Composition for solid phase attachment, solid phase carrier utilizing said composition, and method for producing and using said solid phase carrier

Abstract

The present invention addresses the problem of establishing a means for, when a gene amplification solution is contacted directly with a solid phase part, on which insoluble support particles are immobilized, and then developed on the solid phase to thereby perform detection, suppressing a lowering of the detection sensitivity of a conjugate of a nucleic acid to be detected and the insoluble support particles. The present inventors found that such a large lowering of the detection sensitivity as described above can be suppressed by using a composition, said composition containing insoluble support particles and being to be attached to a solid phase, comprising component (1) that is a water-soluble amphoteric surfactant or a water-soluble nonionic surfactant and component (2) that is an aqueous solvent. The present invention provides: (A) a composition to be attached to a solid phase, said composition containing insoluble support particles formed of, for example, latex; (B) a method for producing a solid phase support using the composition according to the present invention; (C) a solid phase support provided with a sample-contact part containing the composition to be attached to a solid phase from which water has been substantially removed; and (D) a method for using the solid phase support.

Description

[Technical field]

[0001] The present invention relates to a solid phase carrier for detecting a specific substance. In particular, the present invention relates to a solid phase carrier having a sample contact portion that can be directly contacted with a sample for detection of nucleic acid, a method for using the solid phase carrier, a method for producing the solid phase carrier, and a composition for attaching to a solid phase for providing the sample contact portion of the solid phase carrier. [Background technology]

[0002] In recent years, detection of specific nucleic acids as targets and association with the attributes of those nucleic acids have been used for a variety of purposes, including detection of diseases, identification of plant and animal varieties, identification of food quality and origin, parent-child testing, and proof of crime.

[0003] This nucleic acid detection technology has developed rapidly as nucleic acid amplification methods such as PCR have made it possible to easily amplify what are normally minute quantities of nucleic acid to a detectable amount, and a variety of detection methods are available to date.

[0004] Among these, in order to achieve both simplification and high accuracy in nucleic acid detection, techniques for detecting nucleic acids on a solid phase have been provided (Patent Documents 1, 2, and 3).

[0005] The technology of Patent Document 1 is a nucleic acid detection method for detecting double-stranded DNA amplification products obtained by a nucleic acid amplification method, the amplification products having binding sites for specific substances, and the DNA amplification products are concentrated by binding to the specific substances, thereby detecting the desired nucleic acid. The technology disclosed in Patent Document 1 is mainly performed on a development medium (solid phase).

[0006] The technology of Patent Document 2 aims to improve the yield of genuine nucleic acid amplification products produced by introducing a site capable of inhibiting or halting the progress of a polymerase reaction into a part of a primer used to produce a nucleic acid amplification product, thereby improving the sensitivity of solid-phase detection of a target nucleic acid.

[0007] The technology of Patent Document 3 is a method in which a target nucleic acid discrimination probe having a labeled region that specifically hybridizes with the nucleic acid extension region of a genuine nucleic acid amplification product is used in a nucleic acid amplification reaction in order to further improve the yield of producing genuine nucleic acid amplified fragments. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. WO2009 / 034842 [Patent Document 2] International Publication No. WO2013 / 038534 [Patent Document 3] International Publication No. WO2016 / 129609 Summary of the Invention [Problem to be solved by the invention]

[0009] The techniques of Patent Documents 1 to 3 show progress in solid-phase nucleic acid detection technology, and each of them improves the yield of target nucleic acid (target nucleic acid to be detected) amplification. In general, detection of target nucleic acid (target nucleic acid to be detected) on a solid phase is excellent in terms of convenience in that it does not require special facilities or devices, but there are still problems in terms of detection sensitivity.

[0010] To address this issue, a "development sample solution" consisting of a mixture of the amplification reaction solution, the development solution, and insoluble carrier particles such as latex is first prepared in a tube outside the solid phase, and the solid phase (strip) is then immersed in this, causing the complex between the nucleic acid to be detected and the insoluble carrier particles to diffuse and move to the detection area on the solid phase, generating a detection signal (for example, see the examples in Patent Document 3).

[0011] However, this embodiment is cumbersome in practice. Therefore, the present inventors aimed to establish a simplified means for detection by directly contacting the amplification reaction solution with the solid phase portion on which insoluble carrier particles are immobilized and developing the solution on the solid phase. [Means for solving the problem]

[0012] The present inventors have further studied to solve the above problems, and have found that by using a composition containing insoluble carrier particles such as latex and adhering it to a solid phase with a predetermined composition, it is possible to suppress a decrease in detection sensitivity and sufficiently obtain a desired detection signal, even when the solid phase is directly immersed in an amplification reaction solution to cause the above-mentioned bound substance to migrate by diffusion.

[0013] The present invention provides: (A) a composition for containing insoluble carrier particles such as latex and adhering them to a solid phase (the composition of the present invention); (B) a method for producing a solid phase carrier using the composition of the present invention (the production method of the present invention); (C) a solid phase carrier having a sample contact portion containing the composition for solid phase attachment from which moisture has been substantially removed (the solid phase carrier of the present invention); and (D) a method for using the solid phase carrier (the use method of the present invention).

[0014] (A) Composition of the Present Invention The composition of the present invention contains the following components (1) and (2), and is used for attaching insoluble carrier particles capable of binding to a nucleic acid to be detected to a solid phase. (1) A water-soluble amphoteric surfactant or a water-soluble nonionic surfactant, (2) Aqueous Solvent

[0015] In the composition of the present invention, the surfactant (1) is a water-soluble amphoteric surfactant, particularly a water-soluble amphoteric surfactant having cholic acid as the base nucleus, thereby enabling the solid phase carrier of the present invention to be stored at room temperature.

[0016] Furthermore, the composition of the present invention may additionally contain (3) urea or a salt thereof, and may contain, together with or separately from (3), (4) a sugar and / or (5) an amino acid.

[0017] The "solid phase" to which the composition of the present invention is applied is a solid phase carrier for performing chromatography by developing with a solvent as a mobile phase. The composition of the present invention is a composition for attaching "insoluble carrier particles to which a nucleic acid to be detected can be bound" to the solid phase. By using the composition of the present invention, it is possible to suppress a decrease in the sensitivity of chromatography after development with the solvent.

[0018] (B) Production method of the present invention The production method of the present invention is a method for producing a solid phase for nucleic acid detection, which comprises mixing the composition of the present invention with insoluble carrier particles to which a nucleic acid to be detected can bind, attaching the mixture to a solid phase, and drying.

[0019] (C) Solid Phase Carrier of the Present Invention The solid phase carrier of the present invention is a solid phase carrier having a sample contact portion and a detection portion, in which "insoluble carrier particles to which component (1) of the composition of the present invention and the nucleic acid to be detected can bind" are attached to the sample contact portion, and "a substance that generates a signal upon contact with the bound product of the nucleic acid to be detected and the insoluble carrier particles migrating in the solid phase carrier" is attached to the detection portion.

