JP2019154396A - Nucleic acid detection method, target nucleic acid detection probe used therefor, and target nucleic acid detection device - Google Patents

Abstract

To provide a nucleic acid detection method for detecting a target nucleic acid at a high accuracy.SOLUTION: There is provided a target nucleic acid detection method comprising: a target nucleic acid sample applying step where a nucleic acid sample containing a target nucleic acid is applied to a target nucleic acid detection probe which has a predetermined nucleic acid sequence and an artificial nucleic acid sequence and a label molecule and is arranged on a substrate; a nucleic acid degradation step where a completely complementary double stand consisting the predetermined nucleic acid sequence, the artificial nucleic acid sequence and the label molecule, which is formed in a case where the completely complementary double stand may be formed by the predetermined nucleic acid sequence and the artificial nucleic acid sequence, is applied to a nucleic acid degradation enzyme which is specific to the completely complementary double stand and degrades DNA chain of the double stand so as to recognize the completely complementary double stand and degrade the DNA chain of the double stand; and a detection step where any of the label molecule which has been released to move away from the substrate due to degradation by the nucleic acid degradation enzyme or the label molecule in the target nucleic acid detection probe arranged on the substrate is detected.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a nucleic acid detection method, a target nucleic acid detection probe used therefor, and a target nucleic acid detection device.

  Nucleic acids (a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) are known to be involved in various life phenomena, and the number of copies of nucleic acids having a specific base sequence reflects life phenomena. It is believed that For this reason, attempts have been made to grasp the biological state by quantitatively analyzing the copy number of a specific base sequence.

  For example, a nucleic acid having a specific base sequence (hereinafter sometimes referred to as a target nucleic acid) contained in a sample is amplified by polymerase chain reaction (PCR) and detected to detect the target nucleic acid before amplification. A real-time PCR method for estimating the copy number is widely performed.

  In addition, as a method for quantifying the number of target nucleic acids, for example, the number of target nucleic acids that do not require enzyme amplification or nucleic acid amplification is quantified by using hybridization between a sequence complementary to the target nucleic acid and a probe having a detection domain. A method has been proposed (see, for example, Patent Document 1).

  An object of this invention is to provide the nucleic acid detection method which can detect a target nucleic acid with high precision.

As a means for solving the problems, the nucleic acid detection method of the present invention comprises:
A nucleic acid sample containing the target nucleic acid,
A target nucleic acid sample providing step for applying to a target nucleic acid detection probe disposed on a substrate, which has a predetermined nucleic acid sequence and an artificial nucleic acid sequence, and a labeled molecule;
Two complete complementary pairs of the predetermined nucleic acid sequence and the artificial nucleic acid sequence and the target nucleic acid, which are formed when the predetermined nucleic acid sequence and the artificial nucleic acid sequence can form a double strand that is completely complementary to the target nucleic acid. Against the chain
Recognize the fully complementary duplex by specifically recognizing the fully complementary duplex, providing a nucleolytic enzyme that degrades the DNA strand in the duplex to recognize the fully complementary duplex, A nucleic acid decomposing step for decomposing,
A detection step of detecting any of the labeled molecule that has been decomposed by the nucleolytic enzyme and is free to move from the substrate, and the labeled molecule in the target nucleic acid detection probe disposed on the substrate; ,including.

  According to the present invention, it is possible to provide a nucleic acid detection method capable of detecting a target nucleic acid with high accuracy.

FIG. 1 is a diagram showing an example of a nucleic acid detection probe. FIG. 2 is a diagram showing another example of the nucleic acid detection probe. FIG. 3 is a view showing an example of the nucleic acid detection probe of the present invention used in the nucleic acid detection device. FIG. 4 is a diagram illustrating an example of a relationship between a nucleic acid detection probe and a target nucleic acid in a nucleic acid detection device to which a nucleic acid sample has been applied. FIG. 5 is a diagram illustrating an example of a relationship between a nucleic acid detection probe, a target nucleic acid, and a nucleolytic enzyme in a nucleic acid detection device to which a nucleolytic enzyme has been added. FIG. 6 is a diagram showing an example of the relationship between a nucleic acid detection probe treated with a nucleolytic enzyme and a target nucleic acid. FIG. 7 is a diagram showing an example of the nucleic acid detection device of the present invention. FIG. 8 is a diagram showing an example of a nucleic acid detection device processed by the nucleic acid detection method of the present invention.

