JP6736921B2 - FISH staining method - Google Patents

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 and the like. More specifically, the present invention relates to a FISH staining method for labeling a target nucleic acid with fluorescent nanoparticles.

Pathological diagnosis involves slicing a tissue section taken from a patient to prepare a slide, observing the morphology of cells or tissues based on the stained image when stained by a predetermined method, and expressing specific biomolecules. It is a method of diagnosing various events such as whether or not the patient suffers from a specific disease or whether or not a specific therapeutic drug is successful by quantifying and evaluating the level.

For example, using a tissue section prepared by collecting a cancer tissue, a HER2 gene (HER2/neu, c-erbB-2), which is one of oncogenes, and/or a membrane protein produced from the HER2 gene 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 targeted drug "trastuzumab" (trade name "Herceptin" ( A pathological diagnosis for predicting a therapeutic effect by a registered trademark) or an anti-HER2 monoclonal antibody) is widely performed. The fluorescence in situ hybridization (FISH) method is a typical DNA level inspection method.

The FISH method uses, for example, formalin-fixed paraffin-embedded tissue sections and cytology slides, using a fluorescence-labeled nucleic acid probe, the degree of amplification of the expression of DNA, RNA region, which is a hybridizing partner, and translocation of DNA region. It is a method for 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 a phosphor-assembled nanoparticle formed by accumulating a phosphor and a nucleic acid molecule having a predetermined base sequence are bound. It is described that by using such a probe reagent, a fluorescent signal can be stably obtained while suppressing non-specific adsorption. In addition, in the document, (i) the phosphor-assembled nanoparticles and the nucleic acid molecule are bound to the nucleic acid molecule by a covalent bond even if they are bound by a covalent bond using a linker molecule if necessary. The first biomolecule (eg biotin) and the second biomolecule (eg streptavidin) covalently linked to the phosphor-assembled nanoparticles may be indirectly bound by a specific binding, (ii) The site in the nucleic acid molecule for directly or indirectly binding the fluorescent substance-assembled nanoparticles may be introduced at one or more sites by a 5'end or 3'end labeling method, a nick translation method, or the like. What is possible, (iii) it is preferable that the nucleic acid molecule and the phosphor-assembled nanoparticles are bound at a molar ratio of 1:1 to 1:40 when the number of bases in the nucleic acid sequence is 5000 or less. Has been done. For example, as an embodiment using the probe reagent for indirectly binding the nucleic acid molecule and the fluorescent substance-assembled nanoparticles in (i) above, a nucleic acid having a predetermined sequence and a nucleic acid in which a plurality of first biomolecules are linked After the hybridization with the molecule, the fluorescent substance-assembled nanoparticles to which the second biomolecule is linked are added to the reaction system of the hybridization to specifically target the second biomolecule to the first biomolecule. FISH has also been proposed in which a nucleic acid having the above-mentioned predetermined sequence is fluorescently labeled by binding.

On the other hand, as a means to enhance the signal emitted from the labeled substance bound to a specific analyte (analyte) and detect the analyte with high sensitivity, it is possible to detect the analyte in the vicinity of the analyte activated by the enzyme. A sensitization method utilizing 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) activated by an enzyme (eg, horseradish peroxidase) and a detectable substance capable of directly or indirectly generating a detectable or quantifiable signal. A method for detecting or quantifying an analyte by an analyte-dependent enzyme activation system utilizing a conjugate with another label (for example, an enzymatic label, a fluorescent label, or a chemiluminescent label) is disclosed. As an example of the embodiment, the following is described in Example 8. First, an analyte (antibody) is bound to a 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 secondary antibody thus prepared is bound by an 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. In addition, a conjugate of streptavidin and galactosidase is added and allowed to bind to biotin linked to the deposited tyramine. Finally, the analyte is quantified by adding 4-methylumbelliferyl-β-D-galactoside (MUG), reacting with galactosidase, and measuring the fluorescence emitted thereby.

Patent Document 2 exemplifies various labels as the label, but does not specifically disclose the use of phosphor-collected nanoparticles or other phosphor particles. In the above-mentioned examples, the total amount of fluorescence generated from the fluorescent dye generated by the microtiter plate reader is measured, but information such as the position and number of the bright spots of the phosphor-collected nanoparticles is acquired. is not. Further, the method described in Patent Document 2 is basically a means for amplifying a label for detection and quantification of a protein contained in a blood sample or the like as an analyte. No amplification of a label for detection and quantification is described.

On the other hand, in Patent Document 3 (Japanese Patent Laid-Open No. 2013-531801), in an in situ hybridization assay in which a specific gene is detected and quantified, (i) a target nucleic acid sequence is labeled with a hapten (eg, digoxigenin). Binding of the nucleic acid probe prepared above, (ii) reacting a conjugate of an anti-hapten antibody corresponding to the hapten and peroxidase with the hapten to immobilize peroxidase, (iii) in the presence of peroxide , A plurality of conjugates of a hapten with an aryl moiety (eg, tyramine or a tyramine derivative) that can be activated by peroxidase are reacted with the peroxidase and deposited in the vicinity of the immobilized peroxidase (phenol-containing compound such as tyrosine and dimer). Body forming step), (iv) the step of reacting the conjugate of the fluorescent label and the anti-hapten antibody with the hapten of the deposited conjugates, the fluorescence signal of the nucleic acid sequence can be amplified. It is disclosed. 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 via binding with the hapten, and the fluorescent label is avidin. There is no mention of indirect conjugation via conjugation of biotin with biotin. Further, it is not specifically disclosed that the fluorescent substance-assembled nanoparticles are used as the fluorescent label.

Further, in Non-Patent Document 1, in order to detect the HER2 gene on a chromosome, various FISH probes modified with a fluorescent dye molecule and having a base length of about 200 are prepared, and a plurality of them are used in combination to obtain a probe The detectability of the HER2 gene is verified while changing the total base length. As a result, it is described that if the FISH probe has a total length of at least about 3000 (200×15) bases, the bright spots of the fluorescent dye molecule could be visually recognized with a microscope. Conversely, the description in Non-Patent Document 1 indicates that it is difficult to visually recognize with a microscope when the total base length of the probes is 3000 or less.

WO2015/141856 US5196306 Special table 2013-531801

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

In the typical embodiment of the 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 fragments linked to the probe are attached. The nucleic acid having the predetermined sequence is fluorescently labeled with the fluorescent substance-assembled nanoparticles by binding the biomolecule and the second biomolecule corresponding thereto. However, since the fluorescent substance-assembled nanoparticles have a relatively large particle size, the three-dimensional structure is increased between the fluorescent substance-assembled nanoparticles that bind to the probe and the proteins such as histones that surround the nucleic acid. It is considered prone to failure. Due to that effect, the fluorescent substance-assembled nanoparticles become difficult to approach to the probe bound to the target nucleic acid, and conversely, when the fluorescent substance-assembled nanoparticles are bound to the probe in advance, the probe becomes a target nucleic acid. It becomes difficult to approach. From such a viewpoint, in conventional FISH, the binding reactivity when modifying a target nucleic acid using a large number of fluorescent substance-assembled nanoparticles, in other words, the number of fluorescent substance-assembled nanoparticles to be bound and the binding force thereof. There was room for improvement in the labeling rate based on.

On the other hand, if fluorescent nanoparticles with a smaller particle size, such as quantum dots, are used instead of the fluorescent nanoparticles, the problems due to steric hindrance described above may not occur. Since the number of bright spots per particle is smaller than that of particles, 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 the preparation and the better the handleability, but the total amount of the fluorophore and other fluorophores that can be directly bound to the probe is small, and thus the detection of the target nucleic acid is performed. Or it tends to be difficult to quantify.

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

By incorporating a step of amplifying a site to which a fluorescent nanoparticle can bind, by binding with a modified molecule as a starting point to a nucleic acid probe or binding to a molecule around a nucleic acid probe, the present inventors have described above. It has been found that it becomes possible to carry out the FISH staining method for nucleic acids which has solved various problems. That is, the present invention includes the following respective inventions.

