CN117467751B

CN117467751B - Targeting target gene FISH fluorescent probe and self-assembled amplification probe system thereof

Info

Publication number: CN117467751B
Application number: CN202311823098.6A
Authority: CN
Inventors: 董长贵; 李丹阳; 高磊; 苏敏
Original assignee: Beijing Bailige Biotechnology Co ltd
Current assignee: Beijing Bailige Biotechnology Co ltd
Priority date: 2023-12-27
Filing date: 2023-12-27
Publication date: 2024-03-29
Anticipated expiration: 2043-12-27
Also published as: CN117467751A

Abstract

The invention provides a target gene FISH fluorescent probe and a self-assembled amplifying probe system thereof, wherein the fluorescent probe is called a target gene FISH fluorescent probe X, a linear probe sequence of which the length is 15-65bp and of which the target gene is targeted is arranged in the middle of the FISH fluorescent probe X, the left end and the right end of the linear probe sequence are respectively connected with a neck ring sequence of which the length is 5-35bp, RNA bases serving as RNAse cutting sites are introduced at the tail ends of each neck ring sequence, and closed sequences are respectively connected at the tail ends of the RNA bases at the two ends. The invention realizes the improvement of the binding specificity of the probe and the enhancement of the background elimination by developing a novel probe design modification marking mode, can realize the effective binding of long, short and other target sequences, and displays and positions, and has greatly reduced synthesis cost because the amplified signal group is designed as an independent structure.

Description

Targeting target gene FISH fluorescent probe and self-assembled amplification probe system thereof

Technical Field

The invention relates to the field of nucleic acid detection, in particular to a target gene FISH fluorescent probe and a self-assembly amplifying probe system thereof.

Background

Fluorescent in situ hybridization (Fluorescence in situ hybridization, FISH) technology is a method of targeting a target gene by a fluorescently labeled nucleic acid sequence probe and displaying spatial positional information of its target nucleic acid in situ. By combining genetic characteristics of genes and molecular cell biology technology, the number or structure abnormality of chromosomes can be detected at the cellular level, and the method is particularly suitable for chromosome deletion and translocation, and meanwhile, the gene expression difference and the positional relationship can be detected preliminarily and qualitatively. Because of the advantages of certain specificity, in-situ visibility and the like, the gene expression vector is widely applied to clinical applications such as basic scientific research of gene biology, diagnosis of genetic tumor diseases and the like.

Along with the continuous development and optimization of the FISH technology, currently, common sources of FISH probes mainly exist; 1) Short fragment probes (usually 15-30 bp) are obtained by chemically synthesizing oligonucleotide single-chain or double-chain mode, 2) plasmids and Bacterial Artificial Chromosomes (BACs) are linked with a labeled signal group. 3) Double-stranded or single-stranded probes (typically hundreds or thousands of bp in length) are obtained by amplification by enzymatic cloning vectors or PCR methods or in vitro transcription. Such as an oligonucleotide probe (application number 201410045228.2) synthesized by Agilent technologies, inc.

The gene expression abundance is detected by amplifying the genes by a PCR method, the sample mixture is collected, the total RNA gene information is extracted, the specific source cells of the genes cannot be determined, the living cells cannot be detected, and the gene positioning condition cannot be directly displayed.

Long-chain FISH probe detection or chemical synthesis short-chain direct labeling fluorescence detection is obtained through BAC cloning. Short-chain oligonucleotides are used for combining target sequences by synthesizing a plurality of sequences and marking two ends of the sequences, or by targeting combination modes such as ZZ mode, pi mode and the like, and the patent application number is as follows: 202210759816.7.BAC has the disadvantage that BAC clones are not present in all parts of the human genome. Second, BACs based on natural DNA contain a large number of repetitive sequences, such as Alu sequences, and the repetitive-rich probes hybridize to the genome, producing a high background signal. Long-chain detection techniques cannot detect short sequence information, such as miRNA; in addition, the existing Fish probes are relatively weak in binding capacity based on a DNA structure, and a specific blocking method is not arranged, so that the background or non-specific signal of the existing short-chain oligonucleotide probes is high, and the relevant detection mode usually only identifies tissue cells fixed by a fixing solution and cannot carry out calibration detection on relevant genes of living cells.

Along with high-throughput sequencing gene information mining, in addition to gene changes such as related gene deletion, ectopic, mutation recombination and copy number change on DNA chromosomes, and meanwhile, the requirement of observing and detecting gene expression changes in complex cell and tissue environments is met, the traditional FISH technology often faces the problems that the detected genes are single and concentrated on detecting information changes on the gene chromosomes, the newly mined circRNA, microRNA, lncARNA isogenic genes can have insufficient signal amplifying capability, low specificity and low efficiency and background noise, and particularly the detection effect on short-sequence and low-copy number target molecules is relatively poor, the signal intensity can be improved to a certain extent by increasing the number of target probes and accumulated fluorescence intensity, but longer nucleic acid sequences are required for hybridization, so that the application of detecting short-sequence such as microRNA isogenic molecules is greatly limited, and the potential non-specific binding and background signals can be additionally increased. Therefore, there is a need to develop a means for detecting small nucleic acid short sequence RNA (small RNA: microRNA, piRNA), short nucleic acid sequence DNA and variable cutter (alternative splicing) with important functions, such as identifying homologous circRNA and maternal and non-coding RNA, and the like, and a high-signal, high-specificity and low-background FISH detection method is still lacking at present.

Therefore, by combining the above factors, it is necessary to develop an in-situ detection technology which can be positioned, has high signal intensity, strong specificity, low background noise and convenient operation, and a means method for performing differential detection on long and short genes in a living cell sample, a fixed tissue sample, a cell slide or a smear sample is realized.

