JP6977978B2 - Method for detecting target nucleic acid - Google Patents

Description

The techniques disclosed herein relate to methods of detecting a target nucleic acid present in a sample.

Techniques for detecting specific nucleic acids (hereinafter also referred to as target nucleic acids) present in biological samples include analysis at the nucleic acid molecular level of proteins expressed and functioning in specific organs, and information in the nerve, brain or immune system. It is not only important in the study of protein expression control in transmission, but also extremely important in genetic diagnosis such as detection of mutant genes of genetic diseases, diagnosis of cancer, and detection of virus-related genes.

As a rapid and simple method for detecting a target nucleic acid, which has been attracting attention in recent years, there is a gene detection method based on chromatography (Patent Documents 1 to 3). In these methods, a DNA containing a target nucleic acid is amplified by a nucleic acid amplification method using a labeled oligonucleotide primer, a label is introduced, and a sample consisting of this amplification product is applied to a test strip for a chromatograph. Capillary action is used to expand the test strip and capture the amplification product with a probe pre-immobilized on the test strip to detect the target nucleic acid in the presence or absence of a label-based signal.

Japanese Unexamined Patent Publication No. 2004-154075 Japanese Unexamined Patent Publication No. 2003-194817 Japanese Patent No. 3001906

However, since this method detects the target nucleic acid only by its discriminating ability based on the base sequence of the primer, it is precise to detect genotypes and mutations present in the amplification product, for example, including single nucleotide polymorphisms. No sequence can be detected. In addition, although the use of labeled primers is convenient for detection, there is a risk of detecting so-called primer dimers that are amplified by annealing between primers.

The present specification has been made in view of such circumstances, and provides a method for detecting a target nucleic acid, which is more versatile and easier to operate than a conventional method for detecting a target nucleic acid.

Surprisingly, the present inventors prepared a hybrid product of a target nucleic acid contained in the amplification product and a tagged probe that identifies a target sequence in the target nucleic acid, and the hybrid product was referred to as the tag. We have found a method for detection with a capture probe that can hybridize with. Then, according to this method, even a minute target sequence such as SNP, which could not be detected only by the discriminating ability of the primer, can be easily detected with high specificity via a nucleic acid amplification reaction such as PCR. Can be done. The present specification provides the following means based on these findings.

(1) A method for detecting a target nucleic acid containing a target sequence.
An amplification step of performing a nucleic acid amplification reaction on a sample that may contain the target nucleic acid using a nucleic acid amplification reagent to obtain an amplification product, and
The step of detecting the target nucleic acid and
Equipped with
The detection step is a step of detecting a hybridization product obtained by hybridizing a single-stranded nucleic acid containing the target sequence with an identification probe that identifies the target sequence, using a capture probe associated with the target sequence. can be,
The identification probe comprises a first identification sequence that specifically hybridizes to the target sequence and a second identification sequence that specifically hybridizes to the capture probe and does not hybridize to the single-stranded nucleic acid. Have and
The method, wherein the hybridization product has a single chain region comprising the second identification sequence.
(2) The method according to (1), wherein the amplification step is performed using a primer set containing at least one primer provided with a labeling element.
(3) The method according to (2), wherein the labeling element is a labeling substance or a labeling substance binding substance.
(4) The method according to any one of (1) to (3), wherein the amplification step is carried out in the presence of the identification probe.
(5) The method according to any one of (1) to (4), wherein the amplification step amplifies the single-stranded nucleic acid containing the target sequence more than the other single-stranded nucleic acid.
(6) The method according to any one of (1) to (5), wherein the detection step comprises separating the hybridization product by chromatography using a solid phase carrier provided with the capture probe.
(7) The amplification step is performed in the presence of the identification probe using a primer set containing at least one primer provided with the labeling element.
The identification probe has a second identification sequence that specifically hybridizes with a capture probe on the chromatographic solid phase carrier associated with the target nucleic acid, according to any of (1) to (6). The method described.
(8) The method according to any one of (1) to (7), wherein the target sequence contains one or two or more single nucleotide polymorphisms.
(9) The method according to (8), wherein the first identification sequence comprises at least one base that identifies the single nucleotide polymorphism in the substantially center of the identification sequence.
(10) The method according to any one of (1) to (9), wherein the amplification step is carried out in the presence of the identification probe to obtain the hybridization product.
(11) The method according to any one of (1) to (10), wherein the hybridization product is obtained by heat-denaturing the amplification product and cooling it in the presence of the identification probe.
(12) The method according to (11), wherein the identification probe and the single-stranded nucleic acid are cooled to a temperature lower than the melting temperature of any of them within 5 minutes.
(13) The method according to (12), wherein the identification probe and the single-stranded nucleic acid are cooled to a temperature lower than the melting temperature of either of them within 3 minutes.
(14) The method according to any one of (1) to (13), wherein the nucleic acid amplification reaction is any method selected from the group consisting of PCR, LCR, SDA, LAMP, RCA and RPA. Method.
(15) A kit for detecting a target nucleic acid.
Reagents for nucleic acid amplification reaction to amplify target nucleic acid,
A single-stranded nucleic acid containing the target sequence and an identification probe capable of discriminating the target sequence,
A solid-phase carrier for chromatography comprising a capture probe capable of capturing the identification probe,
Equipped with
The kit comprises the identification probe having a first identification sequence that specifically hybridizes to the target sequence and a second identification sequence that specifically hybridizes to the capture probe on the solid phase carrier.
(16) The kit according to (15), wherein the reagent comprises a primer set containing at least one primer comprising a labeling element.

It is a figure which shows the outline of this invention. It is a figure which shows the chromatography device used in Example 1. FIG. It is a figure which shows the two-step chromatography used in Example 1. FIG. It is a figure which shows the detection result by the chromatographic device of Example 1. FIG. It is a figure which shows the detection result by the chromatographic device of Example 2.

The disclosure of the present specification relates to a method for detecting a target nucleic acid, a kit for the same, and the like. FIG. 1 illustrates an outline of a method for detecting a target nucleic acid disclosed in the present specification (hereinafter, also simply referred to as the present detection method).

As shown in FIG. 1, in this detection method, the target nucleic acid is amplified in the amplification step to obtain an amplification product. The identification probe specifically hybridizes to the first identification sequence that specifically hybridizes to the target sequence in the amplification product and the capture probe associated with the target nucleic acid, but does not hybridize to the single-stranded nucleic acid. It has 2 identification sequences.