[1]
A step of hybridizing a target nucleic acid with a nucleic acid probe having a base sequence complementary to the target nucleic acid and having a total base length of 3000 or less (hybridization step);
A step of amplifying a site capable of binding to a fluorescent nanoparticle (binding site amplification step), which is selected from the following steps (1) and (2):
Step (1): Biotin serving as a starting point of amplification is directly or indirectly bound to the nucleic acid probe in advance, and the biotin is bound to avidin contained in the avidin-biotin complex (ABC). Treatment for modifying a nucleic acid probe with biotin contained in the ABC as a site capable of binding to fluorescent nanoparticles (ABC treatment step)
Step (2): Peroxidase is bound to the nucleic acid probe directly or indirectly beforehand, and the peroxidase is contacted with a tyramide modified with a molecule capable of binding to fluorescent nanoparticles in the presence of peroxide. Treatment to deposit a site capable of binding to fluorescent nanoparticles around the nucleic acid probe (Tylamide treatment step)
A method of FISH (fluorescence in situ hybridization) staining of a nucleic acid, 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 indirectly bind biotin (step 1) or peroxidase (step 2) as an origin of amplification 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 hapten-modified nucleic acid probe,
The method for staining a nucleic acid with FISH 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 nucleic acid according to [1] or [2], wherein the hapten is selected from the group consisting of dinitrophenol, digoxigenin and FITC (fluorescein isothiocyanate).

[4]
FISH staining of the nucleic acid according to any one of [1] to [3], wherein after the fluorescent nanoparticle binding step, a step (immobilization step) of preventing the fluorescent nanoparticles from being released from the binding 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-assembled nanoparticles, and the average particle diameter thereof is 20 nm to 300 nm.

[6]
The method for staining a nucleic acid with FISH according to any one of [1] to [4], wherein the fluorescent nanoparticles are inorganic fluorescent substance-assembled nanoparticles, and the average particle size thereof is 20 nm to 300 nm.

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

[8]
In the FISH staining method according to any one of [1] to [7], fluorescent nanoparticles are bound to a plurality of binding sites amplified in the binding site amplification step, FISH-stained pathological specimen.

The FISH staining method according to the present invention labels a target nucleic acid by significantly amplifying the number of sites to which the fluorescent nanoparticles present in the nucleic acid probe or its periphery can bind using a predetermined treatment. The number of fluorescent nanoparticles can be increased, and further, the detachment of the fluorescent nanoparticles due to washing or the like can be suppressed. In this way, by dramatically improving the labeling ability of the fluorescent nanoparticles, the target nucleic acid can be used even when using a nucleic acid probe having a total base length of 3000 or less and relatively easy to design, produce and handle. It is possible to achieve a sufficient brightness (sensitivity) for the detection and quantification of A, and to improve the reproducibility of the staining results between samples.

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 diagram showing an example of a preferred reaction mode 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 is [A] a fluorescent image taken in Example 1 and [B] a fluorescent image taken in Comparative Example 1. FIG. 5 shows a fluorescent image taken in [A] Example 2 and a fluorescent image taken in [B] Comparative Example 2.

The nucleic acid FISH staining method according to the present invention and a pathological specimen stained using the method will be described below.
-Nucleic acid FISH staining method-
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 if necessary, further includes an "immobilization step". 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 hybridized in this step is a nucleic acid probe modified with a predetermined molecule (referred to as “nucleic acid probe modifying molecule”) according to the embodiment of the next binding site amplification step (referred to as “modified nucleic acid probe”). .)

The nucleic acid probe may be composed of one nucleic acid molecule or may be composed of two or more nucleic acid molecules (mixture). That is, the provision 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 2 or more nucleic acid molecules in the latter case. It means that the total of the respective base lengths is 3000 or less. When two or more nucleic acid molecules are used as the nucleic acid probe, the base sequences of the respective nucleic acid molecules are complementary to the base sequences of different (non-overlapping) regions of the target nucleic acid. For example, a set of 10 nucleic acid molecules each having a length of 200 bases can be used as a nucleic acid probe having a total base length of 2000 (200×10).

(Binding site amplification step)
The binding site amplification step performed after the nucleic acid probe contact 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 and 2.
Step 1: Biotin as an origin of amplification is previously bound to the nucleic acid probe directly or indirectly, and the biotin is bound to avidin contained in an avidin-biotin complex (ABC). Thereby, the nucleic acid probe is modified with a plurality of biotins contained in the ABC as sites capable of binding to the fluorescent nanoparticles (ABC processing step).
Step 2: By previously or directly binding peroxidase to the nucleic acid probe and contacting the peroxidase with a tyramide modified with a molecule capable of binding to fluorescent nanoparticles in the presence of peroxide. , A process of depositing a plurality of sites capable of binding to fluorescent nanoparticles around the nucleic acid probe (tilamide treatment process).

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

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

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

The means for "binding to biotin (as a starting point of amplification) and avidin contained in the avidin-biotin complex (ABC)" is not particularly limited, and may be selected from various known means. it can. In general, a substance that supports multiple biotins (eg, peroxidase) is prepared in advance, and then biotin, which is the starting point of amplification, is first contacted with avidin to bind to it, and then the avidin is prepared. The biotin-supported material that has been set aside is contacted so that ABC is formed together with surplus avidin that has not bound to biotin as a starting point of amplification. Alternatively, the biotin-supporting substance and avidin were first contacted with each other to form ABC, and the ABC was contacted with biotin as a base point for amplification, which was unreacted in ABC (not yet bound with biotin). A) avidin may be reacted with the biotin. In the step (1), a plurality of biotins contained in ABC modified with the nucleic acid probe as described above become sites capable of binding to the fluorescent nanoparticles. The biotin-supporting substance is not limited to peroxidase, and a substance (molecule) of an appropriate size capable of supporting a plurality of biotins can be used.

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

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

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

As an example of a means for "preliminarily directly or indirectly binding peroxidase to a nucleic acid probe" in step (2), an implementation using the avidin-biotin complex (ABC) described above in relation to step (1) The form may be mentioned. That is, by binding ABC using peroxidase as a biotin-supporting substance to biotin corresponding to “biotin as a 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 a large number of peroxidases are contained in ABC, the reaction efficiency when contacting tyramide modified with a molecule capable of binding to fluorescent nanoparticles in the presence of peroxide is improved, resulting in more fluorescence. It becomes possible to deposit a site capable of binding with nanoparticles around the nucleic acid probe. When the fluorescent nanoparticle binding step of binding the fluorescent nanoparticles to the site capable of binding to the deposited fluorescent nanoparticles is performed after performing the step (2) of the preferred embodiment, the ABC treatment as described above is performed. As compared with the case of performing the fluorescent nanoparticle binding step of binding the fluorescent nanoparticles to the biotin in ABC after performing the step (1), amplification of the detection sensitivity of about 100 times can be expected, so that the base length of the nucleic acid probe is Is particularly preferable 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 the fluorescent nanoparticles to the binding site amplified in the binding site amplification step. By this step, the target nucleic acid can be labeled with the fluorescent nanoparticles.

When the binding site amplification step is the ABC treatment step of step (1), the “binding site amplified by the binding site amplification step” is “biotin contained in ABC”, and therefore the fluorescent nanoparticles used in the fluorescent nanoparticle binding step are , A molecule that specifically binds to biotin, that is, avidin-modified fluorescent nanoparticles. 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 “tylamide modification molecule”, the fluorescent nanoparticles used in the fluorescent nanoparticle binding step are The fluorescent nanoparticles are modified with a molecule that specifically binds to the modified molecule.

(Immobilization process)
The immobilization step, which is optionally performed after the fluorescent nanoparticle binding step, involves cross-linking the fluorescent nanoparticles bound at a predetermined binding site with the proteins around them, or cross-linking between the proteins around the fluorescent nanoparticles. By making it difficult for the fluorescent nanoparticles that have entered the cell nucleus to go 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 peeled off by subsequent washing treatment. It is a process for.

