WO2020241785A1 - Composition to be attached to solid phase, solid phase support using same, method for producing solid phase support and method for using solid phase support - Google Patents

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

A composition for solid phase adhesion, a solid phase carrier using the composition, and a method for producing and using the solid phase carrier.

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 capable of directly contacting a sample for detection when detecting nucleic acid, a method of using the solid phase carrier, a production method thereof, and sample contact of the solid phase carrier. The present invention relates to a composition for solid phase adhesion for providing a portion.

In recent years, specific nucleic acids have been detected as targets, and the attributes and associations of the nucleic acids have been used in various applications such as disease detection, animal and plant variety identification, food quality and origin identification, paternity testing, and crime proof. It is done.

This nucleic acid detection technology has been rapidly developed by the nucleic acid amplification method such as the PCR method, which makes it possible to easily amplify a trace amount of nucleic acid to a detectable amount, and various techniques have been developed to date. Things are provided.

Among them, in order to achieve both simplification and high accuracy of nucleic acid detection, a technique for detecting this nucleic acid on a solid phase is provided (Patent Documents 1, 2 and 3).

The technique 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, and the amplification product has a binding site for a specific substance and binds to the specific substance. It is a technique for detecting a desired nucleic acid by concentrating the DNA amplification product, and a mode in which this is performed on a developing medium (solid phase) is disclosed as a main form.

The technique of Patent Document 2 improves the yield of producing a genuine nucleic acid amplification product by introducing a site capable of suppressing or stopping the progress of the polymerase reaction into a part of the primers used when producing the nucleic acid amplification product. This is a technique for improving the sensitivity of solid phase detection of a target nucleic acid.

The technique of Patent Document 3 uses a target nucleic acid identification probe having a labeled region that specifically hybridizes with the 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 nucleic acid amplification fragment production. This is the method used.

WO2009 / 034842 International Publication WO2013 / 038534 International Publication WO2016 / 129609 International Publication

The techniques of Patent Documents 1-3 show the progress of nucleic acid detection technology in the solid phase in order, and each of them improves the yield of target nucleic acid (nucleic acid to be detected) amplification. In general, detection of a target nucleic acid (nucleic acid to be detected) in a solid phase is excellent in terms of convenience in that no special equipment or device is required, but there is still a problem in terms of detection sensitivity.

In order to cover this point, a "developed sample solution" in which an amplification reaction solution, a developing solution, and insoluble carrier particles such as latex are mixed is once prepared in a tube outside the solid phase, and a solid phase (strip) is added thereto. By immersion, the conjugate of the nucleic acid to be detected and the insoluble carrier particles is diffused and moved to the detection part on the solid phase to generate a detection signal (for example, Example of Patent Document 3).

However, this aspect is practically complicated.
Therefore, we aimed to establish a simplified means for detecting by directly contacting the amplified reaction solution with the solid phase portion on which the insoluble carrier particles are immobilized and developing the amplified reaction solution on the solid phase portion.

The present inventors further studied to solve the above problems. As a result, by setting the composition of the composition for containing the insoluble carrier particles such as latex and adhering to the solid phase as a predetermined content, the solid phase is directly immersed in the amplification reaction solution and the solid phase is diffused and transferred. It was found that it is possible to sufficiently obtain a desired detection signal by suppressing a decrease in detection sensitivity.

The present invention comprises (A) a composition for containing insoluble carrier particles such as latex and adhering to a solid phase (composition of the present invention), and (B) production of a solid phase carrier using the composition of the present invention. Method (production method of the present invention), (C) a solid phase carrier having a sample contact portion containing the composition for solid phase adhesion from which water has been substantially removed (solid phase carrier of the present invention), and (D. ) A method of using the solid phase carrier (a method of using the present invention).

(A) Composition of the present invention The composition of the present invention is a composition for adhering insoluble carrier particles containing the following components (1) and (2) to which the nucleic acid to be detected can be bound to a solid phase. is there.
(1) Water-soluble amphoteric surfactant or water-soluble nonionic surfactant,
(2) Aqueous solvent

In the composition of the present invention, the surfactant of the present invention is solidified by using the above-mentioned (1) surfactant as a water-soluble amphoteric surfactant, particularly a water-soluble amphoteric surfactant having cholic acid as a mother nucleus. The phase carrier can be stored at room temperature.

Further, the composition of the present invention can additionally contain (3) urea or a salt thereof, and may contain (4) saccharides and / or (5) amino acids together with or separately from the (3). Is possible.

The "solid phase" to which the composition of the present invention is applied is a solid phase carrier for developing a solvent as a mobile phase and performing chromatography. The composition of the present invention is a composition for adhering "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 solvent development.

(B) Production Method of the Present Invention In the production method of the present invention, the composition of the present invention and insoluble carrier particles to which the nucleic acid to be detected can be bound are mixed, the mixture is adhered to a solid phase, and dried, nucleic acid detection. This is a solid phase production method for

(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, and in the sample contact portion, "component (1) of the composition of the present invention". And "insoluble carrier particles to which the detection target nucleic acid can be bound" are attached, and "a substance that generates a signal by contacting the conjugate of the detection target nucleic acid moving on the solid phase carrier and the insoluble carrier particles" is attached to the detection unit. Is a solid phase carrier to which "" is attached.

The solid phase carrier of the present invention is a solid phase carrier for developing a solvent as a mobile phase and performing chromatography. The sample contact is the mechanism in the solid phase carrier for sample contact.

(D) Method of Use of the Present Invention In the method of use of the present invention, "insoluble carrier particles to which the component (1) of the composition of the present invention and the nucleic acid to be detected can be bound" are attached to the sample contact portion. A method of using a solid-phase carrier to which a substance that generates a signal by contacting a conjugate of a nucleic acid to be detected and an insoluble carrier particle that moves in the solid-phase carrier is attached to the detection part, and is attached to the sample contact part. , A sample that may contain the nucleic acid to be detected is brought into contact with the sample, and the conjugate of the nucleic acid to be detected and the insoluble carrier particle moves from the sample contact portion on the solid support, and the detection generated by the contact with the detection portion. It is a method of using a solid phase carrier that detects a signal of a target nucleic acid.

By using the method of the present invention, it is possible to suppress a decrease in the detection sensitivity in the signal due to the movement of the sample in the solid phase carrier as compared with the conventional method. The suppression of this decrease in detection sensitivity is due to the use of the composition of the present invention in the preparation of the sample contact portion of the solid phase carrier as described above.

According to the present invention, when detecting nucleic acid, a solid phase carrier having a sample contact portion capable of directly contacting a sample for detection, a method of using the solid phase carrier, a production method thereof, and a sample contact portion of the solid phase carrier. A composition for solid phase adhesion, for providing the above.

