Methylated biomarker for detecting colorectal cancer or colorectal adenoma

Abstract

The invention relates to a methylation biomarker for detecting colorectal cancer or colorectal adenoma. The methylation biomarker comprises any one gene of SDC2, COL4A2 and C9H9orf50 or any combination of the SDC2, the COL4A2 and the C9H9orf50. Wherein a methylation target detection region of the SDC2 gene is a full-length region of chr8: 96494012-96494130 or a partial region of the full-length region of chr8: 96494012-96494130; a methylation target detection region of the COL4A2 gene is a full-length region of chr13: 110306814-110306983 or a part of the full-length region of the chr13: 110306814-110306983; and the methylation target detection region of the C9H9orf50 gene is the full-length region of chr9: 129620291-129620374 or a part of the full-length region of chr9: 129620291-129620374. According to the kit, methylation signals related to intestinal cancer and adenoma in excrement samples can be more accurately detected through detection in different combination modes of the three markers, the early effective diagnosis rate of colorectal cancer patients can be increased, and therefore the survival rate of the patients is increased, and prognosis is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomarkers, and particularly to a methylation biomarker or a combination thereof for detecting colorectal cancer or colorectal adenoma and its application. Background Art

[0002] Colorectal cancer is one of the malignant tumors with relatively high incidence and mortality rates globally. Early diagnosis and treatment are of great significance for improving the survival rate of patients. Currently, the screening and diagnostic methods for colorectal cancer mainly include fecal occult blood test, colonoscopy, imaging examination, and molecular biomarker detection, etc.

[0003] The fecal occult blood test (FOBT) is a commonly used non-invasive screening method, which has the advantages of simple operation and low cost. However, its sensitivity and specificity are relatively low, especially the detection rate for early colorectal cancer and adenomas is not ideal, and false positive and false negative results are prone to occur. Although colonoscopy is considered the "gold standard" for the diagnosis of colorectal cancer, it is highly invasive, has low patient compliance, and has a certain risk of complications, so it is not suitable as the first choice for large-scale population screening. Imaging examinations such as CT and MRI have limited sensitivity for detecting early small lesions, and the examination costs are relatively high.

[0004] In recent years, molecular biomarkers based on DNA methylation have shown good application prospects in the early diagnosis of colorectal cancer. DNA methylation is an important epigenetic modification and plays a key role in the occurrence and development of tumors. Studies have shown that abnormal methylation of specific genes can be used as biomarkers for the early diagnosis of colorectal cancer.

[0005] Currently, there have been many studies reporting the application of different methylation markers in the diagnosis of colorectal cancer. For example, it has been found that the methylation level of the C9orf50 gene is significantly increased in patients with colorectal cancer or adenomas, and can be used as a marker for diagnosis or auxiliary diagnosis, with relatively high sensitivity and specificity. Another study has shown that the increase in the methylation level of the LRRC4 gene is closely related to the occurrence of colorectal cancer or adenomas, and detecting the methylation status of this gene can effectively improve the detection rate of colorectal cancer or adenomas.

[0006] In addition, the methylation detection of ADHFE1 and SND1 genes has also been applied to the diagnosis of colorectal cancer, which can effectively improve the detection rate of advanced adenomas and the early detection rate of colorectal cancer. It has also been confirmed that the increase in the methylation level of the CpG island of the DOK6 gene can be used for the diagnosis or auxiliary diagnosis of colorectal cancer or precancerous lesions, with relatively high sensitivity and specificity. The methylation status of the C9orf15 gene has also been reported to be used as a marker for the early diagnosis of colorectal cancer and its precancerous lesions, and has good detection effects on advanced colorectal adenomas and colorectal cancer.

[0007] However, there are still some limitations in the clinical application of existing methylation markers. First, the diagnostic efficacy of a single marker is often not ideal, and it is difficult to achieve both high sensitivity and high specificity simultaneously. Second, the detection ability of some markers for precancerous lesions such as early colorectal cancer and adenomas is limited and cannot meet the needs of early screening. In addition, the applicability and stability of existing markers in different populations remain to be verified and may be affected by factors such as age, gender, and race. Finally, the complexity and cost of existing detection methods also limit their application in large-scale population screening.

[0008] Therefore, developing new methylation biomarkers or biomarker combinations to improve the accuracy and reliability of early diagnosis of colorectal cancer and its precancerous lesions has important clinical significance and application value. An ideal biomarker should have high sensitivity and high specificity, be able to effectively distinguish normal populations, adenoma patients, and colorectal cancer patients, and the detection method should be simple, non-invasive, and cost-effective, suitable for large-scale population screening. Summary of the Invention

[0009] The technical problem to be solved by the present invention is that, aiming at the defects existing in the existing colorectal cancer detection methods, such as the poor sensitivity and specificity of fecal occult blood test, colonoscopy being an invasive examination with low patient compliance, the limited detection sensitivity of imaging examination for early micro-lesions, and the poor performance of the detection markers adopted by existing products on the market and the disclosed patents in terms of the sensitivity and specificity of colorectal cancer, especially adenoma detection, and being difficult to accurately identify early lesions, etc., to provide an accurate, non-invasive, convenient colorectal cancer early detection technology with high sensitivity and specificity.

