A method and kit for absolute quantification of DNA methylation without reference

Abstract

This invention belongs to the field of DNA methylation and discloses a reference-free absolute quantification method and kit for DNA methylation. First, a cytosine methylation conversion reagent is used to convert unmethylated C in the DNA sample to U, while 5mC remains unchanged, causing the originally complementary DNA double strands to dissociate into templates. Then, CpG-free primer pairs are used for amplification. Different fluorescent probes are used to detect the amount of methylation in the sense strand and the total amount of template in the antisense strand, achieving absolute quantification of DNA methylation without internal controls or external standardization. This technique also has advantages such as strong resistance to sample degradation, excellent stability to low starting amounts of samples, complete immunity to CNV, simple experimental procedure, and high throughput.

Description

Technical Field

[0001] This invention belongs to the field of DNA detection, specifically relating to a reference-free absolute quantification method and kit for DNA methylation. Background Technology

[0002] DNA methylation, as a key epigenetic modification, plays a central role in regulating gene expression and maintaining genome stability. 1 Abnormal methylation states in specific gene promoter regions are closely related to the occurrence and development of many major diseases, especially tumors, and have become highly promising biomarkers for disease diagnosis, prognosis, and medication guidance. 2-5 Therefore, developing technologies capable of accurately and reliably quantifying DNA methylation levels in trace, degraded samples (such as clinical tissue biopsy samples and cell-free DNA (cfDNA) in plasma) is a major need and technological bottleneck currently facing the field of molecular diagnostics. 6-8 .

[0003] Currently, techniques for quantitative analysis of DNA methylation at specific sites are generally limited by a fundamental physical limitation. After distinguishing the methylation state (usually through bisulfite treatment), these methods require quantification of the "signal molecule" carrying the methylation information. However, since the initial template amount in clinical samples is often unknown, trace, and subject to random loss during extraction and processing, a "reference molecule" must be introduced to calibrate the signal.

[0004] Existing technological approaches all rely on external or uncoupled references. That is, the "signal molecule" and the "reference molecule" are two physically separate and independent molecular entities. For example, the reference molecule might be a housekeeping gene (such as ACTB). 9,10 Or genomic repetitive sequences (such as ALU). 11,12 This decoupling between "signal" and "reference" leads to a series of inherent, insurmountable defects: (1) Random sampling bias: When processing small samples (such as ng-level cfDNA), the proportion of “signal” and “reference” molecules that are randomly assigned to PCR reaction tubes is inherently statistically volatile because they are physically separated. This random sampling error can severely distort the final quantitative results, especially in low-abundance methylation detection close to the clinical decision threshold, and can easily lead to false negatives or false positives.

[0005] (2) Differential degradation and recovery: DNA in clinical samples often exhibits varying degrees of degradation. Bisulfite treatment itself is a violent chemical reaction that can lead to up to 90% DNA breakage and loss. 13Because the "signal" and "reference" molecules differ in their genomic location, sequence composition, and fragment length, they exhibit different degradation kinetics and recovery efficiencies during sample storage, nucleic acid extraction, chemical modification, and recovery before amplification. This variability is unpredictable and uncalibrable, thus introducing systematic quantitative bias.

[0006] (3) Genomic instability interference: Copy number variations (CNVs) are common in the genome in diseases such as tumors. 14 If the chromosomal region containing the selected "reference molecule" is amplified or deleted, while the copy number of the "signal molecule" region is normal, then the quantitative results based on this reference will be severely distorted and will not reflect the true methylation epigenetic state at all.

[0007] In summary, existing DNA methylation quantification technologies, due to their reliance on a "signal-reference physically uncoupled" core design, face fundamental challenges in terms of accuracy, robustness, and reproducibility when dealing with complex clinical samples characterized by low starting amounts, high degradation, and genomic instability. The industry urgently needs a new technological paradigm to fundamentally solve the problem of physical coupling between "signal and reference" and achieve truly absolute quantification. Summary of the Invention

[0008] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a reference-free method and kit for absolute quantification of DNA methylation.

[0009] The technical solution adopted in this invention is: A first aspect of the present invention provides: a reference-free absolute quantification kit for DNA methylation, comprising: Cytosine methylation conversion reagent is used to convert unmethylated cytosine C in the DNA sample into uracil U, while 5-methylcytosine remains unchanged, causing the originally complementary DNA double strands to dissociate, and the sense and antisense strand sequences at the same locus to become no longer complementary; CpG-free primer pairs, wherein the binding sites of the primer pairs are located in the CpG-free regions flanking the CpG site to be tested, can indiscriminately amplify DNA templates from methylated and unmethylated alleles, and the amplification products of the primer pairs contain both transformed sense and antisense strand fragments. A methylation-specific fluorescent probe, wherein the methylation-specific fluorescent probe specifically targets and binds to the positive strand amplification product containing CpG dinucleotides, thereby producing a fluorescence change; A template total amount specific fluorescent probe, which specifically targets and binds to sequences within the antisense strand amplification product that do not contain CpG sites, and then produces a fluorescence change.

[0010] Existing reagents can be used for cytosine methylation conversion, including but not limited to EZ DNAMethylation-Gold Kit (Zymo Research), CpGenome DNA Modification Kit (MerckMillipore), Bisulfite Conversion Kit (Qiagen), or self-prepared cytosine methylation conversion reagents.

[0011] In some instances, the amplification products of primer pairs range in length from 60 to 200 bp. Particularly when processing easily degradable samples (such as formalin-fixed paraffin-embedded (FFPE) tissue DNA or plasma cfDNA), shorter amplicons, preferably 60-120 bp, are strongly recommended to ensure amplification efficiency. The binding region of the primers must be located on a sequence that does not contain any CpG dinucleotides. This design ensures that the binding and extension efficiencies of the primer pairs derived from methylated and unmethylated templates are completely consistent, a prerequisite for achieving unbiased amplification and ensuring accurate quantification.

