WO2024045170A1 - 差异性甲基化区域组合、试剂盒和用途 - Google Patents

差异性甲基化区域组合、试剂盒和用途 Download PDF

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WO2024045170A1
WO2024045170A1 PCT/CN2022/116795 CN2022116795W WO2024045170A1 WO 2024045170 A1 WO2024045170 A1 WO 2024045170A1 CN 2022116795 W CN2022116795 W CN 2022116795W WO 2024045170 A1 WO2024045170 A1 WO 2024045170A1
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seq
sequence
nos
differentially methylated
probe
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French (fr)
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李志隆
汪宇盈
彭佳茜
蒋睿婧芳
孙健泷
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深圳华大基因股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the invention belongs to the field of biomedicine and relates to the combination, kit and use of differentially methylated regions of OPLAH gene and ADHFE1 gene.
  • the kit is a kit for detecting the differentially methylated region combination.
  • the use is for detecting colorectal cancer, or for prognostic risk assessment of colorectal cancer patients.
  • Colorectal cancer is a common malignant tumor that occurs in the colon and rectum of the lower gastrointestinal tract. According to the latest statistics, the number of new cases of colorectal cancer ranks third among men and the second among women worldwide, and the number of deaths from colorectal cancer ranks fourth among men and third among women. bit (Bray et al., 2018). In my country, the prevention and control situation of colorectal cancer is also very severe, ranking third in new cases of malignant tumors (388,000) and fifth in deaths (187,000) (Zheng Rongshou et al., 2019). Colorectal cancer develops slowly and generally goes through processes such as polyps, adenomas, and bowel cancer.
  • Colonoscopy is the gold standard for the diagnosis of intestinal cancer. By inserting a lens and a light source from the anus, it can reach the terminal ileum through the rectum, sigmoid colon and other parts. The image can be transmitted to the monitor in real time for observation by the operating doctor.
  • the images of colonoscopy are intuitive and clear, and various lesions such as cancer, polyps, ulcers, and bleeding can be observed, and polyps can also be removed under the microscope.
  • it is an invasive testing technology with complicated preparation process.
  • the fecal occult blood test is a non-invasive method that detects the presence of gastrointestinal bleeding by detecting blood components (hemoglobin) in the stool, allowing doctors to determine the risk of bowel cancer. This method is fast and simple, and the compliance of patients to be tested is high. However, this detection method is susceptible to false positives due to the influence of liver, blood products, green leafy vegetables, etc. in the diet.
  • CEA has low specificity, is prone to produce more false positives, and has limited sensitivity. Therefore, there is a need to establish an accurate, simple, and economical early screening method for colorectal cancer.
  • DNA methylation is an important gene expression regulatory mechanism that can regulate gene expression and silencing and has a significant impact on the occurrence and development of tumors. Abnormal methylation of cancer-related genes often appears in the early stages of cancer, so DNA methylation signals are considered to be potential early screening markers for tumors.
  • Human feces samples are mixed with intestinal cell DNA, which carries information such as methylation. By detecting and analyzing this information, the possibility of the subject suffering from intestinal cancer can be inferred.
  • Some genes have been proven to be methylation markers for intestinal cancer detection in stool, such as SFRP2, SEPT9, NDRG4 and SDC2 (Hannes M Müller et al., 2004; Jie Chen et al., 2019), but their performance is still unclear. It cannot fully meet clinical needs. For example, the sensitivity and specificity of SFRP2 for intestinal cancer are only 77% (Hannes M Müller et al., 2004).
  • the OPLAH gene encodes 5-hydroxyprolinase and affects the glutamate salvage pathway and abnormal expression. Studies have shown that the down-regulation of expression caused by abnormal hypermethylation of OPLAH affects the metabolism of glutathione and is accompanied by tumorigenesis (Liu Y et al., 2014).
  • ADHFE1 encodes a transhydrogenase, and abnormal regulation caused by hypermethylation of this gene can shorten the cycle of intestinal 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.).
  • DMR differentially methylated region
  • differentially methylated regions which comprise the following differentially methylated regions:
  • the above (1) and (2) can also be called differentially methylated region combinations.
  • the differentially methylated region consists of differentially methylated regions (1) and (2).
  • the differentially methylated region wherein:
  • the base sequence of the differentially methylated region (DMR1) shown in item (1) is as shown in SEQ ID NO:1 or its complementary sequence,
  • the base sequence of the differentially methylated region (DMR2) shown in item (2) is as shown in SEQ ID NO:2 or its complementary sequence.
  • the differentially methylated region is used for detecting colorectal cancer or colorectal cancer precancerous lesions, or for prognostic risk assessment of colorectal cancer patients.
  • the differentially methylated region has been processed as follows:
  • Bisulfite treatment methylation-sensitive endonuclease treatment, methylation-restriction enzyme treatment, or methylation-modifying enzyme treatment.
  • the differentially methylated region of the present invention is an OPLAH gene methylation marker or an ADHFE1 gene methylation marker used for colorectal cancer detection.
  • the differentially methylated region combination is a combination of OPLAH gene methylation markers and ADHFE1 gene methylation markers used for colorectal cancer detection.
  • kits including a reagent for detecting the differentially methylated region described in any one of the present invention or a reagent for methylation sites within the differentially methylated region.
  • the kit wherein the reagents include:
  • the reagents further include:
  • the kit wherein:
  • the sequence of the primer is selected from SEQ ID NOs: 3-79, and the sequence of the probe is selected from SEQ ID NOs: 160-188; and 2) The sequence of the primer is selected from SEQ ID NOs: 80-159, and the sequence of the probe The sequence is selected from SEQ ID NOs: 189-209;
  • the sequence of the primer is selected from SEQ ID NO:30 and SEQ ID NO:32, and the sequence of the probe is selected from SEQ ID NO:170; and 2) The sequence of the primer is selected from SEQ ID NOs:108-109, and the sequence of the probe is selected from SEQ ID NO:108-109.
  • the sequence of the needle is selected from SEQ ID NO:197;
  • 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: 164; and 2) The sequence of the primer is selected from SEQ ID NOs: 144-145, and the sequence of the probe is selected from SEQ ID NOs: 144-145. Since SEQ ID NO:204;
  • the sequence of the primer is selected from SEQ ID NOs: 26-27, and the sequence of the probe is selected from SEQ ID NO: 168; and 2) The sequence of the primer is selected from SEQ ID NOs: 154-155, and the sequence of the probe is selected from SEQ ID NOs: 154-155. Since SEQ ID NO:206;
  • the sequence of the primer is selected from SEQ ID NOs:33-34, and the sequence of the probe is selected from SEQ ID NOs:171; and 2) The sequence of the primer is selected from SEQ ID NOs:156-157, and the sequence of the probe is selected from SEQ ID NOs:156-157. Since SEQ ID NO:209;
  • the sequence of the primer is selected from SEQ ID NOs:56-57, and the sequence of the probe is selected from SEQ ID NO:184; and 2) The sequence of the primer is selected from SEQ ID NOs:158-159, and the sequence of the probe is selected from SEQ ID NOs:158-159. Since SEQ ID NO:203;
  • the sequence of the primers is selected from SEQ ID NOs: 61-62, and the sequence of the probe is selected from SEQ ID NOs: 183; and 2) The sequence of the primers is selected from SEQ ID NOs: 108-109, and the sequence of the probe is selected from SEQ ID NOs: 108-109. Since SEQ ID NO:191;
  • the sequence of the primers is selected from SEQ ID NOs:33-34, and the sequence of the probe is selected from SEQ ID NOs:171; and 2) The sequence of the primers is selected from SEQ ID NOs:108-109, and the sequence of the probe is selected from SEQ ID NOs:108-109. Since SEQ ID NO:191;
  • 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: 164; and 2) The sequence of the primer is selected from SEQ ID NOs: 158-159, and the sequence of the probe is selected from SEQ ID NOs: 158-159. Since SEQ ID NO:203;
  • the sequence of the primers of the internal reference gene in 3) is shown in SEQ ID NOs: 210-211, and the sequence of the probe of the internal reference gene is shown in SEQ ID NO: 212.
  • the kit further contains one or more items selected from the following:
  • Fecal sample sampler nucleic acid extraction reagents, methylation detection sample pre-processing reagents, PCR reagents and fecal occult blood detection reagents;
  • the fecal occult blood detection reagent is a fecal immunochemical detection method detection reagent, a guaiac detection method detection reagent, a tetramethylbenzidine method detection reagent or an ELISA double-antibody sandwich method detection reagent;
  • the fecal immunochemical detection method detection reagent is an immune colloidal gold method fecal occult blood detection reagent.
  • the kit wherein the methylation detection sample pretreatment reagent is bisulfite, methylation-sensitive endonuclease, methylation restriction endonuclease or methylation-modifying enzymes.
  • Another aspect of the present invention relates to the differentially methylated region of the present invention or the detection of the differentially methylated region according to any one of the present invention or the method of detecting methylation sites within the differentially methylated region.
  • the use, wherein the reagent is used to detect the differentially methylated region or methylation within the differentially methylated region in any one of the present invention Primers and probes for the base site.
  • Yet another aspect of the present invention relates to a method for detecting colorectal cancer or colorectal cancer precancerous lesions or a method for assessing the prognostic risk of colorectal cancer patients, comprising the following steps:
  • detecting the content of the fragments in the differentially methylated region for example, detecting the content of the fragments by a PCR method, can represent the content of the differentially methylated region.
  • the method wherein the content of each differentially methylated region or fragments thereof according to any one of the present invention is detected by fluorescence quantitative PCR method.
