8] The method for detecting a target RNA according to any one of [1] to [7], wherein the water-insoluble carrier is a particulate carrier. [9] a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region on the 5'-end or 3'-end of a single-stranded target RNA, and a region SG to which a labeling substance can be bound; a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to a second region that includes the other end of the first region side of the target RNA and does not overlap with the first region, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure; A probe kit comprising:

[10] The probe kit according to [9], further comprising a labeling substance capable of binding to the region SG of the DNA probe.

[11] The probe kit according to [9] or

[10] , wherein the total length of the base sequence D1 and the base sequence D2 is 10 to 100 bases long.

[12] The probe kit according to any one of [9] to

[11] , wherein the linker is a base sequence consisting of DNA and / or RNA.

[13] The probe kit according to

[12] , wherein the linker has a length of 3 to 50 bases.

[14] The probe kit according to any one of [9] to

[13] , wherein the length of the base sequence SD and the length of the base sequence SR are each 3 to 30 bases long.

[15] The probe kit according to any one of [9] to

[14] , further comprising a capture body comprising a water-insoluble carrier and an anti-DNA / RNA chimeric antibody immobilized on the water-insoluble carrier.

[16] The probe kit according to

[15] , wherein the water-insoluble carrier is a particle carrier.

[17] The probe kit according to any one of [9] to

[16] , which is a kit for use in the method for detecting a target RNA according to any one of [1] to [8].

[0018] Although the reason why the above object is achieved by the configuration of the present invention is not entirely clear, the inventors speculate as follows. Specifically, in the present invention, as described above, DNA probes to be hybridized to target RNA are divided into two types: DNA probes to which a labeling substance is bound, and chimeric probes that form a stem-loop structure and whose stem region is a DNA-RNA hybrid. The base sequences of these probes are designed based on the base sequence of the target RNA, and they hybridize to non-overlapping regions (first region and second region) on the target RNA. The stem-loop structure of the chimeric probe adds RNA with a base length equal to the stem region in addition to the base length of the target RNA. It is speculated that this increases the recognition site for anti-DNA-RNA chimeric antibodies, thereby leading to higher sensitivity in detecting the target RNA.

[0019] Furthermore, in conventional methods in which a DNA probe is hybridized to the entire length of a target RNA, the Tm value of the DNA probe tends to increase depending on its base length. Although RNA and DNA hybridization is possible at low temperatures, maintaining sufficient hybridization accuracy typically requires a temperature about 5°C lower than the Tm value (e.g., 55°C or higher). Hybridization at temperatures lower than this increases nonspecificity and reduces accuracy. In contrast, in the present invention, the probe hybridized to the target RNA is divided into two as described above, allowing the Tm value of each probe to be lowered. It is believed that sufficient hybridization accuracy can be maintained even when the steps from hybridization to detection are performed at room temperature (e.g., about 37°C or lower) and isothermal (all steps at the same temperature). Therefore, the inventors believe that, combined with the elimination of the above-mentioned nucleic acid amplification reaction, it is now possible to detect target RNA with high sensitivity in a significantly simpler manner than before. [Effects of the Invention]

[0020] According to the present invention, it is possible to provide a target RNA detection method that can detect even short-chain RNA such as miRNA with high sensitivity and in a simple manner, as well as a probe kit that can be suitably used for this method. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic conceptual diagram showing one embodiment of a target RNA, a DNA probe, a chimeric probe, and a complex thereof according to the present invention. [Figure 2] FIG. 1 is a schematic conceptual diagram showing another embodiment of the target RNA, DNA probe, chimeric probe, and complex thereof according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be described in more detail below by taking preferred embodiments as examples, but the present invention is not limited thereto.

[0023] <Target RNA detection method> The method for detecting a target RNA of the present invention comprises: (I) a first region on the 5'-end or 3'-end side of a single-stranded target RNA, and a second region including the other end of the first region and not overlapping with the first region; a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region of the target RNA and a region SG to which a labeling substance can be bound; a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to the second region of the target RNA, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure. a hybridization step in which the (II) a capture step of capturing a complex of the target RNA, the DNA probe, and the chimeric probe with a capture body comprising a water-insoluble carrier and an anti-DNA / RNA chimeric antibody immobilized on the water-insoluble carrier; (III) a detection step of detecting the complex captured by the capturer; The method includes:

[0024] Preferred embodiments of the target RNA detection method of the present invention will be described in detail below with reference to examples, possibly with reference to Figures 1 and 2, but the present invention is not limited thereto. Figures 1 and 2 are schematic conceptual diagrams showing embodiments of the target RNA, DNA probe, chimeric probe, and complex thereof according to the present invention, respectively. In the following description and drawings, identical or corresponding elements are designated by the same reference numerals, and redundant explanations will be omitted.

[0025] In the present invention, a "base sequence consisting of DNA" refers to a base sequence consisting of deoxyribonucleotides (oligoDNA), a "base sequence consisting of RNA" refers to a base sequence consisting of ribonucleotides (oligoRNA), and a "base sequence consisting of DNA and / or RNA" refers to a base sequence consisting of deoxyribonucleotides, a base sequence consisting of ribonucleotides, or a base sequence consisting of deoxyribonucleotides and ribonucleotides (oligonucleotide).

[0026] Furthermore, the nucleotides constituting the target RNA, DNA probe, and chimeric probe according to the present invention may not be composed solely of natural nucleotides (deoxyribonucleotides and / or ribonucleotides), and may, for example, be composed partly or entirely of the non-natural nucleotides. Furthermore, the method for obtaining the base sequences constituting the DNA probe and chimeric probe according to the present invention is not particularly limited, and a conventionally known method or a method based thereon can be appropriately adopted. For example, the base sequences can be chemically synthesized using a commercially available synthesizer to produce synthetic oligonucleotides.

[0027] Furthermore, in the present invention, a "complementary base sequence" to a certain base sequence (or region) may be a base sequence that can hybridize with each other to form a double strand, and does not have to be completely complementary. In the present invention, when "base sequence (or region, hereinafter the same) X and base sequence (or region, hereinafter the same) Y are complementary," the hybridization conditions are preferably such that the sequence complementarity between base sequence X and base sequence Y is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). Note that the sequence complementarity can be calculated appropriately by those skilled in the art using known methods (e.g., BLAST (NCBI)). Furthermore, the hybridization conditions may be such that the base sequence X is completely complementary to the entire length of the base sequence Y, and at one or more positions, a continuous sequence of 1 to 5 bases (preferably 1 to 3 bases, for example, 3 bases, 2 bases, or 1 base) is inserted, deleted, or substituted.