Such a step is performed by, for example, reacting an amino group of a phosphor-assembled nanoparticle and an amino group of a protein around it, or an amino group of a protein around a phosphor-assembled nanoparticle with a compound such as an aldehyde. Can be carried out by cross-linking (methylene cross-linking). As the immobilizing agent for causing such a crosslinking reaction, dialdehydes such as paraformaldehyde, glutaraldehyde and glyoxal are preferable.

In the present invention, the nucleic acid probe modifying molecule in the hybridization step, the primary antibody, the secondary antibody, the antibody modifying molecule, and the peroxidase modifying molecule in the binding site amplification step are selected according to the embodiment of the binding site amplification step. Embodiments such as the tyramide modifying molecule also vary. Hereinafter, the whole of the present invention when the ABC treatment step of the step (1) is adopted as the binding site amplification step is the "first embodiment", and the entire present invention when the tyramide treatment step of the step (2) is adopted is the "first embodiment". 2 embodiment”, and 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 Step 1: ABC treatment step is performed as the binding site amplification step. In the hybridization step, the modified nucleic acid probe (4) consisting of the nucleic acid probe (2) and the nucleic acid probe modifying molecule (3) hybridizes 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 a starting point for amplification of the monoclonal secondary antibody (20a) and amplification (corresponding to the secondary antibody modified molecule (21)). The modified monoclonal secondary antibody (22a) consisting of biotin (50) binds to the primary antibody (10). Furthermore, avidin-biotin complex (ABC) (80) consisting of avidin (60) and a plurality of biotins (50) supported by peroxidase (70) binds to the modified monoclonal secondary antibody (22a). In the fluorescent nanoparticle binding step, the modified fluorescent nanoparticles (102) composed of the fluorescent nanoparticles (100) and avidin (60) (corresponding to the fluorescent nanoparticle modifying molecule (101)) are contained in the ABC (80). Bind to (50).

FIG. 2 shows an example of the 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. In the hybridization step, the modified nucleic acid probe (4) consisting of the nucleic acid probe (2) and the nucleic acid probe modifying molecule (3) hybridizes to the target nucleic acid (1). In the binding site amplification step, the modified monoclonal secondary antibody (22a), which comprises the modified monoclonal nucleic acid probe (4) bound to the modified monoclonal primary antibody (10) and comprises the secondary monoclonal antibody (20a) and the modified secondary antibody molecule (21). Bind to the monoclonal primary antibody (10), and the modified peroxidase (72) consisting of the peroxidase (70) and the peroxidase modifying molecule (71) binds to the modified monoclonal secondary antibody (22a). Furthermore, in the presence of a peroxide (H ₂ 0 _2), the modified tyramide (92a) before being activated consists before being activated tyramide (90a) and tyramide modified molecule (91) Peroxidase (70 ), it is converted into activated modified tylamide (92b) consisting of activated tylamide (90b) and modified tylamide molecule (91), and is deposited around the nucleic acid probe (2). In the fluorescent nanoparticle binding step, the modified fluorescent nanoparticle (102) consisting of the fluorescent nanoparticle (100) and the fluorescent nanoparticle modifying molecule (101) becomes the tylamide modification molecule (91) contained in the activated modified tylamide (92b). Join.

An example of the 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 one nucleic acid molecule, and the target nucleic acid (1) is hybridized with the plurality of modified nucleic acid probes (4). And (ii) instead of the modified monoclonal secondary antibody (22a) consisting of the monoclonal secondary antibody (20a) and the secondary antibody modifying molecule (21), a polyclonal secondary antibody (20b) and a secondary antibody As shown in FIG. 2, 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). It's different.

(Target nucleic acid)
The nucleic acid targeted in the FISH staining method of the present invention, that is, the nucleic acid to be detected and quantified (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, DNA or RNA having various base sequences is included in the target nucleic acid. Typically, a gene having a specific nucleotide sequence present on a chromosome serves as a target nucleic acid, but a non-chromosomal nucleic acid such as mRNA, tRNA, rRNA, miRNA, siRNA, non-coding RNA may also serve as a target nucleic acid. ..

Biomarkers that can serve as target nucleic acids include biomarkers for diagnosis, biomarkers for determining the stage of disease, biomarkers for disease prognosis, and biomarkers for monitoring for the purpose of observing response to therapeutic treatment. For example, HER2, TOP2A, HER3, EGFR, P53, MET etc. are mentioned as a gene related to cancer growth and the response rate of a molecular target drug. Furthermore, the following are examples of genes known as cancer-related genes. 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, BMI1. , LGR5, EPHB3, RET, EPHB4, RON (MST1R), FGFR1, ROS (ROS1), FGFR2, TIE2 (TEK), FGFR3, TRKA (NTRK1), FGFR4, TRKB (NTRK2), FLT1 (VEGFR1), VEGFR1 and TRKR (TRKTR). In addition, examples of breast cancer-related genes include ATM, BRCA1, BRCA2, BRCA3, CCND1, E-Cadherin, ERBB2, ETV6, FGFR1, HRAS, KRAS, NRAS, NTRK3, p53, and PTEN. Related genes include BCL2, BRD4, CCND1, CDKN1A, CDKN2A, CTNNB1, HES1, MAP2, MEN1, NF1, NOTCH1, NUT, RAF, SDHD, VEGFA, and APC, MSH6, AXIN2. , MYH, BMPR1A, p53, DCC, PMS2, KRAS2 (or Ki-ras), PTEN, MLH1, SMAD4, MSH2, STK11, MSH6. Lung cancer-related genes include ALK, PTEN, CCND1, RASSF1A, CDKN2A. , RB1, EGFR, RET, EML4, ROS1, KRAS2, TP53, MYC, etc. As liver cancer-related genes, Axin1, MALAT1, b-catenin, p16 INK4A, c-ERBB-2, p53, CTNNB1, RB1, Cyclin D1, SMAD2, EGFR, SMAD4, IGFR2, TCF1, KRAS, etc. Kidney cancer related genes include Alpha, PRCC, ASPSCR1, PSF, CLTC, TFE3, p54nrb/NONO, TFEB. Cancer-related genes Then, AKAP10, NTRK1, AKAP9, RET, BRAF, TFG, ELE1, TPM3, H4/D10S170, TPR can be mentioned. Examples of ovarian cancer-related genes include AKT2, MDM2, BCL2, MYC, BRCA1, NCOA4, CDKN2A, p53, ERBB2, PIK3CA, GATA4, RB, HRAS, RET, KRAS, and RNASET2. 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 is composed of a nucleic acid molecule having a base sequence (probe sequence) complementary to part or all of the base sequence of the target nucleic acid and capable of forming a complementary chain with the target nucleic acid. The nucleic acid probe may be composed of naturally occurring molecules such as DNA and RNA, and may be PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid, BNA: sometimes referred to as Bridged Nucleic Acid), etc. It may be composed of an artificial nucleic acid molecule, or may be composed of both of them.

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, 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 by using known means. As described above, when a set of two or more nucleic acid molecules is used as the nucleic acid probe, the base sequence of each nucleic acid molecule is complementary to the base sequences of different (non-overlapping) regions of the target nucleic acid. Designed to.

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

When detecting a normal structural gene, the probe sequence preferably does not include a gene sequence portion having a polymorphism in copy number 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 to 2, so when the copy number estimated from the number of bright spots of the fluorophore is 3 or more. When the copy number is 0, on the contrary, it can be determined that the abnormality of the chromosome in which the gene is amplified has occurred. When the sequence of the polymorphic gene as described above is included in the probe sequence, the number of bright spots of the fluorophore does not match the copy number of the specific gene of interest, which hinders the detection of the copy number as described above. Come.

In addition, for example, a specific base (for example, thymine (T)) in a nucleic acid molecule is replaced with a biotin-labeled nucleotide (for example, Biotin-16-dUTP) by nick translation, and biotin at the substitution site is replaced with an enzyme-modified molecule. A modified enzyme having avidin or an anti-avidin antibody as a primary antibody can be bound. 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, so The probe sequence may be designed by determining a specific base in the nucleic acid molecule so that the strength has a desired degree.

