Differential methylation region combination, kit and application

Abstract

Differential methylation region combinations, kits and uses are provided; the differential methylation region combination comprises the following differential methylation regions: (1) chr8: 145106171-145107467, and (2) chr8: 67344198-67345563, and is characterized in that the chr8: 145106171-145107467 and the chr8: 67344198-67345563 The differential methylation region combination can be used for effectively detecting the colorectal cancer or precancerous lesions of the colorectal cancer or evaluating the prognosis risk of a patient with the colorectal cancer, and has good sensitivity and specificity.

Description

Differentially methylated region combinations, kits, and uses Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a combination of differentially methylated regions of the OPLAH gene and the ADHFE1 gene, a kit, and uses thereof. Specifically, the kit is a kit for detecting this combination of differentially methylated regions. Specifically, the use thereof is for detecting colorectal cancer or for assessing the prognostic risk of colorectal cancer patients. Background Art

[0002] Colorectal cancer, which occurs in the colon and rectum of the lower digestive tract, is a common malignant tumor. According to the latest statistics, worldwide, colorectal cancer ranks third in new cases among men and second among women, and fourth in deaths among men and third among women (Bray et al., 2018). In my country, the prevention and control of colorectal cancer is also very serious, ranking third among new cases of malignant tumors (388,000) and fifth among deaths (187,000) (Zheng Rongshou et al., 2019). Colorectal cancer develops slowly, generally progressing through polyps, adenomas, and colorectal cancer. The progression from adenoma to colorectal cancer can take up to 5-10 years. Intervention in the early stages of colorectal cancer can significantly reduce mortality. The five-year survival rate for patients with stage I colorectal cancer can exceed 90%, while the five-year survival rate for patients with stage IV colorectal cancer is less than 20% (Marzieh Araghi et al., 2020).

[0003] Traditional colorectal cancer early screening techniques primarily include colonoscopy, fecal occult blood testing, and tumor markers such as CEA and CA19-9. These techniques all currently have limitations. Colonoscopy is the gold standard for colorectal cancer diagnosis. A camera and light source are inserted through the anus and passed through the rectum, sigmoid colon, and other sites to the terminal ileum. Images are transmitted in real time to a monitor for the operating physician to observe. Colonoscopy provides intuitive and clear images, allowing for the detection of a variety of lesions, including cancer, polyps, ulcers, and bleeding, and also allows for endoscopic removal of polyps. However, it is an invasive procedure with complex preparation, requiring dietary restrictions and bowel cleansing, leading to low patient compliance. Furthermore, the procedure requires specialized equipment and personnel, requiring hospital-based personnel and anesthesiologists. Some patients experience discomfort and carry a risk of complications (3-5 cases per 1,000 people). Fecal occult blood testing is a non-invasive method that detects blood components (hemoglobin) in the stool to identify the presence of gastrointestinal bleeding, allowing physicians to assess colorectal cancer risk. This method is quick and simple, with high patient compliance. However, it is susceptible to false positives due to dietary influences such as liver, blood products, and green leafy vegetables. Furthermore, its sensitivity is low in patients with early-stage colorectal cancer who experience minimal bleeding, leading to missed diagnoses. Broad-spectrum tumor markers such as CEA have low specificity, are prone to false positives, and have limited sensitivity. Therefore, an accurate, simple, and cost-effective method for early colorectal cancer screening is needed.

[0004] DNA methylation is an important regulatory mechanism for gene expression, regulating both gene expression and silencing, and has a significant impact on the development and progression of tumors. Aberrant methylation of cancer-related genes often occurs early in the development of cancer, and therefore DNA methylation signals are considered potential markers for early cancer screening. Human fecal samples contain DNA from intestinal cells, which carries information such as methylation. By detecting and analyzing this information, the likelihood of a subject developing colorectal cancer can be inferred. Some genes have been shown to be methylation markers for detecting colorectal cancer in stool, such as SFRP2, SEPT9, NDRG4, and SDC2 (Hannes M. Müller et al., 2004; Jie Chen et al., 2019), but their performance does not yet fully meet clinical needs. For example, the sensitivity and specificity of SFRP2 for colorectal cancer are only 77% (Hannes M. Müller et al., 2004).

[0005] The OPLAH gene encodes 5-hydroxyprolinase, which affects the glutamate salvage pathway and aberrant expression. Studies have shown that downregulation of OPLAH expression caused by abnormal hypermethylation affects glutathione metabolism and is associated with tumorigenesis (Liu Y et al., 2014).

[0006] ADHFE1 encodes a transhydrogenase. Abnormal regulation caused by hypermethylation of this gene can shorten the cell cycle of colorectal cancer cells and promote cancer cell proliferation (Tae CH, Ryu KJ, Kim SH, Kim HC, Chun HK, Min BH, et al. Alcohol dehydrogenase, iron containing, 1 promoter hypermethylation associated with colorectal cancer differentiation. BMC Cancer. 2013; 13: 142.).

[0007] Currently, there is a need to develop new markers for colorectal cancer detection.

[0008] Summary of the Invention

[0009] After in-depth research and creative work, the inventors discovered differentially methylated regions (DMRs) in the OPLAH gene and differentially methylated regions in the ADHFE1 gene. The inventors surprisingly discovered that a combination of these DMRs can be used to effectively detect colorectal cancer or colorectal precancerous lesions, or to assess the prognostic risk of colorectal cancer patients, with good sensitivity and specificity. This provides the following invention:

[0010] One aspect of the present invention relates to differentially methylated regions comprising the following differentially methylated regions:

[0011] (1)chr8:145106171-145107467 (DMR1), and

[0012] (2)chr8:67344198-67345563(DMR2).

[0013] (1) above is the differentially methylated region of the OPLAH gene.

[0014] (2) above is the differentially methylated region of the ADHFE1 gene.

[0015] The above (1) and (2) can also be called differentially methylated region combinations.

[0016] In some embodiments of the present invention, the differentially methylated region consists of differentially methylated regions (1) and (2).

[0017] In some embodiments of the present invention, the differentially methylated region, wherein:

[0018] The base sequence of the differentially methylated region (DMR1) shown in item (1) is shown in SEQ ID NO: 1 or its complementary sequence,

[0019] The base sequence of the differentially methylated region (DMR2) shown in item (2) is shown in SEQ ID NO: 2 or its complementary sequence.