[0028] (Target RNA) In the present invention, "target RNA" refers to RNA to be detected by the target RNA detection method of the present invention. In the present invention, the target RNA is not particularly limited as long as it can hybridize with the DNA probe and chimeric probe described below. The target RNA may be single-stranded in the region to which the DNA probe and chimeric probe hybridize, or may be double-stranded partially containing such a single-stranded region, or may have a three-dimensional structure such as a hairpin structure or hammerhead structure.

[0029] Among these, examples of the target RNA according to the present invention include mRNA (messenger RNA), viral RNA, fragmented RNA, miRNA (microRNA), tRNA (transfer RNA), rRNA (ribosomal RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), and siRNA (small interfering RNA). From the viewpoint of particularly effective application of the target RNA detection method of the present invention, RNA molecules having a length of 10 to 100,000 bases are preferred, single-stranded RNA molecules having a length of 15 to 30 bases are more preferred, and miRNA is even more preferred. Note that "miRNA" generally refers to a single-stranded RNA molecule having a length of 19 to 25 bases, which is not translated into protein and is said to be mainly involved in regulating gene expression after transcription in eukaryotes.

[0030] miRNAs are also known as markers for various diseases, and examples of such miRNAs include miR21-5p, Let7a, miR1185-3p, miR6875-5p, miR17-3p, miR421, miR27-a-3p, miR149-3p, miR-192-5p, miR-15b-5p, miR-125b, miR-155-5p, miR-885-5p, and miR-1306-5p. Among these, miR21-5p, Let7a, miR1185-3p, miR6875-5p, miR17-3p, miR421, miR27-a-3p, miR149-3p, miR-192-5p, miR-15b-5p, etc. can be detected with particularly higher sensitivity than conventional detection methods using the target RNA detection method of the present invention.

[0031] Such target RNA is not particularly limited and may be extracted from a sample that may contain the target RNA or artificially synthesized. The sample that may contain the target RNA is also not particularly limited and may include, for example, solutions containing chemically synthesized RNA, various organisms (including cells, tissues, organs, and individuals) and extracts thereof, human and animal body fluids (saliva, nasal aspirate, nasal swab, throat swab, gargle, nasal discharge, tears, sweat, urine, sputum, bronchoalveolar lavage fluid, blood, serum, plasma, cerebrospinal fluid, lymph, semen, amniotic fluid, etc.) and feces, plant biofluids, biological culture solutions, environmental water (rivers, lakes, harbors, waterways, groundwater, purified water, sewage, wastewater, etc.), and suspensions of solids (soil, cinders, etc.). Furthermore, the sample may be diluted or suspended in a diluent, or may have its pH adjusted as appropriate. Examples of the diluent include biochemical buffers such as sodium phosphate buffer, TriS buffer, phosphate buffer, and Good's buffer. Furthermore, as a method for extracting target RNA from the sample, any known method can be appropriately adopted.

[0032] In order to show the correspondence with the DNA probes and chimeric probes described below, the single-stranded portion of the target RNA will hereinafter be referred to for convenience as including a first region and a second region that do not overlap with each other. As shown in Figure 1 (a1), when the first region (111) is set at the 5' end of the target RNA (110), the second region (112) is set at the other end of the first region, i.e., the 3' end of the target RNA. As shown in Figure 2 (a2), when the first region (121) is set at the 3' end of the target RNA (120), the second region (122) is set at the other end of the first region, i.e., the 5' end of the target RNA.

[0033] However, the configuration of the first and second regions is not limited to that shown in Figures 1 and 2. For example, the first regions (111, 121) may not include the terminal portion of the single-stranded portion (110, 120) of the target RNA. In this case, the first regions may be located at a distance of at least one base from the 5' or 3' end of the target RNA. On the other hand, the second regions (112, 122) preferably include the terminal portion of the target RNA (110, 120) (i.e., located one base from the 5' or 3' end of the target RNA). Furthermore, the first and second regions do not have to be adjacent to each other and may be separated by a region (spacer) of at least one base (e.g., preferably 1 to 15 bases long when the target RNA is the miRNA), but they are more preferably adjacent to each other.

[0034] The preferred ranges of the length of the first region and the length of the second region according to the present invention correspond to the preferred ranges of the length of the base sequence D1 and the length of the base sequence D2 below, respectively.

[0035] (DNA probe) The "DNA probe" according to the present invention is an oligonucleotide probe comprising a base sequence D1 consisting of DNA complementary to the first region of the target RNA, and a region SG to which a labeling substance can be bound.

[0036] The total length of the DNA probe according to the present invention can be adjusted depending on the length of the target RNA, but is preferably 5 to 100 bases long, more preferably 10 to 60 bases long, and even more preferably 15 to 40 bases long. For example, when the target RNA is the miRNA, the total length is preferably 15 to 30 bases long.

[0037] [Base sequence D1] The base sequence D1 of the present invention is a base sequence that is complementary to the first region of the target RNA and is composed of DNA. Such base sequence D1 can be appropriately designed and prepared to match the base sequence of the first region of the target RNA of interest so as to satisfy the above-mentioned hybridization conditions, and more preferably so as not to hybridize to the chimeric probe described below or other regions.

[0038] The length of the base sequence D1 according to the present invention is preferably 5 bases or more, more preferably 5 to 50 bases, and, for example, when the target RNA is the miRNA, preferably 5 to 20 bases. If the length of the base sequence D1 is less than the lower limit, hybridization with the first region of the target RNA tends to be difficult, whereas if the length exceeds the upper limit, non-specific reactions tend to occur more easily.

[0039] [Area SG] The region SG according to the present invention is a region consisting of a site to which a labeling substance can be bound. The region SG (site to which a labeling substance can be bound) may be a region consisting of multiple nucleotides, or a region consisting of only a portion of a specific nucleotide. When the region SG is a region consisting of multiple nucleotides, the nucleotides contained in the region SG are preferably deoxyribonucleotides.

[0040] The embodiment of the region SG is not particularly limited. In FIGS. 1 and 2, the base sequence D1 (211, 221) and the region SG (212, 222) are separate regions, with one region SG located adjacent to the 3'-end (212) of the base sequence D1 (211) or the 5'-end (222) of the base sequence D1 (221). However, the region SG may be contained within the base sequence D1, or multiple regions may be present. Furthermore, when the base sequence D1 and the region SG are separate regions, the base sequence D1 and the region SG may be separated by a region (spacer) having a length of 1 to 50 bases (preferably, 1 to 20 bases). Examples of the spacer include poly(A) sequences. Among these, the embodiment of the region SG is preferably such that, when the DNA probe forms a complex (410, 420) with the target RNA and the chimeric probe, the region SG is located at the end of the base sequence D1 opposite to the end where the base sequence D2 described below is located, via a spacer as necessary.

