Tumor detection method and reagent

Abstract

The invention relates to a DNA methylation detection method and a reagent. Specifically, the invention relates to a method for diagnosing whether a tumor exists in a subject and whether a tumor postoperative minimal residual focus exists or not by detecting a methylation marker in free DNA in a sample from the subject to judge the methylation level of the DNA, and judging the postoperative prognosis of the subject suffering from the tumor. Methods of predicting postoperative recurrence or assessing the efficacy of a treatment on a subject having a tumor. The invention also relates to a marker, a kit and a detection reagent for the method.

Description

Tumor detection methods and reagents Technical Field The present invention relates to the field of medical diagnosis, and in particular to the diagnosis, prognosis and efficacy evaluation of tumors, and also to a kit and reagents used for the diagnosis, prognosis and efficacy evaluation. Background Art Cancer has become a major public health problem worldwide. More than 10 million people die of cancer every year. Generally speaking, different treatments can be used for cancer patients according to clinical stages. For example, early and middle stage patients can be treated with surgery, while late stage patients can be treated with radiotherapy, chemotherapy, targeted therapy, immunotherapy, etc. The treatment effect of patients is directly related to the clinical stage. The 5-year survival rate of early-stage patients can reach 90%, while that of late-stage patients may be as low as 10%. Therefore, early detection and diagnosis of tumors through various screening methods can significantly improve the efficacy and prognosis of patients. Take colorectal cancer as an example. It originates from the colorectal mucosal epithelium and is one of the most common digestive system malignancies. Its incidence ranks third among malignant tumors worldwide, and its mortality ranks second. [1] The incidence and mortality of colorectal cancer in my country have shown an upward trend in the past 10 years. [2] Colorectal cancer is mainly caused by the evolution of intestinal polyps over 10-15 years. The early symptoms of the disease are relatively hidden, resulting in some patients having developed to the middle and late stages of the tumor when diagnosed, and the prognosis is poor. [3] The survival rate of colorectal cancer patients decreases significantly with the increase of clinical stage. The five-year survival rate of patients with early localized tumors is 90%, while the survival rate of patients with advanced stage is about 15%. [4] The study showed that secondary prevention based on screening played a 53% role in reducing the mortality rate of colorectal cancer, while tertiary prevention based on clinical treatment played only a 12% role in reducing the mortality rate of colorectal cancer. [5] These evidences show that early screening is the most important means to reduce the mortality rate of colorectal cancer. Therefore, early screening of colorectal cancer is of great significance. Colonoscopy is currently the gold standard for the diagnosis of colorectal cancer, with a high detection rate, but it requires strict intestinal preparation and is invasive, resulting in poor compliance among examinees. In my country, large-scale colonoscopy screening for early screening of colorectal cancer is difficult to implement due to factors such as insufficient colonoscopy resources, differences in physician skills, a large population, and traditional culture, especially in remote and underdeveloped areas. The guaiac fecal occult blood test (gFOBT) and immunochemical fecal occult blood test (FIT) based on stool testing are simple and convenient to operate, but have defects such as low sensitivity and poor specificity. [9] Therefore, the development of new detection technologies for early diagnosis of colorectal cancer has important application value and practical significance. However, even if the tumor is discovered at an early stage and radical surgery is performed, a considerable proportion of patients will still experience recurrence and metastasis. The probability of recurrence and metastasis is usually related to the stage of the tumor. In clinical practice, it is often considered whether to give postoperative adjuvant therapy to reduce the probability of recurrence according to the tumor stage. However, the accuracy (including sensitivity and specificity) of this prognostic assessment based on tumor staging and postoperative dynamic follow-up (including imaging examinations and serological examinations) has a lot of room for improvement. Let's take colorectal cancer as an example. The main treatments for colorectal cancer include surgery and postoperative adjuvant radiotherapy and chemotherapy. However, approximately 35% of colorectal cancer patients experience recurrence and metastasis after surgery, and 80% of them occur within 2 years after surgery.

[0010] Recurrence and metastasis are the main reasons for poor prognosis in patients with colorectal cancer.

[0011] Timely detection of postoperative recurrence and metastasis, followed by clinical intervention, can effectively improve the prognosis of colorectal cancer patients. [12,13] At present, the monitoring of postoperative recurrence and metastasis of colorectal cancer mainly relies on imaging examinations such as CT and protein markers such as carcinoembryonic antigen (CEA). CT examination requires tumor recurrence and metastasis to reach a certain volume before it can be detected, so the sensitivity of early monitoring of recurrence is poor. CEA is only positive in about 50% of patients with postoperative recurrence and metastasis, and the detection sensitivity is low.

[0014] CT and CEA are used for postoperative monitoring of colorectal cancer. When they are discovered, the tumor recurrence and metastasis have often reached a certain extent, and effective clinical intervention cannot be carried out in time. Therefore, the development of effective monitoring technology for postoperative recurrence and metastasis of colorectal cancer can timely intervene clinically in patients with recurrence and metastasis, and improve patient prognosis. With the rapid development of molecular diagnostic technology, liquid biopsy technology that detects circulating tumor DNA (ctDNA) in the blood as a molecular diagnostic marker has received widespread attention. Taking peripheral blood plasma as an example, the free DNA (cfDNA) it contains may come from a variety of organs and cells. White blood cells (including neutrophils, B cells and T cells, etc.) are the main source of cfDNA in plasma, and vascular endothelial cells, hepatic parenchymal cells, etc. can also provide a small amount of cfDNA. [33,34] . The cfDNA derived from these cells can usually be considered normal DNA. In the plasma of cancer patients or individuals with precancerous lesions, there may also be free DNA derived from tumor cells, usually called free tumor DNA (circulating tumor DNA, ctDNA). Compared with tissue biopsy, liquid biopsy has the advantages of being non-invasive, fast, easy to dynamically monitor, and not easily affected by tumor heterogeneity.

[0015] Compared with fecal occult blood test, liquid biopsy has higher sensitivity and specificity and is not easily affected by diet and other factors.

[0016] ctDNA is tumor cell-derived DNA in peripheral blood, which is released into peripheral blood after tumor cell necrosis and apoptosis. It has an average length of about 160bp and a short half-life. It carries genetic and epigenetic changes consistent with tumor tissue, such as gene mutations, copy number variations, methylation abnormalities, etc.

[0017] , can be used for tumor screening, molecular typing, efficacy evaluation, prognosis evaluation, postoperative residual lesion detection and dynamic monitoring, etc. The occurrence and development of tumors is a multi-factor, multi-stage process, and epigenetic changes are deeply involved in the occurrence and development of colorectal cancer. DNA methylation is one of the main changes in epigenetic changes. It refers to the process in which a methyl group is covalently bound to the 5th carbon position of cytosine in the genomic CpG dinucleotide with S-adenosyl methionine (SAM) as a methyl donor under the catalysis of DNA methyltransferase (DNMT). [6] Studies have shown that DNA methylation status begins to change abnormally in the early stages of tumor development [7] DNA methylation mainly participates in the occurrence and development of colorectal cancer through high methylation of CpG islands, which leads to downregulation of corresponding gene expression and low methylation of the genome, which causes genomic instability. [8] Many studies have shown that ctDNA methylation can be used for early diagnosis of cancer, recurrence monitoring, and prognosis assessment. [18,19] . In most cases, especially in early-stage tumors, after radical surgery, and after drug treatment, the relative content of ctDNA in plasma and other body fluids is low, even lower than 0.1%, or even lower than 0.01%. Most of the free DNA in plasma comes from normal cells (leukocytes, vascular endothelial cells, hepatocytes, etc.), and these noise signals greatly increase the difficulty of ctDNA monitoring. Taking DNA methylation markers as an example, if the free DNA from normal cells carries DNA methylation signals, even if the DNA methylation level in these normal cells is low, it may cause false positive signals and affect clinical monitoring results. Therefore, in the screening process of DNA methylation markers, it is necessary to consider both the methylation level in tumor cells and the DNA methylation level in normal cells that may potentially release cfDNA. It is further necessary to consider the degree of cfDNA release by normal cells, so as to formulate a strict and executable strategy. Epi proColon is the first blood test product approved by the U.S. FDA for colorectal cancer screening. It detects the methylation of the SEPTIN9 gene associated with colorectal cancer. Epi proColon uses HeavyMethyl real-time PCR technology to detect the target gene SEPTIN9 and the internal reference gene ACTB in a single qPCR reaction.

