Composition for solid-phase attachment, solid-phase carrier using the composition, and production method and use method of the solid-phase carrier

Abstract

An object of the present invention is to establish the following means: when a gene amplification reaction solution is brought into direct contact with a solid phase portion to which insoluble carrier particles are immobilized and the detection is carried out by spreading it on the solid phase, a decrease in detection sensitivity in a conjugate of a nucleic acid to be detected and the insoluble carrier particles is suppressed. The present inventors have found that by containing component (1) a water-soluble amphoteric surfactant or a water-soluble nonionic surfactant and component (2) an aqueous solvent in the composition of a composition for containing insoluble carrier particles to adhere to a solid phase, the significant decrease in detection sensitivity can be suppressed. The present invention provides (A) a composition for containing insoluble carrier particles such as latex to adhere to a solid phase, (B) a method for producing a solid phase carrier using the composition of the present invention, (C) a solid phase carrier having a sample contact portion containing the composition for adhering to a solid phase from which substantially water has been removed, and (D) a method for using the solid phase carrier.

Description

Technical Field

[0001] The present invention relates to a solid phase carrier for detecting a predetermined substance. In particular, the present invention relates to a solid phase carrier having a sample contact portion that can directly contact a sample for detection when nucleic acid is detected, a method for using the solid phase carrier, a method for producing the solid phase carrier, and a composition for solid phase attachment used to set the sample contact portion of the solid phase carrier. Background Art

[0002] In recent years, the detection of specific nucleic acids and the establishment of associations with the properties of the nucleic acids have been used in various applications such as disease detection, identification of animal and plant species, determination of food quality and origin, paternity testing, and proof of crime.

[0003] The nucleic acid detection technology has been rapidly developing because it is possible to easily amplify a trace amount of nucleic acid to a detectable amount by using nucleic acid amplification methods such as the PCR method, and various technologies have been provided so far.

[0004] Among them, in order to achieve both simplification and high accuracy of nucleic acid detection, technologies for performing nucleic acid detection 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 a double-stranded DNA amplification product obtained by a nucleic acid amplification method. The amplification product has a binding site for a specific substance. The technology is to concentrate the DNA amplification product by binding to the specific substance, thereby performing the desired nucleic acid detection. The method of performing it on a development medium (solid phase) has been disclosed as the main form.

[0006] The technology of Patent Document 2 is a technology that aims to improve the yield of authentic nucleic acid (Japanese: authentic nucleic acid) amplification products generated by introducing a site that can inhibit or stop the polymerase reaction into a part of the primer used when generating nucleic acid amplification products, thereby improving the sensitivity of solid phase detection of target nucleic acids.

[0007] The technology of Patent Document 3 is a method of using a target nucleic acid recognition probe having a label region that specifically hybridizes with a nucleic acid extension region of a genuine nucleic acid amplification product in a nucleic acid amplification reaction in order to further improve the yield of generating a genuine nucleic acid amplification fragment.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: International Publication No. WO2009 / 034842

[0011] Patent Document 2: International Publication No. WO2013 / 038534

[0012] Patent Document 3: International Publication Gazette WO2016 / 129609 Summary of the Invention

[0013] Problems to be Solved by the Invention

[0014] The techniques of Patent Documents 1-3 successively show the progress of nucleic acid detection techniques in the solid phase, respectively improving the yield of amplification of the target nucleic acid (nucleic acid to be detected). Generally, the detection of the target nucleic acid (nucleic acid to be detected) in the solid phase is excellent in simplicity in that it does not require special equipment or devices, but there are still problems in terms of detection sensitivity.

[0015] To compensate for this, a "development sample solution" in which an amplification reaction solution, a developing solution, and insoluble carrier particles such as latex are mixed is temporarily prepared in a test tube outside the solid phase, and the solid phase (strip) is immersed therein, so that the conjugate of the nucleic acid to be detected and the insoluble carrier particles diffuses and moves to the detection part on the solid phase, generating a detection signal (for example, the examples of Patent Document 3).

[0016] However, this method is cumbersome in practice.

[0017] Therefore, the aim is to establish a simplified method of directly contacting the amplification reaction solution with the solid phase part to which insoluble carrier particles are fixed and developing it on the solid phase for detection.

[0018] Means for Solving the Problems

[0019] In order to solve the above problems, the present inventor further conducted research. As a result, it was found that by making the composition of the composition for attaching insoluble carrier particles such as latex to the solid phase have a specified content, even if the solid phase is directly immersed in the amplification reaction solution to carry out the diffusion movement of the conjugate, a decrease in detection sensitivity can be suppressed, and a desired detection signal can be sufficiently obtained.

[0020] The present invention provides (A) a composition for attaching insoluble carrier particles such as latex 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 part containing the composition for attaching the solid phase with substantially water removed (the solid phase carrier of the present invention), and (D) a method for using the solid phase carrier (the method for using the present invention).

[0021] (A) The Composition of the Present Invention

[0022] 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, containing the following components (1) and (2).

[0023] (1) A water-soluble amphoteric surfactant or a water-soluble nonionic surfactant,

[0024] (2) An aqueous solvent

[0025] In the composition of the present invention, by making the surfactant of the above (1) a water-soluble amphoteric surfactant, particularly a water-soluble amphoteric surfactant having cholic acid as a mother nucleus, the storage of the solid phase carrier of the present invention at room temperature becomes possible.

[0026] Furthermore, in addition to the above, the composition of the present invention may contain (3) urea or a salt thereof, and may contain (4) saccharides and / or (5) amino acids together with or separately from the (3).

[0027] The "solid phase" that is the application object of the composition of the present invention is a solid phase carrier for chromatography developed 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 bind" to the solid phase. By using the composition of the present invention, a decrease in the sensitivity of chromatography after solvent development can be suppressed.

[0028] (B) Production method of the present invention

[0029] The production method of the present invention is a production method of a solid phase for nucleic acid detection, in which the composition of the present invention and insoluble carrier particles to which a nucleic acid to be detected can bind are mixed, the mixture is attached to the solid phase, and dried.

[0030] (C) Solid phase carrier of the present invention

[0031] The solid phase carrier of the present invention is a solid phase carrier having a sample contact portion and a detection portion. In the above sample contact portion, "component (1) of the composition of the present invention and insoluble carrier particles to which a nucleic acid to be detected can bind" are attached, and in the above detection portion, "a substance that generates a signal by contacting a conjugate of a nucleic acid to be detected moving in the solid phase carrier and the insoluble carrier particles" is attached.

[0032] The solid phase carrier of the present invention is a solid phase carrier for chromatography developed with a solvent as a mobile phase. The sample contact portion is an mechanism in the solid phase carrier for sample contact.

[0033] (D) Usage method of the present invention

[0034] The method of using the present invention is a method of using a solid-phase carrier. In the sample contact portion, "the insoluble carrier particles to which the component (1) of the composition of the present invention and the nucleic acid to be detected can bind" are attached. In the detection portion, a substance that generates a signal upon contact with the conjugate of the nucleic acid to be detected and the insoluble carrier particles moving in the solid-phase carrier is attached. In this method, a sample that may contain the nucleic acid to be detected is brought into contact with the sample contact portion, and the signal of the nucleic acid to be detected generated by the contact of the conjugate of the nucleic acid to be detected and the insoluble carrier particles, which moves in the solid-phase carrier from the sample contact portion, with the detection portion is detected.

