JP2003250599A - Method for detecting target nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method excellent in operability and capable of rapidly and simply detecting the existence of a target nucleic acid in a sample, and to provide a kit for detecting the target nucleic acid. <P>SOLUTION: This method for detecting the target nucleic acid comprises using a capture probe having bondability to the target nucleic acid and a competitive probe having no bondability to the target nucleic acid but having bondability to the capture probe, wherein the method comprises a step for mixing the competitive probe having a ligand with a sample, a step for introducing the obtained mixture on a water-absorbing substrate fixed with the capture probe, a step for introducing a signal reporter on the water-absorbing substrate, a step for developing the mixture and the signal reporter on the water-absorbing substrate, and a step for detecting a signal from the signal reporter. The method determines that, if the lower signal than that of a control sample is detected, the target nucleic acid is contained in the sample. The kit for detecting the target nucleic acid is provided. <P>COPYRIGHT: (C)2003,JPO

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 gist of the present invention is [1] a capture probe having a binding property to 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) mixing the competitive probe having a ligand with a test sample. Step (b) introducing the mixture obtained in step (a) onto a water-absorbent substrate having a stationary phase formed by immobilizing the capture probe, (c) on the water-absorbent substrate, Introducing a signal reporter having a ligand-binding compound, (d) developing the mixture and the signal reporter on a water-absorbent substrate under stringent conditions, and (e) from the signal reporter in a stationary phase. A step of detecting a signal, and the target nucleic acid is contained in the test sample when the signal is detected low in the stationary phase as compared to a control sample containing no target nucleic acid. Target nucleic acid detection method, and [2] a kit for detecting a target nucleic acid comprising a competitive probe having the ligand according to [1] above, a signal reporter having a ligand-binding compound, and a water-absorbing substrate About.

[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 extent of the competitive probe hybridized to the capture probe is determined by the ligand and the ligand. Detected as a signal from the signal reporter bound through the binding of the binding compound, when the target nucleic acid is not included at all, i.e., the case where only the competitive probe hybridizes to the capture probe as a control, as compared to such a case, It is a method for detecting a target nucleic acid, which determines that the test sample contains the target nucleic acid when the detection level of the signal decreases.

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) a step of mixing the competitive probe having a ligand (hereinafter, may be simply referred to as a competitive probe) and a test sample, (b) on a water-absorbent substrate having a stationary phase obtained by immobilizing the capture probe The step of introducing the mixture obtained in step (a), (c) the step of introducing a signal reporter having a ligand-binding compound (hereinafter, may be simply referred to as a signal reporter) on the water-absorbent substrate,
(D) The step of developing the mixture and the signal reporter on a water-absorbent substrate under stringent conditions, and (e) the step of detecting a signal from the signal reporter in a stationary phase. And
Perform the same procedure as the test sample separately using a control sample that does not contain the target nucleic acid, and target the target sample in the test sample when the signal from the signal reporter is detected lower in the stationary phase than in the control sample. It is determined that the nucleic acid is contained.

In the present specification, "competitive probe having a ligand" and "signal reporter having a ligand-binding compound" are described, but the ligand and the ligand-binding compound may be reversed. In the present specification, from the viewpoint of simplification of description, the case where the competitive probe has a ligand and the signal reporter has a ligand binding compound will be described as an example. A ligand-binding compound refers to a compound capable of binding to a ligand. Further, in the present specification, “having binding properties” means capable of hybridizing with each other under stringent conditions, and “having no binding properties” means being capable of hybridizing with each other under stringent conditions. It means not soybeans. Further, "nucleic acid" includes DNA (deoxyribonucleic acid) and R
NA (ribonucleic acid) is 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, a test sample provided in such a form is mixed with a competitive probe having a ligand [step (a)], and the mixture obtained is introduced into one end of the water-absorbent substrate [step (b)]. And then introducing a signal reporter having a ligand-binding compound [step (c)],
They are immediately developed on the water-absorbent substrate [step (d)].