  • the Ct value of the internal reference gene is used to evaluate the content of human DNA in the sample.
  • the Ct value of the internal reference gene is >37, the human DNA content of the sample is judged to be too low, and the test fails.
  • the Ct value of the internal reference gene is ⁇ 37, the sample is evaluated as qualified. Subsequent analysis can be performed.
  • the method wherein, for the evaluation of qualified samples:
  • the difference ⁇ Ct value between the Ct value of each differentially methylated region and the internal reference gene is further calculated.
  • ⁇ Ct (Ct value of the differentially methylated region or Ct value of the amplified fragment) - Ct value of the internal reference gene
  • the amplified fragment includes one or more methylation sites within the differentially methylated region.
  • Item (1) has a cutoff value of 7, and/or
  • the cutoff value for item (2) is 12.
  • the method further includes the step of evaluating combined with fecal occult blood test results.
  • the method wherein the sample is a human tissue sample, a blood sample, a cell sample, a secretion sample or an excretion sample such as a stool sample.
  • stool samples are used as detection objects.
  • the stool sample contains intestinal exfoliated cells, which meets the needs of the test sample, is non-invasive and more patient-friendly.
  • optional detection methods include but are not limited to: Sanger sequencing, pyrosequencing, 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 detect directly. Pretreatment of nucleic acids is often required during the experiment. Pretreatment methods here include but are not limited to: bisulfite treatment and methylation-sensitive endolysis. Enzyme treatment, methylation restriction enzyme treatment or methylation modifying enzyme treatment, etc. At the same time, third-generation sequencing technology has been proven to be used to directly measure the methylation modification status of DNA.
  • Fluorescent quantitative PCR technology is one of the most commonly used and mature detection technologies in molecular diagnosis. It is fast, accurate and low-cost.
  • This technical solution uses bisulfite treatment combined with fluorescence quantitative PCR, and designs several methylation-specific PCR primers and probes for the OPLAH gene and ADHFE1 gene, which can specifically identify methylated DNA molecules. The methylation status of the sample is detected and the cancer risk of the subject is assessed.
  • the inventor uses a sampling device to collect fecal samples, and the samples undergo nucleic acid extraction and purification steps to obtain nucleic acid samples containing human DNA. Furthermore, the inventor used bisulfite to treat the nucleic acid sample to convert unmethylated cytosine into uracil, so that the methylation site can be detected.
  • the inventors designed several primers and probes as described in Table 1, which can specifically amplify and identify relevant 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 identical to, complementary to, or hybridize to the methylated region pre-treated by methylation modification.
  • the primers and probes provided in this article can effectively reflect the alpha of the relevant sites through the Ct value and/or ⁇ Ct value (the difference between the Ct value of the target gene and the Ct value of the internal reference gene) and/or the curve. methylation status, and then determine the methylation status of the subject's relevant genes. Furthermore, by testing several samples from cancer patients and non-cancer patients, the cutoff value for determining the risk of cancer can be obtained. By comparing the test results with the cutoff value, the risk of cancer of the subject can be assessed.
  • the optimized primers, probes and PCR reaction system can form an OPLAH gene and ADHFE1 gene methylation detection kit, and further the kit can be used for cancer risk assessment.
  • detection methods may include fecal immunochemical detection (FIT), guaiac detection (gFOBT), tetramethylbenzidine method, and ELISA double-antibody sandwich method, etc.
  • FIT fecal immunochemical detection
  • gFOBT guaiac detection
  • tetramethylbenzidine method tetramethylbenzidine method
  • ELISA double-antibody sandwich method etc.
  • FIT uses monoclonal or polyclonal antibodies to directly detect hemoglobin in human feces and is not affected by food intake.
  • Qualitative FIT produces a visible color change when the hemoglobin content in the stool exceeds a certain threshold.
  • Quantitative FIT can measure the value and is defined as positive when it exceeds a certain normal value range.
  • Qualitative FIT detection can use the immunocolloidal gold method, which has the advantages of convenient and fast detection; quantitative FIT detection can use the latex immunoturbidimetric method, which has high sensitivity and specificity.
  • the method for determining the results of the gene-fecal occult blood joint test can be that the genetic test results and the fecal occult blood test can separately customize the judgment thresholds. Based on the above two parts of the test results, the subject is judged to be positive or the candidate is positive.
  • the determination method can also be a logistic regression method, giving different weights to the test results of the OPLAH gene, ADHFE1 gene and fecal occult blood, and calculating them through a logistic function.
  • Different weights ⁇ i reflect the different contributions of independent variables to dependent variables.
  • Different test results Xi can include but are not limited to Ct values, ⁇ Ct values, methylation rates of genetic testing, qualitative fecal occult blood test results, and quantitative fecal occult blood. One or any combination of more than one test result.
  • the kit contains a combined detection reagent for OPLAH and ADHFE1 gene methylation based on the fluorescence quantitative PCR method, which can provide methylation detection results of OPLAH and ADHFE1 genes. Furthermore, the kit may also include a fecal occult blood detection reagent, which can provide fecal occult blood detection results. By calculating the ⁇ Ct value or risk index and comparing it with the preset positive judgment value of the kit, it can be used to determine the risk of colorectal cancer.
  • the combined detection reagent for OPLAH and ADHFE1 gene methylation based on the fluorescence quantitative PCR method contains primers and probes that can specifically amplify the OPLAH and ADHFE1 genes.
  • the reagent can detect the methylation status of the OPLAH and ADHFE1 genes in the nucleic acid after methylation modification treatment.
  • the gene methylation joint detection reagent can be used 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 may be a kit capable of extracting nucleic acids from human biological samples, or a kit containing nucleic acid capture magnetic beads.
  • the methylation modification pretreatment reagent modifies the nucleic acid sequence to produce a difference between methylation-modified cytosine and non-methylation-modified cytosine to facilitate detection.
  • the methylation modification pretreatment reagent can convert unmethylated cytosine into uracil, while the methylated modified cytosine remains unchanged, and the sequence changes can be used for subsequent detection.
  • the fecal occult blood detection reagent preferably uses an immune colloidal gold method, which can easily and quickly obtain qualitative detection results of fecal occult blood.
  • the immunocolloidal gold method fecal occult blood detection reagent can include a sampler, a sample storage and a test strip.
  • the detection test paper is coated with antibodies and colloidal gold. When the target protein labeled by the colloidal gold combines with the antibody pre-coated on the test paper, it will remain on the test paper and form a visible change, thereby determining whether the sample contains the target protein.
  • the positive judgment value is delineated using samples with known clinical information. By testing several positive and negative samples, a classification model is constructed to determine the conditions for a positive judgment.
  • g(y) is the risk index.
  • the risk index is higher than the preset value, it is determined to be positive or the candidate is positive;
  • e is a natural constant
  • ⁇ 0 is a constant term
  • ⁇ i is the coefficient to be found
  • Yet another aspect of the present invention relates to a colorectal cancer diagnosis or evaluation system, including:
  • DMR detection unit obtain content data of the differentially methylated region or fragments thereof according to any one of the present invention; wherein the fragments include one or more of the differentially methylated regions. Methylation sites;
  • Fecal occult blood detection unit obtain fecal occult blood detection result data
  • Data analysis unit input 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;
  • Risk assessment unit Outputs the risk index or judgment results of individual samples.
  • 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 preparing products for detecting colorectal cancer or colorectal cancer precancerous lesions.
  • colon cancer refers to colon cancer and/or rectal cancer, also referred to as “intestinal cancer” for short.
  • the term “early colorectal cancer” refers to stage I or stage II colorectal cancer.
  • detecting differentially methylated regions or detecting fragments thereof refers to detecting the content of differentially methylated regions or detecting the content of fragments thereof; wherein the fragments include the difference One or more methylation sites within a sexually methylated region.
  • detecting methylation sites in a differentially methylated region refers to detecting the content of methylation sites in a differentially methylated region or detecting a differentially methylated region. Methylation rate of methylation sites within.
  • It can detect, diagnose or screen colorectal cancer, or be used for prognostic risk assessment of colorectal cancer, with high sensitivity and/or specificity; preferably, it has both high sensitivity and specificity.
  • the present invention has the advantages of higher sensitivity, completely non-invasive, non-invasive detection process, no need for intestinal preparation, and high acceptance of the test by asymptomatic people.
  • Figure 1 Differentially methylated regions of OPLAH and ADHFE1 genes in intestinal cancer and paracancerous tissue samples.
  • Figure 2 Results of OPLAH and ADHFE1 gene detection reference products.
  • Figure 3 ROC curve of clinical samples for OPLAH and ADHFE1 gene detection.
  • Figure 4 Comparison of ROC curves between genetic testing and gene + FIT joint testing.
  • the criteria for bowel cancer staging are those of the AJCC.
  • Example 1 Real-time fluorescence quantitative PCR combined detection of methylation levels of OPLAH and ADHFE1 genes
  • the inventors discovered the differentially methylated regions of the OPLAH and ADHFE1 genes through targeted methylation capture sequencing.
  • the differentially methylated regions have high methylation levels in cancer tissues and low methylation levels in adjacent tissues.
  • This embodiment takes the fluorescence quantitative PCR method as an example.
  • the inventor designed several methylation-specific primers and probes, which can specifically amplify the methylated nucleic acid sequence. After system primer screening and system optimization, a fluorescence quantitative PCR-based joint detection method for OPLAH and ADHFE1 genes was developed.
