Qpcr-based multi-gene methylation detection system for peripheral blood free DNA

Abstract

Provided is a qPCR-based multi-gene methylation detection system for peripheral blood free DNA. Under the conditions of satisfying the nucleic acid extraction efficiency of peripheral blood free DNA and reducing the occurrence of large fragment contamination during extraction, the co-immunoprecipitation technology is further used to improve the antibody enrichment and purification effects of methylated target nucleic acids, and an appropriate amount of a blocking agent is added to effectively reduce non-methylation enrichment. Methylated target signals are amplified indiscriminately by means of pre-amplification, so that the fluorescence quantitative PCR technology can also realize synchronous detection of a large number of target genes. The system has good openness, can expand the number of detection genes according to actual needs, and can meet the requirements of simultaneously carrying out combined detection of a plurality of target genes to obtain indicators of multiple cancer types and multiple types of diseases.

Description

qPCR-based detection system for multiple gene methylation of cell-free DNA in peripheral blood Technical Field

[0001] This invention relates to the fields of molecular biology and gene detection, and more specifically, to a qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system. Background Technology

[0002] In recent years, liquid biopsy technology, which uses non-solid biological tissues as samples and is based on molecular diagnostic technologies such as PCR and NGS, has developed rapidly and has broad application prospects in many fields, including early cancer screening. Liquid biopsy is a non-invasive blood test that can determine the gene mutation status of tumors by detecting circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) fragments, and exosomes released into the bloodstream from tumors or their metastases.

[0003] Abnormal ctDNA methylation in cell-free DNA of peripheral blood is considered one of the important epigenetic modifications driving cancer development, often occurring in the early stages of cancer. ctDNA methylation is one of the most well-established features used in early cancer screening.

[0004] Currently, most screening methods can only target specific cancer types, failing to meet the needs of users who require screening for multiple cancer types in a single test. Furthermore, there is a lack of mature multi-cancer combined detection technologies in China for pan-cancer testing, and the detection performance of pan-cancer early screening has not yet met clinical requirements. At the same time, the development and detection costs of multi-cancer systems are very high due to the widespread use of high-throughput methylation sequencing or multi-omics sequencing technologies.

[0005] DNA methylation detection methods include bisulfite conversion sequencing (BS-Seq), MeDIP-Seq, RRBS-Seq, WGBS, MBD-Seq, and SMRT. Among them, whole genome bisulfite sequencing (WGBS) is considered the "gold standard" for DNA methylation research. It works by treating DNA with bisulfite, causing unmethylated C to become U, which then becomes T during subsequent PCR and sequencing. Methylated C is unaffected by bisulfite. Subsequent sequencing of the treated genomic DNA library allows for analysis of methylation across the entire genome, providing single-base resolution and precise assessment of the methylation level of individual C bases. It offers broad coverage but is costly.

[0006] qPCR detection platforms based on bisulfite treatment, taking the detection of Septin9 methylation in blood as an example, have a major limitation: relatively low sensitivity in identifying colorectal cancer and precancerous lesions (adenomas), with sensitivity for advanced adenomas ranging from only 7.9% to 38.7%. Increasing the number of methylated genes can improve sensitivity for colorectal cancer, but it still cannot meet the actual clinical needs. Since the amount of blood sample collected in a single clinical session is generally between 3 mL and 20 mL, existing qPCR technology based on bisulfite methylation analysis can only simultaneously detect a limited number (generally no more than 10) of methylated genes in a single ctDNA sample. Currently, the product approved by the National Medical Products Administration for detecting the most genes in a single sample is a combined detection reagent for human Septin9, BCAT1, IKZF1, BCAN, and VAV3 gene methylation, which detects 6 methylated regions of 5 genes by extracting free DNA from 3 mL of plasma. However, this only meets the requirements for detecting one type of cancer and cannot meet the requirements for detecting multiple cancer types and multiple genes.

[0007] Although some teams have attempted to pre-amplify DNA before qPCR based on bisulfite treatment, mainstream methods have significant limitations. A common approach involves pre-mixing primer pools for multiple known target genes after bisulfite treatment, performing 10-15 rounds of pre-amplification, and then performing target fluorescence amplification. This pre-amplification strategy has significant drawbacks: it doesn't effectively increase signal abundance, and the lower signal after bisulfite treatment leads to greater reproducibility bias in each experiment, with pre-amplification exacerbating signal shifts. This strategy is limited to pre-amplification of a small number of target genes and cannot be extended to a wider range of gene detection. In high-throughput sequencing platforms using bisulfite-treated sequencing methods, the base conversion required for library construction and amplification necessitates two library construction strategies: end repair and adapter addition, performed before bisulfite treatment (for double-stranded DNA) and after treatment (for single-stranded DNA). This introduces further bias during library construction. The end-repair process in double-stranded library construction leads to distorted methylation signals, and subsequent bisulfite conversion causes even greater DNA loss; therefore, it is not recommended for methylation NGS library construction. Single-stranded DNA library construction, using single-stranded DNA as a template, is a common method for methylation NGS library construction. However, this method suffers from insufficient single-stranded DNA ligation efficiency and the need to introduce other sequences, leading to biases in subsequent data analysis. Applying the same strategy for pre-amplification on the qPCR platform significantly increases the difficulty of pre-amplification design and reduces experimental reproducibility, further amplifying signal bias. While whole-genome MeDIP-Seq has a library amplification module, there is no evidence to prove that the pre-amplification strategy of this module can be directly transferred to the qPCR platform; uncertainty remains. Achieving indiscriminate amplification of methylation signals on the qPCR platform will also be a technical challenge.

[0008] Therefore, given the limited blood volume, how to avoid excessive loss of trace early molecular signals, improve the extraction and purification efficiency of target nucleic acids in peripheral blood cell-free DNA samples, and simultaneously achieve sensitive detection of methylation markers of multiple cancer-specific genes at low cost, and establish a more sensitive, effective, and universally accessible multi-gene detection system through module assembly optimization, are urgent problems that need to be solved in carrying out early cancer screening and early diagnosis of chronic diseases. Summary of the Invention

[0009] In a first aspect of the present invention, a qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system is provided, comprising:

[0010] 1) cfDNA extraction unit: used to extract cfDNA from plasma samples; preferably, when the yield of double-stranded DNA of the extracted cfDNA product is not less than 0.5 ng per mL of plasma, it enters the cfDNA methylation enrichment and purification unit.

[0011] 2) cfDNA methylation enrichment and purification unit: methylated DNA is enriched and purified by antibody using immunoprecipitation, while an inhibitor is added to reduce non-methylation enrichment, resulting in a methylated DNA enrichment library; preferably, when the recovery rate of methylated quality control products after enrichment is not less than 65% and the recovery rate of non-methylated quality control products is not higher than 1%, it enters the qPCR detection unit.

[0012] 3) qPCR detection unit: used to detect methylated DNA and obtain qPCR detection results.

[0013] Preferably, the method for extracting cfDNA from a plasma sample in the cfDNA extraction unit includes the following steps:

[0014] S11. Place 2 mL of plasma into a centrifuge tube containing 100 μL to 150 μL of proteinase K, then add 100 μL to 150 μL of lysis buffer 1, mix well, and react at 15-60℃ for 10-60 minutes to obtain a mixture.

[0015] S12. Add 4 mL to 8 mL of lysis buffer 2 and 30 μL to 50 μL of magnetic beads to the mixture obtained in S11. Mix well at room temperature and react for 10 to 60 minutes. After centrifugation, transfer to a magnetic rack to adsorb the magnetic beads and discard the solution.

[0016] S13. Add 0.5 mL to 0.8 mL of washing solution 1, mix well, centrifuge, place on a magnetic rack for adsorption, and discard the solution;

[0017] S14. Add 0.5 mL to 0.8 mL of washing solution 2, mix well, transfer to a magnetic rack for adsorption, and discard the solution;

[0018] S15. After centrifugation, remove all residual liquid; allow the magnetic beads to dry in air for 2-5 minutes;

[0019] S16. Add 50μL to 100μL of elution buffer 1, dissolve the DNA, transfer it to a magnetic rack for adsorption, and transfer the supernatant containing cfDNA to a new centrifuge tube to obtain the cfDNA extraction product.

[0020] Preferably, the lysis buffer 1 contains 0.05% to 1% sodium citrate dodecahydrate and 1% to 5% SDS;

[0021] The lysis buffer 2 contains 10%–20% guanidine isothiocyanate, 1%–5% NP40, 3% potassium chloride, 2% sodium chloride, 0.1%–0.3% sodium dihydrogen phosphate, 0.5%–1% tris(hydroxymethyl)aminomethane, 0.05%–0.2% sodium tartrate, and 25% isopropanol.

[0022] Washing solution 1 contains 20%–25% guanidine isothiocyanate, 0.2%–1% SDS, and 30%–50% anhydrous ethanol;

[0023] Washing solution 2 contains 0.1% to 0.8% tris(hydroxymethyl)aminomethane, 0.5% to 1% Trinton X-100, and 70% to 80% anhydrous ethanol;

[0024] Eluent 1 contains 0.05% to 0.2% tris(hydroxymethyl)aminomethane.

[0025] More preferably, the lysis buffer 1 consists of 0.07% sodium citrate dodecahydrate and 4% SDS;

[0026] The components of lysis buffer 2 are 20% guanidine isothiocyanate, 3% NP40, 3% potassium chloride, 2% sodium chloride, 4% sodium lauroyl amino acid, 0.1% sodium dihydrogen phosphate, 0.7% tris(hydroxymethyl)aminomethane, 0.05% sodium tartrate and 25% isopropanol.

[0027] The washing solution 1 also contains 0.2% to 0.5% tris(hydroxymethyl)aminomethane and 1.5% to 5% potassium chloride.

[0028] Preferably, in the cfDNA extraction unit, the magnetic beads are amino-silylated magnetic beads.

