WO2023226939A1 - 用于检测结直肠癌淋巴结转移的甲基化生物标记物及其应用 - Google Patents
用于检测结直肠癌淋巴结转移的甲基化生物标记物及其应用 Download PDFInfo
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Classifications
-
- 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
Definitions
- the present invention relates to the fields of biotechnology and medical diagnosis, and in particular to methylation biomarkers for detecting lymph node metastasis of colorectal cancer and their applications.
- CRC Colorectal cancer
- the mortality rate of colorectal cancer is increasing every year around the world. According to the latest statistics, the incidence rate of CRC is gradually increasing globally, and the mortality rate remains high. CRC shows a younger trend. Its morbidity and mortality have climbed to the third and second places. Among them, CRC lymph node metastasis is the main cause of postoperative recurrence and increased mortality. In China, CRC is also one of the most common malignant tumors, which brings a heavy blow to national health and economic burden.
- CRC World Health Organization's International Agency for Research on Cancer
- CRC computed tomography
- MRI magnetic resonance imaging
- positron emission computed tomography positron emission tomography
- PET-CT emission tomography-computed tomography
- Serological indicators and imaging diagnosis are widely used in clinical practice. Serological indicators mainly include gastrointestinal tract-associated tumor antigen (CA199) and serum carcinoembryonic antigen (CEA). However, their testing performance is limited, and they cannot yet CEA and CA199 are used to identify lymph node metastasis.
- CA199 gastrointestinal tract-associated tumor antigen
- CEA serum carcinoembryonic antigen
- PET-CT which has been widely recognized for tumor diagnosis, especially for small lesions with high sensitivity and specificity, has very limited diagnostic performance on CRC lymph node metastasis.
- PET-CT has a sensitivity of 66%, a specificity of 60%, and an accuracy of 63% for detecting proximal lymph nodes in CRC. Its specificity and accuracy are 29% higher than CT's specificity and 59% accuracy year-on-year. %, but still less than 70% or even higher.
- the purpose of the present invention is to use DNA methylation biomarkers to detect lymph node metastasis and non-metastasis of colorectal cancer.
- the methylation biomarkers provided by the present invention can achieve the purpose of accurate detection/diagnosis. of.
- a first aspect of the present invention provides a methylation biomarker for diagnosis of lymph node metastasis of colorectal cancer, wherein the methylation biomarker includes the methylation sites provided in Appendix Table 1 any one or any combination thereof.
- the methylation biomarker includes any one of the methylation sites provided in Appendix Table 2 or any combination thereof.
- the methylation site includes at least one of chr2:74726037, chr2:74726227 and chr2:74726230.
- the methylation site also includes at least one of chr2:74726181 and chr2:74726179.
- the methylation site also includes at least one of chr20:62272362, chr2:74725882, chr20:62272293, chr2:74726094, and chr20:62272371.
- the methylation sites also include chr2:74726006, chr2:74726124, chr2:74726073, chr20:62272341, chr2:74726131, chr2:74726257, chr20:62272381, chr2:74726274, chr2 0:62272382 ⁇ At least one of chr2:74726083.
- the methylation sites also include chr2:74726332, chr2:74726276, chr2:74726271, chr20:62272199, chr2:74725967, chr2:74726137, chr20:62272313, chr2:74726265, chr2 0:62272387 ⁇ chr2:74725994 ⁇ chr20:62272480 ⁇ chr20:62272478 ⁇ chr2:74726106 ⁇ chr20:62272434 ⁇ chr2:74726216 ⁇ chr2:74726058 ⁇ chr2:74726190 ⁇ chr2:74725992 ⁇ chr 2:74726312, chr20:62272513, chr20:62272388, chr20: At least one of 62272453, chr20:62272448, chr20:62272447, chr2:747255,
- the methylation sites also include chr20:62272487, chr20:62272547, chr13:20693357, chr20:62272310, chr2:74726320, chr19:48983569 ⁇ chr20:62272446 ⁇ chr2:74725984 ⁇ chr20:62272404 ⁇ chr13:20693417 ⁇ chr2:74725926 ⁇ chr20:62272449 ⁇ chr20:62272470 ⁇ chr13:2069357 8.
- the methylation sites include chr13:20693341, chr13:20693342, chr13:20693356, chr13:20693357, chr13:20693359, chr13:20693360, chr13:20693384, chr13:206 93385, chr13:20693414 ⁇ chr13:20693415 ⁇ chr13:20693416 ⁇ chr13:20693417 ⁇ chr13:20693447 ⁇ chr13:20693448 ⁇ chr13:20693459 ⁇ chr13:20693460 ⁇ chr13:20693 468, chr13:20693469, chr13:20693491, chr13:20693492, chr13: 20693564, chr13:20693565, chr13:20693569, chr13:20693570, chr13:20693571, chr13:20693572,
- the methylation biomarkers include sites from the genome region 20693341-20693653 of chromosome 13, sites from the genome region 74725882-74726332 of chromosome 2, and sites from the genome region 62272199-62272548 of chromosome 20. locus and/or locus in the genomic region 48983509-48983698 of chromosome 19.
- the methylation sites include sites from the genomic region 74725882-74726332 of chromosome 2.
- the methylation sites include chr2:74725882, chr2:74725887, chr2:74725895, chr2:74725926, chr2:74725934, chr2:74725967, chr2:74725984, chr2:74725992, chr2:74725994 ⁇ chr2:74726006 ⁇ chr2:74726037 ⁇ chr2:74726058 ⁇ chr2:74726073 ⁇ chr2:74726083 ⁇ chr2:74726094 ⁇ chr2:74726106 ⁇ chr2:74726124 ⁇ chr2:7 4726131, chr2:74726137, chr2:74726179, chr2: 74726181, chr2:74726190 ⁇ chr2:74726216 ⁇ chr2:74726227 ⁇ chr2:74726230 ⁇ chr2:
- the methylation site includes at least one of chr2:74726037, chr2:74726227 and chr2:74726230.
- the methylation site also includes at least one of chr2:74726181 and chr2:74726179.
- the methylation site also includes at least one of chr2:74725882 and chr2:74726124.
- the methylation site also includes at least one of chr2:74726094, chr2:74726073, and chr2:74726006.
- the methylation sites also include chr2:74726332, chr2:74726274, chr2:74726137, chr2:74726257, chr2:74726131, chr2:74726190, chr2:74726271, chr2:74726083, chr2:7 4725967, At least one of chr2:74726106.