  • KAPA HyperPlus Library Preparation Kit KAPA, #KK8510
  • EZ DNA Metlylation-Gold Kit Zymo Research, #D5006
  • the adapters used in the ligation step and the PCR primers used in the PCR step are replaced with adapters and primers suitable for the MGISEQ platform.
  • the adapters need to be methylated (for example, using methylated dCTP to synthesize the adapter) to avoid bisulfite. Process the change sequence.
  • Hybridization, capture and elution were performed using Seq Cap EZ Hybridization and Wash Kit (ROCHE, 5634253001) and SeqCap Epi CpGiant Enrichment Kit (ROCHE, 7138911001). Since the sequencing instrument of the MGI platform is used, the blocking sequence corresponding to the MGI sequencing platform must be used to block the adapter sequence during the hybridization process.
  • PE100 sequencing was performed using MGISEQ-2000 (MGI).
  • Specific site methylation rate methylated read sequencing depth/total sequencing depth.
  • Figure 1 shows the methylation levels of various CpG sites in the OPLAH and ADHFE1 genes in cancer tissues and para-cancerous tissues.
  • the inventors designed a CpG site that can specifically amplify methylated CpG sites with high methylation rates in cancer tissues and low methylation rates in adjacent tissues.
  • the designed primer and probe sequences are shown in Table 1.
  • Fluorescence quantitative PCR can jointly detect the methylation levels of DMR of OPLAH and ADHFE1 genes.
  • the inventors selected optimized primer sets and probes for OPLAH and ADHFE1 gene methylation detection.
  • the primer set described 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).
  • the fluorescence quantitative PCR reaction system includes 1 ⁇ PCR Buffer, 2-5mM magnesium ions, 0.2-0.8mM dNTPs, 0.2 ⁇ M primers for each gene, 50nM probes for each gene and 1 unit of 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.
  • Hongshi 96S qPCR instrument was used for detection. The baseline and threshold were set according to the default settings, and the fluorescence signal was collected at the end of each cycle.
  • the inventors used fully methylated standards and fully non-methylated standards (Merck, #S8001) to verify the detection ability of the method for the methylation levels of OPLAH and ADHFE1 genes.
  • the fully methylated standard is cell line DNA that has been treated with M.SssI methyltransferase, and all CpG sites are methylated; the fully unmethylated standard is cells in which the methyltransferase gene has been knocked out. DNA whose methylation level at CpG sites is less than 5%.
  • the inventors used these two standards and mixed them into references with different methylation levels, including 100%, 50%, 25%, 12%, 6%, 3%, 1% and 0%.
  • Example 2 OPLAH and ADHFE1 gene methylation combined detection kit for bowel cancer detection
  • This embodiment takes the fluorescent quantitative PCR method as an example to disclose a kit for detecting intestinal cancer by jointly detecting the methylation levels of OPLAH and ADHFE1 genes.
  • This kit contains Buffer, dNTPs, enzymes and other reagents required for PCR amplification, as well as OPLAH and ADHFE1 gene primer sets and probes.
  • the primer set described 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).
  • this kit also includes primers and probes for detecting the internal reference gene.
  • 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.
  • the GAPDH gene was 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, and then performed 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. Hongshi 96S qPCR instrument was used for detection. The baseline and threshold were set according to the default settings, and the fluorescence signal was collected at the end of each cycle.
  • the sample test first uses a disposable stool sampler (BGI) to collect the subject's stool sample. Then use the Stool Sample Nucleic Acid Extraction Kit (BGI) to extract DNA from the stool sample. Next, use the methylation detection sample pretreatment kit (BGI) to perform methylation modification pretreatment on the DNA to obtain the transformed DNA. Finally, use this kit to detect the transformed DNA and obtain the Ct values of the internal control, OPLAH and ADHFE1 genes for each sample.
  • BGI disposable stool sampler
  • BGI Stool Sample Nucleic Acid Extraction Kit
  • BGI methylation detection sample pretreatment kit
  • the internal reference gene threshold is 37.
  • the Ct value of the internal reference gene in the sample is greater than 37, it is considered that the human DNA content in the sample is insufficient and the test is determined to have failed.
  • the Ct value of the internal reference gene is less than or equal to 37, the quality inspection is qualified.
  • the gene test is judged to be negative.
  • the ⁇ Ct value of the target gene is further calculated.
  • the positive cutoff value was determined using SPSS for analysis.
  • the inventor used SPSS software to draw a ROC curve for a single gene.
  • the AUCs of the OPLAH and ADHFE1 genes were 0.911 and 0.918 respectively.
  • the inventor used the same method to detect the PPP2R5C and SDC2 genes of the above samples, and obtained the Ct values and ⁇ Ct values of the PPP2R5C and SDC2 genes.
  • the PPP2R5C and SDC2 genes also have significant methylation differences between cancer tissues and non-cancerous tissues.
  • the inventor used SPSS software to analyze the detection effect of the four-gene combination of OPLAH, ADHFE1, PPP2R5C and SDC2.
  • the detection results are shown in Figure 3. It can be seen that the detection effect of the four-gene combination detection is similar to that of the OPLAH and ADHFE1 double-gene detection, AUC is 0.950. Since dual-gene testing can achieve the same effect as four-gene combined testing, considering cost, testing stability, etc., it is determined to use dual-gene combined testing. It was finally determined that when the ⁇ Ct value of the DMR of the OPLAH gene is less than or equal to 7, or the ⁇ Ct value of the DMR of the ADHFE1 gene is less than or equal to 12, the detection performance of clinical samples is best.
  • the specific performance is shown in Table 6.
  • the sensitivity of this kit reaches 86.62 %, the overall specificity (specificity of healthy human samples and polyp samples combined) reached 92.78%, and the sensitivity of advanced adenoma reached 52.63%.
  • This kit also has high sensitivity for early-stage intestinal cancer. As shown in Table 7, the sensitivity of stage I and stage II intestinal cancer reached 88.64% and 89.02% respectively.
  • the total coincidence rate (number of samples with correct negative test of gold standard + number of samples with correct positive test of gold standard)/total number of samples.
  • the gold standard is denoscopy.
  • Example 3 OPLAH and ADHFE1 gene methylation combined with fecal occult blood detection kit for bowel cancer detection Measurement
  • the invention discloses a kit for detecting intestinal 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.
  • the OPLAH and ADHFE1 gene methylation level detection module is based on the fluorescence quantitative PCR method and includes reagents such as Buffer, dNTPs and enzymes required for PCR amplification, as well as OPLAH and ADHFE1 gene primer sets and probes.
  • the primer set described in this example 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).
  • this kit also includes primers and probes for detecting the internal reference gene.
  • 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.
  • the GAPDH gene was 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 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds. Hongshi 96S qPCR instrument was used for detection. The baseline and threshold were set according to the default settings, and the fluorescence signal was collected at the end of each cycle.
  • the sample test first uses a disposable stool sampler (BGI) to collect the subject's stool sample. Then use the Stool Sample Nucleic Acid Extraction Kit (BGI) to extract DNA from the stool sample. Next, use the methylation detection sample pretreatment kit (BGI) to perform methylation modification pretreatment on the DNA to obtain the transformed DNA. Finally, use this kit to detect the transformed DNA and obtain the Ct values of the internal control, OPLAH and ADHFE1 genes for each sample.
  • BGI disposable stool sampler
  • BGI Stool Sample Nucleic Acid Extraction Kit
  • BGI methylation detection sample pretreatment kit
  • the internal reference gene threshold is 37.
  • the Ct value of the internal reference gene in the sample is greater than 37, it is considered that the human DNA content in the sample is insufficient and the test is determined to have failed.
  • the Ct value of the internal reference gene is less than or equal to 37, the quality inspection is qualified.
  • the gene test is judged to be negative.
  • the ⁇ Ct value of the target gene is further calculated.
  • the fecal occult blood module contains test strips that can qualitatively detect fecal hemoglobin content, and its detection principle is the colloidal gold method. Specifically, a FIT sampler is used to collect fecal samples, mix them with the sample preservation solution, take about 100 ⁇ l of the sample on the test paper, and read the test results within a certain period of time.
  • bands should appear on the quality control line, otherwise the test fails; if the quality control line is qualified, observe the test line. If bands appear on the test line, the fecal occult blood test is positive, and if there are no bands, it is negative. The result reading should be completed within the specified time and will be invalidated after timeout.
  • This example uses 318 samples with fecal occult blood detection results among the samples described in Example 2 for analysis.
  • the clinical information of the samples is shown in Table 9.
  • the positive cutoff value was determined using SPSS for analysis.
  • the inventor used SPSS software to draw a ROC curve for the gene detection results.
  • the AUC of the joint detection of OPLAH and ADHFE1 genes was 0.931.
  • the OPLAH and ADHFE1 genes were used in the detection of intestinal cancer. has better detection performance.
  • FIT fecal occult blood test
  • the ROC curve After calculating the detection risk index of each sample, draw the ROC curve to determine the optimal positive cutoff value. As shown in Figure 4, the AUC of gene and FIT joint testing reached 0.975. Gene and FIT joint testing can effectively improve detection performance. As shown in Table 10, when the positive cut-off value is 0.55, the best performance is achieved. At this time, the sensitivity and overall specificity are 95.97% and 91.43% respectively.
  • This kit has good detection ability for advanced adenomas. The output rate reached a sensitivity of 58.62%.
  • This kit has high sensitivity for early-stage intestinal cancer. As shown in Table 11, the sensitivity for stage I and stage II intestinal cancer reached 89.47% and 100% respectively. As shown in Figure 5, compared with dual-gene testing or FIT testing, this kit has higher detection sensitivity for colorectal cancer and advanced adenomas.
