Tumor detection method and reagents

Abstract

The present invention relates to a method and reagents for detecting DNA methylation. Specifically, the present invention relates to a method for diagnosing the presence or absence of a tumor in a subject, a method for diagnosing the presence of microresidual lesions after tumor surgery, a method for determining the postoperative prognosis of a subject with a tumor, a method for predicting postoperative recurrence in a subject with a tumor, or a method for evaluating the therapeutic effect on a subject with a tumor, by detecting a methylation marker in free DNA in a sample derived from the subject and determining the methylation level of the DNA. The present invention also relates to a marker, a kit, and a detection reagent used in the above method.

Description

[Technical Field]

[0001] This invention relates to the field of medical diagnostics, and more specifically to the diagnosis, prognosis, and evaluation of treatment effectiveness of tumors. Furthermore, this invention also relates to kits and reagents for the aforementioned diagnosis, prognosis, and evaluation of treatment effectiveness. [Background technology]

[0002] Globally, cancer is a major public health challenge. More than 10 million people die from cancer worldwide every year. Generally, cancer patients may receive different treatment options depending on the clinical stage of their disease. For example, patients with early or intermediate-stage cancer may be treated with surgery, while patients with later-stage cancer may be treated with radiation, chemotherapy, targeted therapy, immunotherapy, etc.

[0003] The effectiveness of a patient's treatment is directly related to the clinical stage. The five-year survival rate for early-stage patients can reach 90%, while it can be as low as 10% for later-stage patients. Therefore, early detection and diagnosis of tumors through various screening methods can significantly improve the effectiveness of a patient's treatment and their prognosis.

[0004] Taking colorectal cancer as an example, it originates from the mucosal epithelium of the colorectal colon and is one of the most common malignancies of the digestive system. It is the third most common malignancy and the second most common malignancy worldwide. [1] In China, both the incidence and mortality rates of colorectal cancer have been on an upward trend for nearly a decade. [2] Colorectal cancer primarily develops from intestinal polyps over a period of 10 to 15 years. Early symptoms are often latent, and by the time a definitive diagnosis is made, the tumor has progressed to a moderate stage, resulting in a poor prognosis. [3] As the clinical stage progresses, the survival rate for colorectal cancer patients decreases significantly, with a 5-year survival rate of 90% for patients with early-stage localized tumors, and approximately 15% for patients with later-stage tumors. [4] Previous studies have shown that screening-based secondary prevention is effective in reducing colorectal cancer mortality by 53%, while clinical treatment-based tertiary prevention is effective in reducing colorectal cancer mortality by only 12%.[5] These evidences suggest that the initial screening is the most important means to reduce the mortality of colorectal cancer. Therefore, the early screening of colorectal cancer is very important.

[0005] Colonoscopy is the current gold standard for the diagnosis of colorectal cancer, with a high detection rate. However, it requires strict bowel preparation, is invasive, and has poor compliance among subjects. In China, due to factors such as insufficient resources for colonoscopy, differences in the levels of doctors, overpopulation, and traditional culture, it is difficult to conduct early screening of colorectal cancer through large-scale colonoscopy screening, especially in remote and poverty-stricken areas, it is even more difficult to conduct screening over a wide range. The guaiac-based fecal occult blood test (gFOBT) and immunochemical fecal occult blood test (FIT) based on fecal detection are simple to operate, but have drawbacks such as low sensitivity and low specificity. [9] Therefore, the development of new detection technologies for the early diagnosis of colorectal cancer has important application value and practical significance.

[0006] However, even if the tumor is detected relatively early and treated with radical surgery, recurrence and metastasis may still occur in a considerable proportion of patients. The probability of recurrence and metastasis is generally also related to the stage of the tumor. Clinically, it is often considered whether to perform adjuvant postoperative treatment according to the tumor stage to reduce the recurrence probability. However, this prognostic evaluation based on tumor stage classification, as well as the dynamic follow-up after surgery (including imaging examinations and serological examinations), has great room for improvement in accuracy (including sensitivity and specificity).

[0007] Taking colorectal cancer as an example again. The main treatment methods for colorectal cancer include surgery and adjuvant postoperative radiotherapy and chemotherapy, etc. However, about 35% of colorectal cancer patients experience postoperative recurrence and metastasis, and 80% of them occur within 2 years after surgery.

[10] Recurrence and metastasis are the main causes of poor prognosis in colorectal cancer patients.

[11] Early detection of postoperative recurrence and metastasis and subsequent clinical intervention can significantly improve the prognosis of colorectal cancer patients. [12,13]Currently, monitoring for recurrence and metastasis after colorectal cancer surgery relies primarily on imaging tests such as CT scans and the detection of protein markers such as carcinoembryonic antigen (CEA). CT scans have low sensitivity for early recurrence monitoring because they can only detect tumor recurrence when it reaches a certain volume. CEA tests show positive results in approximately 50% of patients with postoperative recurrence and metastasis, indicating low detection sensitivity.

[14] CT and CEA are used for postoperative monitoring of colorectal cancer, but by the time tumor recurrence or metastasis is detected, the disease has already progressed to a certain extent, often leading to delays in appropriate clinical intervention. Therefore, if technologies for effectively monitoring recurrence and metastasis after colorectal cancer surgery are developed, it will be possible to provide early clinical intervention to patients who experience recurrence or metastasis, leading to improved prognosis.

[0008] With the rapid development of molecular diagnostic technologies, liquid biopsy techniques that detect circulating tumor DNA (ctDNA) in the blood as a molecular diagnostic marker are attracting attention. Taking peripheral blood plasma as an example, the free DNA (circulating free DNA, cfDNA) contained within it may originate from a variety of organs and cells. White blood cells (including neutrophils, B cells, and T cells) are the main source of cfDNA in plasma, and vascular endothelial cells and hepatocytes can also provide some cfDNA. [33,34] These cell-derived cfDNAs are generally considered normal DNA. In the plasma of cancer patients and individuals with precancerous lesions, there may also be free DNA derived from tumor cells, usually called circulating tumor DNA (ctDNA). Compared to tissue biopsy, liquid biopsy has the advantages of being non-invasive, rapid, and allowing for easy dynamic monitoring, as well as being less affected by tumor heterogeneity.

[15] Compared to fecal occult blood tests, liquid biopsy offers higher sensitivity and specificity, and is less affected by diet and other factors.

[16] ctDNA is DNA derived from tumor cells in peripheral blood. It is released into the peripheral blood due to tumor cell necrosis and apoptosis, has an average length of about 160 bp, a short half-life, and possesses the same genetic and surface genetic variations (e.g., gene mutations, copy number variations, methylation abnormalities, etc.) as tumor tissue.

[17] It is useful for tumor screening, molecular type analysis, treatment efficacy evaluation, prognosis assessment, detection of residual lesions after surgery, and dynamic monitoring.