(A) Composition of the present invention As described above, the composition of the present invention can contain (3) urea or a salt thereof in addition to the following components (1) and (2). A composition for attaching insoluble carrier particles to which a nucleic acid to be detected can be bound, which can contain (4) saccharides and / or (5) amino acids, together with or separately from 3).

(I) Insoluble carrier particles to which the nucleic acid to be detected can be bound The insoluble carrier particles to which the nucleic acid to be detected can be bound have a function to which the nucleic acid to be detected in the sample in contact with the solid phase carrier of the present invention can be bound. It is an insoluble carrier particle (hereinafter, also referred to as "bonding carrier particle").

Examples of the insoluble carrier particles that form the basis of the bonding carrier particles include latex particles, silica particles, and metal colloidal particles. Examples of the metal colloid include gold colloid, silver colloid, and copper colloid, but gold colloid is preferable. The particle size of the metal colloid is not particularly limited and is generally in the range of 1-50 nm. By agglutinating the metal colloidal particles at the detection part of the solid phase carrier of the present invention, the metal colloidal particles can be easily visually grasped as a color development signal in which the coloring is accumulated.

Latex is a preferred embodiment of the insoluble carrier particles. Latex is also called a polymer emulsion, in which a polymer is dispersed in an aqueous solvent such as water, the aqueous solvent becomes a continuous phase, and polymer particles having a shape similar to a true sphere or a sphere become a discontinuous phase. .. Latex particles are polymer particles that form a discontinuous phase of this latex. In the present specification, "latex" is used as a general expression including latex particles.

Latex is a latex for physical adsorption such as polystyrene latex, ultra-low carboxylic acid modified latex, hydrophilic group localized latex, etc .; carboxylic acid modified latex, amino modified latex, hydroxy modified latex, glycidyl modified latex, aldehyde modified latex, amide. Latex for chemical bonding such as modified latex; colored latex, high specific gravity polystyrene latex, magnetic latex and the like can be mentioned. It can be used in the present invention according to the characteristics of each of these latexes.

One of the suitable latexes in the present invention is a colored latex that is colored blue, red, green, orange, or the like. By agglutinating the colored latex at the detection part of the solid phase carrier of the present invention, the colored latex can be easily visually grasped as a color-developing signal in which the coloring is accumulated.

The particle size of the latex particles is not particularly limited, and can be widely selected from an average particle size of approximately 0.01-1 μm.

It is preferable that the silica particles are colored in the same manner as latex. By agglutinating the colored silica particles at the detection part of the solid phase carrier of the present invention, the colored silica particles can be easily visually grasped as a color-developing signal in which the coloring is accumulated.

The particle size of the silica particles is not particularly limited and can be widely selected from an average particle size of approximately 1 nm-2 μm.

The insoluble carrier particles have a function of being able to bind the nucleic acid to be detected. The function is specified in combination with the binding function of the nucleic acid to be detected. That is, the binding function of the insoluble carrier particles and the binding function of the nucleic acid to be detected described later (hereinafter, also referred to as "pairing carrier particle binding function") work as a set, and the binding function of the insoluble carrier particles is of this set. This is the first part.

Examples of the binding function that works as this set include hydrogen bonding, ionic bonding, electrostatic bonding, hydrophobic bonding, physical interconnection, and the like. Specifically, organic compound-organic compound interaction, protein-protein, etc. Examples include interactions, protein-nucleotide interactions, nucleotide-nucleotide interactions, organic compound-protein interactions, and the like. And, as more specific group elements, avidin (streptavidin) -binding function by biotin system, binding function by nucleic acids using tag base sequence, binding function by FITC and anti-FITC, anti-digoxigenin (DIG) -digoxigenin (DIG), a binding function by an antigen-antibody reaction such as a tag amino acid sequence and a specific antibody can be mentioned.

In addition, the insoluble carrier particles have a binding function (hereinafter, also referred to as “pair detection unit binding function”) that is paired with a binding function in the detection unit described later, in addition to the binding function to the detection target nucleic acid. May be. Examples of the pair detection binding function of the insoluble carrier particles include a binding function similar to the binding function to the above-mentioned nucleic acid to be detected.

The binding carrier particles have either or both of the above-mentioned binding functions as the above-mentioned first part.

The addition of the first portion of the insoluble carrier particles can be carried out by using a conventional method according to the type of the insoluble carrier particles and the portion, whereby the carrier particles for binding can be prepared. ..

It is preferable that the carrier particles for binding are mixed with the composition of the present invention at the time of use. The amount of the carrier particles added to the composition of the present invention can be freely selected depending on the type of the carrier particles and the contents of the measurement system, and is not particularly limited, but is usually 0 as the content in the composition. It is 0.01-2% by mass, preferably 0.02-1% by mass. For example, the content of the colored latex particles used in the examples is preferably 0.01-1% by mass, more preferably 0.02-0.5% by mass, and particularly preferably 0.04-0. It is 2% by mass.

(II) Ingredients of the composition of the present invention (1) Water-soluble amphoteric surfactant or water-soluble nonionic surfactant (ingredient (1))
The above-mentioned water-soluble amphoteric surfactant is not particularly limited, and is an amphoteric surfactant having cholic acid as a mother nucleus, a carboxylic acid type amphoteric surfactant, a sulfonic acid type amphoteric surfactant, and a sulfate ester salt type amphoteric surfactant. Activators and the like can be mentioned. Of these, a water-soluble surfactant having cholic acid as a mother nucleus will be described later.

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

In particular, by using a water-soluble amphoteric surfactant having cholic acid as a mother nucleus as the component (1), the solid phase carrier of the present invention can be controlled at room temperature. The normal temperature is assumed to be about 25 ° C., but practically, it is stored in a non-refrigerated state.

The water-soluble amphoteric surfactant having cholic acid as a mother nucleus is not particularly limited, but is preferably CHAPS (3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonate), CHASPO (3-[(3-Cholamidopropyl)). Dimethylammonio] -2-hydroxypropanesulfonate) is exemplified. These water-soluble surfactants having cholic acid as a mother nucleus can be blended alone or in combination.

A water-soluble amphoteric surfactant having cholic acid as a mother nucleus can be produced by a conventional method, but a commercially available product can also be used.

The content of the above-mentioned surfactant in the composition of the present invention (total amount as one or more kinds of surfactants) is 1 with respect to the composition when the following components (3) to (4) are not blended. .5-20% by mass is preferable, particularly preferably 2-14% by mass, extremely preferably 4-12% by mass, and most preferably 8-10% by mass.