  • the total coincidence rate (number of samples with correct negative test of gold standard + number of samples with correct positive test of gold standard)/total number of samples.
  • the gold standard is denoscopy.

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Abstract

提供差异性甲基化区域组合、试剂盒和用途；所述差异性甲基化区域组合，其包含如下的差异性甲基化区域：(1) chr8: 145106171-145107467和（2) chr8:67344198-67345563；所述的差异性甲基化区域组合能够用于有效地检测结直肠癌或结直肠癌癌前病变，或者用于评估结直肠癌患者的预后风险，具有良好的灵敏度和特异性。

Description

差异性甲基化区域组合、试剂盒和用途 技术领域
本发明属于生物医药领域，涉及OPLAH基因和ADHFE1基因的差异性甲基化区域的组合、试剂盒和用途。具体地，所述试剂盒为检测该差异性甲基化区域组合的试剂盒。具体地，所述用途为用于检测结直肠癌，或者用于结直肠癌患者的预后风险评估的用途。
背景技术
发生在下消化道结肠、直肠部位的结直肠癌是一种常见的恶性肿瘤。据最新统计数字显示，全世界范围内结直肠癌的新发病例数在男性中排第三位、在女性中排第二位，死亡病例数在男性中排第四位、女性中排第三位(Bray等，2018)。在我国，结直肠癌的防控形势也十分严峻，为恶性肿瘤新发病例的第三位(38.8万人)、死亡病例的第五位(18.7万人)(郑荣寿等，2019)。结直肠癌发展缓慢，一般会经历息肉、腺瘤、肠癌等过程，从腺瘤发展成为肠癌时间可长达5-10年。而如在结直肠癌发展早期进行干预，可显著降低死亡率。I期结直肠癌患者的五年生存率可达90％以上，而IV期结直肠癌患者的五年生存率低于20％(Marzieh Araghi等，2020)。
传统的结直肠癌早筛技术主要有肠镜、大便潜血试验、肿瘤标志物CEA和CA19-9等，这些技术手段目前均存在一定的局限性。肠镜检测是肠癌诊断的金标准，通过从肛门插入镜头及光源，循腔经直肠、乙状结肠等部位可达回肠末端，图像可实时传输到显示器上，供操作医生观察。肠镜的图像直观、清晰，可观察到癌、息肉、溃疡、出血等多种病变，还可对息肉等进行镜下摘除等操作。但其为一种侵入性的检测技术，准备过程复杂，需控制饮食及进行肠道清洁，待检者的依从性低。且对设备和人员有要求，需要在医院专业人员和麻醉师操作，部分受检者会有不适感并且有产生并发症的风险(3-5例/1000人)。大便潜血试验是一种无创的方法，通过检测大便中的血液成分(血红蛋白)来判别有无消化道出血，以便医生判断肠癌风险。该方法快捷简便，待检者依从性较高。但该检测方法易受到饮食中的肝脏、血液制品、绿叶蔬菜等影响出现假阳性，同时对出血量较少的早期结直肠癌患者的灵敏度低，易出现漏诊。而CEA等作为广谱的肿瘤标志物，特异度低，易产生较多的假阳性，灵敏度亦有限。因此， 需要建立一种准确、简便、经济的结直肠癌早期筛查方法。
DNA甲基化是一种重要的基因表达调控机制，能够调节基因的表达和沉默，在肿瘤的发生发展中具有重大的影响。癌症相关基因的异常甲基化常出现于癌症发生的早期，因此DNA甲基化信号被认为是有潜力的肿瘤早期筛查标志物。人的粪便样本中混杂有肠道细胞DNA，这些DNA携带有甲基化等信息，通过检测和分析这些信息，可推断受检者罹患肠癌的可能性。已有部分基因被证明了可以作为粪便中肠癌检测的甲基化标志物，例如SFRP2，SEPT9，NDRG4和SDC2等(Hannes M Müller等，2004；Jie Chen等，2019)，但它们的性能尚不能完全满足临床需求，例如SFRP2对肠癌的灵敏度和特异性均仅为77％(Hannes M Müller等，2004)。
OPLAH基因编码5-羟脯氨酸酶，并影响谷氨酸补救路径和异常表达。有研究表明，OPLAH异常高甲基化引起的表达下调，影响谷胱甘肽的代谢并伴随着肿瘤发生(Liu Y等，2014)。
ADHFE1编码一种转氢酶，由该基因的过甲基化引起的异常调控能缩短肠癌细胞周期并促进癌细胞增殖(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.)。
目前，尚需要开发新的用于结直肠癌检测的标志物。
发明内容
本发明人经过深入的研究和创造性的劳动，发现了OPLAH基因的差异性甲基化区域(Differentially methylated region，DMR)以及ADHFE1基因的差异性甲基化区域。本发明人惊奇地发现，所述差异性甲基化区域的组合能够用于有效地检测结直肠癌或结直肠癌癌前病变，或者用于评估结直肠癌患者的预后风险，具有良好的灵敏度和特异性。由此提供了下述发明：
本发明的一个方面涉及差异性甲基化区域，其包含如下的差异性甲基化区域：
(1)chr8:145106171-145107467(DMR1)，和
(2)chr8:67344198-67345563(DMR2)。
上面的(1)为OPLAH基因的差异性甲基化区域。
上面的(2)为ADHFE1基因的差异性甲基化区域。
上面的(1)和(2)也可以称为差异性甲基化区域组合。
在本发明的一些实施方式中，所述的差异性甲基化区域，其由差异性甲基化区域(1)和(2)组成。
在本发明的一些实施方式中，所述的差异性甲基化区域，其中：
第(1)项所示的差异性甲基化区域(DMR1)的碱基序列如SEQ ID NO:1或其互补序列所示，
第(2)项所示的差异性甲基化区域(DMR2)的碱基序列如SEQ ID NO:2或其互补序列所示。
DMR1的碱基序列：
Figure PCTCN2022116795-appb-000001
Figure PCTCN2022116795-appb-000002
DMR2的碱基序列：
Figure PCTCN2022116795-appb-000003
Figure PCTCN2022116795-appb-000004
在本发明的一些实施方式中，所述的差异性甲基化区域，其用于检测结直肠癌或结直肠癌癌前病变，或者用于结直肠癌患者的预后风险评估。
在本发明的一些实施方式中，所述的差异性甲基化区域，其经过了如下处理：
亚硫酸氢盐处理、甲基化敏感性内切酶处理、甲基化限制性内切酶处理或甲基化修饰酶处理。
本发明的差异性甲基化区域是用于结直肠癌检测的OPLAH基因甲基化标志物或ADHFE1基因甲基化标志物。所述差异性甲基化区域组合为用于结直肠癌检测的OPLAH基因甲基化的标志物和ADHFE1基因甲基化的标志物的组合。
本发明的另一方面涉及一种试剂盒，包括检测本发明中任一项所述的差异性甲基化区域的试剂或者所述差异性甲基化区域内的甲基化位点的试剂。
在本发明的一些实施方式中，所述的试剂盒，其中，所述试剂包括：
1)检测第(1)项所示的差异性甲基化区域或者所述差异性甲基化区域内的甲基化位点的引物和探针；
2)检测第(2)项所示的差异性甲基化区域或者所述差异性甲基化区域内的甲基化位点的引物和探针；
优选地，所述试剂还包括：
3)内参基因的引物和探针。
在本发明的一些实施方式中，所述的试剂盒，其中：
1)中引物的序列选自SEQ ID NOs:3-79，探针的序列选自SEQ ID NOs:160-188； 和2)中引物的序列选自SEQ ID NOs:80-159，探针的序列选自SEQ ID NOs:189-209；
优选地：
1)中引物的序列选自SEQ ID NO:30和SEQ ID NO:32，探针的序列选自SEQ ID NO:170；和2)中引物的序列选自SEQ ID NOs:108-109，探针的序列选自SEQ ID NO:197；
1)中引物的序列选自SEQ ID NOs:25-26，探针的序列选自SEQ ID NO:164；和2)中引物的序列选自SEQ ID NOs:144-145，探针的序列选自SEQ ID NO:204；
1)中引物的序列选自SEQ ID NOs:26-27，探针的序列选自SEQ ID NO:168；和2)中引物的序列选自SEQ ID NOs:154-155，探针的序列选自SEQ ID NO:206；
1)中引物的序列选自SEQ ID NOs:33-34，探针的序列选自SEQ ID NO:171；和2)中引物的序列选自SEQ ID NOs:156-157，探针的序列选自SEQ ID NO:209；
1)中引物的序列选自SEQ ID NOs:56-57，探针的序列选自SEQ ID NO:184；和2)中引物的序列选自SEQ ID NOs:158-159，探针的序列选自SEQ ID NO:203；
1)中引物的序列选自SEQ ID NOs:61-62，探针的序列选自SEQ ID NO:183；和2)中引物的序列选自SEQ ID NOs:108-109，探针的序列选自SEQ ID NO:191；
