JP2003199600A - Target nucleic acid detection method - Google Patents

Abstract

[PROBLEMS] To provide a method for quickly and easily detecting a target nucleic acid in a test sample, and a kit for detecting a target nucleic acid used in the detection method. A method of detecting a target nucleic acid using a capture probe having a binding property to a target nucleic acid and a competitive probe having no binding property to the target nucleic acid but having the binding property, (A) A test sample is placed on a water-absorbing substrate having a stationary phase in which the capture probe is immobilized and a reporter phase in which a labeled complex having the competitive probe and a signal reporter is held so as to be able to be developed. And (b) detecting a signal from the signal reporter in the stationary phase, wherein the signal is detected lower in the stationary phase than in the control sample containing no target nucleic acid. A method for detecting a target nucleic acid, which determines that a target nucleic acid is contained in a test sample, and a method for detecting a target nucleic acid comprising a water-absorbing substrate having the stationary phase and a reporter phase. To

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a target nucleic acid and a kit for detecting the target nucleic acid.

[0002]

2. Description of the Related Art Detection of pathogenic microorganisms such as Salmonella, which has been a problem in recent years, in foods and patients often requires many days and complicated operations.
Further, depending on the disease, more rapid diagnosis is often required.

Various immunoassays (immunoassays) such as labeled immunoassays are used for these detections and diagnoses, and recently, rapid and simple tests by an immunochromatographic system have been widely performed. In such a measurement method,
Antibodies against the microorganisms being tested are essential. However, some microorganisms require detection at the genus level rather than at the species level, and thus bacteria having various serotypes can be targets for detection. In such a case, since antibodies against various antigens are required, a great deal of labor and cost are required to secure the antibodies.

Therefore, in recent years, a hybridization method using a DNA probe or the like has been used. For example, in a method for detecting a specific microorganism, hybridization is carried out by targeting bacterial ribosomal RNA (rRNA). Bacterial r
From the viewpoint that the base sequence of RNA reflects the evolution of bacteria, its effectiveness is widely known [DeLong E.
F. et al., Science 243: 1360-1363 (1989)]. Therefore, such a method is an excellent method for detecting a specific microorganism. However, the usual hybridization method includes procedures such as blotting, hybridization, washing, and color development, and the membrane on which any sample is blotted is sealed in a plastic bag and heated, or repeated washing is performed. However, it is hard to say that it is superior in terms of operability.

[0005]

DISCLOSURE OF THE INVENTION The present invention has extremely excellent operability as compared with conventional hybridization methods.
An object of the present invention is to provide a method for detecting a target nucleic acid capable of detecting the presence of the target nucleic acid in a test sample rapidly and simply, and a target nucleic acid detection kit suitably used for the detection method.

[0006]

That is, the present invention is
[1] A capture probe having a binding property with a target nucleic acid,
A method for detecting a target nucleic acid using a competitive probe having no binding property with respect to a target nucleic acid but having binding property with the capture probe, comprising: (a) a stationary phase obtained by immobilizing the capture probe; A step of introducing a test sample onto a water-absorbent substrate having a reporter phase in which a labeled complex having a competitive probe and a signal reporter is foldably held by contact with a liquid, (b) stringent Under the conditions, the step of developing the test sample on the water-absorbent substrate, and (c) detecting the signal from the signal reporter in the stationary phase, and the case of a control sample containing no target nucleic acid In comparison, when the signal is detected low in the stationary phase, it is determined that the target nucleic acid is contained in the test sample, [2] the water-absorbing substrate introduces the test sample. [3] The detection method according to [1] above, wherein the signal reporter is a colored particle, an enzyme or a fluorescent dye. [4] The detection method according to [1] or [2], [4] The detection method according to any one of [1] to [3], wherein the target nucleic acid is a single-stranded nucleic acid, and [5] the single-stranded nucleic acid is a ribosome. The detection method according to [4] above, which is RNA, [6] Ribosomal RN
The present invention relates to the detection method according to the above [5], wherein A is derived from Salmonella or Listeria, and [7] a kit for detecting a target nucleic acid comprising the water-absorbing substrate according to the above [1].

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The method for detecting a target nucleic acid of the present invention (hereinafter, referred to as a detection method) is one in which the detection of a target nucleic acid in a test sample by a conventional hybridization method is performed by a chromatographic method. A major feature. Therefore, according to the present invention, a test sample, a reagent or the like is introduced onto the water-absorbent substrate used in the present invention without performing the complicated operation of the conventional hybridization method (for example,
The presence of the target nucleic acid in the test sample can be detected rapidly and simply by simply adding (dripping).

That is, according to the present invention, the target nucleic acid and the competitive probe are hybridized competitively with the capture probe by a chromatographic method, and the degree of the competitive probe hybridized with the capture probe is determined by the signal bound to the competitive probe. When the signal from the reporter is detected and the target nucleic acid is not contained at all, that is, when only the competing probe hybridizes to the capture probe as a control, the test is performed when the signal detection is decreased as compared to such a case. It is a method for detecting a target nucleic acid, which determines that the sample contains the target nucleic acid.

Specifically, the detection method uses a capture probe having a binding property with a target nucleic acid and a competitive probe having no binding property with the target nucleic acid but having a binding property with the capture probe. A method for detecting a target nucleic acid, comprising:
(A) On a water-absorbing substrate having a stationary phase formed by immobilizing the capture probe and a reporter phase formed by holding a labeled complex having the competitive probe and a signal reporter so as to be developed by contact with a liquid The step of introducing the test sample, (b) developing the test sample on the water-absorbent substrate under stringent conditions, and (c) detecting the signal from the signal reporter in the stationary phase. The process of doing is included. Then, using a control sample containing no target nucleic acid, the same operation as the test sample is separately performed,
It is judged that the target nucleic acid is contained in the test sample when the signal from the signal reporter of the labeled complex is detected low in the stationary phase as compared with the case of the control sample.

In the present specification, "having binding ability" means capable of hybridizing with each other under stringent conditions and "having no binding ability".
Means that they do not hybridize to each other under stringent conditions. Also, "nucleic acid" means DNA
(Deoxyribonucleic acid) and RNA (ribonucleic acid) are included. The nucleic acid includes any nucleic acid such as naturally-occurring nucleic acid, nucleic acid produced by biological method and / or chemical method, amplified nucleic acid, recombinant nucleic acid and the like.