(2) Aqueous solvent (component (2))
The aqueous solvent is a solvent mainly composed of water, and examples thereof include water and various buffer solutions. The buffer solution is not particularly limited as long as it is used in the field of biochemistry, is suitable for storing the binding carrier particles, and does not inhibit the binding reaction of the binding carrier particles when in contact with the sample. .. Specific examples thereof include phosphate buffer, acetate buffer, boric acid buffer, Tris-hydrochloric acid buffer, glycine buffer, Good's buffer and the like.

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

(3) Urea or a salt thereof (component (3))
The type of urea salt is not particularly limited, and hydrochloride and the like are exemplified.

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, most preferably 12-22% by mass, based on the composition. Is 12-21%. When this component (3) is blended, the content of the surfactant (component (1)) (total amount as one or more kinds of surfactants) is preferably 2 to 20% by mass, particularly. It is preferably 4-15% by mass, very preferably 9-12% by mass, and most preferably 10-12% by mass. Urea or urea salt improves the coloration of the coloration signal on the solid phase at least under refrigerated storage by using it in combination with the above-mentioned water-soluble amphoteric surfactant having cholic acid as a mother nucleus. It is possible to improve the color-developing property of the color-developing signal on the solid phase under normal temperature storage by combining with the component (4) and / or the component (5) described later.

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

(4) Sugars (component (4))
Sugars, along with the following amino acids, are one of the preferred additional ingredients in the compositions of the present invention. By blending the composition of the present invention with a combination of the above-mentioned water-soluble amphoteric surfactant having cholic acid as a mother nucleus and a saccharide, at least the coloration property of the coloration signal on the solid phase under refrigerated storage can be obtained. Can be improved. Further, by blending the above-mentioned urea or urea salt and saccharide in combination with the above-mentioned water-soluble amphoteric surfactant having cholic acid as a mother nucleus in the composition of the present invention, the composition is stored in a refrigerator and at room temperature. The color-developing property of the color-developing signal on the solid phase can be improved. When a saccharide is added to the composition of the present invention in combination with the above-mentioned water-soluble amphoteric surfactant having cholic acid as a mother nucleus, it is also preferable to further add it in combination with the following amino acids. In addition, even when a water-soluble nonionic surfactant or a water-soluble amphoteric surfactant other than the water-soluble amphoteric surfactant having cholic acid as a mother nucleus is blended, it is possible to blend sugars in combination. However, there is currently no advantage in the color development of the color-developing signal on the solid phase.

The saccharides that can be blended in the composition of the present invention are not particularly limited, but disaccharides or monosaccharides are preferable.

Examples of the disaccharide include, but are not limited to, maltose, cellobiose, lactulose, lactose, sucrose, trehalose, cellobiose and the like. Among these, trehalose and sucrose are preferable.

Examples of monosaccharides include, but are not limited to, D-glucose, D-mannose, D-galactose, D-fructose and the like. Among these, D-glucose is preferable.

The content of saccharides 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 5-11% by mass with respect to the composition. It is preferably 5-10% by mass.

(5) Amino acid (component (5))
Amino acids, along with the above saccharides, are one of the preferred additional ingredients in the compositions of the present invention. By blending the composition of the present invention with a combination of the above-mentioned water-soluble amphoteric surfactant having cholic acid as a mother nucleus and an amino acid, at least the coloration property of the coloration signal on the solid phase under refrigerated storage can be obtained. Can be improved. By blending the above-mentioned urea or urea salt together with the above-mentioned urea or urea salt in combination with the above-mentioned water-soluble amphoteric surfactant having cholic acid as a mother nucleus, the composition of the present invention is stored in a refrigerator and at room temperature. It is possible to improve the color-developing property of the color-developing signal on the solid phase below. It is also possible to combine amino acids when blending a water-soluble nonionic surfactant or a water-soluble amphoteric surfactant other than the water-soluble amphoteric surfactant having cholic acid as a mother nucleus. However, there is currently no advantage in the color development of the color-developing signal on the solid phase.

The amino acids that can be blended in the composition of the present invention are not particularly limited, and for example, L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-tryptophane, L-phenylalanine, L-proline, etc. Examples thereof include 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 and the like. .. Typical examples include L-arginine, L-histidine, L-aspartic acid, and glycine.

The content of amino acids in the composition of the present invention is preferably 0.1-4% by mass, particularly preferably 0.2-2% by mass, and most preferably 0.4-2% by mass with respect to the composition. Is.

When the above saccharide (component (4)) and / or amino acid (component (5)) is blended, the surfactant of the component (1) in the composition of the present invention (preferably, the above cholic acid is used as the mother nucleus). The content of the water-soluble amphoteric surfactant) is the same as the content of the above-mentioned "in the case of a single surfactant blending" when urea or a urea salt is not blended in the composition. Alternatively, when the urea salt is combined and blended, the content is the same as the above-mentioned "when the urea or urea salt is blended and blended". The combinational combination of the component (4) and / or the component (5) with urea or a urea salt is particularly preferable from the viewpoint of improving the color development of the color-developing signal in the refrigerated storage and normal temperature storage of the solid phase carrier of the present invention. This is an aspect.

(6) Other Ingredients In addition, blocking agents such as BSA, casein, serum and skim milk, stabilizers such as gum arabic, preservatives such as sodium azide, and additives such as chelating agents also substantially exhibit the effects of the present invention. It can be blended in the composition of the present invention within a qualitative and quantitative limit that does not impair.

(III) Others The pH of the composition of the present invention is preferably about 4-9, particularly preferably about 6-9.

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

The solid-phase carrier of the present invention does not inhibit the binding reaction between the nucleic acid to be detected and the insoluble carrier particles, and is also referred to as a conjugate of the nucleic acid to be detected and the insoluble carrier particles generated by this binding reaction (hereinafter, also referred to as “nucleic acid particle conjugate”). It has a material that can be diffused by itself, including at least a sample contact portion and a detection portion. This is a form in which the nucleic acid particle conjugate generated at the sample contact portion can be moved to the detection portion by solvent diffusion. Examples of the material include, but are not limited to, non-woven fabric, filter paper, glass fiber, nitrocellulose filter, polyether sulfone filter, nylon filter, polyvinylidene fluoride filter, and porous material (silica, etc.). It may be single or complex. Further, the shape of the material portion or the entire solid phase carrier may be a shape adopted in ordinary solid phase chromatography, for example, a rectangular sheet shape (fragment shape), a long shape, a thin rod shape, or the like. It is preferably elongated.

