A cfDNA methylation detection method based on a multiple quantitative fluorescent PCR detection system and application thereof

Abstract

The application relates to the field of biotechnology, and discloses a cfDNA methylation detection method based on a multiplex quantitative fluorescent PCR detection system and application thereof. The cfDNA methylation detection method based on the multiplex quantitative fluorescent PCR detection system comprises the following steps: extracting cfDNA and leukocyte gDNA; using a methylation-sensitive restriction endonuclease to digest the cfDNA and the leukocyte gDNA to obtain digestion products; using a multiplex quantitative fluorescent PCR detection system to amplify the digestion products to obtain amplification products, wherein the multiplex quantitative fluorescent PCR detection system comprises 22 primer pairs, which are used for simultaneously amplifying 15 detection sites and 7 internal reference sites; performing capillary electrophoresis detection on the amplification products; and determining the capillary electrophoresis detection result. The cfDNA methylation detection method provided by the application realizes effective detection on multiple methylation sites by combining capillary electrophoresis detection, and realizes high-sensitivity and specific detection on colorectal cancer by combining a corresponding result determination method, so that the application has important clinical application value and wide market prospect.

Description

[0001] This application is a divisional application of the invention patent with application number CN202512015235.9, application date December 30, 2025, authorization date April 23, 2026, and invention title "A Multiplex Fluorescent PCR Detection System and Its Application". Technical Field

[0002] This invention relates to the field of biological gene detection technology, and in particular to a method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system and its application. Background Technology

[0003] Cancer poses a serious threat to human health, and early screening and diagnosis are key to improving treatment outcomes and increasing patient survival rates. Liquid biopsy technology, especially based on the detection of cell-free DNA (cfDNA) in peripheral blood samples, has become one of the important technologies suitable for early cancer screening and diagnosis due to its relatively simple sample processing and theoretically high detection rate for early tumors.

[0004] The copy number of circulating tumor DNA (ctDNA) in the peripheral blood of cancer patients is extremely low, only about 0.1-10 copies / ml in the early stages of cancer or after treatment remission, accounting for 0.01-1.5% of cfDNA. How to effectively detect a small copy number of ctDNA in a high background of cfDNA is the key to realizing liquid biopsy technology for peripheral blood samples.

[0005] DNA methylation, as one of the core mechanisms of epigenetic modification, can be detected early in tumor development and exhibits good stability. ctDNA methylation has become a highly valuable biomarker in tumor liquid biopsy. Existing cfDNA methylation detection technologies typically achieve relative enrichment of methylated nucleic acids through specific methods, followed by detection using techniques such as qPCR, capillary electrophoresis, or next-generation sequencing.

[0006] However, existing technologies still have the following shortcomings: on the one hand, the absolute content of ctDNA is extremely low, and single-site detection is prone to false negatives due to the low target copy number; on the other hand, methods such as qPCR and ddPCR can detect only a few sites per reaction, and detecting multiple sites requires more reaction times, which not only increases workload and cost, but also multiplies the demand for sample volume; although next-generation sequencing can achieve multi-site detection, it is complex to operate, has a long detection cycle, and is costly, which is not conducive to large-scale practical applications.

[0007] Therefore, developing a simple, rapid, and low-cost cfDNA methylation detection method that can simultaneously detect multiple methylation sites in a single reaction has significant clinical application value for the early screening and diagnosis of tumors such as colorectal cancer. Summary of the Invention

[0008] In view of the above-mentioned shortcomings in current colorectal cancer diagnosis, the present invention provides a cfDNA methylation detection method based on a multiplex quantitative fluorescence PCR detection system, which can detect more than ten sites in one reaction, and can perform cfDNA methylation detection for colorectal cancer diagnosis in a relatively simple, rapid and low-cost manner.