[0020] The solid phase carrier of the present invention is a solid phase carrier for performing chromatography by developing a solvent as a mobile phase. The sample contact portion is a mechanism on the solid phase carrier for contacting a sample.

[0021] (D) Method of Use of the Invention The method of use of the present invention is a method of using a solid phase carrier in which "insoluble carrier particles to which component (1) of the composition of the present invention and the nucleic acid to be detected can bind" are attached to the sample contact area, and a substance is attached to the detection area which generates a signal upon contact with the bound complex of the nucleic acid to be detected and insoluble carrier particles that migrates on the solid phase carrier, in which a sample which may contain the nucleic acid to be detected is contacted with the sample contact area, and a signal of the nucleic acid to be detected that is generated by contact between the bound complex of the nucleic acid to be detected and insoluble carrier particles that migrates from the sample contact area on the solid phase carrier and the detection area is detected.

[0022] By carrying out the method of use of the present invention, it is possible to suppress the decrease in detection sensitivity of the signal due to the movement of the sample on the solid phase carrier, as compared with the conventional method. This suppression of the decrease in detection sensitivity is due to the use of the composition of the present invention when preparing the sample contact portion of the solid phase carrier as described above. Effect of the Invention

[0023] The present invention provides a solid phase carrier having a sample contact portion that can be directly contacted with a sample for detection of nucleic acid, a method for using the solid phase carrier, a method for producing the solid phase carrier, and a solid phase attachment composition for providing the sample contact portion of the solid phase carrier. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] (A) Composition of the Present Invention As described above, the composition of the present invention is a composition for attaching insoluble carrier particles to which a nucleic acid to be detected can bind to a solid phase, which can contain the following components (1) and (2) in addition to (3) urea or a salt thereof, and can contain, together with or separately from (3), (4) a sugar and / or (5) an amino acid.

[0025] (I) Insoluble carrier particles to which the nucleic acid to be detected can bind The insoluble carrier particles to which the target nucleic acid can bind are insoluble carrier particles that have the function of being able to bind the target nucleic acid in a sample that comes into contact with the solid phase carrier of the present invention (hereinafter also referred to as "carrier particles for binding").

[0026] Examples of insoluble carrier particles that are the basis of the carrier particles for binding include latex particles, silica particles, metal colloid particles, etc. Metal colloids include gold colloids, silver colloids, copper colloids, etc., with gold colloids being preferred. The particle size of the metal colloid is not particularly limited and is generally in the range of 1-50 nm. The metal colloid particles aggregate in the detection portion of the solid phase carrier of the present invention, and can be easily visually detected as a color signal resulting from the accumulation of color.

[0027] Latex is a preferred embodiment of the insoluble carrier particles. Latex is also called a polymer emulsion, and is a dispersion of a polymer in an aqueous solvent such as water, with the aqueous solvent being the continuous phase and spherical or nearly spherical polymer particles being the discontinuous phase. Latex particles are polymer particles that form the discontinuous phase of this latex. In this specification, "latex" is used as a general expression including latex particles.

[0028] Examples of the latex include latexes for physical adsorption such as polystyrene latex, very low carboxylic acid modified latex, and hydrophilic group localized latex, latexes for chemical bonding such as carboxylic acid modified latex, amino modified latex, hydroxyl modified latex, glycidyl modified latex, aldehyde modified latex, and amide modified latex, colored latex, high specific gravity polystyrene latex, magnetic latex, etc. These latexes can be used in the present invention depending on their respective characteristics.

[0029] One of the latexes suitable for the present invention is colored latex that is colored in blue, red, green, orange, etc. When the colored latex aggregates at the detection portion of the solid phase carrier of the present invention, the accumulated color can be easily detected visually as a color signal.

[0030] The particle size of the latex particles is not particularly limited, and can be selected broadly from an average particle size of about 0.01 to 1 μm.

[0031] The silica particles are preferably colored in the same manner as latex. When the colored silica particles aggregate at the detection portion of the solid phase carrier of the present invention, the accumulated color can be easily detected by visual observation as a color signal.

[0032] The particle size of the silica particles is not particularly limited and can be broadly selected from the average particle size range of about 1 nm to 2 μm.

[0033] The insoluble carrier particles have a function to which the target nucleic acid can bind. The function is specified in combination with the binding function of the target nucleic acid. That is, the binding function of the insoluble carrier particles and the binding function of the target nucleic acid described below (hereinafter also referred to as "binding function to carrier particles") act as a pair, and the binding function of the insoluble carrier particles is the first part of the pair.

[0034] Examples of binding functions acting as this pair include hydrogen bonds, ionic bonds, electrostatic bonds, hydrophobic bonds, physical mutual bonds, etc., and specific examples include organic compound-organic compound interactions, protein-protein interactions, protein-nucleotide interactions, nucleotide-nucleotide interactions, organic compound-protein interactions, etc. Furthermore, more specific pair elements include a binding function based on the avidin (streptavidin)-biotin system, a binding function between nucleic acids using a tag base sequence, etc., a binding function between FITC and anti-FITC, anti-digoxigenin (DIG)-digoxigenin (DIG), a binding function based on an antigen-antibody reaction between a tag amino acid sequence and a specific antibody, etc.

[0035] The insoluble carrier particles may have a binding function (hereinafter also referred to as "binding function to detection part") that pairs with the binding function in the detection part described below, separately from the binding function to the nucleic acid to be detected. The binding function to the detection part of the insoluble carrier particles may be the same as the binding function to the nucleic acid to be detected.

[0036] join for The carrier particle has either or both of the above binding functionalities as the first moiety.

[0037] The addition of the first portion to the insoluble carrier particles can be carried out using a conventional method depending on the type of insoluble carrier particles and the portion in question, thereby preparing carrier particles for binding.

[0038] The carrier particles for binding are preferably mixed with the composition of the present invention when used.The amount of carrier particles for binding added to the composition of the present invention can be freely selected according to the type of carrier particles and other contents of the measurement system, and is not particularly limited, but the content in the composition is usually 0.01-2% by mass, preferably 0.02-1% by mass.For example, in the case of the colored latex particles used in the examples, the content is preferably 0.01-1% by mass, more preferably 0.02-0.5% by mass, and particularly preferably 0.04-0.2% by mass.

[0039] (II) Components of the Composition of the Invention (1) Water-soluble amphoteric surfactant or water-soluble nonionic surfactant (component (1)) The water-soluble amphoteric surfactant is not particularly limited, and examples thereof include amphoteric surfactants having cholic acid as a base nucleus, carboxylic acid type amphoteric surfactants, sulfonic acid type amphoteric surfactants, sulfate ester salt type amphoteric surfactants, etc. Among these, the water-soluble surfactants having cholic acid as a base nucleus will be described later.

[0040] The water-soluble nonionic surfactant is not particularly limited, and examples thereof include polyethylene glycol type nonionic surfactants, polyhydric alcohol fatty acid ester type nonionic surfactants, etc. The water-soluble nonionic surfactants include polyoxyethylene sorbitol ester (Tween 20: registered trademark) used in the examples.