Disclosure of Invention

In order to solve the technical problems, the invention provides a target gene FISH fluorescent probe, which is called a target gene FISH fluorescent probe X, wherein a linear probe sequence of a target gene with the length of 15-65bp is arranged in the middle of the FISH fluorescent probe X, the left end and the right end of the linear probe sequence are respectively connected with a neck ring sequence with the length of 5-35bp, and when the linear probe sequence is combined with the target gene, the neck ring sequences form a neck ring structure; the neck ring sequences respectively form neck ring structures at the left end and the right end of the linear probe sequence, and the neck ring sequences at the left end and the right end can be the same neck ring sequences or different neck ring sequences; introducing RNA base serving as an RNase cleavage site at the tail end of each neck ring sequence, and respectively connecting closed sequences at the tail ends of the RNA base at the two ends, wherein the length of each closed sequence is 5-30bp, the closed sequences at the left end and the right end are respectively and specifically combined with linear probe sequences at the left end and the right end, and the Tm value of the closed sequences after being specifically combined with the linear probe sequences is at least 5 ℃ lower than that of the closed sequences after being specifically combined with the target genes; the linear probe sequence is marked with a fluorescent group for FISH fluorescence detection, and the marked fluorescent group does not influence the specific combination of the linear probe sequence and a target gene; the blocking sequence is marked with a fluorescence quenching group at the free end of the blocking sequence, and the fluorescence quenching group quenches a fluorescent group on the linear probe sequence under the condition that the blocking sequence is combined with the linear probe sequence, so that the background fluorescence of the FISH fluorescent probe when the target gene is not effectively combined is reduced; when hybridization detection is carried out, the linear probe sequences are combined with the target gene, the neck ring structures at the left end and the right end are opened, the specific combination of the closed sequences at each end and the linear probe sequences at each end is also disconnected, the RNase cleavage site is cleaved by adding RNase, the closed sequences are released, and the fluorescence quenching group does not have quenching effect on the fluorescent group on the linear probe sequences.

In one embodiment, the linear probe sequences are fluorescently labeled consecutively or at intervals.

In one embodiment, the rnase cleavage site is a ribonuclease cleavage site, a thermostable ribonuclease cleavage site.

In one embodiment, the neck ring sequence length is 5-15 bp selected from 5'-ATTGATGTATTGTG-3', 5'-TCAAGAG-3', 5'-TTGATATCCG-3', 5'-ttcaagag a-3' or 5'-CCACACC-3'.

In one embodiment, the probe system comprises the FISH fluorescent probe X for targeting the target gene, and further comprises a primary cascade amplification probe Y, wherein the probe Y comprises a 15-65 bp Y probe intermediate sequence and two flanking sequences at two ends of the Y probe, the Y probe intermediate sequence is not combined with the target gene or/and specifically combined with the X probe, the Y probe intermediate sequence marks a fluorescent group, and the flanking sequences at two ends of the Y probe respectively target the neck ring sequences at two ends of the X probe, and the two flanking sequences are specifically combined.

In one embodiment, the probe system further comprises a secondary cascade amplification probe Z, wherein the probe Z comprises a Z probe intermediate sequence of 15-65 bp and flanking sequences at two ends of the Z probe, the Z probe intermediate sequence is specifically combined with the Y probe intermediate sequence, the Z probe intermediate sequence is not combined with a target gene or/and is specifically combined with an X probe, and the Z probe intermediate sequence is marked with a fluorescent group; and flanking sequences at two ends of the Z probe are respectively complementary with flanking sequences at two ends of the next-stage Y probe.

In one embodiment, the X probe has a binding Tm value for the target gene, the Y probe flanking sequences have a binding Tm value for the neck ring sequences on either side of the X probe, and the Z probe has a binding Tm value for the Y probe that differ by no more than + -5deg.C.

In one embodiment, the X probe has a binding Tm value for the target gene, the Y probe flanking sequences have a binding Tm value for the neck ring sequences on either side of the X probe, and the Z probe has a binding Tm value for the Y probe that differ by no more than + -2 ℃.

In the technical proposal of the invention, when designing the double-lock (double-closed) fluorescent marking probe,

1) First, a key target region of a target gene is determined, wherein the target region is a nucleic acid sequence which can show that diseases or traits (such as tumor, genetic diseases, gene abundance, base mutation, deletion, insertion and the like) are characteristic and specific, and the target region comprises a DNA sequence or an RNA sequence with the characteristics.

2) Designing a double-closed fluorescent labeling probe according to a target gene: the target gene is searched in NCBI and other gene databases by utilizing a bioinformatics technology method, characteristic sequences of the target gene are compared and analyzed, a target probe, namely an X probe for short, is designed for the sequence, the sequence length is 15-65 bp, the GC content is 30-65%, the target probe sequence can be continuously or intermittently marked (such as CY3, FAM, CY5, FITC and other fluorescent markers or biotin, digoxin and other indirect signal markers), and related bases in the sequence can be modified (such as modification modes of 2'F, 2' MOE, LNA, 2'-Ara-F, 2' -O-benzyl, UNA, UNG, UNT, UNC, cEt and the like) in order to increase the stability and binding capacity specificity of the sequence.

3) A blocking sequence (Blocking sequence or Locking sequence) of length (5-30 bp) that labels quenching fluorescence (or blocking fluorescence such as labeled BHQ1, BHQ2, BHQ3, TAMRA, eclipse, dabcyl, etc., quenching fluorophores, the selectivity of which needs to be determined based on target probe labeling fluorescence) at its free end, which quenching fluorescence can quench target probe fluorescence in the event that the locking sequence binds to the target probe, reducing background fluorescence of the target probe in the event that the target probe does not bind effectively to the target gene. Meanwhile, when the Blocking sequence is designed, the cytosine (C) or uracil (U) residues on the single-stranded RNA can be specifically degraded by RNase A (Ribonuclease A) (NEB T3018-2) under the high salt concentration (more than or equal to 0.3 and M) after the Blocking sequence is complementary. Specifically, cleavage recognizes a phosphodiester bond formed by a phosphate group on a 5' -ribose on a nucleotide and an adjacent pyrimidine nucleotide 3' -ribose, thereby hydrolyzing 2', 3' -cyclic phosphate to the corresponding 3' -nucleoside phosphate (e.g., pG-pG-pC-pA-pG is cleaved by RNase A to produce pG-pG-pCp and A-pG); or by ribonuclease If (RNase If) (NEB, M0243) is an endonuclease that cleaves on all RNA dinucleotide linkages, leaving 5' hydroxyl and 2', 3' cyclic phosphate; or through thermostable ribonuclease H (RNase H) (NEB, M0523) can selectively hydrolyze phosphodiester bonds of RNA strand in DNA hybrid molecule, and at the same time keep the DNA strand intact and have optimal activity above 65 ℃; or by ribonuclease H (Ribonuclease H) (NEB, M0297), can specifically hydrolyze phosphodiester bonds of RNA hybridized with DNA, and single-or double-stranded DNA cannot be digested. Or after the Cas12a forms a ternary complex with crRNA and target DNA, the complex has single-stranded nucleic acid cutting activity, any single-stranded DNA in the system is cut into fragments (called trans cutting), so that the quenching fluorescent group marked on the residue is released, and the effect on fluorescence quenching of the target probe is eliminated. And unbound single-stranded RNA in the sample can be eliminated. To increase sequence stability and binding specificity, the relevant bases in the sequence may be optionally modified (e.g., 2'F, 2' MOE, LNA, 2'-Ara-F, 2' -O-benzyl, UNA, cEt, etc.).