[0020] . This method extracts cfDNA from plasma, and after sulfite conversion, methylated cytosine remains unchanged, and unmethylated cytosine is converted to un-uracil. On this basis, a non-extendable oligonucleotide sequence is designed as a blocker for the unmethylated SEPTIN9 gene sequence to achieve specific amplification of methylated SEPTIN9. In the reaction system, this method uses two different fluorescent groups to label the target gene and the internal reference gene respectively to achieve specific signal recognition of the target gene SEPTIN9. ACTB is used as an internal reference gene to reflect the template amount in the qPCR system.

[0021] . This technology has the following defects: 1. Only the methylation status of a single site of a gene (SEPTIN9) is detected, which has a low sensitivity. The detection rate of colorectal cancer in asymptomatic people is 48.2%, and the specificity is 91.5%; the sensitivity in patients with stage I-IV colorectal cancer is 35.0%, 63.0%, 46.0%, and 77.4%, respectively.

[0022] , the sensitivity is insufficient, especially in the early stages such as stage I colorectal cancer patients, and there is a serious problem of missed detection when used for early tumor detection. 2. Since the amount of ctDNA in plasma is relatively low, sulfite conversion causes significant damage to the DNA template, resulting in obvious DNA breakage. The purification process after conversion further causes DNA loss. These factors lead to a further decrease in the absolute amount of target methylated DNA molecules in the detection system. Since only the methylation of one target gene, SEPTIN9, is detected, this method is limited in its application in the detection of minimal residual disease (MRD) after colorectal cancer surgery, recurrence monitoring, and prognosis assessment. An existing study has shown that Epi proColon technology can successfully detect 15 out of 21 recurrent patients, and the positive detection rate is slightly lower than that of the imaging method (71.4% vs 85.7%).

[0023] . 3. It is necessary to ensure that the blocker binds efficiently and specifically to the non-methylated sequence in the target region. If the binding efficiency is insufficient, false positive signals will result; if the binding specificity is insufficient, the sensitivity of the target methylated sequence detection will be reduced. 4. The result is a qualitative test. Due to the low content of ctDNA in peripheral blood and the weak signal of single gene detection, only qualitative results can be interpreted and quantitative analysis cannot be performed. ColonAiQ (Chang Aike), a multi-gene methylation detection method developed by Xunyuan Biotechnology, has the functions of early screening for colorectal cancer and postoperative recurrence monitoring. ColonAiQ uses multiplex qPCR technology to detect 6 methylation markers (SEPTIN9, BCAT1, IKZF1, BCAN, SEPT9 region 2, VAV3). After sulfite conversion, pre-amplification, qPCR and other steps, the MHLcot algorithm is used to comprehensively evaluate the samples. The comprehensive sensitivity of ColonAiQ for detecting colorectal cancer stages I-IV is 86%, the sensitivity for progressive adenomas is 42%, and the detection specificity for healthy people is 92%. The R&D team also explored the application potential of Chang Aike in colorectal cancer recurrence monitoring. There was a significant difference in the ColonAiQ score of postoperative samples between the recurrence group and the non-recurrence group (P=0.00017)

[0024] . This technology has the following defects: 1. The sample processing process is complicated and requires pre-amplification. The pre-amplification step can easily cause laboratory PCR product contamination, making it difficult to apply clinically. 2. The MHLcot algorithm is required to interpret the results and integrate the test results of 6 genes. The algorithm is complex and inconvenient for clinical use. 3. Testing the six genes separately increases the workload and cost, and clinical testing is prone to operational errors. GRAIL has developed a blood-based "pan-cancer" early screening method. First, whole-genome bisulfite sequencing (WGBS) was performed on the tissues and buffy coats of a large number of subjects to establish a pan-cancer methylation sequencing database. Through bioinformatics analysis, a targeted methylation sequencing panel for pan-cancer screening was identified, covering 103,456 methylated regions and a total of 1,116,720 CpG sites. This targeted methylation sequencing panel was then used to test the subjects' cfDNA. In the validation cohort, the specificity was 99.8%, and the sensitivity for detecting more than 50 types of cancer in stages I-III was 43.9%. The detection sensitivity increased with the increase in stage, including 18% in stage I, 43% in stage II, and 81% in stage III. In the detection of colorectal cancer patients, the detection rate was 40-45% in stage I, 70-75% in stage II, and 76-80% in stage III.

[0025] . This technology has the following defects: 1. This method uses targeted methylation sequencing, which has high sequencing costs, long cycles, and difficult data analysis. It requires specialized analysis equipment and personnel, and is not easy to promote clinically. 2. This method includes methylation markers for multiple cancers and has poor sensitivity for a single cancer type, such as colorectal cancer. In view of these defects of the existing technology, the single-tube multiple methylation detection method developed by the applicant team in the early stage can realize the quantitative analysis of methylation signals, which can be used for colorectal cancer screening, neoadjuvant chemoradiotherapy efficacy evaluation, postoperative prognosis, postoperative residual lesion detection, dynamic follow-up, early detection of recurrence and metastasis, etc.

