JP2017158497A - FISH staining method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide FISH dyeing method of nucleic acid which can improve the efficiency of a label and increase the sensitivity for detection or determination.SOLUTION: The present invention provides a method comprising: hybridizing to a target nucleic acid a nucleic acid probe having a nucleic acid sequence complementary to the nucleic acid sequence; amplifying a site to which a fluorescent nanoparticle can bind, wherein step (1) a treatment of modifying the nucleic acid probe with biotin as a site capable of binding to the fluorescent nanoparticle after previously directly or indirectly binding biotin as an origin of amplification to the nucleic acid probe, or step (2) a treatment of previously directly or indirectly binding peroxidase to the nucleic acid probe, and depositing a site capable of binding to the fluorescent nanoparticle, around the nucleic acid probe by contacting tyramide modified with a molecule capable of binding to the fluorescent nanoparticle, to the peroxidase in the presence of peroxide is selected; and binding the fluorescent nanoparticle to the binding site; and binding the fluorescent nanoparticle to the binding site.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a fluorescence in situ hybridization (FISH) staining method for quantifying a target nucleic acid in a tissue section used for pathological diagnosis or the like. More specifically, the present invention relates to a FISH staining method for labeling a target nucleic acid using fluorescent nanoparticles.

  In pathological diagnosis, slides are prepared by slicing tissue sections collected from patients, and the morphology of cells or tissues is observed on the basis of stained images when stained by a predetermined method. It is a method of diagnosing various events such as whether a patient suffers from a specific disease or whether a specific therapeutic drug is successful by quantifying and evaluating the level.

  For example, a membrane protein produced from a HER2 gene (HER2 / neu, c-erbB-2) and / or HER2 gene, which is a kind of oncogene, using a tissue section prepared by collecting cancer tissue, By quantifying and evaluating the HER2 protein, which is presumed to function as a receptor for cancer cell growth factor, the prognosis of breast cancer patients can be diagnosed, and the molecular targeted therapeutic drug “Trastuzumab” (trade name “Herceptin” ( A pathological diagnosis for predicting a therapeutic effect by a registered trademark and an anti-HER2 monoclonal antibody is widely performed. A typical DNA level inspection method is a fluorescence in situ hybridization (FISH) method.

  The FISH method is a formalin-fixed paraffin-embedded tissue section, cytodiagnosis slide, etc., which uses a fluorescently labeled nucleic acid probe to hybridize DNA, RNA region expression amplification degree, and DNA region translocation. This is a method of detecting a defect (replacement of the position of a specific gene on a chromosome).

  As a probe reagent for FISH, Patent Document 1 (WO2015 / 141856) discloses a probe reagent in which phosphor-integrated nanoparticles obtained by accumulating phosphors and nucleic acid molecules having a predetermined base sequence are combined. It is described that a fluorescent signal can be stably obtained by using such a probe reagent while suppressing non-specific adsorption. Further, in this document, (i) the phosphor-aggregated nanoparticles and the nucleic acid molecule are linked to the nucleic acid molecule by a covalent bond even if they are covalently bonded using a linker molecule as necessary. It may be indirectly bound by specific binding of one biomolecule (for example, biotin) and a second biomolecule (for example, streptavidin) covalently linked to the phosphor-integrated nanoparticles; (ii) A site in a nucleic acid molecule for directly or indirectly binding phosphor-integrated nanoparticles can be introduced into one or more sites by 5′-end or 3′-end labeling method, nick translation method, etc. (Iii) When the number of bases of the nucleic acid sequence is 5000 or less, it is preferable that the nucleic acid molecule and the phosphor-integrated nanoparticles are bound at a molar ratio of 1: 1 to 1:40. Is described. For example, as an embodiment using a probe reagent that indirectly binds a nucleic acid molecule and phosphor-integrated nanoparticles in (i) above, a nucleic acid in which a plurality of nucleic acids having a predetermined sequence and a plurality of first biomolecules are linked After hybridization with the molecule, the phosphor-integrated nanoparticles linked with the second biomolecule are added to the hybridization reaction system, and the second biomolecule is specifically added to the first biomolecule. FISH has also been proposed in which the nucleic acid having the predetermined sequence is fluorescently labeled by binding.

  On the other hand, as a means to enhance the signal emitted from the label bound to a specific analyte (analyte) and detect the analyte with high sensitivity, it is located in the vicinity of the enzyme activated analyte. A sensitization method using a conjugate of a substrate that can be deposited and a labeling substance (label) or a site for linking it is known.

  For example, US Pat. No. 5,196,306 discloses a substrate (eg, tyramine) that is activated by an enzyme (eg, horseradish peroxidase) and a detectable that can directly or indirectly generate a detectable or quantifiable signal. Disclosed is a method for detecting or quantifying an analyte by an analyte-dependent enzyme activation system using a conjugate with a non-specific label (such as an enzymatic, fluorescent or chemiluminescent label). As an example of the embodiment, the following is described in Example 8. First, an analyte (antibody) is bound to the primary antibody immobilized on a support (polystyrene strip) for enzyme immunoassay by an antigen-antibody reaction, and then horseradish peroxidase is linked to the analyte. The prepared secondary antibody is bound by antigen-antibody reaction. Next, a conjugate of tyramine and biotin is added, and tyramine is activated by horseradish peroxidase to deposit a plurality of conjugates in the vicinity of the primary antibody on the substrate. Furthermore, a conjugate of streptavidin and galactosidase is added and bound to biotin linked to the deposited tyramine. Finally, 4-methylumbelliferyl-β-D-galactoside (MUG) is added, reacted with galactosidase, and the fluorescence emitted thereby is measured to quantify the analyte.

  Patent Document 2 exemplifies various labels as the label, but does not specifically disclose the use of phosphor-integrated nanoparticles or other phosphor particles. In the embodiment described above, the total amount of fluorescence generated from the fluorescent dyes generated by the microtiter plate reader is measured, but information such as the position and number of bright spots of phosphor-integrated nanoparticles, etc. is acquired. is not. In addition, the method described in Patent Document 2 is basically a means for amplifying a label for detection and quantification using a protein contained in a blood sample as an analyte, and a nucleic acid (gene ) Is not described for amplifying labels for detection and quantification.

  On the other hand, in Patent Document 3 (Special Table 2013-531801), in an in situ hybridization assay for detecting and quantifying a specific gene, (i) a target nucleic acid sequence is labeled with a hapten (for example, digoxigenin). (Ii) a step of reacting a conjugate of an anti-hapten antibody corresponding to the hapten and a peroxidase with the hapten to immobilize the peroxidase, and (iii) in the presence of a peroxide. A plurality of conjugates of aryl moieties (for example tyramine or tyramine derivatives) that can be activated by peroxidase and haptens are reacted with the peroxidase and deposited in the vicinity of the immobilized peroxidase (phenol-containing compounds such as tyrosine and dimers). Forming the body), The conjugate of iv) fluorescence-labeled with an anti-hapten antibody, by reacting with the deposited haptens of the plurality of conjugates, such as to be able to amplify the fluorescent signal of the nucleic acid sequence is disclosed. Moreover, quantum dots are exemplified as the fluorescent label.

  The method described in Patent Document 3 is basically a method in which a fluorescent label is modified with an anti-hapten antibody and indirectly bound to a target nucleic acid sequence through the binding of the fluorescent label to the avidin. There is no description about the indirect binding via the binding of biotin to biotin. In addition, the use of phosphor-integrated nanoparticles as a fluorescent label is not specifically disclosed.

  Further, in Non-Patent Document 1, in order to detect the HER2 gene on the chromosome, various FISH probes modified with fluorescent dye molecules and having a base length of about 200 are prepared, and a plurality of them are used in combination. While changing the total base length, the detectability of the HER2 gene is verified. As a result, it is described that if the total length of the FISH probe is at least about 3000 (200 × 15) bases, the bright spot of the fluorescent dye molecule can be visually recognized with a microscope. In other words, the description of Non-Patent Document 1 shows that when the total base length of the probe is 3000 or less, visual recognition with a microscope becomes difficult.

WO2015 / 141856 US5196306 Special table 2013-531801

NATURE METHODS 10, 2, pp.122-124 (2013)

  In a typical embodiment of conventional FISH as disclosed in Patent Document 1, as described above, a probe is bound to a nucleic acid having a predetermined sequence, and one or a plurality of first ligations linked to the probe. The nucleic acid having the predetermined sequence is fluorescently labeled with the phosphor-integrated nanoparticles through binding of the biomolecule and the corresponding second biomolecule. However, since the phosphor-integrated nanoparticles have a relatively large particle size, there is a three-dimensional relationship between the phosphor-integrated nanoparticles that are bound to the probe and between proteins such as histones that surround the nucleic acid. It is likely to cause disability. As a result, it becomes difficult for the phosphor-aggregated nanoparticles to approach the probe bound to the target nucleic acid, and conversely, if the phosphor-aggregated nanoparticle is bound to the probe in advance, the probe will become a target nucleic acid. It becomes difficult to approach. From such a viewpoint, the binding reactivity when modifying a target nucleic acid using a large number of phosphor-integrated nanoparticles in conventional FISH, in other words, the number of phosphor-integrated nanoparticles to be bound and the binding force thereof. There was room for improvement in the tagging rate based on.

  On the other hand, if fluorescent nanoparticles with a smaller particle diameter, such as quantum dots, are used instead of the phosphor-integrated nanoparticles, the problem due to steric hindrance as described above may not occur. Since the bright spot per particle is smaller than the particle, it is desirable to stably bind a larger number of particles to increase the sensitivity for detection or quantification.

  In addition, the shorter the total base length of the probe, the easier it is to prepare and the easier it is to handle, but the total amount of fluorophore and other fluorophores that can be directly bound to the probe decreases, so detection of the target nucleic acid Or it tends to be difficult to quantify.

  The present invention improves the efficiency of labeling for detection or quantification when fluorescent nanoparticles such as phosphor-integrated nanoparticles and inorganic phosphor nanoparticles such as quantum dots are used as a substance for labeling a target nucleic acid. It is an object of the present invention to provide a nucleic acid FISH staining method that can detect or quantify a target nucleic acid even if the total base length of the nucleic acid probe is 3000 or less.

  The present inventors have incorporated the step of amplifying a site to which a fluorescent nanoparticle can be bound by binding starting from a molecule modified with a nucleic acid probe or by binding to a molecule around the nucleic acid probe as described above. The present inventors have found that the FISH staining method for nucleic acids that can solve various problems can be carried out. That is, the present invention includes the following inventions.

[1]
A step (hybridization step) of hybridizing a target nucleic acid with a nucleic acid probe having a base sequence complementary to the base sequence and having a total base length of 3000 or less;
A step of amplifying a site to which fluorescent nanoparticles can be bound, selected from the following step (1) or (2) (binding site amplification step);
Step (1): Biotin as a starting point of amplification is directly or indirectly bound to the nucleic acid probe in advance, and bound to the biotin and avidin contained in the avidin-biotin complex (ABC). Treatment for modifying a nucleic acid probe with biotin contained in ABC as a site capable of binding to fluorescent nanoparticles (ABC treatment step)
Step (2): Peroxidase is directly or indirectly bound to the nucleic acid probe in advance, and in the presence of peroxide, the peroxidase is contacted with tyramide modified with a molecule capable of binding to fluorescent nanoparticles. Treatment for depositing sites capable of binding to fluorescent nanoparticles around the nucleic acid probe (tyramide treatment step)
A method for FISH (fluorescence in situ hybridization) staining of nucleic acids, comprising a step of binding fluorescent nanoparticles to the binding site (fluorescent nanoparticle binding step).