The amplification product is brought into contact with the identification probe to obtain a hybrid product of the single-stranded nucleic acid containing the target sequence and the identification probe. This hybridization product is a partially double-stranded region having a single-stranded region containing a second identification sequence.

The hybridization product in this detection method, that is, the hybridization product obtained by achieving the hybridization between the first identification sequence and the target sequence, is the second capture element for reliably capturing by the capture probe as it is. Can have an identification sequence of. Therefore, according to this detection method, high detection sensitivity and accuracy can be exhibited by using the amplification product of the nucleic acid amplification reaction. Furthermore, according to this detection method, the target nucleic acid can be detected reliably and easily.

Further, when the single-stranded nucleic acid containing the target sequence comprises a labeling element, the obtained hybridization product comprises the capture element and the labeling element as it is, so that the target nucleic acid can be detected more easily.

According to this detection method, a target sequence containing a single nucleotide polymorphism or the like can also be identified by an identification probe, can be obtained as a hybridization product, and can be used in a subsequent detection step. ..

The hybridization product can be obtained, for example, by heating the amplification product produced by the nucleic acid amplification reaction, melting it once, and cooling it in the presence of the identification probe to hybridize the identification probe with the target nucleic acid in the amplification product. Can be done.

In the substep of thermal denaturation and further cooling after the cycle of thermal denaturation-annealing-extension reaction of the so-called nucleic acid amplification reaction in the amplification step, the present inventors surprisingly have a single strand among the amplification products of the target nucleic acid. It has been found that the nucleic acid and the identification probe can hybridize sufficiently specifically, and a hybrid product can be obtained to the extent that it can be detected in the subsequent detection step. According to the present inventors, in order to obtain such a hybridization product, for example, the melting temperature of the discriminating probe and its complementary strand is set rather than the melting temperature of the primer used in the nucleic acid amplification reaction and its complementary strand. Can be set low.

The specific hybridization product between the target nucleic acid and the identification probe thus obtained can be detected on the solid-phase carrier by being captured by, for example, a capture probe immobilized on the solid-phase carrier. Such hybridization products are convenient for separating and capturing the capture probe by chromatography using a solid phase carrier.

Since the discriminating ability of the target sequence by the discriminating probe is sufficiently high, even a single base difference such as SNP can be sufficiently discriminated, and the target sequence is quantitatively hybridized according to the rate of mutation such as homo or hetero. And can be detected.

As used herein, the term "target nucleic acid" means a nucleic acid containing a target sequence to be detected. The origin of the target nucleic acid is not particularly limited, and examples thereof include various biological materials (individuals or parts of individuals such as tissues and organs thereof, excreta, body fluids, etc.) and artificial materials containing artificial nucleic acids.

In this detection method, a nucleic acid-extracted sample obtained by removing a part or all of components other than nucleic acid from biological materials or artificial materials themselves that may contain target nucleic acids or, if necessary, from these various materials is used as a sample. Can be supplied.

A "target sequence" is represented by two or more bases and comprises a sequence feature of a mutation, polymorphism, genus, species or strain consisting of a deletion, insertion and substitution consisting of one or more bases, or a combination thereof. The "target sequence" can typically include various polymorphisms such as single nucleotide polymorphisms, mutations, and sequences for identification of microorganisms.

Hereinafter, the detection method, the kit, and the like disclosed in the present specification will be described in detail with reference to the drawings as appropriate. Here, FIG. 1 is a diagram showing an outline of an example of the present detection method.

(Method for detecting target nucleic acid)
The present detection method can include an amplification step of carrying out a nucleic acid amplification reaction for amplifying a target nucleic acid to obtain an amplification product, and a step of detecting the target nucleic acid.

(Amplification process)
The amplification step is a step of carrying out a nucleic acid amplification reaction using a primer set designed so that the target nucleic acid can be amplified with respect to a sample that may contain the target nucleic acid to obtain an amplification product. Primer sets can be designed to amplify target nucleic acids that may contain the target sequence, for example, as shown in FIG. In this detection method, it is sufficient to acquire a region that may contain the target sequence as an amplification product regardless of the presence or absence of the target sequence.

In this detection method, one or more target nucleic acids can be targeted. Since this detection method detects the target nucleic acid with an identification probe that is not involved in the nucleic acid amplification reaction, high accuracy and accuracy can be ensured even when a plurality of target nucleic acids are targeted for detection. When two or more target nucleic acids are targeted for detection, a plurality of primer sets, identification probes and capture probes are used for amplification and detection of each target nucleic acid.

The primer pair constituting the primer set can have the following features. First, the base length of the target nucleic acid obtained as an amplification product by the primer set can be 80 to 300 bp without particular limitation, but can be, for example, 100 bases or more and 250 bases or less. This is because within this range, it is easy to obtain a hybrid product of the single-stranded DNA strand, which is a single-stranded nucleic acid of the amplification product, and the identification probe. Although not particularly limited, it is more preferably 150 bases or more and 220 bases or less.

The length of each primer constituting the primer set is not particularly limited, and may be 15 to 50 nucleotides or the like. More typically, it is 20 to 30 nucleotides, and can be, for example, about 17 to 25 nucleotides.

The melting temperature (Tm, ° C.) of each primer in the primer set is also not particularly limited, but may be, for example, 45 ° C. or higher and 65 ° C. or lower. Considering the melting temperature difference from the identification probe described later, it is preferably 50 ° C. or higher and 60 ° C. or lower.

It is preferred that any one of the primers in the primer set be provided with a labeling element. Amplification products from such primer sets will include labeling elements. By doing so, the hybridized product can simplify the operation for detection and is convenient for detection in the detection step.

From the viewpoint of imparting a labeling element to the hybridization product, it is preferable to design the primer so that the single-stranded nucleic acid that hybridizes with the identification probe contains the labeling element. That is, when the discriminant probe hybridizes to the antisense strand, the antisense strand is configured to include a labeled element, and when the discriminant probe hybridizes to the sense strand, the sense strand comprises a labeled element. It is configured as follows.

Here, the labeling element includes, for example, a labeling substance and a labeling substance binding substance. As used herein, the term "labeled substance" is a substance that makes it possible to distinguish a substance or molecule to be detected from others. The labeling substance is not particularly limited, but typically includes a labeling substance using fluorescence, radioactivity, an enzyme (for example, peroxidase, alkaline phosphatase, etc.), phosphorescence, chemiluminescence, coloring, and the like.