[0012] Primers can be designed using professional primer design software (such as Primer3Plus, Primer Express 3.0.1, etc.). The annealing temperature (Tm) of the primers should be set in the range of 58-62℃, the GC content should be between 30-70%, and the formation of secondary structures such as primer dimers and hairpins should be strictly avoided.

[0013] The methylation-specific fluorescent probe spans the target CpG site. The probe sequence is completely complementary to the methylated template (retaining CG), but forms a mismatch of at least one base with the unmethylated template (becoming TG). This mismatch is used to achieve specific recognition.

[0014] The 5' end of the methylation-specific fluorescent probe is labeled with a first reporter fluorescent group (preferably FAM), and the 3' end is connected to a non-fluorescent quencher group (NFQ) and a minor groove binder (MGB). The introduction of MGB can significantly increase the probe's Tm value (approximately 15-20 °C) and drastically amplify the effect of single-base mispairing on the melting temperature (leading to a ΔTm as high as 15 °C or more), thereby endowing the probe with extreme single-base discrimination capability.

[0015] The region bound to the template-specific fluorescent probe is CpG-free. Thus, regardless of the methylation state of the original DNA, the antisense strand sequence remains completely constant after bisulfite conversion. The 5' end of the probe is labeled with a second reporter fluorophore (preferably ROX or VIC, whose emission spectrum must not significantly overlap with the first reporter fluorophore), and the 3' end is also linked to MGB-NFQ.

[0016] To ensure that the probe binds to the template preferentially over the primer during the PCR annealing step, the probe's Tm value must be significantly higher than the primer's Tm value, preferably 5-10°C higher. MGB modification helps to easily achieve this requirement.

[0017] To achieve the most efficient 5'→3' exonuclease hydrolysis of Taq DNA polymerase, the 5' end of the probe should be as close as possible to the 3' end of the upstream primer, but they should not overlap, preferably with a gap of more than one base.

[0018] In some instances, the methylation-specific fluorescent probe and the template totality-specific fluorescent probe have different fluorescent groups.

[0019] In some instances, the methylation-specific fluorescent probe and the template totality-specific fluorescent probe are TaqMan hydrolysis probes.

[0020] In some instances, the fluorescent group is selected from FAM, VIC, HEX, TET, JOE, ROX, Cy3, Cy5, and Cy5.5.

[0021] In some instances, the CpG-free primer pairs and probes include: Primer pairs and probes used for quantifying SEPT9 gene methylation, (1) SEPT9-forward primer: 5'-GATTYGTTGTTTATTAGTTATTATGT-3' (2) SEPT9-reverse primer: 5'-AAATAATCCCATCCAACTA-3' (3) SEPT9-Met-probe (signal probe): 5'-TAACCGCGAAATCCGA-3' (4) SEPT9-Ctrl-probe (input probe): 5'-GTTGGATGGGATTATT-3'; or Primer pairs and probes used for quantifying KAZN gene methylation, (1) KAZN-forward primer: 5'-TTGGAAGAGGAGTTATAATGTAGGATGTT-3' (2) KAZN-reverse primer: 5'-CCTCTTAAATCCCTTCAAACCTTTTC-3' (3) KAZN-Met-probe: 5'-ATATTAAGGTAGGTAAGCGTGGTG-3' (4) KAZN-Ctrl-probe: 5'-CATCCTACATTATAACTCCTCTTC-3'; or Primer pairs and probes used for quantifying DUSP3 gene methylation, (1) DUSP3-forward primer: 5'-TTTGTAGGTGGTGGTTTTTGGATAAT-3' (2) DUSP3-reverse primer: 5'-TCTCAATCTTAAAACAATATACCTTTCCAC-3' (3) DUSP3-Met-probe: 5'-GAGTGGTAGGGATATAGACGAGT-3' (4) DUSP3-Ctrl-probe: 5'-AAATTATCCAAAAACCACCACC-3'.

[0022] These features can be combined arbitrarily as long as they do not conflict with each other.

[0023] A second aspect of the present invention provides: a reference-free method for absolute quantification of DNA methylation, comprising the following steps: Take the genomic DNA sample to be tested, convert unmethylated cytosine (C) into uracil (U), while 5-methylcytosine (5mC) remains unchanged, causing the originally complementary DNA double strands to dissociate, and the sense and antisense strand sequences at the same locus become no longer complementary, thus obtaining the transformed DNA template; Amplification and Detection: Amplification primers, methylation-specific fluorescent probes, template-total-specific fluorescent probes, the DNA template, and other PCR reaction components are mixed in a single reaction tube. Amplification and signal acquisition are performed on a real-time quantitative PCR instrument. The absolute methylation level is determined based on the Ct values ​​of the fluorescence of the methylation-specific fluorescent probes and the template-total-specific fluorescent probes. The amplification primers are CpG-free primer pairs with binding sites located in the CpG-free regions flanking the target CpG site. They can indiscriminately amplify DNA templates from methylated and unmethylated alleles. The amplification products of the primer pairs simultaneously contain transformed sense and antisense strand fragments. The methylation-specific fluorescent probes specifically target and bind to the sense strand amplification product containing CpG dinucleotides, producing a fluorescence change. The template-total-specific fluorescent probes specifically target and bind to sequences within the antisense strand amplification product that do not contain CpG sites, producing a fluorescence change. The amount of methylation in the genomic DNA sample to be tested is determined based on the fluorescence changes during the amplification process.

[0024] In some instances, the methylation-specific fluorescent probe and the template totality-specific fluorescent probe have different fluorescent groups.