[0041] In particular, by using a water-soluble amphoteric surfactant having cholic acid as the base nucleus as component (1), the solid phase carrier of the present invention can be stored at room temperature. Room temperature is assumed to be about 25° C., but in practical terms, it is stored in a non-refrigerated state.

[0042] The water-soluble amphoteric surfactant having cholic acid as a base nucleus is not particularly limited, but preferred examples include CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate) and CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate). These water-soluble surfactants having cholic acid as a base nucleus can be formulated alone or in combination.

[0043] The water-soluble amphoteric surfactant having cholic acid as the base nucleus can be produced by a conventional method, but commercially available products can also be used.

[0044] The content of the above surfactants in the composition of the present invention (total amount of one or more surfactants) is determined by the following components (3) to ( 5 When no additive is added, the content is preferably 1.5-20% by mass, particularly preferably 2-14% by mass, extremely preferably 4-12% by mass, and most preferably 8-10% by mass, based on the composition.

[0045] (2) Aqueous solvent (component (2)) The aqueous solvent is a solvent mainly composed of water, and may be water or various buffer solutions. The buffer solution is not particularly limited as long as it is used in the biochemistry field, is suitable for storing the carrier particles for binding, and does not inhibit the binding reaction of the carrier particles for binding when it comes into contact with the sample. Specific examples include phosphate buffer, acetate buffer, borate buffer, Tris-HCl buffer, glycine buffer, Good's buffer, etc.

[0046] The content of the aqueous solvent in the composition of the present invention is the balance of the other components contained.

[0047] (3) Urea or its salts (ingredient (3)) The type of urea salt is not particularly limited, and examples thereof include hydrochloride salts.

[0048] The content of urea or urea salt in the composition of the present invention is preferably 5-35% by mass, particularly preferably 8-25% by mass, extremely preferably 12-22% by mass, and most preferably 12-21% by mass. When this component (3) is blended, the content of the surfactant (component (1)) (total amount of one or more surfactants) is preferably 2-20% by mass, particularly preferably 4-15% by mass, extremely preferably 9-12% by mass, and most preferably 10-12% by mass. Urea or urea salt, particularly when used in combination with the water-soluble amphoteric surfactant having cholic acid as the mother nucleus, can improve the color development of the color signal on the solid phase at least under refrigerated storage, and when combined with the later-described component (4) and / or component (5), can improve the color development of the color signal on the solid phase under room temperature storage.

[0049] The content of the aqueous solvent in the composition of the present invention is the balance of the other components contained.

[0050] (4) Sugars (Component (4)) Saccharides, together with the amino acids described below, are one of the preferred additional components in the composition of the present invention. By incorporating a water-soluble amphoteric surfactant having cholic acid as the nucleus and a saccharide in combination with the composition of the present invention, the color development of the color signal on the solid phase can be improved at least under refrigerated storage. In addition, by incorporating the urea or urea salt and a saccharide in combination with the water-soluble amphoteric surfactant having cholic acid as the nucleus with the composition of the present invention, the color development of the color signal on the solid phase can be improved under refrigerated storage and room temperature storage. When incorporating a saccharide in combination with the water-soluble amphoteric surfactant having cholic acid as the nucleus with the composition of the present invention, it is also preferable to further incorporate it in combination with the amino acids described below. In addition, when incorporating a water-soluble nonionic surfactant or a water-soluble amphoteric surfactant other than the water-soluble amphoteric surfactant having cholic acid as the nucleus, it is possible to combine and incorporate a saccharide, but no advantage in the color development of the color signal on the solid phase has been recognized so far.

[0051] The saccharide that can be incorporated in the composition of the present invention is not particularly limited, but disaccharides or monosaccharides are preferred.

[0052] The disaccharides include, but are not limited to, maltose, cellobiose, lactulose, lactose, sucrose, trehalose, cellobiose, etc. Among these, trehalose and sucrose are preferred.

[0053] Examples of monosaccharides include, but are not limited to, D-glucose, D-mannose, D-galactose, D-fructose, etc. Among these, D-glucose is preferred.

[0054] The content of the sugar in the composition of the present invention is preferably 1-15% by mass, particularly preferably 2.5-12% by mass, extremely preferably 5-11% by mass, and most preferably 5-10% by mass, based on the mass of the composition.

[0055] (5) Amino acids (ingredient (5)) Amino acids, together with the sugars, are one of the suitable additional components in the composition of the present invention. By incorporating a water-soluble amphoteric surfactant having cholic acid as the nucleus in combination with an amino acid in the composition of the present invention, the color development of the color signal on the solid phase can be improved at least under refrigerated storage. By incorporating an amino acid and a sugar in combination with the water-soluble amphoteric surfactant having cholic acid as the nucleus in the composition of the present invention together with the urea or urea salt, the color development of the color signal on the solid phase can be improved under refrigerated storage and room temperature storage. In addition, when incorporating a water-soluble nonionic surfactant or a water-soluble amphoteric surfactant other than the water-soluble amphoteric surfactant having cholic acid as the nucleus, it is possible to combine and incorporate an amino acid, but no advantage in the color development of the color signal on the solid phase has been recognized so far.

[0056] The amino acids that can be incorporated into the composition of the present invention are not particularly limited, and examples thereof include L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-tryptophan, L-phenylalanine, L-proline, glycine, L-serine, L-threonine, L-cysteine, L-tyrosine, L-asparagine, L-glutamine, L-lysine, L-histidine, L-arginine, L-aspartic acid, L-glutamic acid, etc. Typical examples include L-arginine, L-histidine, L-aspartic acid, and glycine.

[0057] The content of the amino acid in the composition of the present invention is preferably 0.1-4 mass % based on the composition, particularly preferably 0.2-2 mass %, and most preferably 0.4-2 mass %.

[0058] When the above-mentioned sugars (component (4)) and / or amino acids (component (5)) are blended, the content of the surfactant (preferably the above-mentioned water-soluble amphoteric surfactant having cholic acid as a base nucleus) of component (1) in the composition of the present invention is the same as the content in the above-mentioned "when surfactant is blended alone" when urea or a urea salt is not blended in the composition, and is the same as the content in the above-mentioned "when urea or a urea salt is blended in combination" when urea or a urea salt is blended in combination. The blending of component (4) and / or component (5) in combination with urea or a urea salt is a particularly preferred embodiment from the viewpoint of improving the color development of the color signal during refrigerated storage and room temperature storage of the solid phase carrier of the present invention.

[0059] (6) Other ingredients In addition, additives such as blocking agents such as BSA, casein, serum, skim milk, etc., stabilizers such as gum arabic, preservatives such as sodium azide, and chelating agents can also be blended into the composition of the present invention, within qualitative and quantitative limits that do not substantially impair the effects of the present invention.

[0060] (III) Other The pH of the composition of the present invention is preferably about 4 to 9, and more preferably about 6 to 9.

[0061] (B) Solid Phase Support and Production Method of the Present Invention As described above, the solid phase carrier of the present invention is a carrier for performing chromatography and has at least a sample contact part and a detection part. The solid phase carrier of the present invention may be used for chromatography by itself, or may be used in a state of being fixed to another support.