- the methylation sites include chr2:74725882, chr2:74725887, chr2:74725895, chr2:74725926, chr2:74725934, chr2:74725967, chr2:74725984, chr2:74725992, chr2:74725994 ⁇ chr2:74726006 ⁇ chr2:74726037 ⁇ chr2:74726058 ⁇ chr2:74726073 ⁇ chr2:74726083 ⁇ chr2:74726094 ⁇ chr2:74726106 ⁇ chr2:74726124 ⁇ chr2:7 4726131, chr2:74726137, chr2:74726179, chr2: 74726181, chr2:74726190, chr2:74726216, chr2:74726227, chr2:74726230,
- the methylation sites include sites from the genomic region 62272199-62272548 of chromosome 20.
- the methylation sites include chr20:62272199, chr20:62272293, chr20:62272309, chr20:62272310, chr20:62272312, chr20:62272313, chr20:62272340, chr20:622 72341, chr20:62272361 ⁇ chr20:62272362 ⁇ chr20:62272371 ⁇ chr20:62272372 ⁇ chr20:62272381 ⁇ chr20:62272382 ⁇ chr20:62272387 ⁇ chr20:62272388 ⁇ chr20:62272403 ⁇ chr20:62272 404, chr20:62272433, chr20:62272434, chr20:62272446, chr20: 62272447 ⁇ chr20:62272448 ⁇ chr20:62272449 ⁇ chr20:62272453 ⁇ chr20
- the methylation site includes at least one of chr20:62272362, chr20:62272293 and chr20:62272371.
- the methylation site also includes at least one of chr20:62272387 and chr20:62272448.
- the methylation site also includes at least one of chr20:62272382 and chr20:62272341.
- the methylation site also includes at least one of chr20:62272199, chr20:62272381, chr20:62272313;
- the methylation sites also include chr20:62272446, chr20:62272478, chr20:62272434, chr20:62272480, chr20:62272513, chr20:62272453, chr20:62272403, chr20:622725 47.
- chr20:62272388 At least one of chr20:62272491.
- the methylation sites include chr20:62272199, chr20:62272293, chr20:62272309, chr20:62272310, chr20:62272312, chr20:62272313, chr20:62272340, chr20:622 72341, chr20:62272361 ⁇ chr20:62272362 ⁇ chr20:62272371 ⁇ chr20:62272372 ⁇ chr20:62272381 ⁇ chr20:62272382 ⁇ chr20:62272387 ⁇ chr20:62272388 ⁇ chr20:62272 403, chr20:62272404, chr20:62272433, chr20:62272434, chr20: 62272446 ⁇ chr20:62272447 ⁇ chr20:62272448 ⁇ chr20:62272449 ⁇ chr20:62272453 ⁇ chr20:62272453 ⁇
- the methylation sites include sites from the genomic region 48983509-48983698 of chromosome 19.
- the methylation sites include chr19:48983509, chr19:48983526, chr19:48983564, chr19:48983569, chr19:48983571, chr19:48983584, chr19:48983596, chr19:489 83601, chr19:48983606 ⁇ chr19:48983608 ⁇ chr19:48983629 ⁇ chr19:48983638 ⁇ chr19:48983653 ⁇ chr19:48983663 ⁇ chr19:48983666 ⁇ chr19:48983671 ⁇ chr19:48983 679 and chr19:48983698 or any combination thereof.
- the methylation site includes at least one of chr19:48983569, chr19:48983526 and chr19:48983663.
- the methylation site also includes at least one of chr19:48983571 and chr19:48983638.
- the methylation site also includes at least one of chr19:48983596 and chr19:48983629.
- the methylation site also includes at least one of chr19:48983666, chr19:48983606, and chr19:48983509.
- the methylation sites include chr19:48983509, chr19:48983526, chr19:48983564, chr19:48983569, chr19:48983571, chr19:48983584, chr19:48983596, chr19:489 83601, chr19:48983606 ⁇ chr19:48983608 ⁇ chr19:48983629 ⁇ chr19:48983638 ⁇ chr19:48983653 ⁇ chr19:48983663 ⁇ chr19:48983666 ⁇ chr19:48983671 ⁇ chr19:48983 679 and chr19:48983698.
- the methylation site includes genes from chromosome 13 The location of the genome region 20693341-20693653.
- the methylation sites include chr13:20693341, chr13:20693342, chr13:20693356, chr13:20693357, chr13:20693359, chr13:20693360, chr13:20693384, chr13:206 93385, chr13:20693414 ⁇ chr13:20693415 ⁇ chr13:20693416 ⁇ chr13:20693417 ⁇ chr13:20693447 ⁇ chr13:20693448 ⁇ chr13:20693459 ⁇ chr13:20693460 ⁇ chr13:20693 468, chr13:20693469, chr13:20693491, chr13:20693492, chr13: 20693564, chr13:20693565, chr13:20693569, chr13:20693570, chr13:20693571, chr13:20693572, chr13:20693
- the methylation biomarker includes at least one of chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037 and chr2:74726230.
- the methylation biomarkers also include at least one of chr22:40504031, chr3:197832359, chr2:74726227, chr3:197828361, chr7:77687875;
- the methylation biomarkers also include chr2:208666424, chr2:74726181, chr2:74726179, chr1:173375804, chr3:197222254, chr19:4745045, chr6:41251759, chr18:7084620, ch r1:166690923 , at least one of chr19:51017312;
- the methylation biomarkers also include chr10:71338666, chr6:161655343, chr2:174424852, chr16:67427066, chr6:42931754, chr1:68295979, chr15:28381565, chr11:4482067 5.chr9:140157146 , chr15:101097210, chr4:4039187, chr14:44168216, chr15:41264369, chr13:19174394, chr21:15497019, chr15:70574296, chr2:74726094, chr2:747260 06.