[0035] By performing the method of using the present invention, it is possible to suppress the decrease in the detection sensitivity of the above signal compared with the conventional method due to the movement of the above sample in the solid-phase carrier. The suppression of the decrease in the detection sensitivity is based on the use of the composition of the present invention when preparing the sample contact portion of the solid-phase carrier as described above.

[0036] Effects of the Invention

[0037] According to the present invention, there are provided a solid-phase carrier having a sample contact portion for directly bringing a sample into contact for detection when detecting a nucleic acid, a method of using the same, a production method, and a solid-phase attachment composition for providing the sample contact portion of the solid-phase carrier. Detailed Description of the Invention

[0038] (A) Composition of the Present Invention

[0039] As described above, the composition of the present invention is a composition for attaching insoluble carrier particles to which the nucleic acid to be detected can bind to a solid phase, which may contain the following components (1) and (2), and may further contain (3) urea or a salt thereof, and may contain (4) saccharides and / or (5) amino acids together with or separately from the (3).

[0040] (I) Insoluble Carrier Particles to which the Nucleic Acid to be Detected can Bind

[0041] The insoluble carrier particles to which the nucleic acid to be detected can bind are insoluble carrier particles having a function of binding to the nucleic acid to be detected in the sample that comes into contact with the solid-phase carrier of the present invention (hereinafter also referred to as "binding carrier particles").

[0042] Examples of the insoluble carrier particles that form the basis of the binding carrier particles include latex particles, silica particles, metal colloid particles, etc. As the metal colloid, gold colloid, silver colloid, copper colloid, etc. can be exemplified, and gold colloid is preferred. There is no particular limitation on the particle size of the metal colloid, and it is generally in the range of 1 - 50 nm. The metal colloid particles aggregate at the detection portion of the solid-phase carrier of the present invention, so that the color development signal accumulated by their coloring can be easily captured visually.

[0043] The latex is a preferred form of the insoluble carrier particles. The latex, also known as a polymer emulsion, is formed by dispersing a polymer in an aqueous solvent such as water, with the aqueous solvent becoming the continuous phase and polymer particles in the shape of spheres or approximate spheres becoming the discontinuous phase. The so-called latex particles are the polymer particles that constitute the discontinuous phase of the latex. In this specification, "latex" is used as the representation of the whole including the latex particles.

[0044] Examples of the latex include latexes for physical adsorption such as polystyrene latex, extremely low carboxylic acid-modified latex, and latex with localized hydrophilic groups; latexes for chemical bonding such as carboxylic acid-modified latex, amino-modified latex, hydroxy-modified latex, glycidyl-modified latex, aldehyde-modified latex, and amide-modified latex; colored latex, high specific gravity polystyrene latex, magnetic latex, etc. According to the respective characteristics of these latexes, they can be used in the present invention.

[0045] As one of the preferred latexes in the present invention, there can be mentioned colored latexes colored with cyan, red, green, orange, etc. By aggregating the colored latex at the detection part of the solid-phase carrier of the present invention, a color development signal of the accumulated coloring can be easily captured visually as such.

[0046] There is no particular limitation on the particle size of the latex particles, and they can be widely selected from an average particle size of approximately 0.01 - 1 μm.

[0047] The silica particles are preferably colored in the same manner as the latex. By aggregating the colored silica particles at the detection part of the solid-phase carrier of the present invention, a color development signal of the accumulated coloring can be easily captured visually as such.

[0048] There is no particular limitation on the particle size of the silica particles, and they can be widely selected from an average particle size of approximately 1 nm - 2 μm.

[0049] The above-mentioned insoluble carrier particles have a function capable of binding to the nucleic acid to be detected. This function is determined by the combination with the binding function possessed by the nucleic acid to be detected. That is, the binding function possessed by the above-mentioned insoluble carrier particles acts in a group with the binding function possessed by the nucleic acid to be detected described later (hereinafter also referred to as "binding function to the carrier particles"), and the binding function of the above-mentioned insoluble carrier particles is the first part of this group.

[0050] As the binding functions that act in a group, for example, hydrogen bonds, ionic bonds, electrostatic binding, hydrophobic binding, physical interactions, etc. can be mentioned. Specifically, organic compound-organic compound interactions, protein-protein interactions, protein-nucleotide interactions, nucleotide-nucleotide interactions, organic compound-protein interactions, etc. can be mentioned. Moreover, as more specific group elements, the binding functions generated by the avidin (streptavidin)-biotin system, the binding functions generated between nucleic acids using a tag base sequence, etc., the binding function generated by FITC and anti-FITC, anti-digoxigenin (DIG)-digoxigenin (DIG), the binding functions generated by antigen-antibody reactions such as a tag amino acid sequence and a specific antibody, etc. can be mentioned.

[0051] In addition, the above insoluble carrier particles can have a binding function (hereinafter also referred to as "binding function to the detection unit") that forms a group with the binding function to the above detection target nucleic acid and the binding function in the detection unit described later. As the binding function of the insoluble carrier particles to the detection unit, the same binding functions as those for the above detection target nucleic acid can be mentioned.

[0052] The binding carrier particles have any one or both of the above binding functions as the above first part.

[0053] The addition of the above first part in the insoluble carrier particles can be carried out using a conventional method suitable for the type of the insoluble carrier particles and this part, whereby binding carrier particles can be prepared.

[0054] The binding carrier particles are preferably mixed and used with the composition of the present invention during use. The addition amount of the binding carrier particles relative to the composition of the present invention can be freely selected according to the type of the carrier particles and the content of other measurement systems, and there is no particular limitation. As the content in the composition, it 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, this 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.

[0055] (II) Components of the composition of the present invention

[0056] (1) Water-soluble amphoteric surfactant or water-soluble nonionic surfactant (component (1))

[0057] The above water-soluble amphoteric surfactant is not particularly limited, and amphoteric surfactants having cholic acid as a mother nucleus, carboxylic acid type amphoteric surfactants, sulfonic acid type amphoteric surfactants, sulfate ester salt type amphoteric surfactants, etc. can be mentioned. Among these, the water-soluble surfactants having cholic acid as a mother nucleus will be described later.

[0058] There is no particular limitation on the above-mentioned water-soluble nonionic surfactant, and examples thereof include polyethylene glycol type nonionic surfactants, polyol fatty acid ester type nonionic surfactants, etc. In this water-soluble nonionic surfactant, polyoxyethylene sorbitan ester (Tween 20: registered trademark) used in the examples is included.

[0059] In particular, by using a water-soluble amphoteric surfactant having cholic acid as a mother nucleus as the component (1), the room temperature management of the solid phase carrier of the present invention becomes possible. The so-called room temperature is assumed to be around 25°C, and in practical terms, it is the storage under non-refrigerated conditions.