[0041] The site to which the labeling substance can be attached is not particularly limited and can be appropriately selected depending on the method for attaching the labeling substance described below. For example, when the labeling substance described below is attached to a DNA probe via the bond between biotin and streptavidin (or avidin), the site to which the labeling substance can be attached can be a site modified with biotin. Such modification with biotin (biotinylation) can be achieved by any conventionally known method or a method based thereon, such as a method in which biotin is introduced into the 5'-end of the base sequence D1 during its synthesis using biotin phosphoramidite to produce the DNA probe; or a method in which a 5'-amino-modified oligo is synthesized and a biotin residue is added to the amino group of the oligo using a biotin-X-NHS ester reagent, but the method is not particularly limited thereto.

[0042] [Labeling substance] In the method for detecting a target RNA of the present invention, a labeling substance is bound to a DNA probe via the region SG, and the target RNA is detected using a signal derived from the labeling substance as an indicator. The labeling substance may be bound to the DNA probe in advance before the hybridization step (I) described below, or may be bound to the DNA probe after the hybridization step (I) or after the capture step (II) described below.

[0043] The labeling substance can be any of those used as labeling substances in known immunoassay methods or methods based thereon, without any particular limitation. For example, enzymes; particles such as latex particles and gold colloid particles; low-molecular-weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); 3 H, 32 P, 35 S, 125 The radioactive substance may be one of these or a combination of two or more of them.

[0044] For example, when an enzyme is used as the labeling substance, various detection methods can be performed depending on the substrate by adding a luminescent substrate, a chromogenic substrate, or a fluorescent substrate such as luminol, luciferin, or lucigenin as a substrate. Examples of the enzyme include peroxidase (POD: horseradish peroxidase (HRP), etc.), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, β-D-glucosidase, and luciferase. Among these, bioluminescent enzymes such as luciferase are preferred from the viewpoint of particularly high sensitivity.

[0045] The method for binding the labeling substance to the region SG may be to directly or indirectly bind the labeling substance to the DNA probe.

[0046] When directly binding, it is preferable to bind the DNA probe in advance before the hybridization step (I) described below, and direct binding can be carried out appropriately by a conventionally known method or a method based thereon.

[0047] In the case of indirect binding, the labeling substance can be indirectly bound to the DNA probe, for example, by binding via an intervening molecule that binds to the DNA probe. The intervening molecule is not particularly limited, and any conventionally known molecule can be used as appropriate. Alternatively, the region SG of the DNA probe may be modified in some way, and a substance that captures the modified portion may be bound to the labeling substance, thereby indirectly binding the labeling substance to the DNA probe. For example, a typical example of the modified portion is biotin, and a typical example of a substance that captures the modified portion is streptavidin or avidin, but these are not limited thereto.

[0048] (chimeric probe) The "chimeric probe" of the present invention is an oligonucleotide probe comprising, in this order, a base sequence D2 consisting of DNA complementary to the second region of the target RNA, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure.

[0049] The total length of the chimeric probe according to the present invention can be adjusted depending on the length of the target RNA, but is preferably 10 to 200 bases long, for example, and when the target RNA is the miRNA, is preferably 15 to 50 bases long.

[0050] [Base sequence D2] The base sequence D2 of the present invention is a base sequence that is complementary to the second region of the target RNA and is composed of DNA. Such base sequence D2 can be appropriately designed and prepared to match the base sequence of the second region of the target RNA of interest so as to satisfy the above-mentioned hybridization conditions, and more preferably so as not to hybridize to the DNA probe or other regions.

[0051] The length of the base sequence D2 according to the present invention is preferably 5 bases or more, more preferably 5 to 50 bases, and, for example, when the target RNA is the miRNA, preferably 5 to 20 bases. If the length of the base sequence D2 is less than the lower limit, hybridization with the second region of the target RNA tends to be difficult, whereas if the length exceeds the upper limit, non-specific reactions tend to occur more easily, or when the target RNA is a short-chain RNA, binding of the base sequence D1 tends to be difficult.

[0052] In the present invention, the total length of base sequence D2 and base sequence D1 is preferably 10 to 100 bases long, and when the target RNA is miRNA, for example, it is preferably 10 to 25 bases long. If the total length is less than the lower limit, hybridization with the target RNA or recognition by an anti-DNA / RNA chimeric antibody tends to be difficult. On the other hand, if the total length exceeds the upper limit, non-specific reactions tend to occur more easily.

[0053] Furthermore, in the present invention, the ratio of the length of base sequence D2 to the length of base sequence D1 is set depending on the base sequence of the target RNA, taking into consideration the length of the target RNA, the sequence complementarity between base sequence D1 and the first region, the sequence complementarity between base sequence D2 and the second region, and the balance between the Tm value obtained from base sequence D1 and the Tm value obtained from base sequence D2, and therefore cannot be generalized. For example, the ratio of the base length of base sequence D2 to the base length of base sequence D1 is preferably 1:9 to 9:1, and when the target RNA is the miRNA, for example, the ratio is preferably 1:4 to 4:1, and more preferably 1:3 to 4:1.

[0054] [Base sequence SD, linker, base sequence SR] The base sequence SD according to the present invention is a base sequence made of DNA, the base sequence SR is a base sequence made of RNA, and the linker is a region that connects the base sequence SD and the base sequence SR.

[0055] When the chimeric probe of the present invention forms a complex (410, 420) with a target RNA and a DNA probe, it contains the nucleotide sequence SD, a linker, and the nucleotide sequence SR, in this order, at the end of the nucleotide sequence D2 opposite to the end where the nucleotide sequence D1 is located, and the nucleotide sequence SD, the linker, and the nucleotide sequence SR form a stem-loop structure. Depending on the positions of the first and second regions on the target RNA and the DNA probe to be combined, the chimeric probe forming such a stem-loop structure may be in the form (310) of nucleotide sequence D1(311)-nucleotide sequence SD(312)-linker(313)-nucleotide sequence SR(314)-5' from the 3' end as shown in Figure 1, or in the form (320) of nucleotide sequence D1(321)-nucleotide sequence SD(322)-linker(323)-nucleotide sequence SR(324)-3' from the 5' end as shown in Figure 2.