In the present invention, the component to be developed on the substrate (hereinafter referred to as the component to be developed) introduced on the water-absorbent substrate is naturally developed by the capillary phenomenon, and therefore the component to be developed is water. It is suitable to use a solution or dispersion as a medium. For example, when the test sample provided in such a form is introduced into one end of the water-absorbent substrate of the present invention [step (a)], the test sample immediately spreads on the water-absorbent substrate [step. (B)] is performed.

The sample to be tested is introduced so that the desired effects of the present invention can be obtained. For example, the introduction may be appropriately performed by dropping or the like, and the introduction amount at that time is not particularly limited as long as the desired effect of the present invention can be obtained. The introduction position is one side where the reporter phase exists from the stationary phase on the water-absorbent substrate, and the position where the reporter phase or the terminal side of the reporter phase is located and a desired expansion migration distance can be secured. Is desirable. The development movement distance is the distance from the introduction position of the test sample to the stationary phase. As the deployment movement distance, preferably 5 to 35
mm, more preferably 10 to 30 mm. Within such a range, the signal detection sensitivity is good and the measurement time is appropriate.

The test sample comes into contact with the reporter phase at the same time as it is introduced, or reaches the reporter phase as it develops. In the reporter phase, a labeled complex having a competitive probe and a signal reporter is mixed with a liquid. Since it is held so that it can be developed by contact, when the medium (preferably water) of the test sample comes into contact with the labeled complex of the reporter phase, the labeled complex is eluted from the reporter phase and released simultaneously and developed It becomes possible, and as a result, it is developed on the water-absorbent substrate together with the test sample.

Since the competitive probe contained in the labeled complex does not bind to the target nucleic acid, they do not hybridize with each other even if the target nucleic acid is present in the test sample. Each body is deployed independently. Since both the target nucleic acid and the competitive probe of the labeled complex have binding properties with the capture probe, when they come into contact with the immobilized capture probe in the stationary phase, they hybridize competitively with the capture probe.

After a certain period of time has passed since the test sample was developed, the signal from the signal reporter is detected in the stationary phase [step (c)].

Further, the same operation as described above is separately performed using a control sample.

When the test sample does not contain any target nucleic acid, only the labeled complex will be captured in the stationary phase through the hybridization between the competitive probe and the capture probe. The signal is detected to the same extent as in the control sample.

On the other hand, when the test sample contains the target nucleic acid, the target nucleic acid is captured in the stationary phase as a result of the competitive hybridization between the target nucleic acid and the competitive probe, and the target nucleic acid is contained in the test sample. The signal from the signal reporter is detected lower than in the control sample due to the reduced or substantially no capture of the labeled complex as compared to the absence of a. Also, if there is no capture of the labeled complex, the signal will not be detected.

Therefore, in the method for detecting a target nucleic acid of the present invention, it is determined that the target nucleic acid is present in the test sample when the signal from the signal reporter is detected lower than that in the control sample. You can

In the present invention, the test sample is not particularly limited as long as it can contain a nucleic acid.
Examples of the test sample include food poisoning-causing bacteria (bacteria,
For example, lysates derived from specific bacterial species such as Salmonella and Listeria, etc., cell lysates of blood and tissues of various organisms, and diluted solutions thereof can be mentioned. To lyse the bacteria or cells or dilute the resulting lysate,
Although not particularly limited, it is preferable to use an aqueous solvent such as water, borate buffer, phosphate buffer or the like. Furthermore, an amplification product obtained by polymerase chain reaction (PCR) using the nucleic acid contained in the lysate or the like as a template can also be used as a test sample.

The target nucleic acid is not particularly limited as long as its sequence is known. For example, specific gene regions encoded on the genome of the causative bacterium of the food poisoning or on the plasmid, or on the genomes of various organisms, RNA contained in the causative bacterium and the like can be mentioned. For example, when the target nucleic acid is a nucleic acid having a base sequence unique to the causative bacterium of food poisoning, if the presence of the target nucleic acid is confirmed in any test sample by the detection method of the present invention, the test sample The presence of the causative bacterium in the source (for example, food) from which is obtained becomes clear. As such a target nucleic acid, a bacterial r whose nucleotide sequence unique to a genus of a specific bacterial species or a phylogenetic group is known.
RNA is preferred. In addition, when the target nucleic acid is a wild type gene or a mutant gene known to be the etiology of a specific disease or to express the pathology of a specific disease, the presence of the gene in a test sample If confirmed, the etiology or pathological condition of the disease of the organism from which the test sample is obtained becomes clear. Furthermore, when a polymorphism exists in a specific gene of an organism, for example, a gene region having a nucleotide sequence characteristic of each gene is used as a target nucleic acid, and the detection method of the present invention is used to detect a test substance derived from the organism. It is also possible to genotype the organism by confirming the presence of the target nucleic acid in a sample.

The form of the target nucleic acid of the present invention may be single-stranded or double-stranded, but is preferably single-stranded. For example, the target nucleic acid of the present invention may be RNA
A single-stranded nucleic acid such as a single-stranded DNA obtained by arbitrarily synthesizing and amplifying by a known method is suitable. As such a single-stranded nucleic acid, for example, from the viewpoint of its usefulness in the case of determining the contamination of food by food-borne bacteria, the rR of the bacterium is
NA is preferable, and rRNA derived from Salmonella or Listeria is more preferable. When the test sample contains a double-stranded nucleic acid, before being subjected to the detection method of the present invention, by a known alkali treatment method or heat treatment method,
It is preferable that the double-stranded nucleic acid is previously dissociated into single strands.

The labeled complex of the present invention comprises a competitive probe and a signal reporter. The competitive probe, specifically, has a base sequence substantially the same as the target nucleic acid, and to the immobilized capture probe of the stationary phase,
There is no particular limitation as long as it is a nucleic acid probe that can specifically hybridize under stringent conditions. Examples of the competitive probe include nucleic acids, modified nucleic acids, nucleic acid analogs and the like. Competing probes can be single-stranded or double-stranded nucleic acids, but are preferably single-stranded nucleic acids. The number of bases is at least about 5 bases, preferably 5 to 100 bases, more preferably 15 to 50 bases, but may be several thousand bases.