Regarding the method of obtaining a nucleic acid probe, if the number of bases of the nucleic acid is several tens to several hundreds, submit the data of the sequence of the nucleic acid including the probe sequence and request the nucleic acid synthesis contract service such as Funakoshi to provide the nucleic acid probe. It is preferable to obtain it. 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 whether the nucleic acid probe is correctly formed by sequencing the base sequence. For example, the confirmation may be performed as follows. It should be noted that the probes designed and prepared by various methods as described above are generally called a DNA probe when the nucleic acid sequence is DNA and an RNA probe when the nucleic acid sequence is RNA.

The first method is to design and synthesize a primer so as to sandwich the probe sequence portion contained in the genomic DNA of the organism to be detected, and use this set of primers for the genomic DNA (or the BAC clone library described above). PCR method using pfu DNA polymerase with high replication accuracy is performed on a genomic library (eg, etc.). Next, the PCR reaction solution is separated by electrophoresis, the 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 (BAC plasmid or the like) is transformed into Escherichia coli (E. coli HST08 Premium Electro-Cells (Takara Bio Inc.) or the like), cultured (amplified), and collected. The nucleic acid can be obtained by performing nucleic acid extraction, cutting out the portion corresponding to the nucleic acid probe with a predetermined restriction enzyme, and performing electrophoresis and nucleic acid purification as described above.

Further, unlike the above-mentioned method for obtaining a nucleic acid probe, a nucleic acid probe is produced 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 is obtained by modifying a nucleic acid probe with a predetermined substance used according to the embodiment of the binding site amplification step, that is, avidin-biotin complex (ABC) in the first embodiment, and in the second embodiment. A molecule for indirectly binding peroxidase. When the nucleic acid probe is a set composed 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 the nucleic acid probe and specifically binding thereto, typically an antibody (where the nucleic acid probe modifying molecule serves as an antigen) is suitable. Such nucleic acid probe modifying molecules include, for example, biotin, avidin and haptens. Haptens include, for example, dinitrophenol, digoxigenin and FITC (fluorescein isothiocyanate). Antibiotin antibody and anti-avidin antibody can be prepared for biotin and avidin, and anti-dinitrophenol antibody, anti-digoxigenin antibody, anti-FITC antibody, etc. can be prepared for haptens such as dinitrophenol, digoxigenin and FITC. it can. Those antibodies can be used as a primary antibody for indirectly binding biotin (first embodiment) or peroxidase (second embodiment) to a nucleic acid probe in the embodiments as described above.

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

(Method for producing modified nucleic acid probe)
The method for introducing the modified molecule into the nucleic acid probe is not particularly limited, and various known methods can be adopted, but from the viewpoint of stability of binding, it is preferable to use a bond having a strong binding force such as a covalent bond. As a method 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, for example, Biotin-dNTP (N is adenine (A), guanine (G), cytosine) is used. (C), thymine (T), or uracil (U) is 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 of the nucleic acid using a reagent, and a modified molecule having a corresponding functional group is reacted to covalently bond Method etc. are mentioned. In the above method (i), if nick translation is used, it is possible to introduce a plurality of modified molecules into a site other than the 5'end or the 3'end of the nucleic acid probe.

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

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

An antibody that specifically binds to the primary antibody is used as the secondary antibody. 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 in the latter case. It becomes possible.

(Antibody modified molecule)
An antibody-modifying molecule is introduced into a primary antibody or a secondary antibody in order to indirectly bind biotin (first embodiment: as a base for binding ABC) or peroxidase (second embodiment) to a 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 a base point of amplification for binding ABC serves as an antibody-modifying molecule, and in the second embodiment (tilamide treatment step), a modified peroxidase is bound. A molecule that specifically binds to the peroxidase-modifying molecule is an antibody-modifying molecule. When modifying a primary antibody, it may be called a primary antibody-modifying molecule, and when modifying a secondary antibody, it may be called a secondary antibody-modifying molecule. It is used as a general term for antibody-modifying molecules.

As the antibody-modifying molecule in the second embodiment, there is a molecule other than the antibody capable of modifying the antibody and specifically binding to the antibody (because the antibody is not used as the peroxidase-modifying molecule to bind to the antibody-modifying molecule, Antibodies are excluded from the molecule to be bound.) As long as it is one, it is not particularly limited. Examples of such an antibody-modifying molecule include biotin and avidin, and biotin is particularly preferable. As described below, for biotin and avidin, avidin and biotin are used as peroxidase-modifying molecules, respectively.

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

(Tyramide modified molecule)
The tyramide modification molecule used in the second embodiment of the present invention is a molecule for binding fluorescent nanoparticles around the nucleic acid probe. The tyramide-modified molecule is a molecule other than an antibody that can modify tylamide and that specifically binds to it (an antibody is not used as a fluorescent nanoparticle-modifying molecule that binds to a tylamide-modified molecule, so an antibody that specifically binds to Excluded.) is not particularly limited as long as it exists. Similar to the antibody-modifying molecule, examples of such a tyramide-modifying molecule include biotin and avidin, and biotin is particularly preferable.

In the second embodiment of the present invention, the antibody-modifying molecule or nucleic acid probe-modifying molecule to which the modified peroxidase binds and the tyramide-modifying molecule to which the modified fluorescent nanoparticle binds may be the same (for example, biotin). , But they may be different (for example, dinitrophenol for the former and biotin for the latter), but if they are the same, even if there is an unreacted nucleic acid probe modified molecule to which the modified peroxidase did not bind, the modified fluorescent nanoparticle exists there. Is preferable, and as a result, the number of fluorescent nanoparticles that label the target nucleic acid can be increased, which is preferable.

(Fluorescent nanoparticle modification molecule)
The fluorescent nanoparticle modifying molecule used in both the first embodiment and the second embodiment of the present invention specifically binds to biotin (first embodiment) or tyramide modifying molecule (second embodiment). Use the molecule. 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. Peroxidase (for example, HRP: horseradish peroxidase) acts on peroxide (for example, hydrogen peroxide) to generate free radicals, and activates (radicalizes) tyramide by a free radical chain reaction. That is, the tyramide can be activated by the treatment of reacting the peroxidase and the tyramide in the presence of peroxide. Although the details of the activated tylamide have not yet been elucidated, it is likely that a plurality of tyramides aggregate with each other to deposit (bond) with molecules around the nucleic acid probe. It may be prepared according to appropriate conditions for such treatment, for example, general conditions such as salt concentration, pH and temperature of the reaction solution.

(Method for producing modified tyramide and modified peroxidase)
Both the modified tyramide and the modified peroxidase can be produced by using a known method (see, for example, US Pat. No. 5,196,306: Patent Document 2 and Table 2013-531801: Patent Document 3). For example, biotin-modified tyramide can be prepared by introducing a reactive functional group into each of tylamide 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 the tyramide and biotin (that is, between the functional groups they have). The conjugate of biotin and tyramide is commercially available and can be easily obtained. Further, for example, a peroxidase modified with avidin can also be prepared by introducing a reactive functional group into peroxidase and avidin using a reagent and reacting these functional groups to form a covalent bond. .. If necessary, a linker may be interposed between peroxidase and avidin (that is, between the functional groups they have).

(Fluorescent nanoparticles)
In the present invention, fluorescent nanoparticles are used as a substance for fluorescently labeling a target gene. The fluorescent nanoparticle is a nano-sized (particle diameter is 1 μm or less) particulate phosphor, and one particle can emit fluorescence having sufficient brightness. Such phosphors can be roughly classified into phosphor-assembled nanoparticles and inorganic fluorescent nanoparticles. In addition, a single fluorescent dye is not used in the present invention because it cannot emit fluorescence having sufficient brightness. The fluorescent substance-assembled nanoparticles include fluorescent dye-encapsulated nanoparticles and inorganic fluorescent substance-assembled nanoparticles. Inorganic phosphor nanoparticles include quantum dots and carbon dots.

As the fluorescent nanoparticles, those that emit fluorescence (color) of a desired wavelength in a dyed image to be photographed may be selected. 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 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 the brightness of a bright spot having an observable brightness by themselves without preparing inorganic phosphor integrated nanoparticles as described below.