[0056] The base sequence SD and the base sequence SR are complementary to each other and form a stem region of a DNA-RNA chimera in the stem-loop structure. The lengths of the base sequence SD and the base sequence SR are not particularly limited, but are preferably each independently 3 to 30 bases long. They are also preferably the same length. If the lengths of the base sequence SD and the base sequence SR are less than the lower limit, hybridization to each other tends to be difficult. On the other hand, if the lengths exceed the upper limit, nonspecific reactions tend to occur, or the anti-DNA-RNA chimera antibody may compete with the target complex, resulting in reduced reactivity.

[0057] The linker forms a loop region in the stem-loop structure that connects the base sequence SD and the base sequence SR in a hairpin loop shape. Such a linker is not particularly limited as long as it is a linear molecule capable of linking the base sequence SD and the base sequence SR. For example, oligonucleotides, oligopeptides, and other well-known linear molecules commonly referred to as linkers can be used. Among these, the linker of the present invention is preferably an oligonucleotide, because it can be easily produced in conjunction with the chemical synthesis of the oligonucleotides, the base sequence SD and the base sequence SR, when producing the chimeric probe. Examples of the oligonucleotide include a base sequence consisting of deoxyribonucleotides, a base sequence consisting of ribonucleotides, and a base sequence consisting of deoxyribonucleotides and ribonucleotides. Because the base sequence SD and the base sequence SR are connected by the linker, the stem structure can be easily formed, allowing RNA consisting of the base sequence SR to be added to the target RNA.

[0058] The length of the linker according to the present invention, when it is an oligonucleotide, is preferably 3 to 50 bases long, more preferably 5 to 20 bases long. If the linker length is less than the lower limit, it tends to be difficult to bend into a hairpin loop, while if it exceeds the upper limit, it tends to be difficult to form a stem structure because self-annealing does not occur preferentially.

[0059] The sequences of the base sequence SD, the linker, and the base sequence SR can be appropriately designed and prepared so that the base sequence SD and the base sequence SR satisfy the above-mentioned hybridization conditions, and more preferably so that they do not hybridize to the DNA probe or other regions.

[0060] (Capture body) The capture body of the present invention is a complex comprising a water-insoluble carrier and an anti-DNA / RNA chimeric antibody immobilized on the water-insoluble carrier, wherein the water-insoluble carrier and the anti-DNA / RNA chimeric antibody are bound directly or indirectly to each other.

[0061] [Non-water-soluble carrier] In the present invention, the "water-insoluble carrier" is not particularly limited as long as it is capable of immobilizing and supporting an anti-DNA / RNA chimeric antibody and is insoluble in water at room temperature and normal pressure, and examples thereof include water-insoluble carriers conventionally known in immunoassays and the like.

[0062] More specifically, examples of the non-water-soluble carrier include particle carriers such as latex particles such as polystyrene latex particles and polyethylene latex particles, metal colloid particles such as gold colloid particles, gelatin particles, and magnetic particles; plate carriers such as polystyrene well plates; and membrane carriers made of fibers such as nitrocellulose, polyethylene, polyethylene terephthalate, nylons, glass, and cellulose. Generally, any of artificial materials, natural materials, and mixtures thereof may be used as long as the material is a commonly used material. Among these, the non-water-soluble carrier according to the present invention is preferably a particle carrier, and more preferably a magnetic particle, from the viewpoint of ease of automation.

[0063] In the present invention, "supporting" is synonymous with solid-phase formation (also referred to as immobilization), and the method of supporting can be any conventionally known method or a method similar thereto, such as a physical adsorption method or a chemical bonding method, as appropriate.

[0064] [Anti-DNA / RNA chimeric antibody] In the present invention, the term "anti-DNA-RNA chimera antibody" refers to an antibody that specifically binds to a double-stranded nucleotide consisting of an oligo-RNA composed of ribonucleotides and an oligo-DNA composed of complementary deoxyribonucleotides (sometimes referred to as a "DNA-RNA chimera" in this specification).

[0065] In the present invention, the term "antibody" includes not only complete antibodies, but also antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, single-chain antibodies, diabodies, etc.) and minibodies formed by combining antibody variable regions. The anti-DNA / RNA chimeric antibody of the present invention may be either a polyclonal or monoclonal antibody, with monoclonal antibodies being more preferred. The anti-DNA / RNA chimeric antibody of the present invention can be produced by appropriately adapting and improving conventionally known production methods, and commercially available antibodies may also be used as appropriate.

[0066] The capture body of the present invention can be produced by binding the anti-DNA / RNA chimeric antibody to the water-insoluble carrier. As a production method, a conventionally known method or a method based thereon can be appropriately adopted, and the anti-DNA / RNA chimeric antibody may be bound to the water-insoluble carrier either directly or indirectly.

[0067] In the case of direct binding, for example, the water-insoluble carrier and / or the anti-DNA / RNA chimeric antibody may have an active group, or the active group may be added as necessary, allowing the anti-DNA / RNA chimeric antibody to be directly bound to the water-insoluble carrier via the active group. Furthermore, when the water-insoluble carrier is a plate, the anti-DNA / RNA chimeric antibody can be directly bound to the water-insoluble carrier by applying the anti-DNA / RNA chimeric antibody to the plate, blocking it as necessary, and then drying it.

[0068] Indirect binding can be achieved by, for example, binding the anti-DNA / RNA chimeric antibody to the water-insoluble support via an intervening molecule that binds to the anti-DNA / RNA chimeric antibody. The intervening molecule is not particularly limited, and examples include a secondary antibody capable of binding to the anti-DNA / RNA chimeric antibody, protein G, protein A, and a molecule having the active group. Alternatively, the anti-DNA / RNA chimeric antibody may be modified in some way, and a substance that captures the modified portion may be immobilized on the water-insoluble support, thereby immobilizing the anti-DNA / RNA chimeric antibody on the water-insoluble support. For example, a typical example of the modified portion is biotin, and a typical example of a substance that captures the modified portion is streptavidin or avidin, but these examples are not limited thereto. Furthermore, commercially available capture bodies may be used as appropriate.