[0025] In some instances, the methylation-specific fluorescent probe and the template totality-specific fluorescent probe are TaqMan hydrolysis probes.

[0026] In some instances, the fluorescent group is selected from FAM, VIC, HEX, TET, JOE, ROX, Cy3, Cy5, and Cy5.5.

[0027] In some instances, based on the CT of the methylation-specific fluorescent probe... meth CT values ​​of specific fluorescent probes relative to total template amount input The value, according to the formula: ΔCT = CT meth -CT input Absolute methylation ratio = 2 -ΔCT The absolute methylation level of the DNA to be tested is calculated by multiplying by 100%.

[0028] A third aspect of the present invention provides the application of the DNA methylation absolute quantification kit described in the first aspect of the present invention in quantifying the absolute amount of DNA methylation in a sample.

[0029] The samples described in the first aspect of this invention are selected from solid tumor samples, including but not limited to solid tumors with methylation gene modifications such as colorectal cancer, gastric cancer, liver cancer, lung cancer, breast cancer, and prostate cancer.

[0030] The beneficial effects of this invention are: Compared with existing technologies, this invention achieves the following significant beneficial effects through its unique "intramolecular control" design: (1) True absolute quantification is achieved: Since the signal and reference are on the same molecule, their stoichiometric ratio is constant at 1:1. Therefore, this method fundamentally eliminates errors caused by variations in template input, differences in amplification efficiency, and batch effects, allowing the calculation of the absolute proportion of methylated molecules without the need for a standard curve. Experimental data show that the quantitative results of this invention exhibit extremely high consistency with the gold standard pyrosequencing method (R0). 2 =0.964), which is far superior to traditional methods.

[0031] (2) Extremely strong resistance to sample degradation: Because the signal and reference are designed within the same short amplicon (e.g., <100 bp), they are physically covalently linked. Therefore, even if the sample DNA is severely fragmented, as long as one amplicon is intact, its internal signal and reference information can be simultaneously preserved and detected. This allows the present invention to maintain quantitative accuracy and robustness when processing severely degraded clinical samples (e.g., FFPE tissue DNA, cfDNA).

[0032] (3) Excellent stability with low initial sample amounts: In trace samples, the physically linked signal and reference either enter the reaction system simultaneously or are lost simultaneously, fundamentally avoiding the imbalance in their ratio caused by random sampling. This allows the present invention to obtain stable quantitative results even with a DNA input as low as 0.1 ng, significantly improving the sensitivity and reliability of the detection. Figure 2 E).

[0033] (4) Completely unaffected by copy number variation (CNV): Since the reference sequence and the signal sequence are located at the same locus, they will be amplified or deleted equally, and their relative proportions will always remain unchanged. This allows the present invention to truly reflect the epigenetic state of the gene without being affected by genomic dosage effects.

[0034] (5) The experimental process is simplified and the detection throughput is increased: The present invention integrates the detection of signal and reference in a single reaction tube, which reduces the number of operation steps, reduces the difference between holes, and makes it easy to achieve automated and high-throughput detection. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the principle of an absolute quantification method for DNA methylation that does not require a reference in some examples of the present invention.

[0036] Figure 2 These are the results of the detection performance and robustness verification of the DNA methylation absolute quantification method that does not require reference in some examples of the present invention.

[0037] Figure 3 The results are benchmarked against bisulfite pyrosequencing and qMSP in clinical samples. Detailed Implementation

[0038] like Figure 1 As shown, a reference-free DNA methylation absolute quantification method (Methylation Reference-Free PCR, MeRFPCR) is based on the following core design idea: utilizing the unique phenomenon that the sense and antisense strands on the same DNA double-stranded molecule lose base complementarity after bisulfite conversion, resulting in sequence asymmetry, the sense strand sequence carrying methylation information is used as the "signal," while the antisense strand sequence, which is physically covalently linked 1:1 within the same amplicon and does not contain CpG sites, is creatively designed as the "input." This achieves perfect physical coupling and an absolute 1:1 stoichiometric ratio between the "signal" and the "reference" within a single molecule.

[0039] The specific implementation steps of this technical solution are as follows: Step 1: Bisulfite treatment of DNA samples Take a sample of genomic DNA to be tested and treat it using a standard bisulfite treatment method (such as the EZ DNAMethylation-Gold™ Kit). This process converts unmethylated cytosine (C) to uracil (U), while 5-methylcytosine (5mC) remains unchanged. After this step, the originally complementary DNA double strands dissociate, and the sense and antisense sequences at the same locus become no longer complementary.

[0040] Step 2: MeRF-based PCR amplification and detection Design a unique PCR reaction system containing the following key components: (1) A pair of CpG-free primers: A pair of upstream and downstream PCR primers were designed with binding sites located in the CpG-free regions flanking the CpG sites to be tested. This design ensures that the primers can amplify DNA templates from methylated and unmethylated alleles indiscriminately, guaranteeing the non-biased amplification. The product (amplifier) ​​amplified by this primer pair contains both the transformed sense and antisense strands.

[0041] (2) A methylation-specific fluorescent probe (signal probe): Design a TaqMan hydrolysis probe labeled with the first fluorescent group (such as FAM). The sequence of this probe is specifically targeted at the sequence of CpG dinucleotides retained on the sense strand after bisulfite conversion (i.e., derived from the methylated DNA template). When the template DNA is unmethylated, the CpG on its sense strand will be converted to TpG, causing the signal probe to be unable to bind and hydrolyze effectively, producing no or only a very low fluorescence signal.

