Detection kit for specific site 5-methylcytosine without bisulfite and detection method thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a bisulfite-free detection kit for specific site 5-methylcytosine and a detection method thereof. According to the method, a TAPbeta treatment method is adopted, 5hmC is sealed through beta-GT, 5mC oxidation is carried out through TET, and 5mC is reduced into dihydrouracil (DHU) through pyridine borane, so that the apparent difference between 5mC and C and 5hmC is converted into single nucleotide polymorphism difference. In addition, a flow microsphere technology based on PNA-assisted click chemical connection starting is established to detect DNA methylation specific sites. A PNA clip is innovatively introduced into click chemical connection, and non-specific connection is inhibited. Target sequence enrichment, signal amplification and signal acquisition are carried out through surface-functionalized magnetic nanoparticles, and the detection sensitivity is improved to fM. The invention provides a new tool for high-specificity and high-sensitivity epigenetic marker detection.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a detection kit and detection method for 5-methylcytosine at a specific site that is free of bisulfite. Background Technology

[0002] DNA methylation is a key epigenetic modification in which a methyl group attaches to the fifth carbon atom of cytosine, forming 5-methylcytosine (5mC). The methylation process includes establishment, maintenance, and elimination. DNMT3a / 3b enzymes add methyl groups to cytosine in repetitive sequences, imprinted sites, and regulatory elements, driving methylation establishment. DNMT1 replicates the methylation pattern during DNA replication, thus maintaining the methylated state. TET enzyme-mediated oxidation converts 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC), achieving methylation elimination.

[0003] In eukaryotes, these processes are crucial for embryonic development, transcriptional regulation, gene imprinting, X chromosome inactivation, and chromatin structure formation, regulating key physiological and pathological processes such as growth, development, and disease progression. In humans, approximately 70% to 80% of CpG sites are methylated, with tissue-specific 5mC levels remaining stable at 4% to 5% (5mC / C). In recent years, significant changes in DNA methylation levels in blood and tissues have been observed in various diseases, particularly cancer, rheumatoid arthritis, and neurological disorders. Recent reports have highlighted the enormous clinical application potential of blood DNA methylation analysis, specifically in the following aspects: First, it can accurately identify the tissue origin of abnormal DNA in the blood and effectively distinguish between healthy and malignant tissues, providing a basis for lesion localization and nature determination; second, it can construct an "age clock" based on methylation patterns to achieve physiological age assessment; third, tumor-derived cell-free DNA (cfDNA) has consistent methylation characteristics with the primary tumor, a characteristic that makes it suitable for cancer screening; furthermore, abnormal DNA methylation is prevalent in the early stages of cancer development and persists throughout the entire cancer progression, therefore, some researchers have proposed that the methylation status of blood DNA may become an important biomarker for cancer diagnosis.

[0004] However, in actual clinical samples, DNA methylation levels are low, and a large amount of nonspecific DNA can mask the target sequence. Most importantly, unmethylated cytosine and its oxidized derivatives (5hmC) have the same base-pairing ability as 5mC, interfering with the accuracy of detection results.

[0005] To detect DNA methylation at specific gene loci, various analytical methods have been developed. These techniques can be broadly categorized into three main types: bisulfite-based methods, affinity-based methods, and methylation-sensitive restriction endonucleases (MSREs).

[0006] Bisulfite-based sequencing technology is a classic method for methylation analysis. Whole-genome bisulfite sequencing (WGBS) has long been the gold standard for single-base resolution methylation analysis, but it is expensive and requires high sequencing depth. Reduced bisulfite sequencing (RRBS) can enrich target sequences, reducing sequencing costs and depth. However, MspI only covers about 10% of CpG-enriched regions, limiting its genome coverage. Although bisulfite conversion is highly efficient, it causes significant DNA damage and has low sample recovery rates. Furthermore, this conversion method is not direct; it relies on comparing pre- and post-conversion sequences to detect 5mC. 95% of cytosine in the genome is unmodified C. In GC-enriched regions, incompletely converted C (CT) increases background noise. These limitations necessitate the development of bisulfite-free DNA methylation methods.

[0007] Affinity enrichment-based detection methods have gained increasing attention due to their ease of operation, high sensitivity, good selectivity, and ease of miniaturization. However, affinity enrichment requires highly specific antibodies or binding proteins to recognize methylation modifications; it can only effectively enrich densely methylated fragments and cannot perform single-base level analysis.

[0008] While enzymatic digestion-based methods have been successfully applied to the detection of DNA methylation, they still face several technical challenges. First, this method can only identify specific DNA sequences, limiting the scope of detection. Second, it cannot provide precise, single-base resolution information about methylation status.

