A rapid in situ hybridization detection method and application thereof

Abstract

The embodiment of the present application relates to the technical field of nucleic acid detection, in particular to a rapid in situ hybridization detection method and application thereof, comprising the following steps: S1, preparation of anti-DNA-RNA hybrid antibody; S2, in situ hybridization detection; through the steps of dewaxing, protease digestion, probe hybridization, anti-DNA-RNA hybrid antibody immunization, DAB (or fast red) color development, hematoxylin staining solution restaining, blue returning, dehydration, transparency, conventional mounting, etc. The anti-DNA-RNA hybrid mouse monoclonal antibody is used in combination, the antibody has high specificity and high sensitivity in combination with DNA-RNA hybrid molecule, can specifically recognize and detect the DNA or RNA sequence specifically recognized by the probe in the cell, and is positively highly expressed in the detection of DNA-RNA hybrid. The antibody can be applied to fluorescence in situ hybridization detection, ELISA detection, antibody chip preparation, flow cytometry and other detection and screening fields, which is conducive to obtaining accurate evaluation and detection results.

Description

Technical Field

[0001] This invention relates to the field of antigen detection technology, specifically a rapid in situ hybridization detection method and its application. Background Technology

[0002] Existing HPV probe (in situ hybridization) and EBER probe (in situ hybridization) detection kits employ fluorescence in situ hybridization (FISH) technology. This involves using a digoxigenin-labeled HPV probe (EBER probe) to hybridize and bind to a specific target sequence in tissue cells. A digoxigenin antibody reacts with the HPV probe (EBER probe) to form an immune complex. Simultaneously, an enhancing secondary antibody binds to the digoxigenin antibody-HPV probe (EBER probe) complex. Finally, HRP catalyzes the formation of a brownish-yellow precipitate of DAB at the binding site, thus revealing the specific target RNA sequence site in the cell under a microscope. This method, using fluorescence in situ hybridization and enhancing secondary antibodies for signal amplification, is time-consuming and has low specificity. In contrast, the rapid FISH method of this invention does not require enhancing secondary antibodies. It directly uses an antibody-AP complex to detect DNA-RNA hybrids, ultimately catalyzing the substrate to emit light. This antibody exhibits a strong binding ability to DNA-RNA hybrids. The use of this antibody shortens the reaction time and improves the sensitivity and specificity of the detection.

[0003] The S9.6 mouse monoclonal antibody was produced in 1986 in mice immunized with bacteriophage φX174, a long DNA-RNA hybrid. The resulting S9.6 exhibits high affinity and specificity for the DNA-RNA hybrid, low affinity for double-stranded RNA (dsRNA), and no affinity for single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Due to its unique properties, S9.6 immunoglobulin provides the basis for numerous applications, including solution recognition of DNA-RNA hybrids, detection of undetected transcriptionally active genomic regions, in vitro and in vivo recognition of R-loop (annealed nascent mRNA from single-stranded genomic DNA) transcriptional cells, and label-free miRNA (a class of non-coding single-stranded RNA molecules approximately 22 nucleotides long encoded by endogenous genes, involved in post-transcriptional gene expression regulation in plants and animals) detection and analysis.

[0004] Although existing S9.6 mouse monoclonal antibodies exhibit higher specificity and sensitivity against DNA-RNA hybrids compared to commercially available antibodies, they still suffer from non-specific recognition and high background values ​​during ELISA detection. Therefore, screening for an anti-DNA-RNA hybrid antibody with low background values ​​is crucial for viral detection and tumor cell detection. In light of this, we propose a rapid in situ hybridization detection method and its application. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid in situ hybridization detection method and its application, which solves the problem that non-specific identification still causes high background values ​​in the existing ELISA detection.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A rapid in situ hybridization detection method and its application, comprising the following steps:

[0008] S1. Preparation of anti-DNA-RNA hybrid antibody; the anti-DNA-RNA hybrid antibody is an anti-DNA-RNA hybrid mouse monoclonal antibody, which can specifically recognize any DNA-RNA hybrid and induce an antigen-antibody immune response; the heavy chain encoding gene used to encode the anti-DNA-RNA hybrid mouse monoclonal antibody in step S1 is SEQ ID 1.

[0009] CAGGTGCAGCTGCAGCAGAGCGGCCCGGAACTGGTGAAACCGGGCGCGAGCGTGAAAATGAGCTGC

[0010] AAAGCGAGCGGCTATAACCTTTACCAGCCTGCGCATTACCGATTGGGTGAAACAGAAACCGGGCCAG

[0011] GGCCTGGAATGGATTGGCGATGTGAGCAGCAAAAAAGAACTGGATAAAAGCCAGGAAAAAAGAAGAA

[0012] GTGCAGACCAAAGCGACCCTGACCAGCGATAAAAGCAGCAGCACCGCGTATATGGAACTGAGCAGC

[0013] CTGACCAGCAAAGATAGCGCGGTGTATTATTGCGCGCGCCAGCATAAAAGCGAAGAAGATATTCTGGCGTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC;

[0014] The light chain encoding gene is SEQ ID2.