[0029] Preferably, the method for enriching methylated fragments in human peripheral blood cell-free DNA in the cfDNA methylation enrichment and purification unit includes the following steps:

[0030] S21. Add an inhibitor to the sample to be tested to reduce the enrichment of unmethylated substances, and obtain a sample with the inhibitor added;

[0031] S22. Mix the immunoprecipitation buffer and the sample with the added blocking agent, incubate at 90-95℃ for 5-20 min, and then cool rapidly to obtain the mixture;

[0032] S23. Add the antibody and magnetic beads to the mixture obtained in step S2 and incubate at 2-8°C for 4-16 hours;

[0033] S24. After brief centrifugation, place the mixture on a pre-cooled magnetic rack and discard the supernatant; wash the magnetic beads with washing solution.

[0034] S25. Elute DNA with elution buffer at 50-60℃ for 15 min, or 95-100℃ for 15 min.

[0035] Preferably, in step S21, the blocking agent is λDNA or human fecal microbial genomic DNA or other non-human methylated genomic DNA.

[0036] Preferably, in step S21, when the blocking agent is λDNA, the mass ratio of methylated DNA to unmethylated DNA is 1:1; when the blocking agent is human fecal microbial genomic DNA, the methylation rate of human fecal microbial genomic DNA is 2.0% to 5.0%.

[0037] Preferably, in the sample with added blocking agent, the mass ratio of cfDNA to blocking agent DNA is 1:10-1:40, wherein the concentration of cfDNA is not less than 0.01 ng / μL.

[0038] Preferably, in step S22, the volume ratio of the immunoprecipitation buffer to the sample with added blocking agent during mixing is (30-35) μL: (55-60) μL.

[0039] Preferably, in step S22, the immunoprecipitation buffer includes 0.1-0.2M sodium phosphate solution, 1-2M sodium chloride solution, and 1%-5% Triton X-100.

[0040] Preferably, step S23 further includes a cleaning step for the magnetic beads and a dilution step for the antibody:

[0041] Cleaning steps for magnetic beads: Take 2-6 μL of 10 mg / mL magnetic bead stock solution and wash the magnetic beads with 10-fold diluted immunoprecipitation buffer. Wash at least twice. For the last wash, resuspend the magnetic beads with 20 μL of 10-fold diluted immunoprecipitation buffer to obtain a magnetic bead suspension with a concentration of 1-3 mg / mL.

[0042] Antibody dilution procedure: Dilute the 1 mg / mL 5-methylcytosine antibody 10-100 times with immunoprecipitation buffer to obtain a solution with an antibody concentration of 0.01-0.1 mg / mL.

[0043] Preferably, in step S23, the antibody, magnetic beads, and the mixture obtained in step S22 are mixed in the following volume ratio: 1:1:(15-20).

[0044] Preferably, in the cfDNA methylation enrichment and purification unit, the magnetic beads are IgG magnetic beads.

[0045] Preferably, in the cfDNA methylation enrichment and purification unit, the antibody is a 5-methylcytosine antibody.

[0046] Preferably, in step S24, the washing solution includes a 10-fold diluted immunoprecipitation buffer and an immunoprecipitation buffer with a high sodium chloride concentration; the immunoprecipitation buffer with a high sodium chloride concentration consists of 0.01-0.02M sodium phosphate solution, 1-4M sodium chloride solution, and 0.1%-0.5% Triton X-100.

[0047] Preferably, in step S25, the elution buffer 2 includes 0.01-0.1M Tris-HCl, 0.01-0.1M EDTA, 0.001-0.1% SDS, and 0.1-1 mg / mL proteinase K.

[0048] Preferably, in the qPCR detection unit, primers and probes are designed for target-specific differentially methylated regions, wherein the differentially methylated regions cover no less than 4 CpG sites.

[0049] Preferably, in the qPCR detection unit, the qPCR reaction uses TSH2B as an internal reference gene, with the chromosome position being: chr6:25726855-25727020.

[0050] Preferably, the internal reference gene also includes GAPDH, with the Hg38 chromosomal coordinates being: chr12: 6534476-6534539.

[0051] Preferably, the qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system further includes:

[0052] 4) Algorithm Analysis Unit: Used to analyze qPCR test results, including: calculating ΔCT by subtracting the CT value of the internal reference gene from the CT value of the target gene; combining the ΔCT values ​​of single or multiple genes, calculating the logistic score of each sample using AI models such as Logistic Regression or Random Forest; selecting an appropriate cut-off value based on different logistic scores; judging a sample as positive when its score is higher than the cut-off value, and judging a sample as negative when its score is lower than the cut-off value.

[0053] And / or, 5) Pre-amplification unit: used to perform pre-amplification reaction on the enriched cfDNA product to obtain a methylated DNA enriched library with indiscriminate amplification; preferably, when the recovery rate of the enriched methylated quality control is not less than 65% and the recovery rate of the non-methylated quality control is not higher than 1%, it enters the pre-amplification unit first and then enters the qPCR detection unit.

[0054] The pre-amplification includes: ligating adapters into the cfDNA extracted by the cfDNA extraction unit; and amplifying the methylated DNA enriched library obtained by the cfDNA methylation enrichment and purification unit.

[0055] In a second aspect, the present invention provides a kit for extracting and enriching methylated fragments from cell-free human peripheral blood DNA, comprising: the lysis buffer 1, lysis buffer 2, washing buffer 1, washing buffer 2, and elution buffer 1 described above, as well as the immunoprecipitation buffer described above, an immunoprecipitation buffer diluted tenfold with a high sodium chloride concentration, and elution buffer 2. Preferably, it further comprises the magnetic beads described above, 5mC antibody, immunomagnetic beads, and blocking agent.

[0056] The present invention provides three aspects of the qPCR-based peripheral blood cell-free DNA multigene methylation detection system, a method for extracting cfDNA from plasma samples in the system, a method for enriching methylated fragments in human peripheral blood cell-free DNA in the system, and the applications of the lysis buffer 1, lysis buffer 2, washing buffer 1, washing buffer 2 and elution buffer 1, as well as the 10-fold diluted immunoprecipitation buffer, immunoprecipitation buffer, 10-fold diluted immunoprecipitation buffer with high sodium chloride concentration and elution buffer 2 in DNA methylation level detection, screening and assessment of disease risk.

[0057] This invention establishes a qPCR detection system for ultra-multi-gene methylation of peripheral blood cell-free DNA using a mild nucleic acid processing technique—non-bisulfite treatment—methodized DNA immunoprecipitation enrichment (MeDIP). This system, while maintaining high extraction efficiency and minimizing large-fragment contamination during extraction, further utilizes immunoprecipitation to enhance the enrichment and purification of methylated target nucleic acids. Through the indiscriminate amplification of methylated target signals by a pre-amplification unit, simultaneous detection of ultra-multi-target genes using quantitative real-time PCR is achieved.

[0058] The system has good openness and can expand the number of genes to be detected according to actual needs, so as to meet the needs of simultaneous joint detection of multiple target genes to obtain indicators of multiple cancer types and multiple disease types.

[0059] Compared to traditional bisulfite treatment, the highly efficient cfDNA extraction and methylation enrichment purification technology avoids excessive loss of trace signals, protects the integrity of early tumor development signals, and can become a better solution for methylation analysis. It significantly reduces damage to peripheral blood cell-free DNA, improves the sensitivity of methylation signals, and enhances the sensitivity of target gene detection.

[0060] The technology system based on real-time fluorescence PCR avoids the high cost of high-throughput sequencing platforms, is simple to operate, has extremely high clinical applicability, and can provide valuable methylation evidence for early screening of single or multiple tumors, early diagnosis of single or multiple diseases, and the development of chronic diseases. Attached Figure Description

[0061] Figure 1 shows the flowchart of this system.

[0062] Figure 2 shows the difference in the area under the main peak of the cfDNA fragment distribution detected by capillary electrophoresis in Test Example 1.

[0063] Figure 3 shows the difference in the position of the main peak of cfDNA fragment distribution detected by capillary electrophoresis in Test Example 1.

[0064] Figure 4 shows the differences in large fragment interference peaks in the distribution of cfDNA fragments detected by capillary electrophoresis in Test Example 1.

[0065] Figure 5 shows the comparison results of the binding efficiency of methylated genes (meDNA) and unmethylated genes (unDNA) in Test Example 2.

[0066] Figure 6 shows the evaluation of the recovery efficiency of unmethylated unDNA under the condition of adding the blocking agent in Test Example 2.

[0067] Figure 7 shows the recovery efficiency evaluation under different detection procedures, including (a) methylated meDNA recovery efficiency evaluation and (b) unmethylated unDNA recovery efficiency evaluation.

[0068] Figure 8 shows the experimental procedure for test example 5.

[0069] Figure 9 shows the evaluation results of the colorectal cancer detection performance of Test Example 5, including (a) the distribution of the combined scores of colorectal cancer (positive) and normal samples (negative); and (b) the receiver operating characteristic (ROC) curve.

[0070] Figure 10 shows the evaluation results of the gastric cancer detection performance of Test Example 5, including (a) the distribution of the combined scores of gastric cancer (positive) and normal samples (negative); and (b) the receiver operating characteristic (ROC) curve.

[0071] Figure 11 shows the evaluation results of the esophageal cancer detection performance of Test Example 5, including (a) the distribution of the combined scores of esophageal cancer (positive) and normal samples (negative); and (b) the receiver operating characteristic (ROC) curve.

[0072] Figure 12 shows the evaluation results of the liver cancer detection performance of Test Example 5, including (a) the distribution of the combined scores of liver cancer (positive) and normal samples (negative); and (b) the receiver operating characteristic (ROC) curve.

[0073] Figure 13 shows the evaluation results of the pancreatic cancer detection performance of Test Example 5, including (a) the distribution of the combined scores of pancreatic cancer (positive) and normal samples (negative); and (b) the receiver operating characteristic (ROC) curve.

[0074] Figure 14 shows the evaluation results of lung cancer detection performance in Test Example 5, including (a) the distribution of combined scores for lung cancer (positive) and normal samples (negative); and (b) the receiver operating characteristic (ROC) curve.