[0069] (complex) As described above, the base sequence D1 of the DNA probe and the base sequence D2 of the chimeric probe are designed based on the base sequences of the first and second regions of the target RNA, respectively. Therefore, by hybridizing these, a complex consisting of the target RNA, the DNA probe, and the chimeric probe (target RNA-DNA probe-chimeric probe complex (sometimes simply referred to as "complex" in this specification)) is formed. When the first region is located on the 5'-end side of the target RNA, as shown in Figure 1 (b1), the base sequence D1 (211) complementary to the first region (111) hybridizes, and the base sequence D2 (311) complementary to the second region (112) hybridizes, thereby forming a complex (410) consisting of the target RNA (110), the DNA probe (210), and the chimeric probe (310). In this case, a stem-loop structure is formed at the 5'-end of the chimeric probe (310) by the nucleotide sequence SD (312), the linker (313), and the nucleotide sequence RD (314), and an RNA equivalent to the nucleotide sequence RD (314) is arranged at the 3'-end of the target RNA (110). When the first region is arranged at the 3'-end of the target RNA, the 5'-end and 3'-end are reversed from those in Figure 1, as shown in (b2) of Figure 2.

[0070] (Hybridization step (I)) The target RNA detection method of the present invention includes, as a first step, a hybridization step in which the DNA probe and the chimeric probe are hybridized to the target RNA.

[0071] In the hybridization step, the target RNA, the DNA probe, and the chimeric probe are contacted with each other to form the complex (target RNA-DNA probe-chimeric probe complex). The contacting method is not particularly limited, and the three may be contacted simultaneously, or the DNA probe and the chimeric probe may be added to the target RNA simultaneously or separately, or one may be added to the DNA probe or the chimeric probe simultaneously or separately.

[0072] A reaction buffer may be added to the reaction system of the hybridization step as appropriate. Examples of the reaction buffer include known buffers with a pH of 5 to 9 (e.g., sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and may also contain surfactants, salts, preservatives, stabilizers (e.g., Mg), proteins, etc. as appropriate.

[0073] In the hybridization step, the amounts of the DNA probe and the chimeric probe used are each independently preferably 10 fM to 10 mM, and more preferably 10 pM to 10 μM in the reaction system.

[0074] In the present invention, the temperature for the hybridization step is, for example, in the range of 15 to 60° C. However, the hybridization step according to the present invention can be carried out at a relatively low temperature that does not require heating, and such a temperature is, for example, in the range of 25 to 40° C. The reaction time is not particularly limited, and may be, for example, in the range of 5 to 60 minutes.

[0075] (Capture step (II)) The target RNA detection method of the present invention includes, as a second step, a capture step of capturing the complex (target RNA-DNA probe-chimeric probe complex) with the capture body.

[0076] In the capture step, the complex is brought into contact with the capture body, and the complex is captured on the water-insoluble carrier via binding between the DNA-RNA chimera in the complex and the anti-DNA-RNA chimera antibody, thereby solidifying the complex. The contact method is not particularly limited; the hybridized complex (e.g., a reaction solution) may be added to the capture body, or the capture body (e.g., a particle suspension) may be added to the complex. Furthermore, since the hybridization step and the capture step can be performed simultaneously as described below, the hybridization step may be performed in the presence of the capture body. An appropriate reaction buffer may be added to the reaction system of the capture step. Examples of the reaction buffer include the same buffers as those listed for the hybridization step.

[0077] In the present invention, the temperature of the capture step is not particularly limited and may be, for example, in the range of 25 to 40°C. The reaction time is also not particularly limited and may be, for example, in the range of 5 to 60 minutes. In the target RNA detection method of the present invention, the temperature of the hybridization step and the temperature of the capture step can be made isothermal, which is preferable from the viewpoint of simplicity. Therefore, the hybridization step and the capture step can be performed simultaneously under the same conditions.

[0078] (Cleaning process) The target RNA detection method of the present invention preferably further includes a washing step following the capture step, in which the complex captured by the capture body is separated from other contaminants not bound to (captured by) the capture body and the contaminants are removed. The method for removing the contaminants is not particularly limited, and any conventionally known method or a method based thereon can be used as appropriate. For example, if the capture body is an antibody-immobilized plate, the liquid phase (supernatant) can be removed from the plate. If the capture body is antibody-immobilized particles, the particles can be recovered by centrifugation or magnetic collection, and the liquid phase (supernatant) can be removed. Furthermore, in the washing step, the injection and removal of a washing solution can be repeated as necessary.

[0079] Examples of the washing solution include known buffer solutions having a pH of 5 to 9 (e.g., sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and may also contain surfactants, salts, preservatives, stabilizers (e.g., Mg), proteins, etc., as appropriate.

[0080] (Detection step (III)) The target RNA detection method of the present invention includes, as a third step, a detection step of detecting the complex captured by the capturer.

[0081] In the detection step, a signal derived from the labeling substance is detected. For example, when the labeling substance is an enzyme, a chromogenic or luminescent substrate corresponding to the enzyme is added and reacted to detect the signal generated. When the DNA probe is one to which the labeling substance has not been pre-bound, a labeling substance capable of binding to region SG of the DNA probe may be added in this detection step. For example, when region SG is a biotinylated site, an example of such a labeling substance is a streptavidin (or avidin)-labeled labeling substance.

[0082] This allows a signal corresponding to the labeling substance to be detected, and the presence or absence of the complex, i.e., the presence or absence of the target RNA in the sample, can be detected based on the presence or absence of the signal. In the present invention, the term "signal" includes color development, reflected light, luminescence, quenching, fluorescence, radiation from radioisotopes, etc., and includes signals that can be confirmed with the naked eye as well as signals that can be confirmed using a measurement method or device appropriate for the type of signal. For example, when a bioluminescent enzyme such as luciferase is used as the labeling substance, luminescence generated by the addition of a substrate such as luciferin can be detected as the signal.

[0083] Furthermore, when target RNA is present in the sample, the amount of target RNA in the sample can be obtained as a signal amount (e.g., luminescence intensity), and if necessary, the amount of target RNA in the sample can be quantified by comparing it with the signal amount in a standard sample.

[0084] <Probe Kit> The present invention also provides a probe kit that can be suitably used in the above-described method for detecting a target RNA of the present invention. a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region on the 5'-end or 3'-end of a single-stranded target RNA, and a region SG to which a labeling substance can be bound; a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to a second region that includes the other end of the first region side of the target RNA and does not overlap with the first region, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure; The kit includes:

[0085] It is preferable that the probe kit of the present invention further comprises at least one selected from the group consisting of a labeling substance capable of binding to the region SG of the DNA probe, and a capture body comprising a water-insoluble carrier and an anti-DNA / RNA chimeric antibody immobilized on the water-insoluble carrier.

[0086] In the probe kit of the present invention, the target RNA, DNA probe, chimeric probe, labeling substance, and capture body, including preferred embodiments thereof, are each as described in the target RNA detection method of the present invention.