1)中引物的序列选自SEQ ID NOs:33-34，探针的序列选自SEQ ID NO:171；和2)中引物的序列选自SEQ ID NOs:108-109，探针的序列选自SEQ ID NO:191；
或者，
1)中引物的序列选自SEQ ID NOs:25-26，探针的序列选自SEQ ID NO:164；和2)中引物的序列选自SEQ ID NOs:158-159，探针的序列选自SEQ ID NO:203；
优选地，3)中内参基因的引物的序列如SEQ ID NOs:210-211所示，内参基因的探针的序列如SEQ ID NO:212所示。
在本发明的一些实施方式中，所述的试剂盒，其还包含选自如下的一项或者多项：
粪便样本采样器、核酸提取试剂、甲基化检测样本前处理试剂、PCR试剂和粪便潜血检测试剂；
优选地，所述粪便潜血检测试剂为粪便免疫化学检测法检测试剂、愈创木脂检测法检测试剂、四甲基联苯胺法检测试剂或ELISA双抗夹心法检测试剂；
优选地，所述粪便免疫化学检测法检测试剂为免疫胶体金法粪便潜血检测试剂。
在本发明的一些实施方式中，所述的试剂盒，其中，所述甲基化检测样本前处理试剂为亚硫酸氢盐、甲基化敏感性内切酶、甲基化限制性内切酶或甲基化修饰酶。
本发明的再一方面涉及本发明的差异性甲基化区域或者检测本发明中任一项所述的差异性甲基化区域或者所述差异性甲基化区域内的甲基化位点的试剂在制备用于检测结直肠癌或结直肠癌癌前病变，或者用于结直肠癌患者的预后风险评估的药物中的用途。
在本发明的一些实施方式中，所述的用途，其中，所述试剂为用于检测本发明中任一项所述的差异性甲基化区域或者所述差异性甲基化区域内的甲基化位点的引物和探针。
本发明的再一方面涉及一种检测结直肠癌或结直肠癌癌前病变或者一种评估结直肠癌患者的预后风险的方法，包括如下步骤：
检测本发明中任一项所述的差异性甲基化区域的含量或者其中的片段的含量；其中，所述片段包含所述差异性甲基化区域内的一个或多个甲基化位点。
本领域技术人员能够理解，检测差异性甲基化区域中片段的含量，例如通过PCR方法检测所述的片段的含量，能够代表差异性甲基化区域的含量。
在本发明的一些实施方式中，所述的方法，其中，通过荧光定量PCR方法检测本发明中任一项所述的各差异性甲基化区域或者其中的片段的含量。
在本发明的一些实施方式中，所述的方法，其中，
采用内参基因Ct值评估样本中人源DNA的含量，当内参基因Ct值>37时，判定样本人源DNA含量过低，本次检测失败，当内参基因Ct值≤37时，评估样本合格，可进行后续分析。
在本发明的一些实施方式中，所述的方法，其中，对于评估合格的样本：
当各差异性甲基化区域的Ct值大于38或无检出Ct值时，判定为检测阴性；
当各差异性甲基化区域的Ct值小于等于38时，进一步计算各差异性甲基化区域与内参基因Ct值的差值△Ct值。
在本发明的一些实施方式中，所述的方法，其中：
如果第(1)项或第(2)项的△Ct≤截断值，判定为结直肠癌高风险，否则判定为 低风险；
其中△Ct＝(所述差异性甲基化区域的Ct值或者扩增片段的Ct值)-内参基因Ct值；
其中，所述扩增片段包含所述差异性甲基化区域内的一个或多个甲基化位点。
在本发明的一些实施方式中，所述的方法，其中：
第(1)项的截断值为7，和/或
第(2)项的截断值为12。
在本发明的一些实施方式中，所述的方法，其中还包括联合粪便潜血检测结果进行评估的步骤。
在本发明的一些实施方式中，所述的方法，其中，所述样本为人体组织样本、血液样本、细胞样本、分泌物样本或排泄物样本例如粪便样本。
根据本发明的一些实施例，采用粪便样本作为检测对象。这样，粪便样本含有肠道脱落细胞，符合检测样本的需求，且具有无创性，对患者更友好。
根据本发明的一些实施例，可选择的检测手段包括但不限于：Sanger测序、焦磷酸测序、高通量测序、核酸质谱、PCR、荧光定量PCR、ddPCR等。由于核酸序列未发生变化，甲基化修饰难以被直接检测，实验过程中往往需要对核酸进行预处理，此处预处理手段包括但不限于：亚硫酸氢盐处理、甲基化敏感性内切酶处理、甲基化限制性内切酶处理或甲基化修饰酶处理等。同时，第三代测序技术已被证实可用于直接测定DNA的甲基化修饰状态，随着技术发展，未来亦可作为检测手段。荧光定量PCR技术是目前分子诊断常用且发展成熟的检测技术之一，具有快速、准确和成本低的特点。本技术方案采用了亚硫酸氢盐处理结合荧光定量PCR的方法，并对OPLAH基因和ADHFE1基因设计了若干甲基化特异性PCR引物和探针，能够特异性地识别甲基化的DNA分子，实现了对样本甲基化状态的检测，进而评估受检者的患癌风险。
通过对结直肠癌组织和对照组织样本进行全基因组甲基化测序和靶向甲基化测序，得到与结直肠癌高度相关的甲基化差异区域，其中DMR1(chr8：145106171-145107467)和DMR2(chr8:67344198-67345563)的甲基化率差异尤为显著。针对所述区域进行引物、探针设计，可用于快捷、准确地检测OPLAH基因和ADHFE1基因的甲基化情况。本发明提供了若干引物和探针序列，如表1所示：
表1：OPLAH基因和ADHFE1基因的引物、探针序列
Figure PCTCN2022116795-appb-000005
Figure PCTCN2022116795-appb-000006
Figure PCTCN2022116795-appb-000007
Figure PCTCN2022116795-appb-000008
Figure PCTCN2022116795-appb-000009
Figure PCTCN2022116795-appb-000010
Figure PCTCN2022116795-appb-000011
Figure PCTCN2022116795-appb-000012
Figure PCTCN2022116795-appb-000013
Figure PCTCN2022116795-appb-000014
Figure PCTCN2022116795-appb-000015
在本文中，本发明人使用采样装置收集粪便样本，样本经过核酸提取和纯化步骤，获得包含人源DNA的核酸样本。进一步地，本发明人采用亚硫酸氢盐对所述核酸样本进行处理，使未发生甲基化的胞嘧啶转化为尿嘧啶，使甲基化位点能够被检测到。针对所述基因，本发明人设计了如表1所述的若干引物和探针，能够特异性地扩增、 识别相关位点。所述引物、探针序列为连续的至少15个碱基长度的片段，而所述引物、探针的核酸序列等同、互补或杂交于经甲基化修饰预处理的所述甲基化区域。使用荧光定量PCR方法，本文提供的引物和探针能够有效地通过Ct值和/或△Ct值(目标基因Ct值与内参基因Ct值的差值)和/或曲线情况反映相关位点的甲基化情况，进而判断受检者相关基因的甲基化情况。进一步地，通过对若干癌症患者和非癌症患者样本的检测，可得到判定患癌风险判定的截断值，通过对比检测结果与截断值，可对受检者患癌风险进行评估。
所述优化后的引物、探针和PCR反应体系，可形成一种OPLAH基因和ADHFE1基因甲基化检测试剂盒，进一步的该试剂盒可用于癌症的风险评估。
关于粪便潜血检测：
对粪便样本中的血红蛋白进行检测，检测手段可包括粪便免疫化学检测法(FIT)、愈创木脂检测法(gFOBT)、四甲基联苯胺法和ELISA双抗夹心法等。
所述粪便潜血的检测方法中，FIT利用单克隆或多克隆抗体直接检测人粪便中的血红蛋白，不受进食食物的影响。定性FIT是在粪便中血红蛋白含量超过一定阈值后会产生可视性的颜色变化，定量FIT则可测量数值，当超过一定的正常值范围后被定义为阳性。定性FIT检测可采用免疫胶体金法，具有检测方便、快捷的优点；定量FIT检测可采用乳胶免疫比浊法，具有较高灵敏度和特异性。
关于联合基因检测和粪便潜血检测的方法：
基因-粪便潜血联合检测的结果判定方法可以是基因检测结果和粪便潜血检测可分别定制判定阈值，综合以上两部分检测结果，受检者被判定为阳性或候选为阳性。
判定方法也可以是采用逻辑回归的方法，对OPLAH基因、ADHFE1基因和粪便潜血的检测结果赋予不同的权重，通过逻辑函数计算。所述逻辑函数为g(y)＝1/(1+e -y)。其中g(y)是风险指数，当风险指数高于预设值时，判定为阳性或候选为阳性；e为自然常数；y＝(θ0+θ1*X1+θ2*X2+...+θn*Xn)，θ0为常数项，θi(i＝1,2,...,n)是待求系数，Xi(i＝1,2,...,n)是基因和粪便潜血检测的结果。不同的权重θi反映了自变量对因变量不同的贡献程度，不同的检测结果Xi可以包括但不限于基因检测的Ct值、△Ct值、甲基化率、粪便潜血定性检测结果和粪便潜血定量检测结果中的一个或一个以上的任意组合。
通过优化判定方法中的参数，可得到更高的检测灵敏度。
试剂盒包含基于荧光定量PCR法的OPLAH和ADHFE1基因甲基化联合检测试剂，可提供OPLAH和ADHFE1基因的甲基化检测结果。进一步地，试剂盒还可包含粪便潜血检测试剂，可提供粪便潜血的检测结果。通过计算△Ct值或风险指数，与试剂盒预设的阳性判断值对比，可用于判断患结直肠癌的风险。
所述基于荧光定量PCR法的OPLAH和ADHFE1基因甲基化联合检测试剂中包含能够特异性扩增OPLAH和ADHFE1基因的引物和探针。所述试剂能够检测经甲基化修饰处理后的核酸中OPLAH和ADHFE1基因的甲基化情况。