[0026] The method designs 10 detection targets in the positive and negative strand regions of the SEPTIN9 gene, performs single-tube multiplex PCR amplification and multiplex signal enrichment and detection based on Taqman probes (see CN112159844B). Due to the application of multiplex PCR reactions, the sensitivity of detection is greatly improved. However, since this method still amplifies and detects multiple regions of a single SEPTIN9 gene, its detection specificity needs to be further improved due to the level of SEPTIN9 gene methylation and the specificity of the marker itself. In some clinical application scenarios, such as postoperative minimal residual disease (MRD) detection, high specificity has better clinical value. Therefore, the art still needs DNA methylation markers with higher tumor specificity, and methods that can perform highly specific quantitative analysis of methylation markers to detect tumors. The art also needs methods for screening DNA methylation markers with high tumor specificity. Summary of the invention The present invention provides a general method for screening markers of highly tumor-specific DNA methylation. The screened markers have extremely high tumor specificity and extremely low background signals in body fluids such as plasma. Liquid biopsy technology based on these markers can achieve a variety of clinical applications (early cancer screening, recurrence prediction, dynamic monitoring and efficacy evaluation, etc.). The present invention also provides methods for further verification and screening of markers. The present invention realizes cancer screening, prognosis evaluation, MRD detection and recurrence monitoring through the combined detection of DNA methylation markers of multiple different genes in the blood. A blood-based multiplex quantitative methylation-specific PCR (mqMSP) detection method was established, which contains 11 colorectal cancer-specific DNA methylation markers. The sensitivity and specificity of this method for colorectal cancer screening were evaluated by testing the plasma of healthy people confirmed by colonoscopy and preoperative colorectal cancer patients. By testing the plasma of colorectal cancer patients within 2 weeks after surgery, the clinical application value of this method for colorectal cancer MRD detection, prognosis assessment and recurrence monitoring was evaluated. In addition, the clinical application value of this method for colorectal cancer recurrence monitoring was evaluated by dynamically monitoring the plasma of patients with colorectal cancer after surgery. Compared with the previous inventions of the applicant's team, the present invention provides a marker screening method with higher specificity. The detection specificity of related DNA methylation markers in healthy people is significantly improved, and it has better detection performance in colorectal cancer MRD detection, prognosis assessment and recurrence monitoring, which can better guide clinical postoperative management. The present invention provides a method for screening highly tumor-specific DNA methylation markers for liquid biopsy to detect tumor signals. The method includes performing DNA methylation analysis on tumor tissue samples, adjacent normal tissue samples, white blood cell samples in the blood, etc., setting a series of screening criteria for DNA methylation levels, and screening out DNA methylation markers that can be used for liquid biopsy, which have high tumor specificity. The combination of markers (at least 2 markers) has good sensitivity and high specificity in a variety of clinical scenarios. Specifically, the present invention relates to the following aspects: On the one hand, the present invention relates to a method for diagnosing whether a tumor or precancerous lesion exists in a subject, whether a tiny residual tumor lesion exists, determining the postoperative prognosis of a patient with a tumor, predicting the postoperative recurrence of a tumor patient, or evaluating the therapeutic effect on a tumor patient, the method comprising detecting a group of DNA methylation markers in free DNA in a sample from the subject to determine the methylation level of the DNA, and if the methylation level is higher than the DNA methylation level of a normal control sample, it is determined that a tumor or precancerous lesion exists in the subject, a tiny residual tumor lesion exists, the postoperative prognosis of a tumor patient is poor, the tumor patient is prone to recurrence after surgery, or the therapeutic effect on the subject is poor, wherein the free DNA in the subject's sample is derived from two major types of cells, namely, tumor or precancerous lesion cells, and non-cancerous cells, wherein the DNA methylation markers are characterized in that the methylation level of each DNA methylation marker in the non-cancerous cell in the subject's sample is relatively low, and at least one DNA methylation marker in the group of methylation markers has a relatively high methylation level in tumor or precancerous lesion cells. In one embodiment, the methylation marker is characterized in that the methylation level in tumor or precancerous lesion cells differs from that in corresponding normal cells by at least 15%, 20%, or even 25%. In one embodiment, the sample is selected from the group consisting of body fluids, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, stool, and tissue samples. In one embodiment, the non-cancerous cells in the subject's sample include cells containing genomic DNA, such as neutrophils, B cells, T cells, vascular endothelial cells, hepatocytes, etc., and when the non-cancerous cells are neutrophils, B cells, T cells, etc., the DNA methylation of each DNA methylation marker in these cells is less than 2%, less than 1.5%, or even less than 1%; when the non-cancerous cells are vascular endothelial cells, hepatocytes, etc., the DNA methylation of each DNA methylation marker in these cells is less than 15%, or even less than 10%. In one embodiment, the methylation level of the methylation marker in non-cancerous cells in a subject's sample is less than 0.1%, and more preferably, is lower than the detection limit or detection threshold of one or more DNA methylation detection methods. In one embodiment, the methylation level of the methylation marker in the peritumoral tissue is low, preferably less than 20%, more preferably less than 10%, more preferably less than 5%, and most preferably less than 1%. In one embodiment, the set of DNA methylation markers comprises at least 2, preferably at least 5, more preferably at least 10 methylation markers. In one embodiment, the methylation marker is two or more selected from ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1 and SFMBT2, preferably all of ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1 and SFMBT2. In one embodiment, the tumor is colorectal cancer. In one embodiment, the detection of the methylation markers is performed using multiplex quantitative methylation-specific PCR, Optionally, the multiplex quantitative methylation-specific PCR includes determination of an internal reference gene ACTB. In one embodiment, the multiplex quantitative methylation-specific PCR uses primers and probes for the methylation markers described above, as well as primers and probes for the internal reference gene ACTB, wherein the primers are selected from the following primer pairs: SEQ ID NO: 4 or a sequence having sufficient identity to SEQ ID NO: 4 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 5 or a sequence having sufficient identity to SEQ ID NO: 5 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 7 or a sequence having sufficient identity to SEQ ID NO: 7 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 8 or a sequence having sufficient identity to SEQ ID NO: 8 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 10 or a sequence having sufficient identity to SEQ ID NO: 10 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 11 or a sequence having sufficient identity to SEQ ID NO: 11 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 13 or a sequence having sufficient identity to SEQ ID NO: 13 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 14 or a sequence having sufficient identity to SEQ ID NO: 14 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 16 or a sequence having sufficient identity to SEQ ID NO: 16 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 17 or a sequence having sufficient identity to SEQ ID NO: 17 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 19 or a sequence having sufficient identity to SEQ ID NO: 19 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 20 or a sequence having sufficient identity to SEQ ID NO: 20 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:22 or a sequence having sufficient identity to SEQ ID NO:22 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:23 or a sequence having sufficient identity to SEQ ID NO:23 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:25 or a sequence having sufficient identity to SEQ ID NO:25 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:26 or a sequence having sufficient identity to SEQ ID NO:26 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:28 or a sequence having sufficient identity to SEQ ID NO:28 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:29 or a sequence having sufficient identity to SEQ ID NO:29 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:31 or a sequence having sufficient identity to SEQ ID NO:31 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:32 or a sequence having sufficient identity to SEQ ID NO:32 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:34 or a sequence having sufficient identity to SEQ ID NO:34 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:35 or a sequence having sufficient identity to SEQ ID NO:35 to ensure sensitivity and specificity of PCR amplification; and SEQ ID NO: 1 or a sequence having sufficient identity with SEQ ID NO: 1 to ensure the sensitivity and specificity of PCR amplification, and SEQ ID NO: 2 or a sequence having sufficient identity with SEQ ID NO: 2 to ensure the sensitivity and specificity of PCR amplification; and, The probe is selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3 or a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3 that has sufficient consistency to ensure the sensitivity and specificity of PCR amplification. On the other hand, the present invention relates to a kit for diagnosing whether a tumor