[0075] Figure 15 shows the evaluation results of the lowest detection limit of test example 6, where (a) is the amplification curve of the concentration gradient reaction of the methylated gene SALL1; and (b) is the amplification curve of the concentration gradient reaction of the methylated gene ITGA4.

[0076] Figure 16 shows the tissue tracing results for nine types of cancer. Detailed Implementation

[0077] This invention, based on non-bisulfite technology and multi-gene qPCR detection, improves the nucleic acid extraction rate and significantly reduces large-fragment contamination during the extraction process by optimizing the reaction conditions for nucleic acid extraction and purification of peripheral blood cell-free DNA (cfDNA). Furthermore, by screening for highly efficient 5-methylcytosine antibodies for methylation immunoprecipitation and adjusting the reaction conditions of methylation antigen-antibody, the enrichment and purification effect of methylated target nucleic acids is further enhanced, increasing the recovery rate of methylated fragments. The addition of appropriate blocking agents effectively reduces non-specific enrichment reactions. Compared with traditional bisulfite treatment, this invention achieves more efficient cfDNA extraction and methylation enrichment and purification, avoiding excessive loss of trace signals and protecting the integrity of early tumor development signals.

[0078] Furthermore, by adding library adapters to peripheral blood cell-free DNA before methylation treatment, followed by A-terminal repair, and then enriching methylated fragments using optimized immunoprecipitation technology, the products were indiscriminately amplified and purified. Multi-target gene probe primer pairs were designed using non-CU transformation principles, enabling real-time quantitative PCR (qPCR) to simultaneously detect methylation levels of multiple genes with high sensitivity and controllable costs down to the hundred-yuan level. This avoids the high costs of high-throughput sequencing, maintains high consistency with clinical results, and has extremely high clinical applicability.

[0079] A qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system includes:

[0080] 1) cfDNA extraction unit: Used to extract cfDNA from plasma samples. When the extracted cfDNA meets the following criteria, it proceeds to the cfDNA methylation enrichment and purification unit:

[0081] The Qubit nucleic acid concentration analyzer detected that the yield of double-stranded DNA in the extracted product was not less than 0.5 ng per mL of plasma;

[0082] 2) cfDNA methylation enrichment and purification unit: Methylated DNA is enriched and purified using immunoprecipitation to obtain a methylated DNA enriched library;

[0083] When the recovery rate of the methylated quality control (a commercially synthesized methylated sequence that is not from any model species and is used as an internal standard) is not less than 65% and the recovery rate of the unmethylated quality control (a commercially synthesized unmethylated sequence that is not from any model species and is used as an internal standard) is not higher than 1%, it is entered into the qPCR amplification unit.

[0084] The formula for calculating the recovery rate is: Recovery efficiency (%) = 2^(Ct) 10％input -Ct 样本 -3.32)×100.

[0085] During testing, 10% of the sample is aspirated as the 10% input sample, and the remaining sample is processed for further processing. Both are amplified together by qPCR. Ct10%input represents the ct value of the amplified 10% input sample; Ctsample represents the Ct value of the amplified sample.

[0086] 3) qPCR detection unit: used to detect methylated DNA; includes:

[0087] I. Design primer and probe combinations targeting differentially methylated regions;

[0088] II. Perform qPCR reaction to obtain qPCR detection results.

[0089] Preferably, each differentially methylated region covers no fewer than 4 CpG sites.

[0090] Preferably, the internal reference gene is TSH2B, with the Hg38 chromosomal coordinates at chr6:25726855-25727020; TSH2B is a human hypermethylated region used to assess the overall level of methylated regions in the sample after MeDIP enrichment.

[0091] Preferably, the internal reference gene also includes GAPDH, a human hypomethylated region used to assess the level of non-specific enrichment of samples after MeDIP. The Hg38 chromosomal coordinates are: chr12: 6534476-6534539.

[0092] The qPCR test results obtained by this system are applicable to a variety of analytical methods, thus providing a data analysis foundation for disease diagnosis and risk assessment. There are no restrictions on the analytical methods or software used; those skilled in the art can choose according to the specific circumstances.

[0093] Preferably, this system also includes:

[0094] 4) Algorithm Analysis Unit: Used to analyze qPCR test results, including: calculating ΔCT by subtracting the CT value of the internal reference gene from the CT value of the target gene; combining the ΔCT values ​​of single or multiple genes, calculating the logistic score of each sample through a logistic regression model; selecting an appropriate cut-off value based on different logistic scores; judging a sample as positive when its score is higher than the cut-off value, and judging a sample as negative when its score is lower than the cut-off value.

[0095] The formula for calculating the logistic score of each gene is: Logistic score = β0 + β1*X1 + β2*X2 + β3*X3 + ... + βn*Xn Gene score = elogistic score / (1 + elogistic score)

[0096] In the formula, β0 represents a constant, β1 represents the weight coefficient of each gene, and X represents each gene.

[0097] The above calculations and analyses can be performed using various computational tools found in commercial software. Alternatively, various AI modeling strategies other than logistic regression can be employed for analysis.

[0098] And / or, 5) Pre-amplification unit: used to amplify more genes; including:

[0099] I. Ligate the extracted cfDNA with a universal adapter;

[0100] II. Amplification of the methylated DNA enrichment library.

[0101] The timing of the connector connection step is crucial in the entire detection system. Adding the connector before MeDIP is more conducive to improving enrichment efficiency, and the library construction process does not affect the authenticity of the methylation signal.

[0102] The principle of this system is as follows: existing qPCR technology based on bisulfite methylation analysis can only simultaneously detect a limited number of methylated genes in a single ctDNA sample. The limiting factor is the low extraction efficiency of current plasma cell-free DNA extraction kits; even the best commercially available kits cannot meet the requirements for simultaneous qPCR detection of multiple genes. Furthermore, the transformed DNA obtained from the mainstream methylation method (bisulfite) of extracted cell-free DNA is predominantly single-stranded, with a mixture of double-stranded and fragmented sections, nicks, and uracil-state nucleotides. This process typically results in the loss of 90% of the DNA template, leaving a large amount of methylation information undetectable in subsequent processes. Simultaneously, incomplete or over-conversion occurs during base transformation, introducing human bias that is further amplified by subsequent PCR amplification, leading to inaccurate signals. The already limited number of cell-free DNA fragments in plasma samples become even more difficult to detect the methylation levels of multiple genes after bisulfite treatment.

[0103] Therefore, to achieve the goal of simultaneously detecting a large number (more than ten) genes using qPCR, the entire system needs to be improved. Firstly, the extraction efficiency of peripheral blood cell-free DNA (cfDNA) must reach a yield of at least 0.5 ng of double-stranded DNA per mL of plasma, meaning the extraction efficiency must be at least twice that of conventional extraction kits. Furthermore, human bias should be minimized to ensure the quality of the extracted product, making it more suitable for downstream enrichment, amplification, and detection procedures.

[0104] Secondly, based on the extracted products meeting the above standards, a methylation fragment recovery rate of over 65% can meet the requirements of qPCR multigene detection. However, the traditional bisulfite conversion method has a recovery rate of less than 10%, failing to meet this standard. Therefore, the system requires amplification of the extracted products via immunoprecipitation to achieve efficient methylation enrichment. Furthermore, the sequence changes after traditional bisulfite treatment, while the gene sequence remains unchanged after methylation immunoprecipitation enrichment. Therefore, pre-amplification can be performed to indiscriminately increase the abundance of methylation signals for subsequent multigene detection.

[0105] This system sets corresponding standards for cfDNA extraction, methylation enrichment and purification processes, aiming to avoid excessive loss of trace signals, protect the integrity of early tumor development signals, improve the sensitivity of methylation signals, enhance the sensitivity of target gene detection, and ultimately meet the needs of qPCR multi-gene detection.

[0106] Example 1

[0107] The specific operations for each unit in this system are as follows:

[0108] 1) In the cfDNA extraction unit, in order to achieve the standard that the yield of double-stranded DNA of the extracted product is not less than 0.5 ng per mL of plasma, the present invention has made systematic adjustments and combinations to the reagents such as lysis buffer, magnetic beads, and washing solution used in the extraction process, as well as the reaction conditions and operating steps.

[0109] In the process of peripheral blood cell-free DNA extraction, lysis buffer 1 (mainly composed of SDS components) is first used to denature the proteins in serum plasma, thereby improving the effect of lysis buffer 2 (mainly composed of guanidine isothiocyanate components) on the second digestion of the sample. At the same time, it also reduces the non-specific adsorption of protein macromolecules to magnetic beads, which is beneficial to improving the nucleic acid yield and purity.

[0110] To address the issue that different anticoagulants in blood collection tubes can have varying degrees of impact on plasma nucleic acid quality, this invention compares different chelating agents such as sodium tartrate, sodium gluconate, and EDTA at different concentrations. Preferably, adding 0.05% to 0.2% sodium tartrate to the lysis buffer 2 achieves compatibility with different types of blood collection tubes.

[0111] Furthermore, 25% isopropanol was added to lysis buffer 2 to enhance the stability of nucleic acid fragments and reduce degradation. The salt environment for nucleic acid lysis was optimized by adjusting the concentration ratio of sodium chloride and potassium chloride.

[0112] Commonly used nucleic acid binding magnetic beads, based on different surface modifications, mainly include aminated magnetic beads, carboxylated magnetic beads, silicified magnetic beads, and amino-silicified magnetic beads. Considering the fragmented and trace amounts of cell-free DNA in human peripheral blood, amino-silicified magnetic beads with a solid content of 1%–5% are preferred because they can bind cfDNA more stably and efficiently, including methylated cfDNA, exhibiting high adsorption efficiency.

[0113] An environment with appropriate concentrations of salt, anhydrous ethanol, and isopropanol is conducive to promoting the adsorption of cfDNA onto magnetic beads, while an appropriate concentration of proteinase K can promote complete protein digestion.

[0114] Washing solution 1 is primarily effective at removing impurities such as proteins, sugars, and isopropanol. Washing solution 2 can further remove salt impurities.