[0042] (3) A template-total-specific fluorescent probe (input probe): Design another TaqMan hydrolysis probe labeled with a second fluorescent group (such as ROX). The sequence of this probe is specifically targeted to a CpG-free sequence on the antisense strand within the same amplicon. Since this region does not contain CpG, the sequence of the antisense strand after bisulfite treatment remains constant regardless of whether the original template is methylated or unmethylated. Therefore, this input probe can identify and quantify all amplified template molecules (including those from methylated and unmethylated sources), thus accurately representing the total amount of effectively amplified template in the reaction system.

[0043] The primers, probes, transformed DNA template, and other PCR reaction components (such as DNA polymerase, dNTPs, etc.) were mixed in a single reaction tube and amplified and signal acquired using a real-time quantitative PCR instrument. The instrument simultaneously recorded the cycle thresholds (Ct values) of the two fluorescent groups (such as FAM and ROX), denoted as CT. meth (Signal probe) and CTinput (Input signal probe).

[0044] Step 3: Calculation of absolute methylation level Since the molar ratio of "signal" (methylated sense strand) to "input" (antisense strand) is naturally 1:1 in every original DNA molecule, this invention can directly calculate the absolute methylation ratio using the difference in Ct values ​​(ΔCT) between the two, without any external calibrators. The calculation formula is as follows: Absolute methylation ratio = 2 -(CTmeth - CTinput) *100% This percentage directly reflects the true proportion of methylated molecules among all amplifiable template molecules.

[0045] Following bisulfite conversion, a primer pair simultaneously amplifies both methylated and unmethylated templates from the same locus. The methylated sense strand retains cytosine at the CpG site and can be recognized by a FAM-labeled methylation-specific probe (signal). The antisense strand contains a constant CpG-free sequence independent of methylation state and can be recognized by a ROX-labeled input probe. This design enforces a 1:1 stoichiometry between signal and input, allowing the absolute methylation level to be calculated using Equation 2. -ΔCT (where ΔCT = CT) meth - CT input This directly yields the results, effectively decoupling the quantitative process from changes in the template input, and achieving absolute quantification without the need for internal parameters and external standardization.

[0046] The technical solution of the present invention will be further illustrated below with examples.

[0047] To enable those skilled in the art to more fully and deeply understand the technical essence of this invention and to implement it, the specific operation process of this invention is described in detail below. The implementation process of this invention mainly includes four core modules: (i) optimization design and selection of oligonucleotides (primers and probes); (ii) DNA sample processing; (iii) establishment and program setting of MeRFPCR amplification reaction system; and (iv) result analysis and calculation of absolute methylation level. (I) Optimization and selection of oligonucleotides (primers and probes)

[0048] The technical effectiveness of this invention relies heavily on a rigorously designed, synergistic combination of primers and probes. Its design follows these unique principles and steps: 1. Identification of target CpG sites and acquisition of flanking sequences

[0049] 1.1. First, determine the target CpG site based on research or diagnostic needs. Utilize public genomic databases (such as UCSC Genome Browser, Ensembl, etc.) to precisely locate the chromosomal coordinates of this CpG site within the genome.

[0050] 1.2. Extract approximately 200-300 bp of genomic DNA sequence upstream and downstream of the CpG site as candidate regions for subsequent primer and probe design. 2. In silico (computer simulation) bisulfite transformation of key sequences

[0051] 2.1. The obtained positive and antisense DNA sequences containing the target CpG site are subjected to computer-simulated bisulfite conversion. This step is crucial: all cytosine (C) in the sequence not in the CpG dinucleotide context are replaced with thymine (T); while cytosine (C) located in the CpG dinucleotide remains unchanged to simulate its methylated protected state.

[0052] 2.2. After this step, two no longer complementary theoretical template sequences are obtained for probe and primer design: one is the transformed sense strand, and the other is the transformed antisense strand. 3. Design principles of MeRFPCR specific primer pairs

[0053] 3.1. Amplicon Length: The preferred PCR amplicon length range is 60-200 bp. In particular, when processing easily degradable samples (such as formalin-fixed paraffin-embedded (FFPE) tissue DNA or plasma cfDNA), it is strongly recommended to design shorter amplicon lengths, preferably 60-120 bp, to ensure amplification efficiency.

[0054] 3.2. CpG-free principle: This is one of the core principles of primer design in this invention. The binding region of the primer must be located on a sequence that does not contain any CpG dinucleotides. This design ensures that the binding and extension efficiencies of primer pairs derived from methylated and unmethylated templates are completely consistent, which is a prerequisite for achieving unbiased amplification and ensuring accurate quantification.

[0055] 3.3. Thermodynamic parameters: Use professional primer design software (such as Primer3Plus, Primer Express3.0.1, etc.) to design the primers, set the annealing temperature (Tm) to be in the range of 58-62℃, the GC content to be between 30-70%, and strictly avoid the formation of secondary structures such as primer dimers and hairpins. 4. Design principles of MeRFPCR dual-probe systems

[0056] This system innovatively uses two probes, each targeting a different strand within the same amplicon. Its design principles are as follows: 4.1. Signal Probe (Methylation Probe): (1) Targeting: The probe is specifically targeted at the positive strand sequence transformed by in silico, and its sequence must cross the target CpG site.

[0057] (2) Specificity: The probe sequence is completely complementary to the methylated template (retaining CG), while it forms a mismatch of at least one base with the unmethylated template (becoming TG). This mismatch is used to achieve specific recognition.

[0058] (3) Modification: The 5' end of the probe is labeled with a first reporter fluorescent group (preferably FAM), and the 3' end is connected with a non-fluorescent quencher group (NFQ) and a minor groove binder (MGB). The introduction of MGB can significantly increase the Tm value of the probe (about 15-20℃) and drastically amplify the effect of single base mispairing on the melting temperature (which can lead to a ΔTm of more than 15℃), thereby giving the probe extreme single base discrimination ability.