[0060] There is no particular limitation on the water-soluble amphoteric surfactant having cholic acid as a mother nucleus. Preferably, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate), CHAPSO (3-[(3-cholamidopropyl)dimethylamino]-2-hydroxypropanesulfonate) can be exemplified. These water-soluble surfactants having cholic acid as a mother nucleus can be blended alone or in combination.

[0061] The water-soluble amphoteric surfactant having cholic acid as a mother nucleus can be produced by a conventional method or a commercially available product can be used.

[0062] Regarding the content of the above surfactant in the composition of the present invention (as the total amount of one or more surfactants), in the case where the following components (3) to (5) are not blended, relative to the composition, it is preferably 1.5 to 20% by mass, particularly preferably 2 to 14% by mass, extremely preferably 4 to 12% by mass, and most preferably 8 to 10% by mass.

[0063] (2) Aqueous solvent (component (2))

[0064] The aqueous solvent is a solvent mainly composed of water, and examples thereof include water or various buffer solutions. As this buffer solution, as long as it is used in the field of biochemistry, is suitable for the preservation of the carrier particles for binding, and does not hinder the binding reaction of the carrier particles for binding when contacting with the sample, there is no particular limitation. Specifically, phosphate buffer solution, acetate buffer solution, borate buffer solution, Tris hydrochloride buffer solution, glycine buffer solution, Good's buffer solution, etc. can be exemplified.

[0065] The content of the aqueous solvent in the composition of the present invention is the balance of the other contained components.

[0066] (3) Urea or its salt (component (3))

[0067] There is no particular limitation on the type of urea salt, and examples thereof include hydrochloride salt, etc.

[0068] Regarding the content of urea or urea salt in the composition of the present invention, relative to the composition, it is preferably 5-35% by mass, particularly preferably 8-25% by mass, extremely preferably 12-22% by mass, and most preferably 12-21%. Moreover, when formulating this component (3), the content of the surfactant (component (1)) (as the 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 can improve the color development property of the color development signal on the solid phase, especially when stored refrigerated, by being used in combination with the water-soluble amphoteric surfactant having cholic acid as the mother nucleus. By combining with the following component (4) and / or component (5), the color development property of the color development signal on the solid phase can be improved when stored at room temperature.

[0069] The content of the aqueous solvent in the composition of the present invention is the balance of the other contained components.

[0070] (4) Sugars (component (4))

[0071] Sugars are one of the components preferably added to the composition of the present invention together with the following amino acids. By formulating the water-soluble amphoteric surfactant having cholic acid as the mother nucleus and sugars in the composition of the present invention, the color development property of the color development signal on the solid phase can be improved, especially when stored refrigerated. In addition, by formulating the above-mentioned urea or urea salt, sugars, and the water-soluble amphoteric surfactant having cholic acid as the mother nucleus in the composition of the present invention, the color development property of the color development signal on the solid phase can be improved when stored refrigerated and at room temperature. When formulating sugars in combination with the water-soluble amphoteric surfactant having cholic acid as the mother nucleus in the composition of the present invention, it is also preferably formulated in combination with the following amino acids. Furthermore, when formulating a water-soluble nonionic surfactant or a water-soluble amphoteric surfactant other than the water-soluble amphoteric surfactant having cholic acid as the mother nucleus, sugars can also be formulated in combination, and no advantages in the color development property of the color development signal on the solid phase produced have been found at present.

[0072] There is no particular limitation on the sugars that can be formulated in the composition of the present invention, and disaccharides or monosaccharides are preferred.

[0073] Examples of disaccharides include maltose, cellobiose, lactulose, lactose, sucrose, trehalose, cellobiose, etc., but are not limited to these. Among these, trehalose and sucrose are preferred.

[0074] Examples of monosaccharides include D-glucose, D-mannose, D-galactose, D-fructose, etc., but are not limited to these. Among these, D-glucose is preferred.

[0075] Regarding the content of saccharides in the composition of the present invention, relative to the composition, it is preferably 1 to 15% by mass, particularly preferably 2.5 to 12% by mass, extremely preferably 5 to 11% by mass, and most preferably 5 to 10% by mass.

[0076] (5) Amino acid (Component (5))

[0077] Amino acids are one of the preferred additional components contained in the composition of the present invention together with the above-mentioned saccharides. By combining, in the composition of the present invention, particularly the water-soluble amphoteric surfactant having cholic acid as the mother nucleus with amino acids, the color development property of the color development signal on the solid phase under at least refrigerated storage can be improved. By combining amino acids and saccharides with the water-soluble amphoteric surfactant having cholic acid as the mother nucleus together with the above-mentioned urea or urea salt in the composition of the present invention, the color development property of the color development signal on the solid phase under refrigerated storage and normal temperature storage can be improved. Further, in the case of combining a water-soluble nonionic surfactant or a water-soluble amphoteric surfactant other than the above-mentioned water-soluble amphoteric surfactant having cholic acid as the mother nucleus, amino acids can be combined and incorporated, and no advantages in the color development property of the color development signal on the resulting solid phase have been found at present.

[0078] There is no particular limitation on the amino acids that can be incorporated into the composition of the present invention. For example, 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. can be mentioned. As typical examples, L-arginine, L-histidine, L-aspartic acid, and glycine can be mentioned.

[0079] Regarding the content of amino acids in the composition of the present invention, relative to the composition, it is preferably 0.1 to 4% by mass, particularly preferably 0.2 to 2% by mass, and most preferably 0.4 to 2% by mass.

[0080] When incorporating the above-mentioned saccharides (Component (4)) and / or amino acids (Component (5)), the content of the surfactant (preferably the above-mentioned water-soluble amphoteric surfactant having cholic acid as the mother nucleus) of Component (1) in the composition of the present invention is the same as the content in the case of "the case of incorporating the surfactant alone" when urea or urea salt is not incorporated into the composition, and is the same as the content in the case of "the case of incorporating urea or urea salt in combination" when urea or urea salt is incorporated in combination. The combined incorporation of Component (4) and / or Component (5) with urea or urea salt is a particularly preferred embodiment from the viewpoint of improving the color development property of the color development signal on the solid phase carrier of the present invention under refrigerated storage and normal temperature storage.

[0081] (6) Other components

[0082] In addition, blockers such as BSA, casein, serum, skim milk, stabilizers such as gum arabic, preservatives such as sodium azide, and additives such as chelating agents can also be incorporated into the composition of the present invention within qualitative and quantitative limits that do not substantially impair the effects of the present invention.

[0083] (III) Others

[0084] The pH of the composition of the present invention is preferably about 4 - 9, particularly preferably about 6 - 9.

[0085] (B) The solid-phase carrier and production method of the present invention

[0086] As described above, the solid-phase carrier of the present invention is a carrier for chromatography, and has at least a sample contact portion and a detection portion. The solid-phase carrier of the present invention can be used for chromatography by itself, or can be used in a state fixed to other supports.