As the quantum dot, a II-VI group compound, a III-V group compound, or a quantum dot containing a IV group element as a component (“II-VI group quantum dot”, “III-V group quantum dot”, “ Any of group IV quantum dots") can be used. Specific examples thereof include particle dots such as CdSe exemplified in International Publication WO2012/133047. These inorganic phosphor nanoparticles may be used alone or in combination of two or more.

Further, it is also possible to use a quantum dot having the above-mentioned quantum dot as a core and a shell provided thereon. Hereinafter, as a notation of a quantum dot having a shell, when the core is CdSe and the shell is ZnS, it is expressed as CdSe/ZnS. Specific examples thereof include CdSe/ZnS and the like exemplified in International Publication WO2012/133047, but are not limited thereto.

The quantum dots may be surface-treated with an organic polymer or the like, if 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 are phosphors that have been attracting attention in recent years because they do not use harmful elements or rare metals unlike conventional quantum dots. Carbon dots include graphene quantum dots, carbon nanodots, polymer dots, and the like. Carbon dots are known substances, and details thereof are described in 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 is a type of graphene quantum dots, are manufactured by oxidizing and cutting 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 to generate carbon dots. A common oxidative cleavage method is oxidation with concentrated acid (nitric acid or sulfuric acid/nitric acid mixture).

There are two steps in manufacturing carbon dots. The first step is a step of converting a graphite-derived substance into a graphite oxide sheet, and this step is usually carried out by changing the Hummres method according to the purpose. The second stage is a step of 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 arc discharge, laser irradiation, and nanolithography using reactive ion etching (RIE), and methods such as electrochemical and hydrothermal/solvothermal oxidation. Are listed.

On the other hand, carbon dots can be produced by performing dehydration and carbonization treatment also 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 treatment methods include hydrothermal, microwave-hydrothermal, plasma-hydrothermal, microwave, combustion, concentrated acid pyrolysis, and carbonization by a microreactor.

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

Examples of the fluorescent dye used for producing the fluorescent dye-assembled nanoparticles include fluorescein dye, rhodamine dye, Alexa Fluor (registered trademark, manufactured by Invitrogen) dye, BODIPY (registered trademark, manufactured by Invitrogen) dye, and cascade. (Registered trademark, Invitrogen) dyes, coumarin dyes, NBD (registered trademark) dyes, pyrene dyes, cyanine dyes, perylene dyes, oxazine dyes, and other low molecular weight organic compounds (polymers such as polymers Fluorescent dyes selected from the group consisting of those which are not compounds). Of these, rhodamine-based dyes such as sulforhodamine 101 and its hydrochloride salt TexasRed (registered trademark) and perylene-based dyes such as perylenediimide are preferable because they have 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-assembled nanoparticles)
The method for producing the phosphor-integrated nanoparticles used in the present invention is not particularly limited, and can be produced by a known method. In general, a manufacturing method can be used in which the phosphors are collected by 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 a method for producing phosphor-assembled nanoparticles produced by using these resins as a matrix are based on a known document (WO2014/136767).

Furthermore, for example, silica nanoparticles encapsulating a fluorescent dye can be synthesized with reference to the synthesis of FITC-encapsulating silica particles described in Langmuir Vol. 8, page 2921 (1992). Various fluorescent dye-encapsulated 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-encapsulating silica nanoparticles described in New Journal of Chemistry, Vol. 33, page 561 (2009).

The average particle size of the fluorescent dye-assembled nanoparticles, the fluorescent substance-assembled nanoparticles such as the inorganic fluorescent substance-assembled nanoparticles is particularly preferably within a range that can be appropriately observed as a bright spot that emits fluorescence when the target gene is labeled. Although not limited, the average particle size of the phosphor-assembled 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.

Quantum dots, the average particle size of the inorganic phosphor nanoparticles such as carbon dots is also 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, but such a The average particle diameter of the inorganic phosphor nanoparticles is 1 nm or more and 300 nm or less, from the viewpoint of making it suitable for fluorescence observation of bright spots, particularly from the viewpoint of easily migrating into the nucleus and labeling the target gene. Is preferable, and more preferably 1 nm or more and 20 nm or less.

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

(Method for producing modified fluorescent nanoparticles)
The modified fluorescent nanoparticles can be prepared using a known method. For example, when fluorescent substance-assembled nanoparticles (fluorescent dye-assembled nanoparticles or inorganic fluorescent substance-assembled nanoparticles) are used as fluorescent nanoparticles, the surface of the fluorescent substance-assembled nanoparticles and the fluorescent nanoparticle-modifying molecule are each treated with a reagent or the like. By introducing a reactive functional group and binding these functional groups to each other, modified phosphor-assembled nanoparticles can be produced. When modifying the surface of the phosphor-assembled nanoparticles, an appropriate reagent or the like is used in consideration of the material (resin, silica, etc.) of the matrix. In addition, if necessary, a linker may be interposed between the avidin and the fluorescent substance-assembled nanoparticles (that is, between the functional groups that they have). Examples of the combination of reactive functional groups include a combination of NHS ester group-amino group and thiol group-maleimide group. For example, (i) the surface of the phosphor-assembled nanoparticles is modified with an amino group in advance using a silane coupling agent, and (ii) streptavidin is reacted with N-succinimidyl-S-acetylthioacetate on the other hand. A maleimide that has an NHS group that reacts with an amino group at one end and a thiol group that reacts with an amino group at one end as a crosslinker, by linking a thiol group protected by an acetyl group to the amino group that streptavidin has. Prepare a polyethylene glycol chain having a group (for example, "SM(PEG)12", Thermo Scientific Co., Ltd.), and (iv) share by reacting the amino group on the surface of the phosphor-collected nanoparticles with the NHS group of the crosslinker After binding, (v) deprotecting the thiol group of streptavidin, and then reacting with the maleimide group of the cross-linker, streptavidin-modified phosphor-assembled nanoparticles are obtained.

When the inorganic phosphor nanoparticles (quantum dots, carbon dots, etc.) are used as the fluorescent nanoparticles, a reagent or the like is used to react the surface of the inorganic phosphor nanoparticles and the fluorescent nanoparticle-modifying molecule with reactive functionalities. A modified inorganic phosphor nanoparticle can be produced by introducing a group or by utilizing a functional group possessed by a carbon dot by a manufacturing method and binding these functional groups to each other.

It should be noted that the number of fluorescent nanoparticle-modifying molecules (the number of modified molecules) that modifies the surface of the fluorescent nanoparticles is schematically illustrated in FIGS. 1 and 2 as one (1:1) for each fluorescent nanoparticle. 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, considering the molecular size of streptavidin, which is a protein having a molecular weight of about 52000, and the average particle size of the fluorescent nanoparticles is relatively large (for example, about 300 nm), It is possible to bind a plurality of streptavidins (for example, hundreds to thousands) to one fluorescent nanoparticle (1: many), and conversely, if the average particle diameter of the fluorescent nanoparticles 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 (many: 1). Generally, it can be said that the larger the number of modified molecules on the surface of the fluorescent nanoparticle, the higher the probability that the fluorescent nanoparticle can bind to the site capable of binding, but it is preferable. However, observation of an appropriate starting point according to the application such as pathological diagnosis The number of modified molecules can be appropriately adjusted within the range where the above can be performed.

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

-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. It is used to prepare a pathological specimen according to the present invention for making a pathological diagnosis, for example, for obtaining information useful for diagnosing or treating a disease. That is, a slide (specimen slide) prepared by using a tissue section collected from a diagnosis target is stained based on the nucleic acid FISH staining method according to the present invention and other staining methods that are combined as necessary, and obtained. For example, by quantifying a specific gene by observing the pathological specimen (a specimen slide that has been subjected to staining or other processing and can be an object of inspection) and/or image processing of its captured image. An examination can be performed to make a pathological diagnosis.

Such a pathological diagnosis is performed until the pathological specimen is prepared by staining the specimen slide (pathological specimen preparation step), that is, the step of performing the nucleic acid FISH staining method according to the present invention and the prepared pathological specimen. Can be roughly divided into a stage (pathological specimen examination stage) up to obtaining desired information.