[0009] Tumor development and progression are multifactorial and multi-step processes, and epigenetic mutations are deeply involved in the development and progression of colorectal cancer. DNA methylation is one of the major changes in epigenetic mutations and refers to the process by which a methyl group is covalently bonded to the carbon atom at the cytosine 5 position of genomic CpG dinucleotides, using S-adenosylmethionine (SAM) as a methyl donor, catalyzed by DNA methyltransferase (DNMT). [6] Research has shown that DNA methylation status undergoes abnormal changes from the early stages of tumor development. [7] DNA methylation, primarily through hypermethylation of CpG islets, leads to corresponding gene expression reduction, while hypomethylation of the genome causes genomic instability, which is involved in the development and progression of colorectal cancer. [8] Numerous studies have shown that ctDNA methylation can be used for early cancer diagnosis, recurrence monitoring, and prognosis assessment. [18,19] .

[0010] The amount of ctDNA in body fluids such as plasma is relatively low, even less than 0.1%, or even less than 0.01%, in most cases, especially in early tumors, after curative surgery, and after drug therapy. The majority of free DNA in plasma originates from normal cells (leukocytes, vascular endothelial cells, hepatocytes, etc.), and these noise signals significantly increase the difficulty of monitoring ctDNA. Taking DNA methylation markers as an example, if free DNA from normal cells has a DNA methylation signal, it can cause a false positive signal even if the DNA methylation level in these normal cells is low, potentially affecting clinical monitoring results. Therefore, when screening for DNA methylation markers, it is necessary to consider not only the methylation level in tumor cells, but also the DNA methylation level of normal cells that may potentially release cfDNA, and further consider the extent to which normal cells release cfDNA, in order to develop a strictly feasible strategy.

[0011] Epi proColon is the first blood detection product for colorectal cancer screening approved by the US FDA to detect methylation of the SEPTIN9 gene associated with colorectal cancer. Epi proColon utilizes HeavyMethyl real-time PCR technology to detect the target gene SEPTIN9 and the internal standard gene ACTB in a single qPCR reaction.

[20] This method extracts cfDNA from plasma, and after sulfite conversion, retains methylated cytosine while converting unmethylated cytosine to uracil. Then, a single, unextendable oligonucleotide sequence is designed as a blocker for the unmethylated SEPTIN9 gene sequence, achieving specific amplification of methylated SEPTIN9. In this method, specific signal recognition of the target gene SEPTIN9 is achieved by labeling the target gene and the internal standard gene with two different fluorescent groups in the reaction system. ACTB, as the internal standard gene, reflects the template amount in the qPCR system.

[21] .

[0012] This technology has the following drawbacks: 1. Because it detects the methylation status of a single site in one gene (SEPTIN9), it has low sensitivity, with a colorectal cancer detection rate of 48.2% and a specificity of 91.5% in the asymptomatic group. The sensitivity in patients with stage I-IV colorectal cancer was 35.0%, 63.0%, 46.0%, and 77.4%, respectively, indicating insufficient sensitivity. In particular, the sensitivity is low in patients with early-stage colorectal cancer such as stage I, posing a serious problem of missed detections when used for early tumor detection.

[0013] 2. Because the ctDNA content in plasma is inherently low, significant DNA template damage occurs due to sulfite conversion, leading to pronounced DNA breaks. Further DNA loss occurs during the purification process after conversion, and these factors further reduce the absolute amount of target methylated DNA molecules in the detection system. Since it detects only the methylation status of a single target gene, SEPTIN9, the application of this method to the detection of minimal residual disease (MRD) after colorectal cancer surgery, recurrence monitoring, and prognosis assessment is limited. Existing studies have shown that Epi proColon technology could detect 15 out of 21 recurrent patients, with a slightly lower positive detection rate than imaging methods (71.4% vs 85.7%).

[23] .

[0014] 3. It is necessary to ensure that the blocker efficiently and specifically binds to the unmethylated sequence in the target region. For example, insufficient binding efficiency can lead to false positive signals, and insufficient binding specificity can reduce the detection sensitivity of the target methylated sequence.

[0015] 4. The results are qualitative. Due to the low content of ctDNA in peripheral blood and the weak detection signal for single genes, only qualitative results can be determined, and quantitative analysis is not possible.

[0016] The multi-gene methylation detection assay ColonAiQ developed by Singlera (KunYuan Biologics) has the functions of early screening and postoperative recurrence monitoring for colorectal cancer. ColonAiQ utilizes multiplex qPCR technology to detect six methylation biomarkers (SEPTIN9, BCAT1, IKZF1, BCAN, SEPT9 region 2, VAV3). After processes such as bisulfite conversion, pre-amplification, and qPCR, it comprehensively evaluates samples using the MHLcot algorithm. ColonAiQ has a total sensitivity of 86% for stages I-IV of colorectal cancer, a sensitivity of 42% for advanced adenomas, and a detection specificity of 92% for the healthy population. The research team explored the potential use of ColonAiQ in monitoring colorectal cancer recurrence and revealed that there is a significant difference (P = 0.00017) in the ColonAiQ score in postoperative samples between the recurrence group and the non-recurrence group of patients.

[24] .

[0017] This technology has the following drawbacks. 1. Sample processing is complex and pre-amplification is required. The pre-amplification process is prone to laboratory PCR product contamination and is difficult for clinical applications.

[0018] 2. It is necessary to interpret the results using the MHLcot algorithm and integrate the detection results of six genes. The algorithm is complex and inconvenient for clinical use.

[0019] 3. Since six genes are detected separately, the detection workload increases, the cost correspondingly increases, and it is more likely to have operation errors in clinical detection.

[0020] GRAIL developed a blood-based "pan-cancer" early screening method. Whole-genome bisulfite sequencing (WGBS) was performed on tissues and buffy coats from a large number of subjects to establish a pan-cancer species-specific methylation sequencing database. Through bioinformatics analysis, a targeted methylation sequencing panel covering 103,456 methylation regions and a total of 1,116,720 CpG sites was identified for pan-cancer species screening. Subsequently, this targeted methylation sequencing panel was used for the detection of cfDNA in subjects and showed 99.8% specificity and 43.9% sensitivity for more than 50 stage I-III cancers in the identification cohort. The detection sensitivity increased with the stage, being 18% for stage I, 43% for stage II, and 81% for stage III. In the detection of colorectal cancer patients, the detection rate was 40-45% for stage I, 70-75% for stage II, and 76-80% for stage III.

[25] .

[0021] This technology has the following drawbacks. 1. This method adopts targeted methylation sequencing, which has high sequencing costs, a long cycle, difficult data analysis, requires dedicated analysis equipment and personnel, and is difficult to popularize clinically.

[0022] 2. This method includes methylation markers for multiple cancers and has low sensitivity for a single cancer type, such as colorectal cancer.