(Nucleic acid detection method)
The nucleic acid detection method of the present invention comprises:
A nucleic acid sample containing the target nucleic acid,
A target nucleic acid sample providing step for applying to a target nucleic acid detection probe disposed on a substrate, which has a predetermined nucleic acid sequence and an artificial nucleic acid sequence, and a labeled molecule;
Two complete complementary pairs of the predetermined nucleic acid sequence and the artificial nucleic acid sequence and the target nucleic acid, which are formed when the predetermined nucleic acid sequence and the artificial nucleic acid sequence can form a double strand that is completely complementary to the target nucleic acid. Against the chain
Recognize the fully complementary duplex by specifically recognizing the fully complementary duplex, providing a nucleolytic enzyme that degrades the DNA strand in the duplex to recognize the fully complementary duplex, A nucleic acid decomposing step for decomposing,
A detection step of detecting any of the labeled molecule that has been decomposed by the nucleolytic enzyme and is free to move from the substrate, and the labeled molecule in the target nucleic acid detection probe disposed on the substrate; ,
In addition, other steps are included as necessary.

When the present inventors examined the nucleic acid detection method which can detect a target nucleic acid with high precision, the following knowledge was acquired.
In the conventional technique using the polymerase chain reaction, there is a problem that slight fluctuations in amplification efficiency and accumulation of differences greatly affect the detection result. Furthermore, when the target nucleic acid is RNA, conversion to cDNA is necessary, and in quantitative analysis, it is necessary to accurately examine the conversion efficiency from RNA to cDNA, and the amount of the original target nucleic acid is accurately detected. There is a problem that it is difficult. Therefore, the technique using the polymerase chain reaction has a problem that the target nucleic acid cannot be quantified with high accuracy.
Furthermore, in the technique of the above-mentioned Patent Document 1, since the target nucleic acid is detected only using hybridization with a probe having a base sequence complementary to the target nucleic acid, erroneous detection due to non-specific hybridization is prevented. There is a problem that can not be. Therefore, there is a problem that the target nucleic acid cannot be quantified with high accuracy.

  In the present invention, a nucleic acid detection probe and a partial nucleic acid are partially recognized by using a nucleolytic enzyme that specifically recognizes a completely complementary double strand between a target nucleic acid and a nucleic acid detection probe and specifically degrades a DNA strand. Since it is possible to prevent erroneous detection when a double strand is formed with a desired target nucleic acid complementary to the target nucleic acid, the target nucleic acid can be detected with high accuracy.

  In the present invention, the nucleic acid means DNA, RNA or the like unless otherwise specified.

In the present invention, the target nucleic acid means a nucleic acid to be analyzed.
The target nucleic acid is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include genomic DNA, messenger RNA, microRNA, plasmid DNA, PCR amplification product, and artificially synthesized base sequence.

<Target nucleic acid sample application step>
The target nucleic acid sample applying step is a step of applying a nucleic acid sample containing a target nucleic acid to a target nucleic acid detection probe that has a predetermined nucleic acid sequence and an artificial nucleic acid sequence, and a labeled molecule, and is arranged on a substrate. It is preferably carried out by the target nucleic acid sample applying means.

  There is no restriction | limiting in particular as a means to provide a nucleic acid sample to the target nucleic acid detection probe arrange | positioned on the base | substrate, According to the objective, it can select suitably, For example, a micropipette, an inkjet apparatus etc. are mentioned.

-Nucleic acid sample-
The nucleic acid sample is not particularly limited as long as it contains the target nucleic acid, and can be appropriately selected according to the purpose.
As a nucleic acid sample, in addition to the target nucleic acid, for example, water, a buffer, a salt, a surfactant, a nucleic acid other than the target nucleic acid, and the like may be contained.

-Probe for detecting target nucleic acid-
The target nucleic acid detection probe has a predetermined nucleic acid sequence, an artificial nucleic acid sequence, and a label molecule.

-Predetermined nucleic acid sequence and artificial nucleic acid sequence-
The predetermined nucleic acid sequence and the artificial nucleic acid sequence include a nucleic acid having a base sequence complementary to the base sequence of the target nucleic acid and a chimeric nucleic acid of the artificial nucleic acid.

  The predetermined nucleic acid sequence and the artificial nucleic acid sequence are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include a sequence having a single-stranded DNA sequence and an artificial nucleic acid sequence.

--- Artificial nucleic acid sequence ---
The artificial nucleic acid sequence is a base sequence composed of an artificial nucleic acid.
An artificial nucleic acid is a non-natural nucleic acid that does not exist in nature, and is an artificially synthesized nucleic acid analog.
Examples of the nucleic acid analog include PNA (Peptide Nucleic Acid) and BNA (Bridged Nucleic Acid). Examples of the BNA include LNA (Locked Nucleic Acid). These may be used individually by 1 type and may use 2 or more types together.