[0009] To achieve the above objectives, the embodiments of the present invention adopt the following technical solutions: A method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system, the method comprising the following steps: Extract cfDNA and leukocyte gDNA; The digestion products were obtained by digesting cfDNA and leukocyte gDNA with a methylation-sensitive restriction endonuclease. The digested products were amplified using a multiplex quantitative PCR detection system to obtain amplified products. The multiplex quantitative PCR detection system includes 22 primer pairs, which are used to simultaneously amplify 15 detection sites and 7 internal reference sites. Capillary electrophoresis detection of amplification products; Determine the results of capillary electrophoresis.

[0010] According to one aspect of the present invention, the 15 detection sites are Cp4PPP2R5C, IKZF1, Cp4A4, Cp3A4, Cp2LRRC4, BCAT1, Cp3NPY, BCAN, Cp1SDC2, Cp3SDC2, Cp1SFRP2, Cp2SFRP2, Cp3PPP2R5C, Cp1NPY, and Cp2RASSF2; the upstream and downstream primer sequences for amplifying the Cp4PPP2R5C site are shown in SEQ ID NO.1 and SEQ ID NO.2; the upstream and downstream primer sequences for amplifying the IKZF1 site are shown in SEQ ID NO.3 and SEQ ID NO.4; the upstream and downstream primer sequences for amplifying the Cp4A4 site are shown in SEQ ID NO.5 and SEQ ID NO.6; the upstream and downstream primer sequences for amplifying the Cp3A4 site are shown in SEQ ID NO.7 and SEQ ID NO.8; and the upstream and downstream primer sequences for amplifying the Cp2LRRC4 site are shown in SEQ ID NO.8. The primer sequences for amplifying the BCAT1 site are shown in SEQ ID NO. 9 and SEQ ID NO. 10; the primer sequences for amplifying the Cp3NPY site are shown in SEQ ID NO. 13 and SEQ ID NO. 14; the primer sequences for amplifying the BCAN site are shown in SEQ ID NO. 15 and SEQ ID NO. 16; the primer sequences for amplifying the Cp1SDC2 site are shown in SEQ ID NO. 17 and SEQ ID NO. 18; the primer sequences for amplifying the Cp3SDC2 site are shown in SEQ ID NO. 19 and SEQ ID NO. 20; the primer sequences for amplifying the Cp1SFRP2 site are shown in SEQ ID NO. 21 and SEQ ID NO. 22; the primer sequences for amplifying the Cp2SFRP2 site are shown in SEQ ID NO. 23 and SEQ ID NO. 24; and the primer sequences for amplifying the Cp3PPP2R5C site are shown in SEQ ID NO. 19 and SEQ ID NO. 20. The primer sequences for amplifying the Cp1NPY site are shown in SEQ ID NO.25 and SEQ ID NO.26; the primer sequences for amplifying the Cp2RASSF2 site are shown in SEQ ID NO.27 and SEQ ID NO.28; and the primer sequences for amplifying the Cp2RASSF2 site are shown in SEQ ID NO.29 and SEQ ID NO.30.

[0011] According to one aspect of the invention, the 15 detection sites are distributed across 10 gene regions, namely PPP2R5C, IKZF1, ALX4, LRRC4, BCAT1, NPY, BCAN, SDC2, SFRP2, and RASSF2.

[0012] According to one aspect of the present invention, the seven internal reference sites are DB2M, TBP, HPRT1, B2M, ACTB, RPL29, and GAPDH; the upstream and downstream primer sequences for amplifying the DB2M site are shown in SEQ ID NO. 31 and SEQ ID NO. 32; the upstream and downstream primer sequences for amplifying the TBP site are shown in SEQ ID NO. 33 and SEQ ID NO. 34; the upstream and downstream primer sequences for amplifying the HPRT1 site are shown in SEQ ID NO. 35 and SEQ ID NO. 36; the upstream and downstream primer sequences for amplifying the B2M site are shown in SEQ ID NO. 37 and SEQ ID NO. 38; the upstream and downstream primer sequences for amplifying the ACTB site are shown in SEQ ID NO. 39 and SEQ ID NO. 40; the upstream and downstream primer sequences for amplifying the RPL29 site are shown in SEQ ID NO. 41 and SEQ ID NO. 42; and the upstream and downstream primer sequences for amplifying the GAPDH site are shown in SEQ ID NO. 43 and SEQ ID NO. 44.