[0023] In view of the drawbacks of these prior technologies, the inventors first developed a single-tube multiplex methylation assay that enables quantitative analysis of methylation signals, screening for colorectal cancer, evaluation of new adjuvant efficacy of chemotherapy, postoperative prognosis, detection of postoperative residual foci, dynamic follow-up, early detection of recurrence and metastasis, etc.

[26] This method involves designing 10 detection targets in the sense and antisense strands of the SEPTIN9 gene, and performing single-tube multiplex PCR amplification, as well as multiplex signal enrichment and detection using a Taqman probe (see CN112159844B). The application of multiplex PCR significantly improved detection sensitivity.

[0024] However, this method, while effective for amplification and detection of multiple regions of a single SEPTIN9 gene, is limited by the methylation level of the SEPTIN9 gene and the specificity of the marker itself. Further improvement in detection specificity is desired. Higher specificity offers greater clinical value in several clinical applications, such as the detection of postoperative microresidical lesions (MRD).

[0025] Therefore, there is still a need in this field for DNA methylation markers with higher tumor specificity, and for methods to quantitatively analyze methylation markers with high specificity to detect tumors. Furthermore, there is also a need in this field for methods to screen for DNA methylation markers with high tumor specificity. [Overview of the project] [Problems that the invention aims to solve]

[0026] This invention provides a versatile method for screening highly tumor-specific DNA methylation markers that have extremely high tumor specificity and extremely low background signal in body fluids such as plasma. Liquid biopsy techniques based on these markers enable diverse clinical applications (such as early cancer screening, recurrence prediction, dynamic monitoring, and treatment efficacy evaluation). This invention further provides methods for verifying and screening the aforementioned markers.

[0027] This invention enables cancer screening, prognosis assessment, MRD detection, and recurrence monitoring by simultaneously detecting DNA methylation markers of multiple different genes in the blood. [Means for solving the problem]

[0028] This invention establishes a blood-based multiplex quantitative methylation-specific PCR (mqMSP) detection method containing 11 intestinal cancer-specific DNA methylation markers. The sensitivity and specificity of this method for colorectal cancer screening were evaluated by detecting preoperative plasma from healthy individuals and colorectal cancer patients confirmed by colonoscopy.

[0029] This invention evaluates the clinical applicability of a method for detecting colorectal cancer MRD, evaluating prognosis, and monitoring recurrence by detecting plasma within two weeks post-surgery in patients with colorectal cancer.

[0030] Furthermore, the present invention evaluates the clinical applicability of the method for monitoring colorectal cancer recurrence by dynamically monitoring postoperative plasma from colorectal cancer patients.

[0031] Compared to the applicant's previous inventions, the present invention provides a marker screening method with higher specificity. The relevant DNA methylation marker exhibits significantly improved detection specificity in the healthy population, resulting in better detection performance in colorectal cancer MRD detection, prognosis assessment, and recurrence monitoring, and enabling better guidance of postoperative clinical management.

[0032] The present invention provides a method for screening highly tumor-specific DNA methylation markers applicable to tumor signal detection via liquid biopsy. The method includes analyzing DNA methylation in tumor tissue samples, adjacent normal tissue samples, leukocyte samples in blood, etc., and establishing a series of DNA methylation level screening criteria to screen for highly tumor-specific DNA methylation markers that can be used in liquid biopsy. The resulting combination of markers (at least two markers) exhibits good sensitivity and high specificity in multiple clinical scenarios.

[0033] Specifically, the present invention relates to the following aspects:

[0034] In one embodiment, the present invention relates to a method for diagnosing the presence or absence of a tumor or precancerous lesion in a subject, diagnosing the presence or absence of tumor microresidual lesions, determining the postoperative prognosis of a tumor patient, predicting postoperative recurrence of a tumor patient, or evaluating the therapeutic effect of a tumor patient, the method comprising detecting a plurality of DNA methylation markers in free DNA in a sample derived from the subject to determine the DNA methylation level. If the methylation level is higher than the DNA methylation level of a normal control sample, it indicates the presence of a tumor or precancerous lesion, the presence of microresidual lesions, a poor postoperative prognosis of the tumor patient, an increased risk of postoperative recurrence of the tumor patient, or poor therapeutic effect in the subject. The free DNA in the sample derived from the subject is derived from two types of cells, namely tumor or precancerous lesion cells and non-cancerous cells. The DNA methylation markers are characterized in that each DNA methylation marker has a lower methylation level in non-cancerous cells in the subject sample, and at least one DNA methylation marker in the group of methylation markers has a higher methylation level in tumor cells or precancerous lesion cells.

[0035] In one embodiment, the methylation marker is characterized in that the methylation level in tumor or precancerous lesion cells is at least 15%, 20%, or 25% or more higher than that of the corresponding normal cells.

[0036] 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, feces, and tissue samples.

[0037] In one embodiment, non-cancerous cells in the subject sample include genomic DNA-containing cells, such as neutrophils, B cells, T cells, vascular endothelial cells, and hepatocytes. 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%, and 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%, and even less than 10%, respectively.

[0038] In one embodiment, the methylation marker has a methylation level of less than 0.1% in non-cancer cells in the subject sample, and more preferably, is below the detection limit or detection threshold of one or more DNA methylation detection methods.

[0039] In one embodiment, the methylation marker has a lower methylation level in the peritumoral tissue, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, and most preferably less than 1%.

[0040] In one embodiment, the DNA methylation marker group includes at least two, preferably at least five, and more preferably at least ten methylation markers.

[0041] In one embodiment, the methylation marker is two or more selected from the group consisting of ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1, and SBMBT2, and preferably all of ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1, and SBMBT2.

[0042] In one embodiment, the tumor is colorectal cancer.

[0043] In one embodiment, the detection of the methylation marker is performed using multiplex quantitative methylation-specific PCR.

[0044] Optionally, the multiplex quantitative methylation-specific PCR may include the measurement of the internal standard gene ACTB.

[0045] In one embodiment, the multiple quantitative methylation-specific PCR uses primers and probes for the methylation marker and primers and probes for the internal standard gene ACTB, and the primers are selected from the following primer pairs.