[0015] GATATTGTGATGACCCAGACCCCGCTGAGCCTGCCGGTGAGCCTGGGCGATCAGGCGAGCATTAGC

[0016] TGCAACATTTGCCGCAACTGCCAGTGCCTGAGCTGCATGGATTGCGGCAAAGATTTTTGGTATCTG

[0017] CAGAAACCGGGCCAGAGCCCGAAACTGCTGATTTATAGCGAAGATCAGAAATATGGCGGCAAAGAT

[0018] GGCGTGCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTG

[0019] GAAGCGGAAGATCTGGGCGTGTATTATTGCGGCAAAGAAAGCCTGGAAAACGAATTTGAAATTTTT

[0020] GGCGGCGGCACCAAACTGGAAATTAAACGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC;

[0021] The heavy chain amino acid sequence of the anti-DNA-RNA hybrid antibody is SEQ ID3, QVQLQQSGPELVKPGASVKMSCKASGYTFTSLRITDWVKQKPGQGLEWIGDVSSKKELDKSQEKEE VQTKATLTSDKSSSTAYMELSSLTSKDSAVYYCARQHKSEEDILAWGQGTTLTVSS;

[0022] The light chain amino acid sequence is SEQ ID4.

[0023] DIVMTQTPLSLPVSLGDQASISCNICRNCQCLSCMDCGKDFWYLQKPGQSPKLLIYSEDQKYGGKD

[0024] GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCGKESLENEFEIFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGS.

[0025] The preparation of the anti-DNA-RNA hybrid antibody in step S1 includes the following steps:

[0026] S1.1 Immunization of animals: 100 μg / ml DNA-RNA hybrid and 100 μg / ml methylated bovine serum albumin were mixed in 10 mM TE buffer at a mass ratio of 1:1. 30 μg of DNA-RNA hybrid was emulsified with Freund's complete adjuvant per Balb / c mouse and injected subcutaneously into the abdomen. Booster immunizations were performed on days 14 and 28 (using the same dose and Freund's incomplete adjuvant emulsification as the primary immunization). Blood was collected from the tail of the mice on day 35 for testing.

[0027] S1.2 Preparation of hybridoma cell lines: lymphocytes from immunized mice were fused and cloned to obtain stable positive hybridoma cell lines that could efficiently secrete antibodies. Total RNA was isolated from the hybridoma cell lines.

[0028] S1.3 Obtaining antibody sequences: Total RNA was reverse transcribed into cDNA, and the nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region were obtained by PCR amplification using specific primers.

[0029] S1.4 Antibody expression and purification; The nucleotide sequence is cloned into the expression vector, and the cultured cells are transiently transfected using the transfection method. After culturing, the supernatant is collected, and the supernatant is purified using Protein A to obtain an antibody with a purity >95%.

[0030] S2, Rapid in situ hybridization detection;

[0031] The rapid in situ hybridization detection in step S2 includes the following detection steps.

[0032] S2.1 Dewaxing; Dry the tissue sections in a 75℃ oven, then dewax them in a dewaxing solution for 5 min × 3 times, treat them with 100% alcohol for 3 min × 2 times, treat them with 95% alcohol for 2 min, treat them with 85% alcohol for 2 min, and soak them in purified water for 2 min; The main component of the dewaxing solution is xylene, and the sections need to be soaked in three xylene solutions for 5 min each time.

[0033] S2.2, Protease Digestion: Shake off the water on the slide, draw circles around the tissue with a water-blocking pen, and wash with PBST for 1 min × 3 times; then add 50-100 μl of protease digestion solution and incubate at 37℃ for 10-20 min; discard the protease digestion solution, and then wash with PBST for 2 min × 3 times; the water-blocking pen is an oil-based pen used to lock in the subsequently added buffer and working solution; the concentration of the PBST washing solution is 0.01 mol / L, and the components of 1 L of solution are as follows: 5.82 g sodium chloride; 38.81 g disodium hydrogen phosphate dodecahydrate; 15.60 g sodium dihydrogen phosphate dihydrate; 200 μl Tween 20; pH value is 7.2-7.4; the protease digestion solution is protease solid dissolved in TBS solution, and the BSA solution is 100 ml of 0.5 mol / L Tris-HCl buffer, 3.5 g of NaCl (0.15 mol / L), and added to a final volume of 1000 ml;

[0034] S2.3, EBER probe hybridization: Depending on the tissue size, add 100-150 μl of EBER probe, place in a humidified chamber, and incubate at 37°C for 1-2 hours. Then wash with PBST for 2 minutes × 3 times. The EBER probe is a DNA sequence complementary to the EB virus-specific target, which is a protected sequence with a length of 35 bp. The length of the EBER probe is 20-100 bp. The EBER probe in step S2.3 has a length of 15 bp, 35 bp, 100 bp, 500 bp, 1000 bp, or 2000 bp. The humidified chamber should be kept away from light during use and should be brown or black in color.