The mixing ratio of the test sample and the competitive probe is
The present invention is not particularly limited as long as the desired effect of the present invention can be obtained, but the amount of target nucleic acid and the amount of competing probe expected in the test sample should be about 1: 1 in molar ratio. Mixing is preferred. The introduction of the mixture is performed 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 dropping 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 preferably one side of the water-absorbent substrate as viewed from the stationary phase, and is a position where a desired expansion movement distance can be secured. The development movement distance is the distance from the introduction position of the mixture to the stationary phase. The deployment movement distance is 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 position of introduction of the signal reporter on the water-absorbent substrate is not particularly limited as long as the desired effect of the present invention can be obtained, and even if it is the same as the position of introduction of the mixture,
Or it may be different. From the viewpoint of expressing the desired effect of the present invention, the signal reporter is preferably introduced in an amount equal to or more than the equivalence of the competitive probe in molar units.

Since the competitive probe does not bind to the target nucleic acid, the target nucleic acid and the competitive probe do not hybridize with each other even if the target nucleic acid is present in the test sample, and the target nucleic acid and the competitive probe are independently developed. It Since both the target nucleic acid and the competitive probe have a binding property 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. On the other hand, the competitive probe and the signal reporter are bound to each other via the bond between the ligand and the corresponding ligand-binding compound to form a so-called labeled complex having the competitive probe and the signal reporter. Therefore, when the competitive probe is captured by the stationary phase through binding with the capture probe, the signal reporter emits a signal depending on the degree of capture of the competitive probe.

In step (e), the mixture, which is the component to be developed, and the signal reporter are developed, and after a certain period of time, the signal from the signal reporter is detected in the stationary phase.

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 competing probe is captured in the stationary phase through the hybridization with the capture probe, so that the signal from the signal reporter is the control sample. It is detected to the same extent as in.

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 because the capture of the competing probe is reduced or virtually no capture occurs as compared to the absence of a. Also, if no competing probe is captured, 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 competitive probe of the present invention specifically has a base sequence substantially the same as that of the target nucleic acid, and is specific to the immobilized capture probe of the stationary phase under stringent conditions. There is no particular limitation as long as it is a nucleic acid probe that can hybridize with. 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. Also,
The number of bases is at least about 5 bases, preferably 5 to 100 bases, more preferably 15 to 50 bases.
Although it has a base length, it may be several thousand bases long.

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. 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. Alternatively, a commercially available product in which a nucleic acid having an arbitrary sequence or modification (label) is synthesized can be purchased.

The signal reporter is not particularly limited as long as it can detect the signal derived from the reporter. 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).

Fluorescein, tetramethylrhodamine, Cy3, Cy5 and the like can be mentioned as fluorescent dyes which can be used as a signal reporter.

The competitive probe having a ligand and the signal reporter having a ligand-binding compound directly or indirectly bind to the competitive probe or signal reporter and the ligand or the ligand-binding compound (hereinafter, they may be referred to as binding targets). It can be obtained by conjugation. The case of indirectly binding means, for example, the case where the binding of the binding target substances is performed via an arbitrary substance. For example, the binding between the binding objects may be performed via the water-dispersible polymer particles.

Further, the binding target does not necessarily have to be bound one-to-one, and for example, a plurality of ligands may be bound directly or indirectly to one competing probe. When a ligand is attached to a competitive probe, the ligand may be attached directly or indirectly to at least one of the 5'end, the 3'end and the interior of the sequence of the competitive probe.

The binding between the binding targets can be suitably carried out using a method utilizing covalent binding, physical adsorption or ionic binding, 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 through a functional group or an ionic group present in a binding target, or a bifunctional organic compound having a water-soluble carbodiimide or another carbon chain group having 1 to 12 carbon atoms as a spacer. To intervene and bind. Further, for example, the binding between the ligand and the competitive probe can be performed by utilizing the binding between a ligand different from the ligand and its corresponding ligand-binding compound. When an enzyme is used as the signal reporter, the degree of freedom of the enzyme can be increased by interposing a spacer of some kind, which is preferable from the viewpoint of increasing the efficiency of the enzymatic reaction. Further, as the signal reporter having the ligand binding compound, a ligand binding compound containing an arbitrary radioisotope as a constituent element can also be used.

Examples of the ligand-ligand binding compound combination preferably used in the present invention include biotin-avidin, biotin-streptavidin, digoxigenin (DIG) -anti-DIG antibody, FITC-anti-FITC antibody and the like. Among them, the combination of biotin-streptavidin and DIG-anti-DIG antibody has high stability of the complex formed by binding them, and is preferably used.