[0087] The solid-phase carrier of the present invention continuously has a raw material that does not hinder the binding reaction between the nucleic acid to be detected and the insoluble carrier particles and is diffusible by itself for the nucleic acid particle conjugate (hereinafter also referred to as "nucleic acid-particle conjugate") generated by the binding reaction, including at least the sample contact portion and the detection portion. It is in a form in which the nucleic acid-particle conjugate generated in the sample contact portion can move to the detection portion by diffusion of the solvent. As such a raw material, non-woven fabric, filter paper, glass fiber, nitrocellulose fiber, polyethersulfone filter, nylon filter, polyvinylidene fluoride filter, porous material (such as silica), etc. can be cited, but are not limited to these, and can be a single substance or can be compounded. In addition, regarding the shape of this raw material portion or the entire solid-phase carrier, shapes adopted in ordinary solid-phase chromatography can be cited, such as rectangular sheets (sheets), long strips, thin rod shapes, etc., and a long strip shape is preferred.

[0088] As described above, the sample contact portion can be provided by preferably coating a composition of the present invention in which insoluble carrier particles capable of binding the above-mentioned binding target nucleic acid are dispersed at a predetermined position on the solid-phase carrier, drying it, and removing moisture. Drying can be natural drying, hot air drying, heater drying, or vacuum drying. There is no particular limitation on the position of the sample contact portion in the solid-phase carrier. As an example of the position of the sample contact portion, the end portion of the solid-phase carrier or its vicinity can be cited. This method is preferable because when the solid-phase carrier of the present invention is brought into contact with a sample that may contain the nucleic acid to be detected, the sample contact portion can face the sample, making it easy for the two to come into contact. The sample contact portion is usually one, or two or more can be provided on the solid-phase carrier.

[0089] After the formation of the nucleic acid particle conjugate in the sample contact part, the detection part can be set by attaching the nucleic acid particle conjugate that diffuses and moves on the solid-phase carrier and generating a binding function that produces a capture signal. This binding function acts in a group with the binding function of the nucleic acid of the nucleic acid particle conjugate or the solid-phase carrier particle, and the desired capture signal is generated by the contact and binding of the two.

[0090] Examples of the binding functions that act in a group include, for example, hydrogen bonds, ionic bonds, electrostatic binding, hydrophobic binding, physical interactions, etc. Specifically, examples include organic compound-organic compound interactions, protein-protein interactions, protein-nucleotide interactions, nucleotide-nucleotide interactions, organic compound-protein interactions, etc. Moreover, as more specific group elements, examples include the binding function generated by the avidin (streptavidin)-biotin system, the binding function generated between nucleic acids using a tag base sequence, etc., the binding function generated by FITC and anti-FITC, the anti-digoxigenin (DIG)-digoxigenin (DIG), the binding function generated by antigen-antibody reactions such as a tag amino acid sequence and a specific antibody, etc.

[0091] As one of the preferred modes of the binding function in the above detection part, an example is the use of a nucleic acid that can hybridize with the tag nucleic acid attached to the "nucleic acid" of the nucleic acid particle conjugate. The so-called hybridization means that for a nucleic acid particle conjugate that is partially or completely complementary to a part of the tag nucleic acid sequence, a double strand (hybridization) is formed between the tag nucleic acid and the nucleic acid in the detection part, so that the nucleic acid particle conjugate can be captured in the detection part. The detection part with a capture signal generation mechanism such as the hybridizable nucleic acid can be provided at one place or two or more places, and a method for performing multiple nucleic acid detections in one operation is achieved. For example, a method of providing the detection part at one place in the downstream direction of the mobile phase from the sample contact part or at two or more places at a predetermined interval can be exemplified. It can also be in the form of an array in which a larger number of detection parts are provided at a predetermined interval. By adopting the form of an array, a complete detection of the nucleic acid to be detected can be performed. In addition, the carrier may include a plurality of divided array regions. These plurality of divided arrays can have the same content or different contents.

[0092] Regarding the method of using the capture signal generation mechanism as the detection part, examples include pasting to maintain the continuity on the solid phase of the mobile phase, and binding of the 3'-end or 5'-end side of the solid-phase carrier that is non-covalently or covalently bound to a substance that is the basis of the signal generation mechanism for the solid phase, such as the above-mentioned hybridizable nucleic acid. This nucleic acid binding can be carried out using existing methods and instruments such as a positioning instrument.

[0093] As long as a certain signal can be generated by capturing the nucleic acid particle conjugate in the detection unit, there is no limitation on the type of the signal. For example, in the mode using colored insoluble carrier particles as the insoluble carrier particles, the nucleic acid particle conjugate in which the colored insoluble carrier particles are bound to the nucleic acid to be detected is captured and accumulated by the detection unit, so that the color of the detection unit becomes darker, and the presence of the nucleic acid to be detected in the sample can be simply detected by visual inspection or relatively simple mechanical analysis.

[0094] In addition to the above, the solid-phase carrier of the present invention can have other mechanisms as needed. For example, an adsorption unit for adsorbing and capturing the components that have not been captured by the detection unit and have moved downstream can be exemplified.

[0095] (C) Method of using the present invention

[0096] (1) Nucleic acid sample and nucleic acid to be detected

[0097] The method of using the present invention includes the following steps: bringing a sample that may contain a nucleic acid to be detected (hereinafter also referred to as "nucleic acid sample") into contact with the sample contact portion of the solid-phase carrier of the present invention.

[0098] 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. There is no particular limitation on its molecular weight, and nucleic acids from oligonucleotides to polynucleotides can be the detection targets. There is also no limitation on the type of nucleic acid, which can be natural nucleic acid or artificial nucleic acid. Furthermore, it can be single-stranded or double-stranded DNA, RNA, DNA / RNA hybrid, DNA / RNA chimera, etc. More specifically, in addition to natural or synthetic nucleic acids such as cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA, synthetic RNA, etc., artificially synthesized nucleic acids such as peptide nucleic acid, morpholino nucleic acid, methylphosphonic acid nucleic acid, S-oligonucleic acid, etc. can be widely targeted. Among these nucleic acids to be detected, DNA is preferred, and the nucleic acid amplification product generated by the nucleic acid amplification method is most preferred. A labeling substance can also be attached to the nucleic acid to be detected as needed. As the label, fluorescent substances, enzymes, radioactive substances, chromogenic substances, etc. can be listed, and the method of attaching the label can adopt a conventional method corresponding to the type of the label.

