Preparation method of fluorescence in situ hybridization kit based on hybridization chain reaction (HCR)

Abstract

The invention belongs to the technical field of molecular biology, and discloses a preparation method of a fluorescence in situ hybridization kit based on a hybridization chain reaction (HCR), the core components of the kit comprise a hybridization probe, an amplification probe, a washing liquid, a confining liquid, protease K, HRP-mouse anti-digoxin, and a TSA developing liquid 520 nm. According to the FISH detection system combined with the HCR signal amplification technology, when a hybridization probe and an amplification probe are simultaneously combined to a position adjacent to a target sequence, a small section of sequence at the top end can trigger a branched HCR reaction to form a macromolecular nucleic acid aggregate. Signal amplification of HCR is achieved through cascade hybridization reaction, detection sensitivity and specificity can be remarkably improved, and meanwhile the problem of background noise caused by a traditional enzymatic method is avoided. The kit disclosed by the invention is wide in sample application range, and can be used for positioning analysis of target RNA of samples such as paraformaldehyde or formalin-fixed animal and plant tissues, paraffin or frozen sections, cells and the like, and high-sensitivity detection of the single cell level of non-coding RNA.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a fluorescence in situ hybridization kit based on hybridization chain reaction (HCR). BACKGROUND

[0002] Fluorescence in situ hybridization (FISH) technology is widely used in molecular biology, developmental biology and medical research. The principle of FISH is to use nucleic acid sequences complementary to the target DNA / RNA sequence as probes, which are directly labeled with fluorescent dyes, or first labeled with biotin, digoxin and other hapten, and then hybridized with target DNA / RNA, and then connected with fluorescent dye markers through immunochemical process. Finally, the hybridization signal is observed under a fluorescence microscope, so as to qualitatively, quantitatively and quantitatively analyze the nucleic acid to be detected in the sample. With the continuous improvement of technology, FISH technology has made great innovations in probe design, signal amplification, imaging and multiplex hybridization. However, the detection of low copy number DNA or RNA targets in cells usually shows poor sensitivity and specificity with the existing FISH-based detection technology, especially for some low copy genes in formalin-fixed, paraffin-embedded (FFPE) tissue sections with significantly degraded RNA.

[0003] Hybridization chain reaction (HCR) is a signal amplification technology first proposed by Dirks et al. in 2004. It is a short-chain-mediated chain displacement amplification process at isothermal and non-enzymatic, that is, through the initiation of specific short-chain nucleic acids, the monomers in the system can change the complementary objects in the double-stranded structure, and finally produce long-chain double helix nucleic acids with incomplete closed phosphate chains. In this process, the presence of oligonucleotides with starting sequences triggers the mutual hybridization of two amplification probes with stem-loop structure. HCR realizes signal amplification through cascade hybridization reaction, which can significantly improve the detection sensitivity and specificity, and at the same time avoid the background noise problem that may be caused by traditional enzymatic signal amplification method. At present, there is no report on the application of HCR signal amplification technology to fluorescence in situ hybridization detection technology. SUMMARY

[0004] Therefore, the present application provides a fluorescence in situ hybridization kit based on hybridization chain reaction (HCR), which aims to realize signal amplification through HCR cascade hybridization reaction and improve detection sensitivity and specificity.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] A fluorescence in situ hybridization kit preparation method based on hybrid chain reaction (HCR), the core components of the kit include hybridization probe, amplification probe, blocking solution, washing solution, proteinase K, HRP-mouse antidigoxin, TSA color developing solution 520nm.

[0007] The hybridization probe is the following five sequences:

[0008] a sequence: 5'-GAGTCTGCTGGTCTCCN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3'

[0009] b sequence: 5'-AGTCTGCTGGTCTCCTN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3'

[0010] c sequence: 5'-GGAGTCTGCTGGTCTCN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3'

[0011] d sequence: 5'-TGGAGTCTGCTGGTCTN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3'

[0012] e sequence: 5'-GTCTGCTGGTCTCCTGN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3';

[0013] Wherein, N1N2N3N4N 5… N21 N 22 N 23 N 24 N 25 It is the complementary sequence of the 25 bp target sequence.