[2]
In the binding site amplification step, in order to bind biotin (step 1) or peroxidase (step 2) as a starting point of amplification indirectly to the nucleic acid probe,
The nucleic acid probe is modified with a hapten,
After binding an anti-hapten polyclonal antibody or an anti-hapten monoclonal antibody as a primary antibody to the nucleic acid probe modified with the hapten,
The method for FISH staining of nucleic acid according to [1], wherein a polyclonal antibody against the primary antibody is bound to the primary antibody as a secondary antibody.

[3]
The method for FISH staining of a nucleic acid according to [1] or [2], wherein the hapten is selected from the group consisting of dinitrophenol, digoxigenin and FITC (fluorescein isothiocyanate).

[4]
The nucleic acid FISH staining according to any one of [1] to [3], wherein after the fluorescent nanoparticle binding step, a step (immobilization step) for preventing the fluorescent nanoparticle from being released from the bonded site is performed. Method.

[5]
The nucleic acid FISH staining method according to any one of [1] to [4], wherein the fluorescent nanoparticles are fluorescent dye-integrated nanoparticles, and an average particle diameter thereof is 20 nm to 300 nm.

[6]
The nucleic acid FISH staining method according to any one of [1] to [4], wherein the fluorescent nanoparticles are inorganic phosphor-integrated nanoparticles, and an average particle diameter thereof is 20 nm to 300 nm.

[7]
[1] to [4], wherein the fluorescent nanoparticles are inorganic phosphor nanoparticles selected from the group consisting of quantum dots and carbon dots, and have an average particle diameter of 1 nm to 300 nm. A method for FISH staining of nucleic acids.

[8]
In the FISH staining method according to any one of [1] to [7], a fluorescent nanoparticle is bound to a plurality of binding sites amplified by the binding site amplification step. Pathological specimen with FISH staining.

  The FISH staining method according to the present invention labels a target nucleic acid by remarkably amplifying the number of sites to which a nucleic acid probe or fluorescent nanoparticles existing in the vicinity thereof can bind using a predetermined treatment. The number of fluorescent nanoparticles can be increased, and furthermore, the fluorescent nanoparticles can be prevented from being detached by washing or the like. Thus, even when using a nucleic acid probe having a total base length of 3000 or less and relatively easy to design, manufacture and handle by dramatically improving the labeling ability with fluorescent nanoparticles, the target nucleic acid The brightness (sensitivity) sufficient for detection and quantification of the sample can be achieved, and the reproducibility of the staining result between samples can also be improved.

FIG. 1 is a schematic diagram showing an example of a reaction mode in the first embodiment of the present invention in which Step 1: ABC treatment step is performed as a binding site amplification step. FIG. 2 is a schematic diagram showing an example of a reaction mode in the second embodiment of the present invention in which Step 2: Tylamide treatment step is performed as the binding site amplification step. FIG. 3 is a schematic view showing an example of a reaction mode preferable as the second embodiment of the present invention (when a polyclonal secondary antibody is used to indirectly bind peroxidase to a nucleic acid probe). FIG. 4 shows [A] a fluorescent image taken in Example 1 and [B] a fluorescent image taken in Comparative Example 1. FIG. 5 shows [A] a fluorescent image taken in Example 2 and [B] a fluorescent image taken in Comparative Example 2.

Hereinafter, the FISH staining method for nucleic acids according to the present invention and the pathological specimens stained using the method will be described.
-Method of FISH staining of nucleic acid-
The FISH staining method according to the present invention includes at least a “hybridization step”, a “binding site amplification step”, and a “fluorescent nanoparticle binding step”, and may further include a “fixation step” as necessary. Good.

(Hybridization process)
The hybridization step is a step of hybridizing a target nucleic acid with a nucleic acid probe having a base sequence complementary to the base sequence and having a total base length of 3000 or less. The nucleic acid probe to be hybridized in this step is referred to as a nucleic acid probe (referred to as a “modified nucleic acid probe”) modified with a predetermined molecule (referred to as a “nucleic acid probe modified molecule”) according to the embodiment of the next binding site amplification step. .)

  The nucleic acid probe may be constituted by one nucleic acid molecule or may be constituted by two or more nucleic acid molecules (mixture). That is, the rule that “the total base length is 3000 or less” means that the base length of one nucleic acid molecule is 3000 or less in the former case, and two or more nucleic acid molecules in the latter case It means that the total of each base length is 3000 or less. When two or more nucleic acid molecules are used as the nucleic acid probe, the base sequence of each nucleic acid molecule is complementary to the base sequence of a different (non-overlapping) region of the target nucleic acid. For example, a set of 10 nucleic acid molecules with a length of 200 bases per strand can be used as a nucleic acid probe with a total base length of 2000 (200 × 10).

(Binding site amplification step)
The binding site amplification step performed after the nucleic acid probe contacting step is a step for amplifying a site to which the fluorescent nanoparticles can bind, and is a step selected from the following steps 1 or 2.
Step 1: Biotin as a starting point of amplification is directly or indirectly bound to the nucleic acid probe in advance, and the biotin and avidin contained in an avidin-biotin complex (ABC) are bound. Thus, the nucleic acid probe is modified with a plurality of biotins contained in the ABC as a site capable of binding to the fluorescent nanoparticles (ABC treatment step).
Step 2: Peroxidase is directly or indirectly bound to the nucleic acid probe in advance, and in the presence of peroxide, the peroxidase is contacted with a tyramide modified with a molecule that can bind to fluorescent nanoparticles. A process of depositing a plurality of sites capable of binding to fluorescent nanoparticles around the nucleic acid probe (tyramide treatment process).

(Step 1: ABC processing step)
As shown in the reaction mode of FIG. 1, in step 1 (ABC treatment step), biotin as a starting point of amplification is bound directly or indirectly to a nucleic acid probe in advance, and the biotin and avidin contained in ABC are included. To the nucleic acid probe is modified with a plurality of biotins contained in ABC as a site capable of binding to the fluorescent nanoparticle.

  In the present specification, “avidin” is a term encompassing analogs of avidin such as streptavidin derived from actinomycetes, neutral avidin obtained by removing a sugar chain from avidin, in addition to the original avidin derived from egg white.

  The means for “binding biotin as a starting point of amplification directly or indirectly in advance to the nucleic acid probe” is not particularly limited, and can be selected from various known means. For example, a method in which biotin is indirectly bound using a primary antibody and a biotin-modified secondary antibody, that is, a modified nucleic acid probe first hybridized to a target nucleic acid is specific for the nucleic acid probe-modified molecule. The reaction mode is preferably such that an antibody (primary antibody) that binds manually is bound, and then an antibody (biotin-modified secondary antibody) corresponding to the primary antibody modified with biotin is bound to the primary antibody. Instead of using the biotin-modified secondary antibody, only the biotin-modified primary antibody may be used and bound to the modified nucleic acid probe. It is also possible to select biotin as the nucleic acid probe modifying molecule and to bind it directly to the nucleic acid probe (by a covalent bond using a linker molecule if necessary). If such a biotin-modified nucleic acid probe is prepared prior to the hybridization step and then used to carry out the hybridization step, the nucleic acid probe directly starts the amplification at the beginning of the binding site amplification step. As a result, biotin was bound. At least one biotin as a starting point for amplification may be used for each nucleic acid probe, but two or more biotins are preferable because the amplification efficiency of the binding site of the fluorescent nanoparticles can be increased.

  The means for “binding with biotin (as a starting point of amplification) and avidin contained in the avidin-biotin complex (ABC)” is not particularly limited, and can be selected from various known means. it can. In general, a substance carrying multiple biotins (for example, peroxidase) is prepared in advance, and then avidin is contacted and bound to biotin as the starting point of amplification, and then the avidin is prepared. The biotin-carrying substance that has been prepared is brought into contact so that ABC is formed together with excess avidin that did not bind to biotin as the starting point of amplification. Alternatively, the biotin-carrying substance and avidin are first brought into contact with ABC to form ABC, and then the ABC is brought into contact with biotin as an amplification base point, which is unreacted in ABC (not yet bound to biotin). A) Avidin may be reacted with the biotin. In step (1), a plurality of biotins contained in ABC, which are modified nucleic acid probes as described above, become sites that can bind to fluorescent nanoparticles. The biotin-carrying substance is not limited to peroxidase, and a substance (molecule) having an appropriate size capable of carrying a plurality of biotins can be used.

(Process 2: Tylamide processing process)
As shown in the reaction mode of FIG. 2, in step 2 (tyramide treatment step), peroxidase is bound directly or indirectly to a nucleic acid probe in advance, and in the presence of peroxide, the peroxidase is bound to fluorescent nanoparticle. This is a process including a process of depositing a plurality of sites capable of binding to the fluorescent nanoparticles around the nucleic acid probe by bringing the tyramide modified with a molecule capable of binding to the particle into contact.

  The means for “peroxidase binding directly or indirectly to the nucleic acid probe in advance” is not particularly limited, and can be selected from various known means. For example, an antibody (primary antibody) that specifically binds to a modified nucleic acid probe hybridized to a target nucleic acid is first bound to the nucleic acid probe-modified molecule, and then antibody modification is performed on the primary antibody. An antibody (modified secondary antibody) corresponding to the primary antibody modified with a predetermined molecule (antibody-modified molecule such as biotin) is bound, and the modified secondary antibody is specifically bound to the antibody-modified molecule. A reaction mode in which peroxidase is indirectly bound to a nucleic acid probe by binding a peroxidase (modified peroxidase) modified with a predetermined molecule for peroxidase modification (peroxidase-modified molecule such as streptavidin) is preferable. Using only the primary antibody modified with biotin or the like (modified primary antibody) without using the modified secondary antibody, binding it to the modified nucleic acid probe, and further binding the peroxidase modified with streptavidin or the like. Also good. Further, by selecting a peroxidase-modified molecule (for example, streptavidin) that specifically binds to a nucleic acid probe-modified molecule (for example, biotin) without using a modified primary antibody and a modified secondary antibody, It can also be combined. At least one peroxidase is sufficient for each nucleic acid probe, but two or more peroxidases are preferable because the amplification efficiency of the binding site of the fluorescent nanoparticles can be increased.

  To “deposit multiple sites capable of binding to fluorescent nanoparticles around nucleic acid probe by contacting peroxidase with Tyramide modified with molecules that can bind to fluorescent nanoparticles in the presence of peroxide” The means is not particularly limited, and can be selected from various known means. In general, first, a tyramide (modified tyramide), a substrate corresponding to peroxidase, modified with a predetermined molecule for tyramide modification (tyramide modified molecule) is prepared, and a peroxide such as hydrogen peroxide is prepared. The modified tyramide may be contacted with peroxidase in the presence of By activating tyramide by the reaction, modified tyramide (tyramide-modified molecule) can be deposited around the modified nucleic acid probe. In step (2), the tyramide-modified molecule deposited around the nucleic acid probe as described above becomes a “site to which fluorescent nanoparticles can bind”.