  Examples of the method for synthesizing an artificial nucleic acid include a solid-phase synthesis method which is a known chemical synthesis method for nucleic acids (for example, see “Chemistry and Education” Vol. 57, No. 7, pages 346 to 347: 2009, etc.). It is done.

  It is known that an artificial nucleic acid has a high binding affinity for DNA or RNA serving as a target nucleic acid. Artificial nucleic acids have nuclease (nucleolytic enzyme) resistance.

--Labeled molecule--
There is no restriction | limiting in particular as a labeled molecule, According to the objective, it can select suitably, For example, a fluorescent molecule, a charged molecule, a radioactive molecule etc. are mentioned.

  The fluorescent molecule is not particularly limited as long as it is a molecule that can emit fluorescence, and can be appropriately selected according to the purpose. Examples thereof include fluorescent dyes, fluorescent proteins, and fluorescently labeled nucleic acid molecules.

The fluorescent dye is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include fluoresceins, azos, rhodamines, coumarins, pyrenes, and cyanines. Of these, fluoresceins, azos, and rhodamines are preferable.
A commercial item can be used as a fluorescent dye. Commercially available products include fluorescein (FLO), FITC, FAM, HEX, TET, NED, TAMRA, ROX, VIC (registered trademark), Texas Red, ROX, PET, ABY (registered trademark), JUN (registered trademark), MUSTANG PURPLE (registered trademark), and the like. These may be used alone or in combination of two or more.

  Examples of the fluorescent protein include GFP.

  There is no restriction | limiting in particular as a charged molecule, According to the objective, it can select suitably, For example, a charged nucleic acid etc. are mentioned.

The radioactive molecule is not particularly limited, and is not particularly limited as long as it contains a radioisotope in the molecule, and can be appropriately selected according to the purpose. For example, a target sequence incorporating ³² P Examples thereof include a nucleic acid fragment having a non-complementary sequence and a peptide containing ³⁵ S.

  There is no restriction | limiting in particular as another member, According to the objective, it can select suitably, For example, a binding molecule etc. are mentioned.

The binding molecule can be imparted to the substrate and the target nucleic acid detection probe, and is not particularly limited as long as the target nucleic acid detection probe can be bound to the substrate, and can be appropriately selected according to the purpose. Examples thereof include avidin and biotin.
In addition to the above, the means for binding the target nucleic acid detection probe to the substrate includes, for example, a binding method in which the target nucleic acid detection probe and the substrate are bonded by a disulfide bond of a thiol group.

<Nucleic acid degradation process>
The nucleic acid degradation step is a process in which a predetermined nucleic acid sequence and an artificial nucleic acid sequence and a target nucleic acid are completely complementary to each other, which is formed when the predetermined nucleic acid sequence and the artificial nucleic acid sequence can form a double strand that is completely complementary to the target nucleic acid. In this step, a nucleolytic enzyme that specifically recognizes and degrades a completely complementary duplex is added to the strand to recognize and degrade the completely complementary duplex.

  There is no restriction | limiting in particular as a means to provide a nucleolytic enzyme, According to the objective, it can select suitably, For example, a micropipetter, an inkjet apparatus, etc. are mentioned.

There is no restriction | limiting in particular as a nucleolytic enzyme, According to the objective, it can select suitably, For example, a double stranded nucleic acid specific decomposing enzyme etc. are mentioned.
The double-stranded nucleic acid-specific degrading enzyme is preferably a duplex-specific nuclease (trade name: Duplex-specific nuclease, lyophilized, Evrogen), which is a double-stranded DNA-specific degrading enzyme derived from the liver of Kamchatka crab. . When the double-stranded nucleic acid-specific degrading enzyme is Duplex-specific nuclease, a completely complementary duplex is distinguished from a completely complementary duplex and a duplex having a single base mismatch (incomplete complement). Can only be disassembled. It is also known that double-stranded nucleic acids are specifically recognized and do not act on single-stranded DNA and RNA, and the double-stranded DNA strand is degraded. For example, in the case of a DNA-DNA double-stranded nucleic acid, the double strand is degraded, and in the case of a hybrid nucleic acid such as DNA-RNA, only a single strand of the DNA strand is degraded. For this reason, even if nonspecific hybridization occurs between the target nucleic acid and the target nucleic acid detection probe, it does not act on the double strand that has caused nonspecific hybridization, and is completely complementary. Only double strands can be detected.