The labeling substance is preferably a luminescent substance, a coloring substance, or a coloring substance that exhibits light emission or color development that can be detected visually (with the naked eye). That is, it is preferably a substance that can directly itself generate a signal that is visible to the naked eye without the need for other components. In particular, it is more suitable for hybridization by chromatography. It can be performed quickly and easily in the detection process. Such substances typically include various colorants such as various pigments and dyes. In addition to precious metals such as gold and silver, various metals or alloys such as copper, or organic compounds (may be complex compounds) containing the metals can be mentioned. Inorganic compounds such as mica, which are similar to colorants, can be mentioned.

Labeling materials of this type typically include various dyes, various pigments, luminols, isolminols, acridinium compounds, olefins, enol ethers, enamines, arylvinyl ethers, dioxene, arylimidazoles, lucigenin, luciferins and eclipses. Chemiluminescent substances can be mentioned. Also included are particles such as latex particles labeled with such labeling materials. Further, a colloid or a sol including a gold colloid or a sol or a silver colloid or a sol can be mentioned. Furthermore, metal particles, inorganic particles and the like can be mentioned.

As described above, the labeling substance may be partially provided with particles. The average particle size of particles such as latex particles constituting a part of the labeling substance is not particularly limited, but is, for example, 0.1 nm or more and 20 μm or less, and can be appropriately selected depending on the pore size of the solid phase carrier and the like.

Preferred particles are particles that can be suspended in an aqueous solution and consist of a water-insoluble polymeric material. Examples thereof include polyethylene, polystyrene, styrene-styrene sulfonate copolymer, acrylic acid polymer, methacrylic acid polymer, acrylonitrile polymer, acrylonitrile-butadiene-styrene, polyvinyl acetate-acrylate, polyvinylpyrrolidone or vinyl chloride-acrylate. Latex particles having an active group, such as a carboxyl, amino or aldehyde group, on their surface can also be mentioned.

Examples of the labeling substance binding substance include substances that can finally bind the labeling substance by utilizing a protein-protein interaction, a low molecular weight compound-protein interaction, a nucleic acid-nucleic acid interaction, and the like. For example, antibodies in an antigen-antibody reaction, biotin in an avidin (streptavidin) -biotin system, digoxigenin in an anti-digoxigenin (DIG) -digoxigenin (DIG) system, haptens represented by FITC in an anti-FITC-FITC system, and each other. Examples thereof include oligonucleotides that can be hybridized. In this case, the labeling substance ultimately used for detection is the labeling substance for the other molecule or substance that interacts with the labeling substance binding substance (eg, antigens such as streptavidin, anti-FITC, oligonucleotides, etc.). It is configured to be provided as a site for binding to a binding substance.

Such labeling substances and labeling substance binding substances are commercially available, and methods for producing labeling substances and labeling substance binding substances and labeling the labeling substances on particles are also known, and those skilled in the art may appropriately use known techniques. Can be obtained. Furthermore, binding of such a labeling substance or a particle labeled with a labeling substance or a labeling substance binding substance to an oligonucleotide such as DNA is also appropriately possible via a functional group such as an amino group, and itself is possible in the art. It is well known.

In the amplification step, a nucleic acid amplification reaction is carried out on a sample that may contain a target nucleic acid together with such a primer set, a DNA polymerase, and nucleotide triphosphates (dNTPs) having various bases. The nucleic acid amplification reaction generally comprises the following steps (1) to (4). Usually, the amplified product can be obtained by repeating (2) to (4) about 15 to 100 times or less, typically about 20 to 50 times. The amplification step may further include, for example, (5) a heat denaturation step and (6) a cooling step for obtaining a hybridization product described later.

(1) Thermal denaturation step before cycle (2) Thermal denaturation step (3) Annealing step (4) Extension reaction step (5) Thermal denaturation step (6) Cooling step

The general conditions of each of the steps (1) to (4) are well known, and those skilled in the art can appropriately set the steps based on the known conditions. For example, (1) the cooling step before the cycle is, for example, about 90 ° C. or higher and 99 ° C. or lower, (2) the heat denaturation step is also 90 ° C. or higher and 99 ° C. or lower, and (3) the annealing step is, for example, a primer. It can be about ± 6 ° C. with respect to the melting temperature of. Further, (4) the extension reaction step can be generally set to 72 ° C., although it depends on the temperature characteristics of the DNA polymerase used.

For example, (5) the heat denaturation step can be carried out at about 90 ° C. or higher and 99 ° C. or lower, like other heat denaturation steps. Further, the heat denaturation step is preferably maintained at such a temperature for preferably 10 seconds or longer, more preferably 20 seconds or longer and 60 seconds or shorter. Further, for example, (6) cooling step is a step of cooling below the melting temperature of the identification probe. The temperature reached in the cooling step (cooling temperature) is, for example, 25 ° C. or lower, preferably 20 ° C. or lower, more preferably 18 ° C. or lower, and further preferably 15 ° C. or lower. The time until the cooling temperature is reached (cooling time) is, for example, 15 minutes or less, preferably 10 minutes or less, more preferably 7 minutes or less, still more preferably 5 minutes or less, still more preferably 3 minutes or less. More preferably, it is a step of cooling in about 1 minute or less. The shorter the cooling time, the shorter the time required for detection. Since both the heat denaturation time and the cooling time may be extremely short, it is effective in shortening the inspection time.

The nucleic acid amplification reaction in the amplification step can be appropriately selected from PCR, LCR (ligase chain reaction), SDA, LAMP, RCA, RPA and the like.

The nucleic acid amplification reaction is also preferably carried out so as to amplify the single-stranded nucleic acid containing the target sequence more than the other single-stranded nucleic acid among the amplification products. It is preferable to use an asymmetric nucleic acid amplification reaction such as so-called asymmetric PCR. By doing so, the hybridization product can be obtained more efficiently and at a high concentration. Such an asymmetric nucleic acid amplification reaction causes the presence of more primers for amplifying the single-stranded nucleic acid to be amplified more than the other primers. For example, in the case of asymmetric PCR, the concentration ratio of the single-stranded nucleic acid primer (A) containing the target sequence to the other single-stranded nucleic acid primer- (B) is set to A: B = 2: 1 to 50: 1. Can be.

(Hybridization product of single-stranded nucleic acid containing target sequence and identification probe)
In this detection method, a hybrid product of a discrimination probe capable of specifically hybridizing to a target sequence of a target nucleic acid or an amplification product thereof and a single-stranded nucleic acid containing the target sequence is provided in the detection step. In this detection method, such hybridization products can be obtained during or after the amplification step. Hereinafter, the hybridization product and its acquisition will be described.