[0010] On the one hand, the present invention provides a methylation biomarker for detecting colorectal cancer or colorectal adenoma, including any one gene or any combination thereof among SDC2, COL4A2, and C9H9orf50. The methylation biomarker can be any one of the SDC2 gene, the COL4A2 gene, or the C9H9orf50 gene, or a combination of any two of these three genes, or all combinations of these three genes. The methylation status of these genes is closely related to the occurrence and development of colorectal cancer or colorectal adenoma. By detecting the methylation level of these genes, it is possible to effectively determine whether the subject has colorectal cancer or colorectal adenoma.

[0011] Specifically, the methylation target detection region of the SDC2 gene is the full-length region of chr8:96494012-96494130 or a partial region thereof. This region is located in the promoter region of the SDC2 gene and shows a hypermethylated state in patients with colorectal cancer or colorectal adenomas. By detecting the methylation level of this region, patients with colorectal cancer or colorectal adenomas can be effectively distinguished from healthy individuals; the methylation target detection region of the COL4A2 gene is the full-length region of chr13:110306814-110306983 or a partial region thereof. This region also shows an abnormal methylation pattern in patients with colorectal cancer or colorectal adenomas and is an effective methylation biomarker; the methylation target detection region of the C9H9orf50 gene is the full-length region of chr9:129620291-129620374 or a partial region thereof. The change in the methylation state of this region is related to the occurrence and development of colorectal cancer or colorectal adenomas and can be used as a biomarker for detecting colorectal cancer or colorectal adenomas.

[0012] In practical applications, it is possible to select to detect the methylation levels of one, two, or all three of the above genes. When multiple genes are selected for detection, the accuracy and reliability of the detection can be improved. For example, it is possible to simultaneously detect the methylation levels of the SDC2 and COL4A2 genes, the methylation levels of the SDC2 and C9H9orf50 genes, the methylation levels of the COL4A2 and C9H9orf50 genes, or simultaneously detect the methylation levels of the three genes SDC2, COL4A2, and C9H9orf50.

[0013] For the methylation target detection region of the SDC2 gene, it is possible to select to detect the full-length region of chr8:96494012-96494130, or it is also possible to select to detect a specific fragment within this region, such as chr8:96494050-96494100. Similarly, for the COL4A2 and C9H9orf50 genes, it is also possible to select to detect the full-length or partial regions of their methylation target detection regions.

[0014] On the other hand, the present invention provides a kit for detecting colorectal cancer or colorectal adenomas, including reagents for detecting the methylation levels of the above methylation markers or their combinations.

[0015] The kit can detect the methylation degree of methylation biomarkers by a variety of methods, including but not limited to fluorescence quantitative PCR, methylation-specific PCR, digital PCR, DNA methylation chip, targeted DNA methylation sequencing, methylation-sensitive restriction enzyme method, direct sequencing method, methylation-sensitive single nucleotide primer extension, bisulfite-conjugated restriction enzyme method, methylation-sensitive single-strand conformation analysis, methylation-sensitive denaturing gradient gel electrophoresis, methylation-specific denaturing high performance liquid chromatography, methylation-specific microarray, methylation-sensitive melting curve analysis, methylation-sensitive dot blot analysis, methylation-specific multiplex ligation-dependent probe amplification, bisulfite sequencing or pyrosequencing.

[0016] Furthermore, the detection sample of the kit is taken from an in vitro blood sample or an in vitro tissue sample of the subject to be tested, preferably a fecal sample.

[0017] Furthermore, the kit includes probes and primers, and the primers and probes include specific primers and probes for the transformed sequences of the following genes or their fragments: any one or more of SDC2, COL4A2 or C9H9orf50.

[0018] Furthermore, the methylation target detection region of the SDC2 gene is the full-length region of chr8:96494012-96494130 or a partial region thereof; the methylation target detection region of the COL4A2 gene is the full-length region of chr13:110306814-110306983 or a partial region thereof; the methylation target detection region of the C9H9orf50 gene is the full-length region of chr9:129620291-129620374 or a partial region thereof.

[0019] Further, the primer nucleotide sequences for detecting the methylated biomarker SDC2 gene or its fragment are nucleotide sequences having at least 80% identity with SEQ ID NO.1 and nucleotide sequences having at least 80% identity with SEQ ID NO.2, and the probe sequence is a nucleotide sequence having at least 80% identity with SEQ ID NO.3; the primer nucleotide sequences for detecting the methylated biomarker COL4A2 gene or its fragment are nucleotide sequences having at least 80% identity with SEQ ID NO.4 and nucleotide sequences having at least 80% identity with SEQ ID NO.5, and the probe sequence is a nucleotide sequence having at least 80% identity with SEQ ID NO.6; the primer nucleotide sequences for detecting the methylated biomarker C9H9orf50 gene or its fragment are nucleotide sequences having at least 80% identity with SEQ ID NO.7 and nucleotide sequences having at least 80% identity with SEQ ID NO.8, and the probe sequence is a nucleotide sequence having at least 80% identity with SEQ ID NO.9.

[0020] On the other hand, the present invention also provides the use of the above methylated biomarker or its combination in the preparation of a kit for detecting, diagnosing, classifying or predicting, monitoring treatment, prognosis or other evaluation of colorectal cancer or colorectal adenoma.

[0021] In the detection application of colorectal cancer or colorectal adenoma, the methylation levels of the three genes SDC2, COL4A2, and C9H9orf50 can be used as biomarkers to develop a kit for early screening. Such a kit can be used for population screening, especially for high-risk populations, such as those with a family history of colorectal cancer, people over 40 years old, etc.