The modified nucleic acid includes a modified sugar group,
A nucleic acid containing a phosphate group or a modified base is mentioned. For example, as an example of a nucleic acid containing a modified base, for example,
Acetylated base (4-acetylcytidine, etc.), carboxylated base (5-carboxymethylaminomethyluridine, etc.), or methylated base (1-
Nucleic acid containing such as methylinosine). Also,
Nucleic acids containing deazaguanine and uracil may be used as competitive probes instead of nucleic acids containing guanine and thymine. In such a case, the thermal stability of the competitive probe when hybridized decreases. Further, a nucleic acid containing 5-methylcytosine may be used as a competitive probe instead of the nucleic acid containing cytosine. In such a case, the thermal stability of the competitive probe when hybridized is improved. Modifications that remove the negative charge from the phosphodiester backbone of nucleic acids also contribute to improving the thermal stability of competing probes. When RNA is used as a competitive probe, it is preferable to modify the sugar group of ribose such as addition of 2′-O-methyl group from the viewpoint of reducing the nuclease sensitivity of the probe.

Examples of the nucleic acid analog include peptide nucleic acid (PNA) in which a standard nucleobase is bound to a peptide-like skeleton composed of repeating N- (2-aminoethyl) glycine units [Nielsen]. , PE
Et al., Science, 254: 1497-1500 (1991)]. PNAs are useful because they are very resistant to degradation by nucleases.

In the present specification, the term "substantially the same nucleotide sequence as the target nucleic acid" means that the sequence identity with the target nucleic acid is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more. A base sequence. The sequence identity refers to sequence similarity of bases between two sequences, and is determined by comparing two appropriately aligned sequences. Specifically, the number of matching sites is determined by determining the same bases present in both sequences, then dividing the number of matching sites by the total number of bases in the sequence regions to be compared,
The obtained numerical value is multiplied by 100 to calculate the sequence identity (%). Such sequence identity can be appropriately determined by using, for example, the BLAST network service.

In the present specification, the stringent condition is not particularly limited as long as it is a condition that significantly reduces the non-specific hybridization reaction. The stringent conditions for performing nucleic acid hybridization are described in, for example, a book (Molecular Cloning, 3rd edition, Sambrook and Russell, Cold Spring Harbor). There is. Factors such as probe length, base composition, sequence identity between hybridizing sequences, temperature, and ionic strength affect the stability of nucleic acid hybrids. For example, stringent conditions such as medium stringency or high stringency can be empirically determined depending on the properties of the competitive probe and the capture probe.

The competitive probe composed of the above-described nucleic acid, modified nucleic acid, nucleic acid analog and the like can be appropriately prepared by a known method (for example, refer to the above-mentioned textbook) so as to have a desired property. Also, a commercially available nucleic acid having an arbitrary sequence and modification can be used.

The signal reporter is not particularly limited as long as it can be directly or indirectly bound to the competitive probe and the signal derived from the reporter can be detected. It is preferable that the signal derived from the reporter can be detected easily and easily by an apparatus or visually, more preferably by visual observation.

Examples of such signal reporters include colored particles, enzymes, fluorescent dyes, ³² P, ³⁵ S and ³ H.
Radioisotopes and the like. Among them, colored particles, enzymes or fluorescent dyes are preferable from the viewpoint of easy detection.

In the present specification, the term "colored particles" means colored particles which can detect a signal as a color emitted directly from the particles. Examples of the particles include metal colloid particles such as gold colloid particles and selenium colloid particles, and colored water-dispersible polymer particles. From the viewpoint of being excellent in dispersibility in water and facilitating immobilization of nucleic acid, enzyme, etc., water-dispersible polymer particles are preferable.

The metal colloidal particles are preferably gold colloidal particles from the viewpoints of easy availability and stability. Further, from the viewpoint of easiness of immobilizing nucleic acid, ligand and the like and easiness of detection, the particle size (average particle size) thereof is preferably 5 to 80 nm, more preferably 10 to 50 nm. The particle size can be obtained, for example, by measuring the absolute particle size of each particle under observation with a transmission electron microscope and averaging the obtained measured values.

The water-dispersible polymer particles can be prepared, for example, by emulsion polymerization of one or more monomers having an unsaturated double bond according to a known method. Examples of such a monomer include olefinic monomers such as ethylene and propylene, vinylic monomers such as vinyl acetate and vinyl chloride, styrene-based monomers such as styrene, methylstyrene and chlorostyrene, and acrylic acid. A (meth) acrylic acid ester-based monomer such as methyl and a diene-based monomer such as butadiene are preferably used.

The water-dispersible polymer particles used in the present invention are not only those composed of homopolymers or copolymers of the above monomers, but also ligands for immobilizing nucleic acids, enzymes and the like on the surface thereof. For the purpose of introducing a spacer or a spacer, particles obtained by adding a functional group or an ionic group may be used,
Further, it may be one obtained by copolymerizing a modifying monomer for the purpose of enhancing the dispersion stability of the particles in an aqueous medium.

As such a functional group or ionic group,
Examples thereof include a carboxyl group, a hydroxyl group, a glycidyl group, an amino group, a formyl group, a carbamoyl group, an isothiocyanate group, an azidocarbonyl group, a hydrazide group, and an acid anhydride group. Of these, a carboxyl group is preferable. To prepare water-dispersible polymer particles having these functional groups and the like, as a monomer component, for example, a monomer having a carboxyl group such as acrylic acid or methacrylic acid, for example, hydroxy acrylate, 2- A monomer having a hydroxyl group such as hydroxyethyl acrylate,
For example, a monomer having a glycidyl group such as glycidyl methacrylate can be emulsion-polymerized with another copolymerizable monomer, if desired, to obtain particles having a carboxyl group, a hydroxyl group and a glycidyl group, respectively. After polymerizing the desired monomer component,
A functional group can be introduced into the obtained particles.

Examples of the modifying monomer include acrylic acid, methacrylic acid, fluorinated methacrylic acid esters such as 2,2,2-trifluoroethyl methacrylate, acrylonitrile, methacrylonitrile, acrylamide, and styrene. Sodium sulfonate, sulfopropyl (meth) acrylate sodium salt, N-vinyl-2-pyrrolidone, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, glycidyl methacrylate and the like are used.

The coloring of the water-dispersed polymer particles is carried out, for example, by
Pigments and dyes represented by Sudan Blue, Sudan Red IV, Sudan III, Oil Orange, and Quinizarine Green, and various fluorescent pigments / pigments can be optionally added during the production of the particles. When colored water-dispersed polymer particles are used as a signal reporter, the signal is directly and rapidly detected by visual inspection or an instrument [for example, a chromatographic scanner (scanning densitometer)]. be able to. From the viewpoint of ease of visual confirmation, water-dispersible polymer particles colored in blue, red, green, or orange are preferable, and the viewpoint of ease of adjustment of dispersion stability and detection sensitivity of a test substance, etc. Therefore, those colored in blue or red are more preferable.