  Implementation using the avidin-biotin complex (ABC) described above in relation to step (1) as an example of means for “peroxidase is directly or indirectly bound to a nucleic acid probe in advance” in step (2) A form is mentioned. That is, by combining ABC using peroxidase as a biotin-carrying substance with biotin corresponding to “biotin as the starting point of amplification” in step (1), for example, a biotin-modified secondary antibody, a plurality of peroxidases contained in ABC Is indirectly bound to the nucleic acid probe. Since ABC contains a large number of peroxidases, the reaction efficiency when contacting Tyramide modified with a molecule capable of binding to fluorescent nanoparticles in the presence of peroxide is improved, and more fluorescence is obtained. Sites capable of binding to the nanoparticles can be deposited around the nucleic acid probe. After performing the step (2) of such a preferred embodiment, when performing the fluorescent nanoparticle binding step of binding the fluorescent nanoparticle to a site capable of binding to the deposited fluorescent nanoparticle, the above-described ABC treatment is performed. Compared with the fluorescent nanoparticle binding step in which fluorescent nanoparticles are bound to biotin in ABC after step (1), the detection sensitivity can be expected to be amplified by about 100 times. Is particularly preferred when is short.

(Fluorescent nanoparticle binding process)
The fluorescent nanoparticle reaction step performed after the binding site amplification step selected from step (1) or (2) is a step of binding fluorescent nanoparticles to the binding site amplified by the binding site amplification step. By this step, the target nucleic acid can be labeled with fluorescent nanoparticles.

  When the binding site amplification step is the ABC treatment step of step (1), since “the binding site amplified by the binding site amplification step” is “biotin contained in ABC”, the fluorescent nanoparticles used in the fluorescent nanoparticle binding step are A molecule that specifically binds to biotin, that is, a fluorescent nanoparticle modified with avidin. When the binding site amplification step is the tyramide treatment step of step (2), since the “binding site amplified by the binding site amplification step” is a “tyramide modified molecule”, the fluorescent nanoparticle used in the fluorescent nanoparticle binding step is Tyramide. The fluorescent nanoparticle is modified with a molecule that specifically binds to the modifying molecule.

(Immobilization process)
The immobilization step optionally performed after the fluorescent nanoparticle binding step is to crosslink the fluorescent nanoparticles bound at a predetermined binding site with the surrounding proteins, or to crosslink the proteins around the fluorescent nanoparticles. By making the fluorescent nanoparticles that have entered the cell nucleus difficult to come out of the cell nucleus, the fluorescent labeling of the target nucleic acid by the fluorescent nanoparticles is made strong and stable so that it cannot be removed by a subsequent washing process or the like. Process.

  Such a process includes, for example, a reaction between an amino group of the phosphor-integrated nanoparticle and an amino group of a protein around it, or an amino group of a protein around the phosphor-integrated nanoparticle and a compound such as aldehyde. It can carry out by bridge | crosslinking by (methylene bridge | crosslinking). As the fixing agent for causing such a crosslinking reaction, dialdehydes such as paraformaldehyde, glutaraldehyde, glyoxal and the like are preferable.

  In the present invention, depending on the embodiment of the binding site amplification step, the nucleic acid probe modification molecule in the hybridization step, the primary antibody in the binding site amplification step, the secondary antibody, the antibody modification molecule, the peroxidase modification molecule Embodiments such as tyramide modified molecules also vary. Hereinafter, the entire present invention when the ABC processing step (1) is adopted as the binding site amplification step is referred to as “first embodiment”, and the entire present invention when the tyramide treatment step (2) is adopted is referred to as “first Matters related to each embodiment will be further described.

  FIG. 1 shows an example of the reaction mode in the first embodiment of the present invention in which the step 1: ABC treatment step is performed as the binding site amplification step. In the hybridization step, the modified nucleic acid probe (4) comprising the nucleic acid probe (2) and the nucleic acid probe modifying molecule (3) is hybridized to the target nucleic acid (1). In the binding site amplification step, the monoclonal primary antibody (10) binds to the modified nucleic acid probe (4) and serves as the origin of amplification of the monoclonal secondary antibody (20a) and (corresponding to the secondary antibody modified molecule (21)). A modified monoclonal secondary antibody (22a) consisting of biotin (50) binds to the primary antibody (10). Furthermore, the avidin-biotin complex (ABC) (80) consisting of avidin (60) and a plurality of biotins (50) carried on peroxidase (70) binds to the modified monoclonal secondary antibody (22a). In the fluorescent nanoparticle binding step, the modified fluorescent nanoparticle (102) composed of the fluorescent nanoparticle (100) and avidin (60) (corresponding to the fluorescent nanoparticle modifying molecule (101)) is biotin contained in ABC (80). Combine with (50).

FIG. 2 shows an example of a reaction mode in the second embodiment of the present invention in which the step 2: tyramide treatment step is performed as the binding site amplification step. In the hybridization step, the modified nucleic acid probe (4) comprising the nucleic acid probe (2) and the nucleic acid probe modifying molecule (3) is hybridized to the target nucleic acid (1). In the binding site amplification step, the monoclonal primary antibody (10) binds to the modified nucleic acid probe (4), and the modified monoclonal secondary antibody (22a) comprises the monoclonal secondary antibody (20a) and the secondary antibody modified molecule (21). Binds to the monoclonal primary antibody (10), and the modified peroxidase (72) comprising the peroxidase (70) and the peroxidase-modified molecule (71) binds to the modified monoclonal secondary antibody (22a). Further, in the presence of peroxide (H ₂ O ₂ ), the pre-activated modified tyramide (92a) consisting of the pre-activated tyramide (90a) and the tyramide-modified molecule (91) is converted to peroxidase (70 ) Is converted into an activated modified tyramide (92b) composed of activated tyramide (90b) and a tyramide modified molecule (91), and deposited around the nucleic acid probe (2). In the fluorescent nanoparticle binding step, the modified fluorescent nanoparticle (102) composed of the fluorescent nanoparticle (100) and the fluorescent nanoparticle modified molecule (101) is converted into the tyramide modified molecule (91) contained in the activated modified tyramide (92b). Join.

  An example of a preferred reaction mode of the second embodiment of the present invention is shown in FIG. FIG. 3 shows that (i) the nucleic acid probe (2) is composed of a plurality of nucleic acid molecules instead of a single nucleic acid molecule, and a plurality of modified nucleic acid probes (4) are hybridized to the target nucleic acid (1). And (ii) a polyclonal secondary antibody (20b) and a secondary antibody instead of the modified monoclonal secondary antibody (22a) consisting of the monoclonal secondary antibody (20a) and the secondary antibody modified molecule (21) A modified polyclonal secondary antibody (22b) consisting of a modified molecule (21) is used, and a plurality of modified polyclonal secondary antibodies (22b) are bound to the monoclonal primary antibody (10) as shown in FIG. It is different.

(Target nucleic acid)
The nucleic acid to be targeted in the FISH staining method of the present invention, that is, the target of detection and quantification (referred to as “target nucleic acid” in the present specification) is not particularly limited, and is the same as in the conventional FISH staining method. In addition, DNAs or RNAs having various base sequences are included in the target nucleic acid. Typically, a gene having a specific base sequence present on a chromosome is a target nucleic acid, but non-chromosomal nucleic acids such as mRNA, tRNA, rRNA, miRNA, siRNA, non-coding RNA, etc. can also be target nucleic acids. .

  Biomarkers that can serve as target nucleic acids include diagnostic biomarkers, biomarkers that determine disease stages, disease prognostic biomarkers, and monitor biomarkers for the purpose of viewing responses to therapeutic treatment. For example, HER2, TOP2A, HER3, EGFR, P53, MET, etc. are mentioned as genes related to cancer growth and the response rate of molecular target drugs. Furthermore, the following are mentioned as a gene known as a cancer related gene. As tyrosine kinase-related genes, ALK, FLT3, AXL, FLT4 (VEGFR3, DDR1, FMS (CSF1R), DDR2, EGFR (ERBB1), HER4 (ERBB4), EML4-ALK, IGF1R, EPHA1, INSR, EPHA2, IRR (INSRR) ), EPHA3, KIT, EPHA4, LTK, EPHA5, MER (MERTK), EPHA6, MET, EPHA7, MUSK, EPHA8, NPM1-ALK, EPHB1, PDGFRα (PDGFRA), EPHB2, PDGFRβ (PDGFRB), PD-L1, PD1-L1 , LGR5, EPHB3, RET, EPHB4, RON (MST1R), FGFR1, ROS (ROS1), FGFR2, TIE2 (TEK), FGFR3, TRKA (NTR) K1), FGFR4, TRKB (NTRK2), FLT1 (VEGFR1), TRKC (NTRK3), and breast cancer-related genes are ATM, BRCA1, BRCA2, BRCA3, CCND1, E-cadherin, ERBB2, ETV6, FGFR1, HRAS, KRAS, NRAS, NTRK3, p53, PTEN Examples of genes related to carcinoid tumors include BCL2, BRD4, CCND1, CDKN1A, CDKN2A, CTNNB1, HES1, MAP2, MEN1, NF1, NOTCH1, HD, RUT, HD Examples of colorectal cancer-related genes include APC, MSH6, AXIN2, MYH, BMPR1A, p53, DCC, PMS2, KRAS2 (or Ki-r). s), PTEN, MLH1, SMAD4, MSH2, STK11, MSH6 Lung cancer-related genes include ALK, PTEN, CCND1, RASSF1A, CDKN2A, RB1, EGFR, RET, EML4, ROS1, KRAS2, TP53, MYC. Examples of genes related to liver cancer include Axin1, MALAT1, b-catenin, p16 INK4A, c-ERBB-2, p53, CTNNB1, RB1, Cyclin D1, SMAD2, EGFR, SMAD4, IGFR2, TCF1, and KRAS. Examples of kidney cancer-related genes include Alpha, PRCC, ASPSCR1, PSF, CLTC, TFE3, p54nrb / NONO, and TFEB. KAP10, NTRK1, AKAP9, RET, BRAF, TFG, ELE1, TPM3, H4 / D10S170, TPR and the like. Examples of ovarian cancer-related genes include AKT2, MDM2, BCL2, MYC, BRCA1, NCOA4, CDKN2A, p53, ERBB2, PIK3CA, GATA4, RB, HRAS, RET, KRAS, and RNASET2. Examples of prostate cancer-related genes include AR, KLK3, BRCA2, MYC, CDKN1B, NKX3.1, EZH2, p53, GSTP1, and PTEN. Examples of bone tumor-related genes include CDH11, COL12A1, CNBP, OMD, COL1A1, THRAP3, COL4A5, and USP6.