[0009] Enzymatic conversion has significant advantages in maintaining DNA integrity and low starting amounts, but it still faces three challenges: about 5% of CpG in high GC regions is affected by residual deamination bias; APOBEC only acts on single-stranded DNA and requires pre-denaturation; and it is an indirect detection strategy. Summary of the Invention

[0010] The purpose of this invention is to provide a detection kit for 5-methylcytosine at a specific site that is free of bisulfite.

[0011] Another object of the present invention is to provide a method for detecting 5-methylcytosine at a specific site without bisulfite.

[0012] In this invention, the specific site 5-methylcytosine refers to the differential site of 5-methylcytosine present on the gene DNA of healthy controls and disease samples. That is, this invention provides a 5-methylcytosine detection kit and related methods.

[0013] This invention employs the TAPβ treatment method to achieve direct 5mC detection without bisulfite treatment. The TAPβ method comprises three steps: 5mC blocking via β-GT, 5mC oxidation via TET, and reduction to dihydrouracil (DHU) via pyridineborane. The transformed sample is then used to establish a PNA-assisted click chemistry-initiated flow cytometry microsphere technique for detecting specific DNA methylation sites.

[0014] To achieve highly specific detection, a novel PNA clip is introduced into click chemical linkages to suppress non-specific linkages. Through the synergistic effect of target sequence enrichment via magnetic nanoparticles, signal amplification, and flow cytometry detection, the detection sensitivity is increased to fM. This invention provides a new tool for the detection of highly specific and sensitive epigenetic markers.

[0015] According to a specific embodiment of the 5-methylcytosine detection kit of the present invention, the kit comprises a 5hmC blocking solution, a TeT enzyme oxidation system, a pyridine borane conversion system, a click chemistry linkage system, and a TdT signal amplification system, wherein, The 5hmC blocking solution consisted of: 0.5 μL of 10× NEB buffer, 1 μL of 50× UDP-Glu, 1 μL of 100 UT4-BGT, and 0.5 μL of water.

[0016] According to a specific embodiment of the 5-methylcytosine detection kit of the present invention, the TeT enzyme oxidation system includes: 1 μL of eTET 2.0 reaction buffer, 3 μL of eTET 2.0 enzyme mixture, and 1×Fe 2+ The solution contains 0.45 μL of water and 3.05 μL of water. Preferably, the TeT enzyme oxidation system also includes a termination reaction buffer and a termination reaction buffer.

[0017] According to a specific embodiment of the 5-methylcytosine detection kit of the present invention, the pyridine borane conversion system includes 1 μL of 6M NaAc solution and 0.1 μL of 1M pyridine borane, and the pH of the NaAc solution is 4.3.

[0018] The 5-methylcytosine detection kit according to a specific embodiment of the present invention comprises a click chemistry reaction system including 1 μL of 50 nM probe A, 1 μL of 50 nM probe B, 1 μL of 10× PBS, 1 μL of 0.1% Tween-20, 1 μL of 3 M NaCl, 1 μL of 1 μM PNA, and 2 μL of water.

[0019] According to a specific embodiment of the 5-methylcytosine detection kit of the present invention, the nucleotide sequence of probe A is: SEQ ID NO.1: 5′-AAAAAAAAAAGTACCTAAGGGGCA-3′; Preferably, the sequence of probe A is: 5′-Biotin-AAAAAAAAAA GT ACC TAA GGG GCA-DBCO-3′.

[0020] The nucleotide sequence of probe B is as follows: SEQ ID NO.2: 5′-GTGCTAATGGGAGAG-3′; Preferably, the sequence of probe B is: 5′-N3-GTG CTA ATG GGA GAG-3′.

[0021] According to a specific embodiment of the 5-methylcytosine detection kit of the present invention, the peptide nucleic acid sequence of PNA is SEQ ID NO.3: 5′-CGGTGCTAAT-3′.

[0022] According to a specific embodiment of the 5-methylcytosine detection kit of the present invention, the preparation method of STV-modified magnetic nanoparticles (STV-MB) in the magnetic nanoparticle enrichment system is as follows: Wash with 1×PBS (20 μL) and add 2 μL (8.4 × 10⁻⁶) of STV-MB magnetic nanoparticles with a particle size of approximately 300 nm. 8 ( / μL) twice, magnetically remove the supernatant, and disperse the 330 nM magnetic nanoparticles with 1×PBS (20 μL). Take 1 μL of STV-MB (8.4×10) 7 (For backup)

[0023] According to a specific embodiment of the 5-methylcytosine detection kit of the present invention, the TdT signal amplification system comprises: 1 μL of 5×TdT buffer solution, 0.02 μL of 20U TdT, 0.5 μL of 10 mM dTTP and 3.5 μL of water.