As described above, for the sample contact portion, the composition of the present invention in which the insoluble carrier particles to which the above-mentioned nucleic acid to be bound can be bound is preferably dispersed is applied to the planned portion of the solid phase carrier, and the composition is dried. It can be provided by removing water. The drying may be natural drying, hot air drying, heater drying, or vacuum drying. The position of the sample contact portion on the solid phase carrier is not particularly limited. An example of the position of the sample contact portion is the end portion of the solid phase carrier or its vicinity. This aspect is preferable because when the solid phase carrier of the present invention is brought into contact with a sample that may contain a nucleic acid to be detected, the sample contact portion can be easily brought into contact with the sample. is there. The sample contact portion is usually one location, but two or more locations can be provided on the solid phase carrier.

The detection unit may be provided by adding a binding function that captures the nucleic acid particle conjugate that has diffused and moved on the solid phase carrier after the nucleic acid particle conjugate is generated in the sample contact portion and generates a capture signal. it can. The binding function and the binding function of the nucleic acid of the nucleic acid particle conjugate or the solid phase carrier particles work as a set, and the desired capture signal is generated by contacting and binding the two.

The binding functions that act as this set include, for example, hydrogen bonding, ionic bonding, electrostatic bonding, hydrophobic bonding, physical interconnection, and the like. Specifically, organic compound-organic compound interaction, protein-protein interaction, etc. Actions, protein-nucleotide interactions, nucleotide-nucleotide interactions, organic compound-protein interactions and the like can be mentioned. And, as more specific group elements, avidin (streptavidin) -binding function by biotin system, binding function by nucleic acids using tag base sequence, binding function by FITC and anti-FITC, anti-digoxigenin (DIG) -digoxigenin (DIG), a binding function by an antigen-antibody reaction such as a tag amino acid sequence and a specific antibody can be mentioned.

One of the preferred embodiments of the binding function in the detection unit is the use of a nucleic acid that can hybridize with the tag nucleic acid added to the "nucleic acid" of the nucleic acid particle conjugate. Hybridizable means that a part or all of the tag nucleic acid sequence is complementary, and the tag nucleic acid and the nucleic acid of the detection part form a double strand (hybridize) in the nucleic acid particle conjugate to form a nucleic acid in the detection part. This means that it is possible to capture particle conjugates. The detection unit to which the capture signal generation mechanism for the hybridizable nucleic acid or the like is added may be one place, but it is an embodiment in which two or more places are provided and various nucleic acids are detected by one operation. It is also possible to do so. For example, an embodiment in which detection units are provided at one location in the downstream direction of the mobile phase from the sample contact portion or at two or more locations at predetermined intervals is exemplified. It is also possible to form an array in which a large number of detection units are provided at predetermined intervals. By using the form of the array, comprehensive detection of the nucleic acid to be detected can be performed. Further, a plurality of partitioned array regions may be provided on the carrier. These plurality of partitioned arrays may have the same content or different contents.

The method of adding the capture signal generation mechanism as a detection unit is to attach the mobile phase while maintaining continuity on the solid phase, or to use a substance that is the basis of the signal generation mechanism for the solid phase, for example, the above-mentioned hybridizable nucleic acid. , Non-covalent or covalent bond to a solid phase carrier on the 3'end or 5'end side, and the like. This nucleic acid binding can be performed using an existing method or an instrument such as a spotter.

The type of signal is not limited as long as it is possible to generate some kind of signal by capturing the nucleic acid particle conjugate in the detection unit. For example, in the embodiment in which colored particles are used 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 in the detection unit, so that the detection unit becomes darker and visually or visually. The presence of the nucleic acid to be detected in the sample can be easily detected by a relatively simple mechanical analysis.

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 trapping a component that moves downstream without being captured by the detection unit is exemplified.

(C) Method of use of the present invention (1) Nucleic acid sample and nucleic acid to be detected In the method of use of the present invention, a sample in which the nucleic acid to be detected may be contained in the sample contact portion of the solid-phase carrier of the present invention (hereinafter referred to as “nucleic acid”). It includes a step of contacting (also referred to as a "nucleic acid sample").

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 detection targets range from oligonucleotides to polynucleotides. The type of nucleic acid is not limited, and it may be a natural nucleic acid or an artificial nucleic acid. Further, it may be single-stranded or double-stranded DNA, RNA, DNA / RNA hybrid, DNA / RNA chimera or the like. More specifically, in addition to natural or synthetic nucleic acids such as cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA, and synthetic RNA, peptide nucleic acid, morpholino nucleic acid, methylphosphonate nucleic acid, S-oligonucleic acid, etc. Artificial synthetic nucleic acids can be widely targeted. Among these nucleic acid to be detected, DNA is preferable, and the nucleic acid amplification product produced by the nucleic acid amplification method is the most suitable. It is also possible to add a labeling substance to the nucleic acid to be detected, if necessary. Examples of the label include a fluorescent substance, an enzyme, a radioactive substance, a coloring substance, and the like, and as a method for adding the label, a conventional method according to the type of the label can be used.

Nucleic acid amplification products are usually artificial nucleic acids produced by subjecting a predetermined portion of a natural nucleic acid, which should be the nucleic acid to be detected, to a nucleic acid amplification method. The natural nucleic acid to be amplified is not particularly limited, and for example, in organisms such as humans and non-human animals such as the onset of a specific disease such as constitution, genetic disease, and cancer, disease diagnosis, treatment prognosis, drug and treatment selection, etc. Nucleic acids to be detected include nucleic acids derived from plants and nucleic acids derived from microorganisms such as pathogenic bacteria and viruses, which contain a base or a base sequence as an index on a gene. When the nucleic acid to be detected is RNA, the RT-PCR method or the like can be used. It is also possible to further amplify a predetermined portion of the nucleic acid amplification product (Nested PCR method, etc.). It is a preferred embodiment that the nucleic acid to be detected, such as a nucleic acid amplification product, has a binding function (anti-carrier particle binding function) for binding to insoluble carrier particles, and the detection unit of the solid phase carrier of the present invention It is also a preferred embodiment to have a binding function (pair detection unit binding function) for binding. The nucleic acid to be detected may have only one of the anti-carrier particle-binding function and the anti-detector-binding function, or may have a combination of the two. Specific examples of these binding functions are selected together with the paired "other binding function". The “other” is a binding function possessed by the insoluble carrier particles in the case of the pair-carrier particle bonding function, and a binding function possessed by the detection unit in the case of the pair-detecting unit binding function.

Regarding the binding function that acts as a set, the "binding function that acts on the nucleic acid to be detected in the insoluble carrier particles" in (A) and (I) and the "binding function that acts as a set in the detection unit" in (B) As already described, the nucleic acid to be detected may have a second part of these sets.