[0062] The solid phase carrier of the present invention has a material that does not inhibit the binding reaction between the target nucleic acid and the insoluble carrier particle, and allows the binding product of the target nucleic acid and the insoluble carrier particle (hereinafter also referred to as "nucleic acid particle binding product") generated by this binding reaction to diffuse, at least including the sample contact part and the detection part. This is a form in which the nucleic acid particle binding product generated at the sample contact part can move to the detection part by solvent diffusion. Examples of the material include nonwoven fabric, filter paper, glass fiber, nitrocellulose filter, polyethersulfone filter, nylon filter, polyvinylidene fluoride filter, and porous material (silica, etc.), but are not limited thereto, and may be a single material or a composite material. In addition, the shape of the material part or the entire solid phase carrier may be a shape adopted in normal solid phase chromatography, such as a rectangular sheet (piece), a long shape, a thin rod, etc., and is preferably a long shape.

[0063] As described above, the sample contact portion is provided at a predetermined location on the solid support, preferably the above-mentioned detection The composition of the present invention, in which insoluble carrier particles capable of binding to the target nucleic acid are dispersed, is applied, and the composition is dried to remove moisture. Drying may be natural drying, hot air drying, heater drying, or vacuum drying. The position of the sample contact part on the solid phase carrier is not particularly limited. An example of the position of the sample contact part is the end of the solid phase carrier or its vicinity. This embodiment is preferable because, when the solid phase carrier of the present invention is brought into contact with a sample that may contain the target nucleic acid, the sample contact part can be directed toward the sample to easily bring them into contact with each other. The sample contact part is usually located at one location, but two or more locations can also be provided on the solid phase carrier.

[0064] The detection section can be provided by adding a binding function that captures the nucleic acid particle-bound substance that has diffused and migrated on the solid phase carrier after the nucleic acid particle-bound substance is generated in the sample contact section, and generates a capture signal. The binding function and the binding function possessed by the nucleic acid of the nucleic acid particle-bound substance or the solid phase carrier particle work as a pair, and the two come into contact and bind to each other to generate a desired capture signal.

[0065] Examples of binding functions acting as this pair include hydrogen bonds, ionic bonds, electrostatic bonds, hydrophobic bonds, physical mutual bonds, etc., and specific examples include organic compound-organic compound interactions, protein-protein interactions, protein-nucleotide interactions, nucleotide-nucleotide interactions, organic compound-protein interactions, etc. Furthermore, more specific pair elements include a binding function based on the avidin (streptavidin)-biotin system, a binding function between nucleic acids using a tag base sequence or the like, a binding function between FITC and anti-FITC, anti-digoxigenin (DIG)-digoxigenin (DIG), a binding function based on an antigen-antibody reaction between a tag amino acid sequence and a specific antibody, etc.

[0066] One of the preferred embodiments of the binding function in the detection section is the use of a nucleic acid capable of hybridizing with a tag nucleic acid added to the "nucleic acid" of the nucleic acid particle-bound substance. "Hybridizable" means that the nucleic acid particle-bound substance can be captured in the detection section by forming a double strand (hybridizing) between the tag nucleic acid and the nucleic acid of the detection section, which is partially or entirely complementary to the tag nucleic acid sequence. The detection section to which the capture signal generating mechanism such as the hybridizable nucleic acid is added can be one location, but it is also possible to provide two or more locations to perform multiple nucleic acid detection in one operation. For example, an embodiment in which the detection section is provided at one location in the downstream direction of the mobile phase from the sample contact section, or at two or more locations at a predetermined interval, is exemplified. Furthermore, it is also possible to provide an array form in which many detection sections are provided at a predetermined interval. By using the array form, comprehensive detection of the nucleic acid to be detected can be performed. In addition, the carrier may be provided with a plurality of partitioned array regions. These multiple partitioned arrays may have the same content or different contents.

[0067] The method of adding the capture signal generating mechanism as a detection part includes attaching the mobile phase to the solid phase while maintaining its continuity, and binding of a substance that is the basis of the signal generating mechanism to the solid phase, such as the above-mentioned hybridizable nucleic acid, to a solid phase carrier via non-covalent or covalent binding at the 3'-end or 5'-end side, etc. This nucleic acid binding can be performed by using existing methods or tools such as a spotter.

[0068] As long as the nucleic acid particle-bound substance is captured in the detection section to generate some kind of signal, the type of the signal is not limited. For example, in an embodiment in which a colored insoluble carrier particle is used as the insoluble carrier particle, the nucleic acid particle-bound substance in which the colored insoluble carrier particle is bound to the nucleic acid to be detected is captured and accumulated in the detection section, so that the color of the detection section becomes dark, and the presence of the nucleic acid to be detected in the sample can be easily detected by visual inspection or relatively simple mechanical analysis.

[0069] In addition to the above, the solid phase carrier of the present invention may have other mechanisms as necessary, such as an adsorption section for adsorbing and trapping components that are not captured by the detection section and move downstream.

[0070] (C) Method of using the present invention (1) Nucleic acid sample and target nucleic acid The method of use of the present invention includes the step of contacting a sample that may contain the nucleic acid to be detected (hereinafter also referred to as a "nucleic acid sample") with the sample contact portion of the solid phase carrier of the present invention.

[0071] The nucleic acid to be detected is a nucleic acid whose presence and / or amount should be detected using the solid phase carrier of the present invention. Its molecular weight is not particularly limited, and anything from an oligonucleotide to a polynucleotide can be detected. The type of nucleic acid is also not limited, and it may be a natural nucleic acid or an artificial nucleic acid. Furthermore, it may be single-stranded or double-stranded DNA, RNA, DNA / RNA hybrid, DNA / RNA chimera, etc. More specifically, natural or synthetic nucleic acids such as cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA, synthetic RNA, etc., as well as artificial synthetic nucleic acids such as peptide nucleic acid, morpholino nucleic acid, methylphosphonate nucleic acid, and S-oligonucleic acid, etc., can be widely targeted. Among these nucleic acids to be detected, DNA is preferred, and nucleic acid amplification products generated by a nucleic acid amplification method are most preferred. It is also possible to add a labeling substance to the nucleic acid to be detected as necessary. Examples of the label include fluorescent substances, enzymes, radioactive substances, and color-developing substances, and the labeling method can be a conventional method according to the type of label.

[0072] A nucleic acid amplification product is usually an artificial nucleic acid produced by subjecting a specific portion of a natural nucleic acid that should be a target nucleic acid to a nucleic acid amplification method. The natural nucleic acid to be amplified is not particularly limited, and includes, for example, bases or base sequences that are genetic indicators in organisms such as humans and non-human animals, such as constitution, genetic disease, onset of a specific disease such as cancer, disease diagnosis, treatment prognosis, and selection of drugs or treatment. Nucleic acids derived from plants and nucleic acids derived from microorganisms such as pathogens and viruses are also included in the target nucleic acid. When the target nucleic acid is RNA, RT-PCR or the like can be used. It is also possible to further amplify a specific portion of the nucleic acid amplification product (nested PCR, etc.). It is a preferred embodiment that the target nucleic acid such as a nucleic acid amplification product has a binding function (a binding function to a carrier particle) for binding to an insoluble carrier particle, and it is also a preferred embodiment that the target nucleic acid has a binding function (a binding function to a detection portion) for binding to the detection portion of the solid phase carrier of the present invention. The target nucleic acid may have only one of the binding function to a carrier particle and the binding function to a detection portion, or may have a combination of the two. These specific examples of binding functions are selected together with the "other binding function" that they form a pair with. In the case of the binding function to the carrier particle, the "other" is the binding function possessed by the insoluble carrier particle, and in the case of the binding function to the detection part, the "other" is the binding function possessed by the detection part.