4) Neck ring structure: the specific sequences of the two side neck rings can be the same or different, but the neck ring structure is required to be formed under the action of the complementary sequence of the targeting probe by adopting 5-15 bp as the neck ring structure (for example, 5'ATTGATGTATTGTG3'; 5 'TCAAGG 3';5 'TTGATATCGG 3'; 5'TTCAAGAGA3';5 'CCACACACCC 3', and the like nucleic acid sequences with cyclic secondary structure characteristics). And complements the downstream amplified signal sequence neck ring structure in a double-stranded form. To increase the stability of the sequence and specificity of the binding capacity, the relevant bases in the sequence may be selectively modified (e.g., 2' F, 2' MOE, 2' Ome, LNA, 2' -Ara-F, 2' -O-benzyl, UNA, cEt, etc. modifications).

5) Self-assembled amplification probe system: adopting a pair of flanking sequences of 5-35 bp to complementarily bind with the neck ring sequences at two sides of the target probe X, and meanwhile, the middle of the flanking sequences contains a sequence Y for complementarily amplifying signals; the complementary sequence Z can be designed for the Y sequence through progressive amplification, flanking sequences can be led out from two sides of the Z sequence and can be reused for downstream amplification binding cascade, the sequences can be continuously or intermittently marked (such as CY3, FAM, CY5, FITC and other fluorescent markers or Biotin and other indirect signal markers), and related bases in the sequences can be modified (such as modification modes of 2'F, 2' MOE, LNA, 2'-Ara-F, 2' -O-benzyl, UNA, cEt and the like) in order to increase the stability and binding capacity specificity of the sequences;

6) Fluorescent group dye: the fluorescent dye (fluorophore) of the label used in the method may be one or more selected from the following dyes. Examples of fluorophores include, but are not limited to, the fluorescent dyes Alexa Fluor 350, alexa Fluor 405, alexa Fluor 488, alexa Fluor 532, alexa Fluor 546, alexa Fluor 555, alexa Fluor 561, alexa Fluor 568, alexa Fluor 594, alexa Fluor 647, alexa Fluor 660, alexa Fluor 680, alexa Fluor 700, alexa Fluor 750, BODIPY FL, coumarin, cy3, cy5, fluorescein (FITC), oregon Green, pacific Blue, pacific Orange, PE-Cyanine7, perCP-Cyanine5.5, tetramethylrhodamine (TRITC); the Super Bright and eFluor dyes eFluor 450, eFluor 506, eFluor 660, PE-eFluor 610, perCP-eFluor 710, APC-eFluor 780, super Bright 436, super Bright 600, super Bright 645, super Bright 702, super Bright 780; DNA dyes DAPI, propidium lodide, SYTO 9, SYTOX Green, TO-PRO-3; qdot probes Qdot 525, qdot 565, qdot 605, qdot 655, qdot 705, and Qdot 800; fluorescent protein markers Allophycocyanin (APC), R-Physoerythhrin (R-PE); expressed fluorescent proteins Cyan Fluorescent Protein (CFP), green Fluorescent Protein (GFP), red Fluorescent Protein (RFP), BUV dyes Brilliant Ultra Violet Polymer Dyes, brilliant Ultra Violet 395, brilliant Ultra Violet 496, brilliant Ultra Violet 563, brilliant Ultra Violet 615, brilliant Ultra Violet 661, brilliant Ultra Violet 737, brilliant Ultra Violet 805, brilliant Violet 421, brilliant Violet 480, brilliant Violet 650, brilliant Violet 711, brilliant Violet 786; novaFluor Dye NovaFluor 510 Dye, novaFluor 530 Dye, novaFluor 555 Dye, novaFluor 585 Dye, novaFluor 610-30S Dye, novaFluor 610-70S Dye, novaFluor 660-40S Dye, novaFluor 660-120S Dye, novaFluor Yellow 570 Dye, novaFluor Yellow 590 Dye, novaFluor Yellow 610 Dye, novaFluor Yellow 660 Dye, novaFluor 690 Dye, novaFluor 700 Dye, novaFluor Yellow 730 Dye, novaFluor d 660 Dye, novaFluor Red 685 Dye, novaFluor 710 Dye, novaFluor Rev 700 Dye; and other organic or inorganic fluorescent dyes such as: texas Red 4-acetamido-4 '-isothiocyanatestilbene-2, 2' -disulfonic acid; acridine and its derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; 5- (2' -aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N- [3-vinyl sulfone/phenyl ] naphthalimide-3,5disulfonate (fluorescein VS) phenyl ] naphthalimide-3,5 disulfonate; n- (4-amino-1-naphthaloyl) maleimide; anthranilamide; brilliant Yellow (Brilliant Yellow); coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151); cyanine and derivatives such as phloxine (cyanosine) bromophthalic trimellite red); 7-diethylamino-3- (4' -isothiocyanatophenyl) -4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetic acid; 4,4 '-diisothiocyanidine dihydro-stilbene-2, 2' -disulfonic acid; 4,4 '-diisocyanatostilbene-2, 2' -disulfonic acid; 5- [ dimethylamino ] naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4- (4' -dimethylaminophenylazo) benzoic acid; 4-dimethylaminophenylazo phenyl-4' -isothiocyanate; eosin and derivatives such as eosin and eosin isothiocyanate; phycoerythrin and derivatives, such as phycoerythrin B and phycoerythrin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4, 6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2'7' -dimethoxy-4 ', 5' -dichloro-6-carboxyfluorescein, fluorescein isothiocyanate, fluorescein chlorotriazine, naphthalenyl fluorescein, and qflitc (XRITC); fluorescent amine; IR 144; IR 1446; lissamine (Lissamine) TM; lissamine rhodamine, fluorescein (Lucifer yellow); isothiocyanato malachite green (Malachite Greenisothiocyanate); 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; basic parafuchsin; nile red; oregon green; phenol red; b-phycoerythrin; o-phthalaldehyde; pyrene and its derivatives such as pyrene, pyrene butyrate and succinimidyl-1-pyrene butyrate; reactive Red 4 (Cibacron bright Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine, 6-carboxyrhodamine (R6G), 4, 7-dichloro rhodamine-lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 sulfonyl chloride derivatives (Texas red), N, N-tetramethyl-6-carboxyrhodamine, tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate; riboflavin; rose acid and terbium chelate derivatives; xanthenes; or a combination thereof. Other fluorophores known to those skilled in the art, or combinations thereof, may also be used.