[0035] S2.4 Immunization with anti-DNA-RNA hybrid antibody: After removing PBST buffer, add 50-100 μl of HRP-labeled anti-DNA-RNA hybrid antibody to each slide and incubate at 37°C for 30 min; after incubation, wash with PBST buffer for 2 min × 3 times; the antibody is purified by Protein G column and diluted 1000 times with antibody dilution buffer before use. The antibody dilution buffer consists of: 2.535-3.501 g sodium chloride; 6-7 ml Tris-HCl buffer; 20-28 ml casein solution; 28-31 μl preservative; the remainder is pure water; the pH of Tris-HCl is 7.4-7.5; the antibody is an anti-DNA-RNA antibody, which is conjugated with horseradish peroxidase (HRP), which can cause DAB chromogenic solution to form a brown precipitate;

[0036] S2.5, DAB staining: Remove the PBST buffer, add 100-150 μl of freshly prepared DAB staining solution (DAB concentrate: DAB buffer = 1:19) to each slide, and incubate at room temperature for 3-5 min; the DAB staining solution should be prepared and used immediately, and the time from preparation to use should not exceed 1 hour; the main component of the DAB staining solution is diaminobenzidine, and both the DAB staining solution and the DAB diluent should be stored away from light;

[0037] S2.6, counterstain with hematoxylin solution; rinse with tap water, add 100-150 μl of hematoxylin solution for counterstaining, incubate for 1-3 min, rinse with tap water to return to blue; dehydrate and clear the sections, and mount them as usual; observe the staining of the tissue under an optical microscope.

[0038] A rapid in situ hybridization detection application, including the application of a rapid in situ hybridization detection method in the detection of anti-DNA-RNA heterozygous antibody protein molecules.

[0039] By employing the above technical solution, the present invention provides a rapid in situ hybridization detection method and its application. It possesses at least the following beneficial effects:

[0040] (1) This invention combines a mouse monoclonal antibody against DNA-RNA hybrids with steps such as dewaxing, protease digestion, EBER probe hybridization, immunization with anti-DNA-RNA hybrid antibody, rapid red staining, hematoxylin staining, blue staining, dehydration, clearing, and conventional mounting. This antibody has high specificity and sensitivity in binding to DNA-RNA hybrid molecules, and can specifically recognize and detect DNA or RNA sequences in cells that are specifically recognized by the probe. It shows high positive expression when detecting DNA-RNA hybrids. Therefore, this antibody can be applied to detection and screening fields such as fluorescence in situ hybridization, ELISA, antibody chip preparation, and flow cytometry, which is beneficial for obtaining accurate assessment and detection results.

[0041] (2) The anti-DNA-RNA hybrid mouse monoclonal antibody of this invention has a highly sensitive and specific binding ability. This antibody can be labeled with alkaline phosphatase or horseradish peroxidase, and has extremely high detection sensitivity and low background value even at extremely low concentrations in the working solution. In the hybridization capture-chemiluminescence method, this antibody can be used simultaneously as the first and second antibody, which has extremely high application value. Attached Figure Description

[0042] The accompanying drawings, which are provided to further illustrate the invention, constitute a part of this application:

[0043] Figure 1 The results of DAB staining detection using the EBER rapid in situ hybridization method in this invention are shown.

[0044] Figure 2 The test results are obtained using existing EBER in situ hybridization methods and reagents.

[0045] Figure 3 The results of rapid red staining detection using the EBER rapid in situ hybridization method in this invention are shown. Detailed Implementation

[0046] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0047] A rapid in situ hybridization detection method and its application, comprising the following steps:

[0048] S1. Preparation of anti-DNA-RNA hybrid antibody; The anti-DNA-RNA hybrid antibody is a mouse monoclonal antibody against DNA-RNA hybrids, which can specifically recognize any DNA-RNA hybrid and react with any DNA-RNA hybrid to produce an antigen-antibody immune response;

[0049] The preparation of the anti-DNA-RNA hybrid antibody in step S1 includes the following steps:

[0050] S1.1 Immunization of animals: 100 μg / ml DNA-RNA hybrid and 100 μg / ml methylated bovine serum albumin were mixed in 10 mM TE buffer at a mass ratio of 1:1. 30 μg of DNA-RNA hybrid was emulsified with Freund's complete adjuvant per Balb / c mouse and injected subcutaneously into the abdomen. Booster immunizations were performed on days 14 and 28 (using the same dose and Freund's incomplete adjuvant emulsification as the primary immunization). Blood was collected from the tail of the mice on day 35 for testing.