[0099] Nucleic acid amplification products are usually artificial nucleic acids generated by performing a nucleic acid amplification method on a specified part of natural nucleic acids that should originally be the nucleic acids to be detected. There are no particular limitations on the natural nucleic acids to be amplified. For example, they include bases or base sequences that are genetic indicators in organisms such as humans and non-human animals for the onset of specific diseases such as physical constitution, genetic diseases, and cancer, disease diagnosis, treatment prognosis, drug, and treatment selection. Nucleic acids from plants, nucleic acids from microorganisms such as pathogenic bacteria and viruses are also included in the nucleic acids to be detected. When the nucleic acid to be detected is RNA, methods such as RT-PCR can be used. In addition, a specified part of the nucleic acid amplification product can be further amplified (Nested PCR method, etc.). It is a preferred mode that the nucleic acid to be detected such as the nucleic acid amplification product has a binding function for binding to insoluble carrier particles (binding function to carrier particles), and it is also a preferred mode that it has a binding function for binding to the detection part of the solid-phase carrier of the present invention (binding function to detection part). In the nucleic acid to be detected, it may have only one of the binding function to carrier particles and the binding function to detection part, or may have both in combination. Specific examples of these binding functions are selected together with the "other binding function" in a group. The so-called "other binding function" is the binding function possessed by the insoluble carrier particles in the case of the binding function to carrier particles, and the binding function possessed by the detection part in the case of the binding function to detection part.

[0100] Regarding the binding functions that act in a group, as the "binding function acting on the nucleic acid to be detected in the insoluble carrier particles" in (A)(I) and the "binding function acting in a group in the detection part" in (B), as already described, the nucleic acid to be detected may have the second part of these groups.

[0101] Similarly to the above, examples of such binding functions include hydrogen bonds, ionic bonds, electrostatic binding, hydrophobic binding, physical interactions, etc. Specifically, examples include organic compound-organic compound interactions, protein-protein interactions, protein-nucleotide interactions, nucleotide-nucleotide interactions, organic compound-protein interactions, etc. Moreover, as more specific group elements, examples include binding functions generated by the avidin (streptavidin)-biotin system, binding functions generated between nucleic acids using tag base sequences, etc., binding functions generated by FITC and anti-FITC antibodies, antigen-antibody reactions such as anti-digoxigenin (DIG) antibody-digoxigenin (DIG), tag amino acid sequences and specific antibodies, etc.

[0102] As nucleic acid amplification methods, PCR method or methods based on the PCR method (RT-PCR method, Nested PCR method, etc.), LAMP method, ICAN method, etc. can be mentioned, but are not limited thereto. It can also be a nucleic acid amplification method provided in the future. Among these nucleic acid amplification methods, the PCR method or methods based on the PCR method are one of the preferred modes.

[0103] The sample raw material obtained as described above is directly or after necessary dilution and addition of drugs to prepare a nucleic acid sample.

[0104] (2) Protocol when the nucleic acid amplification product is used as the nucleic acid to be detected

[0105] As described above, a preferred protocol for the nucleic acid to be detected is the nucleic acid amplification product, and it is more preferred that the nucleic acid amplification product itself has one or more binding functions. There is no limitation on the protocol at this time, and representative protocols will be described.

[0106] As described above, as a nucleic acid amplification method, the PCR method is one of the preferred protocols. Regarding the process of the PCR method itself, it can be carried out according to a conventional method. That is, by using amplification primers capable of amplifying all or a part of the nucleic acid to be detected, and performing the temperature cycle of PCR in the presence of a heat-resistant DNA polymerase, the desired nucleic acid amplification product can be obtained. In the case where the nucleic acid to be detected is RNA, RT-PCR in which reverse transcriptase coexists in the system can be carried out.

[0107] As a method of imparting the above binding function to the nucleic acid amplification product, using nucleic acid amplification primers linked with the desired binding function during gene amplification can be mentioned. Both the reverse primer and the forward primer can have a binding function, or only either one can have a binding function. It is preferred that both have a binding function, one being a binding function to the carrier particle and the other being a binding function to the detection part.

[0108] The binding function can be exemplified by the binding functions described above. As a preferred example, a combination of the binding function generated by the avidin (streptavidin)-biotin system, the binding function generated by FITC and anti-FITC antibody, and the binding function generated between a nucleic acid using a tag base sequence, etc. can be mentioned. In this case, as the insoluble carrier particle, a colored particle is preferably used.

[0109] The binding function produced by the avidin (streptavidin)-biotin system is preferably used as the binding function for carrier particles. That is, by subjecting a nucleic acid amplification product bound to biotin or avidin (streptavidin) obtained by nucleic acid amplification using one of the nucleic acid amplification primers with biotin or avidin (streptavidin) attached to the 5'-end as a nucleic acid to be detected having a binding function for carrier particles to contact an insoluble carrier particle loaded with avidin (streptavidin) or biotin, a nucleic acid particle conjugate bound by the avidin (streptavidin)-biotin system can be obtained. The preparation of insoluble carrier particles, particularly latex, loaded with avidin (streptavidin) or biotin is relatively simple. At the same time, by attaching a tag nucleic acid as a binding function for the detection part to the 5'-end in another nucleic acid amplification primer, a nucleic acid particle conjugate having the tag nucleic acid further on the surface can be obtained in the above-mentioned nucleic acid particle conjugate bound by the avidin (streptavidin)-biotin system. If the above-mentioned nucleic acid particle conjugate having the tag nucleic acid contacts the detection part of a solid-phase carrier of a nucleic acid having a base sequence capable of hybridizing with a part or all of the tag nucleic acid by diffusion of an aqueous solvent, the tag nucleic acid forms a double strand with the nucleic acid of the detection part, and the nucleic acid particle conjugate is captured by the detection part. In this case, if the insoluble carrier particle is colored, the coloring accumulates in the detection part and becomes visible until it becomes obvious, which becomes a signal of the presence of the nucleic acid to be detected in the sample.

[0110] For specific tag sequences and nucleic acid sequences (probes) hybridizing therewith, for example, they can be in accordance with the disclosures of Patent Documents 2 and 3.

[0111] In addition, as shown in Patent Document 2, by introducing a site capable of suppressing or stopping 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, the yield of the generated true nucleic acid amplification product can be increased. Further, as shown in Patent Document 3, by using, instead of the gene amplification primer with the tag sequence attached to the 5'-end, a detection target nucleic acid recognition probe having a labeled region specifically hybridizing with the nucleic acid extension region of the true nucleic acid amplification product and having a tag sequence at the 3'-end in the nucleic acid amplification reaction using the above-mentioned gene amplification primer without the tag sequence attached to the 5'-end and the gene amplification primer with avidin (streptavidin) or biotin labeled at the 5'-end, and using the tag sequence of this probe as the binding function for the detection part, the yield of the generated true nucleic acid amplification product can be increased.

[0112] Examples

[0113] Examples of the present invention are disclosed below. Unless otherwise specified, % is % by mass relative to the compounding object.

[0114] The following disclosure consists of positive controls and examples (including comparative examples).

[0115] [Common reagents]

[0116] The following reagents (a) and (b) are reagents used in both Positive Control 1 and the examples.

[0117] (a) 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")

[0118] (b) A TE solution (10 mM Tris, 1 mM EDTA) of an oligonucleotide labeled with biotin at the 5'-end and having a base sequence 5'-AACGTCCAATAGTAACCAGAGCG (SEQ ID NO: 1) complementary to the capture DNA probe of the following detection section (hereinafter also referred to as "complementary strand oligonucleotide solution")

[0119] [Positive Control 1]

[0120] (1) Preparation of a strip for chromatography (solid phase carrier)

[0121] For glass wool (manufactured by Millipore Corporation) (8 mm × 2 mm) as the solid phase carrier, using a GENESHOT (registered trademark) positioning instrument of Nippon Gaishi Co., Ltd. that employs the ejection unit (inkjet method) described in Japanese Patent Application Laid-Open No. 2003-75305, a TE solution of a capture DNA probe composed of the following base sequence was made into dots, which was used as the "detection section" of the solid phase carrier. As the synthetic oligonucleotide sequence, 5'-CGCTCTGGTTACTATTGGACGTT (SEQ ID NO: 2) was used to prepare a strip for development type chromatography.