- the methylation biomarkers also include chr7:129752313, chr2:74726332, chr1:16553114, chr2:74726124, chr19:33863663, chr2:74726257, chr8:7004715, chr2:74726137, chr2 2:45363303 , chr2:74726276, chr2:74725882, chr10:71338651, chr2:74726083, chr1:161450901, chr2:74726274, chr2:74725967, chr7:77687820, chr7:77687882, ch r12:80462103, chr19:43778881, chr6:71980418, chr8 :136263479 ⁇ chr17:3548648 ⁇ chr11:128590262 ⁇ chr4
- chr2:74726058, chr19:38983253, chr12:1932190, chr3 At least one of :197222253, chr6:144881470, chr1:161450899, chr2:74726216;
- the methylation biomarkers also include chr20:24899315, chr6:144881495, chr4:190963293, chr9:118501681, chr2:28700693, chr10:108412225, chr19:49891736, chr20:6227 2293, chr5: 77165020, chr4:909786, chr16:2076342, chr6:144881587, chr2:32205788, chr13:19174421, chr1:54121906, chr3:197830661, chr17:62690049, chr3:19 7217049, chr6:144881549, chr2:36716536, chr2:74726293, chr5:476865 ⁇ chr17:77047294 ⁇ chr8:106967372 ⁇ chr2:74726190 ⁇ chr
- chr20:62272478 chr5:61065238, chr20:62272382, chr15: 26543473, chr21:44197355, chr20:62272480, chr19:8211090, chr3:97982831, chr8:11062433, chr8:10825314, chr1:147827054, chr20:42427883, chr3: 3225323 ⁇ chr21:15497020 ⁇ chr2:96990550 ⁇ chr8:95627967 ⁇ chr3:38618469, chr21:46238680, chr8:89712289, chr20:62272313, chr10:3870830, chr19:1545977, chr4:31261294, chr8:2181379, chr1:9327169, chr10: 71338662, chr16:886194
- the methylation biomarkers include chr1:147827054, chr1:161450899, chr1:161450901, chr1:16553114, chr1:166690923, chr1:173375804, chr1:219944199, chr1:5412 1906 , chr1:68295979, chr1:9327169, chr10:102648751, chr10:106093878, chr10:108412225, chr10:131724514, chr10:3870825, chr10:3870830, chr10:3870 833, chr10:3870842, chr10:3870873, chr10:3870880, chr10 :62452853 ⁇ chr10:64613369 ⁇ chr10:71338651 ⁇ chr10:71338662 ⁇ chr10:713386
- the diagnosis is to distinguish the presence or absence of lymph node metastasis in a subject with colorectal cancer.
- the colorectal cancer is selected from the group consisting of T1, T2, T3, or T4 colorectal cancer of T stage in the TNM staging.
- a second aspect of the present invention provides the use of a methylation biomarker as described in the first aspect of the present invention in the preparation of a reagent or kit for diagnosing the presence of lymph node metastasis in a subject suffering from colorectal cancer. .
- a third aspect of the present invention provides a kit for diagnosing colorectal cancer lymph node metastasis, wherein the kit includes a method for detecting methylation in a sample to be tested as described in the first aspect of the present invention. Reagents for biomarkers of methylation levels.
- the reagent is a reagent used in a method for detecting methylation levels selected from: fluorescence quantitative PCR, methylation-specific PCR, digital PCR, DNA methylation One or more of chemical chip, targeted DNA methylation sequencing, whole-genome methylation sequencing and DNA methylation mass spectrometry.
- the sample to be tested is selected from one or more of tissue, blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.
- a fourth aspect of the present invention provides a system for diagnosing colorectal cancer lymph node metastasis, wherein the system includes a detection device, a computing device and an output device;
- the detection device includes an injector and a detector, the injector is used to collect samples from subjects suffering from colorectal cancer, and the detector is used to detect in the sample as described in the first aspect of the present invention.
- the computing device includes a memory and a processor, a computer program is stored in the memory, and the processor is configured to execute the computer program stored in the memory to achieve the following determination:
- the methylation level of the methylation biomarker in the sample is different from the methylation level of the methylation biomarker measured in a subject with colorectal cancer in the absence of lymph node metastasis, then It is determined whether the subject corresponding to the sample has colorectal cancer lymph node metastasis.
- the methylation biomarker provided by the present invention can be used to diagnose whether there is lymph node metastasis in subjects with colorectal cancer, and has good sensitivity, specificity and accuracy.
- Figure 1 is a heat map of 341 markers in 19 cases of colorectal cancer with lymph node metastasis and 11 cases of colorectal cancer with non-lymph node metastasis.
- Figure 2 is a heat map of 125 markers.
- the use of “substantially” or “substantially” means that the standard deviation from the theoretical model or theoretical data is within the range of 5%, preferably 3%, and more preferably 1%.
- references to “some specific/preferred embodiments”, “other specific/preferred embodiments”, “implementations”, etc. refer to the specific elements described related to the embodiment (for example, Features, structures, properties and/or characteristics) are included in at least one embodiment described herein and may or may not be present in other embodiments. Additionally, it is to be understood that the described elements may be combined in various embodiments in any suitable manner.
- the "plurality” mentioned in the present invention means two or more.
- “And/or” describes the relationship between related objects, indicating that there can be three relationships.
- a and/or B can mean: A exists alone, A and B exist simultaneously, and B exists alone.
- the character “/” generally indicates that the related objects are in an "or” relationship.
- colon refers to the colon, rectum and/or appendix, ie the entire large intestine.
- cancer also called carcinoma
- carcinoma generally refers to any type of malignant neoplasm, that is, any target cell that displays or has a tendency to develop cancer characteristics when compared to unaffected (healthy) wild-type control cells.
- Morphological and/or physiological changes (based on genetic re-programming). Examples of such changes may involve cell size and shape (becoming larger or smaller), Cell proliferation (increase in cell number), cell differentiation (change in physiological state), apoptosis (programmed cell death), or cell survival. Therefore, the term “colorectal cancer” refers to cancerous growths in the colon, rectum, and appendix.
- colorectal cancer cell The most common type of colorectal cancer cell is adenocarcinoma, accounting for approximately 95% of cases.
- Other types of CRC include lymphoma and squamous cell carcinoma, among others.
- TNM Tumor Node Metastasis
- T Tumor Node Metastasis
- T4 Tumor Node Metastasis
- T1 to T4 where the T4 stage also includes the T4a stage and the T4b stage, where the T4a stage is when the tumor penetrates the visceral peritoneum (including intestinal perforation through the tumor and continuous invasion through the inflammatory area on the visceral peritoneal surface);
- Stage T4b refers to the tumor directly invading or adhering to adjacent organs and structures;
- N Node refers to the involvement of regional lymph nodes.