[0022] In the diagnostic application of colorectal cancer or colorectal adenoma, a kit for auxiliary diagnosis can be developed using these methylated biomarkers. When the results of traditional diagnostic methods such as colonoscopy and imaging examination are unclear, the levels of these methylated biomarkers can be detected to provide additional evidence for diagnosis.

[0023] In the classification or prediction application of colorectal cancer or colorectal adenoma, a kit for classification or prediction can be developed using these methylated biomarkers. Different types or stages of colorectal cancer or colorectal adenoma may exhibit different methylation patterns. By detecting the levels of these methylated biomarkers, the disease can be classified or the development trend of the disease can be predicted.

[0024] In the application of treatment monitoring for colorectal cancer or colorectal adenoma, these methylation markers can be used to develop kits for treatment monitoring. During the treatment process, regularly detecting the levels of these methylation markers can evaluate the treatment effect and timely adjust the treatment plan.

[0025] In the application of prognostic assessment for colorectal cancer or colorectal adenoma, these methylation markers can be used to develop kits for prognostic assessment. By detecting the levels of these methylation markers, the prognosis of patients can be predicted, providing a basis for formulating individualized treatment plans.

[0026] In other applications for evaluating colorectal cancer or colorectal adenoma, such as recurrence risk assessment, metastasis risk assessment, etc., corresponding kits can also be developed using these methylation markers.

[0027] These applications can use a single methylation marker alone or a combination of multiple methylation markers. When using a combination of multiple methylation markers, the accuracy and reliability of detection, diagnosis, classification, or prediction, treatment monitoring, prognosis, or other evaluations can be improved.

[0028] For example, in the early screening of colorectal cancer, the methylation levels of two genes, SDC2 and COL4A2, can be detected simultaneously. By comprehensively analyzing the results of these two markers, the sensitivity and specificity of the screening can be improved.

[0029] In the classification of colorectal cancer, the methylation levels of three genes, SDC2, COL4A2, and C9H9orf50, can be detected simultaneously. By analyzing the methylation patterns of these three markers, colorectal cancer can be divided into different subtypes, providing a basis for individualized treatment.

[0030] In the treatment monitoring of colorectal cancer, the levels of these methylation markers can be regularly detected. By comparing the changes before and after treatment, the treatment effect can be evaluated and subsequent treatment can be guided.

[0031] On the other hand, the present invention also provides a method for detecting, diagnosing, staging, classifying, treating, monitoring, prognosticating, or other evaluating colorectal cancer or colorectal adenoma, comprising the following steps:

[0032] (1) Extracting the genomic DNA and / or cell-free DNA of the biological sample to be tested;

[0033] (2) Performing bisulfite conversion on the DNA;

[0034] (3) Performing co-methylation detection on the bisulfite-converted DNA and a control for the methylation marker or its combination as described in claim 1 or 2 to obtain a methylation map;

[0035] (4) Compare the methylation map of the methylation marker or its combination with the map determination threshold obtained from mathematical modeling based on the dataset to make a judgment on the detection, diagnosis, staging, classification, treatment monitoring, and prognosis of colorectal cancer or colorectal adenoma.

[0036] In step (4), compare the detected methylation map with the pre-established determination threshold to make a judgment. The determination threshold is obtained by analyzing the methylation data of a large number of known samples (including samples of colorectal cancer or colorectal adenoma patients and healthy control samples) using mathematical modeling methods.

[0037] For example, for the SDC2 gene, if its methylation level exceeds the determination threshold, it is judged as positive for colorectal cancer or colorectal adenoma; if its methylation level is lower than the determination threshold, it is judged as negative for colorectal cancer or colorectal adenoma.

[0038] When detecting multiple methylation markers simultaneously, a comprehensive scoring system can be used for judgment. For example, a certain weight can be assigned to each marker, the comprehensive score can be calculated, and then compared with the determination threshold to make a final judgment.

[0039] This method can not only be used for the detection and diagnosis of colorectal cancer or colorectal adenoma, but also for disease staging, classification, treatment monitoring, and prognosis evaluation, etc. For example, by analyzing the methylation maps of colorectal cancer patients at different stages, a model for disease staging can be established; by analyzing the changes in methylation maps before and after treatment, the treatment effect can be evaluated; by analyzing the relationship between methylation maps and the prognosis of patients, the prognosis of patients can be predicted.

[0040] The beneficial effects of the present invention are as follows: Compared with the prior art, the three innovative markers screened by the present invention have higher specificity and sensitivity, can more accurately detect the methylation signals related to intestinal cancer and adenoma in fecal samples, and the present invention is based on fecal sample detection, with significant advantages such as convenient acquisition, non-invasive, and high patient acceptance, which helps to improve the early effective diagnosis rate of colorectal cancer patients, thereby improving the survival rate of patients and improving the prognosis. Detailed implementation manners

[0041] Next, the technical solutions of the present invention will be described clearly and completely in conjunction with the embodiments. Obviously, the described embodiments are a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0042] It should be noted that for the experimental methods without specific conditions in the following embodiments of the present invention, they are generally carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. All common chemical reagents used in the embodiments are commercially available products.

[0043] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0044] To facilitate the understanding of the present technology, some terms and phrases are defined below.