[0122] (2) Developing solution

[0123] The developing solution for Positive Control 1 was the following formulation. In e, a, b, c, and d were dissolved to prepare the developing solution for Positive Control 1.

[0124]

[0125] (3) Detection method for Positive Control 1

[0126] 10 μl (46.5 mass parts) of the developing solution for the above-mentioned positive control 1 was mixed with 1.5 μl (7 mass parts) of the colored latex emulsion (a) and 10 μl (46.5 mass parts) of the 1× complementary strand oligo DNA solution (b) to prepare a test solution with a total volume of 21.5 μl. The test solution formed a nucleic acid particle conjugate produced by the contact of the above (a) and (b) (produced by the binding between streptavidin coated on the colored latex and biotin labeled in the complementary strand oligo DNA).

[0127] The test solution was placed in a tube (2 ml microtube), and the end of the chromatographic strip of the above (1) was immersed therein, and the test solution containing the above nucleic acid particle conjugate was developed toward the detection part. The degree of color development associated with the formation of double-stranded DNA between the complementary strand DNA produced by the contact of the solution with the capture DNA in the detection part and the capture by the detection part was quantified using Image Lab (manufactured by BIO-RAD), and this value was used as the positive control value.

[0128] [Examples (including Comparative Examples)]

[0129] A. Group 1

[0130] (1) Preparation of the conjugate pad (solid-phase carrier)

[0131] For glass wool (manufactured by Millipore Corporation) (8 mm × 2 mm) as the solid-phase carrier, the end of the chromatographic strip prepared for the above-mentioned positive control, which is 2 mm from the end closer to the detection part when viewed in the length direction, was immersed in a mixture of 10 μl of the composition of the following Examples or Comparative Examples and 1.5 μl of the colored latex free solution (a) (the content of the colored latex particles in this mixture was 0.065 mass%) for 10 seconds, and then vacuum dried using a drying device (manufactured by EYELA) for 2 hours. This part was used as the "sample contact part" to prepare the solid-phase carriers for each Example or Comparative Example.

[0132] (2) Test method

[0133] For the solid-phase carriers of the examples prepared in (1) above, the products stored in air at 4°C or 37°C for 2 weeks (hereinafter referred to as "4°C product" and "37°C product" respectively) were used as test articles, and 20 μl of the complementary strand oligonucleotide solution (b) was used as a nucleic acid sample at room temperature, and contact with the sample contact part of the solid-phase carrier was carried out by dipping. The 4°C product was positioned as a cold storage model of the solid-phase carrier, and the 37°C product was positioned as a room temperature storage model (accelerated test). It was further left at room temperature for 20 minutes to sufficiently diffuse the solvent in the solid-phase carrier, and the degree of color development in the detection part was digitized using Image Lab (BIO-RAD), and this value was evaluated as a percentage relative to the above positive control value.

[0134] (Example 1)

[0135] The composition of Example 1 was the following formulation.

[0136]

[0137] <Manufacturing method, etc.>

[0138] Dissolve a, b, c, and d in e to prepare the composition of Example 1 (10 μl), add the colored latex (a) (1.5 μl) thereto and stir to attach it to the sample contact part of the solid-phase carrier, and prepare a solid-phase carrier using the composition of Example 1 according to the above procedure.

[0139] <Test results>

[0140] The degree of color development of the 4°C product was 100%, and the degree of color development of the 37°C product was 80 - 90%.

[0141] (Example 2)

[0142] The composition of Example 2 was the following formulation.

[0143]

[0144] <Manufacturing method, etc.>

[0145] Dissolve a, b, c, and d in e to prepare the composition of Example 2 (10 μl), add the colored latex (a) (1.5 μl) thereto and stir to attach it to the sample contact part of the solid-phase carrier, and prepare a solid-phase carrier using the composition of Example 2 according to the above procedure.

[0146] <Test results>

[0147] The degree of color development of the 4°C product was 100%, and the degree of color development of the 37°C product was 80 - 90%.

[0148] (Example 3)

[0149] The composition of Example 3 is the following formulation.

[0150]

[0151] <Manufacturing method, etc.>

[0152] Dissolve a, b, c, and d in e to prepare the composition of Example 3 (10 μl). Add the colored glue emulsion (a) (1.5 μl) thereto and stir to make it adhere to the sample contact portion of the solid-phase carrier. A solid-phase carrier using the composition of Example 3 was produced according to the above-mentioned procedure.

[0153] <Test results>

[0154] The color development degree of the product at 4°C is 100%, and that at 37°C is 80 - 90%.

[0155] (Example 4)

[0156] The composition of Example 4 is the following formulation.

[0157]

[0158] <Manufacturing method, etc.>

[0159] Dissolve a, b, c, and d in e to prepare the composition of Example 4 (10 μl). Add the colored glue emulsion (a) (1.5 μl) thereto and stir to make it adhere to the sample contact portion of the solid-phase carrier. A solid-phase carrier using the composition of Example 4 was produced according to the above-mentioned procedure.

[0160] <Test results>

[0161] The color development degree of the product at 4°C is 100%, and that at 37°C is 80 - 90%.

[0162] (Example 5)

[0163] The composition of Example 5 is the following formulation.

[0164]

[0165] <Manufacturing method, etc.>

[0166] Dissolve a, b, c, d, e, and f in g to prepare the composition of Example 5 (10 μl). Add the colored glue emulsion (a) (1.5 μl) thereto and stir to make it adhere to the sample contact portion of the solid-phase carrier. A solid-phase carrier using the composition of Example 5 was produced according to the above-mentioned procedure.

[0167] <Test results>

[0168] The color development degree of the product at 4°C is 100%, and the color development degree of the product at 37°C is also 100%.

[0169] (Example 6)

[0170] The composition of Example 6 is the following formulation.

[0171]

[0172] <Manufacturing method, etc.>

[0173] Dissolve a, b, c, d, e, and f in g to prepare the composition of Example 6 (10 μl), add the colored gum emulsion (a) (1.5 μl) thereto and stir, and make it adhere to the sample contact portion of the solid-phase carrier. A solid-phase carrier using the composition of Example 6 was produced according to the above-mentioned procedure.

[0174] <Test results>

[0175] The color development degree of the product at 4°C is 100%, and the color development degree of the product at 37°C is also 100%.

[0176] (Example 7)

[0177] The composition of Example 7 is the following formulation.