[0020] The base sequence of DMR1:

[0021]

[0022]

[0023] The base sequence of DMR2:

[0024]

[0025]

[0026] In some embodiments of the present invention, the differentially methylated region is used to detect colorectal cancer or colorectal precancerous lesions, or to assess the prognostic risk of colorectal cancer patients.

[0027] In some embodiments of the present invention, the differentially methylated regions are processed as follows:

[0028] Bisulfite treatment, methylation-sensitive endonuclease treatment, methylating restriction endonuclease treatment, or methylation-modifying enzyme treatment.

[0029] The differentially methylated region of the present invention is an OPLAH gene methylation marker or an ADHFE1 gene methylation marker for colorectal cancer detection. The differentially methylated region combination is a combination of an OPLAH gene methylation marker and an ADHFE1 gene methylation marker for colorectal cancer detection.

[0030] Another aspect of the present invention relates to a kit comprising a reagent for detecting the differentially methylated region according to any one of the present invention or a reagent for detecting methylation sites within the differentially methylated region.

[0031] In some embodiments of the present invention, the kit, wherein the reagents include:

[0032] 1) Primers and probes for detecting the differentially methylated region shown in item (1) or the methylation sites within the differentially methylated region;

[0033] 2) primers and probes for detecting the differentially methylated regions shown in item (2) or methylation sites within the differentially methylated regions;

[0034] Preferably, the reagent further comprises:

[0035] 3) Primers and probes for internal reference genes.

[0036] In some embodiments of the present invention, the kit, wherein:

[0037] 1) wherein the primer sequence is selected from SEQ ID NOs: 3-79, and the probe sequence is selected from SEQ ID NOs: 160-188; and 2) wherein the primer sequence is selected from SEQ ID NOs: 80-159, and the probe sequence is selected from SEQ ID NOs: 189-209;

[0038] Preferably:

[0039] 1) wherein the primer sequences are selected from SEQ ID NO:30 and SEQ ID NO:32, and the probe sequence is selected from SEQ ID NO:170; and 2) wherein the primer sequences are selected from SEQ ID NOs:108-109, and the probe sequence is selected from SEQ ID NO:197;

[0040] 1) wherein the primer sequence is selected from SEQ ID NOs: 25-26, and the probe sequence is selected from SEQ ID NO: 164; and 2) wherein the primer sequence is selected from SEQ ID NOs: 144-145, and the probe sequence is selected from SEQ ID NO: 204;

[0041] 1) wherein the primer sequence is selected from SEQ ID NOs: 26-27, and the probe sequence is selected from SEQ ID NO: 168; and 2) wherein the primer sequence is selected from SEQ ID NOs: 154-155, and the probe sequence is selected from SEQ ID NO: 206;

[0042] 1) wherein the primer sequence is selected from SEQ ID NOs: 33-34, and the probe sequence is selected from SEQ ID NO: 171; and 2) wherein the primer sequence is selected from SEQ ID NOs: 156-157, and the probe sequence is selected from SEQ ID NO: 209;

[0043] 1) wherein the primer sequence is selected from SEQ ID NOs: 56-57, and the probe sequence is selected from SEQ ID NO: 184; and 2) wherein the primer sequence is selected from SEQ ID NOs: 158-159, and the probe sequence is selected from SEQ ID NO: 203;

[0044] 1) wherein the primer sequence is selected from SEQ ID NOs: 61-62, and the probe sequence is selected from SEQ ID NO: 183; and 2) wherein the primer sequence is selected from SEQ ID NOs: 108-109, and the probe sequence is selected from SEQ ID NO: 191;

[0045] 1) wherein the primer sequence is selected from SEQ ID NOs: 33-34, and the probe sequence is selected from SEQ ID NO: 171; and 2) wherein the primer sequence is selected from SEQ ID NOs: 108-109, and the probe sequence is selected from SEQ ID NO: 191;

[0046] or,

[0047] 1) wherein the primer sequence is selected from SEQ ID NOs: 25-26, and the probe sequence is selected from SEQ ID NO: 164; and 2) wherein the primer sequence is selected from SEQ ID NOs: 158-159, and the probe sequence is selected from SEQ ID NO: 203;

[0048] Preferably, the sequences of the primers for the internal reference gene in 3) are shown as SEQ ID NOs: 210-211, and the sequence of the probe for the internal reference gene is shown as SEQ ID NO: 212.

[0049] In some embodiments of the present invention, the kit further comprises one or more items selected from the following:

[0050] Fecal sample samplers, nucleic acid extraction reagents, methylation test sample pretreatment reagents, PCR reagents, and fecal occult blood test reagents;

[0051] Preferably, the fecal occult blood detection reagent is a fecal immunochemical detection reagent, a guaiac detection reagent, a tetramethylbenzidine detection reagent or an ELISA double antibody sandwich detection reagent;

[0052] Preferably, the fecal immunochemical detection reagent is an immunocolloidal gold fecal occult blood detection reagent.

[0053] In some embodiments of the present invention, in the kit, the methylation detection sample pretreatment reagent is bisulfite, a methylation-sensitive endonuclease, a methylation restriction endonuclease, or a methylation-modifying enzyme.

[0054] Another aspect of the present invention relates to the use of the differentially methylated regions of the present invention or reagents for detecting the differentially methylated regions of any one of the present invention or the methylation sites within the differentially methylated regions in the preparation of drugs for detecting colorectal cancer or colorectal precancerous lesions, or for assessing the prognosis risk of colorectal cancer patients.

[0055] In some embodiments of the present invention, the use, wherein the reagents are primers and probes for detecting the differentially methylated regions or methylation sites within the differentially methylated regions according to any one of the present invention.

[0056] Another aspect of the present invention relates to a method for detecting colorectal cancer or colorectal precancerous lesions or assessing the prognostic risk of colorectal cancer patients, comprising the following steps:

[0057] Detecting the content of the differentially methylated region or the content of a fragment thereof according to any one of the present invention; wherein the fragment comprises one or more methylation sites within the differentially methylated region.

[0058] Those skilled in the art will understand that detecting the content of a fragment in a differentially methylated region, for example, detecting the content of the fragment by PCR, can represent the content of the differentially methylated region.

[0059] In some embodiments of the present invention, the method, wherein the content of each differentially methylated region or a fragment thereof according to any one of the present invention is detected by fluorescent quantitative PCR method.