[0014] The hybridization probe comprises a stem-loop structure and a hairpin structure. The 16-base sequences at the 5' and 3' ends are complementary, forming a stem-loop structure. The 3' end has four more bases than the 5' end, and the middle hairpin structure is 25 bases long. Its sequence is complementary to the target sequence. The 4-6 bases in the stem-loop structure near the 5' end of the hairpin sequence, combined with the 25 bases in the hairpin structure, are also complementary to the target sequence.

[0015] The amplification probes are the following 6 sequences:

[0016] Ha sequence: 5'-CTGGAGAATGTGGGAGTCGTCTGTGAGCACTCTCTTGTGCGACAGCACAAGAGAGTGCTCCAGGAGACAGACGACTCCCACATT-3'

[0017] Hb sequence: 5'-GTGCTCACAGACGACTCCCACATTCTCCAGAATGTGGAGTCTGCTGGTCTCCTG-3'

[0018] Hc sequence: 5'-CACTCCAGTGAGCACTCTCTTGTGGAATCCGAAGCTCAGACCCTGCTGAGCTTCGGATTCCGACAGCACAAGAGAGTGCTCACT-3'

[0019] Hd sequence: 5'-GGATTCCACAAGAGAGTGCTCACTGGAGTGAGTGAGCACTCTCTTGTGCTGTCG-3'

[0020] He sequence: 5'-dig-GGCCAAACAGAATCCGAAGCTCAGACCCTGCTGAGCTTCGGATTCTGT-dig-3'

[0021] Hf sequence: 5'-dig-CTGAGCTTCGGATTCTGTTTGGCCACAGAATCCGAAGCTCAAGCAGGGT-dig-3'

[0022] The six amplification probes contain trigger structure, stem loop structure and hairpin structure, and the GC content is 40%-70%. The amplification probe trigger sequence is 6-8 bp, the hairpin structure is 18 complementary bases, and the stem loop structure is 6 bases. The inner hairpin structure is 15 complementary bases, and the inner stem loop structure is 6 bases. The sequence of the trigger structure of the Ha sequence and the sequence of the stem loop structure of the Hb sequence are complementary. The sequence of the hairpin structure of the Hb sequence and the sequence of the outer hairpin structure of the Ha sequence are the same. The sequence of the trigger structure of the Hc sequence and the sequence of the stem loop structure of the Hd sequence are complementary. The sequence of the hairpin structure of the Hd sequence and the sequence of the outer hairpin structure of the Hc sequence are the same. The 5' and 3' ends of the He sequence and the Hf sequence are modified with digoxin (dig). The sequence of the trigger structure of the He sequence and the sequence of the stem loop structure of the Hf sequence are complementary. The sequence of the hairpin structure of the He sequence and the sequence of the hairpin structure of the Hf sequence are the same. The sequence of the trigger structure of the Hf sequence and the inner stem loop structure in the Hc sequence are complementary.

[0023] The hybridization probe and amplification probe are dissolved in the HCR reaction solution. The HCR reaction solution contains 5 kinds of hybridization probes each 1 umol, 6 kinds of amplification probes each 1 umol, sodium chloride 4.3825g, trisodium citrate 2.205g, tween 20 100uL, 5% glycerol 5mL, ddH2O 95mL.

[0024] The 100ml blocking solution contains blocking chain 1umol concentration, deionized formamide 50mL, 10mg / mL inactivated HRP 50uL, LDS 30mg, sodium chloride 1.753g, trisodium citrate 0.882g, ddH2O 50mL.

[0025] The blocking chain is the following 9 sequences: FB1 sequence: CGTCACGTGGAG, FB2 sequence: CTGGAGAATGTG, FB3 sequence: GTGCTCACAGAC, FB4 sequence: GCTGGTCTCCTG, FB5 sequence: CACTCCAGTGAG, FB6 sequence: GGATTCCACAAG, FB7 sequence: CTTGTGCTGTCG, FB8 sequence: GGCCAAACAGAA, FB9 sequence: GCTCAGCAGGGT.

[0026] The 10L washing solution contains sodium chloride 44g, trisodium citrate 22g, LDS 3g, ddH2O 10L.

[0027] The proteinase K, HRP-mouse anti-digoxin, TSA developing solution 520nm are proteinase K (100X), HRP-mouse anti-digoxin (100X), TSA developing solution 520nm (ready-to-use) respectively.