[0073] The binding functions acting as this pair are as already described in (A)(I) as the "binding function acting on the nucleic acid to be detected in the insoluble carrier particle" and in (B) as the "binding function acting as a pair in the detection portion," and the nucleic acid to be detected may have a second portion of these pairs.

[0074] As in the above, examples of the binding function include hydrogen bonds, ionic bonds, electrostatic bonds, hydrophobic bonds, physical mutual bonds, etc., and specific examples include organic compound-organic compound interactions, protein-protein interactions, protein-nucleotide interactions, nucleotide-nucleotide interactions, organic compound-protein interactions, etc. Furthermore, more specific examples of the binding elements include a binding function based on the avidin (streptavidin)-biotin system, a binding function between nucleic acids using a tag base sequence, etc., a binding function between FITC and an anti-FITC antibody, anti-digoxigenin (DIG) antibody-digoxigenin (DIG), a binding function based on an antigen-antibody reaction between a tag amino acid sequence and a specific antibody, etc.

[0075] Examples of the nucleic acid amplification method include, but are not limited to, the PCR method or a method based on the PCR method (RT-PCR method, nested PCR method, etc.), the LAMP method, the ICAN method, etc. Any nucleic acid amplification method that will be provided in the future may also be used. Among these nucleic acid amplification methods, the PCR method or a method based on the PCR method is one of the preferred embodiments.

[0076] The sample material obtained as described above is used as is or after necessary dilution or addition of chemicals to prepare a nucleic acid sample.

[0077] (2) When the nucleic acid amplification product is the nucleic acid to be detected As described above, a preferred embodiment of the nucleic acid to be detected is a nucleic acid amplification product, and it is more preferred that the nucleic acid amplification product itself has one or more binding functions. In this case, the embodiment is not limited, but a representative embodiment will be described.

[0078] As described above, the PCR method is one of the preferred embodiments of the nucleic acid amplification method. The PCR process itself can be carried out according to a conventional method. That is, a desired nucleic acid amplification product can be obtained by carrying out a temperature cycle of PCR in the presence of a heat-resistant DNA polymerase using an amplification primer capable of amplifying all or a part of the nucleic acid to be detected. When the nucleic acid to be detected is RNA, RT-PCR can be carried out in the presence of a reverse transcriptase in the system.

[0079] As a method for imparting the above-mentioned binding function to the nucleic acid amplification product, a primer for nucleic acid amplification to which a desired binding function is linked can be used during gene amplification. Both the reverse primer and the forward primer may have a binding function, or only one of them may be used, but it is preferable that both have a binding function, one of which has a function to bind to the carrier particle and the other has a function to bind to the detection part.

[0080] The binding function is exemplified by those already mentioned above, and preferred examples include a binding function by the avidin (streptavidin)-biotin system, and a combination of a binding function by FITC and an anti-FITC antibody and a binding function between nucleic acids using a tag base sequence, etc. In this case, it is preferred to use colored particles as the insoluble carrier particles.

[0081] The binding function of the avidin (streptavidin)-biotin system is preferably used as a binding function to carrier particles. That is, the nucleic acid amplification product to which biotin or avidin (streptavidin) is bound, obtained by performing nucleic acid amplification using one of the primers for nucleic acid amplification to which biotin or avidin (streptavidin) is added at the 5' end, is used as a detection target nucleic acid having a binding function to carrier particles, and the nucleic acid amplification product is brought into contact with insoluble carrier particles carrying avidin (streptavidin) or biotin, thereby obtaining a nucleic acid particle binding substance bound by the avidin (streptavidin)-biotin system. Insoluble carrier particles carrying avidin (streptavidin) or biotin, particularly latex, are relatively easy to prepare. At the same time, a tag nucleic acid serving as a paired detection unit binding function is added to the 5' end of the other nucleic acid amplification primer, thereby obtaining a nucleic acid particle-bound product having a tag nucleic acid on its surface in addition to the nucleic acid particle-bound product bound by the avidin (streptavidin)-biotin system. When the nucleic acid particle-bound product having the tag nucleic acid contacts the detection unit of a solid-phase carrier having a nucleic acid with a base sequence capable of hybridizing with a part or all of the tag nucleic acid due to diffusion of an aqueous solvent, the tag nucleic acid and the nucleic acid of the detection unit form a double strand, and the nucleic acid particle-bound product is captured by the detection unit. In this case, if the insoluble carrier particles are colored, the color accumulates in the detection unit and becomes visible, which serves as a signal for the presence of the nucleic acid to be detected in the sample.

[0082] Specific tag sequences and nucleic acid sequences (probes) that hybridize therewith can be determined according to the disclosures of, for example, Patent Documents 2 and 3.

[0083] Also, as shown in Patent Document 2, the yield of the generated genuine nucleic acid amplification product can be improved by introducing a site capable of suppressing or halting the progress of the polymerase reaction between the tag sequence of the nucleic acid amplification primer used in generating the nucleic acid amplification product and the main body of the nucleic acid amplification product. Furthermore, as shown in Patent Document 3, instead of using the gene amplification primer to which the above-mentioned tag sequence is added at the 5' end, a detection target nucleic acid discrimination probe having a tag sequence at the 3' end and a labeling region that specifically hybridizes with the nucleic acid extension region of the genuine nucleic acid amplification product is made to coexist in a nucleic acid amplification reaction using the gene amplification primer to which the above-mentioned tag sequence is not added at the 5' end and a gene amplification primer to which an avidin (streptavidin) or biotin label is added at the 5' end, and the tag sequence of this probe is used as a function for binding to the paired detection portion, thereby improving the yield of the generated genuine nucleic acid amplification product. EXAMPLES

[0084] Examples of the present invention will be disclosed below. Unless otherwise specified, % refers to mass % relative to the blended subject.

[0085] The following disclosure consists of positive controls and examples (including comparative examples).

[0086] [Common Reagents] The following reagents (a) and (b) were used in both the positive control 1 and the examples. (a) 0.5% colored latex solution of streptavidin-coated colored latex (product name SA-Lx: manufactured by Fujikura Kasei Co., Ltd.) (hereinafter, also referred to as "colored latex solution") (b) A TE solution (10 mM Tris, 1 mM EDTA) of an oligo DNA (hereinafter also referred to as "complementary strand oligo") labeled with biotin at the 5' end, which has a base sequence of 5'-AACGTCCAATAGTAACCAGAGCG (SEQ ID NO: 1) complementary to the capture DNA probe in the detection section described below (hereinafter also referred to as "complementary strand oligo solution").