(Nucleic acid probe)
The nucleic acid probe has a base sequence (probe sequence) complementary to part or all of the base sequence of the target nucleic acid, and consists of a nucleic acid molecule that can form a complementary strand with the target nucleic acid. The nucleic acid probe may be composed of a naturally occurring molecule such as DNA or RNA, or PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid, BNA: sometimes called Bridged Nucleic Acid), etc. It may be composed of an artificial nucleic acid molecule or both.

  As the base sequence of the nucleic acid probe (probe sequence), a base sequence of the target nucleic acid, for example, a sequence complementary to all or part of the base sequence on the chromosome related to the biomarker gene such as HER2 is preferably used. . Even when the target nucleic acid is a non-chromosomal nucleic acid, for example, mRNA, tRNA, rRNA, miRNA, siRNA, or non-coding RNA, a sequence complementary to all or part of the base sequence may be used as the probe sequence. A nucleic acid probe having a desired base sequence and base length can basically be prepared using known means. As described above, when a set of two or more nucleic acid molecules is used as a nucleic acid probe, the base sequence of each nucleic acid molecule is complementary to the base sequence of a different (non-overlapping) region of the target nucleic acid. Designed as

  The probe sequence is preferably designed to include a unique sequence in a specific region on the target nucleic acid, eg, chromosome. In addition, when detecting the copy number of a specific gene on a chromosome by FISH, it is necessary to design a probe sequence in consideration of a genomic sequence including an intron before splicing. As a method of obtaining a genome sequence containing a gene to be detected, search using a database name DDBJ (DNA Data Bank of Japan) using the organism name, gene name, chromosome number, etc. as search words. Or by searching for “Cancer cell lines BACS” or the like as a search word. Here, when detecting the copy number of a cancer (original) gene by FISH, the sequence of the BAC clone library of “Cancer cell lines BACS” including the sequence of the cancer (original) gene is suitable.

  As for the probe sequence, when a normal structural gene is detected, it is preferable not to include a gene sequence portion in which the copy number is polymorphic, such as indel, VNTR (Variable Number of Tandem Repeat), and microsatellite. In human (2n = 46) cells, the normal gene copy number per cell (nucleus) is 1-2, so the copy number estimated from the number of bright spots of the phosphor is 3 or more Is abnormal in the chromosome to which the gene is amplified. Conversely, when the copy number is 0, it can be determined that an abnormality in the chromosome in which the gene is defective has occurred. If the polymorphic gene sequence as described above is included in the probe sequence, the number of bright spots of the phosphor will not match the copy number of the specific gene of interest, which will hinder the detection of the copy number as described above. Come on.

  In addition, for example, a specific base (eg, thymine (T)) in a nucleic acid molecule is substituted with a biotin-labeled nucleotide (eg, Biotin-16-dUTP) by nick translation, and biotin at the substitution site is substituted with an enzyme-modified molecule. A modified enzyme having avidin or an anti-avidin antibody can be bound as a primary antibody. In such a case, the number of specific bases in the nucleic acid molecule (thymine (T) in the above example) affects the number of bright spots and the intensity of light emission when FISH is performed. A probe sequence may be designed by determining a specific base in the nucleic acid molecule so that the strength becomes a desired level.

  Regarding the method of obtaining a nucleic acid probe, if the base number of the nucleic acid is several tens to several hundred bases, submit the nucleic acid sequence data including the probe sequence and request a nucleic acid synthesis contract service such as Funakoshi to obtain the nucleic acid probe. It is preferable to obtain. On the other hand, if the nucleic acid has a large number of bases (for example, more than 1000 bases), it can be synthesized as described above, but it takes time, so that the nucleic acid probe is correctly formed by sequencing the base sequence. For example, the following may be performed on the premise of confirmation. Probes designed and created by various techniques are generally called DNA probes if the nucleic acid sequence is DNA, and RNA probes if the nucleic acid sequence is RNA.

  As a first method, a primer is designed and synthesized so as to sandwich a probe sequence portion contained in the genomic DNA of the organism to be detected, and genomic DNA (or the above BAC clone library is used) using this primer set. PCR method using pfu DNA polymerase with high replication accuracy is performed. Next, the PCR reaction solution is separated by electrophoresis, and a band corresponding to the length of the target nucleic acid is cut out and eluted using a nucleic acid purification kit (a kit such as MonoFas (registered trademark) DNA purification kit I). Thus, the target nucleic acid can be obtained.

  As another method, a plasmid containing a nucleic acid probe sequence (such as a BAC plasmid) is transformed into E. coli (E. coli HST08 Premium Electro-Cells (Takara Bio Inc.), etc.), cultured (amplified), and collected. The nucleic acid is extracted, and the portion corresponding to the nucleic acid probe is excised with a predetermined restriction enzyme and subjected to electrophoresis and nucleic acid purification as described above.

  Further, unlike the above-described method for obtaining a nucleic acid probe, a nucleic acid probe is synthesized by artificially synthesizing a probe (nucleic acid) having a sequence that can be used as a probe using an artificial nucleic acid such as PNA or LNA (BNA). Can also be obtained.

(Nucleic acid probe modification molecule)
The nucleic acid probe modifying molecule modifies a nucleic acid probe with a predetermined substance used in accordance with the embodiment of the binding site amplification step, that is, an avidin-biotin complex (ABC) in the first embodiment, and in the second embodiment. A molecule for indirectly binding peroxidase. If the nucleic acid probe is a set of a plurality of nucleic acid molecules, the nucleic acid probe modifying molecule is introduced into each of the nucleic acid molecules.

  As the nucleic acid probe modifying molecule, a substance having a molecular structure capable of modifying a nucleic acid probe and having a specific binding substance, typically an antibody (the nucleic acid probe modifying molecule becomes an antigen) is suitable. Examples of such nucleic acid probe modifying molecules include biotin, avidin and hapten. Examples of haptens include dinitrophenol, digoxigenin, and FITC (fluorescein isothiocyanate). Anti-biotin antibody and anti-avidin antibody can be prepared for biotin and avidin, and antibodies such as anti-dinitrophenol antibody, anti-digoxigenin antibody and anti-FITC antibody are prepared for haptens such as dinitrophenol, digoxigenin and FITC, respectively. it can. These antibodies can be used as primary antibodies for indirectly binding biotin (first embodiment) or peroxidase (second embodiment) to nucleic acid probes in the embodiments as described above.

  In addition, with respect to biotin and avidins, they are substances to which avidin and biotin specifically bind, respectively. Therefore, for example, biotin is selected as the nucleic acid probe-modifying molecule, and if biotin is directly bound to the nucleic acid probe (by a covalent bond using a linker molecule, if necessary), further, an avidin-biotin complex ( Unreacted avidin contained therein (first embodiment) or biotin-modified peroxidase can be bound (second embodiment), and a primary antibody and a modified secondary antibody are used. There is no need.

(Method for producing modified nucleic acid probe)
The method for introducing the modifying molecule into the nucleic acid probe is not particularly limited, and various known methods can be adopted. However, it is preferable to use a bond having a strong binding force such as a covalent bond because of the stability of the bond. As a technique for preparing a nucleic acid probe to which a modified molecule is bound using a covalent bond, (i) a base to which a modified molecule is covalently bound, such as Biotin-dNTP (N is adenine (A), guanine (G), cytosine) (C), thymine (T), or uracil (U)) can be used to attach a modified molecule having a corresponding reactive functional group. Alternatively, (ii) after preparing a nucleic acid probe, a reactive functional group is introduced into the 5 'end or 3' end using a reagent, and a modified molecule having a corresponding functional group is reacted to be covalently bonded. The method etc. are mentioned. In the above method (i), if nick translation is used, it is also possible to introduce a plurality of modified molecules at sites other than the 5 ′ end or 3 ′ end of the nucleic acid probe.

(Primary antibody and secondary antibody)
As described above, when indirectly binding biotin (first embodiment) or peroxidase (second embodiment) to a nucleic acid probe, a primary antibody and a secondary antibody may be used.

  As the primary antibody, an antibody that specifically binds to a nucleic acid probe-modified molecule is used. For example, when biotin (avidin) is used as the nucleic acid probe-modifying molecule, an anti-avidin antibody (anti-biotin antibody) is the primary antibody, and when hapten is used as the nucleic acid probe-modifying molecule, the anti-hapten antibody is the primary antibody. Become. The primary antibody may be a monoclonal antibody or a polyclonal antibody.

  As the secondary antibody, an antibody that specifically binds to the primary antibody is used. The secondary antibody may be a monoclonal antibody or a polyclonal antibody. When a monopolyclonal antibody against the primary antibody is used as the secondary antibody, the secondary antibody binds at a single site of the primary antibody, that is, one secondary antibody binds to one primary antibody. When a polyclonal antibody against the primary antibody is used as the secondary antibody, the secondary antibody binds to the primary antibody at a plurality of sites, that is, a plurality of secondary antibodies bind to one primary antibody. Therefore, in the latter case, more avidin-biotin complex (first embodiment) or modified peroxidase (second embodiment) that specifically binds to the antibody-modified molecule of the secondary antibody is bound than in the former case. It becomes possible.

(Antibody-modifying molecule)
The antibody-modifying molecule is introduced into the primary antibody or the secondary antibody in order to bind biotin (first embodiment: as a base for binding ABC) or peroxidase (second embodiment) indirectly to the nucleic acid probe. And a molecule according to the embodiment of the binding site amplification step is used. That is, in the first embodiment (ABC treatment step), biotin as an amplification base for binding ABC becomes an antibody-modified molecule, and in the second embodiment (tyramide treatment step), a modified peroxidase is bound. Therefore, a molecule that specifically binds to a peroxidase-modified molecule is an antibody-modified molecule. When the primary antibody is modified, the primary antibody modifying molecule is sometimes referred to as the secondary antibody modifying molecule. When the secondary antibody is modified, the primary antibody modifying molecule and the secondary antibody are sometimes referred to simply as the modified antibody molecule. It is used as a general term for antibody-modified molecules.

  The antibody-modifying molecule in the second embodiment includes a molecule other than an antibody that can specifically modify the antibody and that specifically binds to the antibody (the antibody is not used as a peroxidase-modified molecule that binds to the antibody-modifying molecule. The antibody is excluded from the molecules to be bound. Examples of such antibody-modifying molecules include biotin and avidin, and biotin is particularly preferable. As will be described later, for biotin and avidin, avidin and biotin are used as peroxidase-modified molecules, respectively.

(Peroxidase-modified molecule)
As the peroxidase-modified molecule used in the second embodiment of the present invention, a molecule that specifically binds to an antibody-modified molecule or a nucleic acid probe-modified molecule is used. For example, when biotin is used as the antibody modifying molecule or nucleic acid probe modifying molecule, avidin, preferably streptavidin is used as the peroxidase modifying molecule.

(Tylamide modified molecule)
The tyramide-modified molecule used in the second embodiment of the present invention is a molecule for binding fluorescent nanoparticles around the nucleic acid probe. Tyramide-modified molecules can modify tyramide, and molecules other than antibodies that specifically bind to it (no antibodies are used as fluorescent nanoparticle-modified molecules that bind to Tylamide-modified molecules. If it exists, it will not be specifically limited. Examples of such a tyramide-modified molecule include biotin and avidin as in the case of an antibody-modified molecule, and biotin is particularly preferable.