[0051] S1.2 Preparation of hybridoma cell lines: lymphocytes from immunized mice were fused and cloned to obtain stable positive hybridoma cell lines that could efficiently secrete antibodies. Total RNA was isolated from the hybridoma cell lines.

[0052] S1.3 Obtaining antibody sequences: Total RNA was reverse transcribed into cDNA, and the nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region were obtained by PCR amplification using specific primers.

[0053] S1.4 Antibody expression and purification: The nucleotide sequence was cloned into the expression vector, and the cultured cells were transiently transfected using the transfection method. After culturing, the supernatant was collected, and the supernatant was purified using Protein A to obtain an antibody with a purity >95%.

[0054] S2, Rapid in situ hybridization detection;

[0055] The rapid in situ hybridization detection in step S2 includes the following detection steps:

[0056] S2.1 Dewaxing; Dry the tissue sections in a 75℃ oven, then dewax them in a dewaxing solution for 5 min × 3 times, treat them with 100% alcohol for 3 min × 2 times, treat them with 95% alcohol for 2 min, treat them with 85% alcohol for 2 min, and soak them in purified water for 2 min; The main component of the dewaxing solution is xylene, so the sections need to be soaked in three xylene solutions for 5 min each time.

[0057] S2.2, Protease Digestion: Shake off excess water from the slide, draw circles around the tissue with a water-blocking pen, and wash with PBST for 1 min × 3 times; then add 50-100 μl of protease digestion solution and incubate at 37°C for 10-20 min; discard the protease digestion solution, and then wash with PBST for 2 min × 3 times; the water-blocking pen is an oil-based pen used to lock in the subsequently added buffer and working solution; the PBST washing solution concentration is 0.01 mol / L, and the components of 1 L of solution are as follows: 5.82 g sodium chloride; 38.81 g disodium hydrogen phosphate dodecahydrate; 15.60 g sodium dihydrogen phosphate dihydrate; 200 μl Tween 20; pH value is 7.2-7.4; the protease digestion solution is protease solid dissolved in TBS solution, the BSA solution is 100 ml of 0.5 mol / L Tris-HCl buffer, 3.5 g NaCl (0.15 mol / L), and the volume is adjusted to 1000 ml;

[0058] S2.3, EBER probe hybridization; depending on the tissue size, add 100-150 μl of EBER probe, place in a humidified chamber, and incubate at 37°C for 1-2 hours, then wash with PBST for 2 minutes × 3 times; the EBER probe is a DNA sequence complementary to the EB virus-specific target, which is a protected sequence with a length of 35 bp; the length of the EBER probe is 20-100 bp, and the EBER probe in step S2.3 has a length of 15 bp, 35 bp, 100 bp, 500 bp, 1000 bp, or 2000 bp; the humidified chamber should be kept away from light during use, and the chamber color should be brown or black;

[0059] S2.4 Immunization with anti-DNA-RNA hybrid antibody: After removing PBST buffer, add 50-100 μl of HRP-labeled anti-DNA-RNA hybrid antibody to each slide and incubate at 37℃ for 30 min; after incubation, wash with PBST buffer for 2 min × 3 times; the antibody is purified by Protein G column, and used after diluting the antibody 1000 times. The antibody diluent components are: sodium chloride 2.535-3.501 g; Tris-HCl buffer 6-7 ml; casein solution 20-28 ml; preservative 28-31 μl; the remainder is pure water; the pH of Tris-HCl is 7.4-7.5; the antibody is an anti-DNA-RNA antibody, which is conjugated with horseradish peroxidase (HRP), which can cause DAB chromogenic solution to form a brown precipitate;

[0060] S2.5, DAB staining: Remove the PBST buffer, add 100-150 μl of freshly prepared DAB staining solution (DAB concentrate: DAB buffer = 1:19) to each slide, and incubate at room temperature for 3-5 min; the DAB staining solution should be prepared and used immediately, and the time from preparation to use should not exceed 1 hour; the main component of the DAB staining solution is diaminobenzidine, and both the DAB staining solution and the DAB diluent should be stored away from light;

[0061] S2.6, counterstain with hematoxylin solution; rinse with tap water, add 100-150 μl of hematoxylin solution for counterstaining, incubate for 1-3 min, rinse with tap water to return to blue; dehydrate and clear the sections, and mount them as usual; observe the staining of the tissue under an optical microscope.