[0060] In some embodiments of the present invention, the method, wherein,

[0061] The Ct value of the internal reference gene is used to evaluate the content of human DNA in the sample. When the Ct value of the internal reference gene is >37, the human DNA content in the sample is judged to be too low and the test fails. When the Ct value of the internal reference gene is ≤37, the sample is evaluated as qualified and can be subjected to subsequent analysis.

[0062] In some embodiments of the present invention, the method, wherein, for samples that have passed the assessment:

[0063] When the Ct value of each differentially methylated region was greater than 38 or no Ct value was detected, the test was considered negative;

[0064] When the Ct value of each differentially methylated region was less than or equal to 38, the difference ΔCt value between the Ct value of each differentially methylated region and the reference gene was further calculated.

[0065] In some embodiments of the present invention, the method, wherein:

[0066] If the △Ct of item (1) or item (2) is less than the cutoff value, the patient is judged to be at high risk of colorectal cancer, otherwise, the patient is judged to be at low risk;

[0067] Wherein ΔCt=(Ct value of the differentially methylated region or Ct value of the amplified fragment)-Ct value of the reference gene;

[0068] Wherein, the amplified fragment contains one or more methylation sites within the differentially methylated region.

[0069] In some embodiments of the present invention, the method, wherein:

[0070] The cutoff value for item (1) is 7, and / or

[0071] The cutoff value for item (2) is 12.

[0072] In some embodiments of the present invention, the method further comprises the step of evaluating the combined fecal occult blood test results.

[0073] In some embodiments of the present invention, in the method, the sample is a human tissue sample, a blood sample, a cell sample, a secretion sample, or a fecal sample such as a stool sample.

[0074] According to some embodiments of the present invention, stool samples are used as test subjects. In this way, stool samples contain intestinal exfoliated cells, meet the requirements of test samples, are non-invasive, and are more patient-friendly.

[0075] According to some embodiments of the present invention, optional detection methods include but are not limited to: Sanger sequencing, pyrophosphate sequencing, high-throughput sequencing, nucleic acid mass spectrometry, PCR, fluorescence quantitative PCR, ddPCR, etc. Since the nucleic acid sequence has not changed, methylation modification is difficult to be detected directly, and the nucleic acid often needs to be pretreated during the experiment. The pretreatment methods here include but are not limited to: bisulfite treatment, methylation-sensitive endonuclease treatment, methylation restriction endonuclease treatment or methylation modification enzyme treatment, etc. At the same time, third-generation sequencing technology has been proven to be able to directly measure the methylation modification status of DNA. With the development of technology, it can also be used as a detection method in the future. Fluorescence quantitative PCR technology is one of the commonly used and mature detection technologies for molecular diagnosis. It has the characteristics of rapidity, accuracy and low cost. This technical solution adopts a bisulfite treatment combined with fluorescence quantitative PCR method, and designs several methylation-specific PCR primers and probes for the OPLAH gene and ADHFE1 gene, which can specifically identify methylated DNA molecules, realize the detection of the methylation status of the sample, and then evaluate the cancer risk of the subject.

[0076] By performing whole-genome methylation sequencing and targeted methylation sequencing on colorectal cancer tissue and control tissue samples, differentially methylated regions highly associated with colorectal cancer were identified, with the methylation rates of DMR1 (chr8: 145106171-145107467) and DMR2 (chr8: 67344198-67345563) being particularly significant. Primers and probes were designed for these regions, allowing for rapid and accurate detection of methylation in the OPLAH and ADHFE1 genes. The present invention provides several primer and probe sequences, as shown in Table 1:

[0077] Table 1: Primer and probe sequences for OPLAH and ADHFE1 genes

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] In this article, the inventors use a sampling device to collect stool samples, and the samples undergo nucleic acid extraction and purification steps to obtain nucleic acid samples containing human DNA. Furthermore, the inventors use bisulfite to treat the nucleic acid sample to convert unmethylated cytosine into uracil, so that the methylation site can be detected. For the gene, the inventors designed several primers and probes as described in Table 1, which can specifically amplify and identify related sites. The primer and probe sequences are continuous fragments of at least 15 bases in length, and the nucleic acid sequences of the primers and probes are equivalent, complementary or hybridized to the methylated region pretreated with methylation modification. Using the fluorescent quantitative PCR method, the primers and probes provided herein can effectively reflect the methylation of the relevant sites through Ct values ​​and / or ΔCt values ​​(the difference between the Ct value of the target gene and the Ct value of the internal reference gene) and / or curve conditions, thereby judging the methylation of the relevant genes of the subject. Furthermore, by testing samples from several cancer patients and non-cancer patients, a cutoff value for determining the risk of cancer can be obtained. By comparing the test results with the cutoff value, the cancer risk of the subject can be assessed.

[0090] The optimized primers, probes and PCR reaction system can form a methylation detection kit for OPLAH gene and ADHFE1 gene, and the kit can be used for cancer risk assessment.

[0091] About fecal occult blood testing:

[0092] Hemoglobin in stool samples is tested using methods that may include fecal immunochemical test (FIT), guaiac test (gFOBT), tetramethylbenzidine method, and ELISA double antibody sandwich method.

[0093] In the fecal occult blood detection method, FIT uses monoclonal or polyclonal antibodies to directly detect hemoglobin in human stool and is unaffected by food. Qualitative FIT detects a visible color change when the hemoglobin content in stool exceeds a certain threshold, while quantitative FIT measures the value, with a positive result defined as exceeding a certain normal range. Qualitative FIT testing can be performed using the immunocolloidal gold method, which is convenient and rapid; quantitative FIT testing can be performed using the latex immunoturbidimetric method, which offers high sensitivity and specificity.

[0094] Regarding the combined genetic testing and fecal occult blood testing method:

[0095] The result determination method of the combined gene-fecal occult blood test can be that the determination thresholds of the gene test result and the fecal occult blood test can be customized separately, and the subject is determined to be positive or a candidate to be positive based on the above two parts of the test results.

[0096] The determination method can also be a logistic regression method, which assigns different weights to the test results of OPLAH gene, ADHFE1 gene and fecal occult blood, and calculates them through a logistic function. The logistic function is g(y)=1 / (1+e -y Where g(y) is the risk index. When the risk index is higher than a preset value, the patient is considered positive or the candidate is positive; e is a natural constant; y = (θ0 + θ1*X1 + θ2*X2 + ... + θn*Xn), where θ0 is the constant term, θi (i = 1, 2, ..., n) are the coefficients to be determined, and Xi (i = 1, 2, ..., n) are the results of genetic and fecal occult blood tests. Different weights θi reflect the different contributions of the independent variables to the dependent variable. Different test results Xi can include, but are not limited to, any combination of one or more of the Ct value, ΔCt value, methylation rate, qualitative fecal occult blood test results, and quantitative fecal occult blood test results.