[0046] SEQ ID NO:4, or a sequence having sufficient identity with SEQ ID NO:4 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:5, or a sequence having sufficient identity with SEQ ID NO:5 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0047] SEQ ID NO:7, or a sequence having sufficient identity with SEQ ID NO:7 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:8, or a sequence having sufficient identity with SEQ ID NO:8 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0048] SEQ ID NO:10, or a sequence having sufficient identity with SEQ ID NO:10 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:11, or a sequence having sufficient identity with SEQ ID NO:11 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0049] SEQ ID NO:13, or a sequence having sufficient identity with SEQ ID NO:13 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:14, or a sequence having sufficient identity with SEQ ID NO:14 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0050] SEQ ID NO:16, or a sequence having sufficient identity with SEQ ID NO:16 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:17, or a sequence having sufficient identity with SEQ ID NO:17 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0051] SEQ ID NO:19, or a sequence having sufficient identity with SEQ ID NO:19 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:20, or a sequence having sufficient identity with SEQ ID NO:20 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0052] SEQ ID NO:22, or a sequence having sufficient identity with SEQ ID NO:22 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:23, or a sequence having sufficient identity with SEQ ID NO:23 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0053] SEQ ID NO:25, or a sequence having sufficient identity with SEQ ID NO:25 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:26, or a sequence having sufficient identity with SEQ ID NO:26 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0054] SEQ ID NO:28, or a sequence having sufficient identity with SEQ ID NO:28 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:29, or a sequence having sufficient identity with SEQ ID NO:29 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0055] SEQ ID NO:31, or a sequence having sufficient identity with SEQ ID NO:31 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:32, or a sequence having sufficient identity with SEQ ID NO:32 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0056] SEQ ID NO:34, or a sequence having sufficient identity with SEQ ID NO:34 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:35, or a sequence having sufficient identity with SEQ ID NO:35 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0057] SEQ ID NO:1, or a sequence having sufficient identity with SEQ ID NO:1 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:2, or a sequence having sufficient identity with SEQ ID NO:2 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0058] The probe is a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 3, or a sequence that has sufficient identity with a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 3, and that can guarantee the sensitivity and specificity of PCR amplification.

[0059] In another embodiment, the present invention relates to a kit for diagnosing the presence or absence of a tumor or precancerous lesion in a subject, diagnosing the presence or absence of tumor microresiduals, determining the postoperative prognosis of a tumor patient, predicting postoperative recurrence in a tumor patient, or evaluating the therapeutic effect of a tumor patient, the kit comprising a reagent for detecting said marker. Optionally, the kit further comprises a reagent for detecting the internal standard gene ACTB.

[0060] In one embodiment, the reagents for detecting the methylation marker and the internal standard gene are a primer pair and a probe, wherein the primer is the following primer pair

[0061] SEQ ID NO:4, or a sequence having sufficient identity with SEQ ID NO:4 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:5, or a sequence having sufficient identity with SEQ ID NO:5 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0062] SEQ ID NO:7, or a sequence having sufficient identity with SEQ ID NO:7 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:8, or a sequence having sufficient identity with SEQ ID NO:8 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0063] SEQ ID NO:10, or a sequence having sufficient identity with SEQ ID NO:10 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:11, or a sequence having sufficient identity with SEQ ID NO:11 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0064] SEQ ID NO:13, or a sequence having sufficient identity with SEQ ID NO:13 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:14, or a sequence having sufficient identity with SEQ ID NO:14 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0065] SEQ ID NO:16, or a sequence having sufficient identity with SEQ ID NO:16 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:17, or a sequence having sufficient identity with SEQ ID NO:17 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0066] SEQ ID NO:19, or a sequence having sufficient identity with SEQ ID NO:19 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:20, or a sequence having sufficient identity with SEQ ID NO:20 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0067] SEQ ID NO:22, or a sequence having sufficient identity with SEQ ID NO:22 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:23, or a sequence having sufficient identity with SEQ ID NO:23 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0068] SEQ ID NO:25, or a sequence having sufficient identity with SEQ ID NO:25 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:26, or a sequence having sufficient identity with SEQ ID NO:26 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0069] SEQ ID NO:28, or a sequence having sufficient identity with SEQ ID NO:28 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:29, or a sequence having sufficient identity with SEQ ID NO:29 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0070] SEQ ID NO:31, or a sequence having sufficient identity with SEQ ID NO:31 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:32, or a sequence having sufficient identity with SEQ ID NO:32 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0071] SEQ ID NO:34, or a sequence having sufficient identity with SEQ ID NO:34 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:35, or a sequence having sufficient identity with SEQ ID NO:35 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0072] SEQ ID NO:1, or a sequence having sufficient identity with SEQ ID NO:1 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:2, or a sequence having sufficient identity with SEQ ID NO:2 and capable of guaranteeing the sensitivity and specificity of PCR amplification,

[0073] Selected from, and,

[0074] The probe is a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 3, or a sequence that has sufficient identity with a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 3, and that can guarantee the sensitivity and specificity of PCR amplification.

[0075] In another embodiment, the present invention relates to the above-mentioned primer pair.

[0076] In another embodiment, the present invention relates to a nucleic acid molecule to be used as a probe, which is a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 3, or a sequence that has sufficient identity with a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 3, and which can guarantee the sensitivity and specificity of PCR amplification.

[0077] The sequences of primers, probes, and competitors used in each embodiment of the present invention are not limited to those listed in the table and sequence numbers above, but also include sequences having sufficient identity thereto. Such sequences having sufficient identity include sequences that have at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% identity with the sequence indicated by the SEQ ID NO, while still maintaining its function. Among these, sequences in which the 10 consecutive bases at the 3' end have at least 80% identity with the primer sequence of the present invention are preferred. Those skilled in the art can determine sequence identity using conventional means.

[0078] In each embodiment of the present invention, those skilled in the art may also design corresponding primers and / or probes upstream and downstream of the genomic regions corresponding to the primer sequences listed in the table and sequence numbers above, for example, within 1000 bp, more preferably within 500 bp, and even more preferably within 200 bp, to analyze DNA methylation. Analytical methods include methylation-specific PCR, quantitative PCR after methylation-sensitive restriction enzyme digestion, and post-capture analysis of DNA methylation (quantitative PCR, high-throughput sequencing, etc.). [Brief explanation of the drawing]

[0079] [Figure 1] Figure 1 shows the methylation levels in the erythrocyte sedimentation layer (Buffy coat), normal tissue adjacent to cancer, and colorectal cancer tissue (Tumor tissue). [Figure 2] Figure 2 shows a comparison of the detection results for single-target markers and multi-target markers. [Figure 3] Figure 3 shows the detection sensitivity evaluation of the multi-target assay. [Figure 4] Figure 4 shows the detection limit evaluation of the multiple detection method of the present invention. [Figure 5] Figure 5 shows a comparison (one-factor analysis of variance) of the methylation level ΔCq measured between healthy individuals and intestinal cancer patients according to the present invention. [Figure 6] Figure 6 shows the results of the ROC curve analysis (CRC vs. ROC of healthy controls) for subjects with colorectal cancer and healthy controls. [Figure 7] Figure 7 shows the results of an analysis of the relationship between postoperative recurrence and ctDNA status in patients with colorectal cancer. [Figure 8] Figure 8 shows the results of a study on MRD detection and recurrence-free survival (RFS) in 76 patients with stage II colorectal cancer. [Modes for carrying out the invention]