  In the second embodiment of the present invention, the antibody modified molecule or nucleic acid probe modified molecule to which the modified peroxidase binds and the tyramide modified molecule to which the modified fluorescent nanoparticle binds may be the same (for example, both biotin). If they are the same, for example, the former is dinitrophenol and the latter is biotin, but the modified peroxidase does not bind, even if there is an unreacted nucleic acid probe-modified molecule, there will be modified fluorescent nanoparticles. Can bind, and as a result, it is possible to increase the number of fluorescent nanoparticles that label the target nucleic acid, which is preferable.

(Fluorescent nanoparticle modification molecule)
The fluorescent nanoparticle-modified molecule used in both the first embodiment and the second embodiment of the present invention specifically binds to biotin (first embodiment) or tyramide-modified molecule (second embodiment). Use molecules. For example, for biotin, avidin, preferably streptavidin, can be used as the fluorescent nanoparticle modifying molecule.

(Tyramide and peroxidase)
In the second embodiment of the present invention, tyramide (sometimes called tyramide) is used as a substrate, and peroxidase is used as an enzyme having an action of activating the substrate. A peroxidase (for example, HRP: horseradish peroxidase) acts on a peroxide (for example, hydrogen peroxide) to generate a free radical, and activates (radicalizes) tyramide by a free radical chain reaction. That is, tyramide can be activated by a treatment in which peroxidase and tyramide react with the presence of peroxide. Although the details of the activated tyramide are not yet elucidated, a plurality of tyramides are likely to be aggregated and precipitated to bind (deposit) to molecules around the nucleic acid probe. What is necessary is just to prepare according to general conditions, such as suitable conditions for such a process, for example, salt concentration of reaction solution, pH, and temperature.

(Method for producing modified tyramide and modified peroxidase)
Both modified tyramide and modified peroxidase can be prepared by using known methods (see, for example, US Pat. No. 5,196,306: Patent Document 2 and JP2013-531801: Patent Document 3). For example, a tyramide modified with biotin can be prepared by introducing a reactive functional group into each of tyramide and biotin using a reagent and reacting these functional groups with each other to form a covalent bond. If necessary, a linker may be interposed between tyramide and biotin (that is, between functional groups that they have). In addition, the conjugate of biotin and tyramide is commercially available and can be easily obtained. Also, for example, a peroxidase modified with avidin can be prepared by introducing a reactive functional group into each of peroxidase and avidin using a reagent and reacting these functional groups with each other to form a covalent bond. . If necessary, a linker may be interposed between peroxidase and avidin (that is, between functional groups that they have).

(Fluorescent nanoparticles)
In the present invention, fluorescent nanoparticles are used as a substance for fluorescently labeling the target gene. A fluorescent nanoparticle is a nano-sized (a diameter is 1 micrometer or less) particulate-form fluorescent substance, and can emit the fluorescence which has sufficient brightness | luminance with one particle. Such phosphors can be broadly classified into phosphor-integrated nanoparticles and inorganic fluorescent nanoparticles. A single fluorescent dye is not used in the present invention because it cannot emit fluorescence having sufficient luminance. The phosphor-integrated nanoparticles include fluorescent dye-containing nanoparticles and inorganic phosphor-integrated nanoparticles. Inorganic phosphor nanoparticles include quantum dots and carbon dots.

  What is necessary is just to select the fluorescent nanoparticle which emits fluorescence (color) of a desired wavelength in the dye | stained image image | photographed. In addition, when there are a plurality of biomolecules to be fluorescently labeled, a plurality of types of fluorescent nanoparticles that emit fluorescence of different wavelengths corresponding to each of them may be used in combination.

(Inorganic phosphor nanoparticles)
In the present invention, quantum dots and carbon dots can be used as the inorganic phosphor nanoparticles. These inorganic phosphor nanoparticles can constitute brightness of bright spots having observable brightness independently without preparing inorganic phosphor integrated nanoparticles as described below.

  Quantum dots include quantum dots containing II-VI group compounds, III-V group compounds, or IV group elements as components ("II-VI group quantum dots", "III-V group quantum dots", " Any of the “Group IV quantum dots” can be used. Specifically, particle dots such as CdSe exemplified in International Publication WO2012 / 133447 can be mentioned. These inorganic phosphor nanoparticles may be used either alone or in combination.

  Moreover, the quantum dot which made the said quantum dot a core and provided the shell on it can also be used. Hereinafter, as a notation of quantum dots having a shell, when the core is CdSe and the shell is ZnS, it is expressed as CdSe / ZnS. Specific examples include CdSe / ZnS exemplified in International Publication No. WO2012 / 130347, but are not limited thereto.

  As the quantum dots, those subjected to surface treatment with an organic polymer or the like may be used as necessary. For example, commercially available CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like can be used.

  On the other hand, carbon dots, unlike conventional quantum dots, do not use harmful elements or rare metals, and thus are recently attracting attention. Carbon dots include graphene quantum dots, carbon nanodots, polymer dots, and the like. Carbon dots are publicly known substances.For details, see Nano Research DOI10.1007 / s12274-014-0644-3, Shoujun Zhu et al., The photoluminescence mechanism in carbon dots (graphene quantum dots, carbon nanodots and polymer dots): current state and future perspective, 2014.

  For example, carbon dots, which are a kind of graphene quantum dots, are produced by oxidative cutting of various carbon sources (graphite powder, carbon rods, carbon fibers, carbon nanotubes, carbon black, etc.). In this process, the carbon source is cleaved into small molecules, and the surface is modified with acidic groups, thereby producing carbon dots. A common oxidative cleavage method is oxidation with concentrated acid (nitric acid or sulfuric acid / nitric acid mixture).

  Carbon dots are manufactured in two steps. The first step is a step of changing the graphite-derived substance into a graphite oxide sheet, and this step is usually performed by changing the Hummres method according to the purpose. The second stage is a process for producing carbon dots from the graphite oxide sheet by using various known cutting methods.

  Examples of the method for cutting the carbon source into small molecules include physical methods such as nanolithography using arc discharge, laser irradiation and reactive ion etching (RIE), and methods such as electrochemistry and hydrothermal / solvothermal oxidation. Is mentioned.

On the other hand, carbon dots can be produced by dehydration and carbonization from small molecules and polymers having a hydroxy group, a carboxy group, a ketone group and an amino group.
Examples of the dehydration and carbonization methods include hydrothermal, microwave-hydrothermal, plasma-hydrothermal, microwave, combustion, concentrated acid pyrolysis, and carbonization by a microreactor.

(Phosphor integrated nanoparticles)
The phosphor-integrated nanoparticles are obtained by integrating fluorescent dyes or inorganic phosphor nanoparticles (quantum dots, carbon dots, etc.). In the present invention, fluorescent dye integrated nanoparticles and inorganic phosphor integrated nanoparticles can be used as the phosphor integrated nanoparticles. By using such phosphor-integrated nanoparticles, the amount of fluorescence emitted per particle, that is, the brightness of a bright spot for labeling a predetermined biomolecule can be obtained as compared with a single fluorescent dye or inorganic phosphor nanoparticle. Can be increased.

  Examples of the fluorescent dye used for producing the fluorescent dye-integrated nanoparticles include a fluorescein dye, a rhodamine dye, an Alexa Fluor (registered trademark, manufactured by Invitrogen) dye, a BODIPY (registered trademark, manufactured by Invitrogen) dye, and a cascade. (Registered trademark, Invitrogen) Low molecular organic compounds (polymer organics such as polymers) such as dyes, coumarin dyes, NBD (registered trademark) dyes, pyrene dyes, cyanine dyes, perylene dyes, oxazine dyes A fluorescent dye selected from the group consisting of non-compounds). Of these, rhodamine dyes such as sulforhodamine 101 and its hydrochloride, such as TexasRed (registered trademark), and perylene dyes such as perylene diimide are preferred because of their relatively high light resistance. On the other hand, examples of the inorganic phosphor used for producing the inorganic phosphor-integrated nanoparticles include the substances that can be used alone as the inorganic phosphor nanoparticles as described above.

(Method for producing phosphor-integrated nanoparticles)
The production method of the phosphor-integrated nanoparticles used in the present invention is not particularly limited, and can be produced by a known method. In general, a production method can be used in which phosphors are gathered together using a resin or silica as a base material (the phosphors are immobilized inside or on the surface of the base material). For example, the resin used as the matrix of the phosphor-integrated nanoparticles may be a thermosetting resin or a thermoplastic resin. Specific examples of various resins and methods for producing phosphor-integrated nanoparticles produced using these resins as a base conform to known literature (WO2014 / 136776).

  Further, for example, silica nanoparticles encapsulating a fluorescent dye can be synthesized with reference to the synthesis of FITC-encapsulated silica particles described in Langmuir 8, Vol. 2921 (1992). Various fluorescent dye-containing silica nanoparticles can be synthesized by using a desired fluorescent organic dye instead of FITC. Silica nanoparticles encapsulating semiconductor nanoparticles can be synthesized with reference to the synthesis of CdTe-encapsulated silica nanoparticles described in New Journal of Chemistry, Vol. 33, p. 561 (2009).

  The average particle diameter of phosphor-integrated nanoparticles such as fluorescent dye-integrated nanoparticles and inorganic phosphor-integrated nanoparticles is particularly within the range that can be appropriately observed as a bright spot that emits fluorescence when a target gene is labeled. Although not limited, the average particle diameter of the phosphor-integrated nanoparticles is preferably 20 nm or more and 300 nm or less, and more preferably 20 nm or more and 100 nm or less, from the viewpoint of being suitable for fluorescence observation of such bright spots. preferable.

  The average particle diameter of inorganic phosphor nanoparticles such as quantum dots and carbon dots is not particularly limited as long as it is within a range that can be appropriately observed as a bright spot that emits fluorescence when the target gene is labeled. From the viewpoint of making it suitable for fluorescent observation of bright spots, particularly from the viewpoint of facilitating labeling of the target gene by moving into the nucleus, the average particle diameter of the inorganic phosphor nanoparticles is 1 nm or more and 300 nm or less. Is preferably 1 nm or more and 20 nm or less.

  The average particle diameter of the fluorescent nanoparticles can be measured by a method known in the art. For example, a sufficient number (for example, 1000) of fluorescent nanoparticles can be measured using a scanning electron microscope (SEM). After measuring the particle size, it is calculated as the arithmetic average.