[0028] The kit is stored at 4℃ for short-term and at -20℃ for long-term. The effective period is 12 months.

[0029] The detection kit is used for the localization analysis of target RNA in samples such as paraformaldehyde or formalin-fixed animal and plant tissues, paraffin or frozen sections, and cells, and for high-sensitivity detection of non-coding RNA (including miRNA, circRNA, and IncRNA) at the single-cell level.

[0030] Beneficial effects

[0031] This invention is a FISH detection system that combines HCR signal amplification technology. When the hybridization probe and the amplification probe simultaneously bind to adjacent positions of the target sequence, a series of self-assembled DNA hairpin structures are triggered to form long chains of fluorescently labeled polymers, thereby achieving multiple signal amplification and significantly improving detection sensitivity and specificity. It can not only analyze the location of target RNA in samples such as animal and plant tissues, paraffin or frozen sections, and cells, but also perform high-sensitivity detection of low-copy number miRNAs, circRNAs, or lncRNAs.

[0032] Compared with traditional FISH techniques (such as using the biotin-avidin system or enzymatic color development), HCR-FISH has the following advantages:

[0033] 1. No enzyme dependency: Reduce nonspecific background signals.

[0034] 2. High signal-to-noise ratio: geometric signal amplification is achieved through multi-stage hybridization reactions.

[0035] 3. Multiplex detection capability: Hairpin probes labeled with different fluorescence can detect multiple targets simultaneously.

[0036] This invention not only promotes the development of basic research on FISH technology, but also provides new tools and methods for clinical diagnosis and disease monitoring, and will play an even more important role in life science research in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of HCR-FISH signal amplification principle.

[0038] Figure 2 A and Figure 2 B is a diagram of the hybridization probe and a schematic diagram of the hybridization method.

[0039] Figure 3 A magnified probe image.

[0040] Figure 4 It is a schematic diagram of the implementation process of this kit.

[0041] Figure 5 A and Figure 5B is the fluorescence chart of traditional FISH detection of honeybee apd-1 and HCR-FISH detection of honeybee apd-1. DETAILED DESCRIPTION

[0042] In order to better explain the present application, in order to facilitate understanding, the present application is described in detail by specific embodiments, and the prior art is not explicitly emphasized.

[0043] The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0044] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0045] Example 1: This embodiment designs a FISH detection system combined with HCR signal amplification technology, which will describe the signal amplification process of hybridization probe and amplification probe in detail.

[0046] The hybridization probe is the following five probes:

[0047] a sequence: 5'-GAGTCTGCTGGTCTCCN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3'

[0048] b sequence: 5'-AGTCTGCTGGTCTCCTN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3'

[0049] c sequence: 5'-GGAGTCTGCTGGTCTCN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3'

[0050] d sequence: 5'-TGGAGTCTGCTGGTCTN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3'

[0051] e sequence: 5'-GTCTGCTGGTCTCCTGN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3';

[0052] wherein N1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 is the complement of the 25 bp target sequence.

[0053] The hybridization probe structure is described as follows:

[0054] The hybridization probe contains a stem-loop structure and a hairpin structure, the 5' end and the 3' end 16 base sequences are complementary, forming a stem-loop structure, wherein the 3' end is 4 bases longer than the 5' end, the middle hairpin structure is 25 bases, and its sequence is complementary to the target sequence. The 4-6 bases adjacent to the 5' end of the hairpin sequence are combined with the 25 base hairpin structure, which are also complementary to the target sequence.

[0055] The amplification probe is the following six probes:

[0056] Ha sequence: 5'-CTGGAGAATGTGGGAGTCGTCTGTGAGCACTCTCTTGTGCGACAGCACAAGAGAGTGCTCCAGGAGACAGACGACTCCCACATT-3'

[0057] Hb sequence: 5'-GTGCTCACAGACGACTCCCACATTCTCCAGAATGTGGAGTCTGCTGGTCTCCTG-3'

[0058] Hc sequence: 5'-CACTCCAGTGAGCACTCTCTTGTGGAATCCGAAGCTCAGACCCTGCTGAGCTTCGGATTCCGACAGCACAAGAGAGTGCTCACT-3'