(Identification probe)
The identification probe can be designed for one or more target nucleic acids, respectively, and one or more can be prepared. For example, in the case of a single nucleotide polymorphism of T / A, there are two types of target sequences, T type (for example, wild type) and A type (mutant type), and the target nucleic acid is also two types. In this case, a total of two types of identification probes are prepared for the wild-type target sequence and the mutant target sequence, depending on the target sequence, that is, the target nucleic acid. In addition, a plurality of types of identification probes may be subjected to one detection method corresponding to different mutations and polymorphisms.

Here, the discriminant probe can have a first discriminant sequence that specifically hybridizes to the target sequence. By having the identification probe having the first identification sequence, it is possible to hybridize with the single-stranded nucleic acid having the target sequence.

For example, when the target sequence is a single nucleotide polymorphism, the base for detecting at least one single nucleotide polymorphism among one or two or more single nucleotide polymorphisms is any of the range of the base length of the first identification sequence. It may be in the position of. Even if it is set at an arbitrary position, the target sequence can be detected with high accuracy. That is, the base for identification may be present on the central side, may be at the 3'end or 5'end, or may be inside the end.

The length of the first identification sequence in the identification probe is not particularly limited, but is designed to ensure amplification of the target nucleic acid by the primer set. That is, in the (3) annealing step, only the primer is annealed to the target nucleic acid, but at this time, it is preferable that the identification probe does not anneal to the target nucleic acid or the primer. More specifically, if the length of the first identification sequence is too long, there is a tendency to anneal to the target nucleic acid or the like in the (3) annealing step.

Further, the length of the first identification sequence is preferably a length suitable for specifically hybridizing with the target sequence. That is, in (6) cooling step, it is preferable not to hybridize to other than the target sequence non-specifically. More specifically, if the first discriminant sequence is too long, non-specific hybridization will occur, and if the first discriminant sequence is too short, the hybridization itself will be suppressed.

The suitable length of the first identification sequence can be appropriately set according to the type of target sequence and the conditions of (3) annealing step and (6) cooling step. Typically, it can be 10 to 25 nucleotides. For example, the first identification sequence may be about 13 bases or more and 15 bases or less, although it is an example and is not limited.

It is preferred that the identification probe has a melting temperature of the first identification sequence and the target sequence lower than the melting temperature of each primer in the primer set for amplifying the target nucleic acid. By designing the melting temperature of the identification probe sufficiently lower than that of the primer set, it is possible to obtain a hybrid product of the target sequence and the identification probe in (6) cooling step after the heat denaturation step. can. How low the melting temperature of the primer set should be depends on the heat denaturation temperature used for the amplification reaction, the annealing temperature, etc., but the melting temperature of the identification probe is preferably the average of the melting temperatures of each primer of the primer set. The temperature is preferably 10 ° C. or higher, more preferably 15 ° C. or higher, further preferably 16 ° C. or higher, still more preferably 17 ° C. or higher, and even more preferably 18 ° C. or higher. Further, the temperature may be lower than the average melting temperature of the primer set by about 20 ° C.

Further, the identification probe may have its 3'end blocked with a known blocking agent so that a stretching reaction does not occur. For example, the 3'end may be modified with a hydroxyl group. For example, US Pat. No. 5,516,663 describes modifications that can be used to make a probe inextensible.

In addition to the first identification sequence, the identification probe can include a second identification sequence that can specifically hybridize with the capture probe associated with the target sequence. By providing the second identification sequence, it is possible to sufficiently secure the hybridization specificity with the capture probe and enable highly accurate detection.

The second identification sequence is a sequence that does not specifically hybridize with the single-stranded nucleic acid containing the target sequence (that is, one DNA strand constituting the amplification product). By doing so, as shown in FIG. 1, when the identification probe hybridizes with the single-stranded nucleic acid having the target sequence via the first identification sequence, the second identification sequence is the single-stranded nucleic acid. It can form part of a single-stranded region that does not hybridize. The inclusion of the second identification sequence in the single-stranded region facilitates and ensures hybridization with the capture probe. Further, the second identification sequence can be, for example, a sequence designed so as not to artificially cross-hybridize.

(Hybridization product)
The hybridization product constitutes an amplification product. As shown in FIG. 1, the hybridization product of the single-stranded nucleic acid containing the target sequence and the identification probe has a portion having a single-stranded region containing the second identification sequence. It is a double-stranded nucleic acid. Further, since the hybridization product hybridizes with a discrimination probe that is sufficiently short with respect to the length of the single-stranded nucleic acid containing the target sequence, the single-stranded nucleic acid has a labeling element at the 5'end or the like. Also, when the labeling element is a labeling substance binding substance, the labeling substance can be easily bound.

(Acquisition of hybridization product in amplification process)
The hybridization product of the identification probe and the target sequence is produced, for example, by coexisting the identification probe capable of specifically hybridizing to the target sequence of the target nucleic acid or its amplification product in at least (6) cooling step in the growth step. , Can be obtained. According to the present inventors, hybridization based on specific hybridization between the identification probe and the target sequence is performed by coexisting the identification probe in the cooling step even in the presence of an amplification product, a residual primer, or the like. You can get the product. By acquiring the hybridization product in the amplification step, it is efficient without carrying out an additional step.

In such an amplification step, (5) the heat denaturation step heats a liquid (nucleic acid amplification reaction solution) containing an amplification product which may contain a target nucleic acid containing a target sequence so that the amplification product can be melted. Specifically, it can be heated to the temperature already described. Further, in the (6) cooling step, the liquid is cooled so that a hybrid product of the single-stranded nucleic acid containing the target sequence and the identification probe can be obtained. Specifically, it can be cooled at the temperature and time already described.

Further, the identification probe may coexist with a primer or the like in the step of (6) the heat denaturation step prior to the cooling step or the amplification cycle step of (2) to (4) in the step (2) to (4). 1) It may coexist with a primer or the like from the heat denaturation step before the cycle. This is because the identification probe can coexist not only in the cooling step but also in the amplification step when it has one or more suitability as the above-mentioned identification probe. In particular, it is preferable that the hybridization product can be obtained at once without opening the tube used for the amplification step by coexisting the identification probe from the beginning of the amplification step, that is, from the heat denaturation step before the cycle.

Based on the specificity of the identification probe, as a hybridization product, for example, there are two types of target sequences (target nucleic acids), one of which is a T-type (wild type) sequence of T / A single nucleotide polymorphism (SNP). And when the other is a type A (mutant) sequence, it is possible in the SNP whether the T / A is 1: 1 heterozygous or 2: 0 or 0: 2 homozygous. Specific hybridization products can be obtained depending on the combination.