[0024] The beneficial effects of this invention are: This invention establishes a flow cytometry method for detecting DNA methylation at specific sites based on template-free DNA signal amplification induced by mild transformation and specific ligation using magnetic microbeads. This method utilizes biological enzymes and mild chemical transformation to convert 5mC into a U analog, thereby converting 5mC with C and 5hmC into single-base mutations. This transformation avoids DNA damage caused by bisulfite treatment and acts directly and efficiently on 5mC.

[0025] Meanwhile, by incorporating PNA into the click chemistry ligation reaction, this invention effectively suppresses non-specific ligation, thus enabling the click chemistry reaction to achieve a high signal-to-noise ratio with very simple operation. Furthermore, TdT-dependent amplification on magnetic beads does not require a template sequence, greatly simplifying probe design. Leveraging the aforementioned efficient amplification and the powerful signal readout capability of flow cytometry, the method of this invention can detect methylated target sequences as low as 1 fM.

[0026] The method of this invention can distinguish between 5mC and C and 5hmC using single bases, achieving a distinction rate of 1% and exhibiting high specificity. Due to its high sensitivity and specificity, this method has great application potential in biological research and disease diagnosis related to DNA methylation. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This diagram illustrates the principle of flow cytometry microsphere technology for analyzing DNA methylation sites, initiated by mild transformation and chemical linkage.

[0029] Figure 2 This demonstrates the effect of different PNA concentrations on the method specificity; among them, A represents the mean fluorescence intensity (MFI) of flow cytometry at different concentrations of PNA, including C, 5mC, 5hmC, and blank. B represents the signal-to-background ratio (S / B) of C, 5mC, and 5hmC at different concentrations of PNA.

[0030] Figure 3 This shows the effect of different probes (A and B) on the experiment; among them, A represents the MFI at different concentrations of the probe: C, 5mC, 5hmC, and blank. B represents the S / B ratio of C, 5mC, and 5hmC at different probe concentrations.

[0031] Figure 4 The MFI values ​​are shown at different 5mC concentrations. A is a bar chart of MFI values ​​at different 5mC concentrations; B is the MFI value and -lg C 5mC Linear relationship between concentrations.

[0032] Figure 5 This demonstrates the specificity of the method of the present invention in detecting 50 fM 5mC; wherein, A is a bar chart showing the MFI values ​​of 50fM C, 5mC, 5hmC and the blank control; B represents the flow cytometry fluorescence histograms for 50 fM C, 5 mC, 5 hmC, and blank cells.

[0033] Figure 6 This invention demonstrates the method for detecting the MFI value of a mixed sample of 5mC and C or 5hmC; wherein, In Figure A, the red bars represent the MFI values ​​of the 5mC mixture from 0% to 100% for 5mC, and the green bars represent the MFI values ​​of the 5mC mixture from 0% to 100% for 5mC.

[0034] B shows the fluorescence thermograms from left to right for 5mC-C, 5mC-5hmC mixtures from 0% to 100% for 5mC, and 5mC for 5mC. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] The detection kit of the present invention includes the following reagents: (1) 5hmC blocking solution: Take 0.5 μL of 10× NEB buffer, 1 μL of 50× UDP-Glu, 1 μL of 100 U T4-BGT, and 0.5 μL of water, for a total of 3 μL.

[0037] (2) TeT enzyme oxidation system: 1 μL eTET 2.0 reaction buffer, 3 μL eTET 2.0 enzyme mixture, 1×Fe 2+ 0.45 μL of solution and 3.05 μL of water, for a total of 7.5 μL.

[0038] (3) Pyridineborane conversion system: Take 6M NaAc (pH = 4.3) and 1M pyridineborane, with a volume of 1.1 μL between NaAc and pyridineborane: to obtain a mixture of 600 mM NaAc (pH = 4.3) and 100 mM pyridineborane, which is the pyridineborane conversion system.

[0039] (4) Click on the chemical reaction system: 1 μL 50 nM probe A, 1 μL 50 nM probe B, 1 μL 10× PBS, 1 μL 0.1% Tween-20, 1 μL 3 M NaCl, 1 μL 1 μM PNA and 2 μL water, total 8 μL.

[0040] (5) STV-MB enrichment system: Wash with 1×PBS (20 μL) for particles approximately 300 nm in size, and 2 μL (8.4 x 10⁻⁶) of STV-MB. 8 The magnetic beads were dispersed twice with PBS (20 μL) and the supernatant was discarded by magnetic adsorption. The magnetic beads were then dispersed with 1×PBS (20 μL) to obtain STV-MB magnetic nanoparticles (8.4*10⁻⁶ μL). 7 Preferably, the above-mentioned magnetic nanoparticles use streptavidin (STV) functionalized magnetic microbeads.