7) An indirect labeling group: canavalia ectenes A-mercapto; PEG, PLGA, PEI, biotin, carboxyl, amino, polypeptide, HRP-strepitavidin, digoxin, aptamer or other functional groups which can be combined and labeled through hydrogen bonds, chemical bonds, van der Waals forces, structural domains, antigen antibodies and the like.

8) The probe design needs to consider the base complementation Tm value requirement: the Tm value is the temperature at which the ultraviolet absorption value reaches 1/2 of the maximum value during thermal denaturation of a nucleic acid double-stranded complementary structure under a certain reaction condition. Denaturation occurs at relatively high temperatures (70-90 ℃), and the double-stranded structure of nucleic acid breaks down into single strands and becomes random coils. An "hyperchromic effect" is produced in the optical properties, i.e. the value of the ultraviolet absorption (at 260 nm wavelength) increases. The temperature at which the hyperchromatic effect reaches half of the maximum is generally referred to as the melting temperature (or melting point) of the DNA and is denoted by the symbol Tm. The Tm values of DNA of different sequences differ. The higher the GC content in the DNA double strand, the higher the Tm value, and the proportional relation is that under the same GC content, the Tm of DNA is generally higher than that of RAN, DNA is generally higher than that of RNA, and the Tm of the double strand structure of RNA is generally higher in sequence; in addition, the Tm value of the nucleotide in the sequence can be significantly increased by different chemical modifications (such as increasing modification modes of LNA, cEt and the like). As shown in figure 2, in the design process, the Tm values of the X probe and the target gene are combined as much as possible, the Tm values of the flanking sequences of the Y probe and the neck ring sequences at both sides of the X probe are combined, the Tm values of the Z probe and the Y probe are combined, the Tm values of relevant matched bases can be close to or consistent by adjusting the sequence length, GC content, base modification and other modes, and the general deviation is recommended to be not more than +/-5 ℃ (preferably not more than 2 ℃); the Tm value design mainly enables the probe to be effectively combined and complemented with the target gene, so that the target sequence can have more specific combination effect under the condition of proper hybridization buffer and temperature.

9) The X sequence synthesis of the target gene X probe has the form shown in the attached figure 1, and mainly contains the following key information, (1) the probe sequence of the target gene (the range length is 15-65 bp, the GC content is 30-65%, the specificity of the target gene is required to be 100 percent), various fluorescent groups or indirect labeling groups can be labeled, and the related labeling does not influence the binding characteristic with the target gene); (2) A closed sequence (Blocking sequence, the range length is 5-30 bp, the matching degree with the two ends of the target gene probe is 75-100%, and the matching degree is required to be higher when the closed sequence is shorter); (3) The neck ring structure (the range length is 5-35 bp, the GC content is 20-75%, and the matching binding degree of the target gene or other probe region is as low as possible to ensure the neck ring effect of the target gene or other probe region), and an RNase cutting site is introduced between the closed sequence and the neck ring sequence.

10 The primary cascade amplification probe Y is synthesized by Y sequence, and has the form shown in figure 2, and mainly contains the following key information, (1) a Y probe intermediate sequence (the range length is 15-65 bp, the GC content is 30-65%, the non-specific binding requirement of genes related to a target sample and an X probe is lower than 50%, a plurality of fluorescent groups or indirect marking groups can be marked, and the related marking does not influence the binding characteristic of the target gene), (2) the flanking sequences at two ends of the Y probe need to target the neck ring structure of the X probe sequence (the range length is 5-30 bp, the GC content is 20-75%, the matching degree of the flanking sequences at two ends and the X probe sequence is 90-100%, and one or more of the following modifications can be carried out for changing the stability or Tm of the probe: phosphorothioate PS, methoxy 2'Ome at position 2 of the sugar ring, fluoro at position 2 of the sugar ring, methoxyhexyl 2' MOE at position 2 of the sugar ring, locked nucleic acids LNA, cEt and other modifications).

11 The synthesis of the Z sequence of the secondary cascade amplification probe has the form shown in figure 2 and mainly contains the following key information: (1) Z probe intermediate sequence (range length 15-65 bp, GC content 30-65%, Z probe and Y probe intermediate core sequence complementation matching degree requirement 95-100%, and non-specific binding requirement lower than 50% with target sample, X probe related gene, can mark multiple fluorescent groups or indirect mark groups, and related mark does not affect binding characteristic with target gene); (2) The flanking sequences at two ends of the Z probe target the flanking sequences at two ends of the Y probe (the range length is 5-30 bp, the GC content is 20-75%, the complementary matching degree of the flanking sequences at two ends and the flanking sequences of the Y probe is 90-100%, and one or more of Phosphorothioate (PS), sugar ring 2-methoxy (2 ' Ome), sugar ring 2-fluoro (2 ' F), sugar ring 2-methoxyhexyl (2 ' MOE), locked Nucleic Acid (LNA), cEt and other modification modes can be carried out to change the stability or Tm of the probe.

12 According to the experimental requirement, the X probe can be used alone or the X: Y: Z probe can be used together. Especially for the gene with lower abundance, the cascade amplification signal of the Y-Z combination can effectively increase the model strength and improve the detection sensitivity; in addition, the self-closing self-assembly characteristic of the invention ensures that the self-closing self-assembly characteristic of the invention has the probe locking characteristic and reduces the combination condition with a background gene, so that the background signal is effectively controlled.