[0087] [Positive control 1] (1) Preparation of chromatography strips (solid phase carriers) A TE solution of a capture DNA probe consisting of the following base sequence was spotted on a solid support, glass wool (Millipore) (8 mm x 2 mm), using a GENESHOT (registered trademark) spotter from NGK Insulators, Ltd., which uses a discharge unit (inkjet method) described in JP-A-2003-75305, and this was used as the "detection part" of the solid support. A strip for development chromatography was prepared using 5'-CGCTCTGGTTACTATTGGACGTT (SEQ ID NO: 2) as a synthetic oligo DNA sequence.

[0088] (2)Developing solution The developing solution for positive control 1 has the following composition: The developing solution for positive control 1 was prepared by dissolving a, b, c, and d in e. Ingredients Blend amount a CHAPS 8% by mass b BSA 1% by mass c Urea 21% by mass d 20×PBS 5% by mass e Distilled water remaining

[0089] (3) Detection method for positive control 1 10 μl (46.5 parts by mass) of the developing solution of the above positive control 1 was mixed with 1.5 μl (7 parts by mass) of the colored latex solution (a) and 10 μl (46.5 parts by mass) of the 1× complementary oligo DNA solution (b) to obtain a detection solution of 21.5 μl in total. In this detection solution, a nucleic acid particle bond (due to the bond between streptavidin coated on the colored latex and biotin labeled on the complementary oligo DNA) is formed by contact between the above (a) and (b).

[0090] This detection solution was placed in a tube (2 ml microtube), and the end of the chromatography strip (1) above was immersed in it. The detection solution containing the nucleic acid particle-bound substance was then spread toward the detection section. The degree of coloration of the nucleic acid particle-bound substance resulting from the capture of the nucleic acid particle in the detection section by the formation of double-stranded DNA between complementary DNA strands due to contact between the solution and the capture DNA in the detection section was quantified using Image Lab (BIO-RAD), and this value was used as the positive control value.

[0091] [Examples (including comparative examples)] A. Group 1 (1) Preparation of conjugate pad (solid phase support) A chromatographic strip prepared as the positive control above was attached to a solid support of glass wool (Millipore) (8 mm x 2 mm), and a length of up to 2 mm from the end closest to the detection area was immersed in a mixture of 10 μl of the composition of an example or comparative example having the formulation shown below and 1.5 μl of colored latex suspension (a) (the content of colored latex particles in the mixture was 0.065% by mass) for 10 seconds. The mixture was then vacuum dried for 2 hours using a drying apparatus (EYELA), and this portion was used as the "sample contact portion" and as the solid support of each example or comparative example.

[0092] (2) Test method The solid phase carriers of the examples prepared in (1) above were stored in air at 4°C or 37°C for 2 weeks (hereinafter referred to as "4°C product" and "37°C product", respectively) as test samples, and 20 μl of complementary oligonucleotide solution (b) was used as a nucleic acid sample and immersed in the sample contact area of ​​the solid phase carrier. The 4°C product was positioned as a refrigerated storage model of the solid phase carrier, and the 37°C product was positioned as a room temperature storage model (accelerated test). The solid phase carriers were left to stand for another 20 minutes at room temperature to allow sufficient solvent diffusion in the solid phase carrier, and the degree of color development in the detection area was quantified using Image Lab (BIO-RAD), and this value was evaluated as a percentage of the positive control value.

[0093] Example 1 The composition of Example 1 has the following formulation: Ingredients Blend amount a CHAPS 8% by mass b BSA 1% by mass c Urea 21% by mass d 20×PBS 5% by mass e Distilled water remaining

[0094] <Manufacturing method etc.> A, b, c, and d were dissolved in e to prepare the composition of Example 1 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 1 in the manner described above.

[0095] <Test Results> The color development degree of the 4°C sample was 100%, while that of the 37°C sample was 80-90%.

[0096] Example 2 The composition of Example 2 has the following formulation: Ingredients Blend amount a CHAPSO 8% by mass b BSA 1% by mass c Urea 21% by mass d 20×PBS 5% by mass e Distilled water remaining

[0097] <Manufacturing method etc.> A, b, c, and d were dissolved in e to prepare the composition of Example 2 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 2 in the manner described above.

[0098] <Test Results> The color development degree of the 4°C sample was 100%, while that of the 37°C sample was 80-90%.

[0099] Example 3 The composition of Example 3 has the following formulation: Ingredients Blend amount a CHAPS 8% by mass b BSA 1% by mass c Urea 12% by mass d 20×PBS 5% by mass e Distilled water remaining

[0100] <Manufacturing method etc.> A, b, c, and d were dissolved in e to prepare the composition of Example 3 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 3 in the manner described above.

[0101] <Test Results> The color development degree of the 4°C sample was 100%, while that of the 37°C sample was 80-90%.

[0102] Example 4 The composition of Example 4 has the following formulation: Ingredients Blend amount a CHAPSO 8% by mass b BSA 1% by mass c Urea 12% by mass d 20×PBS 5% by mass e Distilled water remaining

[0103] <Manufacturing method etc.> A, b, c, and d were dissolved in e to prepare the composition of Example 4 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 4 in the manner described above.

[0104] <Test Results> The color development degree of the 4°C sample was 100%, while that of the 37°C sample was 80-90%.

[0105] Example 5 The composition of Example 5 has the following formulation: Ingredients Blend amount a CHAPS 8% by mass b BSA 1% by mass c Urea 21% by mass d Trehalose 10% by mass e L-arginine 0.4% by mass f 20×PBS 5% by mass g Distilled water remaining

[0106] <Manufacturing method etc.> A, b, c, d, e, and f were dissolved in g to prepare the composition of Example 5 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 5 in the manner described above.

[0107] <Test Results> The color development degree of the 4°C sample was 100%, and the color development degree of the 37°C sample was also 100%.

[0108] Example 6 The composition of Example 6 has the following formulation: Ingredients Blend amount a CHAPSO 8% by mass b BSA 1% by mass c Urea 21% by mass d Trehalose 10% by mass e L-arginine 0.4% by mass f 20×PBS 5% by mass g Distilled water remaining

[0109] <Manufacturing method etc.> A, b, c, d, e, and f were dissolved in g to prepare the composition of Example 6 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 6 in the manner described above.

[0110] <Test Results> The color development degree of the 4°C sample was 100%, and the color development degree of the 37°C sample was also 100%.

[0111] Example 7 The composition of Example 7 has the following formulation: Ingredients Blend amount a CHAPS 8% by mass b BSA 1% by mass c Urea 12% by mass d Trehalose 10% by mass e L-arginine 0.4% by mass f 20×PBS 5% by mass g Distilled water remaining

[0112] <Manufacturing method etc.> A, b, c, d, e, and f were dissolved in g to prepare the composition of Example 7 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 7 in the manner described above.

[0113] <Test Results> The color development degree of the 4°C sample was 100%, and the color development degree of the 37°C sample was also 100%.