[0115] By selecting, optimizing the combination of, and adjusting the concentration ratios of the above reagents, they work together during the extraction process, resulting in a significant improvement in the efficiency and quality of peripheral blood cell-free DNA nucleic acid extraction.

[0116] The dosage of each reagent during the extraction process can be adjusted adaptively according to the plasma volume ratio. Taking the extraction of cfDNA from 2mL of plasma as an example, the specific method includes the following steps:

[0117] S11. Place the plasma into a centrifuge tube containing 100μL-150μL of proteinase K, then add 100μL-150μL of lysis buffer 1, mix well, and react at 15-60℃ for 10-60 minutes, for example, at 55℃ for 30 minutes to obtain a mixture.

[0118] The lysis buffer 1 contains sodium dodecyl sulfate (SDS); preferably, the lysis buffer 1 contains 0.05% to 1% sodium citrate dodecahydrate and 1% to 5% SDS.

[0119] In this embodiment, the components of lysis buffer 1 are 0.07% sodium citrate dodecahydrate and 4% SDS.

[0120] S12. Add 4 mL to 8 mL of lysis buffer 2 and 30 μL to 50 μL of magnetic beads to the mixture obtained in step 11. Mix well at room temperature (e.g., mix by inversion) and react for 10-60 minutes, for example, 10 minutes. After centrifugation, transfer to a magnetic rack and let stand for 2-10 minutes to adsorb the magnetic beads. Discard the solution.

[0121] The lysis buffer 2 comprises 10%–20% guanidine isothiocyanate, 1%–5% alkoxypolyethylene hydroxyethanol (NP40), 3% potassium chloride, 2% sodium chloride, 0.5%–1% tris(hydroxymethyl)aminomethane, 0.05%–0.2% sodium tartrate, and 25% isopropanol; preferably, the lysis buffer 2 comprises 20% guanidine isothiocyanate, 3% NP40, 3% potassium chloride, 2% sodium chloride, 2%–10% sodium lauroyl amino acid, 0.1%–0.3% sodium dihydrogen phosphate, 0.7% tris(hydroxymethyl)aminomethane, 0.05% sodium tartrate, and 25% isopropanol.

[0122] In this embodiment, the components of lysis buffer 2 are 20% guanidine isothiocyanate, 3% NP40, 3% potassium chloride, 2% sodium chloride, 4% sodium lauroyl amino acid, 0.1% sodium dihydrogen phosphate, 0.7% tris(hydroxymethyl)aminomethane, 0.05% sodium tartrate and 25% isopropanol.

[0123] Preferably, the magnetic beads are amino-silicified magnetic beads with a solid content of 1% to 5%. In this embodiment, the magnetic beads are 2.5% (w / v) silica amino magnetic beads (Silica Magnetic Beads (-NH2)). This invention does not limit the dilution concentration of the magnetic beads in the reaction system during use. Those skilled in the art can dilute the magnetic bead stock solution according to actual conditions, for example, to a magnetic bead suspension of 0.003% to 0.01%.

[0124] S13. Add 0.5 mL to 0.8 mL of washing solution 1, mix well (e.g., invert to mix), centrifuge, and then place on a magnetic rack for adsorption for 1-3 minutes, e.g., 1 minute. Discard the solution. This step can be repeated 1-5 times as needed.

[0125] Washing solution 1 contains 20%–25% guanidine isothiocyanate, 0.2%–1% SDS and 30%–50% anhydrous ethanol; preferably, washing solution 1 contains 25% guanidine isothiocyanate, 0.2%–0.5% tris(hydroxymethyl)aminomethane, 1.5%–5% potassium chloride, 0.5% SDS and 50% anhydrous ethanol.

[0126] In this embodiment, the washing liquid 1 consists of 25% guanidine isothiocyanate, 0.3% tris(hydroxymethyl)aminomethane, 2% potassium chloride, 0.5% SDS and 50% anhydrous ethanol.

[0127] S14. Add 0.5 mL to 0.8 mL of washing solution 2, mix well (e.g., vortex for 5 seconds), then transfer to a magnetic rack for adsorption for 1 minute, and discard the solution. This step can be repeated 1-3 times as needed.

[0128] The washing solution 2 consists of 0.1%–0.8% tris(hydroxymethyl)aminomethane, 0.5%–1% 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether (Trinton X-100), and 70%–80% anhydrous ethanol.

[0129] In this embodiment, the washing liquid 2 consists of 0.3% tris(hydroxymethyl)aminomethane, 0.7% Trinton X-100, and 75% anhydrous ethanol.

[0130] S15. After centrifugation, remove all residual liquid; air dry for 2-5 minutes to allow the magnetic beads to dry, avoiding over-drying.

[0131] S16. Add 50μL to 100μL of elution buffer 1, shake at 37℃ for 5 minutes to fully dissolve the DNA; transfer to a magnetic rack for adsorption for 1-3 minutes, and transfer the supernatant containing cfDNA to a new centrifuge tube to obtain the cfDNA extraction product.

[0132] Eluent 1 contains 0.05% to 0.2% tris(hydroxymethyl)aminomethane.

[0133] In this embodiment, the eluent 1 is 0.1% tris(hydroxymethyl)aminomethane.

[0134] The concentrations of the components in the above-mentioned lysis buffer 1-2, washing buffer 1-2, and elution buffer 1 are expressed as mass percentages.

[0135] 2) cfDNA methylation enrichment and purification unit:

[0136] This unit employs a non-bisulfite conversion, non-enzymatic conversion, and 5mC detection method for the enrichment and purification of cell-free DNA methylation in human peripheral blood. This method uses an antibody to capture methylated DNA, followed by immunoprecipitation of the methylated DNA with the antibody. This method can isolate specific DNA from a sample and requires the antibody to be coupled to a fixation matrix (such as magnetic beads) within a specified time frame for DNA separation. The steps include:

[0137] S21. Add the blocking agent to the sample to be tested to obtain the sample with the added blocking agent.

[0138] The initial cfDNA sample mass is generally not less than 0.5 ng; the mass ratio of cfDNA to DNA methylation protectant is in the range of 1:10-1:40. The volume after mixing has little impact on subsequent reactions, and those skilled in the art can adjust the total volume of the sample mixture with added blocking agent according to the actual situation.

[0139] For example, the sample preparation method for adding the blocking agent is as follows: the cfDNA sample mass starts at 0.5 ng, the cfDNA sample volume is 42 μL, the blocking agent concentration is 50 ng / μL, and the volume of the added blocking agent is 0.1 to 15 μL, so that the mass ratio of cfDNA to blocking agent is in the range of 1:10 to 1:40. Water is added to make the total volume of the sample mixture with added blocking agent reach the selected volume, such as 55-60 μL.

[0140] Preferably, in the sample with added blocking agent, the concentration ratio of cfDNA template to blocking agent is 1:10-1:40.

[0141] Preferably, the blocking agent is λDNA or human fecal microbial genomic DNA or other non-human methylated genomic DNA.

[0142] Preferably, when the blocking agent is λDNA, the mass ratio of methylated DNA to unmethylated DNA is 1:1; when the blocking agent is human fecal microbial genomic DNA, the methylation rate of human fecal microbial genomic DNA is 2.0% to 5.0%.

[0143] S22. Mix the immunoprecipitation buffer and the sample with the added blocking agent (e.g., gently mix by pipetting) and incubate at 90-95°C for 5-20 min. After incubation, cool rapidly to obtain the mixture.

[0144] Preferably, when mixing, the volume ratio of immunoprecipitation buffer to sample with added blocking agent is 33 μL: 57 μL.

[0145] S23. Add the antibody and magnetic beads to the mixture obtained in step S22 and incubate at 2-8°C for 4-16 hours, for example, incubate at 4°C for 8 hours.

[0146] Preferably, step S23 further includes a pretreatment step: a magnetic bead washing step and an antibody dilution step.

[0147] Cleaning steps for magnetic beads: Transfer 2-6 μL of the original magnetic bead solution (10 mg / mL) and wash the magnetic beads with a 10-fold diluted immunoprecipitation buffer. Wash at least twice. For the last wash, resuspend the magnetic beads in 20 μL of a 10-fold diluted immunoprecipitation buffer to obtain a magnetic bead suspension with a concentration of 1-3 mg / mL, preferably 2.5 mg / mL.

[0148] Antibody dilution steps: Dilute the stock 5-methylcytosine antibody with a concentration of 1 mg / mL by 10-100 times with immunoprecipitation buffer to obtain a solution with an antibody concentration of 0.01-0.1 mg / mL, preferably 0.02-0.05 mg / mL.

[0149] Preferably, the antibody, magnetic beads, and the mixture obtained in step S22 are mixed in the following proportions: 1 μL antibody: 1 μL magnetic beads: 15 μL mixture obtained in step S2.

[0150] Preferably, the magnetic beads are 2.8 μm superparamagnetic microbeads with affinity-purified IgG covalently coupled to their surface. For example, Dynabeads... TM M-280 Goat Anti-Mouse IgG Magnetic Beads (Thermo Fisher Scientific (USA), Catalog No.: 11202D), DiaMag anti-mouse IgG coated magnetic beads (Diagenode (Belgium), Catalog No.: C03010022-220), IgG MagBeads Goat anti-Mouse (Yisheng Biotechnology (Shanghai) Co., Ltd., Catalog No.: 47502ES03).

[0151] Preferably, the antibody is a 5-methylcytosine antibody (monoclonal antibody, MeDIP / CHIP grade), such as mouse monoclonal antibody [33D3] to 5-methylcytosine (5-mC) (product name: Anti-5-methylcytosine (5-mC) antibody [33D3], Abogen (Shanghai) Trading Co., Ltd., catalog number: ab10805), 5-Methylcytosine (5-mC) Monoclonal Antibody [33D3] (Epigentek, catalog number: A-1014-010), 5-methylcytosine (5-mC) Antibody-clone 33D3 (Diagenode, catalog number: C15200003).

[0152] S24. After brief centrifugation, place the mixture on a pre-cooled magnetic rack and discard the supernatant; wash the magnetic beads with washing solution.