At the stage of preparing a pathological specimen for FISH, generally, a sample slide preparation step (fixing treatment, dehydration treatment, paraffin embedding treatment, thin sectioning treatment, etc.), specimen pretreatment step (deparaffinization treatment, heat treatment, protease treatment, etc.) ), a staining step (DNA denaturation treatment, FISH staining treatment, post-fixation treatment, nuclear staining treatment, etc.) and a specimen post-treatment step (washing treatment, dehydration treatment, solvent substitution treatment, encapsulation treatment, etc.). The FISH staining method of the present invention can be used to perform the FISH staining treatment and the post-fixation treatment in the above staining step. That is, the hybridization step, the binding site amplification step, and the fluorescent nanoparticle reaction step defined 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 immobilization step can be incorporated as a post-immobilization treatment in the staining step.

Hereinafter, an example of a more specific procedure will be described for the dyeing step when the FISH dyeing method of the present invention is used. The other steps included in the step of preparing a stained specimen for FISH, that is, the specimen preparation step, the specimen pretreatment step and the specimen posttreatment step, and the stained specimen inspection step are common pathological specimen preparations using the conventional FISH staining method. It can be performed in the same manner as the step (see, for example, WO2015/141856: Patent Document 1).

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

(2) FISH Staining Treatment When preparing a pathological specimen using the present invention, the FISH staining treatment includes the hybridization step, binding site amplification step and fluorescent nanoparticle reaction step in the FISH staining method of the present invention as described above. The processing will follow.

(2-1) Hybridization Step The modified nucleic acid probe solution is dropped on the sample slide, and the target nucleic acid contained in the sample is brought into contact with the modified nucleic acid probe solution to perform hybridization. At this time, a buffer solution containing a component that promotes hybridization (it 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 binding site amplification step to be performed next. Such a modified nucleic acid probe is prepared in advance, a solution having an appropriate concentration is prepared, and used in this hybridization step.

Regarding the reaction conditions for hybridization, the GC content (Tm value) of the nucleic acid probe used, the concentration of monovalent cations present in the reaction system of hybridization (M), and the concentration of formamide of the reaction system (% The reaction temperature and reaction time may be set in consideration of the fact that the accuracy in binding the nucleic acid molecule to the nucleotide sequence of the target gene on the chromosome changes due to the above).

(2-2) Binding Site Amplification Step A solution according to the embodiment of the binding site amplification step is sequentially added dropwise to the specimen slide that has undergone the hybridization step, and the nucleic acid probe is used as a starting point for the modified molecule or the nucleic acid probe. The binding to the surrounding molecules amplifies the site to which the fluorescent nanoparticles can bind.

That is, in the case of 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 (eg biotinylated HRP) solution are sequentially added dropwise. In the case of conforming to the second embodiment of the present invention, for example, a peroxide aqueous solution such as hydrogen peroxide solution, a modified peroxidase solution, and a modified tyramide solution are sequentially added dropwise. The modified secondary antibody, modified peroxidase, and modified tyramide are prepared in advance as described above, and a solution having an appropriate concentration is prepared for use in this hybridization step.

Binding of primary antibody to modified nucleic acid probe molecule and binding of modified secondary antibody to primary antibody (first and second embodiments); Binding of ABC to biotin-modified secondary antibody (first embodiment) ); Binding of modified peroxidase to antibody-modifying molecule or nucleic acid probe-modifying molecule (second embodiment); Reaction of modified peroxidase and modified tyramide (second embodiment), etc., respectively, according to known binding conditions or reaction conditions. It suffices to perform it under the set appropriate temperature, time, and other conditions.

(2-3) Fluorescent Nanoparticle Binding Step A solution of modified fluorescent nanoparticles is dropped on the specimen slide that has undergone the binding site amplification step, and biotin (first embodiment) or tyramide modified molecule (second embodiment) contained in ABC is added. ) To attach 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 nanoparticles are fluorescent nanoparticles 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, a solution having an appropriate concentration is prepared, and used in this fluorescent nanoparticle binding step.

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

(3) Post-fixation treatment When a pathological specimen is prepared by 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 been subjected to FISH staining treatment, and the target nucleic acid, the modified nucleic acid probe, the modified fluorescent nanoparticle, and the intermediary for connecting them are provided. Contact with the substance used in the binding site amplification step (modified tyramide, ABC, primary antibody, modified secondary antibody, etc., if necessary), and at an appropriate temperature (usually room temperature) for an appropriate time and reaction I will let you. These reaction conditions can be appropriately adjusted. For example, when a paraformaldehyde solution is used as the immobilizing 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. You can

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

(5) Other treatments The dyeing process may include other process examples, if necessary. For example, before adding the primary antibody used as necessary in the first embodiment and the second embodiment, a blocking treatment using a commercially available blocking agent for suppressing nonspecific adsorption of the antibody in the tissue section is performed. Is preferably performed. Further, for example, after hybridization, it is preferable to perform a washing treatment using a commercially available detergent.

[Preparation Example 1] Preparation of DNP-modified nucleic acid probe for HER2 gene 5×GoTaq reaction buffer (Promega) 5 μL, GoTaq DNA polymerase (5 U/μL, Promega) in a 0.2 mL PCR strip tube (Bio-Rad) 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, dTTP (1 mM, Takara Bio Inc.) 3.75 μL, DNP -11-UTP (1 mM, Perkin-Elmer) 1.25 μL, human HER2 gene Forward primer (sequence: 5′-CGATGTGACTGTCTCCTCCC-3′, 10 μM) 0.5 μL, Reverse primer corresponding to the Forward primer (sequence: 5) A total of 25 μL of'-ATCCTACTCCATCCCAAGGCC-3', 10 μM) 0.5 μL, and milliQ 7 μL were prepared.

After setting the strip tube in a thermal cycler (Chromo4, Bio-Rad), PCR is carried out by the method from Step 1 to Step 5 shown below to prepare a PCR product which becomes a DNP-modified nucleic acid probe of about 200 base pairs. did. The PCR product was purified using the 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 (repeat 39 times)
Step 5: 72°C, 3 minutes

According to the method described above, one type of DNP-modified nucleic acid probe that hybridizes to about 200 base pairs in the HER2 region was prepared. Furthermore, four kinds of probes of about 200 base pairs which hybridize to other places in the HER2 region were prepared in the same manner except that the forward primer and the reverse primer having different base sequences were used, and these five kinds were mixed. It was one HER2-FISH probe. The total base length of the nucleic acid probe that hybridizes to the target HER2 region is about 1000 bases.

[Preparation Example 2] Preparation of avidin-modified fluorescent dye-accumulated melamine resin nanoparticles (i) Synthesis of fluorescent dye-accumulated melamine resin nanoparticles Sulforhodamine 101 ("Sulforhodamine 101", Sigma Aldrich, Texas Red dye) 2.5 mg was 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 solution after stirring, 1.5 g of melamine resin "Nikalac MX-035" (manufactured by Nippon Carbide Industry Co., Ltd.) was added, and the mixture was further heated and stirred for 5 minutes under the same conditions.

Formic acid (100 μL) was added to the stirred solution, and the solution was stirred for 20 minutes while maintaining the temperature of the solution at 60° C., and then the solution was allowed to cool to room temperature. Thus, a dispersion liquid 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 remove the supernatant. Then, the recovered particles X were washed with ethanol and water. The average particle diameter of the particles X was 80 nm.

(Ii) Connection of Maleimide Group to the Surface of Phosphor-Aggregated Melamine Resin Nanoparticles 0.1 mg of washed particles X was dispersed in 1.5 mL of ethanol, and aminopropyltrimethoxysilane "LS-3150" (manufactured by Shin-Etsu Chemical Co., Ltd.) ) 2 μL was added and the mixture was reacted for 8 hours at room temperature with stirring to perform surface amination treatment.

The concentration of the particle X whose surface was aminated was adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and the linker reagent “SM(PEG)12” ( (Thermo Scientific Co., Ltd.) 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 the mixture was centrifuged again under the same conditions. The particles were surface-modified with a PEG chain having a maleimide group at the end by performing washing with the same procedure three times.