Similar to the above, the binding function includes, for example, hydrogen bond, ionic bond, electrostatic bond, hydrophobic bond, physical interconnect, etc. Specifically, organic compound-organic compound interaction, protein- Examples thereof include protein interaction, protein-nucleotide interaction, nucleotide-nucleotide interaction, and organic compound-protein interaction. And, as more specific group elements, the binding function by the avidin (streptavidin) -biotin system, the binding function between nucleic acids using the tag base sequence, the binding function by FITC and the anti-FITC antibody, and the anti-digoxigenin (DIG) antibody. -Digoxigenin (DIG), tag amino acid sequence and binding function by antigen-antibody reaction such as specific antibody can be mentioned.

Examples of the nucleic acid amplification method include, but are not limited to, a PCR method or a method based on the PCR method (RT-PCR method, Next PCR method, etc.), LAMP method, ICAN method, and the like. It may be a nucleic acid amplification method provided in the future. Among these nucleic acid amplification methods, the PCR method or the method based on the PCR method is one of the preferred embodiments.

Nucleic acid samples are prepared by using the sample raw materials obtained as described above as they are, or by adding necessary dilutions and chemicals.

(2) Aspects in which a nucleic acid amplification product is used as a nucleic acid to be detected As described above, a preferred embodiment as a nucleic acid to be detected is a nucleic acid amplification product, and the nucleic acid amplification product itself has one or more binding functions. Is even more suitable. The embodiment in this case is not limited, but a typical embodiment will be described.

As described above, the PCR method is one of the preferred embodiments as the nucleic acid amplification method. The PCR method itself can be performed according to a conventional method. That is, a desired nucleic acid amplification product is obtained by performing a PCR temperature cycle in the presence of a thermostable DNA polymerase using an amplification primer capable of amplifying all or part of the nucleic acid to be detected. Can be done. When the nucleic acid to be detected is RNA, RT-PCR in which reverse transcriptase coexists in the system can be performed.

As a method of imparting the above-mentioned binding function to a nucleic acid amplification product, there is an example of using a nucleic acid amplification primer in which a desired binding function is linked at the time of gene amplification. Both the reverse primer and the forward primer may have a binding function, or only one of them may have a binding function, but both have a binding function, one has a carrier particle binding function, and the other has a pair detection part binding. It is preferable that it is a function.

The above-mentioned binding function has already been exemplified, but preferred examples thereof include a binding function by an avidin (streptavidin) -biotin system, a binding function by FITC and an anti-FITC antibody, and nucleic acids using a tag base sequence. There is a combination of binding functions by. In this case, it is preferable to use colored particles as the insoluble carrier particles.

The binding function by the avidin (streptavidin) -biotin system is preferably used as the binding function for carrier particles. That is, a nucleic acid amplification product to which biotin or avidin (streptavidin) bound obtained by performing nucleic acid amplification using one of the primers for nucleic acid amplification to which biotin or avidin (streptavidin) is added to the 5'end is used as a carrier. As a detection target nucleic acid having a particle-binding function, by contacting this with insoluble carrier particles carrying avidin (streptavidin) or biotin, a nucleic acid particle conjugate bound by the avidin (streptavidin) -biotin system is obtained. Can be done. Insoluble carrier particles carrying avidin (streptavidin) or biotin, especially latex, are relatively easy to prepare. At the same time, by adding a tag nucleic acid as a pair detection part binding function to the 5'end of the other nucleic acid amplification primer, the nucleic acid particle conjugate bound by the above-mentioned avidin (streptavidin) -biotin system is further added. A nucleic acid particle conjugate having a tagged nucleic acid on its surface can be obtained. When the nucleic acid particle conjugate having the tag nucleic acid comes into contact with the detection part of the solid phase carrier having 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 is detected. The nucleic acid of the part forms a double strand, and the nucleic acid particle conjugate is captured by the detection part. In this case, if the insoluble carrier particles are colored, the coloring accumulates in the detection unit and the coloring becomes apparent to the extent that it can be visually recognized, which signals the presence of the nucleic acid to be detected in the sample.

For a specific tag sequence and a nucleic acid sequence (probe) that hybridizes with the tag sequence, for example, the disclosures of Patent Documents 2 and 3 can be followed.

Further, as shown in Patent Document 2, a site capable of suppressing or stopping the progress of the polymerase reaction is introduced between the tag sequence of the nucleic acid amplification primer used when producing the nucleic acid amplification product and the main body of the nucleic acid amplification product. As a result, the yield of producing a genuine nucleic acid amplification product can be improved. Further, as shown in Patent Document 3, instead of using the gene amplification primer to which the tag sequence at the 5'end is added, it has a labeled region that specifically hybridizes with the nucleic acid extension region of the true nucleic acid amplification product. Then, a nucleic acid identification probe to be detected having a tag sequence at the 3'end is added with a gene amplification primer to which the tag sequence at the 5'end is not added and an avidin (streptavidin) or biotin label is added to the 5'end. By coexisting in a nucleic acid amplification reaction using a new gene amplification primer and using the tag sequence of this probe as a pair detection unit binding function, the yield of producing a genuine nucleic acid amplification product can be improved.

Hereinafter, examples of the present invention will be disclosed. Unless otherwise specified,% is mass% with respect to the compounding target.

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

[Common reagents]
The following reagents (a) and (b) are reagents used in both Positive Control 1 and Examples.
(A) 0.5% colored latex liquid of streptavidin-coated colored latex (product name SA-Lx: manufactured by Fujikura Kasei Co., Ltd.) (hereinafter, also referred to as "colored latex liquid")
(B) A biotin-labeled oligo DNA at the 5'end (hereinafter, also referred to as "complementary strand oligo") of the base sequence 5'-AACGTCCAATAGTAACCAGAGCG (SEQ ID NO: 1) complementary to the captured DNA probe of the detection unit below. TE solution (10 mM Tris, 1 mM EDTA) (hereinafter, also referred to as "complementary strand oligo solution")

[Positive control 1]
(1) Preparation of Chromatographic Strip (Solid Phase Carrier) A TE solution of a captured DNA probe consisting of the base sequence shown below was applied to glass wool (manufactured by Millipore) (8 mm x 2 mm) as a solid phase carrier in JP-A-2003. A spot was used as a spot using a DNA SHOT (registered trademark) spotter of Nippon Gaishi Co., Ltd. using a discharge unit (inkprint method) described in -75305, and this was used as a "detector" of a solid phase carrier. As a synthetic oligo DNA sequence, 5'-CGCTCTGGTTACTATTGGACGTT (SEQ ID NO: 2) was used to prepare strips for unfolded chromatography.