In the present invention, various commercially available colored water-dispersible polymer particles can also be preferably used. As commercially available water-dispersed polymer particles,
For example, a homopolymer or copolymer of p-chlorostyrene, a styrene-butadiene copolymer, a styrene-acrylonitrile-butadiene copolymer, or a homopolymer containing styrene or a derivative thereof as a monomer component, or A copolymer can be mentioned. Further, a homopolymer composed of a long-chain alkyl ester of (meth) acrylic acid or a derivative thereof, and a copolymer of these with methyl (meth) acrylate, ethyl (meth) acrylate, glycidyl (meth) acrylate, etc. Used in the present invention. Further, a copolymer of styrene or its derivative and (meth) acrylic acid ester or its derivative is also used. Also, rubber, nylon, polyurethane,
Microcrystalline cellulose or the like may be used.

The water-dispersible polymer particles of the present invention may be composed of one kind of the above-mentioned homopolymer or copolymer, or may be composed of two or more kinds. .

The water-dispersible polymer particles are preferably those which do not cause fusion or aggregation during use or storage. Therefore,
The particles are preferably composed of monomers selected so as to have a desired glass transition point. The glass transition point is preferably 10 ° C. or higher, more preferably room temperature (15 ° C.) or higher. The glass transition point can be measured by, for example, a differential scanning calorimeter.

The particle size (average particle size) of such water-dispersible polymer particles is from the viewpoint of dispersibility and maintenance of developability in a water-absorbent substrate, and also from the viewpoint of immobilizing nucleic acid, ligand and the like. From
The thickness is preferably 3 μm or less, more preferably 2 μm or less, and preferably 0.01 μm or more, more preferably 0.1 μm or more from the viewpoint of easiness of purification when immobilizing nucleic acid, ligand and the like. That is, the particle diameter of the water-dispersed polymer particles is preferably 0.01 to 3 μm, more preferably 0.1 to 2 μm. In addition, the said particle diameter can be measured by the method similar to a metal colloid particle.

When an enzyme is used as the signal reporter, the signal is emitted from the product resulting from the action of the enzyme on its substrate. Preferable examples of the enzyme that can be used in the present invention include enzymes that are commonly used as labels in known enzyme immunoassay methods. Examples thereof include peroxidase, β-D-galactosidase, alkaline phosphatase, glucose oxidase, luciferase, esterase, β-D-glucuronidase and the like. Among them, peroxidase or alkaline phosphatase is preferable from the viewpoint of achieving more sensitive and stable detection.

The substrate of the enzyme is not particularly limited as long as it can detect the enzyme reaction product, but a chromogenic substrate is preferable from the viewpoint of rapid and simple detection of the product. Such a chromogenic substrate can be appropriately selected from chromogenic substrates used in known enzyme immunoassays according to the enzyme used.

As the chromogenic substrate, for example, in the case where the enzyme is peroxidase, it may be a substrate capable of reacting with a combination of peroxidase and hydrogen peroxide to produce a color, for example, 2,2'-azino-bis (3 -Ethylbenzthiazoline) -6-sulfonic acid, o-phenylenediamine, 3,3 ', 5,5'-tetramethylbenzidine (hereinafter referred to as TMB), o-dianididine, 3,
3'-diaminobenzidine, 3-amino-9-ethylcarbazole, 4-chloro-1-naphthol and the like are mentioned, and TMB is preferable from the viewpoint of color developability and nontoxicity.

In the present invention, the substrate is preferably used in a solution state, for example, as an enzyme substrate solution. As the enzyme substrate solution, a solution prepared by dissolving the chromogenic substrate in, for example, water, borate buffer solution, phosphate buffer solution or the like is suitable. Further, the substrate solution may optionally contain other components suitable for carrying out an appropriate enzymatic reaction (for example, hydrogen peroxide).

Examples of fluorescent dyes that can be used as a signal reporter include fluorescein derivatives, tetramethylrhodamine, Cy3 and Cy5.

The labeled complex can be obtained by directly or indirectly binding the signal reporter and the competitive probe as described above. When connecting indirectly,
For example, it refers to the case where the signal reporter and the competitive probe are bound via an arbitrary substance. For example, when an enzyme is used as the signal reporter, the labeled complex may be obtained by, for example, binding the enzyme and the competitive probe through the water-dispersed polymer particles.

The signal reporter is the 5'of the competitive probe.
It may be directly or indirectly bound to at least one of the end, the 3 ′ end and the inside of the sequence. The two do not necessarily have to be bound one-to-one, and for example, a plurality of signal reporters may be bound directly or indirectly to one competing probe.

The labeled complex is prepared as follows. For example, the signal reporter and the competitive probe can be bound to each other preferably by a method utilizing a covalent bond, a physical adsorption or an ionic bond, which is generally known as a binding method between arbitrary substances. From the viewpoint of bond stability, it is preferable to adopt a method utilizing a covalent bond.

For example, a spacer is directly bonded via a functional group or an ionic group existing in a signal reporter or the like, or a water-soluble carbodiimide or another bifunctional organic compound having a carbon chain group having 1 to 12 carbon atoms as a spacer. To intervene and bind. In addition, for example, a ligand is bound to a signal reporter, a compound capable of binding to the ligand (hereinafter referred to as a ligand binding compound) is bound to a competitive probe, and the binding between the ligand and the ligand binding compound is used. It is also possible to combine both by combining. Preferable examples of such a combination of ligand-ligand binding compound include biotin-avidin, biotin-
Streptavidin, digoxigenin (DIG) -anti-D
Examples thereof include IG antibody and fluorescein-anti-fluorescein antibody. Among them, biotin-streptavidin,
The combination of DIG-anti-DIG antibody is more preferably used because the complex formed by binding them is highly stable. For example, when an enzyme is used as the signal reporter, the degree of freedom in the labeled complex can be increased by interposing some kind of spacer, which is preferable from the viewpoint of increasing the efficiency of the enzymatic reaction.