[0062] Example

[0063] The anti-DNA-RNA hybrid antibody obtained using the method in step S1 is labeled with AP and then applied to the HPV chemiluminescence-hybridization capture assay reagent. The assay reagent is used to detect the sample to be tested. The operation steps are as follows:

[0064] Step 1, Denaturation: The cells of the sample to be tested are disrupted, causing protein denaturation, RNA degradation, and DNA denaturation into single-stranded DNA, resulting in a denatured sample. After cell disruption, the sample is reacted with a 1.75 mol / L sodium hydroxide solution at a volume ratio of 1:2 to the sample at 65°C for 30 minutes. This process denatures proteins and degrades RNA, reducing the impact of proteins and RNA on subsequent detection. Furthermore, the sodium hydroxide solution opens the double helix structure of DNA, denaturing it into single-stranded DNA. In this embodiment, a combination of chemical and physical methods is used to efficiently degrade RNA and denature double-stranded DNA into single-stranded DNA. If the sample contains bacteria or viruses, the double-stranded DNA of the bacteria or viruses is denatured into single-stranded DNA.

[0065] Step 2, Hybridization: The denatured sample obtained in Step 1 is mixed with a specific single-stranded RNA probe stored in a nucleic acid preservation solution at pH 3.5-4.0. Hybridization is then performed for 45 minutes at 65°C and a pH range of 7.0-7.4, allowing the specific single-stranded RNA probe to hybridize with the single-stranded DNA in the denatured sample, forming a DNA-RNA hybrid solution. The single-stranded DNA in the denatured sample forms a DNA-RNA hybrid with the single-stranded RNA probe. The specific single-stranded nucleic acid probe is then placed in a solution at pH 3.5- Preservation in a 4.0-pH nucleic acid preservation solution helps to keep specific single-stranded nucleic acid probes stable and prevent degradation by enzymes in the environment without the need for freezing. When a specific single-stranded nucleic acid probe solution with a pH of 3.5-4.0 is mixed with a sample that has been denatured and decomposed into a single-stranded DNA solution with an alkaline pH, a neutral pH solution is formed, and the specific single-stranded nucleic acid probe becomes a single-stranded RNA probe. At 65°C, according to the complementary pairing principle of base sequences, the single-stranded DNA in the denatured sample and the single-stranded RNA probe form a DNA-RNA hybrid.

[0066] Based on 100 mL of nucleic acid preservation solution, the nucleic acid preservation solution comprises the following components in the following amounts: trisodium citrate 5.8-5.95 g; biological buffer 19.56-20.21 g; disodium ethylenediaminetetraacetate 0.173-0.192 g; acidity adjuster 8.2-9.0 mL; preservative 28-33 μl; the balance being pure water; the pH of the preservation solution is 3.5-4.0; the buffer includes at least one of triethanolamine hydrochloride and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; the acidity adjuster includes at least one of glacial acetic acid and polyacrylic acid.

[0067] Specific single-stranded nucleic acid probes are preserved in a nucleic acid preservation solution. This solution allows for the preservation of single-stranded RNA probes without freezing, and also keeps the single-stranded RNA probes structurally stable and resistant to degradation.

[0068] Specific single-stranded RNA probes can be full-length single-stranded RNA probes targeting the target DNA or fragmented single-stranded RNA probes targeting a portion of the target DNA. When the specific single-stranded RNA probe is a full-length single-stranded RNA probe targeting the target DNA, it can be a continuous full-length single-stranded RNA probe targeting the target DNA or a segmented full-length single-stranded RNA probe targeting the target DNA. By adopting the above technical solution, whether it is a full-length single-stranded nucleic acid probe targeting the target DNA or a fragmented single-stranded RNA probe targeting a portion of the target DNA, the bases on it that can bind to single-stranded DNA have a certain degree of specificity. This allows the specific single-stranded RNA probe to pair with the bases on the single-stranded DNA and achieve hybridization between the specific single-stranded RNA probe and single-stranded DNA, obtaining a DNA-RNA hybrid.

[0069] Step 3, capture: The first antibody is fixed on the vector, and then the DNA-RNA hybrid solution obtained in step 2 is added. At a temperature of 42°C, the first antibody is used to capture the hybrids in the DNA-RNA hybrid solution for 60 min, and then the liquid is removed.