[0041] (6) TdT signal amplification system: The 5 μL mixture contained 1×TdT buffer solution, 1 U TdT, 1 mM dTTP, and water. The volumes of 5×TdT buffer solution, 20U TdT, 10 mM dTTP and water were 1 μL, 0.02 μL, 0.5 μL and 3.5 μL, respectively.

[0042] Preferably, the TeT enzyme oxidation system uses the recombinant Ten-Eleven Translocase (TET) enzyme 2.0 kit, and the TeT oxidation reaction system is prepared according to the kit instructions.

[0043] The method of the present invention is applicable to test samples including chemically synthesized DNA sequences, blood samples, and tissue samples, wherein the DNA in blood samples and tissue samples can be extracted using a kit and then tested.

[0044] The detection method of the present invention includes the following steps: (1) 5hmC closed Take 2 μL of the DNA sample to be tested and 3 μL of 5 hmC blocking solution, mix them, and react under the following conditions: 37℃ for 2 h and 65℃ for 20 min.

[0045] (2) TeT enzyme oxidation After the DNA sample to be tested is reacted with 5 hmC blocking solution, it does not need to be purified and can be directly added to the TeT enzyme oxidation system. The volume of the reaction solution and the TeT enzyme oxidation solution obtained in step (1) is 5 μL: 7.5 μL. After the reaction is complete, add the reaction termination solution: reaction termination buffer and reaction termination solution.

[0046] The volumes of the termination reaction buffer and the termination reaction buffer enzyme are 0.5 μL and 0.5 μL respectively.

[0047] (3) Pyridineborane conversion The reaction solution obtained in step (2) was added to the pyridineborane conversion system and incubated in a rotary mixer at 37 ℃ and 850 rpm for 16 h.

[0048] The above reaction solution was purified using the UNIQ-10 oligonucleotide purification kit to obtain the target sequence.

[0049] (4) Click chemical reaction The target sequence obtained in step (3) was mixed with the click chemistry reaction system, with 2 μL and 8 μL of the target sequence mixed with the click chemistry reaction system. The reaction conditions were 85℃ for 3 min and 50℃ for 20 min to obtain the click chemistry linker.

[0050] (5) STV-MB enrichment 1 μL of STV-MB (8.4*10) 7 Add the click chemical linker solution; reaction conditions: 50℃, 250 rpm, shaker for 2 hours.

[0051] Wash the magnetic beads twice with 30 μL 1xPBS to remove unconnected probes, and then add 5 μL 1xPBS to the magnetic beads.

[0052] (6) TdT signal amplification The obtained target sequence was mixed with the signal amplification system at a volume ratio of 5 μL:5 μL. Reaction conditions: incubation at 37 °C for 1 h on a homogenizer at 250 rpm.

[0053] After the reaction was complete, 1 μL of a 100 nM PolyA25-FAM probe was added to the above mixture to bind to the extended dT sequence. The reaction was carried out at 4 °C for 12 h.

[0054] Finally, flow cytometry was used to detect the fluorescence signal.

[0055] This invention achieves a method for the specific transformation of 5mC and the detection of 5-methylcytosine at a specific site without bisulfite by employing an environmentally friendly biotransformation and efficient, mild chemical transformation strategy. The principle is as follows: (1) Selective and mild conversion of 5mC First, 5hmC was blocked with glucose using β-glucose transferase (β-GT) to convert it into 5gmC. Then, 5mC was oxidized to 5caC using TeT oxidase. Finally, 5caC was converted to DHU by pyridineborane. Since DHU has the same pairing ability as T, the converted 5mC forms a single base to distinguish it from C, providing a basis for subsequent detection.

[0056] (2) Detection of DNA methylation sites using flow cytometry microsphere technology based on PNA and chemical linkage. To simplify and sensitively detect target sequences, this invention fully leverages the advantages of click chemical linkage, such as high efficiency, stability, and a wide controllable temperature range, to provide a TdT-amplified flow cytometry method based on PNA and chemical linkage for detecting DNA epigenetic modification sites.

[0057] For the DHU target molecule, probes A and B, which are partially complementary to the DHU target sequence, were designed. Probe A is modified with biotin at its 5' end and with azidocyclooctane (Aza-DBCO) at its 3' end. Probe B is modified with azide (N3) at its 5' end and with a hydroxyl group (-OH) at its 3' end. After probes A and B hybridize with the DHU target, Aza-DBCO and N3 are labeled on adjacent nucleotides.

[0058] Therefore, in the presence of the DHU target, probes A and B are very close, forming a sandwich hybrid, which enables an effective click chemical link between Aza-DBCO and the N3 group.