In addition, for example, when the signal reporter and the competitive probe are bound via the water-dispersed polymer particles, for example, the form of (signal reporter)-(water-dispersed polymer particles)-(competitive probe) is used. In the case of obtaining a labeled complex having, the labeled complex is bound between the signal reporter and the water-dispersed polymer particle and between the water-dispersed polymer particle and the competitive probe as described above. It can be prepared.

The labeled complex is bound to the signal reporter by binding the competitive probe and then subjected to, for example, a membrane separation method or filtration,
It can be obtained by a conventional separation method such as a centrifugation method, for example, by separating and purifying from a solvent or the like which is a reaction field in which the binding is performed. The labeled complex can be stored until use, eg by immersion in water or by lyophilization.

The reporter phase can be formed by holding the obtained labeled complex on the water-absorbent substrate as follows. The labeled complex can be directly or indirectly held on the water-absorbent substrate, but it is preferably held directly from the viewpoint of operation efficiency.

When directly held on the water-absorbent substrate,
For example, the obtained labeled complex is dispersed or dissolved in water, borate buffer solution, phosphate buffer solution, or the like, and is placed in a line at a desired position on the water-absorbent substrate using, for example, a dispenser. Or spotted in a circular shape with a pipette or the like and air-dried at 15 to 80 ° C.

When indirectly held on the water-absorbent substrate,
For example, the obtained labeled complex is dispersed or dissolved in water, borate buffer, phosphate buffer, etc., and impregnated into a pad made of non-woven fabric such as polyester or rayon or glass fiber, Air-dry in the same manner as above, and then at a desired position on the water-absorbent substrate, for example,
This can be performed by adhering the pads by adhesion.

From the viewpoint of facilitating the development of the labeled complex by contact with a liquid, a solvent such as sucrose or trehalose may be contained in the solvent in which the labeled complex is dispersed or dissolved.

The shape of the reporter phase is arbitrary, but it is preferable that the reporter phase is formed in a line shape having the same width as the width of the water-absorbent substrate in the direction perpendicular to the developing direction of the test sample. Further, the amount of the labeled complex retained in the reporter phase is not particularly limited, but the expected amount of the target nucleic acid in the test sample and the amount of the competitive probe in the labeled complex are in a weight ratio of 1: 1. An amount that is a degree is preferable.

From the viewpoint of obtaining the desired effect of the present invention, the position where the reporter phase is formed on the water-absorbent substrate is the position where the test sample is introduced, or the position where the test sample is introduced and the stationary phase. It is preferable that it is between.

When colored particles or fluorescent dyes are used as the signal reporter, the color or fluorescence emitted by them in the stationary phase is used as a signal for visual detection, for example. When using a radioisotope, for example,
The presence of radioactivity in the stationary phase is detected as a signal by autoradiography.

When an enzyme is used as the signal reporter, after introducing the test sample onto the water-absorbent substrate in the step (a) of the detection method of the present invention, the enzyme substrate solution is further applied with the desired effect of the present invention. Introduce as obtained. The introduction position of the enzyme substrate solution may be the same as or different from the introduction position of the test sample.

The time required for the enzyme reaction is preferably 1 after the enzyme substrate solution is introduced, including the time for developing the enzyme substrate solution.
It is about 20 minutes. However, since the time depends on the enzyme, the substrate, the reaction conditions, etc., it is not particularly limited. For example, in a preferred embodiment of the present invention, when a chromogenic substrate is used, the color detected as a signal in the stationary phase on the water-absorbent substrate is the enzyme of the signal reporter in the labeled complex captured in the stationary phase. It is derived from the chromogenic product generated from the substrate by the action of.

In the detection method of the present invention, the component to be developed is developed on the water-absorbent substrate under stringent conditions. Therefore, the component to be developed is introduced onto the water-absorbent substrate as a preferably aqueous dispersion or solution having a composition suitable for the conditions. The “composition that meets the conditions” refers to a composition capable of hybridizing a capture probe with a competitive probe or a target nucleic acid in a test sample, and may be appropriately selected depending on the Tm value of the nucleic acid used, the temperature of the reaction environment, and the like. Good. The composition and temperature conditions are described in detail in the aforementioned publications and the like.

For example, a dispersion liquid or solution of the test sample,
The enzyme substrate solution is diluted with a previously prepared concentrated solution of a buffer solution (hybridization buffer) having the above composition and, if desired, water, adjusted to a desired composition, and then introduced onto a water-absorbent substrate. The components to be developed may be prepared from the beginning so as to have the composition of the hybridization buffer. Water (eg, deionized water, distilled water, etc.) may be used as the control sample.

The development is preferably 1 after introducing the test sample.
It may be performed for about 10 minutes. After introducing the test sample,
A washing solution may be introduced for the purpose of washing away nonspecific adsorption of nucleic acids. As the washing solution, the hybridization buffer is suitable.

As the water-absorbent substrate used in the present invention,
The base material is not particularly limited as long as it has a property of absorbing an aqueous liquid. In the present invention, a water-absorbent base material having an appropriate water-absorbent property so that the component to be developed can be rapidly developed is preferable. Specific examples of such a water-absorbent substrate include non-woven fabric, filter paper, glass fiber cloth, glass filter, nitrocellulose, and porous membrane.

The degree of water absorption of the water-absorbent substrate is, for example,
When one end of a water-absorbent substrate having a thickness of 1 mm, a width of 5 mm, and a length of 100 mm is immersed in water for 1 minute, the water absorption distance from the boundary position between the part immersed in water and the part not immersed is 0.5. It is preferably -5 cm. The degree of water absorption is appropriately adjusted by, for example, coating the surface of the water-absorbent substrate with a hydrophilic polymer such as hydroxypropylmethyl cellulose, polyvinyl alcohol, or hydroxyethyl cellulose, or impregnating the water-absorbent substrate. You can also

The shape of the water-absorbent substrate is not particularly limited as long as the mixture or the like can be developed. For example, a rectangular sheet shape (piece shape) or rod shape is preferable.

The stationary phase on the water-absorbent substrate is capable of binding to the target nucleic acid and the competing probe, that is, the capture probe capable of hybridizing under stringent conditions is directly or on the water-absorbent substrate. It is formed by indirectly fixing. The capture probe has a base sequence complementary to the base sequences of the target nucleic acid and the competitive probe. Here, “complementary” does not require that the base sequence of the capture probe accurately reflects the complementary strand of the base sequences of the target nucleic acid and the competitive probe, but it does not affect the target nucleic acid and the competitive probe under stringent conditions. It is sufficiently similar to the complementary strand to the extent that it can hybridize. The degree of similarity is preferably 80% or more,
It is more preferably 85% or more, still more preferably 95% or more. The “degree of similarity” means the “sequence identity”.
It can be evaluated according to the calculation method.