(Acquisition of hybridization product after amplification step)
The hybridization product can also be obtained after the amplification step. For example, after the steps (1) to (4) in the amplification step are completed, prior to the detection step, the (5) thermal transformation step and the (6) cooling step can be separately performed in the presence of the identification probe. be able to. In this case, after the (4) extension reaction step of the amplification step, a cooling step up to the melting temperature of the primer can be provided.

A person skilled in the art can carry out the amplification step in an appropriately different manner depending on the type of nucleic acid amplification reaction used and the like. In the case of the so-called PCR method, the final extension reaction can be carried out after the above (4) extension reaction step. Further, (4) a cooling step to lower the melting temperature of the primer set after the extension reaction step or the final extension step can be provided.

In addition to the amount (ratio) of the template DNA and the primer in the amplification step, the amount of the identification probe can be appropriately set as needed. Various methods for synthesizing oligonucleotides such as primers and probes are well known to those skilled in the art, and similarly, binding of a labeling element to an oligonucleotide is also well known to those skilled in the art.

(Detection process)
The detection step is a step of detecting the target nucleic acid. Such a detection step can be carried out as a step of detecting the target nucleic acid from the obtained hybridization product. The hybridization product identifies the target sequence and captures the single-stranded nucleic acid containing the target sequence. Therefore, it is preferable to detect the target nucleic acid by detecting the hybridization product as it is.

In order to detect a hybridization product, as described above, the second identification sequence imparted to the identification probe is used to bring the second identification sequence into contact with a capture probe that specifically hybridizes to hybridize. It is preferable to capture and detect by.

Typically, a nucleic acid amplification reaction solution containing a hybridization product can be supplied to a solid-phase carrier on which a capture probe is immobilized to carry out hybridization.

The form of hybridization is not particularly limited, and a nucleic acid amplification reaction solution containing a hybridization product is supplied to a cavity containing a capture probe of a solid-phase carrier on which a capture probe capable of specifically hybridizing with a second identification sequence is immobilized. Hybridization may be carried out. Further, a nucleic acid amplification reaction solution containing a hybridization product may be supplied to a solid-phase carrier on which a capture probe is immobilized, and hybridization may be performed by chromatography. In particular, hybridization by chromatography is in that the hybridization product can be separated and detected by an extremely simple operation such as immersing a part of the solid phase carrier in a nucleic acid amplification reaction solution containing the hybridization product. preferable. Further, the detection time is also preferable in that it can be completed in an extremely short time such as within 15 to 30 minutes. The solid-phase carrier and the like used for hybridization will be described in detail later.

The target nucleic acid can be detected by detecting the hybridization product captured by the capture probe on the solid-phase carrier. The method for detecting the hybrid product is not particularly limited, and various detection methods capable of physicochemically detecting the hybrid product, such as a labeling element provided in the hybrid product, can be adopted.

For example, when a hybridization product comprises a labeling element, more specifically, when the hybridization product comprises an extension derived from a primer comprising the labeling element, the hybridization product may be detected based on the labeling element. can. Detection using the above-mentioned marker elements is well known in the art.

(When the hybridization product contains a labeling substance)
When the labeling element contained in the hybridization product is a labeling substance, if the hybridization product is captured by a capture probe, the hybridization product can be obtained based on the signal of the labeling substance and the position information on the solid phase carrier of the capture probe. Can be detected and identified.

(When the hybridization product has a labeling substance binding substance)
On the other hand, when the labeling element contained in the hybridization product is a labeling substance binding substance such as haptens or oligonucleotides typified by biotin, digoxigenin, FITC, etc., the labeling substance finally used for detection is hybridization. It is fed to the hybridized product prior to, with, or after hybridization of the product to the capture probe.

For example, when the labeling substance is supplied prior to the hybridization of the hybridization product and the capture probe, the nucleic acid amplification reaction solution containing the hybridization product contains, for example, avidin-bound colored beads or hybridization containing the beads. A liquid (in the case of chromatography, a developing liquid; the same applies hereinafter) may be supplied to bind the labeling substance to the hybridization product and then supplied to a solid phase carrier for hybridization with a capture probe. can.

When the labeling substance is supplied along with hybridization, for example, the labeling substance is retained in a part of a solid phase carrier for chromatography that passes immediately after the start of the developing solution. An embodiment in which a labeling substance is bound to a hybridization product passing through the site, further developed, and hybridized with a capture probe can be mentioned.

When supplying the labeling substance after hybridization, for example, a nucleic acid amplification reaction solution containing a hybridization product or the reaction solution and the hybridization solution are provided to a solid phase carrier on which a capture probe is immobilized. An embodiment in which hybridization (chromization) is performed, and then a hybridization solution containing the labeling substance is separately supplied to the solid phase carrier to bind the labeling substance to the hybridization product captured by the capture probe in the solid phase carrier. Can be mentioned.

It is useful for such two-step hybridization, especially hybridization by chromatography. That is, the first chromatography step of supplying the nucleic acid amplification reaction solution containing the hybridization product to the solid phase carrier to perform chromatography, and the solid phase carrier after the first chromatography step are labeled with a labeling substance. A second chromatographic step of supplying the liquid and performing the chromatography can be carried out. By doing so, the target nucleic acid can be detected by a simple procedure.

For example, the first chromatography step is a step for hybridization between a hybridization product and a capture probe, but nucleic acid amplification is performed by inserting a solid phase carrier into a container such as a tube in which the nucleic acid amplification reaction has been completed. It can be carried out by immersing a part of the solid phase carrier in the reaction solution. Further, a solid phase carrier after the first chromatography step is inserted into another container filled or filled with a hybridization liquid (labeling liquid) containing a labeling substance, and a part thereof is immersed in the labeling liquid. A second chromatographic step can be performed to bind the labeling substance to the hybrid product. By doing so, hybridization and detection can be easily and promptly performed after a nucleic acid amplification reaction such as PCR.

Hybridization associated with the detection step can be appropriately selected from conditions well known to those skilled in the art. For example, in the case of hybridization by chromatography, the temperature can be, for example, about 5 to 40 ° C, preferably about 15 to 35 ° C, and particularly about room temperature. The time required for nucleic acid chromatography can be appropriately set according to the shape of the nucleic acid chromatography carrier, the type of the substrate, and the composition of the developing medium. Typically, it can be about 2 to 50 minutes.