[0080] Differential methylation regions (DMRs) exhibiting hypermethylation in colorectal cancer tumor tissue were screened by the inventors' sequencing data analysis and TCGA database data analysis. This invention utilizes fluorescence quantitative methylation-sensitive PCR (qMSP) technology to design qPCR primers and probes based on bisulfite conversion for the 10 DMRs screened above. After eliminating primer pairs showing amplification in bisulfite-converted hemoglobin saturation samples, primer pairs showing good amplification in bisulfite-converted colorectal cancer cell line (HCT116) DNA were selected. Using AutoDimer software, primer pairs that readily formed primer dimers or hairpin structures were eliminated, ultimately resulting in an mqMSP detection technique comprising 11 qMSP assays and one internal standard assay. Analysis of the present invention's technology regarding sensitivity and detection limits confirmed that it can stably detect methylated DNA at a low concentration of 0.02% in a single-tube PCR reaction, and maintains an extremely high probability (approximately 90%) of detecting methylation signals even at a low methylated DNA concentration of 0.01%. Therefore, the present invention may be applicable to the auxiliary diagnosis of colorectal cancer and postoperative MRD evaluation.

[0081] By detecting clinical blood samples using established mqMSP technology, and by detecting plasma samples from 96 healthy control subjects and preoperative plasma samples from 120 colorectal cancer patients, this method was demonstrated to have significant applicability in the screening and auxiliary diagnosis of colorectal cancer.

[0082] Using the established mqMSP method, plasma samples from 143 patients with stage I-III colorectal cancer collected within two weeks post-surgery were detected. Analysis of the relationship between postoperative ctDNA negativity / positivity and recurrence-free survival (RFS) in patients with chronic recurrence regurgitation (CRC) revealed that patients with positive postoperative ctDNA had significantly shorter recurrence times and a worse prognosis. The present invention demonstrates that postoperative ctDNA methylation levels detected by the mqMSP technology are effective as a marker for CRC prognosis and an indicator of the presence of MRD.

[0083] This invention screens and validates numerous methylation markers specific to colorectal cancer, some of which have not been reported in existing literature to be useful in detecting colorectal cancer.

[0084] This invention establishes an updated mqMSP technology utilizing these novel methylation markers. By combining multiple DNA methylation markers for detection, this invention improves detection specificity while maintaining high detection sensitivity compared to existing patented methods (e.g., V1-V4 in CN112159844B). The technology of this invention achieved a specificity of 96.9% in the healthy control group, which is clearly superior to the 83.3% specificity of conventional markers. Compared to conventional detection markers, it has higher specificity at the same level of sensitivity, and the false positive rate is lower in the detection of healthy and asymptomatic groups. In the general population or high-risk groups, the proportion of tumor patients is low, so low specificity in detection leads to an increase in false positive results, causing patient anxiety and additional hospitalization and testing, leading to increased medical costs and resource consumption. Compared to conventional detection markers, the detection markers of this invention have higher detection specificity and superior detection performance. The mqMSP technology established by this invention is useful for screening colorectal cancer, and a positive detection result in a sample suggests that the subject is likely to have colorectal cancer.

[0085] The mqMSP technology established by this invention can be used to evaluate the surgical prognosis of colorectal cancer patients. A positive detection result in a postoperative sample suggests the possibility of microresidual lesions and a potentially poor prognosis. Due to its high specificity, when used for postoperative residual lesion analysis, this invention can be used to determine postoperative prognosis and the selection of adjuvant chemotherapy, resulting in a higher positive predictive value and making the detection of positive results a more reliable indicator for guiding postoperative treatment.

[0086] The mqMSP technology established by this invention is useful for monitoring postoperative recurrence in colorectal patients, and a positive detection result in a sample suggests the possibility of disease progression or metastasis in colorectal patients. [Examples]

[0087] Example 1 Screening for highly specific colorectal cancer DNA methylation markers

[0088] The amount of ctDNA in body fluids such as plasma is relatively low, even less than 0.1%, or even less than 0.01%, in most cases, especially in early tumors, after curative surgery, and after drug therapy. The majority of free DNA in plasma originates from normal cells (leukocytes, vascular endothelial cells, hepatocytes, etc.), and these noise signals significantly increase the difficulty of monitoring ctDNA. Therefore, when screening for DNA methylation markers, it is necessary to consider not only the methylation levels within tumor cells, but also the DNA methylation levels of normal cells that may potentially release cfDNA, and further consider the extent to which normal cells release cfDNA, in order to develop a strictly feasible strategy.

[0089] The inventors systematically analyzed DNA methylation data from the TCGA database (including DNA methylation data from various tumors and normal tissues) and DNA methylation data unique to this research team (performing low-representative Bisulfite sequencing on DNA from 30 pairs of colorectal cancer tumor tissue and adjacent normal tissue, 15 benign polyps, 15 advanced adenoma tissues, and 10 cerebrospinal erythrocytes). Through multiple screenings, specifically requiring a DNA methylation level of less than 1%, with DNA methylation being at least 15% higher in colorectal tumors compared to the corresponding adjacent normal tissue, and less than 15% in normal hepatocytes, they identified potential DNA methylation markers. Ten of these DNA methylation markers are shown in Table 1.

[0090] [Table 1]

[0091] Ten DMR regions were selected as methylation markers, and primers and Taqman probes were designed for detection using MSRE-qPCR. Ten samples each of buffy coat, colorectal cancer, and corresponding adjacent normal tissue were selected. For each DNA sample, mock digestion and actual enzyme digestion (cleavage using a combination of DNA methylation-sensitive internal cleavage enzymes such as HpaII, HhaI, and BstUI) were performed in parallel, and then quantified by SYBR green fluorescence quantitative PCR. The DNA methylation level was calculated based on the following formula.

[0092] Methylation%=2 CtM-CtE ×100%, where CtM and CtE represent the Ct values ​​of the fluorescence quantitative PCR of the sample after simulated enzyme digestion and actual enzyme digestion, respectively.

[0093] The methylation levels of the 10 amplicons listed in Table 1 were verified by MSRE-qPCR, and the results are shown in Figure 1. In 10 buffy coat samples, the methylation levels of the 10 DNA markers were close to 0. In adjacent normal tissues to cancer, the methylation levels of the 10 markers were also extremely low, with most markers showing methylation levels of less than 10%, and even less than 5%, in most normal tissue samples. On the other hand, DNA methylation was high in tumor samples for all markers. These results indicate that the screening method of the present invention has high efficiency, and that the screened markers meet the intended criteria and have potential clinical value.

[0094] As those skilled in the art will understand, the upstream and downstream bases of the selected markers can also typically be used for mqMSP measurement and primer design. Therefore, for the markers listed in the table above—ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1, and SFMBT2—the ranges of up to 500 bp, preferably 400 bp, more preferably 300 bp, even more preferably 200 bp, even more preferably 100 bp, and most preferably within 50 bp, both upstream and downstream of the listed chromosomal locations.