[0059] Hd sequence: 5'-GGATTCCACAAGAGAGTGCTCACTGGAGTGAGTGAGCACTCTCTTGTGCTGTCG-3'

[0060] He sequence: 5'-dig-GGCCAAACAGAATCCGAAGCTCAGACCCTGCTGAGCTTCGGATTCTGT-dig-3'

[0061] Hf sequence: 5'-dig-CTGAGCTTCGGATTCTGTTTGGCCACAGAATCCGAAGCTCAGCAGGGT-dig-3'

[0062] The amplified probe structure is described as follows:

[0063] The Ha sequence comprises a trigger structure, an outer hairpin structure, an inner hairpin structure, an outer stem loop structure, and an inner stem loop structure. The trigger sequence is 6-8 bp, the outer hairpin structure is 18 complementary bases, and the outer stem loop structure is 6 bases. The inner hairpin structure is 15 complementary bases, and the inner stem loop structure is 6 bases. The GC content of the trigger structure, the outer hairpin structure, the inner hairpin structure, the outer stem loop structure, and the inner stem loop structure is 40%-70%. The sequence of the trigger structure is complementary to the sequence of the stem loop structure of the Hb sequence. The sequence of the outer hairpin structure is the same as the sequence of the hairpin structure of the first segment of the Hb sequence.

[0064] The Hb sequence comprises an outer trigger structure, an inner trigger structure, a hairpin structure, and a stem loop structure. The outer trigger sequence is 6-8 bp, the inner trigger sequence is 6-8 bp, the hairpin structure is 18 complementary bases, and the stem loop structure is 6 bases. The GC content of the outer trigger structure, the inner trigger structure, the hairpin structure, and the stem loop structure is 40%-70%. The outer trigger structure is complementary to the outer stem loop structure in the Ha sequence, and the inner trigger structure is complementary to the sequence of the inner hairpin structure in the first segment of the Ha sequence. The sequence of the hairpin structure is the same as the sequence of the outer hairpin structure of the Ha sequence.

[0065] The Hc sequence comprises a trigger structure, an outer hairpin structure, an inner hairpin structure, an outer stem loop structure, and an inner stem loop structure. The trigger sequence is 6-8 bp, the outer hairpin structure is 18 complementary bases, and the outer stem loop structure is 6 bases. The inner hairpin structure is 15 complementary bases, and the inner stem loop structure is 6 bases. The GC content of the trigger structure, the outer hairpin structure, the inner hairpin structure, the outer stem loop structure, and the inner stem loop structure is 40%-70%. The sequence of the trigger structure is complementary to the sequence of the stem loop structure of the Hd sequence. The sequence of the outer hairpin structure is the same as the sequence of the hairpin structure of the Hd sequence.

[0066] The Hd sequence comprises an outer trigger structure, an inner trigger structure, a hairpin structure and a stem loop structure. The outer trigger sequence is 6-8 bp, the inner trigger sequence is 6-8 bp, the hairpin structure is 18 complementary bases and the stem loop structure is 6 bases. The GC content of the outer trigger structure, the inner trigger structure, the hairpin structure and the stem loop structure is 40%-70%. The outer trigger structure is complementary to the inner stem loop structure in the Hc sequence and also complementary to the outer stem loop structure in the Hc sequence. The inner trigger structure is complementary to the inner hairpin sequence in the Hc sequence. The hairpin structure sequence is the same as the outer hairpin structure sequence in the Hc sequence.

[0067] The He sequence comprises a trigger structure, a hairpin structure and a stem loop structure. The trigger sequence is 6-8 bp, the hairpin structure is 18 complementary bases and the stem loop structure is 6 bases. The GC content of the trigger structure, the hairpin structure and the stem loop structure is 40%-70%. The trigger structure sequence is complementary to the stem loop structure sequence in the Hf sequence. The hairpin structure sequence is the same as the hairpin structure sequence in the Hf sequence. The 5' and 3' ends of the He sequence are modified with digoxin (dig).

[0068] The Hf sequence comprises a trigger structure, a hairpin structure and a stem loop structure. The trigger sequence is 6-8 bp, the hairpin structure is 18 complementary bases and the stem loop structure is 6 bases. The GC content of the trigger structure, the hairpin structure and the stem loop structure is 40%-70%. The trigger structure is complementary to the inner stem loop structure in the Hc sequence and also complementary to the stem loop structure sequence in the He sequence. The hairpin structure sequence is the same as the outer hairpin structure sequence in the He sequence. The 5' and 3' ends of the Hf sequence are modified with digoxin (dig).