Examples of the capture probe include nucleic acids, modified nucleic acids, nucleic acid analogs and the like. The capture probe can be a single-stranded or double-stranded nucleic acid, but is preferably a single-stranded nucleic acid. The number of bases is at least about 5 bases, preferably 5 to 10 bases.
0 base length, more preferably 15 to 50 base length,
It may be several thousand bases long.

Examples of the modified nucleic acids and nucleic acid analogs include those mentioned above. The capture probe can be appropriately prepared according to a known method in the same manner as in the case of the competitive probe so as to have a desired property.

The stationary phase is at a desired position on the water-absorbent substrate,
The capture probe may be applied directly on the line by using a dispenser or the like, or may be directly spotted in a circular shape by using a pipette or the like and immobilized, but it is perpendicular to the developing direction of the component to be developed. The width is preferably the same as the width of the water-absorbent substrate in the direction. Further, the occupied area of the stationary phase on the water-absorbent substrate is preferably 0.5 to 3% in percentage with respect to the area of the water-absorbent substrate.

Immobilization of the capture probe directly on the water-absorbent substrate can be appropriately carried out according to the above method for binding the signal reporter and the competitive probe in the preparation of the labeled complex. Alternatively, for example, the capture probe can be simply applied on a water-absorbent substrate, and then immobilized by heating at 120 ° C. for about 30 minutes in an oven for heat treatment.

When the capture probe is indirectly immobilized on a water-absorbent substrate to form a stationary phase, for example, the capture probe is absorbed by a pad made of a nonwoven fabric made of polyester, rayon, glass fiber or the like, and the capture probe is absorbed by water. It is preferable to immobilize the obtained pad on a water-absorbent substrate, for example, by adhering it to a stationary phase by immobilizing it on the water-absorbent substrate, similarly to the case of directly immobilizing on a hydrophilic substrate. Also,
If the adhesion is made detachable by an arbitrary method, it is suitable when the target nucleic acid captured by the stationary phase is recovered and used.

As the stationary phase, even if the content of the target nucleic acid in the test sample is very small, the capture probe has an amount capable of capturing the labeled complex to the extent that the signal difference from that in the control sample can be detected. Is preferably immobilized. The immobilized amount of the capture probe in the stationary phase can be appropriately determined in relation to the amount of the competing probe used, and cannot be determined unconditionally, but usually, the capture probe is preferably 1 to 10.
It is preferable to uniformly immobilize in the range of 0 ng / cm ² to form a stationary phase.

The water-absorbent substrate on which the stationary phase is formed prevents non-specific adsorption of nucleic acids other than the target nucleic acid on the substrate, ease of development, and storage stability of the immobilized capture probe. From the viewpoint of the property, it is preferable to use it by immersing it in a solution containing a blocking agent, a surfactant and sugar (hereinafter referred to as a treatment solution) and drying it according to a known method. Here, examples of the blocking agent used include proteins having adsorptivity to a water-absorbent substrate such as bovine serum albumin, casein, gelatin, skim milk, polyvinyl alcohol, and polyvinylpyrrolidone. As the surfactant, polyoxyethylene (10) octyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene alkyl allyl ether phosphate, polyoxyethylene alkyl ether phosphate, polyoxyethylene alkyl phenyl ether, etc. Is mentioned. Examples of sugars include saccharose and trehalose. The content of the blocking agent in the treatment liquid is preferably 0.1 to 10% by weight.
Is. The content of the surfactant in the treatment liquid is preferably 0.01 to 1% by weight. The sugar content in the treatment liquid is preferably 0.1 to 10% by weight. Also,
The above operation can be optionally carried out after the stationary phase is formed on the water-absorbing substrate, as long as the desired effect of the present invention is not impaired before the use in the detection method of the present invention. .

Further, on the water-absorbent substrate, on the side opposite to the dripping pad portion (portion for dropping the development target component) and / or the portion for introducing the development target component with the stationary phase sandwiched therebetween, You may provide a water-absorbing pad part etc. which are means which can accelerate | stimulate the spreading | diffusion of the component to be spread | deployed on the water-absorption base material by the absorbency of the aqueous liquid. The drip pad portion can be appropriately prepared by, for example, laminating a nonwoven fabric made of polyester, rayon, glass fiber or the like in a desired shape at a desired position on the water absorbent substrate. Further, as the water-absorbing pad used for the water-absorbing pad portion, a material having excellent absorbency of an aqueous liquid is suitable, and for example, a glass fiber non-woven fabric or the like can be used.

The dripping pad section is
It is desirable to be provided at a position where the desired deployment movement distance as described above can be secured.

For example, as an example of the water-absorbent substrate used in the present invention, the water-absorbent substrate shown in FIG. 1 can be cited. The size of the water-absorbent substrate is not particularly limited as long as the desired effect of the present invention can be obtained, for example,
It has a size of about 2 to 10 mm × 30 to 80 mm.

The water-absorbent substrate shown in FIG.
It comprises a water-absorbent substrate 2, a water-absorbent pad 3, a stationary phase 4 and a reporter phase 5. Between the drip pad 1 and the stationary phase 4,
There is a distance that can secure the expansion movement distance within the preferable range.