所述基因甲基化联合检测试剂可搭配不同的粪便样本采样装置、核酸提取试剂和甲基化修饰预处理试剂使用。所述粪便采样装置可包含采样器和样本存储器，用于采集和存储样本。所述核酸提取试剂可以是能够提取人体生物样本中核酸的试剂盒，也可以是含有核酸捕获磁珠的试剂盒。所述甲基化修饰预处理试剂通过对核酸序列进行修饰，使发生甲基化修饰的胞嘧啶与未发生甲基化修饰的胞嘧啶产生便于检测的差异。通常的，所述甲基化修饰预处理试剂能够使未发生甲基化修饰的胞嘧啶转化为尿嘧啶，而发生甲基化修饰的胞嘧啶保持不变，序列的变化可被用于后续的检测。
所述粪便潜血检测试剂优选地，使用免疫胶体金法，可简便、快捷地获得粪便潜血定性检测结果。免疫胶体金法粪便潜血检测试剂可包含采样器、样本存储器和检测试纸。所述检测试纸包被有抗体和胶体金，当被胶体金标记的目标蛋白与试纸上预包被的抗体结合时会滞留在试纸上形成可视变化，从而判断样本中是否含有目标蛋白。
所述阳性判断值使用已知临床信息的样本划定，通过对若干阳性和阴性样本进行检测，构建分类模型，确定判定为阳性的条件。
本发明的再一方面涉及一种结直肠癌的诊断或评估模型，采用逻辑函数g(y)＝1/(1+e - y)进行计算，其中：
g(y)是风险指数，当风险指数高于预设值时，判定为阳性或候选为阳性；
e为自然常数；
y＝(θ0+θ1*X1+θ2*X2+...+θn*Xn)，θ0为常数项，θi(i＝1,2,...,n)是待求系数，Xi(i＝1,2,...,n)是本发明中任一项所述的差异性甲基化区域的检测结果和粪便潜血的检测结果。
本发明的再一方面涉及一种结直肠癌的诊断或评估系统，包括：
1)DMR检测单元：获取本发明中任一项所述的差异性甲基化区域或者其中的片段的含量数据；其中，所述片段包含所述差异性甲基化区域内的一个或多个甲基化位点；
2)粪便潜血检测单元：获取粪便潜血检测结果数据；
3)数据分析单元：将DMR检测单元和粪便潜血检测单元获得的数据输入本发明的结直肠癌的诊断或评估模型进行分析；
4)风险评估单元：输出个体样本的风险指数或者判断结果。
本发明的再一方面涉及本发明的差异性甲基化区域(1)和差异性甲基化区域(2)在制备检测结直肠癌或者结直肠癌癌前病变的产品中的用途。
本发明中，如果没有特别说明，术语“结直肠癌”是指结肠癌和/或直肠癌，也简称为“肠癌”。
本发明中，如果没有特别说明，术语“早期结直肠癌”是指I期或II期结直肠癌。
本发明中，如果没有特别说明，检测差异性甲基化区域或者检测其中的片段，是指检测差异性甲基化区域的含量或者检测其中的片段的含量；其中，所述片段包含所述差异性甲基化区域内的一个或多个甲基化位点。
本发明中，如果没有特别说明，检测差异性甲基化区域内的甲基化位点，是指检测差异性甲基化区域内的甲基化位点的含量或者检测差异性甲基化区域内的甲基化位点的甲基化率。
发明的有益效果
本发明取得了如下的技术效果中的一项或者多项：
(1)能够实现OPLAH基因和ADHFE1基因甲基化状态的快速检测；
(2)能够检测、诊断或筛查结直肠癌，或者用于结直肠癌的预后风险评估，具有较高的灵敏度和/或特异性；优选地，同时兼具较高的灵敏度和特异性。
(3)能够应用于早期结直肠癌或结直肠癌癌前病变的检测、诊断或筛查，具有较高的灵敏度和/或特异性；优选地，同时兼具较高的灵敏度和特异性。
(4)可联合检测粪便中的血红蛋白，进一步提高结直肠癌的检测灵敏度。
(5)与传统检测手段相比，本发明具有更高的敏感性，完全无创，检测过程非侵入性，无需肠道准备、无症状人群对该检测接受度高等优点。
附图说明
图1：OPLAH和ADHFE1基因在肠癌和癌旁组织样本中的差异甲基化区域。
图2：OPLAH和ADHFE1基因检测参考品结果。
图3：OPLAH和ADHFE1基因检测临床样本的ROC曲线。
图4：基因检测与基因+FIT联检ROC曲线对比。
图5：检测灵敏度对比。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
部分相关定义参考中国临床肿瘤学会(CSCO)结直肠癌诊疗指南2021，部分相关内容引用如下面的表2：
表2
Figure PCTCN2022116795-appb-000016
肠癌分期的标准采用AJCC。
实施例1：实时荧光定量PCR联合检测OPLAH和ADHFE1基因的甲基化水平
发明人通过靶向甲基化捕获测序发现了OPLAH和ADHFE1基因的差异甲基化区域。所述差异甲基化区域在癌组织中具有高甲基化水平，而在癌旁组织中的甲基化水平较低。本实施例以荧光定量PCR法为例，针对所述区域，发明人设计了若干甲基化特异性引物和探针，能够特异性地扩增发生甲基化的核酸序列。经过体系引物筛选和体系优化，开发得到一种基于荧光定量PCR的OPLAH和ADHFE1基因联合检测方法。
具体步骤如下：
1、靶向甲基化捕获测序发现差异甲基化区域：
对来自于38人的38对结直肠癌样本和癌旁组织样本(其中就一对结直肠癌样本和癌旁组织样本而言，来自同一人)，使用DNeasy Blood & Tissue Kit(Qiagen，#69506)进行核酸提取，用Qubit 3.0系统(Invitrogen，美国)进行定量。
取200ng提取好的DNA，同时加入1ng Unmethylated lambda DNA(PROMEGA，#D1521)用于后续C-U转化质控，使用超声打断仪将DNA打断，再使用AMPure XP(AGENCOURT，#A63882)对DNA进行片段选择，使DNA片段大小集中在160bp左右。
使用KAPA HyperPlus Library Preparation Kit(KAPA，#KK8510)进行文库构建，使用EZ DNA Metlylation-Gold Kit(Zymo Research，#D5006)对接头连接产物进行亚硫酸氢盐转化，具体操作参考试剂盒说明书，其中接头连接步骤所使用的接头以及PCR步骤所使用的PCR引物更换为适用于MGISEQ平台的接头和引物，接头需要进行甲基化修饰(例如使用甲基化的dCTP合成接头)，以避免亚硫酸氢盐处理改变序列。
使用Seq Cap EZ Hybridization and Wash Kit(ROCHE，5634253001)和SeqCap Epi CpGiant Enrichment Kit(ROCHE，7138911001)进行杂交、捕获和洗脱。由于使用了MGI平台的测序仪器，杂交过程中对接头序列的封闭须使用MGI测序平台对应的封闭序列。
使用MGISEQ-2000(MGI)进行PE100测序。
对测序数据进行质控、过滤不合格测序序列、将测序序列与人类基因组序列的经计算机模拟的亚硫酸氢盐转化型式比对之后，统计所有序列并计算出每个CpG位点 的甲基化率(Non-invasive lung cancer diagnosis and prognosis based on multi-analyte liquid biopsy，Chen Kezhong等，2020)。
特定位点甲基化率＝甲基化reads测序深度/总测序深度。
本发明人发现OPLAH基因和ADHFE1基因存在显著的甲基化差异区域，具有区分癌与非癌的潜力。图1展示了OPLAH和ADHFE1基因中各个CpG位点在癌组织和癌旁组织中的甲基化水平。
2、甲基化引物、探针设计
根据发现的差异甲基化区域，本发明人针对在癌组织中高甲基化率且在癌旁组织中具有较低甲基化率的CpG位点，设计了能够特异性扩增经甲基化修饰预处理后核酸序列的引物和探针，其中一对引物中至少包含一条引物的3’端具有至少一个CpG位点。所设计的引物、探针序列如表1所示。
3、荧光定量PCR能够联合检测OPLAH和ADHFE1基因的DMR的甲基化水平
本发明人选择经优化后的引物组和探针进行OPLAH和ADHFE1基因甲基化检测。本实施例中所述引物组包括：OPLAH基因引物组(SEQ ID NO:30和SEQ ID NO:32)、OPLAH基因探针(SEQ ID NO:170)、ADHFE1基因引物组(SEQ ID NO:108和SEQ ID NO:109)和ADHFE1基因探针(SEQ ID NO:197)。荧光定量PCR反应体系包括1×PCR Buffer、2-5mM镁离子、0.2-0.8mM dNTP、每个基因0.2μM引物、每个基因50nM探针和1单位Taq聚合酶。反应在95℃预变性10min，然后进行95℃变性30秒、55℃退火30秒和72℃延伸30秒的循环40个。采用宏石96S qPCR仪进行检测，基线、阈值按照默认设置，在每个循环结束时收集荧光信号。
本发明人使用全甲基化标准品和全非甲基化标准品(Merck，#S8001)验证所述方法对OPLAH和ADHFE1基因甲基化水平的检测能力。全甲基化标准品是经过M.SssI甲基转移酶处理的细胞系DNA，所有CpG位点均被甲基化修饰；全非甲基化标准品是甲基转移酶基因被敲除的细胞系DNA，其CpG位点的甲基化水平低于5％。本发明人使用这两种标准品，混合成不同甲基化水平的参考品，包括100％、50％、25％、12％、6％、3％、1％和0％。
对每个参考品取20ng进行3次检测，首先使用甲基化检测样本前处理试剂盒(BGI)对DNA进行甲基化修饰前处理，使未发生甲基化修饰的胞嘧啶转化为尿嘧啶。然后使用上述荧光定量PCR反应体系对参考品进行检测。