[0069] The hybridization probe and the amplification probe do not form dimers, do not have mismatches and do not have sequences that specifically bind to other nucleic acids in the entire detection system.

[0070] The signal amplification process is described as follows: in the HCR-FISH detection system, two kinds of DNA monomers, i.e. the starting hybridization probe and the amplification probe, need to be prepared. The starting hybridization probe contains a specific sequence for the target sequence and a starting sequence for triggering the mutual hybridization of the amplification probe. The six HCR amplification probe sequences and the starting hybridization probe sequence are stable stem-loop structure oligonucleotide single-stranded DNA at room temperature. When the starting hybridization probe binds to the target sequence, the trigger chain is exposed, triggering the opening of the amplification probe Ha and causing Ha and Hb to hybridize with each other. This process is the hybridization chain reaction (HCR). At the beginning, the specific sequence of the starting probe binds and exposes the starting sequence. This triggers a cascade reaction of the amplification probe, and the sticky end of the hybridization probe binds to the Hb sequence and opens the stem-loop structure of the amplification probe Hb. The free sequence released after the Hb stem-loop structure is opened can bind to the sticky end of the Ha sequence and open the stem-loop structure of Ha. The two free sequences newly released from Ha can bind to the sticky ends of Hb and Hd sequences and open the stem-loop structures of Hb and Hd, respectively. The free sequence released after the Hd stem-loop structure is opened can bind to the sticky end of the Hc sequence and open the stem-loop structure of Hc. The two free sequences newly released from Hc can bind to the sticky ends of Hd and Hf sequences and open the stem-loop structures of Hd and Hf, respectively. The free sequence released after the Hf stem-loop structure is opened can bind to the sticky end of the He sequence and open the stem-loop structure of He. Thus, a cascade reaction occurs at the binding site of the starting probe, and the fluorescent signal on the amplification probe is concentrated at the target site.

[0071] Example 2: A hybridization chain reaction (HCR) based fluorescence in situ hybridization kit

[0072] A hybridization chain reaction (HCR) based fluorescence in situ hybridization kit, the core components of the kit include hybridization probes, amplification probes, blocking solution, washing solution, proteinase K, HRP-mouse anti-digoxin, TSA developing solution 520 nm. The liquid components in the kit are as follows:

[0073] HCR reaction solution 3 mL

[0074] Blocking solution 3 mL

[0075] Washing solution 10 L

[0076] Proteinase K (100X) 30 uL

[0077] HRP-mouse anti-digoxin (100X) 30 uL

[0078] TSA developing solution 520 nm (ready-to-use) 3 mL

[0079] The above reagent components are described as follows: (all reagents are purchased from SIGMA)

[0080] 1. HCR reaction solution: 1 μmol of each of the five hybridization probes, 1 μmol of each of the six amplification probes, 4.3825 g of sodium chloride, 2.205 g of trisodium citrate, 100 μL of Tween 20, 5 mL of 5% glycerol, and 95 mL of ddH2O.

[0081] 2. Blocking solution (100 mL): 1 μmol blocking chain, 50 mL of deionized formamide, 30 mg of LDS, 1.753 g of sodium chloride, 0.882 g of trisodium citrate, and 50 mL of ddH2O.

[0082] 3. Washing solution (10L): 44g sodium chloride, 22g trisodium citrate, 3g LDS, 10L ddH2O.

[0083] 4. Proteinase K (100X): Prepare to 20 mg / mL in 20 mM Tris-HCl, 50 mM NaCl, 50% Glycerol, 5 mM CaCl2, pH 7.5 buffer.

[0084] 5. HRP-mouse anti-digoxigenin (100X): Prepare to 0.5 mg / mL in 0.01 M sodium phosphate, 0.25 M NaCl, pH 7.6 buffer, and 50% glycerol.

[0085] 6. TSA colorimetric solution 520nm (ready-to-use): 0.1M boric acid buffer, 0.003% H2O2, 0.1% Tween-20, 0.5% Triton X-100, 0.05% proclin-300, and ammonia water adjusted to pH 7 to prepare 5ug / mL TSA colorimetric solution 520nm.

