ICON probe, detection kit and application of human LINE transposon DNA methylation

Abstract

The present invention provides an ICON probe, a detection kit and an application for DNA methylation of a human LINE transposon, and belongs to the technical field of gene detection. The nucleotide sequence of the ICON probe for DNA methylation of a human LINE transposon provided by the present invention is SEQ ID NO: 1, G indicates that Bipyridine is modified on g, and T indicates that the amino group is modified on t. Biotin‑X‑X‑NHS is used to biotin-label the three amino groups on the LINE1 ICON probe. Under the mediation of osmium acid, Bipyridine forms a stable cross-link with the methyl group on the methylated C in the LINE1 of the genome. After denaturation treatment, the LINE1 ICON probe is not eluted. At the same time, the biotin on the LINE1 ICON probe binds to the streptavidin in the tyramide signal amplification detection reagent to amplify the fluorescent signal, thereby achieving the purpose of rapid and visual detection at the in situ level of the cell.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to an ICON probe, a detection kit and an application of human LINE transposon DNA methylation. Background Art

[0002] DNA methylation is an important modification pathway. Under the catalysis of methyltransferase, methyl groups are selectively added to the cytosines of the two nucleotides CG in DNA to form 5-methylcytosine (see Figure 1 ), which is commonly found in the 5'-CG-3' sequence of genes. Methylation mainly occurs on the 5-carbon atom of cytosine in CpG dinucleotides, and its product is 5-methylcytosine (5mC). DNA methylation is mainly established by DNA methyltransferases Dnmt3a and Dnmt3b, and auxiliary factor Dnmt3L; its maintenance is mainly achieved by Dnmt1. About 90% of 5mC is located in repetitive sequences, including tandem repeats (mouse: major and minor satellites; human: satellite 2,3 and alpha satellite), long interspersed nuclear elements (LINE) and short interspersed nuclear elements (mouse: Short interspersed nuclear elements, SINE; human: Alu). The distribution of CpG dinucleotides in the genome is uneven. In some regions, the density of CpG sequences is very high, forming CpG islands. The CpG islands of mammals contain approximately 300 to 3000 bases and are usually located in the promoter region or the first exon region of the gene.

[0003] The DNA methylation status presents a certain distribution pattern in the genome: in the genome of healthy people, CpG sites in CpG islands are usually in a non-methylated state, while CpG sites outside CpG islands are usually in a methylated state; this form of methylation can be stably retained during cell division, and plays an important regulatory role in gene expression, chromatin reconstruction, genomic imprinting, X chromosome inactivation and genome stability without changing the DNA sequence.

[0004] Factors such as chronic inflammation, hepatitis virus, chemicals and insufficient methyl donors caused by malnutrition are known important causes of abnormal DNA methylation. Abnormal DNA methylation levels are closely related to the occurrence and development of many human diseases, including cancer. For example, in tumor cells, abnormal DNA methylation is mainly manifested in: 1) CpG islands in the promoter regions of tumor suppressor genes and DNA repair genes are highly methylated and silent; 2) Oncogene promoter regions are hypomethylated and activated; 3) The overall methylation level of the genome is reduced, which activates transposons, retrotransposons and repetitive sequences, thereby reducing chromosome stability.

[0005] Studies have shown that local hypermethylation of gene promoter regions precedes the malignant proliferation of cells, while genome-wide hypomethylation also occurs with tumorigenesis, and becomes more significant as the tumor malignancy increases. Abnormal DNA methylation is closely related to processes such as tumor cell escape from death, induction of angiogenesis, sustained proliferation signals, changes in energy metabolism, and escape from immune system destruction, and is an important cause of tumor occurrence, development, metastasis, and deterioration.

[0006] Using DNA methylation analysis technology to detect changes in the degree of methylation of specific genes in tissue cells and the overall DNA methylation level (i.e., methylation spectrum) is a potential means for early diagnosis of tumors, disease course monitoring, and efficacy evaluation. Current methylation detection methods include methylation-specific PCR (MSP), methylation-specific real-time fluorescence quantitative PCR, and bisulfite sequencing. None of the above methods can observe the DNA methylation status of a specific sequence in situ in cells. Currently, human LINE transposon DNA methylation detection mostly uses bisulfite sequencing. Sample preparation is time-consuming and requires expensive sequencers, which is not easy to achieve in general laboratories. And it is impossible to observe the DNA methylation status of a specific sequence in situ in cells. The Benke group has previously designed ICON probes for mouse and human satellite sequences, and developed a MeFISH detection method for satellite ICON probes ( Figure 2 )(Yufeng Li, et al. Sequence-specific microscopic visualization of DNAmethylation status at satellite repeats in individual cell nuclei and chromosomes. Nucleic Acids Research. 2013.41(19):e186). Summary of the invention

[0007] In view of this, the object of the present invention is to provide an ICON probe (LINE1 ICON probe) and a detection kit for DNA methylation of human LINE transposon, which can realize the visualization detection of DNA methylation in situ in cells.