[0153] Preferably, the magnetic bead precipitate is washed using at least two washing solutions (such as immunoprecipitation buffer, 1-4M NaCl solution, etc.) at least four times, with centrifugation and discarding of residual liquid after each wash. More preferably, one washing solution is a 10-fold diluted immunoprecipitation buffer; the other washing solution is a 10-fold diluted immunoprecipitation buffer with a high sodium chloride concentration, the components of which are 0.01-0.02M (mol / L) sodium phosphate solution (pH=7.0), 1-4M sodium chloride solution, and 0.1%-0.5% Triton X-100; preferably, the components are 0.02M sodium phosphate solution, 2M sodium chloride solution, and 0.5% Triton X-100.

[0154] More preferably, two washing solutions with different salt ion concentrations are combined to perform a gradient dilution with different salt ion concentrations, further reducing non-specific amplification. Specifically, the device is first washed twice with an immunoprecipitation buffer diluted 10-fold with a low sodium chloride concentration, and then washed twice twice with an immunoprecipitation buffer diluted 10-fold with a high sodium chloride concentration.

[0155] In the above steps, the 10-fold diluted immunoprecipitation buffer consists of a 0.01-0.02 M (mol / L) sodium phosphate solution (pH = 7.0), a 0.1-0.2 M sodium chloride solution, and 0.1%-0.5% Triton X-100; preferably, the components are 0.02 M sodium phosphate solution, 0.2 M sodium chloride solution, and 0.5% Triton X-100.

[0156] The procedure involves diluting the immunoprecipitation buffer tenfold. Those skilled in the art can perform this procedure using standard methods, such as mixing the immunoprecipitation buffer with TE solution (10mM Tris-HCl, 1mM EDTA, pH=8.0) (Beyotime, R0225) or nuclease-free water at a 1:9 volume ratio.

[0157] A 10-fold dilution of immunoprecipitation buffer refers to a buffer solution diluted 10 times.

[0158] S25. Elute the DNA with elution buffer 2 at 50-60℃ for 15 min, or 95-100℃ for 15 min. For example, conditions could be 55℃ for 15 min, or 100℃ for 15 min.

[0159] This invention does not limit the eluent 2; those skilled in the art can choose commercial products or prepare their own as needed.

[0160] Preferably, the eluent 2 comprises 0.01-0.1M Tris-HCl, 0.01-0.1M EDTA, 0.001-0.1% SDS, and 0.1-1 mg / mL proteinase K. More preferably, the eluent 2 comprises 0.05M Tris-HCl, 0.01M EDTA, 0.001% SDS, and 0.4 mg / mL proteinase K.

[0161] The detection data were analyzed, and recovery efficiency was used as the evaluation index. The formula is as follows: Recovery efficiency (%) = 2^(Ct) 10％input -Ct 样本 -3.32)×100.

[0162] During testing, 10% of the sample was aspirated as the 10% input sample, and the remaining sample was used for subsequent processing. Both were then amplified together by qPCR. 10％input This represents the Ct value for amplifying 10% of the input sample; Ct 样本 This represents the Ct value of the amplified sample.

[0163] This embodiment completes the enrichment of methylated DNA according to the following steps:

[0164] S21. Add 50-200 ng of blocking agent, for example, 120 ng, to a 5 ng cfDNA sample. If the blocking agent is λDNA, the mass ratio of methylated λDNA to unmethylated λDNA is 1:1. In this embodiment, add 0.1-15 μL of blocking agent at a concentration of 50 ng / μL to a test sample containing at least 0.5 ng of cfDNA, so that the mass ratio of cfDNA to DNA methylation protectant is in the range of 1:10-1:40. Add water to bring the volume of the sample with added blocking agent to 57 μL.

[0165] S22. Mix 33 μL of immunoprecipitation buffer with 57 μL of sample containing the blocking agent, incubate at 95°C for 10 min, and then cool on an ice box for 15 min. Then, take 7.5 μL of the reaction solution as input and store at 2-8°C. Use the remaining 75 μL of the solution for subsequent experiments.

[0166] S23. Washing the magnetic beads: Transfer 2-6 μL of the original magnetic bead solution (10 mg / mL) and wash the magnetic beads twice with a 10-fold diluted immunoprecipitation buffer. For the last wash, resuspend the magnetic beads in 20 μL of a 10-fold diluted immunoprecipitation buffer to obtain a magnetic bead suspension with a concentration of 2.5-3 mg / mL.

[0167] Diluting the antibody: Dilute the 5-methylcytosine antibody with a stock concentration of 1 mg / mL by 10-100 times with immunoprecipitation buffer to obtain an antibody solution with a concentration of 0.03-0.05 mg / mL.

[0168] Add 5 μL of antibody solution and 5 μL of magnetic bead suspension to 75 μL of the mixture obtained in step S2, and incubate at 4°C for 4-16 hours.

[0169] S24. Add 100 μL of 10-fold diluted immunoprecipitation buffer (on ice), invert to resuspend the magnetic beads, and incubate at 4°C for 5 min (40 rpm). After incubation, centrifuge briefly, place on a pre-cooled magnetic rack, and discard the supernatant after 1 min. Repeat the same steps at least once.

[0170] Add 100 μL of immunoprecipitation buffer diluted 10-fold with high sodium chloride concentration (ice bath), resuspend the magnetic beads by inverting, and incubate at 4°C for 5 min (40 rpm). After incubation, centrifuge briefly, place on a pre-cooled magnetic rack, and discard the supernatant after 1 min. Repeat the same steps at least once.

[0171] The components of a 10-fold dilution of immunoprecipitation buffer with high sodium chloride concentration are: 0.02M sodium phosphate solution, 2M sodium chloride solution, and 0.5% Triton X-100.

[0172] S25. Elute DNA with elution buffer 2 at 55℃ for 15 min, then at 100℃ for 15 min.

[0173] Elution buffer 2 contains 0.01-0.1M Tris-HCl, 0.01-0.1M EDTA, 0.001-0.1% SDS, and 0.1-1 mg / mL proteinase K.

[0174] 3) In the qPCR detection unit: This system can simultaneously detect up to nearly a hundred gene loci.

[0175] When there are a large number of genes to be tested, such as more than ten genes, the methylated DNA enriched library obtained by the cfDNA methylation enrichment and purification unit is pre-amplified. Those skilled in the art can choose to use kits from commercial companies or those developed in-house for library construction, depending on the specific circumstances. For example, kits like the Rapid Plus DNA Lib Prep Kit for illumina (Cat. No. RK20208, ABclonal) or the VAHTS Universal Pro DNA Library Prep Kit for illuminaVazyme (Cat. No. ND608-02, Novizan) can be used for end repair, adding an "A" tail, and connecting to adapters. The adapter-added products were pre-amplified using KAPA HiFi Hot start Ready mix and Library Amplification Primer Mix or Vazyme PCR Primer Mix 3 for Illumina and VAHTS HiFi Amplification Mix. After amplification, the products were purified using VAHTS DNA Clean Beads (Cat.No.N411-03, Vazyme).

[0176] 3.1 Primer and probe design

[0177] Based on cancer-specific methylation differential regions screened by high-throughput sequencing analysis or differentially methylated genes reported in publicly available data literature, primer-probe pairs suitable for fluorescence quantitative PCR detection can be designed.

[0178] To achieve simultaneous detection of a large number of genes, it is preferable that the differentially methylated regions contain at least four CpG sites to enhance the primer's specific recognition of methylated regions and increase sensitivity and specificity. Those skilled in the art can select different differentially methylated regions and design corresponding primer-probe combinations according to actual needs.

[0179] This embodiment designs primer-probe combinations for 16 gene loci associated with colorectal cancer (SALL1, ITGA4, Septin9-1), gastric cancer (OLIG2, RNF180, Septin9-2), esophageal cancer (CDO1, SHOX2, PAX1-2), liver cancer (IRF4, C1QL3), pancreatic cancer (PAX1-1, Septin9-3), and lung cancer (EMX1, BARX1, ZNF781) to simultaneously detect methylation levels. The 16 gene loci regions were identified by the inventors through high-throughput sequencing analysis of methylation profiles in relevant cancer and non-cancer clinical samples, revealing cancer-specific correlations. Septin9-1, Septin9-2, and Septin9-3 represent different regions of the same gene most relevant to their respective cancer types.

[0180] The specific primer and probe sequences are shown in Table 1.

[0181] Table 1

[0182] 3.2 Those skilled in the art can design different qPCR reaction systems and reaction procedures according to the actual situation.

[0183] In this embodiment, the target gene qPCR reaction system is shown in Table 2 below.

[0184] Table 2

[0185] The qPCR reaction procedure is shown in Table 3 below.

[0186] Table 3

[0187] After the reaction is complete, the qPCR detection results are obtained.

[0188] 4) In the algorithm analysis unit, logistic regression equations are used to analyze the offline data. Specifically, ΔCT is calculated by subtracting the CT value of the internal reference gene from the CT value of the target gene. Then, the coefficients of each gene are calculated according to the logistic regression equation. Finally, the logistic score of each gene is calculated using the following formula: Logistic score=β0+β1*X1+β2*X2+β3*X3+β4*X4+β5*X5+........+β n *X n Overall score = elogistic score / (1 + elogistic score) × 1000

[0189] Select an appropriate cut-off value as the basis for judgment.

[0190] In the formula, β0 represents a constant, β1 represents the weight coefficient of each gene, and X represents each gene.

[0191] The above calculations and analyses can be performed using various calculation tools in commercial software, such as SPSS Statistics v27.0.1.

[0192] Example 2

[0193] The system in this embodiment, based on the system in embodiment 1, adds a pre-amplification unit (5). This includes the following steps:

[0194] 5.1 Adapter ligation steps: Ligate universal adapters to the cfDNA obtained from the cfDNA extraction unit;

[0195] 5.2 Amplification of the methylated DNA enrichment library.