or precancerous lesion exists in a subject, whether a small residual tumor lesion exists, judging the prognosis of a patient with a tumor after surgery, predicting the recurrence of a patient with a tumor after surgery, or evaluating the therapeutic effect on a patient with a tumor, which comprises a reagent for detecting the markers described above. Optionally, the kit further comprises a reagent for detecting the internal reference gene ACTB. In one embodiment, the reagents for detecting methylation markers and internal reference genes are primer pairs and probes, wherein the primers are selected from the following primer pairs: SEQ ID NO: 4 or a sequence having sufficient identity to SEQ ID NO: 4 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 5 or a sequence having sufficient identity to SEQ ID NO: 5 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 7 or a sequence having sufficient identity to SEQ ID NO: 7 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 8 or a sequence having sufficient identity to SEQ ID NO: 8 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 10 or a sequence having sufficient identity to SEQ ID NO: 10 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 11 or a sequence having sufficient identity to SEQ ID NO: 11 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 13 or a sequence having sufficient identity to SEQ ID NO: 13 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 14 or a sequence having sufficient identity to SEQ ID NO: 14 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 16 or a sequence having sufficient identity to SEQ ID NO: 16 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 17 or a sequence having sufficient identity to SEQ ID NO: 17 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 19 or a sequence having sufficient identity to SEQ ID NO: 19 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 20 or a sequence having sufficient identity to SEQ ID NO: 20 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:22 or a sequence having sufficient identity to SEQ ID NO:22 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:23 or a sequence having sufficient identity to SEQ ID NO:23 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:25 or a sequence having sufficient identity to SEQ ID NO:25 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:26 or a sequence having sufficient identity to SEQ ID NO:26 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:28 or a sequence having sufficient identity to SEQ ID NO:28 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:29 or a sequence having sufficient identity to SEQ ID NO:29 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:31 or a sequence having sufficient identity to SEQ ID NO:31 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:32 or a sequence having sufficient identity to SEQ ID NO:32 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:34 or a sequence having sufficient identity to SEQ ID NO:34 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:35 or a sequence having sufficient identity to SEQ ID NO:35 to ensure sensitivity and specificity of PCR amplification; and SEQ ID NO: 1 or a sequence having sufficient identity with SEQ ID NO: 1 to ensure the sensitivity and specificity of PCR amplification, and SEQ ID NO: 2 or a sequence having sufficient identity with SEQ ID NO: 2 to ensure the sensitivity and specificity of PCR amplification; and, The probe is selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3, or a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3 that has sufficient consistency to ensure the sensitivity and specificity of PCR amplification. In another aspect, the present invention relates to the primer pair described above. On the other hand, the present invention relates to a nucleic acid molecule used as a probe, which is selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3, or a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3 that has sufficient consistency to ensure the sensitivity and specificity of PCR amplification. In various aspects of the present invention, the primers, probes and competitor sequences used are not limited to those listed in the above-mentioned tables and sequence numbers, but include sequences with sufficient consistency therewith. The sequences with sufficient consistency include those sequences with at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% consistency with the sequences represented by the sequence numbers, and still retain their functions. Among them, it is preferred that the 10 consecutive nucleotides at the 3' end have at least 80% consistency with the primer sequence of the present invention. Those skilled in the art can determine sequence consistency by conventional procedures. In various aspects of the present invention, those skilled in the art can also design corresponding primers and / or probes for analysis of DNA methylation within the upstream and downstream of the genomic regions corresponding to the primer sequences listed in the above-mentioned tables and sequence numbers, such as within 1000 bp, more preferably within 500 bp, and more preferably within 200 bp. Analysis methods include methylation-specific PCR, quantitative PCR after methylation-sensitive restriction endonuclease digestion, post-capture analysis of DNA methylation (quantitative PCR or high-throughput sequencing, etc.), etc. BRIEF DESCRIPTION OF THE DRAWINGS FIG1 shows the methylation levels of some markers in buffy coat, normal tissue adjacent to cancer, and colorectal cancer tissue. Figure 2 shows the comparison of single-target and multi-target marker detection results. Figure 3 shows the evaluation of detection sensitivity of the multi-target assay. FIG4 shows the detection limit evaluation of the multiplex detection method of the present invention. FIG5 shows the comparison of ΔCq results of the present invention for detecting methylation levels in healthy subjects and colorectal cancer patients (one-way analysis of variance). FIG6 shows the results of ROC curve analysis of subjects in colorectal cancer and healthy subjects (ROC of CRC vs healthy controls). FIG7 shows the analysis results of the relationship between postoperative recurrence and ctDNA status in patients with colorectal cancer. FIG8 shows the results of a study on MRD detection and recurrence-free survival (RFS) in 76 patients with stage II colorectal cancer. DETAILED DESCRIPTION Through the analysis of sequencing data and TCGA database data by this research team, differentially methylated regions (DMRs) with high methylation in colorectal cancer tumor tissues were screened out. The present invention uses fluorescent quantitative methylation-sensitive PCR (qMSP) technology to design qPCR primers and probes based on bisulfite conversion for the 10 DMRs screened above, exclude primer pairs that have amplification in bisulfite-converted buffy coat samples, screen out primer pairs that amplify better in bisulfite-converted colon cancer cell line (HCT116) DNA, and use AutoDimer software to exclude primer pairs that are more likely to form primer dimers and hairpin structures, and finally form an mqMSP detection technology containing 11 qMSP assays and 1 internal reference assay. By analyzing the sensitivity and detection limit of the technology of the present invention, it is proved that the technology of the present invention can stably detect methylated DNA as low as 0.02% in a single-tube PCR reaction, and there is still a great probability (about 90%) of detecting methylation signals when the concentration of methylated DNA is as low as 0.01%. Therefore, the present invention has the potential to be applied to auxiliary diagnosis of colorectal cancer and postoperative MRD evaluation. The established mqMSP technology was used to detect clinical blood samples. By testing 96 plasma samples from healthy control subjects and 120 preoperative plasma samples from colorectal cancer patients, it was demonstrated that this method has great application potential in colorectal cancer screening and auxiliary diagnosis. The established mqMSP technology was used to detect plasma samples of 143 patients with stage I-III colorectal cancer within 2 weeks after surgery, and the relationship between ctDNA negative / positive and recurrence-free survival (RFS) of CRC patients after surgery was analyzed. It was found that the recurrence time of patients with postoperative ctDNA positive was significantly shortened, indicating a worse prognosis. It is shown that the mqMSP technology established by the present invention can be used as a marker for CRC prognosis and an indicator of the presence of MRD to detect the ctDNA methylation level in patients after surgery. The present invention screens and verifies a plurality of colorectal cancer-specific methylation markers, some of which have not been reported in the literature to support their use in the detection of colorectal cancer. The present invention uses these new methylation markers to establish an updated version of the mqMSP technology. The present invention combines multiple DNA methylation markers for detection. Compared with the currently disclosed patents such as V1-V4 (CN112159844B), it maintains a high detection sensitivity and improves the specificity of the detection. The specificity of the technology of the present invention in detecting healthy control populations is 96.9%, which is significantly better than the specificity of the original marker of 83.3%. Compared with the original detection markers, it has higher specificity at similar sensitivity, and lower false positives when detected in normal populations and asymptomatic populations. In the general population or high-risk population, due to the low proportion of tumor patients, low-specificity detection will lead to higher false positive test results, causing panic in the subjects and further hospitalization tests, resulting in medical costs and consumption of medical resources. Therefore, compared with the original detection markers, the detection markers of the present invention have higher detection specificity and better detection performance. The mqMSP technology established by the present invention can be used for colorectal cancer screening, and a positive sample test result indicates that the subject may have colorectal cancer. The mqMSP technology established by the present invention can be used to evaluate the surgical prognosis of colorectal cancer patients. A positive test result of a postoperative sample