The hybridization solution is not particularly limited, and may be an aqueous medium containing an organic solvent, if necessary, or a nucleic acid amplification reaction solution may be used as it is. The hybridization solution can contain, for example, a component for making the pH a constant unit. Such components include known buffer components such as acetate buffer consisting of acetic acid and sodium acetate, phosphate buffer consisting of phosphoric acid and sodium phosphate, citric acid buffer consisting of citric acid and sodium citrate, and PBS. Can be mentioned.

In hybridization by chromatography, when the hybrid solution is developed on the solid phase carrier, even if the solid phase carrier is held substantially vertically within the range in which the liquid can be developed by the capillary phenomenon, the solid phase carrier is substantially horizontal. May be retained in.

(Method for detecting target nucleic acid)
Based on the above, the present specification also provides the following methods for detecting the target nucleic acid. That is, in the presence of a step of heating a liquid containing a sample that may contain a target nucleic acid containing a target sequence so that the target nucleic acid can be melted, and an identification probe that can specifically hybridize to the target sequence. After the heating step, the liquid is cooled to obtain a hybrid product of the single-stranded nucleic acid containing the target sequence and the identification probe, and the target nucleic acid is detected by detecting the hybrid product. A method for detecting a target nucleic acid is also provided, comprising a step. According to this method, the target nucleic acid can be easily and surely detected when the sample can already contain a sufficient amount of the target nucleic acid. In this detection method, various embodiments already described can be applied to the acquisition of the target nucleic acid, the identification probe, the hybridization product, and the detection of the hybridization product.

The identification probe needs to be present in the cooling step, but may already be present in the heating step.

As described above, according to this detection method, the target nucleic acid can be detected easily and accurately. In particular, single nucleotide polymorphisms can be detected extremely easily and quickly as compared with the conventional case by adopting hybridization by chromatography in the detection step.

(kit)
The present specification can provide a kit (hereinafter referred to as the present kit) for detecting a target nucleic acid by the present detection method. The kit can include one or more identification probes capable of identifying the target sequence in the target nucleic acid. The identification probe can take various aspects described above. The identification probe preferably includes a second identification sequence in addition to the first identification sequence described above. The kit can include an identification probe depending on the type of target sequence (target nucleic acid) to be identified. For example, the kit for detecting SNPs can include identification probes corresponding to one or more variants of the wild type of SNPs.

Since this kit is used for the amplification product of the nucleic acid amplification reaction or the amplification step thereof, it can further include a primer set capable of amplifying the target nucleic acid. As for the primer set, various embodiments already described can be appropriately adopted. It is preferred that one primer in the primer set comprises a labeling element. The kit can include a set of primers depending on the type of target nucleic acid to be amplified. For example, the kit for detecting SNPs can include a set of primers to amplify the DNA duplex containing the SNP (target sequence).

The kit can also include one or more capture probes that can specifically hybridize to the second discriminant sequence of one or more discriminating probes. The capture probe may be immobilized on a suitable solid phase carrier. By immobilizing the capture probe on the solid-phase carrier, the position information of each capture probe on the solid-phase carrier (for example, a flat plate) in which a plurality of capture probes are immobilized, and the individual capture probes are immobilized. This is because the identification information (color and size) imparted to the solid-phase carrier (for example, beads or the like) facilitates the detection of a plurality of target nucleic acids.

A solid-phase carrier (hybridization device) on which a capture probe is immobilized will be described. As the solid phase carrier, the carrier may be plastic or glass, and the material is not particularly limited. In particular, examples of the solid phase carrier used for hybridization by chromatography include so-called porous materials mainly composed of polymers such as polyether sulfone, nitrocellulose, nylon, and polyvinylidene fluoride. Further, a cellulosic material such as filter paper can also be preferably used.

The shape of the carrier may be flat plate-shaped, but may be bead-shaped, and the shape is not particularly limited. The solid-phase carrier is preferably an array (particularly a microarray) in which the carrier is in the form of a solid-phase plate and a plurality of capture probes are fixed in a fixed arrangement. The array can fix a large number of probes and is convenient for comprehensively detecting various target nucleic acids at the same time. Further, in a form intended for hybridization by chromatography, the capture probe is fixed along the development direction. In such a hybridization device, the contact order between the product and the probe can be controlled by the fixation position of the capture probe.

The capture probe is not particularly limited and can be immobilized on the solid phase carrier by a method known to those skilled in the art. For example, a means for immobilizing a capture probe and a solid-phase carrier by covalent bonding with a linker as necessary; a means for immobilizing by an electrostatic interaction between the capture probe and the solid-phase carrier, etc. Is exemplified. It may be a means for immobilizing by electrostatic interaction between the capture probe and the carrier, and further causing a covalent bond with ultraviolet rays or the like.

Hybridization devices for chromatography do not necessarily have to be composed of a single solid phase carrier. As long as the developing medium can be moved by the capillary phenomenon as a whole, it may be linked by a plurality of solid phase carriers. Further, the overall form of the chromatographic probe immobilized body is not particularly limited. Any form such as a sheet or a thin rod that can develop and diffuse the chromatographic liquid by a capillary phenomenon may be used. Preferably, the oblong body is such that one end along its longitudinal direction is in contact with the chromatographic development medium.

When a hybridization device for chromatography starts unfolding by immersion in a hybridization solution, the unfolding start portion (immersion portion) has a tapered shape in order to facilitate contact with the hybridization solution in the tube. It can also be. A position marker indicating that it is a deployment start portion may be attached to the device.

The kit may include other elements as needed. Other elements include, for example, reagents for amplifying the target nucleic acid, positive control samples and negative control samples of template nucleic acids.

In this example, as a template (sample), homo (TT, CC) and hetero (TC) polymorphisms (wild-type T / mutant C) of the ampicillin resistance gene (β-lactamase) encoded by pUC are used. A DNA fragment (200 bp) is prepared, each DNA is amplified with the primers shown below, and a hybrid product is obtained using an identification probe that distinguishes between the wild type and the mutant type shown below, and separated by chromatography. Was detected.

Primer F: cgaaa aacgt tttcc aatga tgag (24mer) Tm: 54.6 ° C (SEQ ID NO: 1)
Primer R: Bio-tggtt atggc agcac tgcat (20mer) Tm: 57.5 ° C (SEQ ID NO: 2)

Wild-type probe: tag1-tacac tattc tcag Tm: 34.6 ° C (SEQ ID NO: 3)
Mutant probe: tag2-tacac tactc tcag Tm: 37.5 ° C (SEQ ID NO: 4)

The length of the probe was determined in advance to be 14 mer based on the results of the nucleic acid amplification reaction using the primer to form the primer / probe product, anneal to the target, and examine the detection specificity of SNP.