[0008] The invention provides an ICON probe for DNA methylation of a human LINE transposon. The nucleotide sequence of the ICON probe is aaTcgggtcacTcccacccgaatattgcGcttttcagaccggcTtaagaa, G represents that Bipyridine is modified on g, T represents that an amino group is modified on t, and the amino group is labeled with biotin.

[0009] The invention provides a detection kit for DNA methylation of human LINE transposon, comprising the ICON probe, a hybridization buffer and an osmium acid solution.

[0010] Preferably, the hybridization buffer comprises the following components: 4×SSC buffer, 0.5 mM EDTA, 10% dextran sulfate by mass and 25% formamide by volume.

[0011] Preferably, the final concentration of the osmium acid (K2OsO4·2H2O) solution is 5 mM; the solvent in the osmium acid solution is an aqueous solution containing 50 mM Tris-HCl, 0.5 mM EDTA, and 1 M NaCl at pH 7.7.

[0012] Preferably, the detection kit also includes a tyramide signal amplification detection reagent.

[0013] The present invention provides application of the detection kit in detecting DNA methylation of human LINE transposon.

[0014] The present invention provides the use of the ICON probe in preparing a kit for detecting DNA methylation of human LINE transposon.

[0015] The present invention provides a method for detecting DNA methylation of a human LINE transposon, comprising the following steps:

[0016] The ICON probe is hybridized with the cell target DNA, the fluorescence signal is amplified with a tyramide signal amplification detection reagent, the fluorescence signal is observed and recorded, and then the hybridization system is treated with an osmium acid solution to denature, and the fluorescence signal is observed and recorded again. The methylation status of the target DNA in the cell is determined based on the strength of the fluorescence signal.

[0017] Preferably, the denaturation method is to place the hybridization system in a 2×SSC solution containing 70% formamide by volume and incubate at 60-65° C. for 2.5-3.5 min, remove the uncross-linked LINE1 ICON probe, wash, remove moisture, and seal with an anti-fluorescence quenching sealing agent.

[0018] The present invention provides an ICON (Interstrand Complex Formed by Osmisum and Nucleic Acid) probe for DNA methylation of a human LINE transposon, wherein the nucleotide sequence of the ICON probe is aaTcgggtcacTcccacccgaatattgcGcttttcagaccggcTtaagaa, G represents that g is modified with Bipyridine, T represents that t is modified with an amino group, and the amino group is labeled with biotin. The LINE1 ICON probe provided by the present invention can specifically bind to the human LINE transposon gene, and the g in the cg motif is modified with Bipyridine. During detection, under the mediation of osmium acid, Bipyridine forms a stable crosslink with the methyl group of the methylated C on the genomic target sequence, which is stable to heat. After denaturation treatment, the LINE1 ICON probe is not easily eluted. At the same time, the biotin on the LINE1 ICON probe binds to the streptavidin in the tyramide signal amplification detection reagent to amplify the fluorescent signal, thereby achieving the purpose of rapid and visual detection of the cell in situ level. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the reaction of cytosine DNA methylation;

[0020] Figure 2 Schematic diagram of the MeFISH detection method for satellite ICON probes;

[0021] Figure 3 The FISH and MeFISH results of LINE1 ICON probe in cells;

[0022] Figure 4 This is the MeFISH result of the non-TSA-amplified LINE1 ICON probe in human normal breast epithelial cells HBL-100. DETAILED DESCRIPTION

[0023] The invention provides an ICON probe for DNA methylation of a human LINE transposon. The nucleotide sequence of the ICON probe is aaTcgggtcacTcccacccgaatattgcGcttttcagaccggcTtaagaa, G represents that Bipyridine is modified on g, T represents that an amino group is modified on t, and the amino group is labeled with biotin.