[0086] The above reagents should be stored at 4°C for short-term storage and -20°C for long-term storage. They are valid for 12 months. All reagents must be completely thawed and mixed before use.

[0087] Example 3: A method for using a fluorescence in situ hybridization kit based on hybridization chain reaction (HCR) is provided, specifically:

[0088] 1. Deparaffinization to water

[0089] The sections were sequentially placed in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol for 10 min, 90% alcohol for 10 min, 80% alcohol for 10 min, 70% alcohol for 10 min, and rinsed with pure water.

[0090] 2. Repair

[0091] Cell sample: After the slice is completely dried, draw a suitable hydrophobic circle with a brush, and place the slice horizontally in an in-situ hybridization instrument or a wet box. Drop 100 uL of proteinase K repair solution (1X) on the tissue, incubate at 37°C for 10 min, and rinse with pure water to terminate the reaction.

[0092] Frozen section: Cell sample: After the slice is completely dried, draw a suitable hydrophobic circle with a brush, and place the slice horizontally in an in-situ hybridization instrument or a wet box. Drop 100 uL of proteinase K repair solution (1X) on the tissue, incubate at 37°C for 20 min (for a frozen section), and rinse with pure water to terminate the reaction.

[0093] Paraffin section: Place the slide rack loaded with slides into a repair tank, pour 1X citric acid 6.0 repair solution (the sample area must be immersed), cover with a lid, seal with adhesive tape, and place in a microwave oven, medium heat for 8 min, stop for 8 min, and medium-low heat for 8 min. After natural cooling, seal with 3% hydrogen peroxide for 15 min.

[0094] 3. Blocking

[0095] Remove the slice, spin off the liquid on the slice, draw a suitable hydrophobic circle with a brush, and place the slice horizontally in an in-situ hybridization instrument or a wet box. Add 100 uL of blocking solution to each slice, incubate at 37°C for 30 min, and wash once for 5 min each time. Washing step: Place the slide rack loaded with slides into a washing tank, pour in the washing solution (the sample area must be immersed), and place on a shaker for 5 min at a speed of 60 rpm.

[0096] 4. Probe hybridization

[0097] Spin off the liquid on the slice, add 100 uL of HCR reaction solution to each slice, incubate at 37°C for 2-3 h, and pay attention to maintain humidity to prevent dry slices. Wash 5 times for 5 min each time. Washing step: Place the slide rack loaded with slides into a washing tank, pour in the washing solution (the sample area must be immersed), and place on a shaker for 5 min at a speed of 60 rpm.

[0098] 5. HRP-mouse anti-digoxin

[0099] Spin off the liquid on the slice, add 100 uL of HRP-mouse anti-digoxin (1X) to each slice, and incubate in a wet box at 37°C for 40 min; wash 5 times for 5 min each time. Washing step: Place the slide rack loaded with slides into a washing tank, pour in the washing solution (the sample area must be immersed), and place on a shaker for 5 min at a speed of 60 rpm.

[0100] 6. Color development

[0101] Wash off the liquid on the slice, add 100uL TSA color developing solution to each slice, and incubate at room temperature for 10 min. Wash with pure water to terminate the reaction.

[0102] 7. DAPI nuclear staining

[0103] Add 50uL DAPI staining solution to each slice, and incubate in the dark for 5 min. After washing with pure water, add anti-fluorescence quenching mounting agent to mount the slice.

[0104] Example 4: Taking the detection of honeybee apd-1 as an example, the sensitivity of the probe in the kit of Example 1 is tested by comparing the detection results of the method of the application with traditional FISH.

[0105] The specific steps of the detection method of the application are as described in Example 3 above.

[0106] Traditional FISH experimental steps:

[0107] 1. De-waxing to water

[0108] Put the slice into xylene I 15 min-xylene II 15 min-xylene III 15 min-anhydrous ethanol 10 min-90% alcohol 10 min-80% alcohol 10 min-70% alcohol 10 min-pure water washing, in sequence.

[0109] 2. Repair

[0110] Cell sample: After waiting for the slice to completely dry, draw a suitable hydrophobic circle with a brush, and place the slice horizontally in an in-situ hybridization instrument or a wet box. Add 100uL proteinase K repair solution (1X) to the tissue, and incubate at 37℃ for 10 min. Wash with pure water to terminate the reaction.