[0059] 4.2. Input probe: (1) Targeting: The probe is specifically targeted at antisense strand sequences converted by in silico.

[0060] (2) Invariance principle: This is the core of input probe design. The region it binds to must be CpG-free. In this way, regardless of the methylation state of the original DNA, the antisense strand sequence remains completely constant after bisulfite conversion.

[0061] (3) Modification: The 5' end of the probe is labeled with a second reporter fluorescent group (preferably ROX or VIC, whose emission spectrum must not overlap significantly with FAM), and the 3' end is also connected to MGB-NFQ.

[0062] 4.3. Thermodynamic and position coordination principle: (1) To ensure that the probe binds to the template preferentially over the primer during the PCR annealing step, the Tm value of the probe must be significantly higher than that of the primer, preferably 5-10°C higher. MGB modification can help to easily achieve this requirement.

[0063] (2) In order to achieve the most efficient Taq DNA polymerase 5'→3' exonuclease activity hydrolysis, the 5' end of the probe should be as close as possible to the 3' end of the upstream primer, but the two should not overlap, preferably with a gap of more than 1 base. (ii) Processing of DNA samples

[0064] 1. DNA Extraction: Depending on the sample type (e.g., fresh tissue, FFPE tissue, plasma, etc.), genomic DNA is extracted using standardized DNA extraction kits (e.g., InstaGene Matrix, QIAamp DNA Mini Kit, etc.). The concentration and purity (A260 / A280) of the extracted DNA are determined using a micro-spectrophotometer (e.g., NanoDrop).

[0065] 2. Bisulfite Conversion: Accurately collect 200-500 ng of genomic DNA and perform bisulfite conversion using a commercial kit (such as the EZ DNA Methylation Kit). Strictly follow the kit's instruction manual to ensure complete conversion. Elute the converted DNA in 20-30 μL of elution buffer and store at -80°C for later use. (III) Establishment and Procedure Setting of MeRFPCR Amplification Reaction System 1. Preparation of reaction mixture (single tube 20μL system)

[0066] Prepare the reaction mixture on ice. The components and recommended final concentrations are shown in the table below:

[0067] *Note: The final concentration of MgCl2 needs to be optimized according to the specific primer and probe system, and can be adjusted in the range of 1.5-3.0mM. 2. Program setting for real-time quantitative PCR

[0068] Add the prepared reaction solution to a 96-well PCR plate, seal with optical adhesive sealing film, and run on a real-time quantitative PCR instrument (such as the Applied Biosystems QuantStudio 7 Flex). The recommended thermal cycling procedure is as follows: (iv) Results analysis and calculation of absolute methylation level

[0069] 1. Obtaining Ct values: After the PCR reaction is complete, the analysis software will automatically calculate the cycle threshold (Ct) of the FAM channel and ROX / VIC channel in each reaction well, denoted as CT. meth (Target signal) and CT input (DNA input signal).

[0070] 2. Calculation of absolute methylation level: Due to the unique intramolecular self-correcting design of this invention, the absolute methylation level can be directly calculated using ΔCT, as shown in the formula: ΔCT = CT meth - CT input Absolute methylation percentage (%) = 2 -ΔCT × 100% The calculation results directly represent the true percentage of molecules carrying methylation tags among all DNA template molecules that can be successfully amplified, without the need for any external standards or reference reactions, making the results objective and accurate.

[0071] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to these embodiments. Example 1: Construction and validation of a MeRFPCR detection system for SEPT9 gene methylation in colorectal cancer diagnosis. 1. Primer and probe design and synthesis

[0072] The following oligonucleotide sequence was designed and synthesized targeting the CpG islands in the promoter region of the human SEPT9 gene: (1) SEPT9-forward primer: 5'-GATTYGTTGTTTATTAGTTATTATGT-3' (SEQ ID NO.1) (2) SEPT9-reverse primer: 5'-AAATAATCCCATCCAACTA-3' (SEQ ID NO.2) (3) SEPT9-Met-probe (signal probe): 5'-FAM-TAACCGCGAAATCCGA-MGB-NFQ-3' (SEQ ID NO.3) (4) SEPT9-Ctrl-probe (input probe): 5'-ROX-GTTGGATGGGATTATT-MGB-NFQ-3' (SEQ ID NO.4) The amplicon size is 65 bp. 2. MeRF PCR reaction

[0073] The 20 μL reaction system contained: 1×PCR buffer, 500 nM primers, 150 nM probes, 2.25 mM MgCl2, 200 μM dNTPs, 0.75 U HotStarTaq enzyme, and DNA template converted with bisulfite.

[0074] The reaction was performed on an ABI 7500 Fast real-time PCR system with the following thermal cycling program: 95°C for 10 minutes; followed by 50 cycles of 94°C for 30 seconds, 58°C for 1 minute, and 72°C for 1 minute, with fluorescence signals from the FAM and ROX channels acquired at the 72°C step. 3. Performance Verification

[0075] Commercially available 100% methylated human DNA and 0% methylated human DNA were used as controls. Results ( Figure 2 A) shows that SEPT9-Met-probe (FAM) only generates an amplification signal in 100% methylated DNA, while SEPT9-Ctrl-probe (ROX) generates a signal in both types of DNA, confirming the probe's specificity.

[0076] Ten samples with different SEPT9 methylation states were detected using MeRFPCR in two separate amplification runs conducted by the same operator using different batches of reaction solution over three days, with three counting replicates per sample. Additionally, the target gene SEPT9 was amplified in one well using the conventional qMSP method, while an external reference gene (ALU-C4) was amplified in parallel wells, with the same number of batch-to-batch replicates as described above. The concordance of methylation percentage between the two independent batches was calculated, including the Spearman p-value and R-value. 2 Compared with reference-based qMSP detection, MeRFPCR showed significantly lower variability in technical replicates. Figure 2 B).