- N0 When the lymph nodes are not involved, it is expressed as N0. As the degree and scope of lymph node involvement increases, they are represented by N1 to N3 in sequence; M (Metastasis) refers to distant metastasis (usually blood vessel metastasis). Those without distant metastasis are represented by M0, and those with distant metastasis are represented by M1. On this basis, a combination of the three TNM indicators is used to draw a specific period.
- M Metalastasis
- sample refers to any substance, including biological samples, that may contain target molecules that require analysis.
- sample or biological sample refers to any sample obtained from a live or viral (or prion) source or other source of macromolecules and biomolecules, and includes from which nucleic acids, proteins, and /or other macromolecules in any cell type or tissue of the subject.
- a sample or biological sample may be a sample obtained directly from a biological source or a processed sample.
- Samples or biological samples include, but are not limited to, body fluids (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, mucus, amniotic fluid, etc.), exudate, bone marrow Samples, ascites, pelvic flushes, pleural fluid, spinal fluid, lymph fluid, eye fluid, extracts from nasal, throat or genital swabs, cell suspensions from digestive tissue, or extracts from fecal matter, and extracts from human, Tissue and organ samples of animals (e.g., non-human mammals) and plants, and processed samples derived therefrom.
- body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, mucus, amniotic fluid, etc.
- exudate bone marrow Samples, ascites, pelvic flushes, pleural fluid,
- the term "subject” may be a mammal or a cell of the mammal, Tissue, organ or part.
- mammal refers to any kind of mammal, preferably human (including humans, human subjects or human patients).
- Subjects and mammals include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats, and rodents such as mice and rats.
- diagnosis includes the detection or identification of a disease state or condition in a subject, the determination of the likelihood that a subject will develop a given disease or condition, or the determination of the likelihood that a subject suffering from a disease or condition will respond to treatment. properties, determining the prognosis of a subject suffering from a disease or disorder (or its likely progression or regression), and determining the effect of a treatment in a subject suffering from a disease or disorder.
- diagnosis also means distinguishing the presence or absence of lymph node metastasis in a subject with colorectal cancer.
- complementarity refers to a nucleotide (eg, 1 nucleotide) or polynucleotide (eg, a sequence of nucleotides) associated with a base pairing rule.
- sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5'.
- Complementarity can be "partial,” where only some nucleic acid bases match according to base pairing rules. Alternatively, there may be “complete” or “total” complementarity between nucleic acids. The degree of complementarity between nucleic acid strands affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.
- polymerase chain reaction is used to amplify a target sequence and the method consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by the presence of a DNA polymerase Perform the exact thermal cycling sequence below. Both primers are complementary to the corresponding strands of the double-stranded target sequence. To perform amplification, the mixture is denatured and the primers anneal to their complementary sequences within the target molecule. After annealing, the primers are amplified using a polymerase to form a new pair of complementary strands.
- the steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (i.e., denaturation, annealing, and extension constitute one "cycle”; there can be many "cycles") to obtain a high concentration of amplified fragments of the desired target sequence.
- the length of the amplified fragment of the desired target sequence is determined by the relative position of the primers relative to each other and is therefore a controllable parameter. Due to the repetitive aspect of the method, the method is known as "polymerase chain reaction"("PCR”). Since the desired amplified fragment of the target sequence becomes the dominant sequence (in concentration) in the mixture, it is said to be “PCR amplified” and is a "PCR product" or "amplicon”.
- amplifiable nucleic acid refers to a nucleic acid that can be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid” will generally comprise a “sample template”.
- sample template refers to the nucleic acid derived from the sample used to analyze the presence of the "target".
- background template is used to refer to nucleic acids other than the sample template, which may or may not be present in the sample. Background templates are often unintentional. This may be a carryover result, or it may be due to the presence of nucleic acid contaminants that were attempted to be purified away from the sample. For example, nucleic acids from an organism other than the nucleic acid to be detected may be present as background in the test sample.
- primer refers to an oligonucleotide that occurs naturally or synthetically in a purified restriction digest when exposed to conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (e.g., in It can serve as a starting point for synthesis in the presence of nucleotides and an inducer such as DNA polymerase and at the appropriate temperature and pH).
- Primers are preferably single-stranded for maximum efficiency of amplification, but may be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products.
- the primers are oligodeoxyribonucleotides. The primer must be long enough to initiate the synthesis of the extension product in the presence of the inducer. The exact length of the primer will depend on many factors, including temperature, source of primer, and method used.
- probe refers to an oligonucleotide (e.g., a nucleotide sequence) that occurs naturally in a purified restriction digest or is synthesized, recombinant, or produced by PCR amplification, which is capable of interacting with another A target oligonucleotide hybridizes. Probes can be single-stranded or double-stranded. Probes can be used for the detection, identification and isolation of specific genetic sequences (eg, "capture probes"). It is contemplated that in some embodiments, any probe used in the invention may be labeled with any "reporter” such that it is detectable in any detection system.
- amplification generally refers to the process of producing multiple copies of a desired sequence.
- Multiple copies means at least two copies.
- Copy does not necessarily mean perfect sequence complementarity or identity to the template sequence.
- copies may include nucleotide analogs such as deoxyinosine, intentional sequence changes (such as those introduced by primers containing sequences that are hybridizable but not complementary to the template), and/or those that occur during amplification. Sequence error.
- sequence determination and the like include determination of the nucleotide base sequence of a nucleic acid.
- information may include the identification or determination of part or all of the sequence information of the nucleic acid.
- Sequence information can be determined with varying degrees of statistical reliability or confidence.
- the term includes determining the identity and sequence of multiple consecutive nucleotides in a nucleic acid.
- sequencing high-throughput sequencing
- next generation sequencing include sequence determination using methods that determine many (usually thousands to billions) of sequences in an essentially parallel manner.
- nucleic acid sequences i.e. in this method the DNA templates are prepared not for sequencing one at a time but rather in a batch process and in this method many sequences are preferably read in parallel or the use itself can be parallelized Ultra-high-throughput serial process readout.
- Such methods include, but are not limited to, pyrosequencing (e.g., as commercialized by 454 Life Sciences, Inc., Branford, CT); sequencing by ligation (e.g., as commercialized by SOLiDTM technology, commercialized by Life Technologies, Inc., Carlsbad, Calif.