[0111] Frozen section: Cell sample: After waiting for the slice to completely dry, draw a suitable hydrophobic circle with a brush, and place the slice horizontally in an in-situ hybridization instrument or a wet box. Add 100uL proteinase K repair solution (1X) to the tissue, and incubate at 37℃ for 20 min (for frozen sections). Wash with pure water to terminate the reaction.

[0112] Paraffin section: Put the slide rack loaded with slides into a repair tank, pour 1X citric acid 6.0 repair solution (the sample area must be immersed), cover with a lid, seal with adhesive tape, and put into a microwave oven. Medium heat for 8 min, stop for 8 min, and medium-low heat for 8 min. After natural cooling, seal with 3% hydrogen peroxide for 15 min.

[0113] 3. Blocking

[0114] Take out the section, spin the liquid on the section, draw a suitable size hydrophobic circle with a brush, place the section horizontally in the in-situ hybridization instrument or wet box, add 100ul pre-hybridization solution to each section, and incubate at 37℃ for 60min.

[0115] 4. Probe hybridization

[0116] Spin the liquid on the section, add 100uL of hybridization probe to each section, and incubate at 37℃ overnight, paying attention to maintain humidity to prevent drying.

[0117] 5. HRP-mouse anti-digoxin

[0118] Spin the liquid on the section, add 100uL of HRP-mouse anti-digoxin (1X) to each section, and incubate in a wet box at 37℃ for 40min; wash 5 times for 5min each time. Wash step: Place the slide rack containing the slides into the wash tank, pour in the wash solution (the sample area must be immersed), and place on the shaker for 5min at 60rpm.

[0119] 6. Color development

[0120] Spin the liquid on the section, add 100uL of TSA color development solution to each section, and incubate at room temperature for 10min. Rinse with pure water to stop the reaction.

[0121] 7. DAPI nuclear staining

[0122] Add 50uL of DAPI staining solution to each section, incubate in the dark for 5min, rinse with pure water, and then add anti-fluorescence quenching mounting medium to mount the section.

[0123] Compared with the results of traditional FISH detection, the experimental results are shown in Figure 5 A and Figure 5 B, which shows that in the traditional FISH results, the entire tissue shows a positive result without obvious point-like expression, while in the HCR-FISH results, the background is significantly reduced, and there is obvious point-like expression, which shows that the specificity and sensitivity of HCR-FISH are greatly improved.

[0124] The conventional techniques in the above examples are prior art known to those skilled in the art, and therefore will not be described in detail here. The above embodiments are only used to illustrate the present application, and are not limiting on the present application. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application. Therefore, all equivalent technical solutions also belong to the scope of the present application, and the patent protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing a fluorescence in situ hybridization kit based on hybridization chain reaction (HCR), characterized by: The core components of the kit include hybridization probe, amplification probe, washing solution, blocking solution, proteinase K, HRP-mouse anti-digoxigenin, and TSA color development solution 520nm.

2. The method for preparing the kit according to claim 1, wherein: The hybridization probes are the following 5 sequences: a sequence: 5’-GAGTCTGCTGGTCTCCN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3’ b sequence: 5’-AGTCTGCTGGTCTCCTN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3’ c sequence: 5’-GGAGTCTGCTGGTCTCN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3’ d sequence: 5’-TGGAGTCTGCTGGTCTN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3’ e sequence: 5’-GTCTGCTGGTCTCCTGN1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 CAGGAGACCAGCAGACTCCA-3’; Among them, N1N2N3N4N 5… N 21 N 22 N 23 N 24 N 25 It is the complementary sequence of the 25 bp target sequence.

3. The method for preparing the kit according to claim 2, wherein: The hybridization probe comprises a stem-loop structure and a hairpin structure. The 16-base sequences at the 5' and 3' ends are complementary, forming a stem-loop structure, in which the 3' end has 4 more bases than the 5' end, and the middle hairpin structure is 25 bases, whose sequence is complementary to the target sequence. The 4-6 bases in the stem-loop structure near the 5' end of the hairpin sequence and the 25 bases in the hairpin structure are combined together and are also complementary to the target sequence.