[0077] MeRFPCR simulated a methylation gradient of 10-100% by mixing 100% and 0% methylated DNA in different proportions. Within the set methylation gradient range, the detection results showed an excellent linear relationship with the expected ratio (R0). 2 =0.991)( Figure 2 (C) demonstrates the method's accurate quantitative capability.

[0078] 1. Robustness verification of the detection of fragmented DNA samples (Figure 2D) To evaluate the quantitative accuracy of this invention in highly degraded samples, a fragmented DNA model was constructed using an ultrasonic shearing method: fully methylated and fully unmethylated human genomic DNA were mixed in a 1:1 molar ratio (to construct a template with a theoretical methylation level of 50%) and diluted to 20 ng / μL; the DNA samples were physically sheared using a Covaris M220 focused ultrasound resonator: at 4°C, sonication was performed for 30 seconds, followed by a 30-second pause, and this cycle was repeated for a total of 10 minutes. After mixing, the samples were vortexed and briefly centrifuged, and this process was repeated for a total time of 60 minutes (µs60) or 90 minutes (µs90) to achieve different degrees of fragmentation in DNA fragment size; agarose gel electrophoresis was used to detect and confirm that the sheared DNA fragments were enriched at approximately 100 bp; 10 ng of the above fragmented DNA and an unsheared intact DNA control group were taken and subjected to bisulfite conversion in parallel; the MeRFPCR of this invention and the traditional qMSP method based on external references (using exogenous ALU-C4 as a reference) were used for detection, respectively. The results (Figure 2D) show that in cases of severe DNA fragmentation (<100 bp), the traditional qMSP method exhibits quantitative bias due to the inconsistent amplification efficiency between the signal target and the exogenous reference segment; while the MeRFPCR method of this invention shows consistent methylation percentages in both intact and fragmented DNA, demonstrating robustness against degradation.

[0079] 2. Detection stability verification under low initial template (Figure 2E) To verify the detection limit and stability of this invention in low-abundance and trace clinical samples: DNA templates with known methylation levels after bisulfite conversion were serially diluted using low-EDTA TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) to construct a series of dilution gradients with single PCR reaction inputs of 5.0 ng, 1.0 ng, 0.5 ng, and 0.25 ng. For each dilution gradient, six technical replicates of MeRFPCR amplification were performed. The absolute methylation percentage and its standard deviation at different input amounts were statistically analyzed. The results (Figure 2E) showed that when the DNA template input was as low as 0.25 ng, the detection system of this invention could still output a stable absolute methylation percentage, and the coefficient of variation between technical replicates was less than 5%, confirming that this invention has extremely high sensitivity and stability in trace sample detection.

[0080] 3. Quantitative validation of the biological dynamics of demethylated drug treatment (Figure 2F) To demonstrate the responsiveness of this invention in the detection of dynamic epigenetic modifications in biology: the human colorectal cancer cell line SW480 was cultured in DMEM medium containing 10% fetal bovine serum and passaged in a 37°C, 5% CO2 incubator; at a growth rate of 1×10⁻⁶... 5 Cells were seeded at a density of cells / well in 6-well plates. After 24 hours, the experimental group was treated with the demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) to a final concentration of 5 μM for 72 hours, during which the culture medium containing the same concentration of the drug was replaced every 24 hours. The control group was treated with an equal volume of DMSO solvent. Cells were collected, and genomic DNA was extracted using the QIAamp DNA Mini Kit. 50 ng of the extracted genomic DNA was converted to bisulfite. The transformed DNA was quantitatively analyzed using the MeRFPCR system for the SEPT9, KAZN, and DUSP3 genes constructed in this invention. The results showed ( Figure 2 F): After treatment with 5-aza-dC, the absolute methylation levels of the above three loci in SW480 cells were significantly downregulated (P<0.001). This invention successfully and sensitively captured this biological dynamic methylation modification process. Example 2: Construction of a MeRFPCR detection system for KAZN gene methylation

[0081] The following oligonucleotide sequence was designed and synthesized targeting the human KAZN gene (cg06887407): (1) KAZN-forward primer: 5'-TTGGAAGAGGAGTTATAATGTAGGATGTT-3' (SEQ IDNO.5) (2) KAZN-reverse primer: 5'-CCTCTTAAATCCCTTCAAACCTTTTC-3' (SEQ ID NO.6) (3) KAZN-Met-probe: 5'-FAM-ATATTAAGGTAGGTAAGCGTGGTG-MGB-NFQ-3' (SEQ IDNO.7) (4) KAZN-Ctrl-probe: 5'-ROX-CATCCTACATTATAACTCCTCTTC-MGB-NFQ-3' (SEQID NO.8) The amplicon size is 96 bp.

[0082] Using the same method as in Example 1, the system also exhibited excellent specificity and quantitative linearity. Example 3: Construction of a MeRFPCR detection system for DUSP3 gene methylation

[0083] The following oligonucleotide sequence was designed and synthesized targeting the human DUSP3 gene (cg16747321): (1) DUSP3-forward primer: 5'-TTTGTAGGTGGTGGTTTTTGGATAAT-3' (SEQ ID NO.9) (2) DUSP3-reverse primer: 5'-TCTCAATCTTAAAACAATATACCTTTCCAC-3' (SEQ IDNO.10) (3) DUSP3-Met-probe: 5'-FAM-GAGTGGTAGGGATATAGACGAGT-MGB-NFQ-3' (SEQ IDNO.11) (4) DUSP3-Ctrl-probe: 5'-ROX-AAATTATCCAAAAACCACCACC-MGB-NFQ-3' (SEQ IDNO.12) The amplicon size is 84 bp.