(2) Developing solution The developing solution of Positive Control 1 has the following composition. A, b, c, and d were dissolved in e to prepare a developing solution for Positive Control 1.
Ingredients Blended amount a CHAPS 8% by mass
b BSA 1% by mass
c urea 21% by mass
d 20 x PBS 5% by mass
e Distilled water remaining amount

(3) Detection Method of Positive Control 1 In 10 μl (46.5 parts by mass) of the developing solution of Positive Control 1 described above, 1.5 μl (7 parts by mass) of the colored latex solution (a) and a 1 × complementary strand oligo DNA solution. (B) 10 μl (46.5 parts by mass) was mixed to prepare a total amount of 21.5 μl of the detection solution. In this detection solution, a nucleic acid particle conjugate (due to the binding between streptavidin coated on the colored latex and biotin labeled on the complementary strand oligo DNA) is formed by the contact of (a) and (b) above. ing.

This detection solution is placed in a tube (2 ml microtube), and the end of the chromatography strip of (1) above is immersed therein to detect the detection solution containing the nucleic acid particle conjugate. Image Lab shows the degree of coloration of nucleic acid particle conjugates associated with the formation of double-stranded DNA between complementary strand DNAs due to contact between the solution and the captured DNA of the detector. (BIO-RAD) quantified this value and used it as a positive control value.

[Example (including comparative example)]
A. Group 1
(1) Preparation of a conjugate pad (solid phase carrier) A chromatographic strip prepared for the above positive control on glass wool (manufactured by Millipore) (8 mm × 2 mm), which is a solid phase carrier, was viewed in the length direction. From the end near the detection part to 2 mm, a mixture of 10 μl of the composition of the example or comparative example of the following formulation and 1.5 μl of the colored latex suspension (a) (content of colored latex particles in the mixture). Was immersed in 0.065% by mass) for 10 seconds, and vacuum dried for another 2 hours using a drying device (EYELA), and this portion was used as a "sample contact portion" in each Example or Comparative Example. Was used as a solid phase carrier.

(2) Test method The solid phase carrier of the example prepared in (1) above was 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 a test product, and 20 μl of the complementary strand oligo solution (b) as a nucleic acid sample at room temperature, the solid phase carrier was contacted by immersion in the sample contact portion. 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 solvent was sufficiently diffused on the solid phase carrier by leaving it at room temperature for another 20 minutes, and the degree of color development in the detection unit was quantified by Image Lab (BIO-RAD), and this value was used as the above positive control value. Was evaluated as a percentage of.

(Example 1)
The composition of Example 1 has the following composition.
Ingredients Blended amount a CHAPS 8% by mass
b BSA 1% by mass
c urea 21% by mass
d 20 x PBS 5% by mass
e Distilled water remaining amount

<Manufacturing method, etc.>
The composition (10 μl) of Example 1 is prepared by dissolving a, b, c, and d in e, and the colored latex liquids (a) and (1.5 μl) are added thereto and stirred to obtain the composition. It was attached to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 1 in the above manner.

<Test results>
The 4 ° C. product had a coloration degree of 100%, and the 37 ° C. product had a coloration degree of 80-90%.

(Example 2)
The composition of Example 2 has the following composition.
Ingredients Blended amount a CHASPO 8% by mass
b BSA 1% by mass
c urea 21% by mass
d 20 x PBS 5% by mass
e Distilled water remaining amount

<Manufacturing method, etc.>
The composition (10 μl) of Example 2 is prepared by dissolving a, b, c, and d in e, and the colored latex liquids (a) and (1.5 μl) are added thereto and stirred to obtain the composition. It was attached to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 2 in the above manner.

<Test results>
The 4 ° C. product had a coloration degree of 100%, and the 37 ° C. product had a coloration degree of 80-90%.

(Example 3)
The composition of Example 3 has the following composition.
Ingredients Blended amount a CHAPS 8% by mass
b BSA 1% by mass
c urea 12% by mass
d 20 x PBS 5% by mass
e Distilled water remaining amount

<Manufacturing method, etc.>
A, b, c, and d are dissolved in e to prepare the composition (10 μl) of Example 3, and the colored latex liquids (a) and (1.5 μl) are added thereto and stirred to obtain the composition. It was attached to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 3 in the above manner.

<Test results>
The 4 ° C. product had a coloration degree of 100%, and the 37 ° C. product had a coloration degree of 80-90%.

(Example 4)
The composition of Example 4 has the following composition.
Ingredients Blended amount a CHASPO 8% by mass
b BSA 1% by mass
c urea 12% by mass
d 20 x PBS 5% by mass
e Distilled water remaining amount

<Manufacturing method, etc.>
A, b, c, and d are dissolved in e to prepare the composition (10 μl) of Example 4, and the colored latex liquids (a) and (1.5 μl) are added thereto and stirred to obtain the composition. It was attached to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 4 in the above manner.

<Test results>
The 4 ° C. product had a coloration degree of 100%, and the 37 ° C. product had a coloration degree of 80-90%.

(Example 5)
The composition of Example 5 has the following composition.
Ingredients Blended 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 x PBS 5% by mass
g Distilled water remaining amount

<Manufacturing method, etc.>
The composition of Example 5 (10 μl) is prepared by dissolving a, b, c, d, e, and f in g, and the colored latex liquid (a) (1.5 μl) is added thereto and stirred. Then, this was adhered to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 5 in the above manner.

<Test results>
The 4 ° C. product had a 100% coloration degree, and the 37 ° C. product also had a 100% coloration degree.

(Example 6)
The composition of Example 6 has the following composition.
Ingredients Blended amount a CHASPO 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 x PBS 5% by mass
g Distilled water remaining amount

<Manufacturing method, etc.>
A, b, c, d, e, and f are dissolved in g to prepare the composition (10 μl) of Example 6, and the colored latex liquids (a) and (1.5 μl) are added thereto and stirred. Then, this was adhered to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 6 in the above manner.

<Test results>
The 4 ° C. product had a 100% coloration degree, and the 37 ° C. product also had a 100% coloration degree.

(Example 7)
The composition of Example 7 has the following composition.
Ingredients Blended 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 x PBS 5% by mass
g Distilled water remaining amount

<Manufacturing method, etc.>
A, b, c, d, e, and f are dissolved in g to prepare the composition (10 μl) of Example 7, and the colored latex liquids (a) and (1.5 μl) are added thereto and stirred. Then, this was adhered to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 7 in the above manner.

<Test results>
The 4 ° C. product had a 100% coloration degree, and the 37 ° C. product also had a 100% coloration degree.

(Example 8)
The composition of Example 8 has the following composition.
Ingredients Blended amount a CHASPO 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 x PBS 5% by mass
g Distilled water remaining amount

<Manufacturing method, etc.>
A, b, c, d, e, and f are dissolved in g to prepare the composition (10 μl) of Example 8, and the colored latex liquids (a) and (1.5 μl) are added thereto and stirred. Then, this was adhered to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 8 in the above manner.