For example, when the detection method of the present invention is carried out using the water-absorbent substrate, the aqueous solution of the sample to be tested is dropped onto the dropping pad 1 using a pipette or the like. The dropped test sample is spread on the water-absorbent substrate 2 in the direction of the arrow extending from right to left in the figure, and reaches the reporter phase 5. In the reporter phase 5, water, which is the medium of the test sample, is brought into contact with the labeled complex, and the labeled complex is eluted from the reporter phase 5 to enable development. As a result, the labeled complex will be developed together with the test sample. The test sample and the labeled complex then reach the stationary phase 4. In the stationary phase 4, the target nucleic acid and the competitive probe in the labeled complex hybridize competitively with the capture probe. When the test sample does not contain any target nucleic acid (when a control sample is used), only the competing probe of the labeled complex hybridizes with the capture probe in the stationary phase 4 to capture a line-shaped or circular shape. The shape of the immobilized probe is captured and the labeled complex is captured. On the other hand, when the target nucleic acid is contained in the test sample, the capture probe and the target nucleic acid or the competing probe of the labeled complex competitively hybridize, and as a result, the capture amount of the labeled complex in the stationary phase 4 is increased. Decrease (the untrapped labeled complex flows to the water-absorbing pad 3).
Further, if desired, a cleaning liquid is dropped on the dripping pad 1 to be developed to wash the water absorbent substrate 2. At this point, in the case where the signal reporter is the colored particles described above, if the labeled complex is captured on the stationary phase, the position where the capture occurs occurs color, and the signal is detected as a color. In the case where the signal reporter is an enzyme, for example, if a chromogenic substrate that produces a color by the action of the enzyme is further developed from the dropping pad 1,
The stationary phase 4 is also colored, and a signal is detected as a color. The detection level of the signal is compared between the case of the control sample and the case of the test sample, and if the detection level is low in the case of the test sample, it is determined that the target nucleic acid is present in the test sample. The detection method of the present invention is preferably carried out under the condition that the difference in the detection level of the signal can be caught by visual observation, but when it is difficult to catch the difference by visual observation, for example, the signal Is a color,
It is preferable to capture the difference using the chromatographic scanner or the like.

Further, as another aspect of the present invention, a kit for detecting a target nucleic acid useful in the detection method of the present invention is provided. Examples of the kit include those containing a water-absorbing substrate having a stationary phase formed by immobilizing the capture probe and a reporter phase formed by holding the labeled complex so that it can be developed by contact with a liquid. be able to. Furthermore, the kit may optionally have other components, for example, means for obtaining a test sample, appropriate reagents such as enzyme substrate solution and hybridization buffer, and instructions for carrying out the detection method of the present invention. Etc. may be included.

According to the detection method of the present invention, any test sample or the like can be prepared, for example, by simply dropping it onto the water-absorbent substrate having a stationary phase and a reporter phase used in the present invention. It is possible to quickly and easily determine the contamination of food due to the bacteria causing food poisoning, diagnose the etiology or condition of a disease, and genotype an organism.

[0083]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

Production Example 1: Preparation of chromatographic test strip for detecting Salmonella bacterium rRNA In this Production Example, a chromatographic test strip for detecting rRNA of Salmonella bacterium (target nucleic acid: Salmonella bacterium 23S rRNA) was prepared. It was produced by the method shown below. In addition, in this specification, "upstream" means the terminal side of the water-absorbent substrate on which a drip pad to be described later is arranged.
The structure of the obtained test piece is the same as that of the water-absorbent substrate shown in FIG.

Nylon 66 membrane (pore size 5 μm, 6 mm × 40 mm, made by PALL) as the material for the water-absorbent substrate
Was used. At a position 22.5 mm from this upstream end, 1.5 μL of a capture probe (10 pmol / μL, dissolved in sterilized deionized water) consisting of the base sequence described in SEQ ID NO: 1 below was linearly applied using a dispenser. . The capture probe has a nucleotide sequence complementary to the nucleotide sequence of 23S rRNA of Salmonella. After air drying, the capture probe was immobilized on the membrane by heat treatment at 120 ° C. for 30 minutes to form a stationary phase. After immobilization, the membrane was immersed in 1% by weight bovine serum albumin (manufactured by Oriental Yeast), 1% by weight sucrose (manufactured by Wako Pure Chemical Industries) and 0.1% by weight polyoxyethylene sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd.). Blocked and dried at room temperature.

Further, the labeled complex prepared as described in Production Example 2 described below was adjusted to a final concentration of 0.1% by weight with 0.01 M boric acid-1% (w / v) saccharose p.
Dilute with H8.2, and add 1.5 μL thereof at a position 10 mm from the upstream end of the membrane, the width of which is the same as the width of the membrane in the direction perpendicular to the developing direction, and the thickness is 1 mm. A dispenser was applied in a line shape so as to form a reporter phase.

Furthermore, from a point 3 mm from the upstream end of the water-absorbent substrate, a polyester non-woven fabric Hibon 4880C
(Thickness 2.5 mm, size 7 × 12 mm, manufactured by Shinwa) were attached to each other to set a dropping pad.

Further, 5 to 10 m from the downstream end of the water absorbent base material.
Water absorbent material made of glass fiber (15 mm × 30 mm, thickness 5 mm, Whatman)
(Manufactured by Japan) was attached to prepare a test piece. The test piece is 1
00 pieces were produced and used for the subsequent detection. SEQ ID NO: 1 5'-CTTCACCTAC GTG
TCAGC-3 '

Production Example 2: Preparation of labeled complex for detecting Salmonella bacterium rRNA Colored latex particles (particle size 0.18 μm, manufactured by Nitto Denko)
Water-soluble carbodiimide (1-ethyl-3-
(3-Dimethylaminopropyl) carbodiimide hydrochloride 10 mg / mL, 0.01 M borate buffer pH 8.
2, Dojindo Laboratories) 0.5 mL, streptavidin (1 mg / mL, 0.01 M borate buffer pH 8.
2, Roche Diagnostics) (2 mL) was added, and the mixture was reacted at 10 ° C. for 3 hours.
M-Borate buffer (pH 8.2) was added and the mixture was centrifuged and washed to adjust the solid concentration to 5% by weight with the same buffer. In the following description, streptavidin is abbreviated as SA.
The obtained SA-immobilized colored latex suspension was diluted with the borate buffer solution so that the solid content became a final concentration of 0.1% by weight, and the 5'end having a final concentration of 50 pmol / mL was added thereto by a known method. A labeled probe was prepared by adding a biotin-labeled competitive probe having the nucleotide sequence shown in SEQ ID NO: 2 below and reacting at 10 ° C. for 3 hours while gently stirring. A borate buffer solution was added as a washing solution, and centrifugation was performed for washing, and the solid content concentration was adjusted to 5% by weight with the same buffer solution. The competitive probe is a sequence that is complementary to the above-mentioned capture probe and has no binding property to 23S rRNA of Salmonella spp. SEQ ID NO: 25'-GCT
GACACGT AGGTGAAG-3 '

Production Example 3: Preparation of test sample Salmonella and non-Salmonella bacteria shown in Table 1 were mixed with 5 mL of nutrient broth (Nutrie).
nt Broth) (trade name, prepared using Nissui Pharmaceutical's powder medium) at 37 ° C. overnight, ISOGEN (trade name, manufactured by Nippon Gene) was used as an attached manual. The total RNA was extracted according to the procedure described above and dissolved in sterile deionized water to obtain a test sample. RNA obtained
Is O. D. It was appropriately diluted to 1 μg / μL based on the value of 260 (1O.D.260 = 0.04 μg / μL
Conversion).