[0084] Using the same method as in Example 1, the system also exhibited excellent specificity and quantitative linearity. Example 4: Application of the present invention in clinical samples

[0085] Tumor tissue and paired adjacent normal mucosal tissue (>5 cm from the tumor margin) were collected from 10 patients with pathologically confirmed colorectal cancer. Genomic DNA was extracted using the QIAamp DNA Mini Kit. Preoperative peripheral venous blood (5 mL) was collected from each of the 10 colorectal cancer patients in EDTA anticoagulant tubes. Plasma was separated over 2 hours using a two-step centrifugation method: first, centrifugation at 2,000 × g at 4°C for 10 minutes to remove blood cells, collecting the supernatant; then, centrifugation again at 16,000 × g at 4°C for 10 minutes to completely remove cell debris. Using the QIAamp Circulating Nucleic Acid Kit, all cfDNA was extracted from 2 mL of plasma and eluted in 30 μL of elution buffer.

[0086] For the target sites located in the SEPT9 CpG island and the DUSP3 CpG-deficient region, the SEPT9 gene MeRFPCR detection system constructed in Example 1 and qMSP were used for detection, respectively. The absolute methylation levels of the tumor group and the normal group were analyzed using a two-tailed paired t-test with GraphPad Prism software, and a p-value < 0.05 was considered statistically significant.

[0087] The results show: (1) In tissue samples, the methylation levels in tumor tissue detected by MeRFPCR were significantly higher than those in adjacent normal tissue, and the quantitative results were highly consistent with the gold standard pyrosequencing method (R... 2 =0.964)( Figure 3 (A, 3B). (CD) Site-specific discrimination ability. The methylation levels of paired tumor and normal tissues at CpG-rich SEPT9 sites and CpG-sparse DUSP3 sites were analyzed. MeRFPCR stably distinguished tumors from normal tissues at all CpG density sites (C); while qMSP showed instability in its discrimination ability at individual CpG sites (D).

[0088] (2) In cfDNA samples, MeRFPCR successfully detected reliable methylation signals in 6 out of 10 cases, consistent with reported detection rates, and provided reliable absolute quantification values. However, the detection value of qMSP was below 3% in all samples, below the generally accepted clinical positive threshold (methylation level ≥10% is considered positive). Figure 3 E).

[0089] These results fully demonstrate that the present invention has high accuracy and high reliability when processing real and complex clinical samples.

[0090] References: 1 Schübeler, D. Function and information content of DNA methylation. Nature 517, 321-326, doi:10.1038 / nature14192 (2015).

[0091] 2 Davalos, V. & Esteller, M. Cancer epigenetics in clinical practice. CA Cancer J Clin 73, 376-424, doi:10.3322 / caac.21765 (2023).

[0092] 3 Zhang, L. & Li, J. Unlocking the secrets: the power of methylation-based cfDNA detection of tissue damage in organ systems. Clinical epigenetics15, 168, doi:10.1186 / s13148-023-01585-8 (2023).

[0093] 4 Mavrommatis, C. et al. An unbiased comparison of 14 epigeneticclocks in relation to 174 incident disease outcomes. Nature communications16, 11164, doi:10.1038 / s41467-025-66106-y (2025).

[0094] 5 Horvath, S. DNA methylation age of human tissues and cell types.Genome biology 14, R115, doi:10.1186 / gb-2013-14-10-r115 (2013).

[0095] 6 Zhang, Y. et al. Deep oncopanel sequencing reveals within blockposition-dependent quality degradation in FFPE processed samples. Genomebiology 23, 141, doi:10.1186 / s13059-022-02709-8 (2022).

[0096] 7 Steiert, T. A. et al. A critical spotlight on the paradigms ofFFPE-DNA sequencing. Nucleic Acids Res 51, 7143-7162, doi:10.1093 / nar / gkad519(2023).

[0097] 8 Markus, H. et al. Analysis of recurrently protected genomic regionsin cell-free DNA found in urine. Sci Transl Med 13, doi:10.1126 / scitranslmed.aaz3088 (2021).

[0098] 9 Guo, D. et al. The genomic and epigenomic abnormalities of plasmacfDNA as liquid biopsy biomarkers to detect hepatocellular carcinoma: amulticenter cohort study. Journal of hematology & oncology 18, 94, doi:10.1186 / s13045-025-01747-6 (2025).

[0099] 10 Wang, T. et al. A multiplex blood-based assay targeting DNAmethylation in PBMCs enables early detection of breast cancer. Naturecommunications 14, 4724, doi:10.1038 / s41467-023-40389-5 (2023).

[0100] 11 Weisenberger, D. J. et al. Analysis of repetitive element DNAmethylation by MethyLight. Nucleic Acids Res 33, 6823-6836, doi:10.1093 / nar / gki987 (2005).

[0101] 12 Shiovitz, S. et al. CpG island methylator phenotype is associated with response to adjuvant irinotecan-based therapy for stage III coloncancer. Gastroenterology 147, 637-645, doi:10.1053 / j.gastro.2014.05.009(2014).

[0102] 13 Grunau, C., Clark, SJ & Rosenthal, A. Bisulfite genomicsequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 29, E65-65, doi:10.1093 / nar / 29.13.e65 (2001).

[0103] 14 Steele, CD et al. Signatures of copy number alterations inhuman cancer. Nature 606, 984-991, doi:10.1038 / s41586-022-04738-6 (2022).

