Differential methylation region of OPLAH gene, kit and application

Abstract

The invention discloses a differential methylation region of an OPLAH gene, a kit and application. Specifically, the differential methylation region is chr8: 145106171-145107467, and the molecular weight of the differential methylation region is 25000-25000. The differential methylation region can be used for effectively detecting colorectal cancer or precancerous lesions of the colorectal cancer, or evaluating the prognosis risk of a patient suffering from the colorectal cancer.

Description

Differentially methylated regions of OPLAH genes, kits, and uses Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a differentially methylated region of the OPLAH gene, a kit, and uses thereof. Specifically, the kit is a kit for detecting the differentially methylated region. 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, and has high patient compliance. However, it is susceptible to false positives due to dietary changes such as liver, blood products, and green leafy vegetables. Furthermore, it has low sensitivity in patients with early-stage colorectal cancer who experience minimal bleeding, making it prone to missed diagnoses. Broad-spectrum tumor markers such as CEA have low specificity, are prone to generating many false positives, and have limited sensitivity. Therefore, an accurate, simple, and cost-effective method for early screening of colorectal cancer 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] Currently, there is a need to develop new markers for colorectal cancer detection.

[0007] Summary of the Invention

[0008] After in-depth research and creative work, the inventors discovered a differentially methylated region (DMR) in the OPLAH gene. Surprisingly, the inventors discovered that this DMR 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:

[0009] One aspect of the present invention relates to a differentially methylated region as follows:

[0010] chr8:145106171-145107467 (also referred to as the DMR of the present invention).

[0011] The differentially methylated region is the differentially methylated region of the OPLAH gene.

[0012] In some embodiments of the present invention, the base sequence of the differentially methylated region is shown in SEQ ID NO: 1 or its complementary sequence.

[0013] DMR base sequence:

[0014]

[0015]

[0016] 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.

[0017] In some embodiments of the present invention, the differentially methylated regions are processed as follows:

[0018] Bisulfite treatment, methylation-sensitive endonuclease treatment, methylating restriction endonuclease treatment, or methylation-modifying enzyme treatment.

[0019] The differentially methylated region of the present invention is an OPLAH methylation marker for colorectal cancer detection.

[0020] 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.

[0021] In some embodiments of the present invention, the kit, wherein the reagents include:

[0022] (1) primers and probes for detecting the differentially methylated region or the methylation sites within the differentially methylated region;

[0023] Preferably, the reagent further comprises:

[0024] (2) Primers and probes for internal reference genes.

[0025] In some embodiments of the present invention, the kit, wherein:

[0026] (1) wherein the primer sequences are selected from SEQ ID NOs: 2-78, and the probe sequences are selected from SEQ ID NOs: 79-107;

[0027] Preferably, in (1): the sequence of the primer is selected from SEQ ID NOs: 29 and 31, and the sequence of the probe is selected from SEQ ID NO: 89; the sequence of the primer is selected from SEQ ID NOs: 24-25, and the sequence of the probe is selected from SEQ ID NO: 83; the sequence of the primer is selected from SEQ ID NOs: 25-26, and the sequence of the probe is selected from SEQ ID NO: 87; the sequence of the primer is selected from SEQ ID NOs: 32-33, and the sequence of the probe is selected from SEQ ID NO: 90; or the sequence of the primer is selected from SEQ ID NOs: 55-56, and the sequence of the probe is selected from SEQ ID NO: 103;

[0028] Preferably, the sequences of the primers for the internal reference gene in (2) are shown in SEQ ID NOs: 108-109, and the sequence of the probe for the internal reference gene is shown in SEQ ID NO: 110.

[0029] In some embodiments of the present invention, the kit further comprises one or more items selected from the following:

[0030] Fecal sample sampler, nucleic acid extraction reagents, methylation detection sample pretreatment reagents and PCR reagents.

[0031] 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.