[0196] When there are a large number of genes to be tested, for example, more than ten genes, the methylated DNA enriched library obtained by the cfDNA methylation enrichment purification unit is pre-amplified.

[0197] For cfDNA, those skilled in the art can choose to use kits developed by commercial companies or their own libraries, depending on the specific circumstances. For example, kits of the same type, such as the Rapid Plus DNA Lib Prep Kit for illumina (Cat.No.RK20208, ABclonal) or the VAHTS Universal Pro DNA Library Prep Kit for illuminaVazyme (Cat.No.ND608-02, Novizan), can be used for end repair, adding an "A" tail, and connecting to adapters.

[0198] For the products of methylated DNA enrichment libraries, those skilled in the art can choose to use commercially available or self-developed library construction kits, such as KAPA HiFi Hot start Ready mix and Library Amplification Primer Mix or Novizan PCR Primer Mix 3 for Illumina and VAHTS HiFi Amplification Mix, for pre-amplification, depending on the actual situation.

[0199] In this embodiment, a conventional library construction kit (e.g., VAHTS Universal Pro DNA Library Prep Kit for Illumina, Cat. No. ND608-02, Vazyme) was used to perform end repair, add an "A" tail, and ligate with universal adapters according to the instructions to obtain ligation products. The ligation products were then purified using magnetic beads (e.g., VAHTS DNA Clean Beads, Cat. No. N411-03, Vazyme) and then entered the cfDNA methylation enrichment and purification unit.

[0200] After completing the cfDNA methylation enrichment and purification, 12.5 μL of pre-amplification enzyme, 2.5 μL of pre-amplification primers (e.g., VAHTS Universal Pro DNA Library Prep Kit for Illumina, Cat. No. ND608-02, Vazyme), and the S25 elution product from the previous step were combined to form a 25 μL pre-amplification system. The pre-amplification experiment was performed in a PCR instrument using the following amplification program: 95℃ / 3 min; 98℃ / 20 s; 60℃ / 15 s; 72℃ / 30 s (12 cycles); 72℃ / 5 min; 4℃ / hold (heat cap 105℃).

[0201] In the pre-amplification unit, the universal primers have the following sequences:

[0202] F:5'-AATGATACGGCGACCACCGAGATCTACAC-3'

[0203] R:5'-CAAGCAGAAGACGGCATACGAGAT-3'.

[0204] After amplification, the product was purified using VAHTSDNA Clean Beads (Cat.No.N411-03, Vazyme).

[0205] Test Example 1: cfDNA Extraction

[0206] Sixteen plasma samples were extracted using the peripheral blood cell-free DNA extraction method in the cfDNA extraction unit, and also using the VAHTS Free-Circulating DNA Maxi Kit (NWZ reagent) according to its instructions.

[0207] The NWZ reagent components include Proteinase K, VAHTS Particles G, Buffer L / B, Buffer WA, Buffer WB, and Elution Buffer. Compared to this method, this reagent only contains one lysis buffer, Buffer L / B. During use, the lysis buffer L / B, VAHTS Particles G, Proteinase K, and sample are directly mixed together to complete the lysis and adsorption of free DNA. This method includes lysis buffer 1 and lysis buffer 2. First, Proteinase K and lysis buffer 1 are used for preliminary lysis of the sample. Then, lysis buffer 2 and magnetic beads are used to complete the thorough lysis of the sample and the adsorption of free DNA.

[0208] When using β-actin gene detection, the difference in Ct value reflects the content of nucleic acid template; the concentration of double-stranded DNA in the extracted product was quantitatively determined using Qubit4.0, and the results are shown in Table 4.

[0209] Table 4

[0210] The Ct values ​​and concentration detection results of samples 1-16 were summarized. For the same sample, ΔCt and concentration ratio were calculated: ΔCt = Ct value obtained by the method in Example 1 - Ct value obtained by the NWZ reagent method; concentration ratio = concentration obtained by the method in Example 1 / concentration obtained by the NWZ reagent method. The results show that, compared with the NWZ method, the method provided by this invention improves the Ct value of the obtained product by 0.65–1.56, with an average improvement of 1.06, and the concentration of the obtained product is 2.14–4.07 times that of the NWZ method, with an average of 2.65 times.

[0211] The Qsep100 fully automated nucleic acid and protein analyzer was used to perform capillary electrophoresis to analyze the fragment size and distribution of the extracted products.

[0212] The results are shown in Figure 2. The extraction method of Example 1 has a good extraction effect even when the amount of nucleic acid in plasma is extremely small (Figure 2 left); while commercial kits often fail to detect the main peak of cfDNA under the same sample volume (Figure 2 right).

[0213] The results are shown in Figure 3. Using the extraction method of Example 1, the main peak of the cfDNA extracted product was 160-175bp (Figure 3 left), while the main peak of the cfDNA extracted by the commercial kit was 175-190bp (Figure 3 right). This indicates that the cfDNA extracted by the method provided by the present invention is of better quality and is closer to the reference value of 167bp for the length of cell-free DNA fragments in plasma.

[0214] The results are shown in Figure 4. Using the extraction method of Example 1, the obtained cfDNA extract had fewer large fragment contaminations (Figure 4 left). Compared with commercial kits, the error caused by operation was smaller.

[0215] Test Example 2: cfDNA methylation enrichment

[0216] (1) Improve methylation enrichment efficiency

[0217] A control experiment was designed to purify the extract obtained from the cfDNA extraction unit in the cfDNA methylation enrichment and purification unit, with the steps and conditions remaining unchanged:

[0218] Control group: 1) No blocking agent added; 2) The concentrations of each component of the methylation immunoprecipitation buffer were: sodium phosphate 0.15M, sodium chloride 1.5M, and Triton X-100 3%; 3) The washing process involved washing three times with immunoprecipitation buffer diluted tenfold with low sodium chloride concentration.

[0219] This system: 1) Add 120 ng of modified λDNA blocking agent (methylated λDNA: unmethylated λDNA = 1:1) or fecal microbial DNA blocking agent (with a genomic methylation rate of 2% to 5%); 2) The concentrations of each component of the methylation immunoprecipitation buffer are: sodium phosphate concentration of 0.2M, sodium chloride concentration of 2M, and Triton X-100 concentration of 5%; 3) Washing process: First wash twice with immunoprecipitation buffer diluted 10 times with low sodium chloride concentration, and then wash twice with immunoprecipitation buffer diluted with high sodium chloride concentration.

[0220] The results are shown in Figure 5. The average methylation gene binding efficiency of the control group was 43.8%, while the average methylation gene binding efficiency of this system was 66.74%.

[0221] (2) Reduce nonmethylation enrichment

[0222] With the system unchanged, the effect of reducing nonmethylation enrichment was evaluated by adjusting the type and proportion of the blocking agent added.

[0223] Preparation of the blocking agent:

[0224] 1)λDNA

[0225] Unmethylated λDNA is commercially available, while methylated λDNA can be produced by sonicating unmethylated λDNA and then catalyzing it with a DNA methyltransferase (such as CPG Methyltransferase (M.SssI), New England Biolabs).

[0226] 2) Human fecal microbial genomic DNA

[0227] Take an appropriate amount of human feces and extract it using a microbial extraction kit (such as a nucleic acid extraction or purification reagent (magnetic bead method), Jiangsu Mole Biotechnology Co., Ltd.) according to the kit instructions.

[0228] 3) Mix the blocking agent with plasma cfDNA in the proportions shown in Table 5, add exogenous unmethylated DNA (unDNA) (Primer pair for Unmethylated spike-in control, Diagenode), and perform methylation enrichment and qPCR detection.

[0229] Table 5

[0230] 4) Analyze the detection data, using recovery efficiency as the evaluation index, as shown in the following formula:

[0231] Recovery efficiency (%) = 2^(Ct10%input - Ctsample - 3.32) × 100

[0232] The detection results are shown in Figure 6. Adding the blocking agent λDNA or fecal microbial genomic DNA can significantly reduce the enrichment of unmethylated DNA, and the recovery rate is related to the amount of blocking agent used. Under optimal conditions, the recovery rate can reach less than 0.1%. Adding an appropriate amount of blocking agent can improve the detection specificity.

[0233] Test Example 3

[0234] With the system unchanged, the composition of the immunoprecipitation buffer in the cfDNA methylation enrichment and purification unit was adjusted, and the Ct value was detected using the TSH2B gene and GAPDH gene to observe the effect.

[0235] Different combinations of the immunoprecipitation buffer were obtained by adjusting the different components. (Table 6)

[0236] Table 6

[0237] The corresponding concentrations of the immunoprecipitation components after a tenfold dilution are shown in Table 7.

[0238] Table 7

[0239] The test results are shown in Table 8. P1 and P2 are positive samples for colorectal cancer, N1 and N2 are normal negative samples, and PC and NC are positive control (commercial human fully methylated genomic DNA) and negative control (commercial human unmethylated genomic DNA), respectively.

[0240] Table 8

[0241] The dosage of sodium phosphate was optimized using combinations 1, 2, and 3; the dosage of sodium chloride was optimized using combinations 1, 4, and 5; the dosage of Triton X-100 was optimized using combinations 1, 6, and 7; and finally, the dosage of each component was optimized using combinations 8 and 9. In the detection results, a smaller Ct value for TSH2B indicates more enriched methylated fragments and better results, while a larger Ct value for GAPDH indicates less non-specific enrichment and better results. It can be seen that the enrichment effect of all combinations meets the requirements. Among them, combination 9 has the least non-specific detection and a high total enrichment rate. Combination 9, namely, a sodium phosphate concentration of 0.2M, a sodium chloride concentration of 2M, and a Triton X-100 concentration of 5%, is the preferred combination.

[0242] Test Example 4: MeDIP Pre-amplification Analysis

[0243] The extracted cfDNA was ligated with a universal adapter, and MeDIP pre-amplification was achieved using universal primers for signal indiscriminate amplification. The timing of the adapter ligation step is crucial in the entire detection system. This embodiment evaluates the recovery efficiency of methylated and unmethylated signals by comparing two detection methods: adding the adapter before MeDIP and adding MeDIP before adding the adapter, to select the appropriate timing for adapter ligation.