[0097] By optimizing the parameters in the determination method, higher detection sensitivity can be obtained.

[0098] The kit includes a combined OPLAH and ADHFE1 gene methylation detection reagent based on fluorescent quantitative PCR, which provides OPLAH and ADHFE1 gene methylation detection results. Furthermore, the kit also includes a fecal occult blood detection reagent, which provides fecal occult blood detection results. By calculating the ΔCt value or risk index and comparing it with the kit's preset positive judgment value, it can be used to determine the risk of colorectal cancer.

[0099] The fluorescent quantitative PCR-based combined detection reagent for OPLAH and ADHFE1 gene methylation contains primers and probes capable of specifically amplifying OPLAH and ADHFE1 genes. The reagent can detect the methylation status of OPLAH and ADHFE1 genes in nucleic acids that have undergone methylation modification.

[0100] The gene methylation combined detection reagent can be used in conjunction with different stool sample sampling devices, nucleic acid extraction reagents and methylation modification pretreatment reagents. The stool sampling device may include a sampler and a sample storage device for collecting and storing samples. The nucleic acid extraction reagent can be a kit that can extract nucleic acids from human biological samples, or it can be a kit containing nucleic acid capture magnetic beads. The methylation modification pretreatment reagent modifies the nucleic acid sequence so that the cytosine that has undergone methylation modification and the cytosine that has not undergone methylation modification produce a difference that is easy to detect. Generally, the methylation modification pretreatment reagent can convert the cytosine that has not undergone methylation modification into uracil, while the cytosine that has undergone methylation modification remains unchanged, and the sequence changes can be used for subsequent detection.

[0101] The fecal occult blood detection reagent preferably uses the immunocolloidal gold method to easily and quickly obtain qualitative fecal occult blood detection results. The immunocolloidal gold method fecal occult blood detection reagent may include a sampler, a sample storage device, and a test strip. The test strip is coated with antibodies and colloidal gold. When the target protein labeled with colloidal gold binds to the pre-coated antibody on the test strip, it will be retained on the test strip, forming a visual change, thereby determining whether the sample contains the target protein.

[0102] The positive judgment value is defined using samples with known clinical information. A classification model is constructed by testing a number of positive and negative samples to determine the conditions for judging as positive.

[0103] Another aspect of the present invention relates to a diagnosis or evaluation model for colorectal cancer, using a logic function g(y)=1 / (1+e - y ) is calculated, where:

[0104] g(y) is the risk index. When the risk index is higher than the preset value, it is judged as positive or the candidate is positive;

[0105] e is a natural constant;

[0106] y=(θ0+θ1*X1+θ2*X2+...+θn*Xn), θ0 is a constant term, θi (i=1,2,...,n) is the coefficient to be determined, and Xi (i=1,2,...,n) is the detection result of the differentially methylated region and the detection result of fecal occult blood according to any one of the present invention.

[0107] Another aspect of the present invention relates to a system for diagnosing or evaluating colorectal cancer, comprising:

[0108] 1) DMR detection unit: obtaining content data of the differentially methylated region or a fragment thereof according to any one of the present invention; wherein the fragment comprises one or more methylation sites within the differentially methylated region;

[0109] 2) Fecal occult blood detection unit: obtaining fecal occult blood test result data;

[0110] 3) Data analysis unit: inputting the data obtained by the DMR detection unit and the fecal occult blood detection unit into the colorectal cancer diagnosis or evaluation model of the present invention for analysis;

[0111] 4) Risk assessment unit: outputs the risk index or judgment result of individual samples.

[0112] Another aspect of the present invention relates to the use of the differentially methylated region (1) and the differentially methylated region (2) of the present invention in the preparation of a product for detecting colorectal cancer or colorectal precancerous lesions.

[0113] In the present invention, unless otherwise specified, the term "colorectal cancer" refers to colon cancer and / or rectal cancer, also referred to as "intestinal cancer" for short.

[0114] In the present invention, unless otherwise specified, the term "early stage colorectal cancer" refers to stage I or stage II colorectal cancer.

[0115] In the present invention, unless otherwise specified, detecting a differentially methylated region or detecting a fragment thereof refers to detecting the content of the differentially methylated region or detecting the content of a fragment thereof; wherein the fragment contains one or more methylation sites within the differentially methylated region.

[0116] In the present invention, unless otherwise specified, detecting methylation sites in a differentially methylated region refers to detecting the content of methylation sites in the differentially methylated region or detecting the methylation rate of methylation sites in the differentially methylated region.

[0117] Advantageous Effects of the Invention

[0118] The present invention achieves one or more of the following technical effects:

[0119] (1) Ability to quickly detect the methylation status of OPLAH and ADHFE1 genes;

[0120] (2) Able to detect, diagnose or screen colorectal cancer, or used for prognostic risk assessment of colorectal cancer, with high sensitivity and / or specificity; preferably, having both high sensitivity and specificity.

[0121] (3) It can be used for the detection, diagnosis or screening of early colorectal cancer or colorectal precancerous lesions, and has high sensitivity and / or specificity; preferably, it has both high sensitivity and specificity.

[0122] (4) Hemoglobin in feces can be combined with detection to further improve the detection sensitivity of colorectal cancer.

[0123] (5) Compared with traditional detection methods, the present invention has the advantages of higher sensitivity, being completely non-invasive, non-invasive detection process, no need for intestinal preparation, and high acceptance of the test by asymptomatic people. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1: Differentially methylated regions of OPLAH and ADHFE1 genes in colorectal cancer and adjacent adjacent tissue samples.

[0125] Figure 2: Results of OPLAH and ADHFE1 gene test reference products.

[0126] Figure 3: ROC curves of OPLAH and ADHFE1 gene detection in clinical samples.

[0127] Figure 4: Comparison of ROC curves between genetic testing and gene + FIT combined testing.