[0024] In the present invention, the role of the Bipyridine is to form a stable crosslink with the methyl group on C in the target sequence under the action of osmium acid. The g modified by Bipyridine is preferably located in the middle of the ICON probe nucleotide sequence, which is more conducive to stable binding to the target sequence. The role of the biotin modification is to bind to the streptavidin in the tyramide signal amplification detection reagent during detection to produce a cascade amplification signal effect.

[0025] In the present invention, the preparation method of the ICON probe is preferably completed by referring to the probe synthesis method disclosed in the patent publication number WO / 2006 / 132022. In the embodiment of the present invention, the ICON probe is commissioned to be synthesized by Ajinomoto Bio-Pharmaceutical Co., Ltd. (http: / / webpark1516.sakura.ne.jp / icon-probe / ). The probe is biotin labeled and purified using Biotin-XX-NHS (https: / / www.sigmaaldrich.cn / CN / zh / product / sigma / b3295).

[0026] The invention provides a detection kit for DNA methylation of human LINE transposon, comprising the ICON probe, a hybridization buffer and an osmium acid solution.

[0027] In the present invention, the hybridization buffer preferably comprises the following components: 4×SSC buffer, 0.5 mM EDTA, 10% dextran sulfate by mass and 25% formamide by volume.

[0028] In the present invention, the concentration of the osmium acid solution is preferably 25 mM; the solvent in the osmium acid solution is an aqueous solution containing 50 mM Tris-HCl, 0.5 mM EDTA, and 1 M NaCl at pH 7.7.

[0029] In the present invention, the detection kit preferably also includes a tyramide signal amplification detection reagent. The present invention has no particular restrictions on the source of the tyramide signal amplification detection reagent, and a tyramide signal amplification detection reagent known in the art can be used. In an embodiment of the present invention, the tyramide signal amplification detection reagent is purchased from PE Company of the United States (Article No.: NEL700A00KT, instruction manual link https: / / resources.perkinelmer.com / lab-solutions / resources / docs / man_TSABiotinSystem.pdf?_ga=2.68819732.467907597.1667996319-1434938830.1667996319).

[0030] The present invention provides the use of the detection kit in detecting DNA methylation of human LINE transposon. In the present invention, the detection kit can be used in detecting DNA methylation of human LINE transposon for non-disease diagnosis purposes.

[0031] The present invention provides the use of the ICON probe in preparing a kit for detecting DNA methylation of human LINE transposon.

[0032] In the present invention, the cell source of the human LINE transposon preferably includes cancer cells and / or normal somatic cells. The cancer cells preferably include breast cancer cells. The normal somatic cells preferably include normal breast cells.

[0033] The present invention provides a method for detecting DNA methylation of human LINE transposon. The detection mechanism is that in the presence of osmium acid, the methylated cytosine of LINE1 forms a covalent crosslink with the pyridine on the complementary base on the LINE1 ICON probe, and the crosslink will not separate even after denaturation treatment. Therefore, the fluorescent signal observed after osmium acid treatment and denaturation can reflect the methylation state of LINE1, which can be used for detection purposes other than disease diagnosis. Specifically, the following steps are included:

[0034] The LINE1 ICON probe is hybridized with the cell target DNA, the fluorescence signal is amplified with a tyramide signal amplification detection reagent, the fluorescence signal is observed and recorded, and then the hybridization system is treated with an osmium acid solution to denature, and the fluorescence signal is observed and recorded again. The methylation status of the target DNA in the cell is determined according to the strength of the fluorescence signal.

[0035] In the present invention, the hybridization method is to drop a hybridization buffer containing a LINE1 ICON probe on the fixed cells and perform in situ hybridization with a coverslip and a rubber seal. The method for preparing the fixed cells is preferably to place the cells in a hypotonic solution and let it rest for 8 minutes, slowly drip an equal amount of fixative for fixation, gently blow, centrifuge, add 3 ml of fixation again, gently blow, and centrifuge, repeat the previous step, and finally fix and resuspend the cells with 1 ml to obtain fixed cells. The hypotonic solution is preferably a 75mM KCl aqueous solution. The fixative is preferably a mixture of acetic acid and methanol in a volume ratio of 1:3. The number of fixations is preferably 2 to 3 times. The in situ hybridization conditions are preferably maintained at 80°C for 3 minutes. After the in situ hybridization, it is preferably also included to wash in order to remove unbound probes. The solvents used for the wash are preferably 2×SSC and PBS. Now soak and remove the coverslip in a 2×SSC solution, then wash in a 2×SSC solution for 5 minutes, then wash in a PBS solution for 5 minutes, and reorganize 3 times. The final concentration of the LINE1 ICON probe in the hybridization buffer containing the LINE1 ICON probe is preferably 1.25 ng / μl.