In the invention, aiming at the aspects of detection sensitivity, background value, specificity and the like of the current FISH probe and the multiple combination modes of the FISH probe provided by part of companies, the number of the related combined oligonucleotide probes is generally about 20 or more, the experimental cost is high, the detection effect cannot be detected or is poor aiming at special genes (such as microRNA short sequences, circRNA distinguishing parent gene sequences and the like), and no strategy for adopting a blocking group to eliminate nonspecific or background fluorescence is found in the design, synthesis and application of the current FISH probe sequences. Therefore, the invention realizes the improvement of the binding specificity of the probe and the enhancement of the background elimination by developing a novel probe design modification marking mode, can realize the effective binding of long, short and other target sequences, display and positioning, and greatly reduces the synthesis cost because the amplified signal group is designed as an independent structure. The invention is an in-situ detection technology which has the advantages of positioning, high signal intensity, strong specificity, low background noise and convenient operation, and realizes a means method for carrying out differential detection on long and short genes in a living cell sample, a fixed tissue sample, a cell slide or a smear sample.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows a targeting gene of interest, FISH fluorescent probe X;

FIG. 2 is a schematic diagram of the working principle of the double-lock autonomous amplification system FISH probe combined with a target gene according to the invention;

FIG. 3 is a schematic diagram of a fluorescence report of a gene by eliminating a quenching group from a double-locked self-assembled FISH probe according to the present invention;

FIG. 4 is a graph showing the results of an experiment of the enzyme treatment with a blocked probe of the present invention;

FIG. 5 is a graph showing the results of brightness change in the enzyme treatment with the blocked probe of the present invention;

FIG. 6 is a graph of the observation of a 20-fold objective lens of the inventive magnification system test;

FIG. 7 is a diagram of the observation result of a 100-time magnifying glass of the inventive magnification system test;

FIG. 8 is a graph showing the result of the enzyme treatment test of the amplification system of the present invention;

FIG. 9 is a graph showing the results of the fluorescence intensity of the enzyme treatment experiment of the closed probe of the present invention;

FIG. 10 is a graph showing the result of fluorescence intensity of the amplification system after enzyme treatment with a blocked probe of the present invention.

Detailed Description

In order that those skilled in the art will better understand the technical solutions in the present application, the present invention will be further described with reference to examples. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application. In the following examples, unless otherwise indicated, all methods conventional in the art are described.

The main laboratory instrument equipment used in the present invention:

1) Fluorescence microscope or laser confocal microscope and matched analysis software: the required fluorescent microscope configuration includes: the 10 x eyepiece, 10 x, 40 x objective lens and 100 x oil lens, the microscope excitation light or filter lens need to cover the measured fluorescence band, the detailed use and combination of the filter set used can be known by the filter set supplier in order to select a filter set compatible with the labeling fluorescent dye. The companion analysis software may be executed using computer and biological information based image and data acquisition and analysis software known in the art. The determination of the evaluation decision may be made manually (e.g., by viewing the data and manually comparing the signatures), automatically (e.g., by employing data analysis software specifically configured to match the optically detectable signatures), or a combination.

2) In situ hybridization: the denaturation and hybridization processes can be accomplished automatically according to the setup of the experimenter. The temperature control and humidity conditions can be automatically adjusted. The method comprises 3 program modes: denaturation, hybridization, denaturation+hybridization, locking temperature (fixed temperature) denaturation temperature range: 4 ℃ to 99 ℃; denaturation time: 0-60 minutes; the hybridization temperature range is 4 ℃ to 80 ℃; hybridization time range: 0-99 hours; heating plate temperature range: 4 ℃ to 99 ℃; slide volume: 4-24 sheets; run time range: 0-100 hours; the program can be set: 4-40; rapid temperature rise function: raising the temperature from 4 ℃ to 99 ℃ within 1-2 minutes; rapid cooling function: descending from 99 to 4 ℃ within 3-10 minutes; temperature accuracy: + -0.5deg.C.

Embodiment case one: design and labeling for homoU 6 sequence

1. Designing a synthetic probe, designing a detection probe combination aiming at an internal reference Gene U6 (from NCBI: https:// www.ncbi.nlm.nih.gov/obtaining a Homo U6 target sequence Gene ID: 26827), synthesizing the probe by Shanghai Bai Ge biotechnology Co., ltd., synthesizing one or more of the following sequences, preferably (U6-SEQ 1,2, 3), respectively containing a binding region, a blocking region and a neck ring region, and optionally modifying related bases in the sequence (such as 2'F, 2' MOE, LNA, 2'-Ara-F, 2' -O-benzyl, UNA, cEt, and the like, changing different modification modes such as sequence structure, binding capacity, enzymolysis resistance and the like); the selected neck ring structure may be one or more of the following sequences 5'ATTGATGTATTGTG3' (SEQ ID No. 1); 5 'TCAAGG3' (SEQ ID No. 2); 5 'TTGATATCGG 3' (SEQ ID No. 3), 5'TTCAAGAGA3' (SEQ ID No. 4); 5 'CCACACACC 3' (SEQ ID No. 5) and the like have a circular secondary structural feature. The nucleotide sequence is preferably modified by Int Cy3 dT fluorescence; the 5 'end of the nucleic acid sequence is preferably 5' CY5 and the 3 'end of the nucleic acid sequence is preferably modified by 5' BHQ2. The introduction of RNase A cleavage site xy into the sequence represents a combination or a combination of ribonucleic acids rCrA, rCrU, rCrC, rCrG, see Table 1 for specific sequences.

TABLE 1

The sequence with a thickened and lowercase middle part of the negative control probe is a core negative control core sequence, so that nonspecific binding in the experimental process is eliminated, and the sequence is used for optimizing conditions such as reagent buffer, hybridization problem and the like. The negative control fluorescence was generally weaker than the target probe and was significantly different, see in particular table 2.

TABLE 2

。

As shown in FIGS. 1-3, a target sequence X for a target gene, such as U6-SEQ1 for a homo U6 gene, is designed according to FIG. 1, wherein a sequence comprising a red marker is directly targeted to the target gene, blue sequences on both sides of the red sequence are neck ring sequences, two RNA bases represented by xy are used for enzyme digestion of RNase, and black on both ends of the sequence are blocking sequences (i.e., blocking sequences, and quenching effect can be started by attaching a blocking fluorophore).

According to FIG. 2, the target sequence X is complementarily combined with the target gene (represented by black sequence), meanwhile, a first-stage amplifying probe Y sequence (containing fluorescent marker) is added, flanking sequences at two sides of the Y sequence are complementarily combined with neck ring sequences at two sides of the X sequence, meanwhile, a second-stage amplifying probe Z sequence (containing fluorescent marker) is added, the sequence in the middle of the Z sequence is complementarily base-combined with the intermediate sequence of the Y sequence, and meanwhile, flanking sequences at two sides of the Z sequence are complementarily combined with sequencing sequences at two sides of the next-stage Y sequence, so that self-assembly amplification is realized.

According to FIG. 3, the probe assembly system cleaves 2 RNA bases (represented by xy) linking the blocking probe and blocking group by adding RNase, such that the blocking sequence is separated from the overall probe system, and fluorescence signal enhancement amplification is achieved after release of the blocking group, and the following probe X, Y and Z sequences are used in FIG. 3, see Table 3 in particular.