[0013] According to one aspect of the present invention, the multiplex quantitative fluorescent PCR detection system further includes: DNA polymerase, digestion product, 2× amplification buffer, and sterile water.

[0014] According to one aspect of the present invention, the reaction conditions of the multiplex quantitative fluorescence PCR detection system are as follows: pre-denaturation at 95 degrees Celsius for 15 minutes; denaturation at 95 degrees Celsius for 30 seconds, annealing at 60 degrees Celsius for 90 seconds, extension at 72 degrees Celsius for 60 seconds for a total of 35 cycles; final extension at 72 degrees Celsius for 10 minutes, and storage at 4 degrees Celsius.

[0015] According to one aspect of the present invention, the reaction system for methylation-sensitive restriction endonuclease digestion comprises: cfDNA or gDNA, HpaII, HhaI, ExoI, 10× digestion buffer, and sterile water.

[0016] According to one aspect of the invention, the cfDNA or gDNA sample is derived from peripheral blood.

[0017] According to one aspect of the present invention, the method for determining the results of capillary electrophoresis includes the steps of: determining each site and determining the sample based on the results of each site; the determination of each site includes: when no amplification product is detected in leukocyte DNA at a certain site, if the peak height in cfDNA is ≥500 RFU, the site is determined to be strongly positive; if the peak height of the cfDNA amplification product is greater than 100 RFU but less than 500 RFU, the site is determined to be weakly positive; if amplification products are detected in both leukocyte DNA and cfDNA at a certain site, the upregulation fold of the site in cfDNA is calculated; the determination of the sample based on the results of each site includes: calculating the score for positive sites, with 2 points for strong positive sites and 1 point for weak positive sites, and a total score greater than or equal to 6, in which case the sample is determined to be positive.

[0018] The advantages of this invention are as follows: By employing the above technical solution, multiplex quantitative fluorescent PCR amplification combined with capillary electrophoresis detection is used to achieve simultaneous and effective detection of multiple methylation sites, enabling the detection of cfDNA methylation in a relatively simple, rapid, and low-cost manner; a set of sites for cfDNA methylation detection in colorectal cancer is provided, along with corresponding detection methods and result interpretation methods, achieving high sensitivity and specificity in colorectal cancer detection. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The image shows the capillary electrophoresis results of a cfDNA methylation detection method based on a multiplex quantitative fluorescence PCR detection system described in this invention for detecting cfDNA in normal individuals. Figure 2 This image shows the capillary electrophoresis results of a cfDNA methylation detection method based on a multiplex quantitative fluorescence PCR detection system described in this invention for detecting DNA from normal human leukocytes. Figure 3 This image shows the capillary electrophoresis results of a cfDNA methylation detection method based on a multiplex quantitative fluorescence PCR detection system, as described in this invention, for detecting cfDNA in colorectal cancer samples. Figure 4 This image shows the capillary electrophoresis results of leukocyte DNA detection in colorectal cancer samples using a cfDNA methylation detection method based on a multiplex quantitative fluorescence PCR detection system, as described in this invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1 Methylation data from 410 colorectal cancer tissue samples and 50 adjacent normal colorectal cancer tissue samples were obtained from the public database TCGA (The Cancer Genome Altas). Sites with a differential β value greater than 0.6 were selected, representing gene regions that were highly methylated in colorectal cancer tissue samples but relatively hypomethylated in tumor tissue. Sites previously reported in the literature for colorectal cancer methylation detection were also included. Based on this, and considering expected gene function, reported detection results in the literature, and differential β values, 48 ​​sites from 29 gene regions were selected as initial screening sites.