In addition, for example, when the ligand and the competitive probe are indirectly bound via the water-dispersible polymer particles, for example, (ligand)-(water-dispersible polymer particles)-(competitive probe) When a competitive probe having a shape is obtained, it can be prepared by binding between the ligand and the water-dispersed polymer particle and between the water-dispersed polymer particle and the competitive probe as described above.

The competitive probe and signal reporter used in the detection method of the present invention are bound to each other by binding the binding targets constituting them, and then, 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 competitive probe or the like of the present invention may be stored, for example, by immersing it in water or lyophilizing it. When used in the detection method of the present invention, the obtained competitive probe or the like, for example, water, borate buffer,
It is preferable to disperse or dissolve it in a phosphate buffer or the like.

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, in step (c) of the detection method of the present invention, after introducing the signal reporter onto the water-absorbing substrate, an enzyme substrate solution is further added to obtain the desired effect of the present invention. It is introduced so that expression can be obtained. The introduction position of the enzyme substrate solution may be the same as or different from the introduction position of the mixture.

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 changed from the substrate by the action of the enzyme of the signal reporter captured in the stationary phase. Derived from the resulting chromogenic product.

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 or solution of the test sample or competitive probe, an enzyme substrate solution is diluted with a concentrated solution of a buffer solution (hybridization buffer) having the above composition prepared in advance and, if desired, water. Then, after adjusting to a desired composition, it is introduced onto the water-absorbent substrate. Further, the test sample or the like may be prepared so as to have the composition of the hybridization buffer from the beginning. Water (eg, deionized water, distilled water, etc.) may be used as the control sample.

The development may be carried out for about 1 to 10 minutes after the introduction of the signal reporter. The time from the introduction of the mixture of the competitive probe and the test sample to the introduction of the signal reporter is not particularly limited. In addition, after introducing the mixture and / or after introducing the signal reporter, a washing solution may be introduced for the purpose of washing away nonspecific adsorption of nucleic acid. As the washing solution, the hybridization buffer is suitable.

The water-absorbent substrate used in the present invention includes
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, porous membrane and the like.

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 spread, and for example, a rectangular sheet shape (piece shape) or a 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-mentioned method for preparing a competitive probe or the like having the ligand of the present invention. Alternatively, for example, the capture probe may be simply applied directly on the water-absorbent substrate, and then immobilized by heating at 120 ° C. for about 30 minutes while maintaining it in an oven.

When a capture probe is indirectly immobilized on a water-absorbent substrate to form a stationary phase, for example, the capture probe is directly immobilized on a pad made of glass fiber or the like, and the capture probe is directly immobilized on the water-absorbent substrate. It is preferable that the pad is immobilized in the same manner as described above, and the obtained pad is bonded to the water-absorbent substrate by, for example, bonding to form a stationary phase. Further, if the adhesion is made detachable by an arbitrary method, it is suitable when the target nucleic acid captured in the stationary phase is recovered and used.

As the stationary phase, a signal reporter and a competitive probe captured by the signal reporter are detected to such an extent that a difference in signal from the control sample can be detected even if the content of the target nucleic acid in the test sample is very small. It is preferable that the capture probe is immobilized in an amount that can be captured via the probe. The immobilized amount of the capture probe in the stationary phase can be appropriately determined in relation to the amount of the competing probe to be used, and cannot be determined unconditionally, but usually, the capture probe is preferably 1 to 100 n.
It is preferable to uniformly immobilize in the range of g / cm ² to form a stationary phase.

The water-absorptive 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, in addition to the stationary phase, the water-absorbent substrate has a dropping pad portion (a portion for dropping the developing target component) and / or a portion for introducing the developing target component with the stationary phase sandwiched therebetween. On the side opposite to the above, a water-absorbing pad portion or the like, which is a means for accelerating the development of the component to be developed on the water-absorbent substrate by the absorbing power of the aqueous liquid, may be provided. The dripping pad portion can be appropriately prepared, for example, by laminating a nonwoven fabric made of polyester or rayon in a desired shape at a desired position on the water-absorbent substrate. Also,
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 portion is formed on the water-absorbent substrate.
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 3 to 10 mm × 40 to 80 mm.