TABLE 3 Table 3

。

Implementation case two: fluorescent microscope for verifying enzymolysis effect of fluorescent self-closing probe

1. Experimental procedure

1.1. Selecting 96-well plates, and respectively setting test groups (A-E represents the fluorescent images before and after the enzymolysis of U6-SEQ1, B-F represents the fluorescent images before and after the enzymolysis of U6-SEQ2, C-G represents the fluorescent images before and after the enzymolysis of U6-SEQ 3) and cell control groups (D-H represents the fluorescent background images before and after the enzymolysis of normal culture blank cells);

1.2. both probe and enzyme were dissolved in 2 XSSC buffer, 100. Mu.L of U6-SEQ1 and 0.5mg/mL RNase blocking probe were added to test group 1, 100. Mu.L of U6-SEQ2 and 0.5mg/mL RNase were added to test group 2, and 100. Mu.L of U6-SEQ3 and 0.5mg/mL RNase were added to test group 3. Control group 1 was added with 100. Mu.L of 0.5. Mu.M blocking probe U6-SEQ1, control group 2 with 100. Mu. L U6-SEQ2, control group 3 with 100. Mu. L U6-SEQ3, blank group 1 with 100. Mu.L of 2 XSSC buffer, blank group 2 with 100. Mu.L of 2 XSSC buffer and 0.5mg/mL RNase were repeated three times;

1.3. And (3) incubating the 96-well plate in a 37 ℃ incubator for 20 min through light-shielding treatment, and then observing brightness change under a fluorescence microscope.

2. Experimental results

The experimental results are shown in fig. 4, the brightness values of each graph are respectively identified by using imageJ software, and the results are collated and analyzed in the following table 4 and fig. 5, the fluorescence brightness of each probe after enzyme treatment is improved, and the blank groups have no brightness change, which indicates that the closed probes hydrolyze the RNA structure and the quenching groups carried by the RNA structure as expected after enzyme treatment, but the brightness improvement of each group is inconsistent due to different probe sequences and inconsistent sealing effects.

TABLE 4 fluorescent brightness values for closed probe enzyme treatment test

。

Implementation case three: fluorescent microscope for verifying amplification effect of fluorescent self-closing probe

1. Experimental procedure

1.1. The experiments were divided into 4 groups: experimental group 1, experimental group 2, control group 1, control group 2; adherent cells were seeded in 12-well plates (pre-well placed in treated and appropriately sized coverslips) at a density of 1 x 104 cells/well and incubated overnight in an incubator;

the medium was aspirated and washed twice with PBS for 5 min each;

the PBS was removed by pipetting, 200. Mu.L of 4% paraformaldehyde was added to each well, and the wells were fixed at room temperature for 15 min

4% paraformaldehyde was pipetted off and 200. Mu.L of 0.1% Triton-100 (on-the-fly) was added to each well and the cells were treated at room temperature for 15 min;

Absorbing and discarding 0.1% Triton-100, and washing twice with PBS for 5 min each time;

absorbing and discarding PBS, adding 200 mu L of 2 XSSC into each hole, and placing in a 37 ℃ incubator for 30 min;

incubating the hybridization buffer solution in a water bath kettle at 73 ℃ for 30 min in advance until the hybridization buffer solution is clear and transparent, and respectively diluting the closed probe combinations U6-SEQ1 and U6-SEQ2 to 0.5 mu M by using the hybridization buffer solution;

2 XSSC was aspirated, 200. Mu.L of 0.5. Mu.M of the blocking probe U6-SEQ1 and 0.5. Mu.M of the amplification system X/Y was added per well for the experimental group 1, and 200. Mu.L of 0.5. Mu.M of the blocking probe U6-SEQ2 and 0.5. Mu.M of the amplification system X/Y was added per well for the experimental group 2; 200. Mu.L of 0.5. Mu.M blocking probe U6-SEQ1 was added to each well of control 1, and 200. Mu.L of 0.5. Mu.M blocking probe U6-SEQ2 was added to each well of control 2; 200. Mu.L of hybridization buffer was added to the negative control; placing the obtained product in a 37 ℃ incubator for hybridization overnight after taking light-shielding measures;

after hybridization of 16. 16 h, taking out the sample from a 37 ℃ incubator, sucking and discarding the probe mixed solution, adding 200 mu L of 0.5mg/ml RNase into each hole, and incubating for 20 min at the 37 ℃ incubator; RNase was diluted with 2 XSSC;

absorbing and discarding the RNase solution, adding 200 mu L of 0.1% Tween 20 preheated at 42 ℃ into each hole, and washing for 5 min;

0.1% Tween 20 was pipetted and each well was washed with 200. Mu.L of 42℃pre-warmed 2 XSSC for 5 min;

2 XSSC was pipetted, and 200. Mu.L of pre-warmed 1 XSSC at 42℃was added to each well for 5min;

absorbing and discarding the washing liquid, adding 200 mu L of diluted DAPI working solution into each hole, and incubating for 15min at room temperature in a dark place;

absorbing DAPI working solution, washing with PBS for 2 times, and 5min each time;

anti-quencher was added dropwise to the clean slide, and the cell slide was covered with a cell surface facing down on the slide and observed under a fluorescence microscope.

2. Experimental results

The experimental results are shown in FIGS. 6 and 7, and the brightness value changes after enzyme treatment of the pure probe set and the probe set added to the amplification system were identified by using imageJ software, respectively, and the arrangement analysis is shown in Table 5 and FIG. 8. After enzyme treatment, the fluorescent brightness of the probe group added with the amplifying system is higher than that of a simple probe group, which shows that after enzyme treatment, the amplifying system is combined with the probes, so that the fluorescent brightness is obviously enhanced, and meanwhile, the probes with different sequences are designed to be different.