[0095] Example 2 Design of internal standard reactions

[0096] An internal standard reaction was designed for the ACTB gene and used in the mqMSP reaction for co-amplification with multiple methylation marker genes, functioning as a control reaction and serving as an indicator of the amount of DNA in the reaction. By introducing single mutations to the second base from the 3' end of the PCR primer (forward and reverse) sequences in the internal standard reaction, the fluorescence signal of the internal standard in the mqMSP reaction (VIC fluorescence signal in this invention) was reduced, releasing enzymes and substrates in the reaction system for amplification and detection of the target methylation marker region, while simultaneously reducing inhibition of the signal of the detected methylation marker (FAM fluorescence signal in this invention).

[0097] The genomic coordinates of the ACTB region used in this invention are (chr7:5536826-5536901(GRCh38 / hg38)). The primer and probe sequences are shown in Table 2.

[0098] [Table 2]

[0099] Example 3 Control settings

[0100] To ensure quality control of the mqMSP reaction, negative and positive controls were established. These controls underwent bisulfite conversion simultaneously with each batch of cfDNA samples. The experimental results of the negative and positive controls allowed for evaluation of the experiment's success and reliability. The negative control was a mixture of erythrocyte sedimentation rate (ESR) and macula DNA from 40 healthy control subjects. The positive control was a mixture of HCT116 colorectal cancer cell line DNA and SMR DNA in a 1:99 ratio. 30 ng was taken from each reaction and used as a reference sample to evaluate the effectiveness of the experiment.

[0101] 1. Experimental Method (1) DNA samples were extracted from the HCT116 colorectal cancer cell line (derived from the laboratory of the School of Clinical Medicine, Wenzhou Medical University) and human blood sclerotium genome (peripheral blood samples collected with informed consent from applicants to the First Affiliated Hospital of Wenzhou Medical University), and their concentrations were measured using Qubit.

[0102] (2) Preparation of positive control: Mix HCT116 intestinal cancer cell line DNA and erythrocyte sedimentation malformation DNA in a 1:99 ratio, add water to adjust the concentration to 10 ng / μl, dispense the DNA and store at -80°C, and take 30 ng for each reaction.

[0103] (3) Preparation of negative control: Dilute the erythrocyte sedimentation cerebrospinal plaque DNA to 10 ng / μl, dispense the DNA, store at -80°C, and take 30 ng for each reaction.

[0104] (4) Samples from each batch are treated the same way as positive and negative controls during bisulfite treatment and qPCR detection. Each bisulfite-converted DNA is divided into two parts, and a duplicate reaction is performed in qPCR. After the reaction is complete, the Cq values ​​of the duplicate reaction FAM signal and VIC signal are statistically calculated.

[0105] 2. Experimental Results Table 3 shows the standard and algorithm for test results for different measurement methods. (Here, "+" indicates a Cq value ≤ 45, "-" indicates no amplification signal, and ΔCq = VIC mean Cq - FAM mean Cq). The qPCR reaction is verified as valid if the results of the control qPCR reaction meet the criteria listed in the table below.

[0106] [Table 3-1]

[0107] Example 4 Simultaneous detection of multiple methylation markers, and a sensitivity comparison between this detection and that of a single methylation marker.

[0108] 1. Experimental Method (1) The gene CELSR3 was selected as the single gene for detection, and experiments were conducted using its primer and probe sequences. The reaction system was as follows:

[0109] [Table 3-2]

[0110] The reaction conditions are as follows:

[0111] [Table 3-3]

[0112] (2) Method for simultaneous detection of multiple methylation markers Primers were designed for 10 selected colon cancer markers, and primers and probes were designed for the sense and antisense strands of the genomic region ENSG00000232377, respectively. Therefore, the final mqMSP assay included methylation assays for 11 targets, with ACTB assay added as an internal standard. Table 4 shows the primer and probe sequences for detecting target genes in these multi-target methylation assays.

[0113] [Table 4-1]

[0114] [Table 4-2]

[0115] [Table 4-3]

[0116] Preparation of qPCR primer and probe mixture: The initial concentration of each primer is 100 μM, and they are mixed in the following ratios.

[0117] [Table 4-4]

[0118] [Table 4-5]

[0119] The initial concentration of each probe is 100 μM, and they are mixed in the following ratios.

[0120] [Table 4-6]

[0121] [Table 4-7]

[0122] The multiple detection reaction system is as follows:

[0123] [Table 4-8]

[0124] The reaction conditions are as follows:

[0125] [Table 4-9]

[0126] (3) HCT116 cell line DNA and erythrocyte sedimentation sclera DNA were mixed in a 1:99 ratio to prepare the test sample, with a total DNA amount of 30 ng. This 1% HCT116 cell line DNA was treated with bisulfite conversion, and the same sample was compared using two different methods.

[0127] 2. Experimental Results As shown in Figure 2, the same sample differed by only 3.99 Cq in the detection results of the two methods, and a stronger methylation signal was generated when detecting with multiple methylation markers than when detecting with a single methylation marker.

[0128] Example 5 Sensitivity evaluation of detection using multiple biomarkers

[0129] 1. Experimental Method HCT116 intestinal cancer cell line DNA was mixed as a methylated sample and erythrocyte sedimentation malformation DNA as a non-methylated sample in varying ratios (1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0%) to simulate samples with different degrees of methylation. 60 ng of each sample was taken and subjected to sulfite conversion, and the converted DNA was divided into four parts for four duplication reactions by qPCR.

[0130] 2. Experimental Results The detection results for methylated DNA at different ratios showed that the VIC Cq values ​​for each sample were relatively stable, indicating uniformity in the amount of DNA template in the qPCR reaction for each sample. The FAM Cq value gradually increased as the proportion of methylated DNA decreased, suggesting that the FAM signal also decreased as the proportion of methylated DNA decreased (Figure 3, Table 5). Even at a low proportion of methylated DNA of 0.02%, the methylation signal could be stably detected, demonstrating that the detection sensitivity of the present invention is higher compared to conventional multiplex detection methods, which could only detect 0.05% of methylated samples.

[0131] The detection results are as follows:

[0132] [Table 5]

[0133] Example 6 Detection Limit Evaluation of the Present Invention

[0134] 1. Experimental Method (1) 0.02% HCT116 was independently converted 20 times, using 60 ng of DNA in each conversion reaction. The conversion product was divided into two parts and duplicated in the qPCR reaction.

[0135] (2) 0.01% HCT116 was independently converted 20 times, using 60 ng of DNA in each conversion reaction. The conversion product was divided into two parts and duplicated in the qPCR reaction.

[0136] (3) The detection results of positive and negative controls from different dates were statistically compiled and parallel analyzed.