[0104] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A reference-free absolute quantification kit for DNA methylation, characterized in that, include: Cytosine methylation conversion reagent is used to convert unmethylated cytosine C in the DNA sample into uracil U, while 5-methylcytosine remains unchanged, causing the originally complementary DNA double strands to dissociate, and the sense and antisense strand sequences at the same locus to become no longer complementary; CpG-free primer pairs, wherein the binding sites of the primer pairs are located in the CpG-free regions flanking the CpG site to be tested, can indiscriminately amplify DNA templates from methylated and unmethylated alleles, and the amplification products of the primer pairs contain both transformed sense and antisense strand fragments. A methylation-specific fluorescent probe, wherein the methylation-specific fluorescent probe specifically targets and binds to the positive strand amplification product containing CpG dinucleotides, thereby producing a fluorescence change; A template total amount specific fluorescent probe, which specifically targets and binds to sequences within the antisense strand amplification product that do not contain CpG sites, and then produces a fluorescence change.

2. The DNA methylation absolute quantitative kit according to claim 1, characterized in that, The methylation-specific fluorescent probe and the template totality-specific fluorescent probe have different fluorescent groups.

3. The DNA methylation absolute quantitative kit according to claim 2, characterized in that, The methylation-specific fluorescent probe and the template total amount-specific fluorescent probe are TaqMan hydrolysis probes, and / or the fluorescent groups are selected from FAM, VIC, HEX, TET, JOE, ROX, Cy3, Cy5, and Cy5.

5.

4. The DNA methylation absolute quantitative kit according to claim 1, characterized in that, The CpG-free primer pairs and probes include: Primer pairs and probes used for quantifying SEPT9 gene methylation, (1) SEPT9-forward primer: 5'-GATTYGTTGTTTATTAGTTATTATGT-3' (2) SEPT9-reverse primer: 5'-AAATAATCCCATCCAACTA-3' (3) SEPT9-methylation specific fluorescent probe: 5'-TAACCGCGAAATCCGA-3' (4) SEPT9-template total amount specific fluorescent probe: 5'-GTTGGATGGGATTATT-3'; or Primer pairs and probes used for quantifying KAZN gene methylation, (1) KAZN-forward primer: 5'-TTGGAAGAGGAGTTATAATGTAGGATGTT-3' (2) KAZN-reverse primer: 5'-CCTCTTAAATCCCTTCAAACCTTTTC-3' (3) KAZN-methylation specific fluorescent probe: 5'-ATATTAAGGTAGGTAAGCGTGGTG-3' (4) KAZN-template total amount specific fluorescent probe 5'-CATCCTACATTATAACTCCTCTTC-3'; or Primer pairs and probes used for quantifying DUSP3 gene methylation, (1) DUSP3-forward primer: 5'-TTTGTAGGTGGTGGTTTTTGGATAAT-3' (2) DUSP3-reverse primer: 5'-TCTCAATCTTAAAACAATATACCTTTCCAC-3' (3) DUSP3-methylation specific fluorescent probe: 5'-GAGTGGTAGGGATATAGACGAGT-3' (4) DUSP3-Template total specific fluorescent probe: 5'-AAATTATCCAAAAACCACCACC-3'.

5. A reference-free absolute quantification method for DNA methylation, characterized in that, Includes the following steps: Take the genomic DNA sample to be tested, convert unmethylated cytosine (C) into uracil (U), while 5-methylcytosine (5mC) remains unchanged, causing the originally complementary DNA double strands to dissociate, and the sense and antisense strand sequences at the same locus become no longer complementary, thus obtaining the transformed DNA template; Amplification and Detection: Amplification primers, methylation-specific fluorescent probes, template-total-specific fluorescent probes, the DNA template, and other PCR reaction components are mixed in a single reaction tube. Amplification and signal acquisition are performed on a real-time quantitative PCR instrument. The absolute methylation level is determined based on the Ct values ​​of the fluorescence of the methylation-specific fluorescent probes and the template-total-specific fluorescent probes. The amplification primers are CpG-free primer pairs with binding sites located in the CpG-free regions flanking the target CpG site. They can indiscriminately amplify DNA templates from methylated and unmethylated alleles. The amplification products of the primer pairs simultaneously contain transformed sense and antisense strand fragments. The methylation-specific fluorescent probes specifically target and bind to the sense strand amplification product containing CpG dinucleotides, producing a fluorescence change. The template-total-specific fluorescent probes specifically target and bind to sequences within the antisense strand amplification product that do not contain CpG sites, producing a fluorescence change. The amount of methylation in the genomic DNA sample to be tested is determined based on the fluorescence changes during the amplification process.

6. The absolute quantification method for DNA methylation according to claim 4, characterized in that, The methylation-specific fluorescent probe and the template totality-specific fluorescent probe have different fluorescent groups.

7. The method for absolute quantification of DNA methylation according to claim 4 or 5, characterized in that, The methylation-specific fluorescent probe and the template total amount-specific fluorescent probe are TaqMan hydrolysis probes, and / or the fluorescent groups are selected from FAM, VIC, HEX, TET, JOE, ROX, Cy3, Cy5, and Cy5.

5.

8. The method for absolute quantification of DNA methylation according to claim 5, characterized in that, Based on the CT of the methylation-specific fluorescent probe meth CT values ​​of specific fluorescent probes relative to total template amount input The value, according to the formula: ΔCT = CT meth -CT input Absolute methylation ratio = 2 -ΔCT The absolute methylation level of the DNA to be tested is calculated by multiplying by 100%.

9. The application of the DNA methylation absolute quantification kit according to any one of claims 1 to 4 in quantifying the absolute amount of DNA methylation in a sample.

10. The application according to claim 9, characterized in that, The samples were selected from solid tumor samples, including but not limited to colorectal cancer, gastric cancer, liver cancer, lung cancer, breast cancer, and prostate cancer.

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