[0059] To achieve high specificity in distinguishing between DHU and C, a PNA complementary to the C sequence was designed to effectively inhibit the ligation reaction of the C sequence, ensuring accurate detection of the DHU target molecule. To reduce non-specific amplification and enrich the target sequence, the ligation product with 5-biotin and 3-OH groups was anchored to streptavidin (STV)-coated magnetic beads (STV-MBs), and unligated probes were washed away. Thus, a large amount of 3-OH-terminated single-stranded DNA (ssDNA) was introduced onto the MBs, reflecting the target DHU level.

[0060] To improve the detection sensitivity of the method, nucleic acid amplification was further designed. TdT catalyzes the repeated incorporation of dTTP at each 3-OH end, forming a long poly(T)ssDNA tail of thousands of nucleotides. Each poly(T) tail can capture a large amount of poly(A)25-FAM ssDNA, thereby accumulating a large number of fluorophores on the MB surface and achieving signal amplification.

[0061] Finally, flow cytometry was used to detect the fluorescence signal. As each molecular weight organelle (MB) passed through the flow cytometer, the fluorescence signal of a single MB could be analyzed directly and rapidly. Furthermore, the amount of target DHU could be quantitatively reflected by the average fluorescence intensity (MFI) of these MBs. The reaction principle diagram is shown below. Figure 1 As shown.

[0062] It is noteworthy that only the ligation product can bring 3-OH ssDNA to the surface of MBs, leading to TdT extension and fluorescence accumulation. Conversely, C and 5hmC cannot ligate probes A and B due to mismatch, so only probe A, which exposes the 3'-Aza-DBCO group, can anchor to the MB. Therefore, TdT cannot recognize and extend the MB.

[0063] Experimental reagents used in this invention:

[0064] Table 1 shows the DNA sequences used in this invention:

[0065] Example 1: The detection kit of the present invention The detection kit of the present invention includes the following reagents: (1) 5hmC blocking solution: Take 0.5 μL of 10× NEB buffer, 1 μL of 50× UDP-Glu, 1 μL of 100 U T4-BGT, and 0.5 μL of water, for a total of 3 μL.

[0066] (2) TeT enzyme oxidation system: Prepare the recombinant Ten-Eleven Translocase (TET) enzyme 2.0 kit according to the kit instructions: 1 μL eTET reaction buffer, 3 μL eTET enzyme mixture, 1×Fe 2+ 0.45 μL of solution and 3.05 μL of water, for a total of 7.5 μL.

[0067] (3) Pyridineborane conversion system: Take 1 μL of 6M NaAc solution and 0.1 μL of 1M pyridineborane, with a total volume of 1.1 μL, to obtain a mixture of 600 mM NaAc (pH= 4.3) and 100 mM pyridineborane, which is the pyridineborane conversion system.

[0068] (4) Click on the chemical reaction system: 1 μL of 50 nM probe A, 1 μL of 50 nM probe B, 1 μL of 10× PBS, 1 μL of 0.1% Tween-20, 1 μL of 3 M NaCl, 1 μL of 1 μM PNA, and 2 μL of water, for a total of 8 μL.

[0069] (5) STV-MB enrichment system: Wash with 1×PBS (20 μL) for particles approximately 300 nm in size, and 2 μL (8.4 x 10⁻⁶) of STV-MB. 8 The magnetic beads were dispersed twice with PBS (20 μL) and the supernatant was discarded by magnetic adsorption. The magnetic beads were then dispersed with 1×PBS (20 μL) to obtain STV-MB magnetic nanoparticles (8.4*10⁻⁶ μL). 7 ).

[0070] (6) TdT signal amplification system: The 5 μL system contains 1×TdT buffer solution, 1 U TdT, 1 mM dTTP and water, wherein, 1 μL of 5×TdT buffer solution, 0.02 μL of 20U TdT, 0.5 μL of 10 mM dTTP and 3.5 μL of water.

[0071] The detection method using the above kit includes the following steps: (1) 5hmC closed Take 2 μL of the DNA sample to be tested and 3 μL of 5 hmC blocking solution, mix them, and react under the following conditions: 37℃ for 2 h and 65℃ for 20 min.

[0072] (2) TeT enzyme oxidation After the DNA sample to be tested is reacted with 5 hmC blocking solution, it does not need to be purified and is directly added to the TeT enzyme oxidation system. The volume of the reaction solution obtained in step (1) and the TeT enzyme oxidation system is 5 μL: 7.5 μL. After the reaction is complete, add the reaction termination solution: reaction termination buffer and reaction termination enzyme.

[0073] The volumes of the termination reaction buffer and the termination reaction buffer enzyme were 0.5 μL and 0.5 μL, respectively.

[0074] (3) Pyridineborane conversion The reaction solution obtained in step (2) was added to the pyridineborane conversion system and incubated in a rotary mixer at 37 ℃ and 850 rpm for 16 h.