[0032] In some embodiments of the present invention, the kit further comprises PCR primers and / or probes for detecting one or more genes selected from the following:

[0033] ZNF132, TLX2, TFPI2, LONRF2, ITGA4, and ARHGEF4.

[0034] The protein encoded by the ZNF132 gene belongs to the C2H2 zinc finger protein family and is involved in biological development and differentiation processes. Studies have shown that zinc finger proteins play a role in cancer progression (Hajra, KM et al., 2002).

[0035] TLX2 encodes a key factor in the development of the enteric nervous system. Studies have shown that loss of TLX2 function may play a role in the occurrence of gastrointestinal stromal tumors (Kaifi JT et al., 2006).

[0036] The TFPI2 gene encodes a member of the Kunitz-type serine protease inhibitor family. Studies have shown that TFPI2 plays a role in maintaining tumor stability and inhibiting tumor growth (Ewa Sierko et al., 2007).

[0037] LONRF2 encodes the LON peptidase N-terminal domain and RING finger protein 2. Studies have shown that LONRF2 may play an important role in the occurrence of rectal adenocarcinoma (Yang Hua et al., 2017).

[0038] The ITGA4 gene encodes a protein that is a member of the integrin α chain family and is a signaling molecule involved in tumor cell motility (Leila Darzi et al., 2017).

[0039] ARHGEF4 is a functional linker protein that connects the tumor suppressor APC and G protein signaling, and plays a role in colon carcinogenesis (Y Kawasaki et al., 2000).

[0040] 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.

[0041] 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.

[0042] In some embodiments of the present invention, the use, wherein the primers and probes are the primers and probes described in the above item (1).

[0043] In some embodiments of the present invention, the reagents for the above-mentioned use further include primers and probes for internal reference genes; preferably, the sequences of the primers for the internal reference genes are shown in SEQ ID NOs: 108-109, and the sequence of the probes for the internal reference genes is shown in SEQ ID NO: 110.

[0044] 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:

[0045] 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, for example, 1-20, 1-15, 1-10, 1-5, 1, 2, 3, 4 or 5 methylation sites.

[0046] 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.

[0047] In some embodiments of the present invention, the method, wherein the content of the differentially methylated region or a fragment thereof according to any one of the present invention is detected by fluorescent quantitative PCR method.

[0048] In some embodiments of the present invention, the method, wherein,

[0049] 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.

[0050] In some embodiments of the present invention, the method, wherein,

[0051] If the Ct value of the differentially methylated region is ≤35 or if ΔCt is ≤ a cutoff value (e.g., a cutoff value of 5), the patient is judged to be at high risk for colorectal cancer; otherwise, the patient is judged to be at low risk;

[0052] Wherein ΔCt=(Ct value of the differentially methylated region or Ct value of the amplified fragment)-Ct value of the reference gene;

[0053] The amplified fragment contains one or more methylation sites within the differentially methylated region, for example, 1-20, 1-15, 1-10, 1-5, 1, 2, 3, 4 or 5 methylation sites.

[0054] 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.

[0055] 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.

[0056] 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. At the same time, third-generation sequencing technology has been proven to be used to directly measure the methylation modification status of DNA, and 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, which is fast, accurate and low-cost. In a preferred embodiment of the present invention, a method of bisulfite treatment combined with fluorescence quantitative PCR is adopted, and several methylation-specific PCR primers and probes are designed for the OPLAH 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.

[0057] By performing whole-genome methylation sequencing and targeted methylation sequencing on colorectal cancer tissue and control tissue samples, we identified differentially methylated regions highly associated with colorectal cancer. The methylation rate difference in the region chr8:145106171-145107467 was particularly significant. Primers and probes were designed targeting this region to quickly and accurately detect methylation in the OPLAH gene. The present invention provides several primer and probe sequences, as shown in Table 1:

[0058] Table 1: OPLAH gene primers and probes

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] After pretreatment, human biological samples can be used to specifically amplify methylated nucleic acid fragments using optimized primers, probes, and PCR reaction systems. Clear amplification bands appear in highly methylated samples, while weak or no amplification occurs in hypomethylated samples. The methylation status of the sample can be determined by the Ct value and the curve. For the detection of OPLAH methylation status, primers and probes for internal reference genes, including but not limited to those described in Table 2, can also be added to detect human DNA in the sample to obtain the corresponding Ct value. The methylation status of the sample is determined by calculating the difference (ΔCt) between the OPLAH gene Ct value and the internal reference gene Ct value. Furthermore, by testing samples from several cancer patients and non-cancer patients, a cutoff value for determining cancer risk (also known as a positive judgment value, for example, obtained by plotting a receiver operating characteristic (ROC) curve) can be obtained. By comparing the test results with the cutoff value, the cancer risk of the subject can be assessed.

[0066] Table 2: Sequence list of primers and probes for internal reference genes

[0067]

[0068] The optimized primers, probes and PCR reaction system can form an OPLAH gene methylation detection kit, and the kit can be used for cancer risk assessment.

[0069] Another aspect of the present invention relates to the use of the differentially methylated region of the present invention in the preparation of a product for detecting colorectal cancer or colorectal precancerous lesions.

[0070] 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.

[0071] In the present invention, unless otherwise specified, the term "early stage colorectal cancer" refers to stage I or stage II colorectal cancer.

[0072] 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.

[0073] 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.

[0074] Advantageous Effects of the Invention

[0075] The present invention achieves one or more of the following technical effects:

[0076] (1) Ability to quickly detect the methylation status of the OPLAH gene;

[0077] (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.

[0078] (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. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1: Differentially methylated gene regions in colorectal cancer.

[0080] Figure 2A to Figure 2G: Comparison of the performance of colorectal cancer-specific methylation genes in colorectal cancer detection.

[0081] Figure 2A: ROC curve of TFPI2 gene detection in clinical samples.

[0082] Figure 2B: ROC curve of ITGA4 gene detection in clinical samples.

[0083] Figure 2C: ROC curve of ZNF132 gene detection in clinical samples.

[0084] Figure 2D: ROC curve of TLX2 gene detection in clinical samples.

[0085] Figure 2E: ROC curve of OPLAH gene detection in clinical samples.

[0086] Figure 2F: ROC curve of LONRF2 gene detection in clinical samples.

[0087] Figure 2G: ROC curve of ARHGEF4 gene detection in clinical samples. DETAILED DESCRIPTION

[0088] 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.

[0089] 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 3 below:

[0090] Table 3

[0091]

[0092]

[0093] The AJCC staging standard for colorectal cancer was used.

[0094] Example 1: Discovery of DMRs in Colorectal Cancer-Specific Methylated Genes

[0095] 1. Tissue Sample Extraction:

[0096] DNA was extracted from 38 pairs of colorectal cancer and adjacent tissue samples from 38 individuals using the DNeasy Blood & Tissue Kit (Qiagen, #69506). Quantification was performed using the Qubit 3.0 system (Invitrogen, USA).

[0097] 2.DNA fragmentation:

[0098] 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.

[0099] 3. Library Construction:

[0100] 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.

[0101] 4. High-throughput sequencing:

[0102] PE100 sequencing was performed using MGISEQ-2000 (MGI).

[0103] 5. Data Analysis

[0104] The sequencing data were quality-controlled, filtered, aligned, and calculated (Non-invasive lung cancer diagnosis and prognosis based on multi-analyte liquid biopsy, Chen Kezhong et al., 2020), and the methylation rate of each CpG site was calculated. By comparing the methylation rates of each CpG site in colorectal cancer tissue and adjacent adjacent tissues, a hierarchical Bayesian method was used to identify 373 differentially methylated regions (DMRs) between cancer and adjacent adjacent tissues, representing colorectal cancer-specific methylation regions.

[0105] Specific site methylation rate = methylation reads sequencing depth / total sequencing depth.