[0045] In this specification, the term "plurality" refers to two or more. "And / or" describes the association relationship of associated objects and indicates that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0046] In this specification, the term "cancer" (also known as carcinoma) generally refers to any type of malignant neoplasm, that is, any morphological and / or physiological change (based on genetic re-programming) of target cells that shows or has a tendency to exhibit cancer characteristics compared to unaffected (healthy) wild-type control cells. Examples of such changes may involve cell size and shape (becoming larger or smaller), cell proliferation (increase in cell number), cell differentiation (change in physiological state), apoptosis (programmed cell death), or cell survival. Therefore, the term "gastric cancer" refers to a cancerous growth in the gastric parenchyma.

[0047] In this specification, the term "nucleic acid detection" refers to any method for determining the nucleotide composition of a target nucleic acid. Nucleic acid detection assays include, but are not limited to, DNA sequencing methods and probe hybridization methods.

[0048] In this specification, the "methylation level", "methylation degree", "methylation state", "methylation profile", and "methylation status" of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in the nucleic acid molecule. For example, a nucleic acid molecule containing methylated cytosine is considered methylated (for example, the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule without any methylated nucleotides is considered unmethylated.

[0049] As used herein, the term "methylation assay" or "methylation level detection" or "methylation degree detection" refers to any assay for determining the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0050] As used herein, the term "bisulfite reagent" refers to a reagent that in some embodiments comprises bisulfite, disulfite, hydrogensulfite, or a combination thereof. DNA treated with a bisulfite reagent will have its unmethylated cytosine nucleotides converted to uracil, while methylated cytosines and other bases remain unchanged, thus allowing for the discrimination of methylated and unmethylated cytidines, for example, in a CpG dinucleotide sequence.

[0051] As used herein, the term "polymerase chain reaction" is used to amplify a target sequence and consists of the following steps: introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by a precise thermal cycling sequence in the presence of a DNA polymerase. The two primers are complementary to the respective strands of the double-stranded target sequence. To effect amplification, the mixture is denatured, and then the primers anneal to their complementary sequences within the target molecule. After annealing, the primers are extended with a polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (i.e., denaturation, annealing, and extension constitute a "cycle"; there can be many "cycles") to obtain a high concentration of the amplified fragment of the desired target sequence. The length of the amplified fragment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and thus this length is a controllable parameter. Due to the repetitive aspect of this method, it is called the "polymerase chain reaction" ("PCR"). Since the desired amplified fragment of the target sequence becomes the major sequence (in terms of concentration) in the mixture, it is said to be "PCR amplified," and is a "PCR product" or "amplicon."

[0052] As used herein, the term "nucleic acid detection assay" refers to any method for determining the nucleotide composition of a target nucleic acid. Nucleic acid detection assays include, but are not limited to, DNA sequencing methods and probe hybridization methods.

[0053] As used herein, the term "amplifiable nucleic acid" refers to a nucleic acid that can be amplified by any amplification method. An "amplifiable nucleic acid" is expected to generally contain a "sample template."

[0054] As used in this specification, the term "primer" refers to an oligonucleotide that is naturally occurring or synthetically produced in a purified restriction digest and that, when placed under conditions that induce the synthesis of a primer extension product that is complementary to a nucleic acid strand (e.g., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH), is capable of acting as a point of initiation for synthesis. The primer is preferably single-stranded for maximum efficiency in amplification, but may also be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare the extension product. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to prime the synthesis of the extension product in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, primer source, and the method of use.

[0055] As used in this specification, the term "probe" refers to an oligonucleotide (e.g., a nucleotide sequence) that is naturally occurring in a purified restriction digest or that is synthetically produced, recombinantly produced, or produced by PCR amplification and that is capable of hybridizing to another target oligonucleotide. The probe may be single-stranded or double-stranded. The probe can be used for the detection, identification, and isolation of a specific gene sequence (e.g., a "capture probe"). In some embodiments, any probe used in the present invention can be labeled with any "reporter molecule" such that it is detectable in any detection system.

[0056] As used in this specification, the term "sample" refers to any substance that may contain a target molecule that needs to be analyzed, including biological samples. As used herein, a "sample" or "biological sample" refers to any sample obtained from a living or viral (or prion) source or other source of macromolecules and biomolecules and includes any cell type or tissue of a subject from which nucleic acids, proteins, and / or other macromolecules can be obtained. The sample or biological sample can be a sample obtained directly from a biological source or a processed sample. The sample or biological sample includes, but is not limited to, body fluids (e.g., whole blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, mucus, amniotic fluid, etc.), exudates, bone marrow samples, ascites, pelvic washings, pleural fluid, spinal fluid, lymph fluid, ocular fluid, extracts of nasal, laryngeal, or genital swabs, cell suspensions of digestive tissues, or extracts of fecal material, as well as tissue and organ samples from humans, animals (e.g., non-human mammals), and plants, and processed samples derived therefrom.

[0057] As used in this specification, the term "subject" can be a mammal or a cell, tissue, organ, or part of the mammal. In the present invention, a mammal refers to any species of mammal, preferably a human (including a human, a human subject, or a human patient). Subjects and mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, and rodents such as mice and rats.

[0058] In this specification, the term "cell-free DNA" or its synonyms "cfDNA (circulating free DNA)" and "circulating DNA" refer to DNA that is not contained within intact cells in the corresponding body fluid from which the sample is taken or obtained, but instead circulates freely in the body fluid sample. Cell-free DNA is typically fragmented genomic DNA.