[0128] Figure 5: Comparison of detection sensitivity. DETAILED DESCRIPTION

[0129] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0130] Some relevant definitions refer to the Chinese Society of Clinical Oncology (CSCO) Colorectal Cancer Diagnosis and Treatment Guidelines 2021, and some relevant content is cited in Table 2 below:

[0131] Table 2

[0132]

[0133] The AJCC staging standard for colorectal cancer was used.

[0134] Example 1: Real-time fluorescence quantitative PCR combined detection of methylation levels of OPLAH and ADHFE1 genes

[0135] The inventors discovered differentially methylated regions of the OPLAH and ADHFE1 genes through targeted methylation capture sequencing. These differentially methylated regions have high methylation levels in cancerous tissues, but low methylation levels in adjacent tissues. In this example, using fluorescent quantitative PCR as an example, the inventors designed several methylation-specific primers and probes targeting these regions, capable of specifically amplifying methylated nucleic acid sequences. Through system primer screening and system optimization, they developed a method for combined detection of the OPLAH and ADHFE1 genes based on fluorescent quantitative PCR.

[0136] The specific steps are as follows:

[0137] 1. Targeted methylation capture sequencing reveals differentially methylated regions:

[0138] Nucleic acids were extracted from 38 pairs of colorectal cancer samples and adjacent tissue samples from 38 people (each pair came from the same person) using the DNeasy Blood & Tissue Kit (Qiagen, #69506) and quantified using the Qubit 3.0 system (Invitrogen, USA).

[0139] 200 ng of extracted DNA was taken and 1 ng of unmethylated lambda DNA (PROMEGA, #D1521) was added for subsequent CU conversion quality control. The DNA was sheared using an ultrasonic shearer and then fragmented using AMPure XP (AGENCOURT, #A63882) to select the DNA fragments so that the DNA fragment size was concentrated around 160 bp.

[0140] The library was constructed using the KAPA HyperPlus Library Preparation Kit (KAPA, #KK8510), and the adapter ligation products were bisulfite converted using the EZ DNA Metlylation-Gold Kit (Zymo Research, #D5006). For specific operations, refer to the kit instructions. The adapters used in the adapter ligation step and the PCR primers used in the PCR step were replaced with adapters and primers suitable for the MGISEQ platform. The adapters needed to be methylated (for example, using methylated dCTP to synthesize the adapters) to prevent bisulfite treatment from changing the sequence.

[0141] Hybridization, capture, and elution were performed using the Seq Cap EZ Hybridization and Wash Kit (ROCHE, 5634253001) and SeqCap Epi CpGiant Enrichment Kit (ROCHE, 7138911001). Since an MGI sequencing instrument was used, the adapter sequences must be blocked using the corresponding blocking sequences for the MGI sequencing platform during hybridization.

[0142] PE100 sequencing was performed using MGISEQ-2000 (MGI).

[0143] After quality control of the sequencing data, filtering of unqualified sequencing sequences, and alignment of the sequencing sequences with the computer-simulated bisulfite-converted version of the human genome sequence, all sequences were counted and the methylation rate of each CpG site was calculated (Non-invasive lung cancer diagnosis and prognosis based on multi-analyte liquid biopsy, Chen Kezhong et al., 2020).

[0144] Specific site methylation rate = methylation reads sequencing depth / total sequencing depth.

[0145] The present inventors have discovered that there are significant differential methylation regions in the OPLAH and ADHFE1 genes, which have the potential to distinguish cancer from non-cancerous tissues. Figure 1 shows the methylation levels of various CpG sites in the OPLAH and ADHFE1 genes in cancerous tissues and adjacent tissues.

[0146] 2. Methylation primer and probe design

[0147] Based on the differentially methylated regions identified, the inventors designed primers and probes capable of specifically amplifying nucleic acid sequences pretreated with methylation modification, targeting CpG sites with high methylation rates in cancer tissue and low methylation rates in adjacent tissues. At least one primer in a primer pair must have at least one CpG site at its 3' end. The designed primer and probe sequences are shown in Table 1.

[0148] 3. Fluorescence quantitative PCR can jointly detect the methylation levels of DMRs of OPLAH and ADHFE1 genes

[0149] The inventors selected optimized primer sets and probes for OPLAH and ADHFE1 gene methylation detection. The primer sets described in this example include: OPLAH gene primer set (SEQ ID NO: 30 and SEQ ID NO: 32), OPLAH gene probe (SEQ ID NO: 170), ADHFE1 gene primer set (SEQ ID NO: 108 and SEQ ID NO: 109), and ADHFE1 gene probe (SEQ ID NO: 197). The fluorescence quantitative PCR reaction system includes 1× PCR buffer, 2-5mM magnesium ion, 0.2-0.8mM dNTP, 0.2μM primer per gene, 50nM probe per gene, and 1 unit of Taq polymerase. The reaction was pre-denatured at 95°C for 10min, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds. Detection was performed using a Macrostone 96S qPCR instrument, with the baseline and threshold values ​​set as default, and fluorescence signals collected at the end of each cycle.

[0150] The present inventors used a fully methylated standard and a fully unmethylated standard (Merck, #S8001) to validate the method's ability to detect methylation levels in the OPLAH and ADHFE1 genes. The fully methylated standard is DNA from a cell line treated with M.SssI methyltransferase, with all CpG sites methylated. The fully unmethylated standard is DNA from a cell line in which the methyltransferase gene has been knocked out, with methylation levels at CpG sites below 5%. The present inventors used these two standards and mixed them to create reference samples with different methylation levels, including 100%, 50%, 25%, 12%, 6%, 3%, 1%, and 0%.

[0151] For each reference sample, 20 ng was tested three times. First, the DNA was pre-treated for methylation using a methylation detection sample pretreatment kit (BGI) to convert unmethylated cytosine to uracil. The reference sample was then tested using the aforementioned fluorescent quantitative PCR reaction system.

[0152] The test results are shown in Table 3.

[0153] Table 3: OPLAH and ADHFE1 gene detection reference product data

[0154]

[0155] The results showed that all reference samples mixed with methylated templates were detected with a detection limit as low as 1%, while no amplification occurred in reference samples without methylated templates.

[0156] A standard curve was plotted based on the amount of methylated template input and the average Ct value. As shown in Figure 2, the R2 of the standard curve was good, indicating that this method can effectively detect methylation signals of the OPLAH and ADHFE1 genes in samples without nonspecific amplification.