[0070] The first antibody is a substance that specifically recognizes the hybrid. The substance that specifically recognizes the hybrid is a DNA-RNA hybrid structure-specific antibody, a polyclonal antibody or monoclonal antibody or its fragment, a protein, a catalytically inactivated ribonuclease H, a nucleic acid, a nucleic acid aptamer, or an oligonucleotide that specifically binds to the DNA-RNA hybrid to form a triplet structure. The catalytically inactivated ribonuclease H is obtained by treating ribonuclease H to remove its catalytic activity, which is beneficial for retaining its binding to the DNA-RNA hybrid.

[0071] The primary function of the first antibody is to capture the hybrid in the solution. Reacting at 42°C for 60 minutes helps the first antibody to fully capture the hybrid. The second antibody contains a marker that emits fluorescence upon light excitation, or develops color through gold particle aggregation, or through enzyme-catalyzed reactions, thus achieving detection. The first antibody captures the hybrid by binding to the target site on the hybrid, while the second antibody binds to the DNA-RNA hybrid or conjugate. The detection is then achieved through the marker on the second antibody (in this case, the single-stranded RNA probe does not contain any modification), thereby detecting both the DNA-RNA hybrid and the target DNA in the sample.

[0072] In step three, the first antibody is first immobilized on the carrier, which helps to increase the stability of the first antibody during the operation. When it binds to / captures the hybrid, it helps to improve the stability of the product of the first antibody and the hybrid bound to the hybrid on the carrier. After the first antibody and the hybrid are fully bound, other impurities are washed away, leaving the first antibody and the hybrid for subsequent steps.

[0073] Meanwhile, the primary and secondary antibodies were screened independently, primarily to minimize mismatches between specific single-stranded nucleic acid probes and target DNA, and to avoid inactivation of both antibodies. Specifically, when the screening temperature for the primary and secondary antibodies was set at 42℃, the resulting antibodies exhibited good performance and required a shorter reaction time. Compared to 25℃ and 37℃, the higher temperature of 42℃ accelerated molecular thermal motion, thus helping to shorten the reaction time. The higher temperature effectively increased the binding of the primary and secondary antibodies to the hybrid, resulting in a more accurate signal-to-noise ratio. Compared to 45℃, 50℃, and 65℃, as the temperature increased, the activity of the primary and secondary antibodies gradually decreased or even became inactivated, ultimately making it difficult to accurately detect the signal-to-noise ratio using the markers carried on the secondary antibody.

[0074] Step 4, detection: The DNA-RNA hybrid obtained in Step 3 and captured by the first antibody were reacted with the second antibody stored in the protein preservation solution at 42°C for 30 min, washed, and then allowed to stand at room temperature in the dark for 10 min before detection.

[0075] The second antibody is the anti-DNA-RNA hybrid antibody obtained by the method in step S1. The antibody carries a marker, which emits fluorescence when excited by light, or shows color when gold particles aggregate, or emits light or shows color when catalyzed by enzyme reaction.

[0076] In step four, the protein preservation solution, per 100 ml, includes the following components in the following quantities: 2.535-3.501 g sodium chloride; 6-7 ml Tris-HCl buffer; 20-28 ml casein solution; 28-31 μl preservative; the remainder is pure water; the pH of Tris-HCl is 7.4-7.5.

[0077] In step four, a second antibody is introduced, and the second antibody is also labeled with a marker, which is usually a protein. Using this protein solution for preservation not only eliminates the need for preservation at extremely low temperatures, but also allows the second antibody and the labeled marker to maintain good activity, thereby making the detection results more accurate and reliable.

[0078] The second antibody is one of the following: DNA-RNA hybrid structure-specific antibody, polyclonal antibody or monoclonal antibody or fragment thereof, protein, catalytically inactivated ribonuclease H, nucleic acid, nucleic acid aptamer, or oligonucleotide that specifically binds to DNA-RNA hybrid to form a triplet structure;

[0079] Segmented full-length single-stranded RNA probes targeting the DNA are longer than 100 bp.

[0080] The HPV target DNA is 8000bp in length, and the single-stranded RNA probe can be a full-length single-stranded RNA probe of 8000bp or a fragment of single-stranded RNA probe of 100-2000bp. When choosing a fragment of single-stranded RNA probe of 2000bp, four single-stranded RNA probes need to be used in combination, with lengths of 1-2000bp, 2001-4000bp, 4001-6000bp, and 6001-8000bp.

[0081] Both continuous and segmented probes can induce hybridization between full-length single-stranded RNA probes and single-stranded DNA, resulting in DNA-RNA hybrids. Furthermore, segmentation allows for the introduction of markers, such as biotin, at the beginning and end of each segment's specific single-stranded nucleic acid probe. This enables the subsequent use of avidin, which specifically binds to biotin, to specifically capture biotin and perform subsequent detection, leading to better labeling results. When the length of the segmented full-length single-stranded nucleic acid probe targeting the target DNA exceeds 100 bp, for example, between 100 and 2000 bp, it can form base pairs with single-stranded DNA, thus creating a hybrid between the single-stranded DNA and the segmented full-length single-stranded nucleic acid probe targeting the target DNA.