[0075] The above reaction solution was purified using the UNIQ-10 oligonucleotide purification kit to obtain the target sequence.

[0076] (4) Click chemical reaction The target sequence obtained in step (3) was mixed with the click chemistry reaction system in volumes of 2 μL and 8 μL. The reaction conditions were 85 °C for 3 min and 50 °C for 20 min to obtain the click chemistry linker.

[0077] (5) STV-MB enrichment 1 μL of STV-MB (8.4*10) 7 Add the click chemical linker solution; reaction conditions: 50℃, 250 rpm, shaker for 2 hours.

[0078] Wash the magnetic beads twice with 30 μL 1xPBS to remove unconnected probes, and then add 5 μL 1xPBS to the magnetic beads.

[0079] (6) TdT signal amplification The target sequence obtained above was mixed with the signal amplification system at a volume ratio of 5 μL:5 μL. Reaction conditions: incubation at 37 °C for 1 h on a homogenizer at 250 rpm.

[0080] After the reaction was complete, 1 μL of 100 nM PolyA 25-FAM probe was added to the above mixture to enrich the extended dT sequence. The reaction was carried out at 4 °C for 12 h.

[0081] (7) Flow cytometry detection of fluorescence signals Flow cytometry can be used to disperse microbes (MBs), allowing for individual identification of each MB. Under laser irradiation, these MBs fluoresce, forming fluorescently positive MBs. The 5mC sequence is quantified by the average fluorescence intensity of 10,000 particles at 330 V.

[0082] Experimental results can be processed and analyzed using Origin 8 and Graphpad Prism 9.

[0083] Example 2: Investigating the effect of PNA quantity on the method PNA is a synthetic DNA / RNA analog with a backbone of repeating N-2-(aminoethyl)-glycine units. It lacks phosphate groups, thus exhibiting strong binding affinity to DNA, resistance to enzymatic degradation, and high stability. However, a single base mismatch (Tm drop of 15°C) weakens the PNA / DNA binding stability compared to DNA / DNA under these conditions. Two base mismatches do not hybridize, resulting in extremely high specificity.

[0084] This invention designs a PNA sequence complementary to the target sequence C, provided that the following conditions are met: 1. The PNA and complementary sequence should ideally be 10-15 bases apart.

[0085] 2. The purine content in the PNA sequence is less than 60%. The more purines, the worse the solubility.

[0086] 3. Avoid having six purines together in a row, especially four or more Gs together in a row.

[0087] Through continuous design optimization, the PNA sequence CGGTGCTAAT was obtained. This PNA is completely complementary to the target sequence C, and it has a base mismatch with the transformed 5mC.

[0088] This embodiment further examines the effect of changes in the amount of PNA on detection specificity: The effect of PNA amount on specificity was further investigated by varying the amount of PNA from 0 nM to 1 μM (1 μM, 100 nM, 50 nM, 10 nM, 1 nM and 0 nM), with other steps the same as in Example 1.

[0089] Sample preparation for group C: Mix 1 μL of 50 fM chemically synthesized sequence C with 1 μL of 50 fM complementary chemically synthesized sequence C.

[0090] Preparation of 5mC group samples: 1 μL of 50fM chemically synthesized sequence 5mC and 1 μL of 50fM chemically synthesized sequence complementary C were mixed.

[0091] Sample preparation for 5hmC group: 1 μL of 50fM chemically synthesized sequence 5hmC and 1 μL of 50fM chemically synthesized sequence complementary C were mixed.

[0092] Blank group sample preparation: 2 μL water.

[0093] Figure 2 As shown in Figure A, the fluorescence signal generated by 5mC remained essentially unchanged as the PNA concentration increased from 0 nM to 1 μM, indicating that PNA had no effect on the connection of 5mC. However, with increasing PNA concentration, the fluorescence signals of blank, C, and 5mC showed a gradual decreasing trend, and remained unchanged when the PNA concentration was above 100 nM.

[0094] like Figure 2 As shown in Figure B, the highest S / B ratio was obtained when the concentration of PNA was above 100 nM. The S / B ratio refers to the ratio of the MFI value generated by the 50 fM sequence (C, 5 mC, or 5 hmC) to the MFI value of the blank control.

[0095] It is evident that the concentration of PNA plays an important role in improving connection specificity.

[0096] Example 3: Investigating the effect of the connection probe on the method This invention independently designed and optimized probes A and B. Probe A: Biotin-AAAAAAAAAAGTACCTAAGGGGCA-DBCO; Probe B: N3-GTGCTAATGGGAGAG.