[0157] Example 2: OPLAH and ADHFE1 gene methylation combined detection kit for colorectal cancer detection

[0158] This embodiment uses the fluorescent quantitative PCR method as an example to disclose a kit for detecting colorectal cancer by jointly detecting the methylation levels of the OPLAH and ADHFE1 genes.

[0159] This kit contains reagents such as Buffer, dNTP and enzyme required for PCR amplification, and also includes OPLAH and ADHFE1 gene primer sets and probes. The primer sets described in this embodiment include: OPLAH gene primer set (SEQ ID NO:30 and SEQ ID NO:32), OPLAH gene probe (SEQ ID NO:170), ADHFE1 gene primer set (SEQ ID NO:108 and SEQ ID NO:109) and ADHFE1 gene probe (SEQ ID NO:197). In order to monitor whether the sample contains enough human DNA, primers and probes for detecting internal reference genes are also added to this kit. The internal reference gene is generally a housekeeping gene, and its corresponding primer and probe detection results are only related to the total amount of human DNA, and are not affected by the methylation level. In this embodiment, the GAPDH gene is selected as the internal reference gene, and its primer and probe sequences are shown in Table 4. The PCR reaction was pre-denatured at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Detection was performed using a Macrostone 96S qPCR instrument, with baseline and threshold values ​​set as default, and fluorescence signals collected at the end of each cycle.

[0160] Table 4: Sequence list of primers and probes for internal reference genes

[0161]

[0162] A total of 604 stool samples were included in the performance evaluation of the kit, and the clinical sample information is shown in Table 5.

[0163] Table 5: Sample information table

[0164]

[0165]

[0166] The sample test first involves collecting a stool sample from the subject using a disposable stool sampler (BGI). DNA from the stool sample is then extracted using a stool nucleic acid extraction kit (BGI). Next, the DNA is pretreated for methylation modification using a methylation detection sample pretreatment kit (BGI) to obtain converted DNA. Finally, the converted DNA is tested using this kit to obtain Ct values ​​for the internal reference, OPLAH, and ADHFE1 genes for each sample.

[0167] When analyzing results, the reference gene threshold was 37. When the Ct value of the reference gene in a sample was greater than 37, the sample was considered to contain insufficient human DNA and the test was deemed a failure. When the Ct value of the reference gene was 37 or less, the sample passed the quality inspection. For samples that passed the quality inspection, if the Ct value of the target gene was greater than 38 or no Ct value was detected, the gene test was considered negative. If the Ct value of the target gene was 38 or less, the ΔCt value (the difference between the Ct value of the target gene and the Ct value of the reference gene) was further calculated for the target gene.

[0168] The determination of the positive cutoff value was analyzed using SPSS. First, the inventors used SPSS software to draw the ROC curve for each gene. As shown in Figure 3, the AUCs for the OPLAH and ADHFE1 genes were 0.911 and 0.918, respectively. By integrating the test results of the two genes for analysis, a higher AUC was obtained, reaching 0.942. Furthermore, the inventors used the same method to test the PPP2R5C and SDC2 genes of the above samples, and obtained the Ct values ​​and ΔCt values ​​of the PPP2R5C and SDC2 genes. The PPP2R5C and SDC2 genes also showed significant methylation differences in cancer tissues and non-cancerous tissues. Further, the inventors used SPSS software to analyze the detection effect of the four-gene combination of OPLAH, ADHFE1, PPP2R5C and SDC2. The test results are shown in Figure 3. It can be seen that the detection effect of the four-gene combination test is similar to that of the OPLAH and ADHFE1 double gene test, with an AUC of 0.950. Because dual-gene testing can achieve the same results as a four-gene combination test, the dual-gene combined test was chosen for cost and stability considerations. Ultimately, optimal performance in clinical specimens was determined when the ΔCt value of the DMR for the OPLAH gene was ≤7, or when the ΔCt value of the DMR for the ADHFE1 gene was ≤12. Specific performance is shown in Table 6. The kit achieved a sensitivity of 86.62%, an overall specificity (combined specificity for healthy and polyp samples) of 92.78%, and a sensitivity of 52.63% for advanced adenomas. The kit also demonstrated high sensitivity for early-stage colorectal cancer, as shown in Table 7, with sensitivities of 88.64% and 89.02% for stage I and stage II colorectal cancer, respectively.

[0169] Table 6: Clinical sample detection performance

[0170]

[0171] The total compliance rate = (number of samples correctly tested by the gold standard as negative + number of samples correctly tested by the gold standard as positive) / total number of samples. The gold standard is colonoscopy.

[0172] Table 7: Detection sensitivity of colorectal cancer samples at different stages

[0173]

[0174] According to the above method, the inventors also used some other primer and probe combinations in Table 1 to test the above samples, and obtained good detection effects. The specific results are shown in Table 8 below.

[0175] Table 8

[0176]

[0177]

[0178] Example 3: OPLAH and ADHFE1 gene methylation combined with fecal occult blood detection kit for colorectal cancer detection

[0179] The present invention discloses a kit for detecting colorectal cancer by jointly detecting the methylation levels of OPLAH and ADHFE1 genes and fecal occult blood. The kit includes an OPLAH and ADHFE1 gene methylation level detection module and a fecal occult blood detection module.

[0180] 1. OPLAH and ADHFE1 gene methylation level detection module

[0181] The OPLAH and ADHFE1 gene methylation level detection module is based on a fluorescent quantitative PCR method, comprising reagents such as Buffer, dNTPs and enzymes required for PCR amplification, and also comprising OPLAH and ADHFE1 gene primer sets and probes. The primer set described in this embodiment can be selected from Table 1. The primer set selected in this example includes: OPLAH gene primer set (SEQ ID NO: 30 and SEQ ID NO: 32), OPLAH gene probe (SEQ ID NO: 170), ADHFE1 gene primer set (SEQ ID NO: 108 and SEQ ID NO: 109) and ADHFE1 gene probe (SEQ ID NO: 197). In order to monitor whether the sample contains enough human DNA, primers and probes for detecting internal reference genes are also added to this kit. The internal reference gene is generally a housekeeping gene, and its corresponding primer and probe detection results are only related to the total amount of human DNA, and are not affected by the methylation level. In this embodiment, the GAPDH gene is selected as the internal reference gene, and its primer and probe sequences are shown in Table 4. The PCR reaction was pre-denatured at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Detection was performed using a Macrostone 96S qPCR instrument, with baseline and threshold values ​​set as default, and fluorescence signals collected at the end of each cycle.