[0082] Comparative Example

[0083] The anti-DNA-RNA hybrid antibody used in the comparative example was an S9.6 mouse monoclonal antibody, and the remaining steps were the same as in the example.

[0084] The same test samples were tested in the examples and comparative examples, and the results are shown in the table below.

[0085]

[0086] The signal-to-noise ratio in the table is the average value of the concentration detection values ​​ / the average value of the 0 pg / ml detection values. The CV values ​​of different concentration detection values ​​are all below 15%. The signal-to-noise ratio detection results show that the background of the antibody in the example is relatively low, and the signal-to-noise ratio is higher than that of the comparative example.

[0087] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0088] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A rapid in situ hybridization detection method, characterized in that: Includes the following steps, S1. Preparation of anti-DNA-RNA hybrid antibody; S2, Rapid in situ hybridization detection; The preparation of the anti-DNA-RNA hybrid antibody in step S1 includes the following steps: S1.1 Immunization of animals: 100 μg / ml DNA-RNA hybrid and 100 μg / ml methylated bovine serum albumin were mixed in 10 ml TE buffer at a mass ratio of 1:

1. 30 μg of DNA-RNA hybrid was emulsified with Freund's complete adjuvant per Balb / c mouse and injected subcutaneously into the abdomen. Booster immunizations were given on day 14 and day 28. Blood was collected from the tail of the mice on day 35 for testing. S1.2 Preparation of hybridoma cell lines: lymphocytes from immunized mice were fused and cloned to obtain stable positive hybridoma cell lines that could efficiently secrete antibodies. Total RNA was isolated from the hybridoma cell lines. S1.3 Obtaining antibody sequences: Total RNA was reverse transcribed into cDNA, and the nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region were obtained by PCR amplification using specific primers. S1.4 Antibody expression and purification; The nucleotide sequence is cloned into the expression vector, and the cultured cells are transiently transfected using the transfection method. After culturing, the supernatant is collected, and the supernatant is purified using protein A to obtain an antibody with a purity >95%. The heavy and light chain encoding gene sequences for the anti-DNA-RNA hybrid mouse monoclonal antibody in step S1 are SEQ ID 1 and SEQ ID 2, respectively; the heavy and light chain amino acid sequences for the anti-DNA-RNA hybrid mouse monoclonal antibody in step S1 are SEQ ID 3 and SEQ ID 4, respectively; the sequence of SEQ ID 1 is: CAGGTGCAGCTGCAGAGCGGCCCGGAACTGGTGAAACCGGGCGCGAGCGTGAAAATGAGCTGCAAAGCGAGCGGCTATAACCTTTACCAGCCTGCGCATTACCGATTGGGTGAAACAGAAACCGGGCCAGGGCCTGGAATGGATTGGCGATGTGAGCAGCAAAAAAGAACTGGATAAAAGC CAGGAAAAAGAAGAAGTGCAGACCAAAGCGACCCTGACCAGCGATAAAAGCAGCAGCACCGCGTATATGGAACTGAGCAGCCTGACCAGCAAAGATAGCGCGGTGTATTATTGCGCGCGCCAGCATAAAAGCGAAGAAGATATTCTGGCGTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC; The sequence of SEQ ID2 is: GATATTGTGATGACCCAGACCCCGCTGAGCCTGCCGGTGAGCCTGGGCGATCAGGCGAGCATTAGCTGCAACATTTGCCGCAACTGCCAGTGCCTGAGCTGCATGGATTGCGGCAAAGATTTTTGGTATCTGCAGAAACCGGGCCAGAGCCCGAAACTGCTGATTTATAGCGAAGATCAGAAATATGGCGGCAAAGATGGCGTGCCGGAT CGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATCTGGGCGTGTATTATTGCGGCAAAGAAAGCCTGGAAAACGAATTTGAAATTTTTGGCGGCGGCACCAAACTGGAAATTAAACGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC; The sequence of SEQ ID3 is: QVQLQQSGPELVKPGASVKMSCKASGYTFTSLRITDWVKQKPGQGLEWIGDVSSKKELDKSQEKEEVQTKATLTSDKSSSTAYMELSSLTSKDSAVYYCARQHKSEEDILAWGQGTTLTVSS; The sequence of SEQ ID4 is: DIVMTQTPLSLPVSLGDQASISCNICRNCQCLSCMDCGKDFWYLQKPGQSPKLLIYSEDQKYGGKDGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCGKESLENEFEIFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGS.