4. The method for preparing the kit according to claim 1, wherein: The amplification probes are the following 6 sequences: Ha sequence: 5'-CTGGAGAATGTGGGAGTCGTCTGTGAGCACTCTCTTGTGCGACAGCACAAGAGAGTGCTCCAGGAGACAGACGACTCCCACATT-3' Hb sequence: 5'-GTGCTCACAGACGACTCCCACATTCTCCAGAATGTGGAGTCTGCTGGTCTCCTG-3' Hc sequence: 5'-CACTCCAGTGAGCACTCTCTTGTGGAATCCGAAGCTCAGACCCTGCTGAGCTTCGGATTCCGACAGCACAAGAGAGTGCTCACT-3' Hd sequence: 5'-GGATTCCACAAGAGAGTGCTCACTGGAGTGAGTGAGCACTCTCTTGTGCTGTCG-3' He sequence: 5'-dig-GGCCAAACAGAATCCGAAGCTCAGACCCTGCTGAGCTTCGGATTCTGT-dig-3' Hf sequence: 5'-dig-CTGAGCTTCGGATTCTGTTTGGCCACAGAATCCGAAGCTCAAGCAGGGT-dig-3'.

5. The method for preparing the kit according to claim 4, wherein: The six amplification probes contain a trigger structure, a stem-loop structure, and a hairpin structure, with a GC content of 40%-70%. The amplification probe trigger sequence is 6-8bp, with 18 complementary bases in the hairpin structure and 6 bases in the stem-loop structure, and 15 complementary bases in the internal hairpin structure and 6 bases in the internal stem-loop structure. The Ha sequence trigger structure sequence is complementary to the stem-loop structure sequence of the Hb sequence; the Hb sequence hairpin structure sequence is identical to the external hairpin structure sequence of the Ha sequence; the Hc sequence trigger structure sequence is complementary to the stem-loop structure sequence of the Hd sequence; the Hd sequence hairpin structure sequence is identical to the external hairpin structure sequence of the Hc sequence; the He sequence and the Hf sequence are modified with digoxigenin (dig) at the 5' and 3' ends; the He sequence trigger structure sequence is complementary to the stem-loop structure sequence of the Hf sequence; the He sequence hairpin structure sequence is identical to the Hf sequence hairpin structure sequence; and the Hf sequence trigger structure is complementary to the internal stem-loop structure in the Hc sequence.

6. The method for preparing a kit according to any one of claims 2 to 5, characterized in that: The hybridization probe and the amplification probe were dissolved in an HCR reaction solution, which contained 1 μmol of each of the five hybridization probes, 1 μmol of each of the six amplification probes, 4.3825 g of sodium chloride, 2.205 g of trisodium citrate, 100 μL of Tween 20, 5 mL of 5% glycerol, and 95 mL of ddH2O.

7. The method for preparing a kit according to claim 1, wherein: The 100 ml blocking solution contains 1 μmol of blocking chain, 50 mL of deionized formamide, 50 μL of 10 mg / mL inactivated HRP, 30 mg of LDS, 1.753 g of sodium chloride, 0.882 g of trisodium citrate, and 50 mL of ddH2O.

8. The method for preparing the kit according to claim 7, wherein: The closed chains are the following 9 sequences: FB1 sequence: CGTCACGTGGAG, FB2 sequence: CTGGAGAATGTG, FB3 sequence: GTGCTCACAGAC, FB4 sequence: GCTGGTCTCCTG, FB5 sequence: CACTCCAGTGAG, FB6 sequence: GGATTCCACAAG, FB7 sequence: CTTGTGCTGTCG, FB8 sequence: GGCCAAACAGAA, FB9 sequence: GCTCAGCAGGGT.

9. The method for preparing a kit according to claim 1, wherein: The 10L washing solution contains 44g of sodium chloride, 22g of trisodium citrate, 3g of LDS, and 10L of ddH2O.

10. The method for preparing a kit according to claim 1, wherein the detection kit is characterized in that it is used for localization analysis of target RNA in samples such as paraformaldehyde- or formalin-fixed animal and plant tissues, paraffin- or frozen sections, and cells, and for high-sensitivity detection of non-coding RNA (including miRNA, circRNA, and lncRNA) at the single-cell level.

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