[0137] 2. Experimental results: After more than 20 conversions and detections of 0.02% and 0.01% methylated samples, all 0.02% methylated DNA samples showed a detection result ΔCq higher than the positive threshold (ΔCq = -4), resulting in a 100% positive signal detection rate. However, two of the 0.01% methylated DNA samples showed detection results lower than the positive threshold, resulting in a 90% positive signal detection rate (Table 6, Figure 4). Therefore, the detection limit of this multiple detection method is considered to be DNA at the 0.02% methylation level. This detection limit is clearly superior to the conventional detection limit of 0.05% (CN112159844B).

[0138] [Table 6-1]

[0139] Example 7 Use of the present invention in the auxiliary diagnosis of colorectal cancer

[0140] 1. Experimental Method (1) Between 2016 and 2022, 120 colorectal cancer patients and 96 healthy control subjects were recruited, and 10 mL of venous blood was collected from each subject.

[0141] (2) Whole blood was separated twice by centrifugation to obtain plasma, which was stored at -80°C.

[0142] (3) Peripheral blood free DNA (cfDNA) is processed by Apostle MiniMax TM Extraction was performed using a High Efficiency cfDNA Extraction Kit (Apostle, Cat: A17622CN), and quantification was performed using Qubit.

[0143] (4) 10-50 ng of the cfDNA sample to be detected, and 30 ng each of the positive and negative controls were taken. The DNA was converted to bisulfite using the EZ methylation-Gold kit (Zymo, Cat: D5006), and after conversion, it was eluted in 21 μL of deenzyme-free water.

[0144] (5) The cfDNA after the above bisulfite conversion was subjected to multiplex real-time fluorescence quantitative PCR detection using the multiplex methylation detection method of the present invention (marker, primer and probe are the same as in Example 3(2)), and the qPCR reaction system was prepared as follows.

[0145] [Table 6-2]

[0146] The reaction conditions were set as follows in the ABI 7500 QPCR instrument.

[0147] [Table 6-3]

[0148] (6) The criteria for interpreting the test results are as follows (in the table, ΔCq = VIC mean Cq - FAM mean Cq). If the detection control results from the same batch as the subject sample are verified to be valid, the interpretation of the subject sample qPCR results is as shown in Table 7.

[0149] [Table 7]

[0150] 2. Experimental Results The clinical characteristics and positive detection rates of the 216 recruited subjects are shown in Table 8.

[0151] [Table 8]

[0152] Based on the above detection results, the detection sensitivity of the method of the present invention for colorectal cancer was 73.3%, and the specificity was 96.9%. The detection rates for stages I to IV were 51.5%, 82.1%, 76.3%, and 100%, respectively. The detection results showed that the plasma methylation levels of colorectal cancer patients were significantly higher than those of healthy subjects, and this difference was statistically significant (Figure 5). Furthermore, the methylation level was related to the tumor stage, with higher tumor stages resulting in higher plasma methylation levels (Figure 5). From the ROC curve analysis, when detecting colorectal cancer using the method of the present invention, the majority of the selected samples were patients with stages I-III, which are more difficult to detect. Although patients with stage IV accounted for 8.3% (10 / 120), the area under the curve (AUC) was 0.9152, which is significantly higher than the 0.8912 of the conventional invention (CN112159844B, which had a higher proportion of stage IV patients), indicating that the diagnostic accuracy of this method is higher (Figure 6). More importantly, this method exhibits a high detection specificity of 96.9%, demonstrating superior clinical performance when used clinically, particularly in diagnoses requiring high specificity. In this example, the three false-positive cases had ΔCq values ​​of -3.75, -3.71, and -3.69, respectively, which were very close to the cutoff value (-4). By adopting a stricter cutoff value (e.g., -3), the specificity can be improved to 100%.

[0153] Example 8 Use of DNA methylation markers in prognosis and recurrence prediction of colorectal cancer

[0154] 1. Experimental Method (1) 160 subjects (all patients who underwent curative surgery for stage I-III colorectal cancer) were recruited, and blood samples were collected from patients within two weeks post-surgery (median time: 5 days post-surgery).

[0155] (2) According to the present invention, DNA after the above-mentioned bisulfite conversion was subjected to multiplex real-time fluorescence quantitative PCR detection (the experimental method was the same as in Example 6).

[0156] 2. Experimental Results Of the 160 subjects, 116 did not experience recurrence within 2 years post-surgery, and 44 experienced recurrence within 2 years post-surgery. Of the 116 patients who did not experience recurrence within 2 years, only 5 tested positive for postoperative MRD detection, with a specificity of 95.7%. Of the 44 patients who experienced recurrence within 2 years, MRD was detected in plasma samples taken 5 days post-surgery in 27 patients, with a sensitivity of 61.4%. Of the 32 patients who tested positive for postoperative MRD, 27 experienced recurrence within 2 years, resulting in a positive prediction rate of 84.3%.

[0157] Survival curves were plotted according to each patient's recurrence status and postoperative ctDNA status (Figure 7). Patients with positive postoperative ctDNA results had a significantly shorter relapse-free survival (RFS) than patients with negative ctDNA (P<0.0001).

[0158] There are unique considerations when it comes to the postoperative treatment of stage II colorectal cancer. Adjuvant chemotherapy is recommended for high-risk stage II patients, but generally not for low-risk stage II patients. According to NCCN guidelines, risk factors for stage II colorectal cancer include poorly differentiated or undifferentiated tumor, lymphatic / vascular invasion, nerve invasion, bowel obstruction, lymph node detection <12, or T4N0M0, or T3 with local perforation, approaching the incisal margin, uncertain incisal margin, or positive incisal margin.

[0159] The inventors conducted a subgroup analysis of 76 colorectal cancer patients, and the results are shown in Figure 8.

[0160] 1) There was no significant difference in the amount of cfDNA in plasma within two weeks post-surgery between patients with recurrent and recurrent stage II colorectal cancer (A).

[0161] 2) Peripheral blood target methylation levels in postoperative MRD detection in recurrent patients were significantly higher than in non-recurrent patients (B).

[0162] 3) Of the 13 patients who tested positive for MRD two weeks post-surgery, 12 experienced recurrence within two years, with a positive prediction rate of 92.3%. However, 7.9% (5 / 63) of patients who tested negative also experienced recurrence within two years (RFS, HR, 16, P<0.0001) (C, D).

[0163] 4) In both clinically low-risk and high-risk subgroups, MRD detection results at 2 weeks post-surgery could predict recurrence within 2 years (E,F).

[0164] These data suggest that high levels of ctDNA methylation are associated with a poor prognosis, and that the detection of ctDNA methylation can effectively predict the surgical outcome of patients.