[0059] As used in the present invention, the term "sensitivity" refers to the proportion of positive results detected from samples that are confirmed positive. Its calculation formula is: Sensitivity = (Detected positives / True positives), where true positives are those confirmed positive using a recognized gold standard. And "specificity" refers to the proportion of normal results detected in a certain number of normal human samples. Its calculation formula is Specificity = (Detected negatives / True negatives).

[0060] In this specification, diagnosis includes the detection or identification of the disease state or condition of a subject, determining the likelihood that a subject will develop a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis (or its possible progression or regression) of a subject with a disease or condition, and determining the effect of treatment on a subject with a disease or condition.

[0061] By studying the methylation differences of DNA methylation modifications in fecal samples among patients with colorectal cancer and colorectal adenomas, the present invention successfully screened out 3 novel markers and their combinations that can be used as diagnostic markers for non-invasive screening of colorectal cancer and adenomas. Compared with the markers disclosed in the prior art, these 3 novel markers and their combinations have higher detection specificity and sensitivity, and can more accurately detect the methylation signals related to colorectal cancer and adenomas in fecal samples. By applying these 3 innovative markers to the methylation-specific site PCR technology system, the sensitivity and specificity of fecal colorectal cancer methylation detection for colorectal cancer and adenomas can be significantly improved, making it suitable for large-scale popularization and application.

[0062] Example 1 Identification of Methylation Markers for Colorectal Cancer and Adenomas

[0063] The screening process and steps for methylation markers of colorectal cancer and adenomas are as follows:

[0064] 1. Gene Screening and Selection

[0065] By researching domestic and foreign literature, cohort studies, and existing products, genes with better specificity and sensitivity for colorectal cancer and adenomas were screened, and finally SDC2, COL4A2, and C9H9orf50 were determined as the genes for detecting methylation;

[0066] 2. Determination of CG Site Selection on Genes

[0067] Perform gene chip sequencing (450K / 850K) on cancer tissues and adjacent tissues (normal tissues), detect the methylation levels of the CG sites of the aforementioned genes, and screen out significantly different sites by comparing the methylation levels of these CG sites in cancer tissues and adjacent tissues, which are used as the basis for detecting sites;

[0068] 3. Design and screening of primer probes

[0069] Design primer probes for the aforementioned screened gene regions. In addition to the conventional primer probe design principles, the following additional requirements need to be met: The probe focuses on distinguishing mutations, and important CG sites need to be placed on the probe. Screen the designed primer probes at the cell line level, tissue level, and fecal level in sequence to obtain the biomarker combination with the best effect. The design of the screened primer probes is shown in the following table:

[0070] Table 1

[0071]

[0072] Example 2 Detection method of methylation markers for colorectal cancer and adenoma diagnosis

[0073] Perform methylation library construction and sequencing on the collected samples. The process and steps are as follows:

[0074] 1. Nucleic acid extraction

[0075] a. After centrifuging the 50 mL tube containing the sample, aspirate the sample (aspirate 1.35 ml of the supernatant) into a 2 mL centrifuge tube, add 0.45 mL of the impurity removal buffer, invert several times and then vortex for 10 seconds, and centrifuge at 13000 rpm for 2 minutes. Take 1.2 mL of the middle supernatant for standby;

[0076] b. Add 1.2 mL of lysis buffer, 1.2 mL of the centrifuged sample supernatant, 60 μL of proteinase K, and 10 μL of ribonuclease A solution to a 5 mL or 15 mL centrifuge tube in sequence. Place the centrifuge tube on a thermostatic mixer at 60 °C and 1200 rpm for 20 minutes;

[0077] c. Add 1.45 mL of isopropanol and 50 μL of magnetic bead suspension to the lysate, and use a thermostatic mixer to vortex at room temperature for 10 minutes (Note: The magnetic beads need to be vortexed for 30 seconds before use);

[0078] d. Place the centrifuge tube on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely adsorbed on the side wall of the centrifuge tube, discard the solution completely;

[0079] e. Remove the centrifuge tube from the magnetic stand, add 2 mL of Wash Buffer 1 (check if absolute ethanol has been added before use), and place it on a thermostatic mixer at room temperature with a rotation speed of 1600 rpm for 1 minute of shaking and mixing (ensure that the magnetic beads are in a mixed state during the shaking process). Then place the centrifuge tube on the magnetic stand and let it stand for 1 minute. After the magnetic beads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand to wash off the impurities on the centrifuge tube cap, and then completely discard the solution (keep the centrifuge tube fixed on the magnetic stand);

[0080] f. Repeat the above step e;

[0081] g. Remove the centrifuge tube from the magnetic stand, add 2 mL of Wash Buffer 2 (check if absolute ethanol has been added before use), vortex for 5 seconds, and then place it on a thermostatic mixer at room temperature with a rotation speed of 1600 rpm for 1 minute of shaking and mixing (ensure that the magnetic beads are in a mixed state during the shaking process). Then place the centrifuge tube on the magnetic stand and let it stand for 1 minute. After the magnetic beads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand to wash off the impurities on the centrifuge tube cap, and then completely discard the solution (keep the centrifuge tube fixed on the magnetic stand);

[0082] h. Repeat step g;

[0083] i. Keep the centrifuge tube fixed on the magnetic stand, use a pipette to further remove the solution at the bottom and on the cap of the centrifuge tube, and then place it at room temperature for 5 - 10 minutes to completely volatilize the ethanol (visually observe that the surface of the magnetic beads becomes matte and the magnetic beads have no cracks).