[0087] In the probe kit of the present invention, other components such as a buffer solution, a blocking agent, a preservative, and an antiseptic may be added to the target RNA, the DNA probe, the chimeric probe, the labeling substance, and the capture body (e.g., antibody-immobilized particles).

[0088] Furthermore, the probe kit of the present invention may further include the diluent, the reaction buffer, the washing solution, reagents for extracting and purifying target RNA, reagents necessary for detecting each labeled substance (e.g., substrates necessary for each enzymatic reaction), other buffer solutions, reagents such as pH adjusters, standard RNA, control reagents, instructions for use, etc. [Example]

[0089] The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. The target RNA, DNA probe, and chimeric probe used in each example and comparative example were as follows, respectively.

[0090] <mir6875-5p> (1) As target RNA1, miR6875-5p (21 bases long, SEQ ID NO: 1) shown in Table 1 below was synthesized by a conventional method and used.

[0091] (2) The DNA probes used were synthesized by standard methods based on the base sequence of target RNA1: a DNA probe hybridizing to the entire length of target RNA1 (miR6875-5p-full length, SEQ ID NO: 2); a DNA probe hybridizing to the 14 bases at the 3' end of target RNA1 (miR6875-5p-short (14 nt), SEQ ID NO: 3); a DNA probe hybridizing to the 13 bases at the 3' end of target RNA1 (miR6875-5p-short (13 nt), SEQ ID NO: 4); and a DNA probe hybridizing to the 12 bases at the 3' end of target RNA (miR6875-5p-short (12 nt), SEQ ID NO: 5). Each of these DNA probes had poly(A) and biotin added to the 5' end.

[0092] (3) The chimeric probe used was a chimeric probe (miR-6875-5p-Chimera(7nt) (serving as miR-6875-5p-Chimera(7nt)A LOOP5nt and miR-6875-5p-Chimera(7nt)stem8nt), SEQ ID NO: 6) synthesized by standard methods. This probe hybridizes to the 7 bases at the 5' end of target RNA1 based on its base sequence and forms a stem-loop including the stem region of the DNA / RNA chimera (i.e., forming the structure 320 in Figure 2(a2)). Furthermore, chimeric probes were also used, synthesized by standard methods, in which the base length of miR-6875-5p-Chimera (7 nt) was increased to 8 bases (miR-6875-5p-Chimera (8 nt), SEQ ID NO: 7) and the base length was increased to 9 bases (miR-6875-5p-Chimera (9 nt), SEQ ID NO: 8) that hybridized with the base sequence at the 5' end of target RNA1.

[0093] (4) In addition, chimeric probes synthesized by standard methods were also used: a chimeric probe in which the number of A's in the loop portion (A × 5) of miR-6875-5p-Chimera (7 nt) was 10 (miR-6875-5p-Chimera (7 nt) A LOOP 10 nt, SEQ ID NO: 9); and a chimeric probe in which the number of A's was 20 (miR-6875-5p-Chimera (7 nt) A LOOP 20 nt, SEQ ID NO: 10).

[0094] (5) Furthermore, a chimeric probe (miR-6875-5p-Chimera (7 nt) stem 10 nt, sequence number 19) was also used, synthesized by standard methods, in which the number of G and C in the stem portion (G × 8, C × 8) of miR-6875-5p-Chimera (7 nt) was 10 bases each.

[0095] <mir149-3p> (1) As target RNA2, miR149-3p (miR149, 21 bases long, SEQ ID NO: 11) shown in Table 1 below was synthesized by a conventional method and used.

[0096] (2) The DNA probes used were synthesized by standard methods based on the base sequence of target RNA 2: a DNA probe (miR149-full length, SEQ ID NO: 12) that hybridizes to the entire length of target RNA 2, and a DNA probe (miR149-short, SEQ ID NO: 13) that hybridizes to the 14 bases at the 3' end of target RNA 2. Each of these DNA probes had poly(A) and biotin added to the 5' end.

[0097] (3) The chimeric probe used was a chimeric probe (miR149-Chimera, sequence number 14) synthesized by conventional methods. This probe hybridizes to the 7 bases at the 5' end of target RNA2 based on its base sequence and forms a stem-loop containing the stem region of the DNA / RNA chimera (i.e., forms the structure 320 in Figure 2(a2)).

[0098] <mir1268b> (1) As target RNA3, miR1268b (20 bases long, SEQ ID NO: 15) shown in Table 1 below was synthesized by a conventional method and used.

[0099] (2) The DNA probes used were synthesized by standard methods based on the base sequence of target RNA 3. These probes hybridize to the entire length of target RNA 3 (miR1268b-full length, SEQ ID NO: 16) and to the 13 bases at the 3' end of target RNA 3 (miR1268b-short, SEQ ID NO: 17). Each of these DNA probes had poly(A) and biotin added to the 5' end.

[0100] (3) The chimeric probe used was a chimeric probe (miR1268b-Chimera, sequence number 18) synthesized by conventional methods. This probe hybridizes to the 7 bases at the 5' end of target RNA3 based on its base sequence and forms a stem-loop containing the stem region of the DNA / RNA chimera (i.e., forms the structure 320 in Figure 2(a2)).

[0101] [Table 1]

[0102] (Test Example 1) Confirmation of the effect of the chimeric probe by bioluminescent enzyme immunoassay (1) First, an anti-DNA / RNA antibody was immobilized on magnetic particles for immobilizing antibodies by a known method, and then diluted with a phosphate buffer containing sodium azide to prepare a 1.5 mg / mL suspension of the magnetic particles.

[0103] (2) Then, each target RNA was diluted to 1 × 10 6 or 1 x 10 7 A hybridization solution containing 100 cp / μL of each probe and 10 nM of each probe was prepared by mixing them at room temperature (approximately 25°C). The target RNAs and probes were used in the combinations shown in Table 2 below, and when a DNA probe and a chimeric probe were combined, the hybridization solution was prepared so that the concentration of each was 10 nM. Next, 200 μL of the hybridization solution, 20 μL of the antibody-immobilized magnetic particle suspension, and 40 μL of buffer (Tris-HCl (pH 8.0) solution containing salt and surfactant) were mixed and reacted at 37°C for 15 minutes.

[0104] (3) Hereinafter, target RNA was detected using the fully automated biochemiluminescence immunoassay device "BLEIA (registered trademark) 'Eiken' H. pylori antigen (manufactured by Eiken Chemical Co., Ltd.)" in accordance with the protocol attached to "BLEIA (registered trademark) 'Eiken' H. pylori antigen (manufactured by Eiken Chemical Co., Ltd.)."