检测结果如表3所示。
表3：OPLAH和ADHFE1基因检测参考品数据
Figure PCTCN2022116795-appb-000017
结果显示，所有混入甲基化模板的参考品均被检出，检测限可低至1％。而未加入甲基化模板的参考品则无发生扩增。
根据投入的甲基化模板量和平均Ct值，绘制标准曲线，如图2所示，标准曲线的R2良好。说明本方法能够有效检出样本中的OPLAH和ADHFE1基因甲基化信号，且无非特异扩增。
实施例2：OPLAH和ADHFE1基因甲基化联合检测试剂盒用于肠癌检测
本实施例以荧光定量PCR方法为例，公开一种通过联合检测OPLAH和ADHFE1基因甲基化水平来实现肠癌检测的试剂盒。
本试剂盒包含PCR扩增所需要的Buffer、dNTP和酶等试剂，还包括OPLAH和ADHFE1基因引物组和探针。本实施例中所述引物组包括：OPLAH基因引物组(SEQ ID NO:30和SEQ ID NO:32)、OPLAH基因探针(SEQ ID NO:170)、ADHFE1基因引物组(SEQ ID NO:108和SEQ ID NO:109)和ADHFE1基因探针(SEQ ID NO:197)。为了监测样本中是否含有足够的人源DNA，本试剂盒中还加入了用于检测内参基因的引物和探针。所述内参基因一般为管家基因，其对应的引物和探针检测结果仅与人源DNA总量有关，而不受甲基化水平影响。本实施例中选用GAPDH基因作为内参基因，其引物和探针序列如表4所示。PCR反应在95℃预变性10min，然后进 行95℃变性30秒、55℃退火30秒和72℃延伸30秒的循环40个。采用宏石96S qPCR仪进行检测，基线、阈值按照默认设置，在每个循环结束时收集荧光信号。
表4：内参基因引物、探针序列表
Figure PCTCN2022116795-appb-000018
本发明共纳入604例粪便样本对本试剂盒进行性能评估，临床样本信息如表5所示。
表5：样本信息表
Figure PCTCN2022116795-appb-000019
Figure PCTCN2022116795-appb-000020
样本检测首先使用一次性粪便样本采样器(BGI)采集受检者的粪便样本。再使用粪便样本核酸提取试剂盒(BGI)提取粪便样本DNA。下一步使用甲基化检测样本前处理试剂盒(BGI)对DNA进行甲基化修饰预处理，得到转化后的DNA。最后，使用本试剂盒对转化后的DNA进行检测，获得每个样本的内参、OPLAH和ADHFE1基因的Ct值。
结果分析时，内参基因阈值为37，当样本内参基因Ct值大于37时，认为样本中人源DNA含量不足，判定为检测失败。当内参基因Ct值小于等于37时，质检合格。对质检合格的样本，当目标基因Ct值大于38或无检出Ct值时，判定该基因检测阴性。当目标基因Ct值小于等于38时，进一步计算该目标基因的△Ct值(目标基因Ct值与内参基因Ct值的差值)。
阳性截断值的确定使用SPSS进行分析，首先本发明人对单个基因使用SPSS软件绘制了ROC曲线，如图3所示，OPLAH和ADHFE1基因的AUC分别为0.911和0.918。通过整合两个基因的检测结果进行分析，得到了更高的AUC，达到了0.942。进一步地，本发明人使用相同方法对上述样本的PPP2R5C和SDC2基因进行检测，得到了PPP2R5C和SDC2基因的Ct值和△Ct值。所述PPP2R5C和SDC2基因在癌组织和非癌组织中亦存在较显著的甲基化差异。进一步地本发明人使用SPSS软件分析了OPLAH、ADHFE1、PPP2R5C和SDC2四基因组合的检测效果，检测结果如图3所示，可见四基因组合检测与OPLAH和ADHFE1双基因检测的检测效果相近，AUC为0.950。由于双基因检测即可达到四基因组合检测的效果，从成本、检测稳定性等方面考虑，确定使用双基因联合检测的方法。最终确定当OPLAH基因的DMR的△Ct值小于等于7，或ADHFE1基因的DMR的△Ct值小于等于12时，临床样本检测性能最佳，具体性能如表6所示，本试剂盒灵敏度达到86.62％，总体特异性(健康人样本和息肉样本合计的特异性)达到92.78％，进展期腺瘤的灵敏度达到52.63％。本试剂盒对早期肠癌亦有较高灵敏度，如表7所示，I期、II期肠癌灵敏度分别达到了88.64％和89.02％。
表6：临床样本检测性能
Figure PCTCN2022116795-appb-000021
其中总符合率＝(金标准阴性检测正确的样本数+金标准阳性检测正确的样本数)/样本总数。金标准是指肠镜检测。
表7：各分期肠癌样本的检测灵敏度
Figure PCTCN2022116795-appb-000022
按照上述方法，本发明人还使用表1中的其它一些引物、探针组合对上述样本进行了测试，均获得良好的检测效果，具体结果见下面的表8。
表8
Figure PCTCN2022116795-appb-000023
Figure PCTCN2022116795-appb-000024
实施例3：OPLAH和ADHFE1基因甲基化联合粪便潜血检测试剂盒用于肠癌检 测
本发明公开一种通过联合检测OPLAH和ADHFE1基因甲基化水平以及粪便潜血来实现肠癌检测的试剂盒。所述试剂盒包括OPLAH和ADHFE1基因甲基化水平检测模块，以及粪便潜血检测模块。
1.OPLAH和ADHFE1基因甲基化水平检测模块
所述OPLAH和ADHFE1基因甲基化水平检测模块基于荧光定量PCR方法，包含PCR扩增所需要的Buffer、dNTP和酶等试剂，还包括OPLAH和ADHFE1基因引物组和探针。本实施例中所述引物组可从表1中挑选，本例中选择的引物组包括：OPLAH基因引物组(SEQ ID NO:30和SEQ ID NO:32)、OPLAH基因探针(SEQ ID NO:170)、ADHFE1基因引物组(SEQ ID NO:108和SEQ ID NO:109)和ADHFE1基因探针(SEQ ID NO:197)。为了监测样本中是否含有足够的人源DNA，本试剂盒中还加入了用于检测内参基因的引物和探针。所述内参基因一般为管家基因，其对应的引物和探针检测结果仅与人源DNA总量有关，而不受甲基化水平影响。本实施例 中选用GAPDH基因作为内参基因，其引物和探针序列如表4所示。PCR反应在95℃预变性10min，然后进行95℃变性30秒、55℃退火30秒和72℃延伸30秒的循环40个。采用宏石96S qPCR仪进行检测，基线、阈值按照默认设置，在每个循环结束时收集荧光信号。
样本检测首先使用一次性粪便样本采样器(BGI)采集受检者的粪便样本。再使用粪便样本核酸提取试剂盒(BGI)提取粪便样本DNA。下一步使用甲基化检测样本前处理试剂盒(BGI)对DNA进行甲基化修饰预处理，得到转化后的DNA。最后，使用本试剂盒对转化后的DNA进行检测，获得每个样本的内参、OPLAH和ADHFE1基因的Ct值。
结果分析时，内参基因阈值为37，当样本内参基因Ct值大于37时，认为样本中人源DNA含量不足，判定为检测失败。当内参基因Ct值小于等于37时，质检合格。对质检合格的样本，当目标基因Ct值大于38或无检出Ct值时，判定该基因检测阴性。当目标基因Ct值小于等于38时，进一步计算该目标基因的△Ct值(目标基因Ct值与内参基因Ct值的差值)。
2.粪便潜血模块
所述粪便潜血模块包含可定性检测粪便血红蛋白含量的试纸，其检测原理为胶体金法。具体地，使用FIT采样器采集粪便样本，与样本保存液混匀后取约100μl样本于试纸上，在一定时间内读取检测结果。
分析结果时，质控线应出现条带，否则检测失败；若质控线合格，则观察检测线，若检测线出现条带，则粪便潜血试验为阳性，无条带则为阴性。结果读取应在规定时间内完成，超时作废。
3.OPLAH和ADHFE1基因甲基化检测联合粪便潜血检测的试剂盒的实现与性能评估
本实施例使用实施例2所述样本中具有粪便潜血检测结果的318例样本进行分析，样本的临床信息如表9所示。
表9：样本信息表
Figure PCTCN2022116795-appb-000025
Figure PCTCN2022116795-appb-000026
阳性截断值的确定使用SPSS进行分析，首先本发明人对基因检测结果使用SPSS软件绘制了ROC曲线，如图4所示，OPLAH和ADHFE1基因联检的AUC为0.931，OPLAH和ADHFE1基因在肠癌检测中具有较好的检测性能。进一步地，本发明人将粪便潜血检测(FIT)结果纳入逻辑回归分析，按照公式g(y)＝1/(1+e -y)计算得到检测风险指数，其中g(y)是风险指数，当风险指数高于预设值时，判定为阳性或候选为阳性；e为自然常数；y＝(θ0+θ1*X1+θ2*X2+...+θn*Xn)，θ0为常数项，θi(i＝1,2,...,n)是待求系数，Xi(i＝1,2,...,n)是基因和粪便潜血检测的结果。计算得到每个样本的检测风险指数后，绘制ROC曲线确定最佳阳性截断值。如图4所示，基因和FIT联检的AUC达到了0.975，基因与FIT联检能够有效提高检测性能。如表10所示，当阳性截断值取检测风险指数0.55时，达到最佳性能，此时灵敏度和总体特异性分别为95.97％和91.43％，本试剂盒对进展期腺瘤具有较好的检出率，达到了58.62％的灵敏度。本试 剂盒对早期肠癌具有较高灵敏度，如表11所示，I期、II期肠癌灵敏度分别达到了89.47％和100％。如图5所示，与双基因检测或FIT检测对比，本试剂盒对结直肠癌和进展期腺瘤都具有更高的检测灵敏度。