[0036] In the present invention, the method of amplifying the fluorescence signal with a tyramide signal amplification detection reagent preferably adds TNB buffer to the hybridization system, incubates at room temperature for 30 minutes, then adds HRP reagent (1:500) at room temperature for 30 minutes, washes with TNT buffer for 5 minutes, 3 times; drips Tyramide working solution (1:50) and incubates at room temperature for 10 minutes, washes with TNT buffer for 5 minutes, 3 times; drips SA-FITC solution (1:250) and incubates at 37°C for 40 minutes; washes with TNT buffer for 5 minutes, 3 times; and seals with anti-fluorescence quenching sealing agent (DAPI). The purpose of observing the fluorescence signal before osmium acid treatment is to illustrate that there is no difference in the fluorescence signal intensity of FISH between different cells, because the signal of FISH has nothing to do with whether it is methylated or not, and also to illustrate that the low signal after osmium acid treatment is not caused by FISH.

[0037] In the present invention, the denaturation method is preferably to place the hybridization system in a 2×SSC solution containing 70% formamide by volume and incubate at 60-65° C. for 2.5-3.5 min, remove the uncrosslinked LINE1ICON probe, wash, remove moisture, and seal with an anti-fluorescence quenching sealing agent (DAPI). The incubation temperature during the denaturation is preferably 63° C. The incubation time is preferably 3 min.

[0038] In the examples of the present invention, normal breast cells (HBL-100) and breast cancer cells (MCF-7 and MDA-MB-231) were used as detection objects, respectively, and the detection method provided by the present invention was used to implement target gene methylation detection. The results showed that a strong fluorescence signal could still be detected in the normal breast cell line HBL-100, while no fluorescence signal was detected in the two breast cancer cells, which was consistent with the sequencing results previously reported.

[0039] In the present invention, after the fluorescence signal of the LINE1 ICON probe provided by the present invention is amplified by using a TSA kit, the fluorescence signal can be detected by using an ordinary fluorescence microscope (Olympus IX71), thereby avoiding the problem that the conventional probe method cannot be detected by an ordinary fluorescence microscope and must rely on a confocal microscope.

[0040] The following is a detailed description of an ICON probe, a detection kit and an application of human LINE transposon DNA methylation provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] Design and synthesis of a LINE1 ICON probe for DNA methylation of human LINE transposon

[0043] Human LINE transposon was used as the target gene, and the ICON probe for DNA methylation of human LINE transposon was designed using the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ): aaTcgggtcacTcccacccgaatattgcGcttttcagaccggcTtaagaa (SEQ ID NO: 1), G indicates that g is modified with Bipyridine, T indicates that t is modified with amino (amino-NH2), 50 nt, and was commissioned to be synthesized by Ajinomoto Biopharmaceutical Co., Ltd. The probe was biotin labeled and purified using Biotin-XX-NHS (https: / / www.sigmaaldrich.cn / CN / zh / product / sigma / b3295), and the specific method is as follows:

[0044] 1. Reagent Preparation

[0045] 1. LINE1 ICON probe labeled with three amino groups (30 μg dissolved in 20 μl H2O) 2. Add 15 μl DMSO to the 0.5 mg Biotin-XX-NHS bottle and mix thoroughly until dissolved. Note: Biotin-XX-NHS should be used immediately after dissolution.

[0046] 3. 99.9% DMSO (stored at 4°C, placed at room temperature before use)

[0047] 4. Microcon YM-30 Centrifuge Filter (Millipore cat.No.42422)

[0048] 5. 25mg / ml NaHCO3 Buffer pH 8.5 (can be stored at -20℃ for 6 months, measure pH value before each use)

[0049] 2. Mix the following reagents at room temperature

[0050] 15μl Biotin-XX-NHS in DMSO (0.5mg)

[0051] 5μl NaHCO3 Buffer

[0052] 3 μl RNase-Free Water

[0053] 7μl Aminoallyl-oligo

[0054] Total 30 μl

[0055] 3. After thorough mixing, incubate at room temperature for 1 hour, away from light, and mix occasionally.