indicates that there may be a small residual lesion, indicating that the prognosis may be poor. The high specificity of the present invention enables it to be used for postoperative residual lesion analysis, for judging postoperative prognosis and the selection of postoperative adjuvant chemotherapy, with a higher positive predictive value, and its positive test is more reliable as an indicator for guiding postoperative treatment. The mqMSP technology established in the present invention can be used for postoperative recurrence monitoring of colorectal patients. A positive sample test result indicates that colorectal cancer patients may have disease recurrence, progression or metastasis. Embodiment 1 Screening of highly specific DNA methylation markers for colorectal cancer In most cases, especially in early-stage tumors, after radical surgery, and after drug treatment, the relative content of ctDNA in plasma and other body fluids is low, even lower than 0.1%, or even lower than 0.01%. Most of the free DNA in plasma comes from normal cells (leukocytes, vascular endothelial cells, hepatocytes, etc.), and these noise signals greatly increase the difficulty of ctDNA monitoring. Therefore, in the process of screening DNA methylation markers, it is necessary to consider both the methylation level in tumor cells and the DNA methylation level in normal cells that may potentially release cfDNA. It is further necessary to consider the degree of cfDNA release by normal cells, so as to formulate a strict and executable strategy. The inventor systematically analyzed the DNA methylation data in the TCGA database (including DNA methylation data of various tumors and normal tissues) and the team's own DNA methylation data (reduced representation bisulfite sequencing of 30 pairs of paired colorectal tumors and adjacent normal tissues, 15 benign polyps, 15 progressive adenoma tissues, and 10 buffy coat DNA), and obtained potential DNA methylation markers through multiple filtering, requiring the DNA methylation level to be less than 1%, DNA methylation in colorectal tumors to be at least 15% higher than that in paired adjacent normal tissues, and less than 15% in normal liver cells. Among them, 10 DNA methylation markers are shown in Table 1: Table 1 The genes and chromosomal locations of the 10 methylation markers screened 10 DMRs regions were selected as methylation markers, and primers and Taqman probes were designed for MSRE-qPCR detection. 10 cases of buffy coat, colorectal cancer and paired adjacent normal tissues were selected, and each DNA sample was subjected to mock digestion and real digestion (DNA methylation sensitive endonuclease (HpaII, HhaI, BstUI, etc.) digestion in parallel), and then SYBR green-fluorescence quantitative PCR was used for quantification, and the DNA methylation level was calculated using the following formula: Methylation % = 2 CtM-CtE ×100%; CtM and CtE represent the fluorescence quantitative PCR Ct values ​​of the samples after simulated enzyme digestion and real enzyme digestion, respectively. The methylation levels of the 10 amplicons in Table 1 were verified by MSRE-qPCR, and the results are shown in Figure 1. It can be seen that in the 10 buffy coat samples, the methylation levels of these 10 DNA markers are close to 0; in the normal tissues adjacent to the cancer, the methylation levels of these 10 markers are also extremely low, and the methylation of most markers in most normal tissue samples is less than 10%, or even less than 5%. All markers have high DNA methylation in tumor samples. This shows that the inventor's screening strategy is efficient, the screened markers are in line with expectations, and have potential clinical significance. It will be appreciated by those skilled in the art that, for the selected marker, the bases upstream and downstream thereof can also be used to design mqMSP assays and primers. Therefore, the ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1 and SFMBT2 markers listed in the above table also include a range of 500 bp, preferably 400 bp, preferably 300 bp, preferably 200 bp, preferably 100 bp, and more preferably within 50 bp upstream and downstream of the listed chromosome position. Embodiment 2 Design of internal reference reaction An internal reference reaction is designed for the ACTB gene, which is used for co-amplification with multiple methylation marker genes in the mqMSP reaction as a quality control reaction and as an indicator of the amount of DNA in the reaction. A mutation is introduced into the second to last base at the 3' end of the PCR primer (forward and reverse) sequence in the internal reference reaction to reduce the internal reference fluorescence signal (VIC fluorescence signal in the present invention) in the mqMSP reaction, release the enzyme and substrate in the reaction system for amplification and detection of the target methylation marker region, and reduce the inhibition of the methylation marker signal to be detected (FAM fluorescence signal in the present invention). The genomic coordinates of the ACTB region used in the present invention are (chr7:5536826-5536901 (hg38)), and the primer and probe sequences are shown in Table 2: Table 2 Primer and probe sequences for ACTB reaction Embodiment 3 Quality control product setting Negative and positive quality control products were set up for quality control of mqMSP reactions. The quality control products were subjected to bisulfite conversion at the same time as each batch of cfDNA samples. The experimental results of the positive and negative quality control products can indicate whether the experiment was successful and how reliable it is. The negative quality control product was a mixture of DNA from the buffy coat of 40 healthy control subjects. The positive quality control product was a mixture of DNA from the HCT116 colon cancer cell line and DNA from the buffy coat at a ratio of 1:99. 30 ng was taken as a reference sample for each reaction to evaluate the effectiveness of the experiment. 1. Experimental Methods (1) HCT116 colorectal cancer cell line (from the laboratory of Wenzhou Medical University School of Laboratory Medicine) and human buffy coat genome (from peripheral blood samples collected from volunteers of the First Affiliated Hospital of Wenzhou Medical University with informed consent) were extracted and their concentrations were measured by Qubit. (2) Preparation of positive quality control: HCT116 colorectal cancer cell line DNA and buffy coat DNA were mixed at a ratio of 1:99, and water was added to adjust the concentration to 10 ng / μl. The DNA was divided into aliquots and stored at -80°C, with 30 ng taken for each reaction. (3) Preparation of negative control: Dilute the buffy coat DNA to 10 ng / μl. Store the DNA in aliquots at -80°C, taking 30 ng for each reaction. (4) Each batch of samples should be treated with bisulfite and qPCR for both positive and negative controls. Each bisulfite-converted DNA is divided into two and reacted in duplicate in qPCR. After the reaction is completed, the Cq values ​​of the FAM and VIC signals of the duplicate reactions are calculated. 2. Experimental results For different assay formats, the standards and algorithms for the test results are shown in Table 3: where "+" represents a Cq value ≤ 45, and "-" represents no amplification signal, where ΔCq = VIC average Cq - FAM average Cq. If the qPCR reaction results of the quality control product meet the standards listed in the table below, the qPCR reaction is verified to be valid. Table 3 Quality control result judgment table Embodiment 4 Combined detection of multiple methylation markers and comparison of their sensitivity with that of a single methylation marker 1. Experimental Methods (1) Gene CELSR3 was selected as a single gene detection scheme, and its primer and probe sequences were used for the experiment. The reaction system was as follows: The reaction conditions are as follows: (2) Combined detection of multiple methylation markers Primers were designed for the 10 selected colon cancer markers, and primers and probes were designed for the positive and negative strands of the genomic region ENSG00000232377, respectively. Therefore, the final mqMSP assay contains 11-multiple target methylation assay reactions, and the ACTB assay is added as an internal reference reaction. The primer and probe sequences of the target genes in the multiple target methylation assay are shown in Table 4. Table 4 Primer and probe sequences for multiple methylation gene assays Preparation of qPCR primer and probe mix: The initial concentration of each primer was 100 μM and they were mixed in the following proportions: The initial concentration of each probe was 100 μM and they were mixed in the following proportions: The multiplex detection reaction system is as follows: The reaction conditions are as follows: (3) HCT116 cell line DNA and buffy coat DNA were mixed at a ratio of 1:99 as the sample to be tested, with a total DNA amount of 30 ng. The 1% HCT116 cell line DNA was treated with bisulfite conversion, and the same sample was compared using the two methods. 2. Experimental results As shown in Figure 2, the detection results of the same sample by the two methods differed by 3.99 Cq, and the combined detection of multiple methylation markers produced a stronger methylation signal than the detection of a single methylation marker. Embodiment 5 Evaluation of Assay Sensitivity for Multi-Target Assays 1. Experimental Methods HCT116 colorectal cancer cell line DNA (as a methylated sample) and buffy coat DNA (as an unmethylated sample) were mixed in different proportions (1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01% and 0%) to simulate samples with different degrees of methylation. 