[0182] The sample test first involves collecting a stool sample from the subject using a disposable stool sampler (BGI). DNA from the stool sample is then extracted using a stool nucleic acid extraction kit (BGI). Next, the DNA is pretreated for methylation modification using a methylation detection sample pretreatment kit (BGI) to obtain converted DNA. Finally, the converted DNA is tested using this kit to obtain Ct values ​​for the internal reference, OPLAH, and ADHFE1 genes for each sample.

[0183] When analyzing results, the reference gene threshold was 37. When the Ct value of the reference gene in a sample was greater than 37, the sample was considered to contain insufficient human DNA and the test was deemed a failure. When the Ct value of the reference gene was 37 or less, the sample passed the quality inspection. For samples that passed the quality inspection, if the Ct value of the target gene was greater than 38 or no Ct value was detected, the gene test was considered negative. If the Ct value of the target gene was 38 or less, the ΔCt value (the difference between the Ct value of the target gene and the Ct value of the reference gene) was further calculated for the target gene.

[0184] 2. Fecal occult blood module

[0185] The fecal occult blood module includes a test strip that can qualitatively detect fecal hemoglobin content. Its detection principle is the colloidal gold method. Specifically, a fecal sample is collected using a FIT sampler. After mixing with sample preservation fluid, approximately 100 μl of the sample is placed on the test strip. The test result is read within a certain period of time.

[0186] When analyzing the results, a band should appear on the control line; otherwise, the test fails. If the control line passes, observe the test line. If a band appears on the test line, the fecal occult blood test is positive; if no band appears, the fecal occult blood test is negative. Results should be read within the specified time; any delay will result in a failure.

[0187] 3. Implementation and performance evaluation of a kit for OPLAH and ADHFE1 gene methylation detection combined with fecal occult blood detection

[0188] This example uses 318 samples with fecal occult blood test results from the samples described in Example 2 for analysis. The clinical information of the samples is shown in Table 9.

[0189] Table 9: Sample information table

[0190]

[0191]

[0192] The positive cutoff value was determined by SPSS analysis. First, the inventors used SPSS software to draw the ROC curve for the gene detection results. As shown in Figure 4, the AUC of the OPLAH and ADHFE1 gene joint detection was 0.931. The OPLAH and ADHFE1 genes have good detection performance in colorectal cancer detection. Furthermore, the inventors included the fecal occult blood test (FIT) results in the logistic regression analysis. According to the formula g(y) = 1 / (1 + e -y ) to calculate the test risk index, where g(y) is the risk index. When the risk index exceeds a preset value, the test is considered positive or candidate positive; e is a natural constant; y = (θ0 + θ1*X1 + θ2*X2 + ... + θn*Xn), where θ0 is the constant term, θi (i = 1, 2, ..., n) are the coefficients to be determined, and Xi (i = 1, 2, ..., n) are the results of the genetic and fecal occult blood tests. After calculating the test risk index for each sample, a receiver operating characteristic (ROC) curve was plotted to determine the optimal positive cutoff value. As shown in Figure 4, the AUC for the combined genetic and FIT test reached 0.975, indicating that combined genetic and FIT testing can effectively improve test performance. As shown in Table 10, optimal performance was achieved when the positive cutoff value was 0.55, with a sensitivity and overall specificity of 95.97% and 91.43%, respectively. This kit has a good detection rate for advanced adenomas, achieving a sensitivity of 58.62%. This test kit has a high sensitivity for early-stage colorectal cancer. As shown in Table 11, the sensitivity for stage I and stage II colorectal cancer reached 89.47% and 100%, respectively. As shown in Figure 5, compared with dual-gene testing or FIT testing, this test kit has a higher sensitivity for both colorectal cancer and advanced adenomas.

[0193] Table 10: Clinical sample detection performance

[0194]

[0195] The total compliance rate = (number of samples correctly tested by the gold standard as negative + number of samples correctly tested by the gold standard as positive) / total number of samples. The gold standard is colonoscopy.

[0196] Table 11: Detection sensitivity of colorectal cancer samples at different stages

[0197] Sample stage Number of positive samples Total number of samples Sensitivity Phase I 171989.47% Phase II 3232100.00% Phase III 434595.56% Phase IV 121392.31% Unknown 394097.50% Total 14314995.97%

[0198] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. Differentially methylated regions, including: (1)chr8:145106171-145107467, and (2)chr8:67344198-67345563.

2. The differentially methylated region according to claim 1, wherein: The base sequence of the differentially methylated region shown in item (1) is shown in SEQ ID NO: 1 or its complementary sequence, and / or The base sequence of the differentially methylated region shown in item (2) is shown in SEQ ID NO: 2 or its complementary sequence.

3. The differentially methylated region according to any one of claims 1 to 2, which is used to detect colorectal cancer or colorectal precancerous lesions, or to assess the prognostic risk of colorectal cancer patients.

4. The differentially methylated region according to any one of claims 1 to 3, which has been processed as follows: Bisulfite treatment, methylation-sensitive endonuclease treatment, methylating restriction endonuclease treatment, or methylation-modifying enzyme treatment. 5 . A kit comprising a reagent for detecting the differentially methylated region according to any one of claims 1 to 4 or a reagent for detecting methylation sites within the differentially methylated region.

6. The kit according to claim 5, wherein The reagents include: 1) Primers and probes for detecting the differentially methylated region shown in item (1) or the methylation sites within the differentially methylated region; 2) primers and probes for detecting the differentially methylated regions shown in item (2) or methylation sites within the differentially methylated regions; Preferably, the reagent further comprises: 3) Primers and probes for internal reference genes.