(Method for producing modified fluorescent nanoparticles)
The modified fluorescent nanoparticles can be prepared using a known method. For example, when using phosphor-integrated nanoparticles (fluorescent dye-integrated nanoparticles or inorganic phosphor-integrated nanoparticles) as the fluorescent nanoparticles, the surface of the phosphor-integrated nanoparticles and the fluorescent nanoparticle-modified molecules are respectively separated using reagents. Modified phosphor-integrated nanoparticles can be produced by introducing reactive functional groups and bonding these functional groups together. When modifying the phosphor-integrated nanoparticle surface, an appropriate reagent or the like is used in consideration of the base material (resin, silica, etc.). Moreover, you may interpose a linker between avidin and fluorescent substance integration | stacking nanoparticle as needed (that is, between the functional groups which they have). Examples of combinations of reactive functional groups include NHS ester group-amino group, thiol group-maleimide group combination, and the like. For example, (i) the surface of the phosphor-aggregated nanoparticles is previously modified with an amino group using a silane coupling agent, and (ii) on the other hand, N-succinimidyl-S-acetylthioacetate is reacted with streptavidin. , A thiol group protected with an acetyl group is linked to the amino group of streptavidin, and (iii) a maleimide having an NHS group that reacts with an amino group at one end and a thiol group at the other end as a crosslinker A polyethylene glycol chain having a group (for example, “SM (PEG) 12”, Thermo Scientific Co., Ltd.), and (iv) reacting the amino group on the phosphor-integrated nanoparticle surface with the NHS group of the crosslinker for sharing (V) then deprotecting the thiol group of streptavidin and then reacting with the maleimide group of the crosslinker By phosphor integrated nanoparticles modified with streptavidin is obtained.

  In the case of using inorganic fluorescent nanoparticles (quantum dots, carbon dots, etc.) as fluorescent nanoparticles, reactive functionalities are respectively applied to the surface of the inorganic fluorescent nanoparticles and the fluorescent nanoparticle-modified molecules using reagents, etc. A modified inorganic phosphor nanoparticle can be produced by introducing a group or utilizing a functional group of a carbon dot by a production method and bonding these functional groups to each other.

  Note that the number of fluorescent nanoparticle modifying molecules (the number of modifying molecules) that modify the surface of the fluorescent nanoparticle is schematically depicted as one (1: 1) per fluorescent nanoparticle in FIGS. However, it varies depending on conditions such as the particle size of the fluorescent nanoparticles, and is not limited to 1: 1. For example, when streptavidin is used as the fluorescent nanoparticle-modifying molecule, if the molecular size of streptavidin, which is a protein having a molecular weight of about 52000, is taken into consideration, the average particle diameter of the fluorescent nanoparticles is relatively large (for example, about 300 nm). It is possible to bind a plurality of (for example, several hundred to several thousand) streptavidin per fluorescent nanoparticle (1: many), and conversely, if the average particle size of the fluorescent nanoparticle is relatively small (for example, It is conceivable that a plurality of fluorescent nanoparticles are bound to one streptavidin, that is, the number of modifying molecules per fluorescent nanoparticle is less than 1 (multiple: 1). In general, the higher the number of modifying molecules on the surface of the fluorescent nanoparticle, the higher the probability that the fluorescent nanoparticle can bind to the site capable of binding, which is preferable, but observation of an appropriate starting point depending on the use such as pathological diagnosis The number of modifying molecules can be appropriately adjusted within the range where

-Pathological specimen-
The pathological specimen according to the present invention is obtained by FISH staining of a nucleic acid in which fluorescent nanoparticles are bound to a plurality of binding sites amplified by a binding site amplification step (ABC treatment step or tyramide treatment step) in the FISH staining method of the present invention. This is a pathological specimen.

-Pathological diagnosis-
The use of the FISH staining method of the present invention is not particularly limited, but basically has the same use as a conventional pathological specimen, and typically quantifies a specific gene contained in a nucleic acid. The present invention is used for preparing a pathological specimen according to the present invention for performing pathological diagnosis, for example, for obtaining information useful for diagnosis or treatment of a disease. That is, a slide (specimen slide) prepared using a tissue section collected from a diagnosis target is stained based on the FISH staining method of nucleic acids according to the present invention and other staining methods combined as necessary. Quantify specific genes by microscopic observation of the pathological specimens (specimen slides that have been subjected to staining or other processing and can be examined) and / or image processing of the captured images A pathological diagnosis can be performed by examining.

  Such pathological diagnosis includes a stage from staining a specimen slide to preparing a pathological specimen (pathological specimen preparation stage), that is, a stage in which the method for FISH staining of nucleic acid according to the present invention is performed, and a prepared pathological specimen. Can be roughly divided into stages (pathological specimen inspection stage) until the desired information is acquired.

  In the pathological specimen preparation stage for FISH, generally specimen slide preparation process (fixing process, dehydration process, paraffin embedding process, slicing process, etc.), specimen pretreatment process (deparaffinization process, heat treatment, protease process, etc.) ), Staining steps (DNA denaturation treatment, FISH staining treatment, post-fixation treatment, nuclear staining treatment, etc.) and specimen post-treatment steps (washing treatment, dehydration treatment, solvent replacement treatment, encapsulation treatment, etc.). The FISH dyeing | staining method of this invention can be utilized in order to perform the FISH dyeing | staining process and post-fixation process in the said dyeing | staining process. That is, the hybridization step, the binding site amplification step, and the fluorescent nanoparticle reaction step specified by the FISH staining method of the present invention are further performed as a procedure for the FISH staining process in the staining step and by the FISH staining method of the present invention. Preferably, the fixing step can be incorporated as a post-fixing treatment in the staining step.

  Hereinafter, an example of a more specific procedure will be described for the dyeing process when the FISH staining method of the present invention is used. Other processes included in the preparation stage for stained specimens for FISH, that is, specimen preparation process, specimen pre-treatment process and specimen post-treatment process, and stained specimen inspection stage, are general pathological specimen preparations using conventional FISH staining methods. It can be performed in the same manner as in the step (for example, see WO2015 / 141856: Patent Document 1).

(Dyeing process)
(1) DNA denaturation treatment First, DNA denaturation treatment is performed on the specimen slide that has undergone the specimen pretreatment step. The denaturation treatment of DNA may be performed by heat denaturation according to a conventional method, or may be performed by treatment with a denaturing solution such as a formamide solution. In addition, when performing hybridization included in the FISH staining process described below, dissociation (denaturation) of double-stranded DNA of a chromosome and promotion of binding (hybridization) between the single-stranded DNA and a nucleic acid probe DNA denaturation treatment and hybridization may be performed simultaneously by using a hybridization buffer containing a component that can be produced (for example, IQFISH FFPE Hybridization Buffer, Agilent Technologies).

(2) FISH staining treatment When a pathological specimen is prepared using the present invention, the FISH staining treatment is performed in the hybridization step, the binding site amplification step, and the fluorescent nanoparticle reaction step in the FISH staining method of the present invention as described above. The processing is the same.

(2-1) Hybridization Step Hybridization is performed by dropping the modified nucleic acid probe solution onto the sample slide and bringing the target nucleic acid contained in the sample into contact with the modified nucleic acid probe solution. At this time, a buffer solution containing a component that promotes hybridization (may further contain a component that denatures DNA as described above) can be added.

  The modified nucleic acid probe is a nucleic acid probe modified with a predetermined molecule according to the embodiment of the subsequent binding site amplification step. Such a modified nucleic acid probe is prepared in advance and an appropriate concentration solution is prepared for use in this hybridization step.

  Regarding the hybridization reaction conditions, the GC content (Tm value) of the nucleic acid probe used, the concentration of monovalent cations present in the hybridization reaction system (M), and the formamide concentration (% ) And the like, the reaction temperature and reaction time may be set appropriately in consideration of changes in accuracy when the nucleic acid molecule binds to the base sequence of the target gene on the chromosome.

(2-2) Binding Site Amplification Step A solution according to the embodiment of the binding site amplification step is sequentially dropped on the specimen slide that has undergone the hybridization step, and by binding starting from the molecule modified to the nucleic acid probe, or the nucleic acid probe By binding to surrounding molecules, a site where fluorescent nanoparticles can be bound is amplified.

  That is, when conforming to the first embodiment of the present invention, for example, a primary antibody solution, a biotin-modified secondary antibody solution, a streptavidin solution, and a biotinylated peroxidase (for example, biotinylated HRP) solution are sequentially dropped. In the case of conforming to the second embodiment of the present invention, for example, an aqueous peroxide solution such as hydrogen peroxide solution, a modified peroxidase solution, and a modified tyramide solution are sequentially dropped. The modified secondary antibody, modified peroxidase, and modified tyramide are each prepared in advance as described above, and a solution having an appropriate concentration is prepared and used in this hybridization step.

  Binding of primary antibody to nucleic acid probe-modified molecule and binding of modified secondary antibody to primary antibody (first embodiment and second embodiment); Binding of ABC to biotin-modified secondary antibody (first embodiment) ); Binding of modified peroxidase to antibody-modified molecule or nucleic acid probe-modified molecule (second embodiment); reaction of modified peroxidase and modified tyramide (second embodiment), respectively, according to known binding conditions or reaction conditions What is necessary is just to carry out under the set appropriate temperature, time, and other conditions.

(2-3) Fluorescent Nanoparticle Binding Step A solution of modified fluorescent nanoparticles is dropped onto the specimen slide that has undergone the binding site amplification step, and biotin (first embodiment) or tyramide modified molecule (second embodiment) contained in ABC ) To bind the modified fluorescent nanoparticles to them. By finishing this step, individual target nucleic acids can be finally labeled with a large number of fluorescent nanoparticles.

  The modified fluorescent nanoparticle is a fluorescent nanoparticle modified with a predetermined molecule according to the embodiment of the binding site amplification step as described above. Such modified fluorescent nanoparticles are prepared in advance, and an appropriate concentration solution is prepared for use in this fluorescent nanoparticle binding step.

  Binding of the modified fluorescent nanoparticles to biotin (first embodiment) or tyramide-modified molecule (second embodiment) contained in ABC is performed at an appropriate temperature set according to known binding conditions or reaction conditions, respectively. It may be performed under time and other conditions.

(3) Post-fixation treatment When preparing a pathological specimen using the present invention, the post-fixation treatment is a treatment according to the fixation step in the FISH staining method of the present invention as described above.

  That is, an immobilizing agent having an appropriate concentration such as a paraformaldehyde solution is dropped on a specimen slide that has undergone the FISH staining treatment, and the target nucleic acid, the modified nucleic acid probe, the modified fluorescent nanoparticle, and intervening them are connected. Contact with the substance used in the binding site amplification step (modified tyramide, ABC, primary antibody, modified secondary antibody, etc., if necessary) and reaction at an appropriate temperature (usually room temperature). I will let you. These reaction conditions can be appropriately adjusted. For example, when a paraformaldehyde solution is used as a fixing agent, the concentration is about 1 to 5 w / w%, the temperature is about room temperature, and the time is about 10 to 15 minutes. Can do.

(4) Nuclear staining step After the FISH staining treatment or an optional post-fixation treatment, usually, nuclear staining is further performed to count the number of cells. As a nuclear staining reagent, DAPI is generally used, but Hoechst 33258, Hoechst 33342 or other nuclear staining reagents can also be used.

(5) Other treatments Other examples of steps can be included in the dyeing step as needed. For example, before adding the primary antibody used as necessary in the first embodiment and the second embodiment, a blocking treatment is performed using a commercially available blocking agent in order to suppress nonspecific adsorption of the antibody in the tissue section. It is preferable to carry out. For example, it is preferable to perform a washing process using a commercially available detergent after hybridization.