60 ng of each sample was taken for sulfite conversion. The converted DNA was divided into four and four reactions were performed in qPCR. 2. Experimental results The results of different proportions of methylated DNA detection showed that the VIC Cq values ​​of different samples were relatively stable, indicating that the amount of DNA template in the qPCR reaction of each sample was consistent. The FAM Cq value gradually increased with the decrease of the methylated DNA ratio, indicating that the FAM signal weakened with the decrease of the methylated DNA ratio (Figure 3, Table 5). When the methylated DNA ratio was as low as 0.02%, the methylation signal could still be stably detected. Compared with the original multiple detection method that could only detect 0.05% of methylated samples, the detection sensitivity of the present invention is higher. The test results are as follows: Table 5 Evaluation of detection sensitivity of multi-target assay Embodiment 6 Detection Limit Evaluation of the Present Invention 1. Experimental Methods (1) 0.02% HCT116 was transformed 20 times independently, using 60 ng DNA in each transformation reaction. The transformation product was divided into two and duplicate reactions were performed in the qPCR reaction. (2) 0.01% HCT116 was transformed 20 times independently, using 60 ng DNA in each transformation reaction. The transformation product was divided into two and duplicate reactions were performed in the qPCR reaction. (3) Collect statistics on the test results of positive and negative quality control products on different dates and conduct simultaneous analysis. 2. Experimental results: The conversion and detection of 0.02% and 0.01% methylated samples were repeated for more than 20 times. The ΔCq of the detection results of the 0.02% methylated DNA samples were all higher than the positive threshold (ΔCq=-4), and the positive signal detection rate was 100%. However, 2 detection results of the 0.01% methylated DNA samples were lower than the positive threshold, and the positive signal detection rate was 90% (Table 6, Figure 4). Therefore, it can be considered that the detection limit of the multiple detection method is 0.02% methylated DNA. This detection limit is significantly better than the original (CN112159844B) detection limit of 0.05%. Table 6 Detection limit evaluation of multiple detection methods Embodiment 7 The present invention is used for auxiliary diagnosis of colorectal cancer 1. Experimental Methods (1) From 2016 to 2022, 120 subjects with colorectal cancer and 96 healthy control subjects were enrolled, and 10 mL of venous blood was collected from the subjects. (2) Plasma was obtained by separating whole blood by two centrifugations and stored at -80°C. (3) Peripheral blood free DNA (cfDNA) was extracted using the Apostle cell-free DNA extraction kit (Apostle, Cat: A17622CN) and quantified using Qubit. (4) Take 10-50 ng of the cfDNA sample to be tested, and 30 ng of each of the positive quality control and negative quality control. Use the EZ methylation-Gold kit (Zymo, Cat: D5006) to perform bisulfite conversion on the DNA, and elute it in 21 μL of enzyme-free water after conversion. (5) Using the multiple methylation detection method of the present invention (markers, primers and probes are the same as those in Example 3 (2)), multiple real-time fluorescence quantitative PCR detection was performed on the above-mentioned bisulfite-converted cfDNA. The qPCR reaction system was prepared as follows: The reaction conditions were set up on the ABI 7500 QPCR instrument as follows: (6) The interpretation criteria for the test results are as follows (ΔCq = VIC average Cq - FAM average Cq in the table). When the results of the quality control products tested in the same batch as the subject samples are verified to be valid, the interpretation of the qPCR results of the test samples is as shown in Table 7: Table 7 Interpretation of qPCR results of test samples 2. Experimental results The clinical characteristics and positive detection rates of the 216 subjects are shown in Table 8: Table 8 Performance analysis of this method for colorectal cancer diagnosis According to the above test results, the sensitivity of this method for colorectal cancer detection is 73.3%, and the specificity is 96.9%, wherein the detection rates of stages I-IV are 51.5%, 82.1%, 76.3%, and 100%, respectively. The test results show that the plasma methylation level of patients with colorectal cancer is significantly higher than that of healthy subjects, and the difference is statistically significant (Figure 5). Moreover, the methylation level is related to the tumor stage, and the later the tumor stage, the higher the plasma methylation level (Figure 5). The ROC curve analysis results show that when the method of the present invention is used to detect colorectal cancer, although the vast majority of the selected samples are patients with stages I-III, which are more difficult to detect, only 8.3% (10 / 120) of stage IV patients are used, and the area under the curve (AUC) is 0.9152, which is significantly higher than the original invention (CN112159844B, in which the proportion of stage IV patients is higher) of 0.8912, indicating that this method has higher diagnostic accuracy (Figure 6). More importantly, the specificity of the method is as high as 96.9%, which has better clinical performance in clinical applications, especially when the diagnosis requires higher specificity. The ΔCq values ​​of the three false-positive patients were -3.75, -3.71 and -3.69, which are very close to the cutoff value (-4). When the inventors adopt a stricter cutoff value (such as -3), the specificity can be increased to 100%. Embodiment 8 Application of DNA methylation markers in prognosis assessment and recurrence prediction of colorectal cancer 1. Experimental Methods (1) A total of 160 subjects were enrolled, all of whom were patients with stage I-III colorectal cancer who underwent radical surgery. Blood samples were collected from the patients within 2 weeks after surgery (mean time was 5 days after surgery). (2) The present invention is used to perform multiplex real-time fluorescence quantitative PCR detection on the above-mentioned DNA converted with bisulfite (the experimental method is the same as that of Example 6). 2. Experimental results 160 subjects, 116 of whom did not relapse within 2 years after surgery, and 44 relapsed within 2 years after surgery. Among the 116 patients who did not relapse within 2 years, only 5 were positive for postoperative MRD detection, with a specificity of 95.7%; among the 44 patients who relapsed within 2 years, 27 had MRD detected in plasma samples 5 days after surgery, with a sensitivity of 61.4%. Among the 32 patients who were MRD-positive after surgery, 27 relapsed within 2 years, with a positive predictive value of 84.3%. A survival curve was drawn based on the recurrence of each patient and the postoperative ctDNA status (Figure 7). The relapse free survival (RFS) of patients with positive postoperative ctDNA results was significantly shorter than that of patients with negative ctDNA (P < 0.0001). Stage II colorectal cancer has certain particularities in postoperative treatment. Postoperative adjuvant chemotherapy is recommended for high-risk stage II patients, while postoperative adjuvant chemotherapy is generally not recommended for low-risk stage II patients. According to the NCCN guidelines, risk factors for stage II colorectal cancer include poorly differentiated or undifferentiated tumors, lymphatic / vascular invasion, nerve invasion, intestinal obstruction, <12 detected lymph nodes; or T4N0M0; or T3 with local perforation or close to the resection margin, uncertain resection margin or positive resection margin, etc. The inventors conducted a subgroup analysis on 76 patients with stage II colorectal cancer, and the results are shown in FIG8 : 1) There was no significant difference in the amount of cfDNA in plasma between patients with and without recurrence of stage II colorectal cancer within 2 weeks after surgery (A); 2) The target methylation level of peripheral blood in postoperative MRD detection of relapsed patients was significantly higher than that of non-relapsed patients (B); 3) Of the 13 patients who tested positive for MRD 2 weeks after surgery, 12 relapsed within 2 years, with a positive predictive value of 92.3%, while 7.9% (5 / 63) of patients who tested negative relapsed within 2 years (RFS; HR, 16; P<0.0001) (C, D). 4) In both the clinical low-risk and high-risk subgroups, the MRD test results 2 weeks after surgery could predict recurrence within 2 years (E, F). These data indicate that high levels of ctDNA methylation indicate a poor prognosis, and that ctDNA methylation detection results can effectively predict the patient's surgical prognosis. The DNA methylation screening method of the present invention can screen out highly tumor-specific DNA methylation markers, thereby designing highly specific multiple DNA methylation-specific fluorescent quantitative PCR (mqMSP). Based on the high specificity of the markers and the detection method, the inventor predicted recurrence 2 years after surgery by detecting plasma ctDNA within 2 weeks after surgery (average 5 days), and obtained a very high positive predictive value (84.3% overall positive predictive value for stages I-III, 92.3% positive predictive value for stage II). These extremely high positive predictive values ​​can be used to conduct more active follow-up of patients with positive micro-residual lesions after surgery, and even adopt more active chemotherapy strategies to reduce the probability of recurrence in patients, or to detect postoperative recurrence early to increase the chance of secondary surgery, ultimately benefiting patients. Those skilled in the art will appreciate that the detection rate of recurrence can be further improved by performing regular and continuous follow-up of patients after surgery, obtaining peripheral blood plasma samples, and performing the detection method. References [1]SUNG H,FERLAY J,SIEGEL R L,et al.Global Cancer Statistics 2020:GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J].CA Cancer J Clin,2021,71(3):209-49. [2]CHEN W,ZHENG R,BAADE P D,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-32. [3]DEKKER E,TANIS P J,VLEUGELS J L A,et al.Colorectal cancer[J].Lancet,2019,394(10207):1467-80. [4]MILLER K D,NOGUEIRA L,DEVASIA T,et al.Cancer treatment and survivorship statistics,2022[J].CA Cancer J Clin,2022,72(5):409-36. [5]EDWARDS B K,WARD E,KOHLER B A,et al.Annual report to the nation on the status of cancer,1975-2006,featuring colorectal cancer trends and impact of interventions(risk factors,screening,and treatment)to reduce future rates[J].Cancer,2010,116(3):544-73. [6]PORTELA A,ESTELLER M.Epigenetic modifications and human disease[J].Nature Biotechnology,2010,28(10):1057-68. [7]JONES P A,BAYLIN S B.The fundamental role of epigenetic events in cancer[J].Nat Rev Genet,2002,3(6):415-28. [8]LAO V V,GRADY W M.Epigenetics and colorectal cancer[J].Nature Reviews Gastroenterology&Hepatology,2011,8(12):686-700. [9]MOUSAVINEZHAD M,MAJDZADEH R,AKBARI SARI A,et al.The effectiveness of FOBT vs.FIT:A meta-analysis on colorectal cancer screening test[J].Med J Islam Repub Iran,2016,30:366.