<Detection process>
The detection step is a step of detecting either a labeled molecule that has been decomposed by a nucleolytic enzyme and is free to move from the substrate, or a labeled molecule in a target nucleic acid detection probe arranged on the substrate. It can implement suitably by a means.

There is no particular limitation on the method for detecting a labeled molecule that has been degraded by a nucleolytic enzyme and can be freely moved from the substrate, and can be appropriately selected according to the purpose. For example, it can be freely moved from the substrate. Examples include a method of recovering a labeled molecule and detecting the recovered labeled molecule.
In addition, the method for detecting a labeled molecule in a target nucleic acid detection probe that has been degraded by a nucleolytic enzyme and placed on a substrate is not particularly limited and can be appropriately selected depending on the purpose. Examples include a method of washing the substrate after the step and detecting a labeled molecule remaining on the substrate.

  There is no restriction | limiting in particular as a detection means, According to the objective, it can select suitably, For example, a spectrofluorimeter, an electric potential measuring device, a radiation measuring device etc. are mentioned.

(Target nucleic acid detection probe)
The target nucleic acid detection probe of the present invention is a target nucleic acid detection probe used in the target nucleic acid detection method of the present invention, and has a predetermined nucleic acid sequence, an artificial nucleic acid sequence, and a labeled molecule, and further if necessary. And other members.

-Predetermined nucleic acid sequence and artificial nucleic acid sequence-
As the predetermined nucleic acid sequence and the artificial nucleic acid sequence, those similar to those described in the target nucleic acid detection method of the present invention can be used, and the description thereof is omitted.

-Labeled molecule-
As the labeling molecule, the same as that described in the target nucleic acid detection method of the present invention can be used, and the description thereof is omitted.

-Other components-
As other members, the same members as those described in the target nucleic acid detection method of the present invention can be used, and the description thereof is omitted.

(Target nucleic acid detection device)
The target nucleic acid detection device of the present invention has the target nucleic acid detection probe of the present invention on a substrate, and further includes other members as necessary.

  There is no restriction | limiting in particular as a base | substrate, According to the objective, it can select suitably, For example, glass, a silicon | silicone, an acryl, quartz, a polycarbonate, a polystyrene etc. are mentioned.

The substrate preferably has a molecule on the surface to which the binding molecule specifically binds.
The binding molecule can be imparted to the substrate and the target nucleic acid detection probe, and is not particularly limited as long as the target nucleic acid detection probe can be bound to the substrate, and can be appropriately selected according to the purpose. Examples thereof include avidin and biotin.
In addition to the above, the means for binding the target nucleic acid detection probe to the substrate includes, for example, a binding method in which the target nucleic acid detection probe and the substrate are bonded by a disulfide bond of a thiol group.

  Other members are not particularly limited and may be appropriately selected depending on the purpose. For example, information on detectable label molecules, a nucleic acid sequence complementary to a target nucleic acid included in a target nucleic acid detection device, and an artificial Examples thereof include barcodes having information on nucleic acid sequences, QR codes (registered trademark), memories, IC chips, radio frequency identifiers (hereinafter also referred to as “RFID”), color coding, printing, and the like.

  Hereinafter, embodiments of the target nucleic acid detection method of the present invention using a target nucleic acid detection device having the target nucleic acid detection probe of the present invention will be described in detail with reference to the drawings. In the following description, the case where RNA is used as the target nucleic acid will be described.

<Embodiment>
1 to 3 are diagrams illustrating an example of a target nucleic acid detection probe.
As shown in FIG. 1, the target nucleic acid detection probe 10 is a single-stranded nucleic acid having a label molecule 11, a predetermined nucleic acid sequence 12 and an artificial nucleic acid sequence 13. As shown in FIG. 2, the target nucleic acid detection probe 10 may have a binding molecule 14 for binding to the substrate 15 at the end opposite to the labeled molecule 11. When the binding molecule 14 is present, it is preferable that a molecule that specifically binds to the binding molecule 14 is provided on the surface of the substrate 15.
As shown in FIG. 3, the target nucleic acid detection probe 10 disposed on the substrate 15 is fixed to the substrate 15 via a binding molecule 14. By arranging in advance a molecule that specifically binds to the binding molecule 14 on the substrate 15, the position where the target nucleic acid detection probe 10 is arranged can be controlled.