<Test results>
The 4 ° C. product had a 100% coloration degree, and the 37 ° C. product also had a 100% coloration degree.

(Example 9)
The composition of Example 9 has the following composition.
Ingredients Blended 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 x PBS 5% by mass
g Distilled water remaining amount

<Manufacturing method, etc.>
A, b, c, d, e, and f are dissolved in g to prepare the composition (10 μl) of Example 9, and the colored latex liquid (a) (1.5 μl) is added thereto and stirred. Then, this was adhered to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 9 in the above manner.

<Test results>
The 4 ° C. product had a 100% coloration degree, and the 37 ° C. product also had a 100% coloration degree.

(Example 10)
The composition of Example 10 has the following composition.
Ingredients Blended 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 x PBS 5% by mass
g Distilled water remaining amount

<Manufacturing method, etc.>
A, b, c, d, e, and f are dissolved in g to prepare the composition (10 μl) of Example 10, and the colored latex liquids (a) and (1.5 μl) are added thereto and stirred. Then, this was adhered to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 10 in the above manner.

<Test results>
The 4 ° C. product had a 100% coloration degree, and the 37 ° C. product also had a 100% coloration degree.

(Example 11)
The composition of Example 11 has the following composition.
Ingredients Blended amount a Tween20 8% by mass
b BSA 1% by mass
c urea 12% by mass
d 20 x PBS 5% by mass
e Distilled water remaining amount

<Manufacturing method, etc.>
The composition (10 μl) of Example 11 is prepared by dissolving a, b, c, and d in e, and the colored latex liquids (a) and (1.5 μl) are added thereto and stirred to obtain the composition. It was attached to the sample contact portion of the solid phase carrier to prepare a solid phase carrier using the composition of Example 11 in the above manner.

<Test results>
The product at 4 ° C. had a degree of coloration of 100%, and the product at 37 ° C. had a degree of coloration of 0%.

B. Group 2
Based on the results of the examples in Group 1 above, the scope of the study was further expanded. The “method for producing a conjugate pad (solid phase carrier)” in the test product of this group 2 is the same as that of group 1. Further, in the same manner as in Group 1, the "4 ° C. product" (refrigerated storage model) and the "37 ° C. product" (normal temperature storage model) were used as test products (examples or comparative examples). Preparation of each test product was carried out according to Example 1-11 of Group 1. In the test, 20 μl of the complementary strand oligo solution (b) was used as a nucleic acid sample at room temperature, and the sample contact portion of the solid phase carrier was contacted by immersion and left at room temperature for another 20 minutes to sufficiently solvent the solid phase carrier. Diffusion was performed. Then, the degree of color development in the detection unit was evaluated based on visual observation. The evaluation criteria are as follows.

<Evaluation criteria>
Coloring on a solid phase of a 4 ° C. product using the composition of the above-mentioned Example 1 was performed separately, and this was used as a positive control 2 to evaluate the frameworks marked with ◎, ○, △, and × below. It was.
⊚: Higher coloration than Positive Control 2 ○: Same coloration as Positive Control 2 Δ: Slightly inferior to Positive Control 2, but does not hinder visual identification of color signals None ×: Poor coloration, which hinders the identification of color signals

<Example 12-17, Comparative Example 1-2: CHAPS alone example>
The composition of Example 12 has the following composition.
Ingredients Blended amount a CHAPS 2% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Distilled water remaining amount

The composition of Examples 13-17 and Comparative Example 1-2 is the same as that of Example 12 except for the amount of CHAPS. Table 1 shows the blending amount of CHAPS and the evaluation results in these Examples and Comparative Examples.

<Example 18-22, Comparative Example 3-4: CHASPO alone example>
The composition of Example 18 has the following composition.
Ingredients Blended amount a CHASPO 2% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Distilled water remaining amount

The composition of Examples 19-22 and Comparative Example 3-4 is the same as that of Example 18 except for the amount of CHASPO. Table 2 shows the blending amount of CHASPO and the evaluation results in these Examples and Comparative Examples.

<Example 23-25, Comparative Example 5: CHAPS / CHASPO mixed example>
The composition of Example 23 has the following composition.
Ingredients Blended amount a CHAPS 1% by mass
b CHASPO 1% by mass
c BSA 1% by mass
d 20 x PBS 5% by mass
e Distilled water remaining amount

The composition of Examples 24-25 and Comparative Example 5 is the same as that of Example 23 except for the amounts of CHASPS and CHAPSO. Table 3 shows the blending amounts of CHASPS and CHASPO and the evaluation results in these Examples and Comparative Examples.

Based on the results of Examples 12-25 above, CHAPS and CHAPSO, which are water-soluble double-sided activators based on cholic acid, are added to the composition of the present invention alone or in combination with other substantially additional components. It has been clarified that the solid phase carrier of the present invention can be stored in a practical state under refrigerated storage or at room temperature even when blended without the compound.

<Example 26-30: CHAPS fixed / urea fluctuation example>
The composition of Example 26 has the following composition.
Ingredients Blended amount a CHAPS 8% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Urea 8% by mass
e Distilled water remaining amount

The composition of Example 27-30 is the same as that of Example 26 except for the amount of urea compounded. Table 4 shows the blending amount of urea and the evaluation results in these examples. The evaluation in this test of the 37 ° C. product in the example in which the urea content was 21% by mass (formulation of Example 1: Positive Control 2) was "◯", which was equivalent to that of the 4 ° C. product.

<Example 31-34, Comparative Example 6: Urea fixation, CHAPS fluctuation example>
The composition of Example 31 has the following composition.
Ingredients Blended amount a CHAPS 2% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Urea 21% by mass
e Distilled water remaining amount

The composition of Examples 32-34 and Comparative Example 6 is the same as that of Example 31 except for the amount of CHAPS. Table 5 shows the blending amount of CHAPS and the evaluation results in these Examples and Comparative Examples.

From the results of Examples 26-34 described above, it was clarified that when urea is blended in combination, it is possible to further improve the color-developing property of the color-developing signal on the solid phase under refrigerated storage. ..