- sequencing by synthesis using modified nucleotides e.g., TruSeqTM and HiSeqTM technologies commercialized by Illumina, Inc., San Diego, CA, HeliScopeTM commercialized by Helicos Biosciences Corporation, Cambridge, MA; and PacBio RS commercialized by Pacific Biosciences of California, Inc., Menlo Park, CA
- sequencing by ion detection technology e.g., Ion TorrentTM technology, Life Technologies, Carlsbad, CA
- DNA nanosphere sequencing Complete Genomics, Inc., Mountain View, CA
- highly parallel sequencing methods such as nanopore-based sequencing technology (e.g., developed by Oxford Nanopore Technologies, LTD, Oxford, UK).
- DNA methylation or “methylation” refers to the conversion of methyl methyl in an organism using S-adenosylmethionine (SAM) as the methyl donor under the catalysis of DNA methyltransferase.
- SAM S-adenosylmethionine
- DNA methylation can also occur in cytosine in nucleotide sequences such as CHG and CHH, where H is adenine, cytosine, or thymine. DNA methylation can also occur at non-cytosine, such as N6-methyladenine. In addition, DNA methylation can also be in the form of 5-hydroxymethylcytosine. In most cases, DNA methylation is induced by methylation modifications on the opposite DNA strand or other CpG sites near DNA bases.
- DNA methylation site and “methylation site” refer to single or multiple base positions where DNA methylation modification may occur. For example, CpG sites, CHG sites or CHH sites. In some cases, DNA methylation sites are equivalent to CpG sites.
- methylated nucleotide or “methylated nucleotide base” refers to the presence of a methyl moiety on the nucleotide base, where the methyl moiety does not exist in recognized typical nucleotides. in the base.
- cytosine does not contain a methyl moiety on its pyrimidine ring
- 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide and 5-methylcytosine is.
- thymine contains a methyl moiety at position 5 of its pyrimidine ring.
- methylation level refers to the presence or absence of one or more methyl groups in the nucleic acid molecule.
- nucleotide bases For example, a nucleic acid molecule that contains methylated cytosine is considered methylated (eg, the methylation status of the nucleic acid molecule is methylated). Nucleic acid molecules that do not contain any methylated nucleotides are considered unmethylated.
- methylation status may optionally be represented or indicated by a "methylation value” (eg, indicating methylation frequency, fraction, ratio, percentage, etc.).
- Methylation values can be quantified, e.g., after restriction digestion with methylation-dependent restriction enzymes, or by comparing amplification profiles after bisulfite reaction, or by comparing bisulfite treatment and Unprocessed nucleic acid sequences are generated. Therefore, values such as methylation values represent methylation status and can therefore be used as quantitative indicators of methylation status in multiple copies of a locus.
- methylation frequency or “methylation percent (%)” refers to the number of instances of a molecule or locus that is methylated relative to the number of instances of the molecule or locus that is unmethylated.
- methylation status describes the state of methylation of a nucleic acid (eg, a genomic sequence).
- methylation status refers to the methylation-related properties of a nucleic acid fragment at a specific genomic locus. Such characteristics include, but are not limited to: whether any cytosine (C) residue within this DNA sequence is methylated , the position of methylated C residues, the frequency or percentage of methylated C throughout any particular region of the nucleic acid, and allelic differences in methylation due to, for example, differences in the origin of the allele.
- C cytosine
- methylation status also refer to the relative, absolute concentration or pattern of methylated C or unmethylated C throughout any particular region of nucleic acids in a biological sample .
- a cytosine (C) residue within a nucleic acid sequence is methylated, it may be said to be “hypermethylated” or have “increased methylation,” whereas if a cytosine (C) residue within a DNA sequence is If the base is not methylated, it may be said to be “hypomethylated” or have “reduced methylation”.
- cytosine (C) residue within a nucleic acid sequence is methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.), the sequence is considered to be different from another nucleic acid sequence. Compared to hypermethylation or having increased methylation.
- a cytosine (C) residue within a DNA sequence is unmethylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.)
- the sequence is considered to be identical to another nucleic acid sequence.
- the sequence is hypomethylated or has reduced methylation compared to the sequence.
- methylation pattern refers to the collective sites of methylated and unmethylated nucleotides on a certain region of a nucleic acid. Two nucleotides can have the same or similar methylation frequency or methylation percentage, but when the number of methylated and unmethylated nucleotides is the same or similar throughout the region but methylated and unmethylated Methylated nucleotides have different methylation patterns at different positions.
- sequences differ in the degree, frequency, or pattern of methylation (e.g., one has increased or decreased methylation relative to another), the sequences are said to be “differentially methylated” or said to have “ differentially methylated” or having “different methylation status.”
- the term “differential methylation” refers to the level or pattern of nucleic acid methylation in a cancer-positive (or cancer metastasis-positive, e.g., colorectal cancer lymph node metastasis) sample compared with that in a cancer-negative sample (or cancer metastasis-negative, e.g., colorectal cancer) Differences in nucleic acid methylation levels or patterns compared with those without lymph node metastasis.
- DMR Differentially Methylated Region
- cancer status or cancer metastasis status, such as colorectal cancer lymph node metastasis
- DMRs that include a greater number or frequency of methylation sites may be referred to as hypermethylated DMRs.
- DMRs that include a smaller number or frequency of methylated sites under selected conditions of interest, such as cancer states (or cancer metastasis states, such as colorectal cancer lymph node metastasis) may be termed hypomethylated DMRs .
- DMR as a colorectal cancer methylation biomarker can be called colorectal cancer DMR.
- DMR as a methylation biomarker of colorectal cancer lymph node metastasis can be called colorectal cancer lymph node metastasis DMR.
- a DMR can be a single nucleotide that is a methylation site.
- bisulfite reagent refers to a reagent that in some embodiments includes bisulfite (bisulfite), disulfite (disulfite), hydrogen sulfite (hydrogen sulfite), or a combination thereof,
- bisulfite bisulfite
- disulfite disulfite
- hydrogen sulfite hydrogen sulfite
- a combination thereof After DNA is treated with bisulfite reagent, its unmethylated cytosine nucleotides will be converted into uracil, while the methylated cytosine and other bases will remain unchanged, so it can distinguish, for example, CpG dinuclear Methylated and unmethylated cytidines in nucleotide sequences.
- Some aspects of the present invention provide a methylation biomarker for diagnosis of lymph node metastasis of colorectal cancer, wherein the methylation biomarker includes the methylation sites provided in Appendix Table 1 below. Any one of the points or any combination thereof.