4 to 8 are diagrams showing an example of the nucleic acid detection method of the present invention.
FIG. 4 is a diagram illustrating an example of a relationship between a target nucleic acid detection probe to which a nucleic acid sample is applied and a target nucleic acid, and FIG. 5 illustrates a target nucleic acid detection probe to which a nucleolytic enzyme is applied, a target nucleic acid, and a nucleic acid. FIG. 6 is a diagram showing an example of a relationship with a degrading enzyme, FIG. 6 is a diagram showing an example of a relationship between a target nucleic acid detection probe treated with a nucleolytic enzyme and a target nucleic acid, and FIG. 7 is a diagram showing a target nucleic acid. FIG. 8 is a diagram illustrating an example of a detection device, and FIG. 8 is a diagram illustrating an example of a nucleic acid detection device processed by the nucleic acid detection method of the present invention.

As shown in FIG. 4, when a nucleic acid sample containing the target nucleic acid 16 is applied to the target nucleic acid detection device, the target nucleic acid detection probe having the target nucleic acid 16, the predetermined nucleic acid sequence 12, and the artificial nucleic acid sequence 13 in the nucleic acid sample. Hybridization occurs when 10 can form a double strand. Thus, the target nucleic acid 16 hybridizes to both the nucleic acid sequence 12 and the artificial nucleic acid sequence 13.
Next, when a nucleic acid degrading enzyme 17 that specifically recognizes a completely complementary double-stranded nucleic acid and degrades the DNA strand in the double-stranded DNA is applied to the target nucleic acid detection device, as shown in FIG. The nucleic acid sequence 12 forming a double strand that is completely complementary to the nucleic acid 12 is degraded. At this time, since the target nucleic acid 16 is RNA, it remains without being degraded by the nuclease 17. In addition, the artificial nucleic acid sequence 13 that forms a double strand that is completely complementary to the target nucleic acid 16 is not degraded by the nucleolytic enzyme 17. Therefore, as shown in FIG. 6, only the nucleic acid sequence 12 that forms a double strand that is completely complementary to the target nucleic acid 16 is degraded, and the labeled molecule 11 is released from the substrate 15. At this time, the target nucleic acid 16 is held on the substrate 15 by the high affinity of the artificial nucleic acid sequence 13 to the target nucleic acid 16.

Next, either the labeled molecule 11 that can move freely from the substrate 15 after the nucleic acid degradation step or the labeled molecule 11 in the target nucleic acid detection probe 10 disposed on the substrate 15 is detected. It is possible to detect using this method. Here, the case where the method of detecting the target molecule 11 in the target nucleic acid detection probe 10 arranged on the substrate 15 is used is shown.
As shown in FIG. 7, after the nucleic acid decomposition step, the target nucleic acid detection probe 10 remains on the substrate 15 even after the labeled molecule 11 is released. Although it is not known whether a double strand is formed, the target nucleic acid 16 is specific as shown in FIG. 8 by washing the surface of the substrate 15 and washing away the labeled molecules 11 released from the target nucleic acid detection probe 10. Since the spot 17 of the target nucleic acid detection probe 10 that has been hybridized automatically disappears, the amount of the target nucleic acid in the nucleic acid sample can be quantified by counting the number of spots 17.

  In the nucleic acid detection method of the present invention, since the target nucleic acid detection probe having an artificial nucleic acid is used, the target nucleic acid once hybridized continues to be retained after the nucleic acid degradation step. Thus, it can be prevented that the target nucleic acid detection probe is hybridized again and detected again. Therefore, highly accurate quantitative analysis can be performed.

  Furthermore, since the nucleic acid detection method of the present invention uses a double-stranded nucleolytic enzyme that specifically recognizes and degrades a completely complementary double-stranded nucleic acid, it is nonspecific by a target nucleic acid that is not complementary to the target nucleic acid detection probe. When the target hybridization occurs, the target nucleic acid detection probe is not decomposed, so that the target nucleic acid can be specifically detected, and the target nucleic acid can be detected with high accuracy.