The water-absorbent substrate shown in FIG.
It consists of a water-absorbent substrate 2, a water-absorbent pad 3 and a stationary phase 4.
There is a distance between the dripping pad 1 and the stationary phase 4 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 mixture of the sample to be tested and the competitive probe is dropped on the dropping pad 1 using a pipette or the like. Further, if desired, a cleaning liquid is dropped on the dripping pad 1 to be developed to wash the water absorbent substrate 2. Then, similarly, a signal reporter is dropped. The components to be spread that have been dropped are spread on the water-absorbent substrate 2 in the direction of the arrow extending from the right to the left in the figure, and after a certain period of time, reach the stationary phase 4. In the stationary phase 4, the target nucleic acid and the competitive probe hybridize competitively with the capture probe. When the test sample does not contain any target nucleic acid (when a control sample is used), in the stationary phase 4, only the competitive probe hybridizes to the capture probe, and the ligand and the ligand-binding compound bind to the competitive probe. The signal reporter is captured in the shape of the capture probe immobilized such as a line or a circle through the binding of. On the other hand, when the test sample contains the target nucleic acid, the capture probe hybridizes competitively with the target nucleic acid or the competitive probe, and as a result, the capture amount of the competitive probe in the stationary phase 4 decreases, and thus the signal. The capture amount of the reporter decreases (competitive probes that are not captured flow to the water absorption 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 they are captured by the stationary phase 4, the position where the capture occurs will be colored, and the signal will be detected as a color. When the signal reporter is an enzyme, for example, when a color-developing substrate whose color is obtained by the action of the enzyme is further developed from the dropping pad 1, the stationary phase 4 is colored similarly 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
It is preferable to carry out under the condition that the difference in the detection level of the signal can be detected by visual observation, but if it is difficult to detect the difference by visual observation, for example, if the signal is a color, the chromatographic scanner is used. It is preferable to capture the difference by using such as.

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. The kit can include a kit comprising a water-absorbent substrate having the stationary phase, a competitive probe having a ligand, and a signal reporter having a ligand-binding compound. 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 added, for example, by dropping it onto the water-absorbent substrate having a stationary phase used in the present invention. Determination of food contamination, diagnosis of disease etiology or condition, genotyping of organisms, etc. can be performed quickly and easily.

[0078]

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 Specimen for Detecting Salmonella Bacterial rRNA In this Production Example, a chromatographic test strip for detecting rRNA of Salmonella genus bacterium (target nucleic acid: Salmonella bacterium 23S rRNA) is used. 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, heat treatment was carried out at 120 ° C. for 30 minutes to immobilize the capture probe on the membrane 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.

A non-woven fabric made of polyester Hibon 4880C (thickness: 2.5 mm, size: 7 × 12 mm, made by Shinwa) was attached from 3 mm from the upstream end of the water-absorbent substrate to set a dropping pad.

From the downstream end of the water-absorbent substrate, 5-10
mm water absorbent material (15 mm x 30 mm, thickness 1 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 Biotin-Binding Competitive Probe and Streptavidin-Binding Signal Reporter The 5'end of the competitive probe consisting of the base sequence shown in SEQ ID NO: 2 below was labeled with biotin by a known method. The competitive probe is a nucleic acid probe having a base sequence complementary to the above-mentioned capture probe and having no binding property to 23S rRNA of Salmonella bacteria. SEQ ID NO: 25'-GCTGACACGT AGG
TGAAG-3 '

A water-soluble carbodiimide (1-ethyl-3- (3-) was added to an aqueous dispersion of colored latex particles (particle diameter 0.18 μm, manufactured by Nitto Denko) used as a signal reporter.
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, manufactured by Roche Diagnostics) 2 mL, and added 10
After reacting at 0 ° C. for 3 hours, 0.01 M-borate buffer solution pH 8.2 was added as a washing solution, centrifugation was performed to wash, and the solid content concentration was adjusted to 5% by weight with the same buffer solution. In addition,
In the following description, streptavidin is abbreviated as SA.

Production Example 3: Preparation of Test Samples 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).

[0086]

[Table 1]

Example 1: Specific detection of the presence of Salmonella sp. RRNA by chromatographic method The competitive probe prepared in Production Examples 2 and 3 was mixed with a test sample, and a developing composition liquid having the following composition was prepared. Was prepared. The control sample was sterile deionized water.