[0084] j. Remove the centrifuge tube from the magnetic stand, add 50 - 150 μL of Elution Buffer. Vortex to completely suspend the magnetic beads in the eluent, then transfer it to a 2 mL centrifuge tube and place it on a thermostatic mixer at 56 °C with a rotation speed of 1600 rpm for 10 minutes of oscillatory elution;

[0085] k. Place the centrifuge tube on the magnetic stand and let it stand for 2 minutes. After the magnetic beads are completely adsorbed on the side wall of the centrifuge tube, use a pipette to transfer the eluent to a new centrifuge tube and store it at -20 °C for standby (or proceed with bisulfite conversion).

[0086] 2. Rapid bisulfite conversion and purification

[0087] a. Add 130 μl of CTConversionReagent to 20 μl of DNA (1 ng - 2 μg, with better results for 10 ng - 1 μg). Vortex or pipette to mix well, and then briefly centrifuge to collect the reaction solution at the bottom of the tube; Note: It is normal for a small amount of crystals to precipitate in CTConversionReagent. It can be heated at 60 °C or vortexed until all crystals are completely dissolved. After equilibrating to room temperature, use it, which will not affect the kit effect;

[0088] b. Place the PCR tube in a PCR instrument and perform the reaction according to the following program:

[0089] Table 2

[0090] Temperature Time Hot lid at 105 °C On 95℃ 10 min 20℃ Hold (<20 h)

[0091] c. Add 600 μl of E-Binding Buffer, 10 μl of E-Binding Beads and the transformation product from the previous step to a 1.5 ml nuclease-free centrifuge tube. Pipette 6 - 8 times or vortex at low speed for 30 sec to mix well. Incubate at room temperature for 10 min (it is recommended to incubate with shaking on a mixer, which helps to improve the recovery rate). Note: If the E-Binding Beads are stored at 0 - 4 °C, they need to be equilibrated to room temperature before each use. Since the E-Binding Beads tend to settle, they should be shaken well before each use;

[0092] d. Centrifuge the 1.5 ml nuclease-free centrifuge tube briefly and then place it on a magnetic stand. After the solution becomes clear (about 3 min), carefully remove the supernatant;

[0093] e. Add 400 μl of E-Wash Buffer (with the specified volume of absolute ethanol added) to the 1.5 ml nuclease-free centrifuge tube. Pipette 6 - 8 times or vortex at low speed for 30 sec to mix well. Centrifuge briefly and then place it on a magnetic stand. After the solution becomes clear (about 3 min), carefully remove the supernatant;

[0094] f. Add 200 μl of E-Desulphonation Buffer to the 1.5 ml nuclease-free centrifuge tube. Pipette 6 - 8 times or vortex at low speed for 30 sec to mix well. React at room temperature (15 - 25 °C) for 15 min (it is recommended to react with shaking on a mixer, which helps to improve the recovery rate). Centrifuge briefly and then place it on a magnetic stand. After the solution becomes clear (about 3 min), carefully remove the supernatant. Note: The sample should not stay in the E-Desulphonation Buffer for more than 25 min. The residence time includes the time for resuspending the magnetic beads, reacting and removing the supernatant;

[0095] g. Add 400 μl of E-Wash Buffer (with the specified volume of absolute ethanol added) to the 1.5 ml nuclease-free centrifuge tube. Pipette 6 - 8 times or vortex at low speed for 30 sec to mix well. Centrifuge briefly and then place it on a magnetic stand. After the solution becomes clear (about 3 min), carefully remove the supernatant;

[0096] h. Repeat the operation in step g once, discard all the supernatant, and rinse twice in total (Note: A 10 μl pipette can be used to discard the residual liquid to shorten the drying time);

[0097] i. Dry at 55 °C for 5 - 15 min or at room temperature for 20 - 30 min to fully remove the residual liquid in the tube until there is no reflection on the surface of the magnetic beads;

[0098] j. Add 25 μl of E-Elution Buffer, pipette 6 - 8 times or vortex at low speed for 30 sec to fully resuspend the magnetic beads, incubate at 55 °C for 4 min, centrifuge briefly and place on the magnetic stand. After the solution becomes clear (about 3 min), transfer the supernatant to a new 1.5 ml Nuclease-free centrifuge tube;

[0099] k. Store the transformation product at -30 to -15 °C. For long-term storage, place it at -85 to -65 °C, and avoid repeated freezing and thawing.

[0100] 3. PCR (PCR System Construction + PCR)

[0101] PCR System Construction: Add 11 μl of PCRMix reaction solution to a PCR tube and mix with 9 μl of the transformation product, vortex to mix well, and centrifuge. The components of the PCRMix reaction solution are shown in the following table:

[0102] Table 3

[0103] Reaction solution components Volume (μL) 400 mM Tris-HCl pH 8.3 0.95 2 M KCl 0.4 200 mM MgCl2 0.3045 1% Tween-20 0.4 dNTPs 0.3 20% NaN3 0.05 Taq 1 Anti-Taq 1 1 M sorbitol 0.42 Primer Probe mix 2 Water 4.1755 Total 11

[0104] Place the constructed PCR system in a PCR instrument and perform the reaction as described in the following table:

[0105] Table 4

[0106]