[0023] Peripheral blood samples were collected from 10 patients with stage IV colorectal cancer and 20 healthy individuals. cfDNA and leukocyte gDNA were extracted using standard methods, digested with methylation-sensitive restriction endonucleases, and the distribution specificity and abundance of each initial screening locus in plasma cfDNA or leukocyte gDNA were determined by qPCR. Based on the qPCR results, loci were further screened: loci showing detectable signals (CT < 32) in more than 20% of patient cfDNA and with a relative abundance greater than that in patient leukocyte gDNA (ΔΔCT > 4) were selected, while loci showing high detectable signals (CT > 30) in more than 20% of healthy individuals' cfDNA were removed. Finally, 15 loci from 11 gene regions met the criteria.

[0024] A multiplex quantitative PCR detection system was established to amplify the above 15 sites in one reaction, and the amplified products were subsequently detected by capillary electrophoresis using a sequencer.

[0025] The target fragment size in cfDNA is in the range of 150-200 bp, so the amplicon length needs to be smaller than this range, preferably around 80-120 bp, and the products should be distinguishable by size. Adding internal control sites to the system enables quantitative detection, and all sites should be located in the same fluorescence channel. After multiple rounds of modification and optimization, a multiplex fluorescent PCR detection system containing 15 detection sites and 7 internal control genes was obtained, which can effectively quantify cfDNA samples.

[0026] The constructed system was used to detect colorectal cancer samples and normal samples, and the judgment method was as follows: For each locus, the following criteria are applied: If no amplification product is detected in leukocyte DNA at a certain locus, but a clear amplification product is present in cfDNA (peak height ≥ 500 RFU), the locus is considered strongly positive; if the peak height of the cfDNA amplification product is greater than 100 RFU but less than 500 RFU, the locus is considered weakly positive. If amplification products are detected in both leukocyte DNA and cfDNA at a certain locus, the upregulation fold of that locus in cfDNA is calculated, which is the ratio of the relative peak height in cfDNA to the relative peak height in leukocyte DNA: = (locus peak height in cfDNA / average peak height of products at each control locus in cfDNA) / (locus peak height in leukocyte DNA / average peak height of products at each control locus in leukocyte DNA). If the upregulation fold is ≥ 5, the locus is considered strongly positive; if the upregulation fold is greater than 1.5 but less than 5, the locus is considered weakly positive. The sample is judged based on the results of each point: a score is calculated for each positive site: 2 points for each strong positive site and 1 point for each weak positive site. A total score of 6 or higher is considered a positive result for the sample.

[0027] Example 2 cfDNA methylation was detected in peripheral blood samples from 81 patients with colorectal cancer, including 12 patients in stage I, 20 patients in stage II, 21 patients in stage III, and 28 patients in stage IV, as well as peripheral blood samples from 40 healthy individuals.

[0028] Take 10 ml of freshly collected peripheral blood and centrifuge at 1600-2000×g for 10 minutes at 4℃. After centrifugation, use a sterile pipette to transfer the upper plasma layer to a new centrifuge tube and centrifuge at 16,000×g for 10 minutes at 4℃ to remove residual cells and platelets. Transfer the supernatant (i.e., cell-free plasma) to a new tube for cfDNA extraction. After discarding most of the plasma and red blood cell layer, retain the white blood cell layer and a small number of red blood cells. Wash the white blood cell layer with sterile PBS buffer 1-2 times to remove residual plasma and platelets, finally obtaining white blood cell clumps for gDNA extraction.

[0029] Extracting cfDNA from plasma: Take a certain volume of cell-free plasma, mix it with proteinase K and lysis buffer, vortex thoroughly, and incubate in a 56℃ water bath for 15-30 minutes to completely digest the protein; add binding buffer and mix well; transfer the mixture to a silica membrane adsorption column, centrifuge at ≥6000×g for 1-2 minutes at room temperature to allow cfDNA to specifically bind to the membrane; discard the filtrate, add two different washing buffers in sequence, centrifuge and discard the waste liquid; place the adsorption column in a new collection tube, open the cap and let it air dry for 1-2 minutes to evaporate residual ethanol, add 50-100μL of preheated elution buffer or sterile water to the center of the membrane, let it stand at room temperature for 5 minutes, centrifuge at the maximum speed (≥12000×g) for 1-2 minutes, and collect the eluent as cfDNA.