[0106] Figure 1 is a heat map showing 373 DMRs discovered based on 38 pairs of colorectal cancer tissues and adjacent normal tissues, with colorectal cancer tissues on the right and adjacent normal tissues on the left. The discovered DMRs include 371 hypermethylated DMRs and 2 hypomethylated DMRs. Each grid represents the DMR methylation rate at that site in the corresponding sample, ranging from 0 to 1. The closer the methylation rate is to 0, the darker the color. The inventors found that the methylation rate difference of the DMR of the OPLAH gene (chr8: 145106171-145107467) was particularly significant.

[0107] Example 2: Screening of colorectal cancer-specific methylated genes based on methylation-specific PCR

[0108] To further simplify the detection of colorectal cancer-specific methylated genes, methylation-specific primers and probes were designed and screened for the DMR screened in Example 1. Ultimately, it was determined that this DMR had high sensitivity and specificity in colorectal cancer detection.

[0109] The specific operations are as follows:

[0110] 1. Candidate genes:

[0111] The present invention validated the differentially methylated regions of the following genes: TFPI2 (chr7: 93519162-93520360), LONRF2 (chr2: 100937780-100939327), ITGA4 (chr2: 182321761-182322766), ZNF132 (chr19: 58951214-58952044), ARHGEF4 (chr2: 131720924-131797968), TLX2 (chr2: 74742305-74743437) and OPLAH (chr8: 145106171-145107467).

[0112] 2. Design of methylation-specific primers and probes:

[0113] Primers and probes were designed for the candidate genes. The nucleic acid sequences were first converted to bisulfite-treated sequences. The sites for primer and probe design were then determined based on the methylation differences at the CpG sites and the detection AUC. At least one primer in the primer pair had at least one CpG site at its 3' end.

[0114] 3. Primer and probe screening:

[0115] The primers and probes designed in step 2 were used to amplify the bisulfite-treated methylated and unmethylated standards. Primers and probes were screened by evaluating their specificity, accuracy, detection limit, and amplification efficiency. The primers and probes for the OPLAH gene DMR obtained are listed in Table 1.

[0116] 4. Performance evaluation of colorectal cancer-specific methylation genes in colorectal cancer detection:

[0117] Using the primers and probes screened in Step 3, stool samples from patients with and without colorectal cancer were tested to compare the effectiveness of each gene in colorectal cancer detection. First, stool DNA was extracted using a nucleic acid extraction kit (BGI), followed by bisulfite treatment. Finally, quantitative PCR was performed using the primers and probes designed above to obtain Ct values. Receiver-operating characteristic (ROC) curves were constructed using SPSS software to determine the detection area under the curve (AUC) and cutoff value for each gene.

[0118] Among them, the internal reference gene primers are shown in SEQ ID NO: 108 and SEQ ID NO: 109, the OPLAH gene DMR primers are shown in SEQ ID NO: 29 and SEQ ID NO: 31, the internal reference gene probe is shown in SEQ ID NO: 110, and the OPLAH gene probe is shown in SEQ ID NO: 89.

[0119] After statistical analysis, the results are shown in Figures 2A to 2G.

[0120] The results showed that compared with other candidate genes or regions, the detection of OPLAH gene based on the DMR of the present invention had higher sensitivity and specificity for detecting colorectal cancer, reaching 78.49% and 94.64% respectively, with an AUC of 0.925.

[0121] Example 3: A colorectal cancer detection kit based on OPLAH gene DMR

[0122] This example aims to develop a colorectal cancer detection kit based on the DMR of the OPLAH gene. The performance of the OPLAH gene methylation marker for colorectal cancer detection was determined by testing stool samples from 90 colorectal cancer patients, 98 healthy individuals, and 12 polyps patients.

[0123] The specific steps are as follows:

[0124] Sample collection: A stool sample was collected from the subject using a disposable stool sampler (BGI).

[0125] Nucleic acid extraction: DNA from stool samples was extracted using a stool nucleic acid extraction kit (BGI).

[0126] Bisulfite conversion: DNA was subjected to CU conversion using the Methylation Detection Sample Preparation Kit (BGI).