[0091]

[Table 1]

Example 1: Specific detection of the presence of Salmonella sp. RRNA by chromatographic method Using the test sample prepared in Production Example 3, a developing composition liquid having the following composition was prepared. The control sample was sterile deionized water.

[Composition of Development Composition Liquid] 2 x SSC 50% (w / v) formamide 100 μg / mL salmon sperm DNA 0.1% (w / v) SDS RNA

2 × SSC is 33.3 mM NaC.
1, 33.3 mM sodium citrate, RN
A concentration condition is 0 (control sample), 0.1, 1 μg / mL
Experiments were conducted as three types.

100 μL of the developing composition liquid kept at 37 ° C. was dropped on the dropping pad of the test piece prepared in Production Example 1. After development for 10 minutes, the presence or absence of color development in the stationary phase was visually observed. The results are shown in Table 2. The criteria used in Table 2 are as follows.

[Criteria] +: There is coloring derived from latex ±: Light coloring from latex -: No coloring

[0097]

[Table 2]

As shown in Table 2, when the developing composition does not contain Salmonella bacterium RNA, or when it contains bacterium RNA other than Salmonella bacterium, only the labeled complex is the competitive probe. It was considered that the protein was captured via hybridization with the capture probe of the stationary phase, and a signal was detected as the color of the colored latex particles in the stationary phase. On the other hand, 1 μg of salmonella RNA
When the developing composition liquid containing a concentration of / mL was developed, the signal was not detected in the stationary phase. This is because the 23S rRNA of Salmonella bacteria and the competitive probe competitively hybridized with the capture probe, so that the amount of the labeled complex captured in the stationary phase was decreased, and as a result, the signal derived from the colored latex particle was not detected. It seems to be due to. When the RNA of Salmonella bacteria in the developing composition liquid is 0.1 μg / mL,
A weak signal was detected on the stationary phase. It is considered that this is because a part of the labeled complex was captured by the stationary phase because the amount of 23S rRNA of Salmonella bacterium that competes with the competitive probe decreased. In any case, compared to the case of the control sample, the target nucleic acid, Salmonella spp. 23Sr
Since a low signal was detected in the stationary phase in the test sample containing Salmonella bacterial RNA containing RNA, it can be seen that the presence of the target nucleic acid in the test sample can be detected by the detection method of the present invention.

These reactions proceeded only by dropping the developing composition liquid, and the results could be obtained in about 10 minutes. Whereas a general hybridization method requires a complicated operation such as washing and color development and a time of about 1 to 2 days, the detection method of the present invention adopts a chromatographic method to develop a composition for development. The operation can be completed only by dropping the liquid. Therefore, it is clear that the present invention is effective as a rapid and simple method for detecting a target nucleic acid in a test sample.

Sequence Listing Free Text SEQ ID NO: 1 is the sequence of a probe designed based on the nucleotide sequence of 23S rRNA of Salmonella bacteria.

SEQ ID NO: 2 is 23 of Salmonella spp.
It is the sequence of a probe designed based on the base sequence of SrRNA.

[0102]

Industrial Applicability According to the present invention, a method for detecting a target nucleic acid capable of detecting the presence of the target nucleic acid in a sample to be tested, which is excellent in operability as compared with the conventional hybridization method and can be detected quickly and simply, Also provided is a kit for detecting a target nucleic acid that is preferably used in the detection method. According to the detection method of the present invention, for example, determination of food contamination due to food poisoning causative bacteria, diagnosis of disease etiology or condition, genotyping of organisms, etc. can be carried out quickly and easily.

[0103]

[Sequence list]                             SEQUENCE LISTING <110> NITTO DENKO CORPORATION <120> Method of detecting target gene <130> ND-13-018 <160> 2

[0104] <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Designed probe based on nucleotide sequence of Salmonella 23SrRNA. <400> 1 cttcacctac gtgtcagc 18

[0105] <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Designed probe based on nucleotide sequence of Salmonella 23SrRNA. <400> 2 gctgacacgt aggtgaag 18

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an example of a water-absorbent substrate having a stationary phase and a reporter phase, which is used in the present invention.

[Explanation of symbols]

1 dripping pad 2 Water-absorbent substrate 3 water absorption pad 4 stationary phase 5 Reporter phase

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // C12N 15/09 C12N 15/00 A (72) Inventor Quantum Morioka 1-1-2 Shimohozumi, Ibaraki-shi, Osaka Nitto D term in Denko Corporation (reference) 4B024 AA11 CA09 CA11 HA14 4B063 QA18 QQ03 QQ08 QQ16 QQ58 QR55 QR82 QS34

Claims (7)

[Claims] 1. A method for detecting a target nucleic acid using a capture probe having binding ability to a target nucleic acid and a competitive probe having no binding ability to the target nucleic acid but having binding ability to the target nucleic acid. And (a) a water-absorbing substrate having a stationary phase formed by immobilizing the capture probe and a reporter phase formed by holding a labeled complex having the competitive probe and a signal reporter so as to be developed by contact with a liquid. The step of introducing a test sample, (b) the step of developing the test sample on the water-absorbent substrate under stringent conditions, and (c) the signal from the signal reporter in the stationary phase. A step of detecting the target nucleic acid is determined to be contained in the test sample when the signal is detected low in the stationary phase as compared with a control sample containing no target nucleic acid. For detecting specific nucleic acid. 2. The detection method according to claim 1, wherein the water-absorbent substrate has a reporter phase at a position for introducing a test sample or between a position for introducing a test sample and a stationary phase. . 3. The detection method according to claim 1, wherein the signal reporter is a colored particle, an enzyme or a fluorescent dye. 4. The method according to claim 1, wherein the target nucleic acid is a single-stranded nucleic acid.
3. The detection method according to any one of 3 above. 5. The detection method according to claim 4, wherein the single-stranded nucleic acid is ribosomal RNA. 6. The detection method according to claim 5, wherein the ribosomal RNA is derived from Salmonella or Listeria. 7. A kit for detecting a target nucleic acid, which comprises the water-absorbing substrate according to claim 1.