(1) Preparation of Chromatographic Strip The chromatographic strip has a line shape of a wild-type capture probe that captures a wild-type identification probe and a mutant capture probe that captures a mutant identification probe, as shown in FIG. Fixed to. That is, the capture probe solution was applied to a Hi-Flow Plus membrane sheet (60 mm x 600 mm) manufactured by Merck Millipore, and NGK Insulators, Ltd. (registered) using the ejection unit (inkjet method) described in JP-A-2003-75305. Spotted using a trademark) spotter. A sequence in which polyT (20)) was added to the 3'end side of the capture probe was used as a probe, and a UV irradiation device (XL-1500UV Crosslinker) manufactured by Crosslink was used at a wavelength of 300 mJ containing a component of 280 nm. / cm ² about the irradiation of ultraviolet light was performed immobilized performed.

(2) Amplification of target gene Various pUC fragments were used as templates (samples). The amplification reaction solution shown below contains wild-type DNA: mutant DNA at 1: 0, 0.75: 0.25, 0.5: 0.5, 0.25 so that 1 pg of wild-type and mutant template DNA is contained in total. : 0.75, 0: 1) are included in each ratio.

In addition, biotin was bound to the 5'end of the R primer. A tag (tag1,2: DNA) having a base sequence complementary to the corresponding capture probe of the chromatographic strip prepared in (1) was added to the 5'end side of the wild-type and mutant-type identification probes.

Takara Bio's Premix ExTaq was used as the gene amplification enzyme, and Thermogen's Quick Bath was used as the thermal cycler.

The amplification reaction solution was as shown below.
(Amplified reaction solution)
Sterilized water 1.0 μL
SYBR Premix ExTaq (Tli RNaseH Plus) 5.0 μL
Primer mixture (2.5 μM each) 1.0 μL
Identification probe solution (100 nM each) 2.0 μL
Genomic DNA (1 pg / μL) or DNA-free water 1.0 μL
Total 10.0 μL

Using a thermal cycler, the gene amplification reaction (heating at 97 ° C for 2 minutes, then 97 ° C for 5 seconds, 60 ° C for 5 seconds, and 72 ° C for 5 seconds) was repeated 30 times under the following conditions, and 1 at 97 ° C. After minutes, it was cooled to 15 ° C. in about 1 minute. As a result, it was considered that a hybridization product of each template DNA and the identification probe was generated in the amplification reaction solution.

(3) Chromatographic hybridization and detection As shown in FIG. 2, the lower end of the strip was immersed in this amplification reaction solution and developed (first chromatography). Then, the lower end of the test strip developed with the amplified reaction solution was brought into contact with the following developing solution filled in a 1.5 mL tube and developed again (second chromatography) (see FIG. 2).
(Development liquid)
Developing liquid 5.0 μL
Pure water 5.0 μL
Latex liquid 1.0 μL
11.0 μL in total

The developing solution was completely sucked up to the upper part of the strip in about 15 minutes, and the reaction was completed. After the reaction was completed, the coloration of the detection line on the strip was visually confirmed. The results are shown in FIG.

As shown in FIG. 3, it was possible to detect a line of color intensity according to the ratio of the wild type and the mutant type. Therefore, an amplification step involving additional heat denaturation and cooling substeps results in the formation of hybrid products of each amplified fragment with the corresponding identification probe in each PCR tube, depending on the proportion of DNA of each type. It was thought that it was done.

Therefore, according to this detection method, it was found that the target sequence or target nucleic acid contained in the sample can be detected easily and quantitatively with high specificity.

In this example, pUC18 is used as a template (sample), the ampicillin resistance gene (β-lactamase) sequence is amplified by asymmetric PCR with the primers shown below, and the identification probe that distinguishes the wild type and the mutant type shown below, respectively. The hybridization product was obtained and separated and detected by chromatography.

Primer F: cgaaa aacgt tttcc aatga tgag (24mer) Tm: 54.6 ° C (SEQ ID NO: 1)
Primer R: Bio-tggtt atggc agcac tgcat (20mer) Tm: 57.5 ° C (SEQ ID NO: 2)

Wild-type probe: tag1-tacac tattc tcag Tm: 34.6 ° C (SEQ ID NO: 3)
Mutant probe: tag2-tacac tactc tcag Tm: 37.5 ° C (SEQ ID NO: 4)

(1) Preparation of Chromatographic Strip The chromatographic strip has a line shape of a wild-type capture probe that captures a wild-type identification probe and a mutant capture probe that captures a mutant identification probe, as shown in FIG. Fixed to. That is, the capture probe solution was applied to a Hi-Flow Plus membrane sheet (60 mm x 600 mm) manufactured by Merck Millipore, and NGK Insulators, Ltd. (registered) using the ejection unit (inkjet method) described in JP-A-2003-75305. Spotted using a trademark) spotter. A sequence in which polyT (20)) was added to the 3'end side of the capture probe was used as a probe, and a UV irradiation device (XL-1500UV Crosslinker) manufactured by Crosslink was used at a wavelength of 300 mJ containing a component of 280 nm. / cm ² about the irradiation of ultraviolet light was performed immobilized performed.

In addition, biotin was bound to the 5'end of the R primer. A tag (tag1,2: DNA) having a base sequence complementary to the corresponding capture probe of the chromatographic strip prepared in (1) was added to the 5'end side of the wild-type and mutant-type identification probes.

Takara Bio's SYBR Premix ExTaq (Tli RNaseH Plus) was used as the gene amplification enzyme, and Thermogen's Quick Bath was used as the thermal cycler.

The amplification reaction solution was as shown below.
(Amplified reaction solution)
Sterilized water 1.0 μL
SYBR Premix ExTaq (Tli RNaseH Plus) 5.0 μL
R primer (2.5 μM) 1.0 μL
F primer (2.5, 1.25, 0.6, 0.25, 0.1, 0.05 μM) 1.0 μL
Identification probe solution (1 μM each) 1.0 μL
pUC18DNA (1 pg / μL) 1.0 μL
Total 10.0 μL

Using a thermal cycler, the gene amplification reaction (heating at 97 ° C for 2 minutes, then 97 ° C for 5 seconds, 60 ° C for 5 seconds, and 72 ° C for 5 seconds) was repeated 40 times under the following conditions, and 1 at 97 ° C. After minutes, it was cooled to 15 ° C. in about 1 minute. As a result, it was considered that a hybridization product of each template DNA and the identification probe was generated in the amplification reaction solution.