[0030] Genomic DNA extraction from leukocytes: Resuspend leukocyte clumps in PBS or lysis buffer, add proteinase K and strong lysis buffer, vortex to mix, and incubate in a 56°C water bath for at least 1 hour or overnight until the solution is clear and free of clumps, ensuring complete digestion of cell nuclei and chromosomal proteins; add anhydrous ethanol (or isopropanol) and mix to precipitate DNA, transfer the mixture to a silica membrane adsorption column, centrifuge to allow gDNA to bind to the membrane; discard the filtrate, add two different washing buffers in sequence and centrifuge, then discard the waste liquid; add 100-200 μL of preheated elution buffer to the center of the membrane, let stand at room temperature for 5 minutes, then centrifuge to collect the eluent, which is gDNA.

[0031] The extracted cfDNA or DNA was digested with methylation-sensitive restriction endonucleases. The digestion system used is shown in Table 1 below: Table 1

[0032] The 10× enzyme digestion buffer contains 500mM sodium acetate, 100mM magnesium acetate, 1g / ml bovine serum albumin, and 200nM Tris-Ac, and the pH of the 10× enzyme digestion buffer is 7.9.

[0033] Reaction conditions: digest at 30℃ for 30 minutes, digest at 37℃ for 30 minutes, digest at 60℃ for 30 minutes, and then store at 4℃.

[0034] The digested products were amplified by multiplex fluorescent PCR, and the target gene and internal control gene were detected simultaneously. The multiplex amplification detection system included PCR reaction premix and internal standard. The main components of the PCR reaction premix included hot-start Taq enzyme and amplification buffer, and all primers were mixed according to the experimentally determined proportions to prepare a primer mixture. A 50 μL basic PCR reaction system was used, where the primer mix had a primer concentration of 100 μmol / L. The target gene primer and probe sequences used are shown in Table 2 below. Table 2

[0035] The primer sequences used for the internal reference gene are shown in Table 3 below: Table 3

[0036] Prepare the PCR amplification system according to the components in Table 4 below, vortex to mix well, and then aliquot according to the number of samples: Table 4

[0037] Place each reaction tube into the PCR amplification instrument's reaction chamber and set the reaction volume to 20 µL. Perform PCR amplification according to the following program: pre-denaturation at 95°C for 15 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 90 seconds, extension at 72°C for 60 seconds, for a total of 35 cycles; final extension at 72°C for 10 minutes, and store at 4°C.

[0038] The amplified products were detected by capillary electrophoresis using a gene analyzer. QD550 internal standard and formamide were mixed at a ratio of 2.5:100. 12.5 μL of the mixture was added to a 96-well plate, followed by 1 μL of the amplified product sample or allele standard. The mixture was incubated for several minutes, denatured at 95°C for 3 minutes, and immediately placed on ice for 3 minutes. After centrifugation, the plate was placed on an ABI 5000xL sequencer for detection.

[0039] The results of capillary electrophoresis are shown in the figure below. Figures 1-4 As shown, Figure 1 This is the cfDNA result for a normal person. Figure 2 The results of the normal human leukocyte DNA test show that most of the detection sites other than the internal control site have no amplification products or very low signal intensity. For the few detection sites that have amplification products, the relative intensity of the expected product relative to the internal control is basically consistent with the relative intensity of the corresponding site in the same sample of leukocyte DNA test. Figure 3 For colorectal cancer sample cfDNA results, Figure 4 The results of leukocyte DNA testing in colorectal cancer samples show strong amplification signals at multiple detection sites (indicated by arrows), with relative signal intensity much higher than that at corresponding sites in leukocyte DNA from the same patient.