(Iii) Linking of thiol group to streptavidin On the other hand, preparation of streptavidin having a sulfhydryl group was performed as follows. First, with respect to streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) 40 μL adjusted to 1 mg/mL, N-succinimidyl-S-acetylthioacetate (N-succinimidyl S-acetylthioacetate, SATA, pirace) 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, a known thiol group treatment was performed to generate a free thiol group (-SH) from the protected thiol group and to add a thiol group (-SH) to streptavidin.

This streptavidin solution is desalted with a gel filtration column (Zaba Spin Desalting Columns: Funakoshi), and is capable of binding to a particle X surface-modified with a PEG chain having a thiol group linked thereto, that is, having a maleimide group at the end. I got avidin.

(Iv) Binding of Fluorine-Accumulated Melamine Resin Nanoparticles to Streptavidin Particle X surface-modified with a PEG chain having a maleimide group, and streptavidin having a thiol group linked thereto in PBS containing 2 mM of EDTA. The maleimide group and the thiol group were covalently bonded by mixing and reacting for 1 hour. The reaction was stopped by adding 10 mM mercaptoethanol. 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 streptavidin-modified fluorescent dye-accumulated melamine resin nanoparticles.

[Example 1] Second embodiment using fluorescent dye-assembled nanoparticles (tilamide treatment step, using polyclonal secondary antibody)
(Deparaffinization treatment)
Deparaffinization was performed by treating a sample slide of a HER2-positive stained control specimen (“HER2 Dual ISH 3-in-1 control slide” manufactured by Roche) in the following order (1) to (5). (1) Immerse in xylene at room temperature for 5 minutes. This operation is repeated once again (two times in total). (2) Immerse the specimen slide in 100% ethanol at room temperature for 2 minutes. This operation is repeated once again (two times in total). (3) Immerse the sample slide in 70% ethanol at room temperature for 2 minutes. (4) Immerse the specimen slide in milliQ water for 2 minutes at room temperature.

(Heat treatment)
In order to improve the accessibility of the DNA probe, the sample slide was subjected to heat treatment in the order of (1) to (3) below to remove the proteins of the cell membrane and the nuclear membrane. (1) Treatment with MES buffer (HER2 FISH PharmDx “Dako” Vial 1 diluted 20 times with milliQ water) at 95 to 99° C. for 10 minutes, and then at room temperature for 15 minutes. (2) Immerse the specimen slide in Tris wash buffer (HER2 FISH PharmDx "Dako" Via 6 diluted 20 times with milliQ water) for 3 minutes to wash. (3) Repeat this washing operation once again.

(Protease treatment)
The 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, attach the lower end of the slide glass to a paper towel, and remove excess washing buffer. (2) 5 to 6 drops of protease solution (HER2 FISH PharmDx "Dako" Vial 2A) are dropped on the specimen slide and reacted at room temperature for 10 minutes. This dipping treatment is for degrading proteins of cell membrane and nuclear membrane, particularly collagen. (3) The sample slide is immersed in Tris wash buffer (HER2 FISH PharmDx "Dako" Via 6 diluted 20 times with milliQ water) for 3 minutes and washed. (4) Repeat this washing operation once again.

(dehydration)
After air-drying the sample slide, it was first immersed in 70% ethanol for 2 minutes at room temperature and then in 100% (dehydrated) ethanol for 2 minutes at room temperature. Then, the sample 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 dehydrated specimen slide, a hybridization treatment using the DNP-modified nucleic acid probe obtained in Preparation Example 1 was performed. .. (1) 1 μL of a DNP-modified nucleic acid probe prepared at 5 ng/μL and 9 μL of IQFISH FFPE Hybridization Buffer (Agilent Technology) are added to the hybridization region of a sample slide. Immediately after mixing by pipetting on the slide, a 22 mm×22 mm cover glass is covered, and the DNP-modified nucleic acid probe solution is uniformly spread. Avoid bubbles in the hybridization area. (2) Seal the cover glass with a paper bond (KOKUYO). (3) The sample slide is placed on a hybridizer (Dako), and denatured and hybridized at 80° C. for 10 minutes and then at 45° C. overnight.

(Washing of specimen slide)
The sample slides subjected to the above-mentioned hybridization treatment were subjected to the following treatments (1) to (6) in this order to wash the specimen slides. (1) Post-hybridization wash buffer: SSCwash Buffer (HER2 FISH PharmDx "Dako" Via 2A diluted 20-fold with milliQ water) is placed in a Coplin jar. Preheat in a warm bath until the post-hybridization wash buffer is 63°C. (2) Prepare another coplin jar 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 coplin jar containing post-hybridization wash buffer maintained at room temperature and remove the coverslips. (4) The specimen slide is immersed in a post-hybridization washing buffer solution kept at 63° C. and immersed for 10 minutes for washing. (5) Immerse in Tris wash buffer (HER2 FISH PharmDx "Dako" Via 6 diluted 20 times with milliQ water) for 3 minutes at room temperature to wash. Repeat this wash again. (6) Take out the sample slide from the Coplin jar and air-dry it in the dark.

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

(Binding of DNP-modified nucleic acid probe and primary antibody)
The DNP-modified nucleic acid probe and the primary antibody were bound by performing the following treatments (1) to (4) in this order on the specimen slide subjected to the blocking treatment. (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, and then wipe off the Antibody Diluent, Background Reducing liquid on the slide other than the cell periphery with a filter paper etc. Only wet condition. (2) 100 μL of a solution of an anti-DNP rabbit monoclonal antibody (Anti-DNP rabbit mAb manufactured by Roche) as a primary antibody was dropped on a specimen slide and reacted in a humid 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 immersed in PBS for 5 minutes for washing. (4) The PBS washing operation is repeated twice more.

(Binding of primary antibody and biotin-labeled secondary antibody)
The binding of the primary antibody and the biotin-labeled secondary antibody is performed by performing the following treatments (1) to (4) in this order on the sample slide treated with the above-mentioned DNP-modified nucleic acid probe and the primary antibody. Processed. (1) Shake the slide on the Kim towel up and down so that the corner hits the Kim towel, drop the solution on the slide, and wipe off the solution on the slide other than around the cells with a filter paper etc. to make only the cell surface wet. .. (2) As a biotin-labeled secondary antibody, 100 μL of a solution of biotin-labeled anti-mouse/rabbit polyclonal IgG antibody (Anti-mouse and rabbit Ab, manufactured by Roche) was dropped on the specimen slide, and reacted at room temperature for 60 minutes in a wet box It is bound to the primary antibody. (3) The solution on the slide is dropped on a Kim towel and then immersed in PBS for 5 minutes for washing. (4) The PBS washing operation is repeated twice more.

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

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

(B1) Following the treatment of (A4) above, shake the slide on a Kim towel up and down so that the corners hit the Kim towel, and drop the solution on the slide. Then, add the horseradish peroxidase solution on the slide other than around the cells to a filter paper, etc. Wipe off with to make only the cell surface wet. (B2) 3 drops of the biotin-labeled tyramide solution contained in the kit (it is considered that hydrogen peroxide is also contained in this solution) is dropped on the sample slide, and the reaction is performed in a humid box at room temperature for 15 minutes. (B3) Immerse in TBST for 5 minutes for cleaning. (B4) This washing operation is repeated twice more.

(Binding of melamine resin particles with fluorescent dye)
The binding treatment of the fluorescent dye-accumulated melamine resin particles was performed by performing the following treatments (1) to (4) in this order on the specimen slide treated with the above-mentioned thyramide. (1) Shake the slide on the Kim towel up and down so that the corners hit the Kim towel, drop the solution on the slide, and wipe off the biotin-labeled tyramide solution on the slide other than around the cells with a filter paper, etc. to wet only the cell surface. State. (2) 80 μL of a solution of streptavidin-modified fluorescent dye-accumulated melamine resin nanoparticles obtained in Preparation Example 2 whose concentration was adjusted to 0.1 nM using Antibody Diluent, Background Reducing Solution was dropped onto a specimen slide, The reaction is carried out at room temperature for 60 minutes to bond the biotin contained in tyramide and the streptavidin modified with the fluorescent dye-accumulated 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) This washing operation is repeated twice more.