[Preparation Example 1] Preparation of DNP-modified nucleic acid probe for HER2 gene 5 μL of 5 × GoTaq reaction buffer (Promega) in a 0.2 mL PCR strip tube (Bio-Rad), GoTaq DNA polymerase (5 U / μL, Promega) 0 .25 μL, Human Genomic DNA (100 ng / μL, Takara Bio Inc.) 0.25 μL, dATP, dGTP, dCTP (all 10 mM, Takara Bio Inc.) 0.5 μL each, dTTP (1 mM, Takara Bio Inc.) 3.75 μL, DNP -11-UTP (1 mM, PerkinElmer) 1.25 μL, human HER2 gene Forward primer (sequence: 5′-CGATGTGACTGTCTCCTCCC-3 ′, 10 μM) 0.5 μL, Revers corresponding to the Forward primer e primer (sequence: 5′-ATCCTACTCCATCCCAAGGCC-3 ′, 10 μM) 0.5 μL and milliQ 7 μL in total 25 μL solution was prepared.

After setting the strip tube on a thermal cycler (Chromo4, Bio-Rad), PCR is performed by the method from Step 1 to Step 5 shown below to prepare a PCR product that becomes a DNP-modified nucleic acid probe of about 200 base pairs. did. The PCR product was purified using a PCR purification kit (Qiagen).
Step 1: 95 ° C, 3 minutes Step 2: 95 ° C, 30 seconds Step 3: 56 ° C, 30 seconds Step 4: Return to Step 2 (repeated 39 times)
Step 5: 72 ° C, 3 minutes

  One type of nucleic acid probe modified with DNP, which hybridizes to about 200 base pairs in the HER2 region by the above method, was prepared. Furthermore, except that Forward primer and Reverse primer of different base sequences were used, four types of probes of about 200 base pairs that hybridize to other places in the HER2 region were prepared, and these five types were mixed. One HER2-FISH probe was used. The total base length of the nucleic acid probe that hybridizes to the target HER2 region is about 1000 bases.

[Production Example 2] Production of avidin-modified fluorescent dye-integrated melamine resin nanoparticles (i) Synthesis of fluorescent dye-integrated melamine resin nanoparticles Sulforhodamine 101 ("Sulforhodamine 101", Sigma Aldrich, TexasRed dye) 2.5 mg pure After dissolving in 22.5 mL of water, the solution was stirred for 20 minutes while maintaining the temperature of the solution at 70 ° C. with a hot stirrer. To the stirred solution, 1.5 g of melamine resin “Nicalak MX-035” (manufactured by Nippon Carbide Industries Co., Ltd.) was added, and the mixture was further heated and stirred under the same conditions for 5 minutes.

  100 μL of formic acid was added to the stirred solution and stirred for 20 minutes while maintaining the temperature of the solution at 60 ° C., and then the solution was left to cool to room temperature. As a result, a dispersion of fluorescent dye (Texas Red) -integrated melamine resin nanoparticles (hereinafter abbreviated as particle X) was obtained. The cooled dispersion was dispensed into a plurality of centrifuge tubes and centrifuged at 12,000 rpm for 20 minutes to precipitate the dispersed particles X, and the supernatant was removed. Thereafter, the recovered particles X were washed with ethanol and water. The average particle diameter of the particles X was 80 nm.

(Ii) Coupling of maleimide groups to the surface of phosphor-integrated melamine resin nanoparticles Particles 0.1 mg after washing were dispersed in 1.5 mL of ethanol, and aminopropyltrimethoxysilane “LS-3150” (manufactured by Shin-Etsu Chemical Co., Ltd.) 2 μL was added, and the surface amination treatment was performed by reacting at room temperature with stirring for 8 hours.

  The concentration of the particle X with the aminated surface was adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and the linker reagent “SM (PEG) 12” (to the final concentration of 10 mM) was added to this solution. Thermo Scientific) was mixed and reacted for 1 hour at room temperature with stirring.

  The reaction solution was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again under the same conditions. Washing by the same procedure was performed three times to obtain particles X surface-modified with a PEG chain having a maleimide group at the terminal.

(Iii) Linkage of thiol group to streptavidin On the other hand, streptavidin having a sulfhydryl group was prepared as follows. First, 40 μL of streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL, N-succinimidyl-S-acetylthioacetate (N-succinimidyl S-acetylthioacetate, SATA, Pirce) adjusted to 64 mg / mL 70 μL) was reacted at room temperature for 1 hour. That is, the introduction of protected thiol group to the amino group of streptavidin _{(-NH-CO-CH 2 -S} -CO-CH 3).

  Then, the process which produces | generates a free thiol group (-SH) from the protected thiol group by well-known hydroxylamine process, and adds a thiol group (-SH) to streptavidin was performed.

  This streptavidin solution is desalted with a gel filtration column (Zaba Spin Desaling Columns), and the streptavidin can be bound to particles X having thiol groups linked, that is, surface-modified with PEG chains having maleimide groups at the ends. Avidin was obtained.

(Iv) Binding of phosphor-integrated melamine resin nanoparticles and streptavidin In a PBS containing 2 mM EDTA, particle X surface-modified with a PEG chain having a maleimide group and streptavidin linked with a thiol group The maleimide group and the thiol group were covalently bonded by mixing and reacting for 1 hour. 10 mM mercaptoethanol was added to stop the reaction. After the obtained solution was concentrated with a centrifugal filter, unreacted streptavidin and the like were removed using a gel filtration column for purification to obtain fluorescent dye-integrated melamine resin nanoparticles modified with streptavidin.

[Example 1] Second embodiment using fluorescent dye-integrated nanoparticles (Tylamide treatment step, using polyclonal secondary antibody)
(Deparaffinization)
A sample slide (“HER2 Dual ISH 3-in-1 control slide” manufactured by Roche) was processed in the order of the following (1) to (5) to perform deparaffinization. (1) Immerse in xylene at room temperature for 5 minutes. Repeat this operation once more (perform twice). (2) The specimen slide is immersed in 100% ethanol at room temperature for 2 minutes. Repeat this operation once more (perform twice). (3) Immerse the specimen slide in 70% ethanol at room temperature for 2 minutes. (4) The specimen slide is immersed in milliQ water for 2 minutes at room temperature.

(Heat treatment)
In order to improve the reachability of the DNA probe, the sample slide was subjected to heat treatment in the following order (1) to (3) to remove proteins from the cell membrane and the nuclear membrane. (1) Treat with MES buffer (HER2 FISH PharmDx “Dako” Vial 1 diluted 20-fold with milliQ water) at 95 to 99 ° C. for 10 minutes, and then treat at room temperature for 15 minutes. (2) The specimen slide is immersed in Tris wash buffer (HER2 FISH PharmDx “Dako” Vial 6 diluted 20 times with milliQ water) for 3 minutes and washed. (3) Repeat this washing operation once more.

(Protease treatment)
Protease treatment was performed on the treated specimen slide by performing the following treatments (1) to (4) in this order. (1) Take out the heat-treated specimen slide and attach the lower end of the slide glass to a paper towel to remove excess washing buffer. (2) 5-6 drops of protease solution (HER2 FISH PharmDx “Dako” Vial 2A) is dropped on the specimen slide and allowed to react at room temperature for 10 minutes. This immersion treatment is for degrading proteins of cell membrane and nuclear membrane, particularly collagen. (3) The specimen slide is immersed in Tris wash buffer (HER2 FISH PharmDx “Dako” Vial 6 diluted 20 times with milliQ water) for 3 minutes and washed. (4) Repeat this washing operation once more.

(dehydration)
After the specimen slide was air-dried, it was first immersed in 70% ethanol at room temperature for 2 minutes, and then immersed in 100% (dehydrated) ethanol at room temperature for 2 minutes. Thereafter, the specimen slide was taken out, air-dried with cold air, and left at room temperature for 10 minutes or more.

(Denaturation and hybridization)
By performing the following treatments (1) to (3) in this order on the specimen slide subjected to the dehydration treatment, a hybridization treatment using the DNP-modified nucleic acid probe obtained in Preparation Example 1 was performed. . (1) Add 1 μL of a DNP-modified nucleic acid probe adjusted to 5 ng / μL and 9 μL of IQFISH FFPE Hybridization Buffer (Agilent Technology) to the hybridization region of the specimen slide. Immediately after mixing by pipetting on the slide, cover with a 22 mm × 22 mm cover glass to spread the DNP-modified nucleic acid probe solution uniformly. Prevent bubbles from entering the hybridization area. (2) Seal the cover glass with a paper bond (KOKUYO). (3) Place a specimen slide on a hybridizer (Dako), and perform denaturation and hybridization at 80 ° C. for 10 minutes and then at 45 ° C. overnight.

(Cleaning specimen slides)
By performing the following steps (1) to (6) in this order on the sample slide subjected to the above hybridization treatment, the sample slide was washed. (1) Post-hybridization washing buffer: SSCwash Buffer (HER2 FISH PharmDx “Dako” Vial 2A diluted 20-fold with milliQ water) is placed in a coplinger. Preheat in a warm bath until post-hybridization wash buffer is 63 ° C. (2) Prepare another coplinger containing the post-hybridization washing buffer and keep it at room temperature. (3) Remove the paper bond seal with tweezers. Place the specimen slide in a copliner containing a post-hybridization wash buffer maintained at room temperature and remove the cover glass. (4) The specimen slide is placed in a post-hybridization washing buffer kept at 63 ° C. and immersed for 10 minutes for washing. (5) Immerse in Tris wash buffer (HER2 FISH PharmDx “Dako” Vial 6 diluted 20 times with milliQ water) at room temperature for 3 minutes and wash. This washing is repeated again. (6) Remove the specimen slide from the copliner and air dry under light shielding.

(blocking)
Blocking was performed by placing 100 μL of Antibody Diluent, Background Reduction (Dako) on a slide and immersing in a wet box at room temperature for 30 minutes.

(Binding of DNP-modified nucleic acid probe and primary antibody)
By performing the following processes (1) to (4) in this order on the sample slide subjected to the blocking process, a binding process between the DNP-modified nucleic acid probe and the primary antibody was performed. (1) Shake the slide up and down on the Kim Towel so that the corner hits the Kim Towel, drop the solution on the slide, wipe off the Antibody Diluent and Back Reduction Liquid on the slide other than the cell periphery with filter paper etc. Only wet state. (2) 100 μL of a solution of anti-DNP rabbit monoclonal antibody (Roche Anti-DNP rabbit mAb) as a primary antibody is dropped on a specimen slide and reacted in a wet box at room temperature for 60 minutes to bind to a DNP-modified nucleic acid probe. Let (3) The solution on the slide is dropped on a Kim towel and then washed by immersing in PBS for 5 minutes. (4) Repeat the PBS washing operation two more times.

(Binding of primary antibody and biotin-labeled secondary antibody)
By performing the following steps (1) to (4) in this order on the specimen slide subjected to the binding treatment of the DNP-modified nucleic acid probe and the primary antibody, the primary antibody and the biotin-labeled secondary antibody are bound. Processed. (1) Shake the slide up and down on the Kim Towel so that the corner hits the Kim Towel and drop the solution on the slide. Then wipe the solution on the slide other than the periphery of the cell with filter paper to make only the cell surface wet. . (2) 100 μL of a biotin-labeled anti-mouse / rabbit polyclonal IgG antibody (Roche Anti-mouse and rabbit Ab) solution as a biotin-labeled secondary antibody is dropped onto a specimen slide and allowed to react at room temperature for 60 minutes in a wet box. Bind to primary antibody. (3) The solution on the slide is dropped on a Kim towel and then washed by immersing in PBS for 5 minutes. (4) Repeat the PBS washing operation two more times.