[0114] Example 8 The composition of Example 8 has the following formulation: Ingredients Blend amount a CHAPSO 8% by mass b BSA 1% by mass c Urea 12% by mass d Trehalose 10% by mass e L-arginine 0.4% by mass f 20×PBS 5% by mass g Distilled water remaining

[0115] <Manufacturing method etc.> A, b, c, d, e, and f were dissolved in g to prepare the composition of Example 8 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 8 in the manner described above.

[0116] <Test Results> The color development degree of the 4°C sample was 100%, and the color development degree of the 37°C sample was also 100%.

[0117] Example 9 The composition of Example 9 has the following formulation: Ingredients Blend amount a CHAPS 8% by mass b BSA 1% by mass c Urea 12% by mass d D(+)-glucose 10% by mass e L-arginine 0.4% by mass f 20×PBS 5% by mass g Distilled water remaining

[0118] <Manufacturing method etc.> A, b, c, d, e, and f were dissolved in g to prepare the composition of Example 9 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 9 in the manner described above.

[0119] <Test Results> The color development degree of the 4°C sample was 100%, and the color development degree of the 37°C sample was also 100%.

[0120] Example 10 The composition of Example 10 has the following formulation: Ingredients Blend amount a CHAPS 8% by mass b BSA 1% by mass c Urea 12% by mass d Sucrose 10% by mass e L-arginine 0.4% by mass f 20×PBS 5% by mass g Distilled water remaining

[0121] <Manufacturing method etc.> A, b, c, d, e, and f were dissolved in g to prepare the composition of Example 10 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 10 in the manner described above.

[0122] <Test Results> The color development degree of the 4°C sample was 100%, and the color development degree of the 37°C sample was also 100%.

[0123] Example 11 The composition of Example 11 has the following formulation: Ingredients Blend amount a Tween20 8% by mass b BSA 1% by mass c Urea 12% by mass d 20×PBS 5% by mass e Distilled water remaining

[0124] <Manufacturing method etc.> A, b, c, and d were dissolved in e to prepare the composition of Example 11 (10 μl), to which the colored latex liquid (a) (1.5 μl) was added and stirred, and this was attached to the sample contact portion of the solid phase carrier, thereby preparing a solid phase carrier using the composition of Example 11 in the manner described above.

[0125] <Test Results> The color development degree of the 4°C sample was 100%, while the color development degree of the 37°C sample was 0%.

[0126] B. Group 2 In response to the results of the examples of Group 1, the study was expanded. The "method of preparing the conjugate pad (solid phase carrier)" for the test products of Group 2 was the same as that of Group 1. In addition, the test products (examples or comparative examples) were "4°C product" (refrigerated storage model) and "37°C product" (room temperature storage model) in the same manner as in Group 1. The preparation of each test product was performed in accordance with Examples 1-11 of Group 1. In the test, 20 μl of complementary oligo solution (b) was used as a nucleic acid sample at room temperature, and the sample contact part of the solid phase carrier was immersed in contact with the sample, and the solid phase carrier was left to stand for another 20 minutes at room temperature to allow sufficient solvent diffusion in the solid phase carrier. The degree of coloring in the detection part was then evaluated based on visual observation. The evaluation criteria are as follows.

[0127] <Evaluation criteria> Color development on a solid phase at 4°C using the composition formulated in Example 1 above was separately performed, and this was used as positive control 2, and evaluation was performed according to the framework indicated below as ◎, ○, △, and ×. ◎: Higher color development than positive control 2 ○: Color development is equivalent to that of positive control 2 △: The coloring is slightly inferior to that of the positive control 2, but there is no problem in visually distinguishing the color signal. ×: Poor color development, and color signals are difficult to distinguish

[0128] <Examples 12-17, Comparative Example 1-2: CHAPS alone> The composition of Example 12 has the following formulation: Ingredients Blend amount a CHAPS 2% by mass b BSA 1% by mass c 20×PBS 5% by mass d Distilled water remaining

[0129] The compositions of Examples 13-17 and Comparative Examples 1-2 were similar to those of Example 12, except for the amount of CHAPS. The amount of CHAPS and the evaluation results in these Examples and Comparative Examples are shown in Table 1.

[0130] [Table 1]

[0131] <Examples 18-22 and Comparative Examples 3-4: CHAPSO alone> The composition of Example 18 has the following formulation: Ingredients Blend amount a CHAPSO 2% by mass b BSA 1% by mass c 20×PBS 5% by mass d Distilled water remaining

[0132] The compositions of Examples 19-22 and Comparative Examples 3-4 were similar to those of Example 18, except for the amount of CHAPSO. The amount of CHAPSO and the evaluation results in these Examples and Comparative Examples are shown in Table 2.

[0133] [Table 2]

[0134] <Examples 23-25 ​​and Comparative Example 5: CHAPS / CHAPSO Mixture> The composition of Example 23 has the following formulation: Ingredients Blend amount a CHAPS 1% by mass b CHAPSO 1% by mass c BSA 1% by mass d 20×PBS 5% by mass e Distilled water remaining

[0135] The compositions of Examples 24-25 and Comparative Example 5 were similar to those of Example 23, except for the amounts of CHAPS and CHAPSO. The amounts of CHAPS and CHAPSO and the evaluation results in these Examples and Comparative Example are shown in Table 3.

[0136] [Table 3]

[0137] The results of Examples 12-25 above reveal that the solid support of the present invention can be stored in a usable state under refrigeration or at room temperature even when CHAPS and CHAPSO, which are water-soluble amphoteric surfactants with cholic acid as the base nucleus, are incorporated alone or in combination into the composition of the present invention without any other substantial additional components.

[0138] <Examples 26-30: CHAPS fixation and urea change> The composition of Example 26 has the following formulation: Ingredients Blend amount a CHAPS 8% by mass b BSA 1% by mass c 20×PBS 5% by mass d Urea 8% by mass e Distilled water remaining

[0139] The formulation of the compositions of Examples 27-30 was the same as that of Example 26, except for the amount of urea. The amount of urea and the evaluation results in these Examples are shown in Table 4. Note that the evaluation in this test of the 37°C product of the example in which the amount of urea was 21 mass% (formulation of Example 1: positive control 2) was equivalent to the 4°C product, and was given a grade of "○".

[0140] [Table 4]

[0141] <Examples 31-34, Comparative Example 6: Urea fixation, CHAPS change examples> The composition of Example 31 has the following formulation: Ingredients Blend amount a CHAPS 2% by mass b BSA 1% by mass c 20×PBS 5% by mass d Urea 21% by mass e Distilled water remaining

[0142] The compositions of Examples 32-34 and Comparative Example 6 were similar to those of Example 31, except for the amount of CHAPS. Table 5 shows the amount of CHAPS and the evaluation results in these Examples and Comparative Example.

[0143] [Table 5]

[0144] The results of Examples 26 to 34 above demonstrated that when urea was combined, it was possible to further improve the color development of the color signal on the solid phase during refrigerated storage.