[0056] 3. Purification

[0057] 1. Pre-wash the Microcon YM-30 Centrifuge Filter with 200 μl RNase-Free water and centrifuge according to the instructions.

[0058] 2. Add 400 μl RNase-Free water to the above probe mixture, mix well and add to the filter. Place a 1.5 ml EP tube under the filter to collect waste liquid.

[0059] 3. Centrifuge according to the instructions of Microcon YM-30 and discard the waste liquid in the EP tube.

[0060] 4. Add 400 μl RNase-Free water to the filter, centrifuge according to the instructions of Microcon YM-30, and discard the waste liquid in the EP tube.

[0061] 5. Repeat step 4 (2 times).

[0062] 6. According to the Microcon YM-30 instructions, collect the marked probes in a new EP tube and store them at -20 degrees for later use.

[0063] Example 2

[0064] A kit for detecting DNA methylation of human LINE transposon

[0065] 1. LINE1 ICON probe for human LINE transposon DNA methylation prepared in Example 1

[0066] 2. Tyramide signal amplification (TSA) kit (FITC fluorescence, Shanghai Zhenhe Biotechnology);

[0067] 3. Hybridization Buffer

[0068] The hybridization buffer includes the following components: 4×SSC, 0.5 mM EDTA, 10% dextran sulfate by weight, and 25% formamide by volume.

[0069] Example 3

[0070] A method for detecting DNA methylation of human LINE transposon

[0071] 1. Test materials

[0072] Breast cancer cells: breast cancer MCF-7 cells and MDA-MB-231 cells

[0073] Normal mammary epithelial cells: HBL-100.

[0074] 2. Detection Method

[0075] 1) Cell preparation;

[0076] Normal breast epithelial cells HBL-100, breast cancer cells MCF-7 and MDA-MB-231 were routinely resuscitated and cultured in a 5% CO2 incubator at 37°C. When the cell coverage in the culture flask reached 80% to 90%, the cells were subcultured. The cells were collected at the third generation of culture, washed with PBS, and centrifuged. The cells were gently resuspended in a hypotonic solution (75mM KCl) and allowed to stand at room temperature for 8 minutes. Then, an equal amount of methanol-acetic acid (1:3, v / v) fixative was slowly added dropwise for fixation. After gently blowing, centrifugation, 3 ml of acetic acid / methanol 1:3 mixture was added again, gently blown, and centrifuged. Repeat the previous step, and finally resuspended the cells with 1 ml of acetic acid / methanol 1:3 mixture and stored at -20°C for later use.

[0077] 2) Hybridization of ICON probe with cell target DNA and washing;

[0078] 4 μl of the hybridization mixture containing the labeled probe (final concentration: 4x SSC, 0.5 mM EDTA, 10% dextransulfate, 25% FA, 1.25 ng / μl LINE1 ICON probe) was dropped on the slide of pretreated cells and tissues, and sealed with a cover slip and rubber; the slide was placed in an incubator at 80°C for 3 min to denature the genomic DNA and incubated, and then placed in a wet box at room temperature for 2 h; after hybridization, the cover slip was removed by immersion in 2×SSC at 37°C, washed with 2×SSC for 5 min, twice, and washed with PBS for 5 min, and repeated 3 times.

[0079] 3) The TSA tyramide signal amplification kit amplifies the fluorescence signal, washes it, and observes and records the fluorescence signal under a fluorescence microscope for the first time; the specific steps are as follows:

[0080] Add TNB buffer in the kit and incubate at room temperature in a wet box for 30 minutes; add HRP reagent (1:500) and incubate at 4°C overnight in a wet box; wash with TNT buffer for 5 minutes, 3 times; add Tyramide working solution (1:50) and incubate at room temperature for 10 minutes; wash with TNT buffer for 5 minutes, 3 times; add SA-FITC solution (1:250) and incubate at 37°C for 40 minutes; wash with TNT buffer for 5 minutes, 3 times; seal with DAPI. Observe FISH signals under a fluorescence microscope for the first time.

[0081] 4) Osmium acid solution treatment;

[0082] 10 μl of osmium acid solution was dropped on a glass slide and incubated in a 37°C wet box for 30 min. The osmium acid (K2OsO4) solution was a solution with a final concentration of 25 mM K2OsO4, and the solvent was an aqueous solution containing 50 mM Tris-HCl, 0.5 mM EDTA (pH 7.7), and 1 M NaCl.