[0165] The DNA methylation screening method of the present invention can select highly tumor-specific DNA methylation markers, thereby enabling the design of highly specific multiple DNA methylation-specific fluorescence quantitative PCR (mqMSP). Based on the high specificity of the marker and detection method, the inventors predicted recurrence at 2 years post-surgery by detecting plasma ctDNA within 2 weeks (5 days) post-surgery and obtained very high positive prediction values ​​(84.3% positive prediction value for stages I to III as a whole, and 92.3% positive prediction value for stage II). These very high positive prediction values ​​are expected to lead to a reduction in the probability of recurrence in patients with minimal residual lesions after surgery by enabling more aggressive follow-up and the use of more aggressive chemotherapy, or by enabling early detection of postoperative recurrence and increasing the opportunity for secondary surgery, ultimately benefiting the patient.

[0166] Those skilled in the art will understand that by regularly and continuously monitoring postoperative patients, obtaining peripheral blood plasma samples, and performing detection using this method, the detection rate of recurrence can be further improved.

[0167] 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.

[10] 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.

[11] 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.

[12] VAN DEN BERG I, VAN DE WEERD S, ROODHART J M L, et al. Improving clinical management of colon cancer through CONNECTION, a nation-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.

[13] 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.

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

[15] 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.

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

[17] 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.

[18] 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.

[19] 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.

[20] 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.

[21] 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.

[22] 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.

[23] 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.

[24] 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.

[25] 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.

[26] 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.

[27] 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

[28] 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.

[29] 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.

[30] 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.

[31] 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.

[32] 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.

[33] 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

[34] 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 the presence or absence of tumors or precancerous lesions in a subject, diagnosing the presence or absence of tumor microresiduals, determining the postoperative prognosis of a tumor patient, predicting postoperative recurrence in a tumor patient, or evaluating the effectiveness of treatment in a tumor patient, This includes detecting multiple DNA methylation markers in free DNA in a sample derived from the subject to determine the level of DNA methylation, If the methylation level is higher than that of a normal control sample, it indicates the presence of a tumor or precancerous lesion, the presence of microresidual lesions, poor postoperative prognosis in tumor patients, increased risk of postoperative recurrence in tumor patients, or poor treatment efficacy in the subject. The free DNA in the sample derived from the subject originates from two types of cells: tumor or precancerous lesion cells and non-cancerous cells. The method is characterized in that each DNA methylation marker has a lower methylation level in non-cancer cells in the subject sample, and at least one DNA methylation marker in the group of methylation markers has a higher methylation level in tumor cells or precancerous lesion cells.

2. The method according to claim 1, characterized in that the methylation marker is such that the methylation level in tumor or precancerous lesion cells is at least 15% lower than that of corresponding normal cells.

3. The method according to 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, feces, and tissue samples.

4. Non-cancerous cells in the subject's sample include genomic DNA-containing cells, such as neutrophils, B cells, T cells, vascular endothelial cells, and hepatocytes. When the aforementioned 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%. The method according to any one of claims 1 to 3, characterized in that, 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 methylation marker is one in which the methylation level in non-cancer cells in the subject sample is less than 0.1%. More preferably, the method according to any one of claims 1 to 4, characterized in that it is below the detection limit or detection threshold of one or more DNA methylation detection methods.

6. The method according to any one of claims 1 to 5, characterized in that the methylation marker has a lower methylation level in the peritumoral tissue, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, and most preferably less than 1%.

7. The method according to any one of claims 1 to 6, characterized in that the DNA methylation marker group comprises at least two, preferably at least five, and more preferably at least ten methylation markers.

8. The methylation markers are two or more selected from the group consisting of ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1, and SFMBT2. Preferably, the method according to any one of claims 1 to 7, characterized in that it is all of ZEB2, MSC, ENSG00000232377, IRF4, C12orf42, FBN1, AKR1B1, CELSR3, EMBP1 and SFMBT2.

9. The method according to any one of claims 1 to 8, characterized in that the tumor is colorectal cancer.

10. The detection of the methylation marker is performed using multiplex quantitative methylation-specific PCR. The method according to any one of claims 1 to 9, optionally comprising measuring the internal standard gene ACTB in the multiplex quantitative methylation-specific PCR.

11. The multiple quantitative methylation-specific PCR described above uses a primer and probe for the methylation marker described in claim 8, and a primer and probe for the internal standard gene ACTB. The aforementioned primer is a primer pair as follows: SEQ ID NO:4, or a sequence having sufficient identity with SEQ ID NO:4 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:5, or a sequence having sufficient identity with SEQ ID NO:5 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:7, or a sequence having sufficient identity with SEQ ID NO:7 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:8, or a sequence having sufficient identity with SEQ ID NO:8 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:10, or a sequence having sufficient identity with SEQ ID NO:10 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:11, or a sequence having sufficient identity with SEQ ID NO:11 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:13, or a sequence having sufficient identity with SEQ ID NO:13 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:14, or a sequence having sufficient identity with SEQ ID NO:14 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:16, or a sequence having sufficient identity with SEQ ID NO:16 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:17, or a sequence having sufficient identity with SEQ ID NO:17 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:19, or a sequence having sufficient identity with SEQ ID NO:19 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:20, or a sequence having sufficient identity with SEQ ID NO:20 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:22, or a sequence having sufficient identity with SEQ ID NO:22 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:23, or a sequence having sufficient identity with SEQ ID NO:23 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:25, or a sequence having sufficient identity with SEQ ID NO:25 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:26, or a sequence having sufficient identity with SEQ ID NO:26 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:28, or a sequence having sufficient identity with SEQ ID NO:28 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:29, or a sequence having sufficient identity with SEQ ID NO:29 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:31, or a sequence having sufficient identity with SEQ ID NO:31 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:32, or a sequence having sufficient identity with SEQ ID NO:32 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:34, or a sequence having sufficient identity with SEQ ID NO:34 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:35, or a sequence having sufficient identity with SEQ ID NO:35 and capable of guaranteeing the sensitivity and specificity of PCR amplification, SEQ ID NO:1, or a sequence having sufficient identity with SEQ ID NO:1 and capable of guaranteeing the sensitivity and specificity of PCR amplification, and SEQ ID NO:2, or a sequence having sufficient identity with SEQ ID NO:2 and capable of guaranteeing the sensitivity and specificity of PCR amplification, Selected from, and, The method according to 10, characterized in that the probe is a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3, or a sequence that has sufficient identity with a sequence selected from SEQ ID NO: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 and 3, and that can guarantee the sensitivity and specificity of PCR amplification.

12. A kit for diagnosing the presence or absence of tumors or precancerous lesions in a subject, diagnosing the presence or absence of tumor microresiduals, determining the postoperative prognosis of tumor patients, predicting postoperative recurrence in tumor patients, or evaluating the therapeutic effect of tumor patients, comprising a reagent for detecting the marker described in any one of claims 1 to 11, Optionally, the kit further comprises a reagent for detecting the internal standard gene ACTB.

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

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

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

Images