[0107] Example 3 Performance Verification

[0108] 1. Performance Verification of the Methylation Region of the SDC2 Gene and the Corresponding Primers and Probes

[0109] Set up the following 4 groups of experiments for verification. Only the primers and probes for the SDC gene are different in each group of experiments, and the rest of the detection steps are the same as those in Example 2. The specific settings are as follows:

[0110] Group A: The primers and probes for the SDC2 gene screened in Example 1 of the present invention;

[0111] Group B: The primers and probes for the SDC2 gene in Example 4 of CN114250275B;

[0112] Group C: SDC2 gene primer-probe combination 3 in Example 2 of CN114085905B;

[0113] Group D: qMSP primers and probes in the SDC2 gene in Example 1 of CN110317871B;

[0114] Among them, the primer and probe sequences of groups A, B, C, and D are as follows:

[0115] Table 5

[0116]

[0117] Using the detection method described in Example 2 for the above four groups of primer-probes, the detection performance was verified in intestinal cancer cell samples and normal cell samples respectively, and the test results are shown in the following table:

[0118] Table 6

[0119]

[0120] According to the test results, it can be seen that the CT value of group A is significantly better than the other three groups. Therefore, the SDC2 gene methylation target detection region and primer-probe sequence screened by the present invention are not only different from the publicly disclosed schemes in the prior art, but also have better detection performance.

[0121] 2. Detection performance verification of the biomarker combination for different sample types

[0122] The three biomarkers and their combinations screened by the present invention were horizontally compared with two commercially available kits at the cell line, tissue, and feces levels, and the following 5 groups of tests were set up for detection performance verification. The specific settings are as follows:

[0123] Group A: Primer-probes screened for the SDC2 gene in Example 1 of the present invention;

[0124] Group B: Primer-probes screened for the COL4A2 gene in Example 1 of the present invention;

[0125] Group C: Primer-probes screened for the C9H9orf50 gene in Example 1 of the present invention;

[0126] Group D: Commercially available reagent, with the detected biomarkers being TFPI2 + SDC2;

[0127] Group E: Commercially available reagent, with the detected biomarker being SDC2;

[0128] (1) Detection performance verification of samples at the cell line level

[0129] The detection performance verification was carried out on the above five groups of tests in normal cell samples and intestinal cancer cell samples respectively. Among them, the detection method described in Example 2 was used for groups A - C, and the detection method described in its product manual was used for groups D - E. The test results are shown in the following table:

[0130] Table 7 Performance comparison of different markers in normal cells and intestinal cancer cell lines

[0131]

[0132] *Interpretation criteria for multiple marker combinations: If the detection result of any one marker is positive, it is judged as positive.

[0133] According to the test results, it can be seen that the commercially available group E actually makes mistakes in the interpretation of normal cells. However, for the three markers SDC2, COL4A2, and C9H9orf50 screened by the present invention, the interpretation results of each marker alone and any combination are consistent with the true results, and the specificity is significantly better than that of the commercially available reagent (the positive and negative of all CTs are bounded by 38, that is, CT < 38 is judged as positive, and CT > 38 is judged as negative).

[0134] (2) Detection performance verification at the tissue sample level

[0135] The detection performance verification was carried out on the above five groups of tests in normal tissues, precancerous tissues, and intestinal cancer tissue samples respectively. Among them, the detection method described in Example 2 was used for groups A - C, and the detection method described in its product manual was used for groups D - E. The test results are shown in the following table:

[0136] Table 8 Performance comparison of different markers in normal tissues, precancerous tissues, and intestinal cancer tissues

[0137]

[0138] *Interpretation criteria for multiple marker combinations: If the detection result of any one marker is positive, it is judged as positive.

[0139] From the test results, it can be seen that for normal tissue samples, the three markers screened by the present invention are consistent with the clinical results of the commercially available reagent. However, in the detection of 10 precancerous tissue samples and cancer tissue samples, the three markers screened by the present invention not only have a better positive detection rate of individual markers than the individual markers of the commercially available reagent, but also the positive detection rate of any marker combination is better than that of the commercially available reagent.

[0140] (3) Detection performance verification at the fecal sample level

[0141] The detection performance verification was carried out on the above five groups of tests in normal feces, precancerous feces, and intestinal cancer feces samples respectively. Among them, the detection method described in Example 2 was used for groups A - C, and the detection method described in its product manual was used for groups D - E. The test results are shown in the following table:

[0142] Table 9 Performance Comparison of Different Markers in Normal Stool, Precancerous Stool, and Colorectal Cancer Stool

[0143]

[0144]

[0145] *Interpretation criteria for combinations of multiple markers: If the test result of any one marker is positive, it is judged as positive.

[0146] From the test results, it can be seen that for normal tissue samples, the three markers screened by the present invention and the commercially available reagents are all consistent with the clinical results. However, in the detection of 10 precancerous tissue samples and cancer tissue samples, the three markers screened by the present invention not only have a better positive detection rate of individual markers than the individual markers of the commercially available reagents, but also have a better positive detection rate for any combination of their markers than the commercially available reagents.