[0097] The HAIRPIN plot of probe A shows that its self-hybridization does not exceed two Gs, indicating that self-hybridization is not severe. When probe A hybridizes with the target sequence C, the A base at the 3' end of probe A mismatches with the detection site C. When probe A hybridizes with the transformed target sequence 5mC, the A base at the 3' end of probe A perfectly matches the DHU (pairing ability T) transformed from the detection site 5mC.

[0098] The HAIRPIN plot of probe B shows that self-hybridization does not exceed two GCs, indicating that self-hybridization is not severe. Probe B achieves a perfect match when hybridizing with target sequence C. Probe B also achieves a perfect match when hybridizing with the transformed target sequence 5mC.

[0099] When probe A hybridizes with probe B and C, a single base mismatch prevents click chemolinking between probe A and probe B. However, when probe A and probe B hybridize with transformed 5mC, a perfect match occurs, resulting in efficient click chemolinking between probe A and probe B.

[0100] This embodiment further examines the effect of probe quantity on detection specificity.

[0101] The concentrations of the ligation probes (A and B) were varied from 1 nM to 25 nM (1 nM, 5 nM, and 25 nM) to further investigate the effect of the amount of ligation probes on the experiment. The sequences used are shown in Table 1, and the other steps are the same as in Example 1.

[0102] Sample preparation for group C: Mix 1 μL of 50 fM chemically synthesized sequence C with 1 μL of 50 fM complementary chemically synthesized sequence C.

[0103] Preparation of 5mC group samples: 1 μL of 50fM chemically synthesized sequence 5mC and 1 μL of 50fM chemically synthesized sequence complementary C were mixed.

[0104] Sample preparation for 5hmC group: 1 μL of 50fM chemically synthesized sequence 5hmC and 1 μL of 50fM chemically synthesized sequence complementary C were mixed.

[0105] Blank group sample preparation: 2 μL water.

[0106] like Figure 3 As shown in Figure A, when the ligation probe concentration is 1 nM, the fluorescence signals of the C, 5mC, 5hmC sequences, and the blank are all very low, possibly because the low concentration of the ligation probe reduces the ligation efficiency. The signal difference between 5mC and C and 5hmC is small. When the probe concentration is increased to 5 nM, the fluorescence signal produced by 5mC increases sharply. At the same time, the fluorescence signals of the blank and C and 5hmC also show an increasing trend, but the increase in signal is not as large as that of 5mC.

[0107] like Figure 3 As shown in Figure B, the highest S / B ratio was obtained when the probe concentration was 5 nm. When the probe concentration was 25 nM, the signal at 5 mC did not change much, but C, 5 hmC, and blank increased, thus the S / B ratio decreased.

[0108] It is evident that the concentration of the ligation probe plays a crucial role in improving ligation specificity.

[0109] Example 4: Examining the sensitivity of the method of the present invention for detecting 5mC. Different concentrations of the synthetic 5mC series dilutions were tested. The 5mC concentrations were 1 fM, 2.5 fM, 10 fM, 50 fM, 100 fM, and 250 fM, respectively. The sequences used are shown in Table 1. Other steps were the same as in Example 1.

[0110] like Figure 4 As shown in Figure A, the MFI value of MB gradually increases as the 5mC concentration increases from 1 fM to 250 fM.

[0111] The concentration of 5mC can be quantitatively reflected by the MFI value of each sample. From Figure 4 As shown in B, the MFI value increases exponentially with increasing 5mC concentration. The linear regression equation is Y = 146lg C +2440.74, correlation coefficient R 2 =0.996.

[0112] In addition, to verify the precision of the method, the standard deviation (SD) value of each data point was calculated by three parallel measurements. In Figure B, the error bars represent the standard deviation of three repeated measurements for each concentration (the voltage of the measurement is 330 V). The standard deviation at each concentration is less than 10%.

[0113] Therefore, the measurement method of the present invention can achieve a high sensitivity of 1 fM.

[0114] Example 5: Examining the specificity of the method of the present invention Three identical sequences were selected and modified with C, 5mC, and 5hmC at the same position. The resulting sequences are shown in Table 1. The three sequences were then specifically detected using the method of this invention.

[0115] C sequence: CCT CAG GCT CTC CCA TTA GCA C C G CCC CTT AGG TAC AGT TAC ACT G; 5mC sequence: CCT CAG GCT CTC CCA TTA GCA C 5mC G CCC CTT AGG TAC AGT TAC ACTG; 5hmC sequence: CCT CAG GCT CTC CCA TTA GCA C 5hmC G CCC CTT AGG TAC AGT TACACT G; C complementary sequence: CAG TGT AAC TGT ACC TAA GGG GCG GTG CTA ATG GGA GAG CCT GAGG.