<Example 35-45: Amino acid / sugar compounding example>
(Example 35)
The composition of Example 35 had the following composition, and the evaluation of the product at 4 ° C. was “⊚” and the evaluation of the product at 37 ° C. was “◯”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Urea 21% by mass
e L-arginine 0.4% by mass
f Remaining amount of distilled water

(Example 36)
The composition of Example 36 had the following composition, and the evaluation of the product at 4 ° C. was “⊚”, and the evaluation of the product at 37 ° C. was also “⊚”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Urea 21% by mass
e Trehalose 10% by mass
f Remaining amount of distilled water

(Example 37)
The composition of Example 37 had the following composition, and the evaluation of the product at 4 ° C. was “⊚”, and the evaluation of the product at 37 ° C. was also “⊚”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x 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 amount

(Example 38)
The composition of Example 38 had the following composition, and the evaluation of the product at 4 ° C. was “⊚” and the evaluation of the product at 37 ° C. was “◯”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d L-arginine 0.4% by mass
e Distilled water remaining amount

(Example 39)
The composition of Example 39 had the following composition, and the evaluation of the product at 4 ° C. was “⊚” and the evaluation of the product at 37 ° C. was “◯”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Trehalose 10% by mass
e Distilled water remaining amount

(Example 40)
The composition of Example 40 had the following composition, and the evaluation of the product at 4 ° C. was “⊚” and the evaluation of the product at 37 ° C. was “◯”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Trehalose 10% by mass
e L-arginine 0.4% by mass
f Remaining amount of distilled water

(Example 41)
The composition of Example 41 had the following composition, and the evaluation of the product at 4 ° C. was “⊚” and the evaluation of the product at 37 ° C. was “◯”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Glycine 0.4% by mass
e Distilled water remaining amount

(Example 42)
The composition of Example 42 had the following composition, and the evaluation of the product at 4 ° C. was “⊚” and the evaluation of the product at 37 ° C. was “◯”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d L-histidine 0.4% by mass
e Distilled water remaining amount

(Example 43)
The composition of Example 43 had the following composition, and the evaluation of the product at 4 ° C. was “⊚” and the evaluation of the product at 37 ° C. was “◯”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d L-aspartic acid 0.4% by mass
e Distilled water remaining amount

(Example 44)
The composition of Example 44 had the following composition, and the evaluation of the product at 4 ° C. was “⊚” and the evaluation of the product at 37 ° C. was “◯”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Sucrose 10% by mass
e Distilled water remaining amount

(Example 45)
The composition of Example 45 had the following composition, and the evaluation of the product at 4 ° C. was “⊚” and the evaluation of the product at 37 ° C. was “◯”.
Ingredients Blended amount a CHAPS 10% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d D (+)-glucose 10% by mass
e Distilled water remaining amount

According to the results of Examples 35-45 above, when saccharides and / or amino acids are blended in combination, it is possible to further improve the coloration of the coloration signal on the solid phase at least under refrigerated storage. In the urea compounding examples (Examples 36 and 37), examples of improving the color development of the coloration signal under normal temperature storage were also observed.

<Example 46-47: Formulation example of Tween 20>
(Example 46)
The composition of Example 46 had the following composition, and the evaluation of the product at 4 ° C. was “◯” and the evaluation of the product at 37 ° C. was “x”.
Ingredients Blended amount a Tween20 8% by mass
b BSA 1% by mass
c 20 x PBS 5% by mass
d Urea 21% by mass
e Distilled water remaining amount

(Example 47)
The composition of Example 47 had the following composition, and the evaluation of the product at 4 ° C. was “◯” and the evaluation of the product at 37 ° C. was “x”.
Ingredients Blended amount a Tween20 10% by mass
b BSA 1% by mass
c 20 x 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 amount

According to Examples 46-47 above, it was clarified that even when a water-soluble nonionic surfactant was used, the color development signal could be sufficiently confirmed on the solid phase under refrigerated storage. However, it was reconfirmed that the color development signal under normal temperature storage is very weak and practical use is difficult at normal temperature storage.

Claims (18)

The following components (1) and (2):
(1) Water-soluble amphoteric surfactant or water-soluble nonionic surfactant,
(2) Aqueous solvent;
A composition for adhering insoluble carrier particles to which a nucleic acid to be detected can be bound to a solid phase. The composition according to claim 1, wherein the amphoteric surfactant is a water-soluble amphoteric surfactant having cholic acid as a mother nucleus. (1) A water-soluble amphoteric surfactant having cholic acid as a mother nucleus is CHAPS (3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonate) or CHASPO (3-[(3-Cholamidopropyl) dimethylammonio] -2. -Hydroxypropanesulfonate), the composition according to claim 1 or 2. The composition according to any one of claims 1-3, further containing (3) urea or a salt thereof. The composition according to any one of claims 1-4, further containing (4) saccharides and / or (5) amino acids. (4) The composition according to claim 5, wherein the saccharide is trehalose, D-glucose, or sucrose. (5) The composition according to claim 5 or 6, wherein the amino acid is L-arginine, L-histidine, L-aspartic acid, or glycine. A solid phase for nucleic acid detection, wherein the composition according to any one of claims 1-7 is mixed with insoluble carrier particles to which the nucleic acid to be detected can be bound, the mixture is attached to the solid phase, and dried. Production method. The production method according to claim 8, wherein the insoluble carrier particles are latex particles. A solid-phase carrier having a sample contact portion and a detection portion, in which the water-soluble amphoteric surfactant or the water-soluble nonionic surfactant can bind to the nucleic acid to be detected. A solid-phase carrier to which insoluble carrier particles are attached, and a substance that generates a signal by contacting a conjugate of the nucleic acid to be detected moving in the solid-phase carrier and the insoluble carrier particles is attached to the detection unit. The solid phase carrier according to claim 10, further comprising a sample contact portion of the solid phase carrier at or near the end of the solid phase carrier. The solid phase carrier according to claim 10 or 11, wherein the amphoteric surfactant is a water-soluble amphoteric surfactant having cholic acid as a mother nucleus. A water-soluble amphoteric surfactant or a water-soluble nonionic surfactant and insoluble carrier particles to which the nucleic acid to be detected can be bound are attached to the sample contact portion, and the solid phase is attached to the detection portion. A method of using a solid-phase carrier to which a substance that generates a signal by contacting a conjugate of a nucleic acid to be detected moving in a carrier and an insoluble carrier particle is attached, and the nucleic acid to be detected is contained in the sample contact portion. Contact a possible sample and detect the signal of the detection target nucleic acid generated by the contact between the detection target nucleic acid and the insoluble carrier particle, which moves from the sample contact portion on the solid support, and the above detection portion. , How to use solid phase carrier. 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 insoluble carrier particles. The nucleic acid to be detected has a binding function for binding to the detection part of the solid phase carrier. The method for using a solid phase carrier according to claim 13 or 14. The method for using a 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. The method for using a 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. The method for using a solid phase carrier according to any one of claims 13 to 17, wherein the amphoteric surfactant is a water-soluble amphoteric surfactant having cholic acid as a mother nucleus.