[Composition of the developing composition liquid] 2 x SSC 50% (w / v) formamide 100 μg / mL salmon sperm DNA 0.1% (w / v) SDS 1 pmol/mL competitive probe 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, 100 μL of a signal reporter solution prepared by suspending SA-bonded colored latex particles described in Production Example 2 in 2 × SSC to a final concentration of 0.02% by weight was added dropwise to the same dropping pad. 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.
Shown in. The criteria used in Table 2 are as follows.

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

[0092]

[Table 2]

As shown in Table 2, when the developing composition solution does not contain Salmonella genus bacterial RNA, or when it contains RNA of bacteria other than Salmonella genus bacteria, only the competitive probe is a stationary phase capture probe. It was considered that they were hybridized and captured, and a signal was detected as the color of the colored latex particles on the stationary phase. On the other hand, when the developing composition liquid containing Salmonella RNA at a concentration of 1 μg / mL was developed, the signal was not detected in the stationary phase. This is a 23SrRN of Salmonella
This is probably because A and the competitive probe competitively hybridized with the capture probe, so that the capture amount of the competitive probe in the stationary phase decreased, and as a result, the signal derived from the colored latex particles was not detected.
In addition, RNA of Salmonella spp.
When it was set to 1 μg / mL, a weak signal was detected in the stationary phase. It is considered that this is because a part of the competitive probe was captured by the stationary phase because the amount of 23S rRNA of Salmonella bacteria that competes with the competitive probe decreased. In any case, a lower signal was detected in the stationary phase in the test sample containing Salmonella bacterial RNA containing the target nucleic acid, Salmonella bacterial 23S rRNA, as compared with the case of the control sample. It is understood that the method can detect the presence of the target nucleic acid in the test sample.

These reactions proceed by merely adding the developing composition solution and the signal reporter solution to about 20
I was able to get the result in 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 a liquid or the like.
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.

[0097]

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.

[0098]

[Sequence list]                             SEQUENCE LISTING <110> NITTO DENKO CORPORATION <120> Method of detecting target gene <130> ND-14-009 <150> JP 2001-401212 <151> 2001-12-28 <160> 2

[0099] <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

[0100] <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, which is used in the present invention.

[Explanation of symbols] 1 dripping pad 2 Water-absorbent substrate 3 water absorption pad 4 stationary phase

Continued front page    (72) Inventor Quantum Asai             1-1-2 Shimohozumi, Ibaraki City, Osaka Prefecture Nitto Denko             Within the corporation F term (reference) 4B024 AA11 CA01 CA09 CA11 GA19                       HA12                 4B029 AA07 AA21 AA23 BB02 BB20                       CC03 CC11 CC13 FA13 FA15                 4B063 QA01 QA18 QQ54 QR32 QR38                       QR55 QR57 QR66 QR82 QS12                       QS34 QS39 QX01

Claims (6)

[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. , (A) a step of mixing the competitive probe having a ligand with a test sample, (b) on a water-absorbent substrate having a stationary phase formed by immobilizing the capture probe,
Introducing the mixture obtained in step (a), (c) Introducing a signal reporter having a ligand-binding compound on the water-absorbent substrate, (d) Water-absorbent substrate under stringent conditions Comprising the step of developing the mixture and the signal reporter above, and (e) detecting the signal from the signal reporter in the stationary phase, as compared to a control sample without target nucleic acid, in the stationary phase A method for detecting a target nucleic acid, which comprises determining that a target nucleic acid is contained in a test sample when the signal is detected low. 2. The detection method according to claim 1, wherein the signal reporter is a colored particle, an enzyme or a fluorescent dye. 3. The detection method according to claim 1, wherein the target nucleic acid is a single-stranded nucleic acid. 4. The detection method according to claim 3, wherein the single-stranded nucleic acid is ribosomal RNA. 5. The detection method according to claim 4, wherein the ribosomal RNA is derived from Salmonella or Listeria. 6. A kit for detecting a target nucleic acid, which comprises a competitive probe having the ligand according to claim 1, a signal reporter having a ligand-binding compound, and a water-absorbing substrate.