[0116] like Figure 5 As shown in Figure A, only the fluorescence signal induced by 5mC was clearly distinguishable from the blank control. The fluorescence signals of the C and 5hmC sequences were almost identical to the blank control. The interference of signals generated by C and 5hmC at the same concentration on 5mC detection was calculated to be 1% and 1.4%, respectively.

[0117] The results show that the method of the present invention has good specificity and can distinguish well even with epigenetic differences.

[0118] Example 6: Detection of 5mC in a mixed sample To further verify the high specificity of this method, 5mC was detected in sequences containing high concentrations of C or 5hmC. In this study, the mixing ratio of 5mC and C or 5hmC ranged from 0% to 100%, with a total DNA concentration of 100 fM.

[0119] (1) The preparation process of the 5mC-C mixed sample of 5mC and C is as follows: 0% 5mC: 100fM C and 0fM 5mC; 1% 5mC: 99fM of C and 1fM of 5mC; 10% 5mC: 90fM C and 10fM 5mC; 25% 5mC: 75fM C and 25fM 5mC; 50% 5mC: 50fM C and 50fM 5mC; 100% 5mC: 0fM C and 100fM 5mC.

[0120] (2) The preparation process of the mixed sample 5mC-5hmC is as follows: 0% is 100fM of 5hmC and 0fM of 5mC; 1% 5mC: 99fM of 5mC and 1fM of 5mC; 10% 5mC: 90fM 5mC and 10fM 5mC; 25% 5mC: 75fM 5mC and 25fM 5mC; 50% 5mC: 50fM 5hmC and 50fM 5mC; 100% 5mC: 0fM 5mC and 100fM 5mC.

[0121] like Figure 6 As shown in Figure A, the higher the ratio of 5mC in the DNA mixture, the higher the fluorescence value. When the ratio is 0%, i.e., C or 5hmC is 100 fM, it can be distinguished from 1% 5mC.

[0122] from Figure 6 As shown in Figure B, the fluorescence signal gradually increases as the proportion of 5mC increases from 0% to 100%, and the signal of 1% 5mC in the mixture is consistent with that of pure 5mC. This indicates that C and 5hmC do not cause any non-specific amplification signals, and the detectable fluorescence reaction in the DNA mixture is indeed caused solely by 5mC. It is noteworthy that even concentrations of C higher than 100 times do not interfere with the detection of 5mC.

[0123] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

The 1,5-methylcytosine detection kit is characterized by, The kit includes a 5hmC blocking solution, a TeT enzyme oxidation system, a pyridine borane conversion system, a click chemistry ligation system, and a TdT signal amplification system, wherein... The 5hmC blocking solution consisted of: 0.5 μL of 10× NEB buffer, 1 μL of 50× UDP-Glu, 1 μL of 100 U T4-BGT, and 0.5 μL of water.

2. The 5-methylcytosine detection kit according to claim 1, characterized in that, The TeT enzyme oxidation system includes: 1 μL eTET 2.0 reaction buffer, 3 μL eTET 2.0 enzyme mixture, and 1×Fe 2+ 0.45 μL of solution and 3.05 μL of water.

3. The 5-methylcytosine detection kit according to claim 1, characterized in that, The pyridineborane conversion system consisted of 1 μL of 6M NaAc solution and 0.1 μL of 1M pyridineborane, with the NaAc solution having a pH of 4.

3.

4. The 5-methylcytosine detection kit according to claim 1, characterized in that, The click chemilinking system consists of 1 μL of 50 nM probe A, 1 μL of 50 nM probe B, 1 μL of 10× PBS, 1 μL of 0.1% Tween-20, 1 μL of 3 M NaCl, 1 μL of 1 μM PNA, and 2 μL of water.

5. The 5-methylcytosine detection kit according to claim 4, characterized in that, The nucleotide sequence of probe A is: 5′-AAAAAAAAAAGTACCTAAGGG GCA-3′; The nucleotide sequence of probe B is 5′-GTGCTAATGGGAGAG-3′.

6. The 5-methylcytosine detection kit according to claim 4, characterized in that, The peptide nucleic acid sequence of PNA is 5′-CGGTGCTAAT-3′.

7. The 5-methylcytosine detection kit according to claim 1, characterized in that, It also includes an STV-modified magnetic nanoparticle enrichment system: STV-MB was washed with 1×PBS, the supernatant was discarded by magnetic adsorption, and 330 nM magnetic nanoparticles were dispersed with 1×PBS to obtain an STV-modified magnetic nanoparticle enrichment system.

8. The 5-methylcytosine detection kit according to claim 1, characterized in that, The TdT signal amplification system consists of: 1 μL of 5×TdT buffer solution, 0.02 μL of 20U TdT, 0.5 μL of 10 mM dTTP, and 3.5 μL of water.

Images