[0010] SARGENT D,SOBRERO A,GROTHEY A,et al.Evidence for cure by adjuvant therapy in colon cancer:observations based on individual patient data from 20,898 patients on 18 randomized trials[J].J Clin Oncol,2009,27(6):872-7.

[0011] WANG R,SU Q,YAN Z P.Reconsideration of recurrence and metastasis in colorectal cancer[J].World J Clin Cases,2021,9(24):6964-8.

[0012] VAN DEN BERG I,VAN DE WEERD S,ROODHART J M L,et al.Improving clinical management of colon cancer through CONNECTION,anation-wide colon cancer registry and stratification effort(CONNECTION II trial):rationale and protocol of a single arm intervention study[J].BMC Cancer,2020,20(1):776.

[0013] PURANDARE N C,DUA S G,ARORA A,et al.Colorectal cancer-patterns of locoregional recurrence and distant metastases as demonstrated by FDG PET / CT[J].Indian J Radiol Imaging,2010,20(4):284-8.

[0014] C G,KARLSSON W K,POMMERGAARD H-C,et al.The diagnostic accuracy ofcarcinoembryonic antigen to detect colorectal cancer recurrence–A systematic review[J].International Journal of Surgery,2016,25:134-44.

[0015] NORMANNO N,CERVANTES A,CIARDIELLO F,et al.The liquid biopsy in the management of colorectal cancer patients:Current applications and future scenarios[J].Cancer Treatment Reviews,2018,70:1-8.

[0016] LADABAUM U,DOMINITZ J A,KAHI C,et al.Strategies for Colorectal Cancer Screening[J].Gastroenterology,2020,158(2):418-32.

[0017] HEITZER E,HAQUE I S,ROBERTS C E S,et al.Current and future perspectives of liquid biopsies in genomics-driven oncology[J].Nature Reviews Genetics,2019,20(2):71-88.

[0018] WAN J C M,MASSIE C,GARCIA-CORBACHO J,et al.Liquid biopsies come of age:towards implementation of circμlating tumour DNA[J].Nature Reviews Cancer,2017,17(4):223-38.

[0019] LUO H Y,WEI W,YE Z Y,et al.Liquid Biopsy of Methylation Biomarkers in Cell-Free DNA[J].Trends in Molecμlar Medicine,2021,27(5):482-500.

[0020] COTTRELL S E,DISTLER J,GOODMAN N S,et al.A real-time PCR assay for DNA-methylation using methylation-specific blockers[J].Nucleic Acids Research,2004,32(1):e10.

[0021] POTTER N T,HURBAN P,WHITE M N,et al.Validation of a Real-Time PCR-Based Qualitative Assay for the Detection of Methylated SEPT9 DNA in Human Plasma[J].Clinical Chemistry,2014,60(9):1183-91.

[0022] CHURCH T R,WANDELL M,LOFTON-DAY C,et al.Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer[J].Gut,2014,63(2):317-25.

[0023] SUN J,FEI F,ZHANG M Q,et al.The role of(m)SEPT9 in screening,diagnosis,and recurrence monitoring of colorectal cancer[J].Bmc Cancer,2019,19(1):450.

[0024] CAI G X,CAI M Y,FENG Z Q,et al.A Mμltilocus Blood-Based Assay Targeting Circμlating Tumor DNA Methylation Enables Early Detection and Early Relapse Prediction of Colorectal Cancer[J].Gastroenterology,2021,161(6):205356.

[0025] LIU M C,OXNARD G R,KLEIN E A,et al.Sensitive and specific mμlti-cancer detection and localization using methylation signatures in cell-free DNA[J].Ann Oncol,2020,31(6):745-59.

[0026] JIN S,ZHU D,SHAO F,et al.Efficient detection and post-surgical monitoring of colon cancer with a mμlti-marker DNA methylation liquid biopsy[J].Proc Natl Acad Sci U S A,2021,118(5):e2017421118.

[0027] XIE H,WU Z Y,LI Z H,et al.Significance of ZEB2 in the immune microenvironment of colon cancer[J].Frontiers in Genetics,2022,13:995333

[0028] BARAΜLT L,AMATU A,SIRAVEGNA G,et al.Discovery of methylated circμlating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer[J].Gut,2018,67(11):1995-2005.

[0029] YOUNG G P,SYMONDS E L,NIELSEN H J,et al.Evaluation of a panel of tumor-specific differentially-methylated DNA regions in IRF4,IKZF1 and BCAT1 for blood-based detection of colorectal cancer[J].Clin Epigenetics,2021,13(1):14.

[0030] LIND G E,DANIELSEN S A,AHLQUIST T,et al.Identification of an epigenetic biomarker panel with high sensitivity and specificity for colorectal cancer and adenomas[J].Molecμlar Cancer,2011,10:85.

[0031] CEBOLA I,CUSTODIO J,MUNOZ M,et al.Epigenetics override pro-inflammatory PTGS transcriptomic signature towards selective hyperactivation of PGE(2)in colorectal cancer[J].Clin Epigenetics,2015,7(1):74.

[0032] ZHANG Y F,WU Q,XU L H,et al.Sensitive detection of colorectal cancer in peripheral blood by a novel methylation assay[J].Clin Epigenetics,2021,13(1):90.

[0033] MOSS J,MAGENHEIM J,NEIMAN D,et al.Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease[J].Nature Communications,2018 9,5068

[0034] AUCAMP J,BRONKHORST A J,BADENHORST C P,et al.The diverse origins of circulating cell-free DNA in the human body:a critical re-evaluation of the literature[J].Biol Rev Camb Philos Soc.2018 93(3):1649-1683

Claims

1. A method for diagnosing whether a subject has a tumor or precancerous lesion, whether a tiny residual tumor lesion exists, determining the postoperative prognosis of a patient with a tumor, predicting the postoperative recurrence of a patient with a tumor, or evaluating the therapeutic effect on a patient with a tumor, the method comprising detecting a group of DNA methylation markers in free DNA in a sample from the subject to determine the methylation level of the DNA, and if the methylation level is higher than the DNA methylation level of a normal control sample, it is determined that the subject has a tumor or precancerous lesion, a tiny residual tumor lesion, a poor postoperative prognosis for a patient with a tumor, a high probability of postoperative recurrence for a patient with a tumor, or a poor therapeutic effect on the subject, wherein the free DNA in the subject's sample is derived from two major types of cells, namely, tumor or precancerous lesion cells, and non-cancerous cells, wherein the DNA methylation markers are characterized in that: the methylation level of each DNA methylation marker in the non-cancerous cell in the subject's sample is relatively low, and at least one DNA methylation marker in the group of methylation markers has a relatively high methylation level in a tumor or precancerous lesion cell.

2. The method of claim 1, wherein the methylation marker is characterized in that the methylation level in tumor or precancerous lesion cells differs from that in corresponding normal cells by at least 15%.

3. The method according to claim 1 or 2, characterized in that The sample is selected from the group consisting of body fluids, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, stool and tissue samples.

4. The method according to any one of claims 1 to 3, wherein the non-cancerous cells in the subject's sample include cells containing genomic DNA, such as neutrophils, B cells, T cells, vascular endothelial cells, hepatocytes, etc., and when the non-cancerous cells are neutrophils, B cells, T cells, etc., the DNA methylation of each DNA methylation marker in these cells is less than 1%; when the non-cancerous cells are vascular endothelial cells, hepatocytes, etc., the DNA methylation of each DNA methylation marker in these cells is less than 10%.

5. The method according to any one of claims 1-4, wherein the methylation level of the methylation marker in non-cancerous cells in the subject's sample is less than 0.1%, and more preferably lower than the detection limit or detection threshold of one or more DNA methylation detection methods.

6. The method according to any one of claims 1-5, wherein the methylation level of the methylation marker in the peritumoral tissue is low, preferably lower than 20%, more preferably lower than 10%, more preferably lower than 5%, and most preferably lower than 1%.