- the methylation biomarkers include sites from the genome region 20693341-20693653 of chromosome 13, sites from the genome region 74725882-74726332 of chromosome 2, and genome region 62272199 of chromosome 20.
- the methylation biomarker includes any one of the methylation sites provided in Appendix Table 2 below or any combination thereof.
- the methylation site includes at least any one of chr2:74726037, chr2:74726227, and chr2:74726230.
- the methylation site also includes at least one of chr2:74726181 and chr2:74726179.
- the methylation sites include at least chr2:74726230, chr2:74726227, chr2:74726037, chr2:74726181 and chr2:74726179.
- the methylation site also includes at least one of chr20:62272362, chr2:74725882, chr20:62272293, chr2:74726094, and chr20:62272371.
- the methylation sites also include chr2:74726006, chr2:74726124, chr2:74726073, chr20:62272341, chr2:74726131, chr2:74726257, chr20:62272381 , chr2:74726274, chr20:62272382, at least one of chr2:74726083.
- the methylation sites also include chr2:74726332, chr2:74726276, chr2:74726271, chr20:62272199, chr2:74725967, chr2:74726137, chr20:62272313 , chr2:74726265, chr20:62272387, chr2:74725994, chr20:62272480, chr20:62272478, chr2:74726106, chr20:62272434, chr2:74726216, chr2:74726058 , chr2:74726190, chr2:74725992, chr2:74726312, chr20 ch At least one of r2:74725934 and chr20:62272361;
- the methylation sites also include chr20:62272487, chr20:62272547, chr13:20693357, chr20:62272310, chr2:74726320, chr19:48983569, chr20: 62272446, chr2:74725984, chr20:62272404, chr13:20693417, chr2:74725926, chr20:62272449, chr20:62272470, chr13:20693578, chr2:74725887, chr1 3:20693448, chr13:20693607, chr13:20693414, chr20:62272340, chr13:20693653 ⁇ chr13:20693415 ⁇ chr13:20693492 ⁇ chr20:62272372 ⁇ chr19:48983571 ⁇ ch
- the methylation sites include the methylation sites provided in Appendix Table 2 below.
- the methylation site includes any group selected from the following site combinations or any combination thereof:
- chr2:74726230 chr2:74726227, chr2:74726037, chr2:74726181, chr2:74726179, chr20:62272362, chr2:74725882, chr20:62272293, chr2:74726 094, chr20:62272371, chr2:74726006, chr2:74726124 , chr2:74726073, chr20:62272341, chr2:74726131, chr2:74726257, chr20:62272381, chr2:74726274, chr20:62272382, chr2:74726083, chr2:74726332, chr2:74726276 ⁇ chr2:74726271 ⁇ chr20:62272199 ⁇ chr2 :74725967 ⁇ chr
- chr20:62272494 chr20:62272403, chr2:74725934, chr20 :62272361 ⁇ chr20:62272487 ⁇ chr20:62272547 ⁇ chr13:20693357 ⁇ chr20:62272310 ⁇ chr2:74726320 ⁇ chr19:48983569 ⁇ chr20:62272446 ⁇ chr2:74725984 ⁇ chr 20:62272404, chr13:20693417, chr2:74725926, chr20:62272449 , chr20:62272470, chr13:20693578, chr2:74725887, chr13:20693448, chr13:20693607, chr13:20693414, chr20:62272340, chr13:20693653, chr13:2069 3415, chr13:20693492, chr20:62
- 125marker group includes all methylation sites in Appendix Table 2.
- the methylation sites include sites from chromosome 2 genomic region 74725882-74726332. Specifically, the methylation sites include chr2:74725882, chr2:74725887, chr2:74725895, chr2:74725926, chr2:74725934, chr2:74725967, chr2:74725984, chr2:74725992, chr2:747 25994, chr2: 74726006, chr2:74726037, chr2:74726058, chr2:74726073, chr2:74726083, chr2:74726094, chr2:74726106, chr2:74726124, chr2:74726131, chr2:7472 6137 ⁇ chr2:74726179 ⁇ chr2:74726181 ⁇ chr2:74726
- the methylation site includes at least one of chr2:74726037, chr2:74726227 and chr2:74726230. In some specific embodiments, the methylation sites include at least chr2:74726037, chr2:74726227 and chr2:74726230.
- the methylation site also includes at least one of chr2:74726181 and chr2:74726179.
- the methylation site also includes at least one of chr2:74725882 and chr2:74726124.
- the methylation site also includes at least one of chr2:74726094, chr2:74726073, and chr2:74726006.
- the methylation sites also include chr2:74726332, chr2:74726274, chr2:74726137, chr2:74726257, chr2:74726131, chr2:74726190, chr2:74726271 , chr2:74726083, chr2:74725967, at least one of chr2:74726106.
- the methylation sites include chr2:74725882, chr2:74725887, chr2:74725895, chr2:74725926, chr2:74725934, chr2:74725967 ⁇ chr2:74725984 ⁇ chr2:74725992 ⁇ chr2:74725994 ⁇ chr2:74726006 ⁇ chr2:74726037 ⁇ chr2:74726058 ⁇ chr2:74726073 ⁇ chr2:74726083 ⁇ chr2:7 4726094, chr2:74726106, chr2:74726124, chr2: 74726131, chr2:74726137, chr2:74726179, chr2:74726181, chr2:74726190, chr2:74726216, chr2:74726227, chr2:74726262;
- the methylation site includes any group selected from the following site combinations or any combination thereof:
- 3marker group chr2:74726037, chr2:74726227, chr2:74726230;
- 34marker group chr2:74725882, chr2:74725887, chr2:74725895, chr2:74725926, chr2:74725934, chr2:74725967, chr2:74725984, chr2:74725992, chr2:7472599 4.
- the methylation sites include sites from the genomic region 62272199-62272548 of chromosome 20; specifically, the methylation sites include chr20:62272199, chr20:62272293 , chr20:62272309, chr20:62272310, chr20:62272312, chr20:62272313, chr20:62272340, chr20:62272341, chr20:62272361, chr20:62272362, chr20:622 72371, chr20:62272372, chr20:62272381, chr20:62272382, chr20 :62272387 ⁇ chr20:62272388 ⁇ chr20:62272403 ⁇ chr20:62272404 ⁇ chr20:62272433 ⁇ chr20:62272434 ⁇ chr20:62272446 ⁇ chr20:6227
- the methylation site includes at least one of chr20:62272362, chr20:62272293 and chr20:62272371. In some specific embodiments, the methylation sites include at least chr20:62272362, chr20:62272293 and chr20:62272371.