[0127] qPCR assay: The reaction system consisted of 1x PCR buffer, 2–5 mM magnesium ion, 0.2–0.8 mM dNTPs, 0.2 μM internal reference gene primers (SEQ ID NO:108, SEQ ID NO:109), 0.2 μM OPLAH gene primers (SEQ ID NO:29, SEQ ID NO:31), 50 nM internal reference gene probe (SEQ ID NO:110), 50 nM OPLAH gene probe (SEQ ID NO:89), and 1 unit Taq polymerase. The reaction was pre-denatured at 95°C for 10 min, 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 baseline and threshold settings as default, and fluorescence signals were collected at the end of each cycle.

[0128] Results Analysis: The Ct value of the internal reference gene is used to assess the human DNA content in the sample. When the Ct value is >37, the sample is judged to have too low a human DNA content and the test fails. When the Ct value is ≤37, subsequent analysis can be performed. The positive judgment value of the kit is set as a high risk of colorectal cancer when the Ct value of the OPLAH gene (represented by the DMR) is ≤35 or a ΔCt (Ct value of the OPLAH gene DMR minus the Ct value of the internal reference gene) is ≤5; otherwise, the risk is low. Based on these judgment rules, 66 samples from colorectal cancer patients were classified as high risk, 89 samples from healthy individuals were classified as low risk, and 11 samples from polyps were classified as low risk. Performance statistics are shown in Table 4.

[0129] Table 4: Detection performance of the colorectal cancer detection kit based on the OPLAH gene DMR

[0130]

[0131] 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 results. The specific results are shown in Table 5 below.

[0132] Table 5

[0133] Primer sequence number Probe sequence number Colorectal cancer Sensitivity Specificity 24, 2583 72.22% 92.73% 25, ​​2687 74.44% 90.91% 32, 3390 78.89% 84.55% 55, 56103 74.44% 91.82%

[0134] 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, as follows: chr8:145106171-145107467.

2. The differentially methylated region according to claim 1, wherein the base sequence is shown in SEQ ID NO: 1 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 claim 1 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 or the methylation sites within the differentially methylated region; Preferably, the reagent further comprises: (2) Primers and probes for internal reference genes.

7. The kit according to claim 6, wherein: (1) wherein the primer sequences are selected from SEQ ID NOs: 2-78, and the probe sequences are selected from SEQ ID NOs: 79-107; Preferably, in (1): the sequence of the primer is selected from SEQ ID NOs: 29 and 31, and the sequence of the probe is selected from SEQ ID NO: 89; the sequence of the primer is selected from SEQ ID NOs: 24-25, and the sequence of the probe is selected from SEQ ID NO: 83; the sequence of the primer is selected from SEQ ID NOs: 25-26, and the sequence of the probe is selected from SEQ ID NO: 87; the sequence of the primer is selected from SEQ ID NOs: 32-33, and the sequence of the probe is selected from SEQ ID NO: 90; or the sequence of the primer is selected from SEQ ID NOs: 55-56, and the sequence of the probe is selected from SEQ ID NO: 103; Preferably, the sequences of the primers for the internal reference gene in (2) are shown in SEQ ID NOs: 108-109, and the sequence of the probe for the internal reference gene is shown in SEQ ID NO:

110.

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 sampler, nucleic acid extraction reagents, methylation detection sample pretreatment reagents and PCR reagents.

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. The kit according to any one of claims 5 to 9, further comprising PCR primers and / or probes for detecting one or more genes selected from the group consisting of: ZNF132, TLX2, TFPI2, LONRF2, ITGA4, and ARHGEF4.

11. 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.

12. The use according to claim 11, 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.

13. 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.

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

15. The method according to claim 14, 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.

16. The method according to any one of claims 14 to 15, wherein: If the Ct value of the differentially methylated region is ≤35 or if ΔCt is ≤ the cutoff value, the patient is judged to be at high risk for 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 13 to 16, 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.

18. 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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