7. The method according to any one of claims 1-6, wherein the set of DNA methylation markers comprises at least 2, preferably at least 5, and more preferably at least 10 methylation markers.

8. The method according to any one of claims 1 to 7, wherein the methylation marker is two or more selected from ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1 and SFMBT2, preferably all of ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1 and SFMBT2.

9. The method according to any one of claims 1-8, wherein the tumor is colorectal cancer.

10. The method according to any one of claims 1 to 9, characterized in that The detection of the methylation markers is performed using multiplex quantitative methylation-specific PCR. Optionally, the multiplex quantitative methylation-specific PCR includes determination of an internal reference gene ACTB.

11. The method according to claim 10, characterized in that The multiplex quantitative methylation-specific PCR uses primers and probes for the methylation markers of claim 8, and primers and probes for the internal reference gene ACTB, wherein the primers are selected from the following primer pairs: SEQ ID NO: 4 or a sequence having sufficient identity to SEQ ID NO: 4 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 5 or a sequence having sufficient identity to SEQ ID NO: 5 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 7 or a sequence having sufficient identity to SEQ ID NO: 7 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 8 or a sequence having sufficient identity to SEQ ID NO: 8 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 10 or a sequence having sufficient identity to SEQ ID NO: 10 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 11 or a sequence having sufficient identity to SEQ ID NO: 11 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 13 or a sequence having sufficient identity to SEQ ID NO: 13 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 14 or a sequence having sufficient identity to SEQ ID NO: 14 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 16 or a sequence having sufficient identity to SEQ ID NO: 16 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 17 or a sequence having sufficient identity to SEQ ID NO: 17 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 19 or a sequence having sufficient identity to SEQ ID NO: 19 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 20 or a sequence having sufficient identity to SEQ ID NO: 20 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:22 or a sequence having sufficient identity to SEQ ID NO:22 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:23 or a sequence having sufficient identity to SEQ ID NO:23 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:25 or a sequence having sufficient identity to SEQ ID NO:25 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:26 or a sequence having sufficient identity to SEQ ID NO:26 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:28 or a sequence having sufficient identity to SEQ ID NO:28 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:29 or a sequence having sufficient identity to SEQ ID NO:29 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:31 or a sequence having sufficient identity to SEQ ID NO:31 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:32 or a sequence having sufficient identity to SEQ ID NO:32 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:34 or a sequence having sufficient identity to SEQ ID NO:34 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:35 or a sequence having sufficient identity to SEQ ID NO:35 to ensure sensitivity and specificity of PCR amplification; and SEQ ID NO: 1 or a sequence having sufficient identity with SEQ ID NO: 1 to ensure the sensitivity and specificity of PCR amplification, and SEQ ID NO: 2 or a sequence having sufficient identity with SEQ ID NO: 2 to ensure the sensitivity and specificity of PCR amplification; and, The probe is selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3 or a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3 that has sufficient consistency to ensure the sensitivity and specificity of PCR amplification.

12. A kit for diagnosing whether a subject has a tumor or precancerous lesion, whether a microscopic residual tumor lesion exists, determining the prognosis of a patient with a tumor after surgery, predicting the recurrence of a patient with a tumor after surgery, or evaluating the therapeutic effect on a patient with a tumor, comprising a reagent for detecting the marker according to any one of claims 1 to 11; Optionally, the kit further comprises a reagent for detecting an internal reference gene ACTB.

13. The kit according to claim 12, characterized in that The reagents for detecting methylation markers and internal reference genes are primer pairs and probes, wherein the primers are selected from the following primer pairs: SEQ ID NO: 4 or a sequence having sufficient identity to SEQ ID NO: 4 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 5 or a sequence having sufficient identity to SEQ ID NO: 5 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 7 or a sequence having sufficient identity to SEQ ID NO: 7 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 8 or a sequence having sufficient identity to SEQ ID NO: 8 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 10 or a sequence having sufficient identity to SEQ ID NO: 10 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 11 or a sequence having sufficient identity to SEQ ID NO: 11 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 13 or a sequence having sufficient identity to SEQ ID NO: 13 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 14 or a sequence having sufficient identity to SEQ ID NO: 14 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 16 or a sequence having sufficient identity to SEQ ID NO: 16 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 17 or a sequence having sufficient identity to SEQ ID NO: 17 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 19 or a sequence having sufficient identity to SEQ ID NO: 19 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 20 or a sequence having sufficient identity to SEQ ID NO: 20 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:22 or a sequence having sufficient identity to SEQ ID NO:22 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:23 or a sequence having sufficient identity to SEQ ID NO:23 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:25 or a sequence having sufficient identity to SEQ ID NO:25 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:26 or a sequence having sufficient identity to SEQ ID NO:26 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:28 or a sequence having sufficient identity to SEQ ID NO:28 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:29 or a sequence having sufficient identity to SEQ ID NO:29 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:31 or a sequence having sufficient identity to SEQ ID NO:31 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:32 or a sequence having sufficient identity to SEQ ID NO:32 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:34 or a sequence having sufficient identity to SEQ ID NO:34 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:35 or a sequence having sufficient identity to SEQ ID NO:35 to ensure sensitivity and specificity of PCR amplification; and SEQ ID NO: 1 or a sequence having sufficient identity with SEQ ID NO: 1 to ensure the sensitivity and specificity of PCR amplification, and SEQ ID NO: 2 or a sequence having sufficient identity with SEQ ID NO: 2 to ensure the sensitivity and specificity of PCR amplification; and, The probe is selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3, or a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3 that has sufficient consistency to ensure the sensitivity and specificity of PCR amplification.

14. A primer pair selected from: SEQ ID NO: 4 or a sequence having sufficient identity to SEQ ID NO: 4 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 5 or a sequence having sufficient identity to SEQ ID NO: 5 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 7 or a sequence having sufficient identity to SEQ ID NO: 7 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 8 or a sequence having sufficient identity to SEQ ID NO: 8 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 10 or a sequence having sufficient identity to SEQ ID NO: 10 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 11 or a sequence having sufficient identity to SEQ ID NO: 11 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 13 or a sequence having sufficient identity to SEQ ID NO: 13 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 14 or a sequence having sufficient identity to SEQ ID NO: 14 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 16 or a sequence having sufficient identity to SEQ ID NO: 16 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 17 or a sequence having sufficient identity to SEQ ID NO: 17 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO: 19 or a sequence having sufficient identity to SEQ ID NO: 19 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO: 20 or a sequence having sufficient identity to SEQ ID NO: 20 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:22 or a sequence having sufficient identity to SEQ ID NO:22 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:23 or a sequence having sufficient identity to SEQ ID NO:23 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:25 or a sequence having sufficient identity to SEQ ID NO:25 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:26 or a sequence having sufficient identity to SEQ ID NO:26 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:28 or a sequence having sufficient identity to SEQ ID NO:28 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:29 or a sequence having sufficient identity to SEQ ID NO:29 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:31 or a sequence having sufficient identity to SEQ ID NO:31 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:32 or a sequence having sufficient identity to SEQ ID NO:32 to ensure sensitivity and specificity of PCR amplification; SEQ ID NO:34 or a sequence having sufficient identity to SEQ ID NO:34 to ensure sensitivity and specificity of PCR amplification, and SEQ ID NO:35 or a sequence having sufficient identity to SEQ ID NO:35 to ensure sensitivity and specificity of PCR amplification; and SEQ ID NO: 1 or a sequence that is sufficiently identical to SEQ ID NO: 1 to ensure the sensitivity and specificity of PCR amplification, and SEQ ID NO: 2 or a sequence that is sufficiently identical to SEQ ID NO: 2 to ensure the sensitivity and specificity of PCR amplification.

15. A nucleic acid molecule used as a probe, which is selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3, or a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3 that has sufficient consistency to ensure the sensitivity and specificity of PCR amplification.

Images