[0105] That is, first, after the reaction in (2) above, the solid phase containing the magnetic particles was washed five times with the washing solution for BLEIA (registered trademark)-1200, and after removing the washing solution, 80 μL of labeled streptavidin for BLEIA (registered trademark)-1200 was added, stirred, and reacted at 37°C for 15 minutes.

[0106] The solid phase containing the magnetic particles was then washed five times with the washing solution, after which the washing solution was removed. 50 μL of BLEIA®-1200 BL Luminescence Reagent 1 was added and stirred, followed by 50 μL of BLEIA®-1200 BL Luminescence Substrate, and the luminescence intensity (PC) of luciferase at wavelengths of 480 to 650 nm (λmax: 560 nm) was measured. Furthermore, for each target RNA and probe combination, the luminescence intensity was measured in the same manner as above, except that no target RNA was added, and this was used as a control (NC).

[0107] (4) For each combination of target RNA and probe (Examples 1 to 5, Comparative Examples 1 to 3), the S / N ratio was calculated using the following formula: S / N ratio = PC / NC. The S / N ratio (vs. Comparative Examples) was also calculated for the combination of the DNA probe and chimeric probe (Examples) compared to the combination of the DNA probe and chimeric probe (Comparative Examples). The results are shown in Table 2 below.

[0108] [Table 2]

[0109] As shown in Table 2, when the DNA probe and chimeric probe were used in combination (Examples 1 to 5), the S / N ratio, which indicates the detection intensity, was significantly higher for each target RNA, approximately 12 times higher for target RNA 1 (miR6875-5p), approximately 96 times higher for target RNA 2 (miR149-3p), and approximately 23 times higher for target RNA 3 (miR1268b), compared to when the DNA probe was used alone (Comparative Examples 1 to 3). This confirmed that the use of a DNA probe and chimeric probe in combination enables highly sensitive detection of miRNAs.

[0110] (Test Example 2) Quantitative confirmation using chimeric probes Quantitativeness when using a chimeric probe was confirmed by the same method as in Test Example 1. Specifically, target RNA1 (miR6875-5p) was added at 1 × 10 4 ~1×10 9 Except for adjusting the concentration of each compound to cp / μL, the luminescence intensities (PC and NC) were measured and the S / N ratios calculated in the same manner as in Comparative Example 1 and Example 3 of Test Example 1. The results are shown in Table 3 below.

[0111] [Table 3]

[0112] As shown in Table 3, when the DNA probe and the chimeric probe were used in combination (Example 3), the S / N ratio increased depending on the concentration of target RNA1, as was the case when the DNA probe was used alone (Comparative Example 1).

[0113] (Test Example 3) Examination of the length of the loop region of the chimeric probe by bioluminescent enzyme immunoassay The length of the loop region (A LOOP) of the chimeric probe was examined by the same method as in Test Example 1. Specifically, target RNA1 (miR6875-5p) was added to 1 × 10 6 Hybridization solutions containing 10 cp / μL and 10 nM of each probe were prepared, and the luminescence intensities (PC and NC) were measured and the S / N ratios calculated in the same manner as in Test Example 1, except that the probes were used in the combinations shown in Table 4 below. The S / N ratios (relative to the comparative examples) when the DNA probe and the chimeric probe were used in combination (Examples 6 to 8) were calculated relative to the S / N ratios when the DNA probe was used alone (Comparative Example 4). The results are shown in Table 4 below.

[0114] [Table 4]

[0115] As shown in Table 4, similarly to Test Example 1, when the DNA probe and chimeric probe were used in combination (Examples 6 to 8), the S / N ratio was higher than when the DNA probe was used alone (Comparative Example 4). Among these, the highest S / N ratio was obtained when the loop region (A LOOP) of the chimeric probe was 5 bases long (Example 6). This is presumably because a shorter loop region makes it easier to form a stem region.

[0116] (Test Example 4) Stem length examination by bioluminescent enzyme immunoassay The length of the stem region of the chimeric probe was examined by the same method as in Test Example 1. Specifically, target RNA1 (miR6875-5p) was added to 1 × 10 6 Hybridization solutions containing 10 cp / μL and 10 nM of each probe were prepared, and the luminescence intensities (PC and NC) were measured and the S / N ratios calculated in the same manner as in Test Example 1, except that the probes were used in the combinations shown in Table 5 below. The S / N ratios (relative to the comparative examples) were also calculated for the cases where the DNA probe and the chimeric probe were used in combination (Examples 9 and 10) relative to the case where the DNA probe was used alone (Comparative Example 4). The results are shown in Table 5 below.

[0117] [Table 5]

[0118] As shown in Table 5, similar to Test Example 1, when the DNA probe and chimeric probe were used in combination (Examples 9-10), the S / N ratio was higher in all cases than when the DNA probe was used alone (Comparative Example 4), and was not affected by the length (number of bases) of the stem region.

[0119] (Test Example 5) Confirmation of the effect of the chimeric probe by enzyme immunoassay Target RNA was detected according to the protocol included with the "miREIA-miRNA (microRNA) Enzyme Immunoassay Kit (BioVendor)." Specifically, measurements were performed using the kit according to the protocol, except that target RNA3 (miR1268b) was used as the miRNA and the biotinylated DNA probes were the DNA and chimeric probes in the combinations listed in Table 6 below. Specifically, the probes were preheated in a thermal cycler at 85°C for 3 minutes, 4°C for 2 minutes, and 37°C for 5 minutes, followed by incubation on an antibody-coated plate. The antibody-coated plate was then washed, incubated with streptavidin-HPR (horseradish peroxidase) conjugate, washed again, and TMB substrate was added to measure the luminescence intensity. Each probe was added to a concentration of 0.17 μM when preparing the DNA probe working solution.

[0120] The luminescence intensity was measured at wavelengths of 450 nm and 630 nm, and the difference between these values ​​(luminescence intensity at wavelength 450 nm - luminescence intensity at wavelength 630 nm) was defined as PC. A control (NC) value was obtained in the same manner as above, except that no target RNA was added. Furthermore, the S / N ratio was calculated using the following formula: S / N ratio = PC / NC. The S / N ratio (relative to the comparative example) was also calculated when the DNA probe and chimeric probe were used in combination (Example 11) compared to when the DNA probe was used alone (Comparative Example 5). The results are shown in Table 6 below.