表10：临床样本检测性能
Figure PCTCN2022116795-appb-000027
其中总符合率＝(金标准阴性检测正确的样本数+金标准阳性检测正确的样本数)/样本总数。金标准是指肠镜检测。
表11：各分期肠癌样本的检测灵敏度
样本分期 检测阳性数 样本总数 灵敏度
I期 17 19 89.47％
II期 32 32 100.00％
III期 43 45 95.56％
IV期 12 13 92.31％
未知 39 40 97.50％
总体 143 149 95.97％
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (21)

  1. 差异性甲基化区域，包括：
    (1)chr8:145106171-145107467，和
    (2)chr8:67344198-67345563。
  2. 根据权利要求1所述的差异性甲基化区域，其中：
    第(1)项所示的差异性甲基化区域的碱基序列如SEQ ID NO:1或其互补序列所示，和/或
    第(2)项所示的差异性甲基化区域的碱基序列如SEQ ID NO:2或其互补序列所示。
  3. 根据权利要求1至2中任一权利要求所述的差异性甲基化区域，其用于检测结直肠癌或结直肠癌癌前病变，或者用于结直肠癌患者的预后风险评估。
  4. 根据权利要求1至3中任一权利要求所述的差异性甲基化区域，其经过了如下处理：
    亚硫酸氢盐处理、甲基化敏感性内切酶处理、甲基化限制性内切酶处理或甲基化修饰酶处理。
  5. 一种试剂盒，包括检测权利要求1至4中任一权利要求所述的差异性甲基化区域的试剂或者所述差异性甲基化区域内的甲基化位点的试剂。
  6. 根据权利要求5所述的试剂盒，其中，所述试剂包括：
    1)检测第(1)项所示的差异性甲基化区域或者所述差异性甲基化区域内的甲基化位点的引物和探针；
    2)检测第(2)项所示的差异性甲基化区域或者所述差异性甲基化区域内的甲基化位点的引物和探针；
    优选地，所述试剂还包括：
    3)内参基因的引物和探针。
  7. 根据权利要求6所述的试剂盒，其中：
    1)中引物的序列选自SEQ ID NOs:3-79，探针的序列选自SEQ ID NOs:160-188；和2)中引物的序列选自SEQ ID NOs:80-159，探针的序列选自SEQ ID NOs:189-209；
    优选地：
    1)中引物的序列选自SEQ ID NO:30和SEQ ID NO:32，探针的序列选自SEQ ID NO:170；和2)中引物的序列选自SEQ ID NOs:108-109，探针的序列选自SEQ ID NO:197；
    1)中引物的序列选自SEQ ID NOs:25-26，探针的序列选自SEQ ID NO:164；和2)中引物的序列选自SEQ ID NOs:144-145，探针的序列选自SEQ ID NO:204；
    1)中引物的序列选自SEQ ID NOs:26-27，探针的序列选自SEQ ID NO:168；和2)中引物的序列选自SEQ ID NOs:154-155，探针的序列选自SEQ ID NO:206；
    1)中引物的序列选自SEQ ID NOs:33-34，探针的序列选自SEQ ID NO:171；和2)中引物的序列选自SEQ ID NOs:156-157，探针的序列选自SEQ ID NO:209；
    1)中引物的序列选自SEQ ID NOs:56-57，探针的序列选自SEQ ID NO:184；和2)中引物的序列选自SEQ ID NOs:158-159，探针的序列选自SEQ ID NO:203；
    1)中引物的序列选自SEQ ID NOs:61-62，探针的序列选自SEQ ID NO:183；和2)中引物的序列选自SEQ ID NOs:108-109，探针的序列选自SEQ ID NO:191；
    1)中引物的序列选自SEQ ID NOs:33-34，探针的序列选自SEQ ID NO:171；和2)中引物的序列选自SEQ ID NOs:108-109，探针的序列选自SEQ ID NO:191；
    或者，
    1)中引物的序列选自SEQ ID NOs:25-26，探针的序列选自SEQ ID NO:164；和2)中引物的序列选自SEQ ID NOs:158-159，探针的序列选自SEQ ID NO:203；
    优选地，3)中内参基因的引物的序列如SEQ ID NOs:210-211所示，内参基因的探针的序列如SEQ ID NO:212所示。
  8. 根据权利要求5至7中任一权利要求所述的试剂盒，其还包含选自如下的一项 或者多项：
    粪便样本采样器、核酸提取试剂、甲基化检测样本前处理试剂、PCR试剂和粪便潜血检测试剂；
    优选地，所述粪便潜血检测试剂为粪便免疫化学检测法检测试剂、愈创木脂检测法检测试剂、四甲基联苯胺法检测试剂或ELISA双抗夹心法检测试剂；
    优选地，所述粪便免疫化学检测法检测试剂为免疫胶体金法粪便潜血检测试剂。
  9. 根据权利要求8所述的试剂盒，其中，所述甲基化检测样本前处理试剂为亚硫酸氢盐、甲基化敏感性内切酶、甲基化限制性内切酶或甲基化修饰酶。
  10. 权利要求1至4中任一权利要求所述的差异性甲基化区域或者检测权利要求1至4中任一权利要求所述的差异性甲基化区域或者所述差异性甲基化区域内的甲基化位点的试剂在制备用于检测结直肠癌或结直肠癌癌前病变，或者用于结直肠癌患者的预后风险评估的药物中的用途。
  11. 根据权利要求10所述的用途，其中，所述试剂为用于检测权利要求1至4中任一权利要求所述的差异性甲基化区域或者所述差异性甲基化区域内的甲基化位点的引物和探针。
  12. 一种检测结直肠癌或结直肠癌癌前病变或者一种评估结直肠癌患者的预后风险的方法，包括如下步骤：
    检测权利要求1至4中任一权利要求所述的差异性甲基化区域的含量或者其中的片段的含量；其中，所述片段包含所述差异性甲基化区域内的一个或多个甲基化位点。
  13. 根据权利要求12所述的方法，其中，通过荧光定量PCR方法检测权利要求1至4中任一权利要求所述的各差异性甲基化区域或者其中的片段的含量。
  14. 根据权利要求13所述的方法，其中，
    采用内参基因Ct值评估样本中人源DNA的含量，当内参基因Ct值>37时，判 定样本人源DNA含量过低，本次检测失败，当内参基因Ct值≤37时，评估样本合格，可进行后续分析。
  15. 根据权利要求14所述的方法，其中，对于评估合格的样本：
    当各差异性甲基化区域的Ct值大于38或无检出Ct值时，判定为检测阴性；
    当各差异性甲基化区域的Ct值小于等于38时，进一步计算各差异性甲基化区域与内参基因Ct值的差值△Ct值。
  16. 根据权利要求13至15中任一权利要求所述的方法，其中：
    如果第(1)项或第(2)项的△Ct≤截断值，判定为结直肠癌高风险，否则判定为低风险；
    其中△Ct＝(所述差异性甲基化区域的Ct值或者扩增片段的Ct值)-内参基因Ct值；
    其中，所述扩增片段包含所述差异性甲基化区域内的一个或多个甲基化位点。
  17. 根据权利要求12至16中任一权利要求所述的方法，其中还包括联合粪便潜血检测结果进行评估的步骤。
  18. 根据权利要求12至17中任一权利要求所述的方法，其中，所述样本为人体组织样本、血液样本、细胞样本、分泌物样本或排泄物样本例如粪便样本。
  19. 一种结直肠癌的诊断或评估模型，采用逻辑函数g(y)＝1/(1+e -y)进行计算，其中：
    g(y)是风险指数，当风险指数高于预设值时，判定为阳性或候选为阳性；
    e为自然常数；
    y＝(θ0+θ1*X1+θ2*X2+...+θn*Xn)，θ0为常数项，θi(i＝1,2,...,n)是待求系数，Xi(i＝1,2,...,n)是权利要求1至4中任一权利要求所述的差异性甲基化区域的检测结果和粪便潜血的检测结果。
  20. 一种诊断或评估结直肠癌的系统，包括：
    1)DMR检测单元：获取权利要求1至4中任一权利要求所述的差异性甲基化区域或者其中的片段的含量数据；其中，所述片段包含所述差异性甲基化区域内的一个或多个甲基化位点；
    2)粪便潜血检测单元：获取粪便潜血检测结果数据；
    3)数据分析单元：将DMR检测单元和粪便潜血检测单元获得的数据输入权利要求19所述的结直肠癌的诊断或评估模型进行分析；
    4)风险评估单元：输出个体样本的风险指数或者判断结果。
  21. 权利要求1至4中任一权利要求所述的差异性甲基化区域在制备检测结直肠癌或者结直肠癌癌前病变的产品中的用途。
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