2. The rapid in situ hybridization detection method according to claim 1, characterized in that: The anti-DNA-RNA hybrid antibody in step S1 is an anti-DNA-RNA hybrid mouse monoclonal antibody that can specifically recognize any DNA-RNA hybrid.

3. The rapid in situ hybridization detection method according to claim 1, characterized in that: The rapid in situ hybridization detection in step S2 includes the following detection steps. S2.1 Dewaxing: Dry the tissue sections in a 75℃ oven, dewax them in a dewaxing solution for 5 min × 3 times, treat them with 100% ethanol for 3 min × 2 times, treat them with 95% ethanol for 2 min, treat them with 85% ethanol for 2 min, and soak them in pure water for 2 min. S2.2, Protease digestion; Shake off the water on the slide, draw circles around the tissue with a water-resistant pen, and wash with phosphate-Tween buffer (PBST) for 1 min × 3 times; Add 50-100 μl of protease digestion solution and incubate at 37°C for 10-20 min; Discard the protease digestion solution and wash with PBST for 2 min × 3 times. S2.3, EBER probe hybridization: Depending on the tissue size, add 100-150 μl of EBER probe, place in a humidified chamber, and incubate for hybridization at 37 ℃ for 1-2 h, then wash with PBST for 2 min × 3 times. S2.4 Immunization with anti-DNA-RNA hybrid antibody; Remove PBST buffer, add 50-100 μl of horseradish peroxidase (HRP) labeled anti-DNA-RNA hybrid antibody to each slide, and incubate at 37℃ for 30 min; After incubation, wash with PBST buffer for 2 min × 3 times; S2.5, DAB staining: Remove PBST buffer, add 100-150 μl of freshly prepared DAB staining solution to each slide, DAB concentrate:DAB buffer = 1:19, and incubate at room temperature for 3-5 min. S2.6, Counterstain with hematoxylin solution; rinse with tap water, add 100-150 μl of hematoxylin solution for counterstaining, incubate for 1-3 min, rinse with tap water to return to blue; dehydrate and clear the sections, and mount them as usual; observe the staining of the tissue under an optical microscope.

4. The rapid in situ hybridization detection method according to claim 3, characterized in that: The main component of the dewaxing solution in step S2.1 is xylene, and the slices need to be soaked in three cans of xylene solution for 5 minutes in sequence.

5. The rapid in situ hybridization detection method according to claim 4, characterized in that: The water-blocking pen mentioned in step S2.2 is an oil-based pen used to lock in the subsequently added buffer solution and working solution; The concentration of the PBST washing solution in step S2.2 is 0.01 mol / L. The components of 1 L of solution are as follows: 5.82 g sodium chloride; 38.81 g disodium hydrogen phosphate dodecahydrate; 15.60 g sodium dihydrogen phosphate dihydrate; 200 μl Tween 20; adjust the pH to 7.2-7.

4. The protease digestion solution in step S2.2 is protease solid dissolved in TBS solution. The BSA solution is 100 ml of 0.5 mol / L Tris-HCl buffer, 3.5 g of NaCl, 0.15 mol / L, and the volume is adjusted to 1000 ml.

6. The rapid in situ hybridization detection method according to claim 4, characterized in that: The EBER probe in step S2.3 is a DNA sequence complementary to the specific target of EB virus. This sequence is a protective sequence, and its length is selected from one of 15bp, 35bp, 100bp, 500bp, 1000bp, and 2000bp. The humidification chamber in step S2.3 is kept in a light-proof state during use, and the color of the humidification chamber is brown or black.

7. The rapid in situ hybridization detection method according to claim 4, characterized in that: The antibody in step S2.4 is purified using a Protein G column and then diluted 1000-fold with antibody diluent before use. The antibody diluent consists of: 2.535-3.501 g sodium chloride; 6-7 ml Tris-HCl buffer; 20-28 ml casein solution; 28-31 μl preservative; and the remainder is pure water; the pH of the Tris-HCl solution is 7.4-7.

5. The antibody in step S2.4 is an anti-DNA-RNA antibody, which is conjugated with horseradish peroxidase (HRP). This HRP can cause the DAB chromogenic solution to form a brown precipitate.

8. The rapid in situ hybridization detection method according to claim 4, characterized in that: The DAB colorimetric solution in step S2.5 must be prepared and used immediately, and the preparation time should not exceed 1 hour. The main component of the DAB colorimetric solution in step S2.5 is diaminobenzidine. Both the DAB colorimetric solution and the DAB diluent must be stored away from light.

9. A rapid in situ hybridization detection application, characterized in that: This includes the application of a rapid in situ hybridization detection method as described in any one of claims 1-8 in the detection of anti-DNA-RNA hybrid antibody protein molecules.

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