- the methylation site also includes at least one of chr20:62272387 and chr20:62272448.
- the methylation site also includes at least one of chr20:62272382 and chr20:62272341.
- the methylation site also includes at least one of chr20:62272199, chr20:62272381, and chr20:62272313.
- the methylation sites also include chr20:62272446, chr20:62272478, chr20:62272434, chr20:62272480, chr20:62272513, chr20:62272453, chr20:62272403 , chr20:62272547, chr20:62272388, at least one of chr20:62272491.
- the methylation sites include chr20:62272199, chr20:62272293, chr20:62272309, chr20:62272310, chr20:62272312, chr20:62272313, chr20:62272340 , chr20:62272341, chr20:62272361, chr20:62272362, chr20:62272371, chr20:62272372, chr20:62272381, chr20:62272382, chr20:62272387, chr20:622 72388, chr20:62272403, chr20:62272404, chr20:62272433, chr20 :62272434 ⁇ chr20:62272446 ⁇ chr20:62272447 ⁇ chr20:62272448 ⁇ chr20:62272449 ⁇ chr20
- the methylation site includes any group selected from the following site combinations or any combination thereof:
- 3marker group chr20:62272362, chr20:62272293, chr20:62272371;
- 5marker group chr20:62272362, chr20:62272293, chr20:62272371, chr20:62272387, chr20:62272448;
- the methylation sites include sites from the genomic region 48983509-48983698 of chromosome 19; specifically, the methylation sites include chr19:48983509, chr19:48983526, chr19: 48983564, chr19:48983569, chr19:48983571, chr19:48983584, chr19:48983596, chr19:48983601, chr19:48983606, chr19:48983608, chr19:48983629, chr19:48983638 ⁇ chr19:48983653 ⁇ chr19:48983663 ⁇ chr19:48983666 ⁇ Any one of chr19:48983671, chr19:48983679 and chr19:48983698 or any combination thereof.
- the methylation site includes at least one of chr19:48983569, chr19:48983526 and chr19:48983663. In some specific embodiments, the methylation sites include at least chr19:48983569, chr19:48983526 and chr19:48983663.
- the methylation site also includes at least one of chr19:48983571 and chr19:48983638.
- the methylation site also includes at least one of chr19:48983596 and chr19:48983629.
- the methylation site also includes at least one of chr19:48983666, chr19:48983606, and chr19:48983509.
- the methylation sites include chr19:48983509, chr19:48983526, chr19:48983564, chr19:48983569, chr19:48983571, chr19:48983584, chr19:48983596, chr19:48983601, chr19:48983606, chr19:48983608, chr19:48983629, chr19:48983638, chr19:48983653, chr19:48983663, chr19:48983666, chr19:48983 671, chr19:48983679 and chr19:48983698.
- the methylation site includes any group selected from the following site combinations or any combination thereof:
- 3marker group chr19:48983569, chr19:48983526, chr19:48983663;
- 18marker group chr19:48983509, chr19:48983526, chr19:48983564, chr19:48983569, chr19:48983571, chr19:48983584, chr19:48983596, chr19:48983601, chr1 9:48983606, chr19:48983608, chr19:48983629, chr19:48983638 , chr19:48983653, chr19:48983663, chr19:48983666, chr19:48983671, chr19:48983679, chr19:4898369.
- the methylation sites are from the genomic region 20693341-20693653 of chromosome 13; specifically, the methylation sites include chr13:20693341, chr13:20693342, chr13:20693356, chr13 :20693357 ⁇ chr13:20693359 ⁇ chr13:20693360 ⁇ chr13:20693384 ⁇ chr13:20693385 ⁇ chr13:20693414 ⁇ chr13:20693415 ⁇ chr13:20693416 ⁇ chr13:20693417 ⁇ chr13:20693447, chr13:20693448, chr13:20693459, chr13:20693460 ,chr13:20693468, chr13:20693469 ⁇ chr13:20693491 ⁇ chr13:20693492 ⁇ chr13:20693564 ⁇ chr13:20693565 ⁇ chr13:20693569 ⁇ chr13
- the methylation biomarker includes at least one of chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, and chr2:74726230.
- the methylation biomarkers may include at least chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037 and chr2:74726230.
- the methylation biomarkers also include at least one of chr22:40504031, chr3:197832359, chr2:74726227, chr3:197828361, and chr7:77687875.
- the methylation biomarkers also include chr2:208666424, chr2:74726181, chr2:74726179, chr1:173375804, chr3:197222254, chr19:4745045, chr6: At least one of 41251759, chr18:7084620, chr1:166690923, chr19:51017312.
- the methylation biomarkers also include chr10:71338666, chr6:161655343, chr2:174424852, chr16:67427066, chr6:42931754, chr1:68295979, chr15: 28381565 ⁇ chr11:44820675 ⁇ chr9:140157146 ⁇ chr15:101097210 ⁇ chr4:4039187 ⁇ chr14:44168216 ⁇ chr15:41264369 ⁇ chr13:19174394 ⁇ chr21:15497019 ⁇ ch r15:70574296 ⁇ chr2:74726094 ⁇ chr2:74726006 ⁇ chr13:97991180 ⁇ chr15:101098460, chr19:13944958, chr4:55379617, chr2:74726131, chr9:37949852, chr8:10825518, At least one of
- the methylation biomarkers also include chr7:129752313, chr2:74726332, chr1:16553114, chr2:74726124, chr19:33863663, chr2:74726257, chr8: 7004715, chr2:74726137, chr22:45363303, chr2:74726276, chr2:74725882, chr10:71338651, chr2:74726083, chr1:161450901, chr2:74726274, chr2:74 725967 ⁇ chr7:77687820 ⁇ chr7:77687882 ⁇ chr12:80462103 ⁇ chr19:43778881, chr6:71980418, chr8:136263479, chr17:3548648, chr11:128590262, chr4
- the methylation biomarkers also include chr20:24899315, chr6:144881495, chr4:190963293, chr9:118501681, chr2:28700693, chr10:108412225, chr19 :49891736 ⁇ chr20:62272293 ⁇ chr5:77165020 ⁇ chr4:909786 ⁇ chr16:2076342 ⁇ chr6:144881587 ⁇ chr2:32205788 ⁇ chr13:19174421 ⁇ chr1:54121906 ⁇ chr3:197 830661, chr17:62690049, chr3:197217049, chr6:144881549 , chr2:36716536, chr2:74726293, chr5:476865, chr17:77047294, chr8:106967372, chr2:74726190
- the methylation biomarkers include all methylation sites in Appendix Table 1.