(Tylamide treatment)
Using the kit of GenPoint (Dako), the following treatments (A1) to (A4) and (B1) to (B4) were performed in this order to perform a tylamide treatment.

  (A1) Shake the slide up and down on the Kim towel so that the corner hits the Kim towel, drop the solution on the slide, wipe the solution on the slide other than the periphery of the cell with filter paper etc., and make only the cell surface wet. . (A2) The streptavidin-modified horseradish peroxidase (HRP) solution included in the kit is diluted 100-fold with the diluent in the kit, and this is added dropwise to 90 μL of the specimen slide and reacted at room temperature in a wet box for 15 minutes. I do. (A3) Washing is performed by immersing in TBST (mixed solution of Tris-buffered saline and Tween 20) for 5 minutes. (A4) This washing operation is repeated twice more.

  (B1) Subsequent to the treatment (A4) above, the slide is shaken up and down on the Kim towel so that the corner hits the Kim towel, and the solution on the slide is dropped, and then the horseradish peroxidase solution on the slide other than the periphery of the cells is removed with filter paper, etc. Wipe off with a wet surface. (B2) Three drops of a biotin-labeled tyramide solution (which is considered to contain hydrogen peroxide) contained in the kit is dropped on the specimen slide and reacted at room temperature for 15 minutes in a wet box. (B3) Wash by immersing in TBST for 5 minutes. (B4) This washing operation is repeated twice more.

(Binding of fluorescent dye-integrated melamine resin particles)
The following treatments (1) to (4) were performed in this order on the sample slide that had been subjected to the Tylamide treatment, thereby binding the fluorescent dye-integrated melamine resin particles. (1) Shake the slide up and down on the Kim towel so that the corner hits the Kim towel, drop the solution on the slide, wipe the biotin-labeled Tylamide solution on the slide other than the periphery of the cell with filter paper, etc., and wet only the cell surface. State. (2) 80 μL of a solution in which the concentration of the streptavidin-modified fluorescent dye-integrated melamine resin nanoparticles obtained in Preparation Example 2 was adjusted to 0.1 nM using an Antibody Diluent, Background Reduction solution was dropped on the specimen slide, The reaction is carried out at room temperature for 60 minutes to bind the biotin possessed by tyramide and streptavidin modified with the fluorescent dye-integrated melamine resin particles. (3) The solution on the slide is dropped on a Kim towel, and the specimen slide is immersed in PBS for 5 minutes for washing. (4) Repeat this washing operation two more times.

(Rear fixing)
A post-fixation process was performed by performing the following processes (1) to (3) in this order on the specimen slide subjected to the binding process of the fluorescent dye-integrated melamine resin particles. (1) Shake the slide up and down on the Kim Towel so that the corner hits the Kim Towel and drop the solution on the slide. Then wipe the solution on the slide other than the periphery of the cell with filter paper to make only the cell surface wet. . (2) Drop 150 μL of 4% paraformaldehyde solution on the specimen slide and immerse in a humid box for 10 minutes at room temperature. (3) The solution on the slide is dropped on a Kim towel and washed by immersing in Tris EDTA buffer (Wako) for 5 minutes.

(Nuclear staining)
Nuclei using DAPI (4 ', 6-Diamidino-2-Phenylindole, Dihydrochloride) by performing the following treatments (1) to (4) in this order on the specimen slide subjected to the post-fixation treatment. Staining was performed. (1) Shake the slide up and down on the Kim Towel so that the corner hits the Kim Towel and drop the solution on the slide. Then wipe the solution on the slide other than the periphery of the cell with filter paper to make only the cell surface wet. . (2) 150 μL of staining solution containing 0.01% β-mercaptoethanol and 1.43 μM DAPI (Invitrogen) is added to the hybridization region of the specimen slide, and allowed to react at room temperature in a wet box for 15 minutes. Stain cell nuclei. (3) The solution on the specimen slide is dropped onto a Kim towel and washed by immersing in Tris EDTA buffer (Wako) at room temperature for 5 minutes. (4) The specimen slide is immersed in a PBS buffer for 5 minutes and washed.

(Enclosed)
By performing the following processes (1) to (2) in this order on the specimen slide subjected to the nuclear staining process, an encapsulation process was performed. (1) Shake the slide up and down on the Kim Towel so that the corner hits the Kim Towel and drop the solution on the slide. Then wipe the solution on the slide other than the periphery of the cell with filter paper to make only the cell surface wet. . (2) Aquatex (MERCK) is added drop by drop onto the specimen slide in a non-dried state, and then covered with a cover glass, and the specimen slide is stored in a shaded state until signal measurement.

(Observation)
The specimen slide prepared by the above steps and subjected to FISH staining and DAPI staining was observed using a fluorescence microscope “BZ-X710” (manufactured by Keyence Corporation), and a fluorescence image was obtained. The magnification of the objective lens was 100 times, and the excitation time during imaging of DAPI and fluorescent dye-integrated melamine resin nanoparticles was 20 ms and 500 ms, respectively. The photographed fluorescence image is shown in FIG.

[Comparative Example 1] (Polyclonal secondary antibody, streptavidin-modified HRP)
Stained specimen slides were prepared and observed in the same procedure as in Example 1 except that the “tyramide treatment” was not performed. In this embodiment, fluorescent dye-integrated melamine resin particles modified with streptavidin are bound to biotin modified to the secondary antibody. The photographed fluorescent image is shown in FIG.

[Example 2] Second embodiment using quantum dots (tyramide treatment step, using polyclonal secondary antibody)
Example 1 except that streptavidin-modified quantum dots “SA-QD625” (Life technologies) were used instead of streptavidin-modified fluorescent dye-integrated melamine resin nanoparticles as modified fluorescent nanoparticles. In the same procedure, a stained specimen slide was prepared and observed. However, the excitation time during imaging of the quantum dots was 500 ms. Quantum dots were used. The photographed fluorescence image is shown in FIG.

[Comparative Example 2]
Stained specimen slides were prepared and observed in the same procedure as in Example 2 except that the “tyramide treatment” was not performed. In this embodiment, quantum dots modified with streptavidin bind to biotin modified to the secondary antibody. The photographed fluorescence image is shown in FIG.

[Discussion]
In FIG. 4 [A], a bright spot in the nucleus stained with DAPI (blue in the color photograph) (representing the presence of the amplified HER2 gene, red in the color photograph) that cannot be detected in FIG. 4 [B] is detected. You can see that it is made. Note that bright spots are also present outside the nucleus as non-specific staining, which can be reduced by optimizing the blocking solution or the like.

  In FIG. 5 [A], it can be seen that bright spots in the nucleus stained with DAPI (representing the presence of the amplified HER2 gene) that cannot be detected in FIG. 5 [B] can be detected. Note that bright spots are also present outside the nucleus as non-specific staining, which can be reduced by optimizing the blocking solution or the like. In FIG. 5 [A], quantum dots (average particle diameter of 20 nm) smaller than fluorescent dye-integrated nanoparticles (average particle diameter of 80 nm) were used, and therefore, signals in the nucleus (scattered bright spots) compared to FIG. 4 [A]. Increase is observed.

DESCRIPTION OF SYMBOLS 1 Target nucleic acid 2 Nucleic acid probe 3 Nucleic acid probe modified molecule 4 Modified nucleic acid probe 10 Monoclonal primary antibody 20a Monoclonal secondary antibody 20b Polyclonal secondary antibody 21 Secondary antibody modified molecule 22a Modified monoclonal secondary antibody 22b Modified polyclonal secondary antibody 50 Biotin 60 Avidin 70 Peroxidase 71 Peroxidase-modified molecule 72 Modified peroxidase 80 Avidin-biotin complex (ABC)
90a Tylamide before activation 90b Activated tyramide 91 Tylamide modified molecule 92a Modified tyramide 92b before activation 92b Activated modified tyramide 100 Fluorescent nanoparticle 101 Fluorescent nanoparticle modified molecule 102 Modified fluorescent nanoparticle

Claims (8)

A step (hybridization step) of hybridizing a target nucleic acid with a nucleic acid probe having a base sequence complementary to the base sequence and having a total base length of 3000 or less;
A step of amplifying a site to which fluorescent nanoparticles can be bound, selected from the following step (1) or (2) (binding site amplification step);
Step (1): Biotin as a starting point of amplification is directly or indirectly bound to the nucleic acid probe in advance, and bound to the biotin and avidin contained in the avidin-biotin complex (ABC). Treatment for modifying a nucleic acid probe with biotin contained in ABC as a site capable of binding to fluorescent nanoparticles (ABC treatment step)
Step (2): Peroxidase is directly or indirectly bound to the nucleic acid probe in advance, and in the presence of peroxide, the peroxidase is contacted with tyramide modified with a molecule capable of binding to fluorescent nanoparticles. Treatment for depositing sites capable of binding to fluorescent nanoparticles around the nucleic acid probe (tyramide treatment step)
A method for FISH (fluorescence in situ hybridization) staining of nucleic acids, comprising a step of binding fluorescent nanoparticles to the binding site (fluorescent nanoparticle binding step). In the binding site amplification step, in order to bind biotin (step 1) or peroxidase (step 2) as a starting point of amplification indirectly to the nucleic acid probe,
The nucleic acid probe is modified with a hapten,
After binding an anti-hapten polyclonal antibody or an anti-hapten monoclonal antibody as a primary antibody to the nucleic acid probe modified with the hapten,
The nucleic acid FISH staining method according to claim 1, wherein a polyclonal antibody against the primary antibody is bound to the primary antibody as a secondary antibody.   The nucleic acid FISH staining method according to claim 1 or 2, wherein the hapten is selected from the group consisting of dinitrophenol, digoxigenin and FITC (fluorescein isothiocyanate).   The nucleic acid FISH staining method according to any one of claims 1 to 3, wherein after the fluorescent nanoparticle binding step, a step (immobilization step) for preventing the fluorescent nanoparticle from being released from the bonded site is performed.   The nucleic acid FISH staining method according to any one of claims 1 to 4, wherein the fluorescent nanoparticles are fluorescent dye-integrated nanoparticles, and an average particle diameter thereof is 20 nm to 300 nm.   The nucleic acid FISH staining method according to any one of claims 1 to 4, wherein the fluorescent nanoparticles are inorganic phosphor-integrated nanoparticles, and an average particle diameter thereof is 20 nm to 300 nm.   The nucleic acid according to any one of claims 1 to 4, wherein the fluorescent nanoparticles are inorganic phosphor nanoparticles selected from the group consisting of quantum dots and carbon dots, and the average particle diameter thereof is 1 nm to 300 nm. FISH staining method.   8. The FISH staining of nucleic acid according to claim 1, wherein fluorescent nanoparticles are bound to a plurality of binding sites amplified by the binding site amplification step in the FISH staining method according to claim 1. A pathological specimen.