[0178]

[0179] <Manufacturing method, etc.>

[0180] Dissolve a, b, c, d, e, and f in g to prepare the composition of Example 7 (10 μl), add the colored gum emulsion (a) (1.5 μl) thereto and stir, and make it adhere to the sample contact portion of the solid-phase carrier. A solid-phase carrier using the composition of Example 7 was produced according to the above-mentioned procedure.

[0181] <Test results>

[0182] The color development degree of the product at 4°C is 100%, and the color development degree of the product at 37°C is also 100%.

[0183] (Example 8)

[0184] The composition of Example 8 is the following formulation.

[0185]

[0186]

[0187] <Manufacturing method, etc.>

[0188] Dissolve a, b, c, d, e, and f in g to prepare the composition of Example 8 (10 μl). Add the colored glue emulsion (a) (1.5 μl) thereto and stir to make it adhere to the sample contact portion of the solid-phase carrier. Prepare the solid-phase carrier using the composition of Example 8 according to the above-mentioned procedure.

[0189] <Test Results>

[0190] The color development degree of the product at 4°C is 100%, and the color development degree of the product at 37°C is also 100%.

[0191] (Example 9)

[0192] The composition of Example 9 has the following formulation.

[0193]

[0194] <Manufacturing Method, etc.>

[0195] Dissolve a, b, c, d, e, and f in g to prepare the composition of Example 9 (10 μl). Add the colored glue emulsion (a) (1.5 μl) thereto and stir to make it adhere to the sample contact portion of the solid-phase carrier. Prepare the solid-phase carrier using the composition of Example 9 according to the above-mentioned procedure.

[0196] <Test Results>

[0197] The color development degree of the product at 4°C is 100%, and the color development degree of the product at 37°C is also 100%.

[0198] (Example 10)

[0199] The composition of Example 10 has the following formulation.

[0200]

[0201] <Manufacturing Method, etc.>

[0202] Dissolve a, b, c, d, e, and f in g to prepare the composition of Example 10 (10 μl). Add the colored glue emulsion (a) (1.5 μl) thereto and stir to make it adhere to the sample contact portion of the solid-phase carrier. Prepare the solid-phase carrier using the composition of Example 10 according to the above-mentioned procedure.

[0203] <Test Results>

[0204] The color development degree of the product at 4°C is 100%, and the color development degree of the product at 37°C is also 100%.

[0205] (Example 11)

[0206] The composition of Example 11 has the following formulation.

[0207]

[0208] <Method of Preparation, etc.>

[0209] Dissolve a, b, c, and d in e to prepare the composition of Example 11 (10 μl). Add the colored gum emulsion (a) (1.5 μl) thereto and stir to make it adhere to the sample contact portion of the solid-phase carrier. The solid-phase carrier using the composition of Example 11 was produced according to the above-mentioned procedure.

[0210] <Test Results>

[0211] The color development degree of the product at 4°C was 100%, and that of the product at 37°C was 0%.

[0212] B. Group 2

[0213] Based on the results of the examples of Group 1 above, the object was further expanded and studied. The "method for producing the conjugate pad (solid-phase carrier)" in the test articles of this Group 2 was the same as that of Group 1. In addition, in the same manner as in Group 1, the "product at 4°C" (refrigerated storage model) and the "product at 37°C" (room temperature storage model) were used as test articles (examples or comparative examples). The production of each test article was carried out according to Examples 1-11 of Group 1. In the test, at room temperature, 20 μl of the complementary strand oligonucleotide solution (b) was used as the nucleic acid sample, and the sample contact portion of the solid-phase carrier was contacted by dipping, and it was further left at room temperature for 20 minutes to sufficiently carry out the solvent diffusion in the solid-phase carrier. Then, the degree of color development in the detection portion was evaluated based on visual observation. The evaluation criteria are as follows.

[0214] <Evaluation Criteria>

[0215] In addition, color development on the solid phase of the product at 4°C using the composition of Example 1 above was carried out, and it was used as the positive control 2, and the evaluation of the frames marked as ◎, ○, △, and × below was carried out.

[0216] ◎: Higher color development property compared to the positive control 2

[0217] ○: Color development property equivalent to that of the positive control 2

[0218] △: Slightly poorer color development property compared to the positive control 2, but there is no obstacle to the recognition of the color development signal by visual inspection

[0219] ×: Poor color development property, and there is an obstacle to the recognition of the color development signal

[0220] <Examples 12 - 17, Comparative Examples 1 - 2: CHAPS Alone Examples>

[0221] The composition of Example 12 has the following formulation.

[0222]

[0223] The formulation of the compositions of Examples 13 - 17 and Comparative Examples 1 - 2 was the same as that of Example 12 except for the amount of CHAPS formulated. The amounts of CHAPS formulated and the evaluation results in these Examples and Comparative Examples are shown in Table 1.

[0224]

Table 1

[0225] CHAPS amount Product evaluation at 4°C Product evaluation at 37°C Example 12 2％ ○ ○ Example 13 4％ ○ ○ Example 14 8％ ○ ○ Example 15 10％ ○ ○ Example 16 12％ ○ ○ Example 17 14％ ○ ○ Comparative Example 1 0.5％ × × Comparative Example 2 1％ × ×

[0226] <Examples 18 - 22, Comparative Examples 3 - 4: CHAPSO single examples>

[0227] The composition of Example 18 had the following formulation.

[0228]

[0229] The formulation of the compositions of Examples 19 - 22 and Comparative Examples 3 - 4 was the same as that of Example 18 except for the amount of CHAPSO formulated. The amounts of CHAPSO formulated and the evaluation results in these Examples and Comparative Examples are shown in Table 2.

[0230]

Table 2

[0231] CHAPSO amount Product evaluation at 4°C Product evaluation at 37°C Example 18 2％ ○ ○ Example 19 4％ ○ △ Example 20 8％ ○ ○ Example 21 10％ ○ ○ Example 22 12％ ○ ○ Comparative Example 3 0.5％ × × Comparative Example 4 1％ × ×

[0232] <Examples 23 - 25, Comparative Example 5: CHAPS·CHAPSO mixed examples>

[0233] The composition of Example 23 had the following formulation.

[0234]

[0235] The formulation of the compositions of Examples 24 - 25 and Comparative Example 5 was the same as that of Example 23 except for the amounts of CHAPS and CHAPSO formulated. The amounts of CHAPS and CHAPSO formulated and the evaluation results in these Examples and Comparative Examples are shown in Table 3.

[0236]

Table 3

[0237]

[0238] From the results of Examples 12 - 25 described above, it can be seen that when CHAPS and CHAPSO, which are water - soluble amphoteric surfactants having a bile acid nucleus, are formulated alone or in combination in the composition of the present invention without other substantial additional components, the solid phase carrier of the present invention can be stored in a practical state under refrigerated storage or at room temperature.

[0239] <Examples 26 - 30: Examples of CHAPS immobilization and urea modification>

[0240] The composition of Example 26 has the following formulation.

[0241]

[0242] The formulations of the compositions of Examples 27 - 30 are the same as those of Example 26, except for the amount of urea. The amounts of urea and the evaluation results in these examples are shown in Table 4. Note that the evaluation of the product at 37°C in the example with 21% by mass of urea (formulation of Example 1: positive control 2) is the same as that of the product at 4°C and is "○".