[0121] [Table 6]

[0122] As shown in Table 6, even with enzyme immunoassay, the S / N ratio was lower than that of bioluminescent enzyme immunoassay without preheating, but when the DNA probe was used alone (Comparative Example 5), i.e., when the DNA probe and chimeric probe were used in combination (Example 11), the S / N ratio was higher than that of the method in which the DNA-RNA complex was detected by ELISA using a conventional anti-DNA / RNA antibody. This confirmed that the use of a DNA probe and chimeric probe in combination by enzyme immunoassay also enables highly sensitive detection of miRNA. [Industrial Applicability]

[0123] As described above, the present invention makes it possible to provide a target RNA detection method that can detect even short-stranded RNAs such as miRNAs with high sensitivity and in a simple manner, as well as a probe kit that can be suitably used for this method. [Explanation of symbols]

[0124] 110, 120...target RNA, 111, 121...first region, 112, 122...second region, 210, 220...DNA probe, 211, 221...base sequence D1, 212, 222...region SG, 310, 320...chimeric probe, 311, 321...base sequence D2, 312, 322...base sequence SD, 313, 323...linker, 314, 324...base sequence RD, 410, 420...target RNA-DNA probe-chimeric probe complex. [Sequence List Free Text]

[0125] SEQ ID NO:2 <223> miR6875-5p-full length SEQ ID NO:3 <223> miR6875-5p-short(14nt) SEQ ID NO:4 <223> miR6875-5p-short(13nt) SEQ ID NO:5 <223> miR6875-5p-short(12nt) SEQ ID NO:6 <223> miR-6875-5p-Chimera(7nt) <223> 21-28 indicate RNA SEQ ID NO:7 <223> miR-6875-5p-Chimera(8nt) <223> 22-29 indicate RNA SEQ ID NO:8 <223> miR-6875-5p-Chimera(9nt) <223> 23-30 indicate RNA SEQ ID NO:9 <223> miR-6875-5p-Chimera(7nt)A LOOP10nt <223> 26-33 indicate RNA SEQ ID NO:10 <223> miR-6875-5p-Chimera(7nt)A LOOP20nt <223> 36-43 indicate RNA SEQ ID NO:12 <223> miR149-full length SEQ ID NO:13 <223> miR149-short SEQ ID NO:14 <223> miR149-Chimera <223> 21-28 indicate RNA SEQ ID NO:16 <223> miR1268b-full length SEQ ID NO:17 <223> miR1268b-short SEQ ID NO:18 <223> miR1268b-Chimera <223> 21-28 indicate RNA SEQ ID NO:19 <223> miR-6875-5p-Chimera(7nt) stem10nt <223> 23-32 indicate RNA

Claims

1. 1. A method for detecting a target RNA, comprising: (I) a first region on the 5'-end or 3'-end side of a single-stranded target RNA, and a second region including the other end of the first region and not overlapping with the first region; a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region of the target RNA and a region SG to which a labeling substance can be bound; and a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to the second region of the target RNA, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure. a hybridization step in which the (II) a capturing step of capturing a complex of the target RNA, the DNA probe, and the chimeric probe with a capturing body comprising a water-insoluble carrier and an anti-DNA / RNA chimeric antibody immobilized on the water-insoluble carrier; (III) a detection step of detecting the complex captured by the capturer; Including, the length of the first region is 5 to 50 bases; the second region is 5 to 50 bases in length; the first region and the second region are adjacent to each other, In the DNA probe, when the DNA probe forms a complex with the target RNA and the chimeric probe, the region SG is located at an end of the base sequence D1 opposite to the end where the base sequence D2 is located. A method for detecting a target RNA, comprising:

2. 2. The method for detecting a target RNA according to claim 1, wherein the detection step is a step of detecting the complex using a signal derived from a labeling substance bound to the region SG as an indicator.

3. 3. The method for detecting a target RNA according to claim 1, wherein the target RNA is a single-stranded RNA molecule having a length of 19 to 25 bases.

4. The method for detecting a target RNA according to any one of claims 1 to 3, wherein the linker is a base sequence consisting of DNA and / or RNA.

5. 5. The method for detecting a target RNA according to claim 4, wherein the linker has a length of 3 to 50 bases.

6. 6. The method for detecting a target RNA according to claim 1, wherein the length of the base sequence SD and the length of the base sequence SR are each 3 to 30 bases long.

7. The method for detecting a target RNA according to any one of claims 1 to 6, wherein the temperature of the hybridization step is 15 to 60°C.

8. The method for detecting a target RNA according to any one of claims 1 to 7, wherein the water-insoluble carrier is a particulate carrier.

9. a DNA probe comprising a base sequence D1 consisting of DNA complementary to a first region on the 5'-end or 3'-end of a single-stranded target RNA, and a region SG to which a labeling substance can be bound; a chimeric probe comprising, in this order, a base sequence D2 consisting of DNA complementary to a second region that includes the other end of the first region side of the target RNA and does not overlap with the first region, a base sequence SD consisting of DNA, a linker, and a base sequence SR consisting of RNA, wherein the base sequence SD and the base sequence SR are complementary to each other, and the base sequence SD, the linker, and the base sequence SR form a stem-loop structure; Including, the length of the first region is 5 to 50 bases; the second region is 5 to 50 bases in length; the first region and the second region are adjacent to each other; In the DNA probe, when the DNA probe forms a complex with the target RNA and the chimeric probe, the region SG is located at an end of the base sequence D1 opposite to the end where the base sequence D2 is located. A probe kit comprising:

10. The probe kit according to claim 9, further comprising a labeling substance capable of binding to the region SG of the DNA probe.

11. 11. The probe kit according to claim 9, wherein the total length of the base sequence D1 and the base sequence D2 is 10 to 100 bases long.

12. The probe kit according to any one of claims 9 to 11, wherein the linker is a base sequence made of DNA and / or RNA.

13. The probe kit according to claim 12, wherein the linker has a length of 3 to 50 bases.

14. The probe kit according to any one of claims 9 to 13, wherein the length of the base sequence SD and the length of the base sequence SR are each 3 to 30 bases long.

15. The probe kit according to any one of claims 9 to 14, further comprising a capture body comprising a water-insoluble carrier and an anti-DNA / RNA chimeric antibody immobilized on the water-insoluble carrier.

16. The probe kit according to claim 15, wherein the water-insoluble carrier is a particle carrier.

17. The probe kit according to any one of claims 9 to 16, characterized in that it is a kit for use in the method for detecting a target RNA according to any one of claims 1 to 8.

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