[0040] The method for judging the test results is as follows: First, determine the location: If no amplification product is detected in leukocyte DNA at a certain site, and there is a clear amplification product in cfDNA (peak height ≥ 500 RFU), the site is considered strongly positive. If the peak height of the cfDNA amplification product is greater than 100 RFU but less than 500 RFU, the site is considered weakly positive. When amplification products are detected in both leukocyte DNA and cfDNA at a certain site, the upregulation fold of that site in cfDNA is calculated, which is the ratio of the relative peak height in cfDNA to the relative peak height in leukocyte DNA. This ratio is calculated as: (Cyclical peak height in cfDNA / Average peak height of control sites in cfDNA) / (Cyclical peak height in leukocyte DNA / Average peak height of control sites in leukocyte DNA). If the upregulation fold is ≥5, the site is considered strongly positive; if the upregulation fold is greater than 1.5 but less than 5, the site is considered weakly positive. The sample is judged based on the results of each point: Calculate the score for positive sites: 2 points for each strong positive site and 1 point for each weak positive site. A total score of 6 or higher is considered a positive result for the sample.

[0041] The colorectal cancer samples and healthy human samples were evaluated according to the standards, and the results are shown in Table 5 below: Table 5

[0042] Based on this standard, the sensitivity of this detection system for colorectal cancer patients is 91.4% (74 / 81), and the specificity is 97.5% (39 / 40).

[0043] This demonstrates that the cfDNA methylation detection method, combined with capillary electrophoresis, enables the simultaneous and effective detection of multiple methylation sites, achieving high sensitivity and specificity for colorectal cancer detection, indicating the effectiveness of the overall detection process.

[0044] The advantages of this invention are as follows: By employing the above technical solution, multiplex quantitative fluorescent PCR amplification combined with capillary electrophoresis detection is used to achieve simultaneous and effective detection of multiple methylation sites, enabling the detection of cfDNA methylation in a relatively simple, rapid, and low-cost manner; a set of sites for cfDNA methylation detection in colorectal cancer is provided, along with corresponding detection methods and result interpretation methods, achieving high sensitivity and specificity in colorectal cancer detection.

[0045] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for detecting cfDNA methylation based on a multiplex quantitative fluorescent PCR detection system, characterized in that, The method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system includes the following steps: Extract cfDNA and leukocyte gDNA; The digestion products were obtained by digesting cfDNA and leukocyte gDNA with a methylation-sensitive restriction endonuclease. The digested products were amplified using a multiplex quantitative PCR detection system to obtain amplified products. The multiplex quantitative PCR detection system includes 22 primer pairs, which are used to simultaneously amplify 15 detection sites and 7 internal reference sites. Capillary electrophoresis detection of amplification products; Determine the results of capillary electrophoresis.

2. The method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system according to claim 1, characterized in that, The 15 detection sites are Cp4PPP2R5C, IKZF1, Cp4A4, Cp3A4, Cp2LRRC4, BCAT1, Cp3NPY, BCAN, Cp1SDC2, Cp3SDC2, Cp1SFRP2, Cp2SFRP2, Cp3PPP2R5C, Cp1NPY, and Cp2RASSF2. The primer sequences for amplifying the Cp4PPP2R5C site are shown in SEQ ID NO.1 and SEQ ID NO.2; the primer sequences for amplifying the IKZF1 site are shown in SEQ ID NO.3 and SEQ ID NO.4; the primer sequences for amplifying the Cp4A4 site are shown in SEQ ID NO.5 and SEQ ID NO.6; the primer sequences for amplifying the Cp3A4 site are shown in SEQ ID NO.7 and SEQ ID NO.8; and the primer sequences for amplifying the Cp2LRRC4 site are shown in SEQ ID NO.9 and SEQ ID NO.