[0243]

Table 4

[0244] Urea amount Product evaluation at 4°C Product evaluation at 37°C Example 26 8％ ○ ○ Example 27 9％ ○ ○ Example 28 12％ ○ ○ Example 29 30％ ○ △ Example 30 31％ ○ △

[0245] <Examples 31 - 34, Comparative Example 6: Examples of urea immobilization and CHAPS modification>

[0246] The composition of Example 31 has the following formulation.

[0247]

[0248] The formulations of the compositions of Examples 32 - 34 and Comparative Example 6 are the same as those of Example 31, except for the amount of CHAPS. The amounts of CHAPS and the evaluation results in these examples and comparative example are shown in Table 5.

[0249]

Table 5

[0250] CHAPS amount Product evaluation at 4°C Product evaluation at 37°C Example 31 2％ △ △ Example 32 4％ ○ ○ Example 33 10％ ◎ ○ Example 34 11％ ◎ ○ Comparative Example 6 1％ × ×

[0251] From the results of Examples 26 - 34 above, it can be seen that when urea is combined and formulated, the color development of the color signal on the solid phase under refrigerated storage can be further improved.

[0252] <Examples 35 - 45: Examples of amino acid and sugar formulation>

[0253] (Example 35)

[0254] The composition of Example 35 has the following formulation. The evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is "○".

[0255]

[0256] (Example 36)

[0257] The composition of Example 36 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is also "◎".

[0258]

[0259] (Example 37)

[0260] The composition of Example 37 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is also "◎".

[0261]

[0262] (Example 38)

[0263] The composition of Example 38 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is "○".

[0264]

[0265] (Example 39)

[0266] The composition of Example 39 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is "○".

[0267]

[0268] (Example 40)

[0269] The composition of Example 40 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is "○".

[0270]

[0271] (Example 41)

[0272] The composition of Example 41 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is "○".

[0273]

[0274] (Example 42)

[0275] The composition of Example 42 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is "○".

[0276]

[0277] (Example 43)

[0278] The composition of Example 43 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is "○".

[0279]

[0280] (Example 44)

[0281] The composition of Example 44 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is "○".

[0282]

[0283] (Example 45)

[0284] The composition of Example 45 is the following formulation, the evaluation of the product at 4°C is "◎", and the evaluation of the product at 37°C is "○".

[0285]

[0286] From the results of the above Examples 35 - 45, it can be seen that when sugars and / or amino acids are combined and formulated, the color development property of the color development signal on the solid phase under at least refrigerated storage can be further improved, and an example of the improvement in the color development property of the above color development signal under normal temperature storage was also found in the urea formulation examples (Examples 36 and 37).

[0287] <Examples 46 - 47: Formulation Examples of Tween 20>

[0288] (Example 46)

[0289] The composition of Example 46 is the following formulation, the evaluation of the product at 4°C is "○", and the evaluation of the product at 37°C is "×".

[0290]

[0291] (Example 47)

[0292] The composition of Example 47 is the following formulation, the evaluation of the product at 4°C is "○", and the evaluation of the product at 37°C is "×".

[0293]

[0294] From the above Examples 46 - 47, it can be seen that when a water-soluble nonionic surfactant is used, the color development signal can be sufficiently confirmed on the solid phase under refrigerated storage. However, the color development signal under normal temperature storage is very weak, and it is again confirmed that it is difficult to be practical for normal temperature storage. Sequence Listing <110> Fujikura Kasei Co., Ltd. TBA Co., Ltd. <120> Composition for solid-phase attachment, solid-phase carrier using the composition, production method and usage method of the solid-phase carrier <130> PFKK12PCT <150> JP 2019-100728 <151> 2019-05-29 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Oligo DNA <400> 1 aacgtccaat agtaaccaga gcg 23 <210> 2 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Oligo DNA <400> 2 cgctctggtt actattggac gtt 23

Claims

1. Production method of glass wool solid-phase carrier for nucleic acid detection, wherein, Mix a composition for attaching insoluble carrier particles to a solid phase with latex particles, attach the mixture to a glass wool solid phase carrier, and dry. Among them, the latex particles can bind to a nucleic acid to be detected. The composition contains the following components (1) and (2): (1) A water-soluble amphoteric surfactant having cholic acid as a mother nucleus. (2) An aqueous solvent. The water-soluble amphoteric surfactant having cholic acid as a mother nucleus is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate) and / or CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate). The content of the water-soluble amphoteric surfactant having cholic acid as a mother nucleus in the composition is 2-14% by mass.

2. The production method of the glass wool solid-phase carrier for nucleic acid detection according to claim 1, wherein, The composition further contains (3) urea or a salt thereof.

3. The production method of the glass wool solid-phase carrier for nucleic acid detection according to claim 1, wherein, The composition further contains (4) saccharides and / or (5) amino acids.

4. The production method of the glass wool solid-phase carrier for nucleic acid detection according to claim 3, wherein, (4) The saccharides are trehalose, D-glucose, or sucrose.

5. The production method of the glass wool solid-phase carrier for nucleic acid detection according to claim 3, wherein, (5) The amino acids are L-arginine, L-histidine, L-aspartic acid, or glycine.

6. A glass wool solid-phase carrier for nucleic acid detection, which is a glass wool solid-phase carrier for nucleic acid detection produced by the method according to any one of claims 1-5, and has a sample contact part and a detection part. In the sample contact part, CHAPS and / or CHAPSO and latex particles are attached, wherein, The latex particles can bind to a nucleic acid to be detected. In the detection unit, a binding function for generating a capture signal by attaching a conjugate of a nucleic acid to be detected and latex particles that move in the solid phase carrier is added.

7. The glass wool solid-phase carrier according to claim 6, wherein A sample contact portion of the solid phase carrier is provided at or near the end of the solid phase carrier.

8. Use method of the glass wool solid-phase carrier for nucleic acid detection according to claim 6 or 7, wherein, Bring a sample that may contain a nucleic acid to be detected into contact with the sample contact portion, and detect a capture signal generated by the binding of a conjugate of a nucleic acid to be detected and latex particles that move in the solid phase carrier from the sample contact portion to the detection unit.

9. The method for using the glass wool solid phase carrier according to claim 8, wherein, The nucleic acid to be detected has a binding function for binding to latex particles.

10. The method for using the glass wool solid phase carrier according to claim 8, wherein, The nucleic acid to be detected has a binding function for binding to the detection unit of the solid phase carrier.

11. The method for using the glass wool solid phase carrier according to claim 9, wherein, The nucleic acid to be detected has a binding function for binding to the detection unit of the solid phase carrier.

12. The method for using the glass wool solid phase carrier according to any one of claims 8-11, wherein, The nucleic acid to be detected is a nucleic acid amplification product generated by a nucleic acid amplification method.

13. The method for using the glass wool solid phase carrier according to claim 12, wherein, The nucleic acid amplification method is the PCR method or a method based on the PCR method.

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