Table 5 amplification System enzyme treatment test fluorescent brightness values

Embodiment four: the multifunctional enzyme-labeled instrument verifies the effect of sealing enzymolysis fluorescence intensity

1. Experimental procedure

Selecting a 96-hole black ELISA plate, and respectively setting an experimental group and a negative control group, wherein each group is subjected to 6 repetitions (the highest value and the lowest value are removed);

Experimental group probe dilution system: the experiments were divided into 3 groups, the first group being a negative control (no probe with hybridization solution only); the second group is a group (not enzymatically hydrolyzed) in which the closed probe is not added with RNA A enzyme; the third group is a closed probe and RNA A enzyme group (after enzymolysis), each group is 6 parallel holes, and each hole is added with 100 mu L of probe dilution system. The closed probe group is: U6-SEQ1, U6-SEQ2 and U6-SEQ3. A second set of specific probe dilution systems: according to the grouping of the blocking probes, 597ul of hybridization solution can be added into a 1.5mL centrifuge tube, and 1:1 of each group of blocking probes is respectively added to 3ul of total volume, namely the concentration of the blocking probes in the total system is 0.5 mu M; each set of blocking probes was finally brought to a total volume of 600ul. 100. Mu.L of 0.5. Mu.M blocking probes U6-SEQ1, U6-SEQ2 and U6-SEQ3 were added to each well, one set of 6 parallel wells. Third set of specific probe dilution systems: according to the closed probe grouping, 594uL of hybridization solution can be added into a 1.5mL centrifuge tube, 3uL of RNA A enzyme with initial concentration of 100mg/mL is added, namely the concentration of RNA A enzyme in the total system is 0.5ug/uL; adding 1:1 to 3ul of total volume of each group of blocking probes respectively, namely, the concentration of the blocking probes in the total system of each blocking probe group is 0.5 mu M; each set of blocking probes was finally brought to a total volume of 600ul. Each well was charged with 100. Mu.L of blocking probes U6-SEQ1, U6-SEQ2 and U6-SEQ3 containing 0.5ug/uL of RNase and 0.5 uM of blocking probe combination, respectively, and a set of 6 parallel wells.

And incubating the 96-hole black ELISA plate in a 37 ℃ incubator for 20 min, and then detecting fluorescence intensity in an ELISA instrument.

2. Statistics and analysis of experimental data

The change of fluorescence intensity values before and after enzyme treatment of each graph is respectively identified by using enzyme-labeled instrument software, and is subjected to arrangement analysis, and is shown in table 6 and fig. 9, the fluorescence brightness of each probe combination after enzyme treatment is improved, and a negative control group has no brightness change, which indicates that the closed probe hydrolyzes the RNA structure and the quenching group carried by the RNA structure after enzyme treatment if expected, but the brightness improvement of each group is inconsistent due to the different probe sequences and the influence of salt concentration.

TABLE 6 fluorescence intensity of blocked probe enzyme treatment assay

Fifth embodiment: multifunctional enzyme-labeled instrument verification amplification system detection fluorescence intensity effect

1. The experimental steps are as follows:

1) Selecting a 96-hole black ELISA plate, and respectively setting an experimental group and a negative control group, wherein each group is subjected to 6 repetitions (the highest value and the lowest value are removed);

2) Experimental group probe dilution system: the experiments were divided into 3 groups, the first group being a negative control (no probe with hybridization solution only); the second group is a closed probe and RNA A enzyme group (after enzymolysis); the third group was a closed probe plus RNA A enzyme group plus SEQY+SEQZ (Y: Z=1:1) amplification system, 6 parallel wells per group, each well added with 100. Mu.L of probe dilution system, respectively. The closed probe group is: U6-SEQ1, U6-SEQ2 and U6-SEQ3. A second set of specific probe dilution systems: according to the closed probe grouping, 594uL of hybridization solution can be added into a 1.5mL centrifuge tube, 3uL of RNA A enzyme with initial concentration of 100mg/mL is added, namely the concentration of RNA A enzyme in the total system is 0.5ug/uL; adding 1:1 to 3ul of total volume of each group of blocking probes respectively, namely, the concentration of the blocking probes in the total system of each blocking probe group is 0.5 mu M; each set of blocking probes was finally brought to a total volume of 600ul. Each well was charged with 100. Mu.L of blocking probes U6-SEQ1, U6-SEQ2 and U6-SEQ3 containing 0.5ug/uL of RNase and 0.5 uM of blocking probe combination, respectively, and a set of 6 parallel wells. Third set of specific probe dilution systems: according to the closed probe grouping, 591uL of hybridization solution can be added into a 1.5mL centrifuge tube, and 3uL of RNA A enzyme with initial concentration of 100mg/mL is added, namely the concentration of RNA A enzyme in the total system is 0.5ug/uL; adding 1:1 to 3ul of total volume of each group of blocking probes respectively, namely, the concentration of the blocking probes in the total system of each blocking probe group is 0.5 mu M; then adding 1:1 of the SEQY+SEQZ closed system to 3ul of the total volume (for example, 1.5ul of the SEQZ+1.5 ul of the SEQY) respectively, namely, the concentration of the closed system in the amplifying system is 0.5 mu M; each set of closed probe amplification systems was finally brought to a total volume of 600ul, respectively. 100. Mu.L of a blocking probe enzyme amplification system U6-SEQ1+ SEQY + SEQZ, U6-SEQ2+ SEQY + SEQZ and U6-SEQ3+ SEQY + SEQZ containing 0.5ug/uL of RNA A enzyme and 0.5 mu.M of a blocking probe and an amplification system combination are added to each well respectively, and a group of 6 parallel wells are formed.

3) And incubating the 96-hole black ELISA plate in a 37 ℃ incubator for 16 hours, and then detecting fluorescence intensity in an ELISA instrument.

2. Statistics and analysis of experimental data

The change of the fluorescence intensity values before and after the enzyme treatment of each graph is respectively identified by using enzyme-labeled instrument software, and is subjected to arrangement analysis, as shown in the following table 7 and fig. 10, after the enzyme treatment of each probe combination, the fluorescence intensity is detected by using an enzyme-labeled instrument after the SEQY+SEQZ amplification system is added, and the negative control group has no brightness change, so that the fluorescent brightness is obviously enhanced by combining the amplification system with the probes after the enzyme treatment of the closed probe, and the fluorescence intensity of the amplification system group is about 10 times higher than that of the single probe group after the enzymolysis, but the performance of the probes with different sequences is different.

TABLE 7 detection of fluorescence intensity after closed probe enzyme treatment and amplification System

。

Sample detection using the present invention: cell samples carried in relevant tissues or body fluids such as hematological disorders, genetic disorders, solid tumors (including bone marrow biopsies), and the like: such as chromosomes of bone marrow or chromosomes prepared from intermittent cells, peripheral blood, amniotic fluid, villus, fetal umbilical cord blood, urine, body fluids (e.g., hydrothorax, ascites, saliva), and the like, tissue, cell or body fluid specimens containing sufficient cancer cells or DNA.