[0145] <Examples 35-45: Combination of amino acids and sugars> Example 35 The composition of Example 35 had the following formulation, and the product at 4° C. was rated as "A", and the product at 37° C. was rated as "O". Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d Urea 21% by mass e L-arginine 0.4% by mass f Distilled water remaining

[0146] (Example 36) The composition of Example 36 had the following formulation, and was evaluated as "A" when stored at 4°C and as "A" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d Urea 21% by mass e Trehalose 10% by mass f Distilled water remaining

[0147] (Example 37) The composition of Example 37 had the following formulation, and was evaluated as "A" when stored at 4°C and as "A" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d Urea 21% by mass e Trehalose 10% by mass f L-arginine 0.4% by mass g Distilled water remaining

[0148] (Example 38) The composition of Example 38 had the following formulation, and was evaluated as "A" when stored at 4°C and as "O" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d L-arginine 0.4% by mass e Distilled water remaining

[0149] (Example 39) The composition of Example 39 had the following formulation, and was evaluated as "A" when stored at 4°C and as "O" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d Trehalose 10% by mass e Distilled water remaining

[0150] (Example 40) The composition of Example 40 had the following formulation, and was evaluated as "A" when stored at 4°C and as "O" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d Trehalose 10% by mass e L-arginine 0.4% by mass f Distilled water remaining

[0151] (Example 41) The composition of Example 41 had the following formulation, and was evaluated as "A" when stored at 4°C and as "O" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d Glycine 0.4% by mass e Distilled water remaining

[0152] (Example 42) The composition of Example 42 had the following formulation, and was evaluated as "A" when stored at 4°C and as "O" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d L-histidine 0.4% by mass e Distilled water remaining

[0153] (Example 43) The composition of Example 43 had the following formulation, and was evaluated as "A" when stored at 4°C and as "O" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d L-Aspartic acid 0.4% by mass e Distilled water remaining

[0154] (Example 44) The composition of Example 44 had the following formulation, and was evaluated as "A" when stored at 4°C and as "O" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d Sucrose 10% by mass e Distilled water remaining

[0155] (Example 45) The composition of Example 45 had the following formulation, and was evaluated as "A" when stored at 4°C and as "O" when stored at 37°C. Ingredients Blend amount a CHAPS 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d D(+)-glucose 10% by mass e Distilled water remaining

[0156] The results of Examples 35-45 above revealed that when sugars and / or amino acids are combined, it is possible to further improve the color development of the color signal on the solid phase, at least during refrigerated storage, and examples of improved color development of the above-mentioned color signal during room temperature storage were also observed in the cases where urea was combined (Examples 36 and 37).

[0157] <Examples 46-47: Blending example of Tween 20> (Example 46) The composition of Example 46 had the following formulation, and the product at 4° C. was rated as “◯”, and the product at 37° C. was rated as “×”. Ingredients Blend amount a Tween20 8% by mass b BSA 1% by mass c 20×PBS 5% by mass d Urea 21% by mass e Distilled water remaining

[0158] (Example 47) The composition of Example 47 had the following formulation, and the product at 4° C. was rated as “◯”, and the product at 37° C. was rated as “×”. Ingredients Blend amount a Tween20 10% by mass b BSA 1% by mass c 20×PBS 5% by mass d Urea 21% by mass e Trehalose 10% by mass f L-arginine 0.4% by mass e Distilled water remaining

[0159] The above Examples 46-47 demonstrated that even when a water-soluble nonionic surfactant was used, the color signal was sufficiently visible on the solid phase when stored in a refrigerator. However, the color signal was very weak when stored at room temperature, and it was reaffirmed that storage at room temperature is difficult to use.

Claims

1. The following components (1) and (2): (1) A water-soluble amphoteric surfactant having a cholic acid nucleus, (2) aqueous solvent; A composition for attaching insoluble carrier particles to which a nucleic acid to be detected can be bound to a solid phase, comprising: (1) a water-soluble amphoteric surfactant having a cholic acid moiety, and the content of the water-soluble amphoteric surfactant in the composition is 1.5-20 mass %.

2. The composition described in claim 1, wherein the content of (1) water-soluble amphoteric surfactant having cholic acid as a parent nucleus in the composition is 2-14 mass%.

3. (1) The composition according to claim 1 or 2, wherein the water-soluble amphoteric surfactant having cholic acid as a base nucleus is CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate) or CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate).

4. The composition according to any one of claims 1 to 3, further comprising (3) urea or a salt thereof, and the content of (1) the water-soluble amphoteric surfactant having a cholic acid nucleus in the composition is 2 to 20 mass%.

5. The composition described in Claim 4, wherein the content of (1) water-soluble amphoteric surfactant having cholic acid as a parent nucleus in the composition is 4-15 mass%.

6. The composition according to any one of claims 1 to 5, further comprising (4) a sugar and / or (5) an amino acid.

7. (4) The composition according to claim 6, wherein the sugar is trehalose, D-glucose, or sucrose.

8. (5) The composition according to claim 6 or 7, wherein the amino acid is L-arginine, L-histidine, L-aspartic acid, or glycine.

9. A method for producing a solid phase for nucleic acid detection, comprising mixing the composition according to any one of claims 1 to 8 with insoluble carrier particles capable of binding a nucleic acid to be detected, attaching the mixture to a solid phase, and drying the mixture.

10. The method of claim 9 , wherein the insoluble carrier particles are latex particles.

11. A solid phase carrier having a sample contact portion and a detection portion, wherein the sample contact portion is provided with a water-soluble amphoteric surfactant having cholic acid as a base moiety and insoluble carrier particles to which the target nucleic acid can bind, and the detection portion is provided with a binding function for capturing the bound product of the target nucleic acid and the insoluble carrier particles that migrate on the solid phase carrier, thereby generating a capture signal.

12. 12. The solid support according to claim 11, wherein the sample contact portion of the solid support is at or near the end of the solid support.

13. A method for using a solid phase carrier in which a water-soluble amphoteric surfactant with cholic acid as the base nucleus and insoluble carrier particles to which the target nucleic acid can bind are attached to the sample contact area, and the detection area is added with a binding function that captures the complex between the target nucleic acid and the insoluble carrier particles that migrates on the solid phase carrier and generates a capture signal, the method comprising the steps of: contacting the sample contact area with a sample that may contain the target nucleic acid; and detecting a capture signal generated by the binding between the complex between the target nucleic acid and the insoluble carrier particles that migrates from the sample contact area on the solid phase carrier and the detection area.

14. 14. The method for using the solid phase carrier according to claim 13, wherein the nucleic acid to be detected has a binding function for binding to insoluble carrier particles.

15. 15. The method for using a solid phase carrier according to claim 13, wherein the nucleic acid to be detected has a binding function for binding to a detection portion of the solid phase carrier.

16. The method for using the solid phase carrier according to any one of claims 13 to 15, wherein the nucleic acid to be detected is a nucleic acid amplification product produced by a nucleic acid amplification method.

17. 17. The method for using the solid phase carrier according to claim 16, wherein the nucleic acid amplification method is a PCR method or a method based on the PCR method.

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