(3) Chromatographic hybridization and detection
The lower end of the test strip was brought into contact with the following developing solution filled in a 1.5 mL tube to develop it (see FIG. 5).
(Development liquid)
PCR product 10.0 μL
Developing liquid 10.0 μL
Latex liquid 1.0 μL
21.0 μL in total

The developing solution was completely sucked up to the top of the strip in about 15 minutes, and the reaction was completed. After the reaction was completed, the coloration of the detection line on the strip was visually confirmed. The results are shown in FIG.

As shown in the figure, it was confirmed that the wild-type detection line became stronger as the primer F concentration decreased. Therefore, it was considered that the accumulation amount of the amplified fragment by one difference increased with the decrease of the primer F concentration, and the formation of the hybridization product between the amplified fragment and the discrimination probe was promoted.

Therefore, it was found that by using this detection method in combination with asymmetric PCR, the target sequence or target nucleic acid contained in the sample can be easily detected with high sensitivity.

SEQ ID NO: 1, 2: Primer SEQ ID NO: 3, 4: Probe

Claims (17)

A method for detecting a target nucleic acid containing a target sequence.
An amplification step of performing a nucleic acid amplification reaction on a sample that may contain the target nucleic acid to obtain an amplification product containing a single-stranded nucleic acid containing the target sequence.
A detection step of detecting the target sequence using a capture probe associated with the target sequence, and a detection step.
Equipped with
Prior to the detection step, an identification having a first identification sequence that specifically hybridizes to the target sequence and a second identification sequence that specifically hybridizes to the capture probe and does not hybridize to the single-stranded nucleic acid. Obtaining a hybridization product of the probe and the single-stranded nucleic acid,
The detection step is a step of detecting the hybridization product by using chromatography using a solid phase carrier provided with the capture probe associated with the target sequence.
Method. The amplification step is the following step (a) ;
(A) said performing said nucleic acid amplification reaction in the presence of the identification probes, a process comprising obtaining said hybridized product by subsequently cooling the amplification reaction, the method of claim 1. Further, after the amplification step, the following step ( b );
( B ) A step of obtaining the hybridization product by heating and cooling the amplification reaction solution in the presence of the identification probe added to the amplification reaction solution after the amplification step.
The method according to claim 1. The method according to any one of claims 1 to 3 , wherein the amplification step further comprises a step of amplifying the single-stranded nucleic acid more than the other single-stranded nucleic acid. A method for detecting a target nucleic acid containing a target sequence.
An amplification step of performing a nucleic acid amplification reaction on a sample that may contain the target nucleic acid to obtain an amplification product containing a single-stranded nucleic acid containing the target sequence.
A detection step of detecting the target sequence using a capture probe associated with the target sequence, and a detection step.
Equipped with
The amplification step is a step of amplifying the single-stranded nucleic acid containing the target sequence more than the other single-stranded nucleic acid.
Prior to the detection step, the single-stranded nucleic acid is specifically hybridized to the capture probe and the first identification sequence that specifically hybridizes to the target sequence with respect to the amplification reaction solution of the nucleic acid amplification reaction. By adding an identification probe having a second identification sequence that does not hybridize to, a hybrid product of the single-stranded nucleic acid and the identification probe is obtained.
Said detecting step is a step of detecting using chromatography using solid phase support comprising the capture probe associated with said hybridized product in the target sequence,
Method. The amplification step is any of claims 1 to 5 , wherein the amplification step is a step of obtaining an amplification product containing the single-stranded nucleic acid having the labeling element by using a primer set containing at least one primer having the labeling element. The method described in Crab. The method according to claim 6 , wherein the labeling element is a labeling substance or a labeling substance binding substance. The method according to any one of claims 1 to 7 , wherein the target sequence comprises one or more single nucleotide polymorphisms. The method according to claim 8 , wherein the first identification sequence comprises at least one base that identifies the single nucleotide polymorphism in the substantially center of the first identification sequence. The method of claim 9 , wherein the identification probe and the single-stranded nucleic acid are cooled to a temperature lower than the melting temperature of either of them within 5 minutes. The method of claim 10 , wherein the identification probe and the single-stranded nucleic acid are cooled to a temperature lower than the melting temperature of either of them within 3 minutes. The method according to any one of claims 1 to 11 , wherein the nucleic acid amplification reaction is any method selected from the group consisting of PCR, LCR, SDA, LAMP, RCA and RPA. A kit for detecting a target nucleic acid containing a target sequence,
A reagent for a nucleic acid amplification reaction that amplifies an amplification product containing a single-stranded nucleic acid containing the target sequence, and
An identification probe that can identify the target sequence and
A solid-phase carrier for chromatography comprising a capture probe capable of capturing the identification probe,
Equipped with
The identification probe has a first identification sequence that specifically hybridizes to the target sequence and a second identification sequence that specifically hybridizes to the capture probe and does not hybridize to the single-stranded nucleic acid. It is acquired configured to be able to hybridize products of the identification probe and the single-stranded nucleic acid identifies the target sequence prior to detection by chromatography kit. The nucleic acid amplification reaction is carried out in the presence of the identification probe, and then the hybridization reaction solution is cooled to obtain the hybridization product, or the identification probe added to the amplification reaction solution after the nucleic acid amplification reaction. 13. The kit according to claim 13, wherein the hybridization product is obtained by cooling the amplification reaction solution in the presence of. The kit of claim 13 or 14 , wherein the reagent comprises a primer set comprising at least one primer comprising a labeling element. The kit according to claim 13 or 14 , wherein the reagent comprises a primer set that amplifies the single-stranded nucleic acid containing the target sequence more than the other single-stranded nucleic acid. A kit for detecting a target nucleic acid containing a target sequence,
Reagents for nucleic acid amplification reaction to amplify target nucleic acid,
An identification probe that can identify the target sequence and
A solid-phase carrier for chromatography comprising a capture probe capable of capturing the identification probe,
Equipped with
The identification probe is a first identification sequence that specifically hybridizes to the target sequence and a second identification sequence that specifically hybridizes to the capture probe and does not hybridize to a single-stranded nucleic acid containing the target sequence. And have
It said reagent includes a primer set such as to increase the amplification than before Symbol single-stranded nucleic acid and the other single-stranded nucleic acid,
A kit comprising the presence of the identification probe in the amplification reaction solution of the nucleic acid amplification reaction so that a hybrid product of the identification probe and the single-stranded nucleic acid can be obtained prior to detection by chromatography.

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