[0244] The detection results are shown in Figure 7. The recovery rate of methylated meDNA was 74.2% and the recovery rate of unmethylated unDNA was 0.67% in the MeDIP process after adding the adapter; the recovery rate of methylated meDNA was 70.38% and the recovery rate of unmethylated unDNA was 0.49% in the MeDIP process after adding the adapter. The method of adding the adapter first, without significant difference in specificity, improved the recovery efficiency of methylation signals. The adapter addition step leads to the loss of some cfDNA template; the detection process of adding the adapter first, followed by MeDIP, can effectively reduce the proportion of methylation signal loss, increase the richness of methylation signals, and obtain more effective data.

[0245] Test Example 5: qPCR Detection and Algorithm Analysis

[0246] Three hundred cancer samples were selected, including 71 cases of colorectal cancer, 57 cases of gastric cancer, 29 cases of esophageal cancer, 56 cases of liver cancer, 17 cases of pancreatic cancer, and 70 cases of lung cancer, along with 124 clinical samples from healthy individuals and those with benign diseases, totaling 424 clinical samples. 5–10 mL of peripheral blood was collected from each sample for analysis using this system. The methylated enriched products were used as templates for nucleic acid amplification, amplifying 16 different gene methylation regions in 16 reaction wells. Alternatively, after interference-free multigene amplification testing in the same reaction well, 1–4 fluorescence channels could be selected for multiplex gene amplification, depending on the number of multiplex fluorescence channels available. In this study, only single-gene amplification was performed, meaning one gene was amplified per reaction well.

[0247] According to the system in Example 1, gene combinations were analyzed using logical formulas. The analysis revealed the following: SALL1, ITGA4, and Septin9-1 gene loci showed methylation differences, associated with colorectal cancer; OLIG2, RNF180, and Septin9-2 gene loci showed methylation differences, associated with gastric cancer; CDO1, SHOX2, and PAX1-2 gene loci showed methylation differences, associated with esophageal cancer; IRF4 and C1QL3 gene loci showed methylation differences, associated with liver cancer; PAX1-1 and Septin9-3 gene loci showed methylation differences, associated with pancreatic cancer; and EMX1, BARX1, and ZNF781 gene loci showed methylation differences, associated with lung cancer. The optimal combination and corresponding cancer type were selected for sensitivity-specificity analysis. When analyzing the performance of a single cancer type, only samples of that specific cancer type were analyzed; for example, when analyzing colorectal cancer performance, only colorectal cancer samples from 300 clinical cancer samples were analyzed.

[0248] The specific statistics are as follows:

[0249] Of the 195 colorectal clinical samples selected, 71 were positive for colorectal cancer and 124 were negative. The methylation levels of SALL1, ITGA4, and Septin9-1 were obtained using this system. The detection performance was analyzed using logistic regression and ROC curves. Colorectal cancer: logistic score = 13.84 - 2.38 × ΔCt(SALL1) - 0.20 × ΔCt(ITGA4) - 3.44 × ΔCt(Septin9-1) Overall score = logistic score / (1 + logistic score) × 1000

[0250] After calculation using the logistic regression formula, the cut-off value was selected as 430.23, and the area under the curve (AUC) was 0.94. Its performance was 88.7% sensitivity and 93.5% specificity (Figure 9).

[0251] Of the 181 gastric-related clinical samples selected, 57 were positive for gastric cancer and 124 were negative. After processing by this system's detection process, the detection performance was analyzed using logistic regression formulas and ROC curves in conjunction with the methylation levels of OLIG2, RNF180, and Septin9-2.

[0252] Gastric cancer: logistic score = -0.363 - 0.71 × ΔCt(OLIG2) - 0.141 × ΔCt(RNF180) - 1.679 × ΔCt(Septin9-2) Overall score = logistic score / (1 + logistic score) * 1000

[0253] After calculation using the logistic regression formula, a cut-off value of 371.41 was selected, and the area under the curve (AUC) was 0.955. The performance was 87.7% sensitivity and 92.7% specificity. (Figure 10)

[0254] Of the 153 esophageal-related clinical samples selected, 29 were positive for esophageal cancer and 124 were negative. After processing by this system's detection process, the detection performance was analyzed using logistic regression formulas and ROC curves in conjunction with the methylation levels of CDO1, SHOX2, and PAX1-2.

[0255] Esophageal cancer: logistic score = 1.237 - 0.666 × ΔCt(CDO1) - 0.148 × ΔCt(SHOX2) - 2.542 × ΔCt(PAX1-2) Overall score = logistic score / (1 + logistic score) * 1000

[0256] After calculation using the logistic regression formula, a cut-off value of 536.62 was selected, and the area under the curve (AUC) was 0.943. The performance was 82.8% sensitivity and 91.9% specificity. (Figure 11)

[0257] Of the 180 liver-related clinical samples selected, 56 were positive for liver cancer and 124 were negative. After processing by this system's detection process, the detection performance was analyzed using logistic regression formula and ROC curve analysis in conjunction with IRF4 and C1QL3 methylation levels.

[0258] Liver cancer: logistic score = 4.748 - 0.096 × ΔCt(IRF4) - 1.328 × ΔCt(C1QL3) Overall score = logistic score / (1 + logistic score) * 1000

[0259] After calculation using the logistic regression formula, a cut-off value of 435.8 was selected, and the area under the curve (AUC) was 0.932. The performance was 83.9% sensitivity and 91.1% specificity. (Figure 12)

[0260] Of the 141 pancreatic-related clinical samples selected, 17 were positive for pancreatic cancer and 124 were negative. After processing by this system's detection process, the detection performance was analyzed using logistic regression formula and ROC curve analysis in conjunction with the methylation levels of PAX1-1 and Septin9-3.

[0261] Pancreatic cancer: logistic score = 1.259 - 2.185 × ΔCt(PAX1-1) - 0.357 × ΔCt(Septin9-3) Overall score = logistic score / (1 + logistic score) * 1000

[0262] After calculation using the logistic regression formula, a cut-off value of 446.40 was selected, and the area under the curve (AUC) was 0.917. The performance in pancreatic cancer was 82.4% sensitivity and 87.1% specificity. (Figure 13)

[0263] Of the 194 lung-related clinical samples selected, 70 were positive for lung cancer and 124 were negative. After processing by this system's detection process, the detection performance was analyzed using logistic regression formula and ROC curve analysis in conjunction with the methylation levels of EMX1, BARX1, and ZNF781.

[0264] Lung cancer: logistic score = 0.495 - 0.598 × △Ct(EMX1) - 0.023 × △Ct(BARX1) - 2.096 △Ct(ZNF781) Overall score = logistic score / (1 + logistic score) * 1000

[0265] After calculation using the logistic regression formula, a cut-off value of 430.49 was selected, and the area under the curve (AUC) was 0.93. The lung cancer performance was 81.4% sensitivity and 90.3% specificity. (Figure 14)

[0266] Analysis of the lowest detection limit in Test Example 6

[0267] Using methylated positive genomic DNA solutions enriched by methylation immunoprecipitation as the detection target, three concentration gradients were set up, with a methylation ratio of 1% and methylated DNA of 5, 50, and 500 copies / reaction, respectively. The detection study samples were prepared according to the three groups shown in Table 9. Primers and probes SALL1 and ITGA4 shown in Table 1 were used. The target gene qPCR reaction system is shown in Table 2, and the qPCR reaction program is shown in Table 3.

[0268] Table 9

[0269] As shown in Figure 15, methylated SALL1 and ITGA4 can be amplified well, and at 100% level, the minimum reaction concentration can reach 5 copies / reaction; at 95% level, the minimum reaction concentration of some genes can reach 2-3 copies / reaction, which is better than the minimum detection limit of methylation reported by domestic and foreign methylation gene detection products. The methylation immunoprecipitation enrichment system of this invention can detect lower levels of methylated cfDNA signals in plasma.

[0270] Test Case 7: Tumor Tissue Source Tracing Analysis

[0271] Based on the MeDIP pre-amplification detection system of Example 2, multi-gene joint detection can be achieved. The results of multi-gene joint detection can be used to trace cancer tissue origins using machine learning models. The experimental procedure is shown in Figure 8.

[0272] This embodiment includes 570 samples diagnosed with cancer by clinical pathology, covering 9 major cancer types, including 72 cases of lung cancer, 135 cases of colorectal cancer, 64 cases of gastric cancer, 41 cases of esophageal cancer, 111 cases of liver cancer, 23 cases of pancreatic cancer, 37 cases of breast cancer, 21 cases of ovarian cancer, and 66 cases of nasopharyngeal carcinoma, for tissue source tracing and modeling.

[0273] Nine cancer tissue origination training and independent validation sets were allocated in a 7:3 ratio. Feature difference analysis was performed between each cancer and the other eight cancers, and features with an AUC greater than or equal to 0.6 or less than or equal to 0.4 were selected for tissue origination modeling. A random forest model was used to build tissue origination models for each cancer type, and tissue origination scores were assigned to the independent validation set samples to obtain the predicted tissue location of the cancer. The tissue origination model achieved an accuracy of 65.3% in predicting the TPO1 location and 82.1% in predicting the TPO2 location, demonstrating good performance in cancer origination. Specific model prediction results are shown in Figure 16.

[0274] Traditional extraction and bisulfite treatment methods suffer from low cfDNA extraction and recovery efficiency, as well as significant losses during the intense methylation process, resulting in a severe reduction in methylation signals. Therefore, current methods typically only amplify six methylation regions (in three qPCR reaction wells). In this invention, a qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system improves cfDNA extraction recovery efficiency by 2.6 times and methylation enrichment efficiency by 10 to 30 times. The signal richness of cfDNA methylation in early tumor signals is increased by 26 to 78 times compared to traditional purification systems. Furthermore, the pre-amplification system further amplifies trace signals without offset, enhancing signal intensity and ensuring sufficient nucleic acid template for fluorescent PCR amplification analysis.