7. The kit according to claim 6, wherein: 1) wherein the primer sequence is selected from SEQ ID NOs: 3-79, and the probe sequence is selected from SEQ ID NOs: 160-188; and 2) wherein the primer sequence is selected from SEQ ID NOs: 80-159, and the probe sequence is selected from SEQ ID NOs: 189-209; Preferably: 1) wherein the primer sequences are selected from SEQ ID NO:30 and SEQ ID NO:32, and the probe sequence is selected from SEQ ID NO:170; and 2) wherein the primer sequences are selected from SEQ ID NOs:108-109, and the probe sequence is selected from SEQ ID NO:197; 1) wherein the primer sequence is selected from SEQ ID NOs: 25-26, and the probe sequence is selected from SEQ ID NO: 164; and 2) wherein the primer sequence is selected from SEQ ID NOs: 144-145, and the probe sequence is selected from SEQ ID NO: 204; 1) wherein the primer sequence is selected from SEQ ID NOs: 26-27, and the probe sequence is selected from SEQ ID NO: 168; and 2) wherein the primer sequence is selected from SEQ ID NOs: 154-155, and the probe sequence is selected from SEQ ID NO: 206; 1) wherein the primer sequence is selected from SEQ ID NOs: 33-34, and the probe sequence is selected from SEQ ID NO: 171; and 2) wherein the primer sequence is selected from SEQ ID NOs: 156-157, and the probe sequence is selected from SEQ ID NO: 209; 1) wherein the primer sequence is selected from SEQ ID NOs: 56-57, and the probe sequence is selected from SEQ ID NO: 184; and 2) wherein the primer sequence is selected from SEQ ID NOs: 158-159, and the probe sequence is selected from SEQ ID NO: 203; 1) wherein the primer sequence is selected from SEQ ID NOs: 61-62, and the probe sequence is selected from SEQ ID NO: 183; and 2) wherein the primer sequence is selected from SEQ ID NOs: 108-109, and the probe sequence is selected from SEQ ID NO: 191; 1) wherein the primer sequence is selected from SEQ ID NOs: 33-34, and the probe sequence is selected from SEQ ID NO: 171; and 2) wherein the primer sequence is selected from SEQ ID NOs: 108-109, and the probe sequence is selected from SEQ ID NO: 191; or, 1) wherein the primer sequence is selected from SEQ ID NOs: 25-26, and the probe sequence is selected from SEQ ID NO: 164; and 2) wherein the primer sequence is selected from SEQ ID NOs: 158-159, and the probe sequence is selected from SEQ ID NO: 203; Preferably, the sequences of the primers for the internal reference gene in 3) are shown as SEQ ID NOs: 210-211, and the sequence of the probe for the internal reference gene is shown as SEQ ID NO:

212.

8. The kit according to any one of claims 5 to 7, further comprising one or more selected from the group consisting of: Fecal sample samplers, nucleic acid extraction reagents, methylation test sample pretreatment reagents, PCR reagents, and fecal occult blood test reagents; Preferably, the fecal occult blood detection reagent is a fecal immunochemical detection reagent, a guaiac detection reagent, a tetramethylbenzidine detection reagent or an ELISA double antibody sandwich detection reagent; Preferably, the fecal immunochemical detection reagent is an immunocolloidal gold fecal occult blood detection reagent.

9. The kit according to claim 8, wherein The methylation detection sample pretreatment reagent is bisulfite, methylation-sensitive endonuclease, methylation restriction endonuclease or methylation modification enzyme.

10. Use of the differentially methylated region described in any one of claims 1 to 4 or a reagent for detecting the differentially methylated region described in any one of claims 1 to 4 or the methylation sites within the differentially methylated region in the preparation of a drug for detecting colorectal cancer or colorectal precancerous lesions, or for assessing the prognostic risk of colorectal cancer patients.

11. The use according to claim 10, wherein The reagents are primers and probes for detecting the differentially methylated region according to any one of claims 1 to 4 or the methylation sites within the differentially methylated region.

12. A method for detecting colorectal cancer or colorectal precancerous lesions or assessing the prognostic risk of colorectal cancer patients, comprising the following steps: Detecting the content of the differentially methylated region or the content of a fragment thereof according to any one of claims 1 to 4; wherein, The fragment comprises one or more methylation sites within the differentially methylated region.

13. The method according to claim 12, wherein: The content of each differentially methylated region or a fragment thereof according to any one of claims 1 to 4 is detected by fluorescent quantitative PCR method.

14. The method according to claim 13, wherein The Ct value of the internal reference gene is used to evaluate the content of human DNA in the sample. When the Ct value of the internal reference gene is >37, the human DNA content in the sample is judged to be too low and the test fails. When the Ct value of the internal reference gene is ≤37, the sample is evaluated as qualified and can be subjected to subsequent analysis.

15. The method according to claim 14, wherein For samples that pass the assessment: When the Ct value of each differentially methylated region was greater than 38 or no Ct value was detected, the test was considered negative; When the Ct value of each differentially methylated region was less than or equal to 38, the difference ΔCt value between the Ct value of each differentially methylated region and the reference gene was further calculated.

16. The method according to any one of claims 13 to 15, wherein: If the △Ct of item (1) or item (2) is less than the cutoff value, the patient is judged to be at high risk of colorectal cancer, otherwise, the patient is judged to be at low risk; Wherein ΔCt=(Ct value of the differentially methylated region or Ct value of the amplified fragment)-Ct value of the reference gene; Wherein, the amplified fragment contains one or more methylation sites within the differentially methylated region.

17. The method according to any one of claims 12 to 16, further comprising the step of evaluating the result of a combined fecal occult blood test.

18. The method according to any one of claims 12 to 17, wherein: The sample is a human tissue sample, a blood sample, a cell sample, a secretion sample, or a fecal sample such as a stool sample.

19. A colorectal cancer diagnosis or assessment model using the logistic function g(y) = 1 / (1+e -y ) is calculated, where: g(y) is the risk index. When the risk index is higher than the preset value, it is judged as positive or the candidate is positive; e is a natural constant; y=(θ0+θ1*X1+θ2*X2+...+θn*Xn), θ0 is a constant term, θi (i=1,2,...,n) is the coefficient to be determined, and Xi (i=1,2,...,n) is the detection result of the differentially methylated region and the detection result of fecal occult blood according to any one of claims 1 to 4.

20. A system for diagnosing or evaluating colorectal cancer, comprising: 1) A DMR detection unit: obtaining content data of the differentially methylated region or a fragment thereof according to any one of claims 1 to 4; wherein the fragment comprises one or more methylation sites within the differentially methylated region; 2) Fecal occult blood detection unit: obtaining fecal occult blood test result data; 3) a data analysis unit: inputting the data obtained by the DMR detection unit and the fecal occult blood detection unit into the colorectal cancer diagnosis or evaluation model of claim 19 for analysis; 4) Risk assessment unit: outputs the risk index or judgment result of individual samples.

21. Use of the differentially methylated region according to any one of claims 1 to 4 in the preparation of a product for detecting colorectal cancer or colorectal precancerous lesions.

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