1. The primer sequences for amplifying the BCAT1 site are shown in SEQ ID NO. 10; the primer sequences for amplifying the Cp3NPY site are shown in SEQ ID NO. 13 and SEQ ID NO. 14; the primer sequences for amplifying the BCAN site are shown in SEQ ID NO. 15 and SEQ ID NO. 16; the primer sequences for amplifying the Cp1SDC2 site are shown in SEQ ID NO. 17 and SEQ ID NO. 18; the primer sequences for amplifying the Cp3SDC2 site are shown in SEQ ID NO. 19 and SEQ ID NO. 20; the primer sequences for amplifying the Cp1SFRP2 site are shown in SEQ ID NO. 21 and SEQ ID NO. 22; the primer sequences for amplifying the Cp2SFRP2 site are shown in SEQ ID NO. 23 and SEQ ID NO. 24; and the primer sequences for amplifying the Cp3PPP2R5C site are shown in SEQ ID NO. 25 and SEQ ID NO.

16. As shown in NO.26; the upstream and downstream primer sequences for amplifying the Cp1NPY site are shown in SEQ ID NO.27 and SEQ ID NO.28; the upstream and downstream primer sequences for amplifying the Cp2RASSF2 site are shown in SEQ ID NO.29 and SEQ ID NO.

30.

3. The method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system according to claim 2, characterized in that, The 15 detection sites are distributed across 10 gene regions, namely PPP2R5C, IKZF1, ALX4, LRRC4, BCAT1, NPY, BCAN, SDC2, SFRP2, and RASSF2.

4. The method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system according to claim 1, characterized in that, The seven internal reference sites are DB2M, TBP, HPRT1, B2M, ACTB, RPL29, and GAPDH. The primer sequences for amplifying site DB2M are shown in SEQ ID NO. 31 and SEQ ID NO. 32; the primer sequences for amplifying site TBP are shown in SEQ ID NO. 33 and SEQ ID NO. 34; the primer sequences for amplifying site HPRT1 are shown in SEQ ID NO. 35 and SEQ ID NO. 36; the primer sequences for amplifying site B2M are shown in SEQ ID NO. 37 and SEQ ID NO. 38; and the primer sequences for amplifying site ACTB are shown in SEQ ID NO. 39 and SEQ ID NO.

40. The upstream and downstream primer sequences for amplifying the RPL29 site are shown in SEQ ID NO.41 and SEQ ID NO.42; the upstream and downstream primer sequences for amplifying the GAPDH site are shown in SEQ ID NO.43 and SEQ ID NO.

44.

5. The method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system according to claim 1, characterized in that, The multiplex quantitative fluorescence PCR detection system also includes: DNA polymerase, digestion products, 2× amplification buffer, and sterile water.

6. The method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system according to claim 1, characterized in that, The reaction conditions of the multiplex quantitative fluorescence PCR detection system are as follows: pre-denaturation at 95 degrees Celsius for 15 minutes; denaturation at 95 degrees Celsius for 30 seconds, annealing at 60 degrees Celsius for 90 seconds, extension at 72 degrees Celsius for 60 seconds for a total of 35 cycles; final extension at 72 degrees Celsius for 10 minutes, and storage at 4 degrees Celsius.

7. The method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system according to claim 1, characterized in that, The reaction system for methylation-sensitive restriction endonuclease digestion includes: cfDNA or gDNA, HpaII, HhaI, ExoI, 10× digestion buffer, and sterile water.

8. The method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system according to claim 7, characterized in that, The cfDNA or gDNA samples were obtained from peripheral blood.

9. The method for detecting cfDNA methylation based on a multiplex quantitative fluorescence PCR detection system according to claim 1, characterized in that, The method for determining the results of capillary electrophoresis includes the following steps: determining each site and determining the sample based on the results of each site; The determination of each site includes: if no amplification product is detected in leukocyte DNA at a certain site, and the peak height in cfDNA is ≥500 RFU, the site is determined to be strongly positive; if the peak height of the cfDNA amplification product is greater than 100 RFU but less than 500 RFU, the site is determined to be weakly positive; if amplification products are detected in both leukocyte DNA and cfDNA at a certain site, the upregulation fold of that site in cfDNA is calculated; the determination of the sample based on the results of each site includes: calculating the score of the positive site, with a strong positive site scoring 2 points and a weak positive site scoring 1 point, and a total score greater than or equal to 6, the sample is determined to be positive.

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