[0083] 5) Denaturation and washing;

[0084] The slides were incubated at 63°C for 3 min in 2×SSC solution containing 70% formamide to remove uncrosslinked probe molecules, then washed with PBS, the water was removed by aspiration, and the slides were sealed with anti-fluorescence quenching sealing medium (containing DAPI).

[0085] 6) Observe and record the fluorescence signal again using a fluorescence microscope

[0086] Results Figure 3 . The left side shows the FISH images of the LINE1 probe in normal breast cells HBL-100 and breast cancer cells (MCF-7 and MDA-MB-231). There is no difference in the intensity of the FISH signal in the three cells. The right side shows the MeFISH image of LINE1 in the three cells after treatment with osmium acid and heat denaturation. A strong fluorescence signal can still be detected in the normal breast cell line HBL-100, while no fluorescence signal is detected in the two breast cancer cells. After FISH, no MeFISH fluorescence signal was detected in MCF-7 cells without osmium acid added to the treatment solution, indicating that due to the lack of osmium acid, no stable cross-linking was formed between the LINE1ICON probe and the target sequence.

[0087] The above results suggest that the fluorescence signal in HBL-100 is a methylated LINE1 signal, which is consistent with the sequencing results reported (PLoS One.2014Jun 27;9(6):e100429.doi:10.1371 / journal.pone.0100429.eCollection2014.), that is, LINE1 is highly methylated in normal cells and hypomethylated in breast cancer cells.

[0088] Comparative Example 1

[0089] The LINE1 ICON probe prepared in Example 1 was hybridized with the target cells (the type is the same as described in Example 3), reacted with Streptavidin-FITC (Thermo Fisher SA1001, https: / / www.thermofisher.cn / order / catalog / product / SA1001?SID=srch-srp-SA1001), and the FISH fluorescence signal was detected. Then, the cells were treated with osmium acid, denatured, and the MeFISH fluorescence signal was detected in sequence according to the method of Example 3.

[0090] The MeFISH results of the LINE1 ICON probe in human normal breast epithelial cells HBL-100 are shown in Figure 4Even if it is a normal mammary epithelial cell after denaturation, the MeFISH fluorescence signal cannot be detected under the Olympus IX71 fluorescence microscope. This shows that when the LINE1 ICON probe detection is omitted, the step of amplifying the fluorescence signal by the TSA tyramide signal amplification kit is not conducive to accurately characterizing the detection results.

[0091] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A kit for detecting DNA methylation of human LINE transposon, characterized in that: Includes ICON probe, hybridization buffer, osmium acid solution and tyramide signal amplification detection reagent; The nucleotide sequence of the ICON probe is aaTcgggtcacTcccacccgaatattgcGcttttcagaccggcTtaagaa, G indicates that g is modified with Bipyridine, T indicates that t is modified with an amino group, and the amino group is labeled with biotin.

2. The detection kit according to claim 1, characterized in that: The hybridization buffer comprises the following components: 4×SSC buffer, 0.5 mM EDTA, 10% dextran sulfate by mass and 25% formamide by volume.

3. The detection kit according to claim 1, characterized in that: The final concentration of the osmium acid solution is 5 mM; the solvent in the osmium acid solution is an aqueous solution containing 50 mM Tris-HCl at pH 7.7, 0.5 mM EDTA and 1 M NaCl.

4. Use of the detection kit according to any one of claims 1 to 3 in detecting DNA methylation of human LINE transposon for non-disease diagnosis purposes.

5. A method for detecting DNA methylation of human LINE transposon for non-disease diagnosis purposes, characterized in that: The following steps are involved: The ICON probe described in claim 1 is hybridized with the cell target DNA, the fluorescence signal is amplified with a tyramide signal amplification detection reagent, the fluorescence signal is observed and recorded, and then the hybridization system is treated with an osmium acid solution to denature, and the fluorescence signal is observed and recorded again, and the methylation status of the target DNA in the cell is determined according to the strength of the fluorescence signal.

6. The method according to claim 5, characterized in that: The denaturation method is to place the hybridization system in a 2×SSC solution containing 70% formamide by volume and incubate at 60-65° C. for 2.5-3.5 minutes to remove the uncrosslinked LINE1 ICON probe and seal the slice with an anti-fluorescence quenching sealing agent.

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