[0147] From the above test results, it can be seen that for 9 colorectal cancer stool samples, the positive detection rates of any combination of the three markers screened by the present invention and the commercially available reagent D are both 100%, while the commercially available reagent E only reaches 5 / 9 (55%); for 10 advanced adenoma (AA) stool samples, the positive detection rates of any two combinations of the three markers screened by the present invention are both 8 / 10 = 80%, and the positive detection rate of the three - marker combination is 10 / 10 = 100%, which are all better than the positive detection rates of the commercially available reagent D (5 / 10 = 50%) and the commercially available reagent E (1 / 10 = 10%); for 10 non - advanced adenoma (NAA) stool samples, the positive detection rates of any two combinations of the three markers screened by the present invention are both 5 / 10 = 50%, and the positive detection rate of the three - marker combination is 6 / 10 = 60%, which are significantly better than the positive detection rates of the commercially available reagent D (3 / 10 = 30%) and the commercially available reagent E (1 / 10 = 10%); and for negative stool samples, any combination of the three markers screened by the present invention and the commercially available reagents are all 100% negative, and the specificity is consistent with the commercially available reagents and the clinical results.

[0148] According to the analysis of the test results, it is known that the detection of any combination of the three markers screened by the present invention has good detection sensitivity and specificity in cell lines, tissues, and stool samples.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A methylation biomarker for detecting colorectal cancer or colorectal adenoma, characterized in that, The methylation biomarkers include any one gene or any combination thereof among SDC2, COL4A2, and C9H9orf50.

2. The methylation biomarker according to claim 1, wherein The methylation target detection region of the SDC2 gene is the full-length region of chr8:96494012-96494130 or a partial region thereof; and / or, The methylation target detection region of the COL4A2 gene is the full-length region of chr13:110306814-110306983 or a partial region thereof; and / or, The methylation target detection region of the C9H9orf50 gene is the full-length region of chr9:129620291-129620374 or a partial region thereof.

3. A kit for detecting colorectal cancer or colorectal adenoma, characterized in that, The kit includes reagents for detecting the methylation level of the methylation marker or its combination as described in claim 1 or 2.

4. The kit according to claim 3, characterized in that, The methylation degree of the methylation biomarker is detected by one or more of the following methods: Quantitative fluorescence PCR, methylation-specific PCR, digital PCR, DNA methylation chip, targeted DNA methylation sequencing, methylation-sensitive restriction enzyme method, direct sequencing method, methylation-sensitive single nucleotide primer extension, bisulfite-conjugated restriction enzyme method, methylation-sensitive single-strand conformation analysis, methylation-sensitive denaturing gradient gel electrophoresis, methylation-specific denaturing high performance liquid chromatography, methylation-specific microarray, methylation-sensitive melting curve analysis, methylation-sensitive dot blot analysis, methylation-specific multiplex ligation-dependent probe amplification, bisulfite sequencing, or pyrosequencing.

5. The kit according to claim 3, wherein The test sample of the kit is taken from an in vitro blood sample or an in vitro tissue sample of the subject to be tested; Preferably, the test sample is taken from the feces of the subject to be tested.

6. The kit according to claim 3, wherein The kit includes probes and primers, and the primers and probes include specific primers and probes for the sequences after transformation of the following genes or their fragments: any one or more of SDC2, COL4A2, or C9H9orf50.

7. The kit according to claim 6, wherein The methylation target detection region of the SDC2 gene is the full-length region of chr8:96494012-96494130 or a partial region thereof; and / or, The methylation target detection region of the COL4A2 gene is the full-length region of chr13:110306814-110306983 or a partial region thereof; and / or, The methylation target detection region of the C9H9orf50 gene is the full-length region of chr9:129620291-129620374 or a partial region thereof.

8. The kit according to any one of claims 4-7, characterized in that, The primer nucleotide sequence for detecting the methylation marker SDC2 gene or its fragment is a nucleotide sequence having at least 80% identity with SEQ ID NO.1 and a nucleotide sequence having at least 80% identity with SEQ ID NO.2, and the probe sequence is a nucleotide sequence having at least 80% identity with SEQ ID NO.3; And / or, the primer nucleotide sequences for detecting the methylated marker COL4A2 gene or its fragment are nucleotide sequences having at least 80% identity with SEQ ID NO.4 and nucleotide sequences having at least 80% identity with SEQ ID NO.5, and the probe sequence is a nucleotide sequence having at least 80% identity with SEQ ID NO.6; And / or, the primer nucleotide sequences for detecting the methylated marker C9H9orf50 gene or its fragment are nucleotide sequences having at least 80% identity with SEQ ID NO.7 and nucleotide sequences having at least 80% identity with SEQ ID NO.8, and the probe sequence is a nucleotide sequence having at least 80% identity with SEQ ID NO.

9.

9. Use of the methylated marker or its combination according to any one of claims 1 or 2 in the preparation of a kit for detecting, diagnosing, classifying, monitoring treatment, predicting prognosis or other evaluation of colorectal cancer or colorectal adenoma.

10. A method for detecting, diagnosing, staging, classifying, monitoring treatment, prognosis or other evaluation of colorectal cancer or colorectal adenoma, characterized in that, Comprising the following steps: (1) Extracting genomic DNA and / or cell-free DNA of a biological sample to be tested; (2) Performing bisulfite conversion on the DNA; (3) Performing co-methylation detection of the bisulfite-converted DNA and a control using the methylated marker or its combination according to claim 1 or 2 to obtain a methylation map; (4) Comparing the methylation map of the methylated marker or its combination with a map determination threshold obtained from mathematical modeling based on a data set to make a judgment on the detection, diagnosis, staging, classification, treatment monitoring, and prognosis of colorectal cancer or colorectal adenoma.

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