[0275] The methylation detection system of this invention overcomes the systematic limitations of traditional methods, enabling simultaneous detection of multiple genes in a single blood sample. Early screening and source tracing analysis of nine common cancer types have achieved optimal simultaneous detection of multiple genes through the amplification of 57 target genes, demonstrating good performance. Optionally, after interference-free multi-gene amplification testing in the same reaction well, one to four fluorescence channels can be selected for multiplex gene amplification based on the number of multiplex fluorescence channels. In other words, the methylation detection system of this invention can expand the detection of more cancer types and genes through multiplex qPCR, enabling simultaneous detection of more than ten cancer types and at least one hundred gene loci.

[0276] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system, including: 1) cfDNA extraction unit: Used to extract cfDNA from plasma samples. When the yield of double-stranded DNA of the extracted cfDNA product is not less than 0.5 ng per mL of plasma, it enters the cfDNA methylation enrichment and purification unit. 2) cfDNA methylation enrichment and purification unit: methylated DNA is enriched and purified by antibody using immunoprecipitation, while an inhibitor is added to reduce non-methylation enrichment, thus obtaining a methylated DNA enriched library. When the recovery rate of enriched methylated quality control products is not less than 65% and the recovery rate of unmethylated quality control products is not higher than 1%, they are entered into the qPCR detection unit. 3) qPCR detection unit: used for detecting methylated DNA in target gene regions using fluorescent probes to obtain qPCR detection results.

2. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system according to claim 1, wherein, It also includes a pre-amplification unit: pre-amplifying the methylated DNA enrichment library to obtain an amplified methylated DNA enrichment library; When the recovery rate of enriched methylated quality control is not less than 65% and the recovery rate of unmethylated quality control is not higher than 1%, the sample is first sent to the pre-amplification unit and then to the qPCR detection unit. The pre-amplification includes: ligating adapters into the cfDNA extracted by the cfDNA extraction unit; The methylated DNA enriched library obtained from the cfDNA methylation enrichment and purification unit was amplified.

3. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system according to claim 1, wherein, The cfDNA extraction unit extracts cfDNA from plasma samples, including the following steps: S11. Place 2 mL of plasma into a centrifuge tube containing 100 μL to 150 μL of proteinase K, then add 100 μL to 150 μL of lysis buffer 1, mix well, and react at 15-60℃ for 10-60 minutes to obtain a mixture. S12. Add 4 mL to 8 mL of lysis buffer 2 and 30 μL to 50 μL of magnetic beads to the mixture obtained in S11. Mix well at room temperature and react for 10 to 60 minutes. After centrifugation, transfer to a magnetic rack to adsorb the magnetic beads and discard the solution. S13. Add 0.5 mL to 0.8 mL of washing solution 1, mix well, centrifuge, place on a magnetic rack for adsorption, and discard the solution; S14. Add 0.5 mL to 0.8 mL of washing solution 2, mix well, transfer to a magnetic rack for adsorption, and discard the solution; S15. After centrifugation, remove all residual liquid; allow the magnetic beads to dry in air for 2-5 minutes; S16. Add 50μL to 100μL of elution buffer 1, dissolve the DNA, transfer it to a magnetic rack for adsorption, and transfer the supernatant containing cfDNA to a new centrifuge tube to obtain the cfDNA extraction product. The lysis buffer 1 contains 0.05%–1% sodium citrate dodecahydrate and 1%–5% SDS; The lysis buffer 2 contains 10%–20% guanidine isothiocyanate, 1%–5% NP40, 3% potassium chloride, 2% sodium chloride, 0.1%–0.3% sodium dihydrogen phosphate, 0.5%–1% tris(hydroxymethyl)aminomethane, 0.05%–0.2% sodium tartrate, and 25% isopropanol. Washing solution 1 contains 20%–25% guanidine isothiocyanate, 0.2%–1% SDS, and 30%–50% anhydrous ethanol; Washing solution 2 contains 0.1% to 0.8% tris(hydroxymethyl)aminomethane, 0.5% to 1% Trinton X-100, and 70% to 80% anhydrous ethanol; Eluent 1 contains 0.05% to 0.2% tris(hydroxymethyl)aminomethane.

4. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system as described in claim 2, characterized in that, The components of lysis buffer 1 are 0.07% sodium citrate dodecahydrate and 4% SDS; The components of lysis buffer 2 are 20% guanidine isothiocyanate, 3% NP40, 3% potassium chloride, 2% sodium chloride, 4% sodium lauroyl amino acid, 0.1% sodium dihydrogen phosphate, 0.7% tris(hydroxymethyl)aminomethane, 0.05% sodium tartrate and 25% isopropanol. Washing solution 1 also contains 0.2% to 0.5% tris(hydroxymethyl)aminomethane and 1.5% to 5% potassium chloride; In the cfDNA extraction unit, the magnetic beads are amino-silylated magnetic beads.

5. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system according to claim 1, wherein, The method for enriching methylated fragments in cell-free human peripheral blood DNA in the cfDNA methylation enrichment and purification unit includes the following steps: S21. Add the blocking agent to the sample to be tested to obtain a sample with the added blocking agent; S22. Mix the immunoprecipitation buffer and the sample with the added blocking agent, incubate at 90-95℃ for 5-20 min, and then cool rapidly to obtain the mixture; S23. Add the antibody and magnetic beads to the mixture obtained in step S22 and incubate at 2-8°C for 4-16 hours; S24. After brief centrifugation, place the mixture on a pre-cooled magnetic rack and discard the supernatant; wash the magnetic beads with washing solution. S25. Elute DNA with elution buffer at 50-60℃ for 15 min, or 95-100℃ for 15 min. In step S21, the DNA methylation inhibitor is λDNA or human fecal microbial genomic DNA or other non-human methylated genomic DNA; When the blocking agent is λDNA, the mass ratio of methylated DNA to unmethylated DNA is 1:1; when the blocking agent is human fecal microbial genomic DNA, the methylation rate of human fecal microbial genomic DNA is 2.0% to 5.0%. In samples with added blocking agents, the concentration ratio of cfDNA to DNA methylation blocking agent is 1:10-1:40, with the concentration of cfDNA not less than 0.01 ng / μL.

6. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system according to claim 5, wherein, In step S22, during mixing, the volume ratio of immunoprecipitation buffer to the sample with added blocking agent is (30-35) μL : (55-60) μL; Step S23 also includes a cleaning step for the magnetic beads and a dilution step for the antibody: Cleaning steps for magnetic beads: Take 2-6 μL of 10 mg / mL magnetic bead stock solution and wash the magnetic beads with 10-fold diluted immunoprecipitation buffer. Wash at least twice. For the last wash, resuspend the magnetic beads with 20 μL of 10-fold diluted immunoprecipitation buffer to obtain a magnetic bead suspension with a concentration of 1-3 mg / mL. Antibody dilution procedure: Dilute the 1 mg / mL 5-methylcytosine antibody 10-100 times with immunoprecipitation buffer to obtain a solution with an antibody concentration of 0.01-0.1 mg / mL; The antibody, magnetic beads, and the mixture obtained in step S2 are mixed in the following volume ratio: 1:1:(15-20).

7. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system according to claim 5, wherein, The immunoprecipitation buffer solution includes 0.1-0.2M sodium phosphate solution, 1-2M sodium chloride solution, and 1%-5% Triton X-100. In step S24, the washing solution includes a 10-fold diluted immunoprecipitation buffer and an immunoprecipitation buffer with a high sodium chloride concentration; the immunoprecipitation buffer with a high sodium chloride concentration consists of 0.01-0.02M sodium phosphate solution, 1-4M sodium chloride solution, and 0.1%-0.5% Triton X-100. In step S25, the elution buffer 2 includes 0.01-0.1M Tris-HCl, 0.01-0.1M EDTA, 0.001-0.1% SDS, and 0.1-1 mg / mL proteinase K.

8. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system according to claim 5, wherein, In the cfDNA methylation enrichment and purification unit, the magnetic beads are IgG magnetic beads; the antibody is 5-methylcytosine antibody.

9. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system according to claim 1, wherein, In the pre-amplification unit, the universal primers have the following sequences: F: 5'-AATGATACGGCGACCACCGAGATCTACAC-3' R: 5'-CAAGCAGAAGACGGCATACGAGAT-3'.

10. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system of claim 1, wherein, In the qPCR detection unit, primers and probes are designed for target-specific differentially methylated regions, which cover no less than 4 CpG sites.

11. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system of claim 1, wherein, In the qPCR detection unit, TSH2B is used as an internal reference gene for the qPCR reaction.

12. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system of claim 11, wherein, The internal reference gene is TSH2B, and its coordinate chromosome position on Hg38 is: chr6:25726855-25727020.

13. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system according to claim 11 or 12, wherein, The internal reference gene also includes GAPDH, with the chromosome coordinates of Hg38 as follows: chr12: 6534476-6534539.

14. The qPCR-based peripheral blood cell-free DNA multi-gene methylation detection system according to claim 1, wherein, Also includes: Algorithm analysis unit: used to analyze qPCR detection results, including: calculating ΔCT by subtracting the CT value of the internal reference gene from the CT value of the target gene; By combining the ΔCT values ​​of single or multiple genes, the logistic score of each sample is calculated using AI models such as logistic regression or random forest. An appropriate cutoff value is selected based on the different logistic scores. When the sample score is higher than the cutoff value, it is judged as positive; when the sample score is lower than the cutoff value, it is judged as negative.

15. A kit for extracting and enriching methylated fragments in human peripheral blood cell-free DNA, comprising: The lysis buffer 1, lysis buffer 2, washing buffer 1, washing buffer 2 and elution buffer 1 as described in claim 3 or 4, and the immunoprecipitation buffer, the immunoprecipitation buffer diluted tenfold with a high sodium chloride concentration and the elution buffer 2 as described in claim 4.

16. The kit of claim 15, wherein It also includes the magnetic beads as described in claim 3 or 4, and the 5mC antibody, immunomagnetic beads, and blocking agent as described in claim 5.

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