As an aspect of this invention, it is as follows, for example.
<1> A nucleic acid sample containing a target nucleic acid,
A target nucleic acid sample providing step for applying to a target nucleic acid detection probe disposed on a substrate, which has a predetermined nucleic acid sequence and an artificial nucleic acid sequence, and a labeled molecule;
Two complete complementary pairs of the predetermined nucleic acid sequence and the artificial nucleic acid sequence and the target nucleic acid, which are formed when the predetermined nucleic acid sequence and the artificial nucleic acid sequence can form a double strand that is completely complementary to the target nucleic acid. Against the chain
Recognize the fully complementary duplex by specifically recognizing the fully complementary duplex, providing a nucleolytic enzyme that degrades the DNA strand in the duplex to recognize the fully complementary duplex, A nucleic acid decomposing step for decomposing,
A detection step of detecting any of the labeled molecule that has been decomposed by the nucleolytic enzyme and is free to move from the substrate, and the labeled molecule in the target nucleic acid detection probe disposed on the substrate; ,
A method for detecting a target nucleic acid, comprising:
<2> The target nucleic acid detection method according to <1>, wherein the nucleolytic enzyme is Duplex-specific nuclease.
<3> A target nucleic acid detection probe used in the nucleic acid detection method according to any one of <1> to <2>,
A target nucleic acid detection probe comprising a predetermined nucleic acid sequence, an artificial nucleic acid sequence, and a label molecule.
<4> The probe for detecting a target nucleic acid according to <3>, which has a binding molecule.
<5> The target nucleic acid detection probe according to any one of <3> to <4>, wherein the labeling molecule is a fluorescent molecule.
<6> The target nucleic acid detection probe according to any one of <3> to <4>, wherein the labeling molecule is a charged molecule.
<7> The above-described <3> to <6>, wherein the artificial nucleic acid sequence is a sequence of at least one of PNA (Peptide Nucleic Acid), BNA (Bridged Nucleic Acid), and LNA (Locked Nucleic Acid). The target nucleic acid detection probe described.
<8> The target nucleic acid detection probe according to any one of <3> to <7>, wherein the target nucleic acid is at least one of DNA and RNA.
<9> A target nucleic acid detection device comprising the target nucleic acid detection probe according to any one of <3> to <8> on a substrate.

  The target nucleic acid detection method according to any one of <1> to <2>, the target nucleic acid detection probe according to any one of <3> to <8>, and the target nucleic acid detection according to <9> According to the device, the above-mentioned problems, that is, false detection due to nonspecific hybridization can be solved, and the object of the present invention can be achieved.

10 Probe for detecting target nucleic acid 11 Labeled molecule 12 Nucleic acid sequence 13 Artificial nucleic acid sequence 14 Binding molecule 15 Base 16 Target nucleic acid 17 Nucleolytic enzyme

Special table 2017-535269 gazette

Claims (9)

A nucleic acid sample containing the target nucleic acid,
A target nucleic acid sample providing step for applying to a target nucleic acid detection probe disposed on a substrate, which has a predetermined nucleic acid sequence and an artificial nucleic acid sequence, and a labeled molecule;
Two complete complementary pairs of the predetermined nucleic acid sequence and the artificial nucleic acid sequence and the target nucleic acid, which are formed when the predetermined nucleic acid sequence and the artificial nucleic acid sequence can form a double strand that is completely complementary to the target nucleic acid. Against the chain
Recognize the fully complementary duplex by specifically recognizing the fully complementary duplex, providing a nucleolytic enzyme that degrades the DNA strand in the duplex to recognize the fully complementary duplex, A nucleic acid decomposing step for decomposing,
A detection step of detecting any of the labeled molecule that has been decomposed by the nucleolytic enzyme and is free to move from the substrate, and the labeled molecule in the target nucleic acid detection probe disposed on the substrate; ,
A method for detecting a target nucleic acid, comprising:   The method for detecting a target nucleic acid according to claim 1, wherein the nucleolytic enzyme is a duplex-specific nuclease. A target nucleic acid detection probe used in the nucleic acid detection method according to claim 1,
A probe for detecting a target nucleic acid, comprising a predetermined nucleic acid sequence and an artificial nucleic acid sequence, and a labeled molecule.   The probe for detecting a target nucleic acid according to claim 3, which has a binding molecule.   The probe for detecting a target nucleic acid according to any one of claims 3 to 4, wherein the labeled molecule is a fluorescent molecule.   The probe for detecting a target nucleic acid according to any one of claims 3 to 4, wherein the label molecule is a charged molecule.   The target nucleic acid detection according to any one of claims 3 to 6, wherein the artificial nucleic acid sequence is a sequence of at least one of PNA (Peptide Nucleic Acid), BNA (Bridged Nucleic Acid), and LNA (Locked Nucleic Acid). probe.   The probe for detecting a target nucleic acid according to any one of claims 3 to 7, wherein the target nucleic acid is at least one of DNA and RNA. 9. A target nucleic acid detection device comprising the target nucleic acid detection probe according to claim 3 on a substrate.