(Fixed after)
The post-fixation treatment was performed by performing the following treatments (1) to (3) in this order on the specimen slide to which the binding treatment of the fluorescent dye-accumulated melamine resin particles was performed. (1) Shake the slide on the Kim towel up and down so that the corner hits the Kim towel, drop the solution on the slide, and wipe off the solution on the slide other than around the cells with a filter paper etc. to make only the cell surface wet. .. (2) 150 μL of 4% paraformaldehyde solution is dropped on the specimen slide, and immersed in a wet box at room temperature for 10 minutes. (3) The solution on the slide is dropped on a Kim towel and immersed in Tris EDTA buffer (Wako) for 5 minutes for washing.

(Nuclear stain)
Nucleus using DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) is obtained by performing the following treatments (1) to (4) in this order on the specimen slide that has undergone the post-fixation treatment. Dyeing treatment was performed. (1) Shake the slide on the Kim towel up and down so that the corner hits the Kim towel, drop the solution on the slide, and wipe off the solution on the slide other than around the cells with a filter paper etc. to make only the cell surface wet. .. (2) 150 μL of a staining solution containing 0.01% β-mercaptoethanol and 1.43 μM DAPI (Invitrogen) was added to the hybridization region of the sample slide, and reacted at room temperature for 15 minutes in a wet box, Stain cell nuclei. (3) The solution on the sample slide is dropped on a Kim towel and immersed in Tris EDTA buffer (Wako) for 5 minutes at room temperature for washing. (4) Immerse the sample slide in PBS buffer for 5 minutes to wash it.

(Enclosed)
The encapsulation process was performed by performing the following processes (1) and (2) on the sample slide that had been subjected to the nuclear staining process in this order. (1) Shake the slide on the Kim towel up and down so that the corner hits the Kim towel, drop the solution on the slide, and wipe off the solution on the slide other than around the cells with a filter paper etc. to make only the cell surface wet. .. (2) In the non-dried state, after adding Aquatex (MERCK Co., Ltd.) drop by drop onto the sample slide, the sample slide is covered with a cover glass, and the sample slide is protected from light and stored until signal measurement.

(Observation)
The specimen slide, which had been subjected to the FISH staining and the DAPI staining, produced through the above steps was observed using a fluorescence microscope "BZ-X710" (manufactured by KEYENCE CORPORATION), and a fluorescence image was acquired. The magnification of the objective lens was 100 times, and the excitation times at the time of imaging the DAPI and the fluorescent dye integrated melamine resin nanoparticles were 20 ms and 500 ms, respectively. The captured fluorescence image is shown in FIG.

[Comparative Example 1] (polyclonal secondary antibody, streptavidin-modified HRP)
A stained specimen slide was prepared and observed by the same procedure as in Example 1 except that the "tyramide treatment" was not performed. In this embodiment, the fluorescent dye-accumulated melamine resin particles modified with streptavidin are bound to the biotin modified to the secondary antibody. The captured fluorescence image is shown in FIG. 4[B].

Example 2 Second Embodiment Using Quantum Dots (Tyramide Treatment Step, Using Polyclonal Secondary Antibody)
Example 1 and Example 1 except that streptavidin-modified fluorescent dye-assembled melamine resin nanoparticles were replaced with streptavidin-modified quantum dots "SA-QD625" (Life technologies) as modified fluorescent nanoparticles. By the same procedure, a stained specimen slide was prepared and observed. However, the excitation time during imaging of the quantum dots was set to 500 ms. The quantum dots used were. The captured fluorescence image is shown in FIG.

[Comparative example 2]
A stained specimen slide was prepared and observed by the same procedure as in Example 2 except that the "tyramide treatment" was not performed. In addition, in this embodiment, the quantum dots modified with streptavidin are bound to the biotin modified to the secondary antibody. The captured fluorescence image is shown in FIG. 5[B].

[Discussion]
In FIG. 4[A], bright spots (representing the presence of amplified HER2 gene, red in the color photograph) in the nucleus stained with DAPI (blue in the color photograph), which cannot be detected in FIG. 4[B], are detected. You can see that it is done. In addition, bright spots exist outside the nucleus as non-specific stains, but these can be reduced by optimizing the blocking solution and the like.

In FIG. 5[A], it can be seen that bright spots (representing the presence of the amplified HER2 gene) in the nucleus stained with DAPI, which cannot be detected in FIG. 5[B], can be detected. In addition, bright spots exist outside the nucleus as non-specific stains, but these can be reduced by optimizing the blocking solution and the like. In FIG. 5[A], since quantum dots (average particle size 20 nm) smaller than the fluorescent dye-assembled nanoparticles (average particle size 80 nm) were used, signals in the nucleus (scattered bright spots) were compared to FIG. 4[A]. Is observed.

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 Tylamide 91 Tylamide Modification Molecule 92a Modified Tylamide Before Activation 92b Activation Modified Tylamide 100 Fluorescent Nanoparticle 101 Fluorescent Nanoparticle Modified Molecule 102 Modified Fluorescent Nanoparticle

Claims (7)

A step of hybridizing a target nucleic acid with a nucleic acid probe having a base sequence complementary to the target nucleic acid and having a total base length of 3000 or less (hybridization step);
A step of amplifying a site capable of binding to a fluorescent nanoparticle (binding site amplification step), which is selected from the following steps (1) and (2):
Step (1): Biotin serving as a starting point of amplification is directly or indirectly bound to the nucleic acid probe in advance, and the biotin is bound to avidin contained in the avidin-biotin complex (ABC). Treatment for modifying a nucleic acid probe with biotin contained in the ABC as a site capable of binding to fluorescent nanoparticles (ABC treatment step)
Step (2): Peroxidase is bound to the nucleic acid probe directly or indirectly beforehand, and the peroxidase is contacted with a tyramide modified with a molecule capable of binding to fluorescent nanoparticles in the presence of peroxide. Treatment to deposit a site capable of binding to fluorescent nanoparticles around the nucleic acid probe (Tylamide treatment step)
Look including a step (fluorescent nanoparticles coupled step) of bonding the fluorescent nanoparticle to the binding site,
After the fluorescent nanoparticle binding step, a step (immobilization step) of preventing the fluorescent nanoparticles from being released from the binding site is performed,
A method for FISH (fluorescence in situ hybridization) staining of a nucleic acid, wherein the immobilization step is a step of cross-linking the fluorescent nanoparticles with surrounding proteins or a step of cross-linking proteins around the fluorescent nanoparticles . In the binding site amplification step, in order to indirectly bind biotin (step 1) or peroxidase (step 2) as an origin of amplification 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 hapten-modified nucleic acid probe,
The FISH staining method for nucleic acid according to claim 1, wherein a polyclonal antibody against the primary antibody is bound as a secondary antibody to the primary antibody. The method for FISH staining of nucleic acid according to claim 2 , wherein the hapten is selected from the group consisting of dinitrophenol, digoxigenin and FITC (fluorescein isothiocyanate). The fluorescent nanoparticle is a fluorescent dye integrated nanoparticles, an average particle diameter of 20 nm to 300 nm, FISH staining methods of nucleic acid according to any one of claims 1-3. The fluorescent nanoparticles are inorganic phosphor integrated nanoparticles, an average particle diameter of 20 nm to 300 nm, FISH staining methods of nucleic acid according to any one of claims 1-3. The fluorescent nanoparticles are inorganic phosphor nanoparticles selected from the group consisting of quantum dots and carbon dots, the average particle diameter of 1 nm to 300 nm, nucleic acid according to any one of claims 1 to 3 FISH staining method. FISH staining of nucleic acid, characterized in that, in the FISH staining method according to any one of claims 1 to 6 , fluorescent nanoparticles are bound to a plurality of binding sites amplified by the binding site amplification step. A method for manufacturing a damaged pathological specimen.