The invention is applicable to the detection of types of gene changes in a variety of diseases or functional alterations including, but not limited to, the following:

1) Related genetic changes on DNA chromosome: gene information and positional changes such as gene rearrangements, inversions, deletions, mutations, strand breaks, insertions, and translocations are used to pinpoint changes in related DNA genes on chromosomes based on clinically validated knowledge of disease or functional relationships. In some embodiments, hybridization of one or more sets of probes to a target chromosome can provide a multi-color pattern by anticipating binding of a target gene to a set of labeled probes on the chromosome, hybridization, elution, visualization by fluorescent visualization or substrate reaction, analysis of the pattern of results of binding and feedback of probes, indicating whether genetic information and positional changes such as gene rearrangements, inversions, deletions, mutations, strand breaks, insertions, and translocations have occurred on the relevant chromosome genome.

2) Each type of RNA expression and positional variation: for example miRNA, lncRNA, mRNA, circRNA, piRNA, snRNA, tRNA, or various RNA or DNA target sequences exogenously introduced into cells. And carrying out fine localization or quantitative analysis on the change of the endogenous expressed or exogenously introduced RNA genes according to the knowledge of the relationship between the clinical verification and the diseases or functions. In some embodiments, hybridization of one or more sets of probes to a target chromosome can provide a multi-color pattern, analysis of the resultant pattern of feedback of probe-bound target RNA by binding of the target gene to the set of labeled probes on the desired RNA, hybridization, elution, development by fluorescent or substrate reaction,

3) The present invention may be used for a variety of clinical or non-clinical diagnostic and biological gene detection, gene function research and analysis purposes, and in particular, the methods described above may be used for diagnosing or studying various types of genetic abnormalities, cancers and other mammalian diseases, including, but not limited to, tumors and cancers: such as prostate cancer, lung cancer, renal cancer, bladder cancer, colorectal cancer, oral cancer, esophageal cancer, nasopharyngeal cancer, gastric cancer, breast cancer, leukemia, lymphoma, pancreatic cancer cholangiocarcinoma, glioma, skin cancer, etc.; parkinson's disease; alzheimer's disease; epilepsy; amyotrophic lateral sclerosis; multiple sclerosis; a stroke; autism; cat's syndrome; patau syndrome; 1p36 deficiency syndrome; edwardsies syndrome; angelman syndrome; klinefelter syndrome; prader-Willi syndrome; turner syndrome; down syndrome; palatoglycardia syndrome and other diseases.

It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.

Claims

1. The target gene FISH fluorescent probe is characterized in that the fluorescent probe is called a target gene FISH fluorescent probe X, a linear probe sequence of the target gene with the length of 15-65bp is arranged in the middle of the FISH fluorescent probe X, the left end and the right end of the linear probe sequence are respectively connected with a neck ring sequence with the length of 5-35bp, and when the linear probe sequence is hybridized with the target gene, the neck ring sequence forms a neck ring structure; the neck ring sequences respectively form neck ring structures at the left end and the right end of the linear probe sequence, and the neck ring sequences at the left end and the right end can be the same neck ring sequences or different neck ring sequences; introducing RNA base serving as an RNase cleavage site at the tail end of each neck ring sequence, and respectively connecting closed sequences at the tail ends of the RNA base at the two ends, wherein the length of each closed sequence is 5-30bp, the closed sequences at the left end and the right end are respectively and specifically combined with linear probe sequences at the left end and the right end, and the Tm value of the closed sequences after being specifically combined with the linear probe sequences is at least 5 ℃ lower than that of the closed sequences after being specifically combined with the target genes;

The linear probe sequence is marked with a fluorescent group for FISH fluorescence detection, and the marked fluorescent group does not influence the specific combination of the linear probe sequence and a target gene;

the blocking sequence is marked with a fluorescence quenching group at the free end of the blocking sequence, and the fluorescence quenching group quenches a fluorescent group on the linear probe sequence under the condition that the blocking sequence is combined with the linear probe sequence, so that the background fluorescence of the FISH fluorescent probe when the target gene is not effectively combined is reduced;

when hybridization detection is carried out, the linear probe sequences are combined with the target gene, the neck ring structures at the left end and the right end are opened, the specific combination of the closed sequences at each end and the linear probe sequences at each end is also disconnected, the closed sequences are released by adding RNase to cut the RNase cutting sites, and the fluorescence quenching groups do not have quenching effect on the fluorescence groups on the linear probe sequences;

the rnase cleavage site is a thermostable rnase cleavage site;

the length of the neck ring sequence is 5-15 bp, and is selected from 5'-ATTGATGTATTGTG-3', 5 '-TCAAGG-3', 5'-TTGATATCCG-3', 5'-TTCAAGAGA-3' or 5 '-CCACACACC-3'.

2. The FISH fluorescent probe of claim 1, wherein the linear probe sequences are fluorescently labeled consecutively or at intervals.

3. The self-assembled amplification probe system for the FISH fluorescence detection comprises the FISH fluorescence probe X for targeting the target gene according to claim 1, and further comprises a primary cascade amplification probe Y, wherein the probe Y comprises a Y probe intermediate sequence of 15-65 bp and flanking sequences at two ends of the Y probe, the Y probe intermediate sequence is not combined with the target gene or/and specifically combined with the X probe, the Y probe intermediate sequence marks a fluorescent group, and the flanking sequences at two ends of the Y probe respectively target the neck ring sequences at two ends of the X probe, and the two sequences are specifically combined.

4. The self-assembled amplification probe system according to claim 3, further comprising a secondary cascade amplification probe Z, wherein the probe Z comprises a Z probe intermediate sequence of 15-65 bp and flanking sequences at two ends of the Z probe, the Z probe intermediate sequence is specifically combined with the Y probe intermediate sequence, the Z probe intermediate sequence is not combined with a target gene or/and is specifically combined with an X probe, and the Z probe intermediate sequence marks a fluorescent group; and flanking sequences at two ends of the Z probe are respectively complementary with flanking sequences at two ends of the next-stage Y probe.

5. The self-assembled amplification probe system of claim 4, wherein the Tm values for the binding of the X probe to the target gene, the Tm values for the binding of the Y probe flanking sequences to the neck ring sequences on either side of the X probe, and the Tm values for the binding of the Z probe to the Y probe differ by no more than +5 ℃.

6. The self-assembled amplification probe system of claim 5, wherein the Tm values for the binding of the X probe to the target gene, the Tm values for the binding of the Y probe flanking sequences to the neck ring sequences on either side of the X probe, and the Tm values for the binding of the Z probe to the Y probe differ by no more than +2 ℃.