- the methylation biomarkers may include any one of the following site combinations or any combination thereof:
- 5marker group chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230;
- 50marker group chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230, chr22:40504031, chr3:197832359, chr2:74726227, chr3:19 7828361, chr7:77687875, chr2:208666424, chr2:74726181 , chr2:74726179, chr1:173375804, chr3:197222254, chr19:4745045, chr6:41251759, chr18:7084620, chr1:166690923, chr19:51017312, chr10:7133866 6.
- 100marker group chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230, chr22:40504031, chr3:197832359, chr2:74726227, chr3:1 97828361, chr7:77687875, chr2:208666424, chr2:74726181 , chr2:74726179, chr1:173375804, chr3:197222254, chr19:4745045, chr6:41251759, chr18:7084620, chr1:166690923, chr19:51017312, chr10:7133866 6.
- 200marker group chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230, chr22:40504031, chr3:197832359, chr2:74726227, chr3:1 97828361, chr7:77687875, chr2:208666424, chr2:74726181 , chr2:74726179, chr1:173375804, chr3:197222254, chr19:4745045, chr6:41251759, chr18:7084620, chr1:166690923, chr19:51017312, chr10:7133866 6.
- 341marker group includes all methylation sites in Appendix Table 1.
- the diagnosis is to distinguish the presence or absence of lymph node metastasis in a subject with colorectal cancer.
- the subject is a mammal; preferably, the mammal is a human; further preferably, the subject is a colorectal cancer patient.
- the colorectal cancer is selected from the group consisting of T1, T2, T3 or T4 colorectal cancer of the T stage in the TNM staging.
- the use of the above-mentioned methylation biomarker in preparing a reagent or kit for diagnosing the presence of lymph node metastasis in a subject suffering from colorectal cancer is provided.
- a reagent for detecting the methylation level of the above-mentioned methylation biomarker is provided in the preparation of a reagent or kit for diagnosing the presence of lymph node metastasis in a subject suffering from colorectal cancer. use.
- kits for diagnosing colorectal cancer lymph node metastasis includes a method for detecting the methylation level of the above-mentioned methylation biomarker in the sample to be tested. reagents.
- the reagents are reagents used in the method of detecting methylation levels selected from the following: fluorescence quantitative PCR (qPCR), methylation-specific PCR (MSP), One or more of digital PCR (ddPCR), DNA methylation chip, targeted DNA methylation sequencing, whole genome methylation sequencing (WGBS), DNA methylation mass spectrometry (MassArray) .
- qPCR fluorescence quantitative PCR
- MSP methylation-specific PCR
- ddPCR digital PCR
- DNA methylation chip DNA methylation chip
- targeted DNA methylation sequencing targeted DNA methylation sequencing
- WGBS whole genome methylation sequencing
- MassArray DNA methylation mass spectrometry
- the sample to be tested is selected from one or more of tissue, blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant. kind.
- the sample to be tested is tissue, such as colorectal cancer tissue.
- Some aspects of the present invention provide a system for colorectal cancer lymph node metastasis diagnosis, wherein the system includes a detection device, a computing device and an output device;
- the detection device includes an injector and a detector, the injector is used to collect samples from subjects suffering from colorectal cancer, and the detector is used to detect the above-mentioned methylation biomarkers in the samples. methylation level;
- the computing device includes a memory and a processor, a computer program is stored in the memory, and the processor is configured to execute the computer program stored in the memory to achieve the following determination:
- the methylation level of the methylation biomarker as described above in the sample is different from the methylation level of the methylation biomarker determined in a subject with colorectal cancer in the absence of lymph node metastasis, Then it is determined that the subject corresponding to the sample has colorectal cancer lymph node metastasis.
- the output device is used to output the detection results of the detection device and/or the discrimination results of the computing device, and the output device includes at least one of a display, a printer, and an audio output device.
- the computing device includes at least one of a computer host, a central processing unit, and a network server.
- a method for diagnosing colorectal cancer lymph node metastasis includes the following steps.
- the samples include 19 fresh frozen tissue samples of colorectal cancer tissue with lymph node metastasis (CRC LN+) and 11 fresh frozen tissue samples of colorectal cancer tissue without lymph node metastasis (CRC LN-).
- CRC LN+ 19 fresh frozen tissue samples of colorectal cancer tissue with lymph node metastasis
- CRC LN- 11 fresh frozen tissue samples of colorectal cancer tissue without lymph node metastasis
- the extracted tissue DNA (50ng) was subjected to bisulfite conversion, so that the unmethylated cytosine in the DNA was deaminated and converted into uracil, while the methylated cytosine remained unchanged, and bisulfite conversion was obtained.
- the specific operations of the final DNA transformation were carried out according to the instructions of Zymo Research's EZ DNA Methylation-Lightning Kit.
- the hybridization capture kit is xGen Lockdown Reagents from IDT. Please follow the instructions.
- Table 1 Annotation information and AUC of 341 marker genes
- STMN3 gene (chr20:62272199-62272548) has 38 CpG sites.
- the average methylation levels of these 125 markers in 19 subjects with lymph node metastasis and 11 subjects with lymph node non-metastasis are as follows: Table 2 shows that, as mentioned above, the average methylation level is the average of the ⁇ values at each methylation site in 19 metastatic and 11 non-metastatic subjects respectively, using DSS software.
- the p-value obtained by differential analysis shows that these regions and the CpG sites within the region have good ability to distinguish metastasis from non-metastasis.
- Figure 2 shows the heat map of the above 125 markers.
- Table 2 below shows the average methylation levels of these 125 markers in 19 lymph node metastasis and 11 lymph node non-metastasis subjects, showing the difference between metastasis and The diffs of these markers between non-transfers were all above ⁇ 0.1, and the p-values were all less than 0.05.
- the 3 marker group includes: chr2:74726037, chr2:74726227, chr2:74726230;
- the 5marker group includes: chr2:74726037, chr2:74726227, chr2:74726230, chr2:74726181, chr2:74726179;
- the 7marker group includes: chr2:74726037, chr2:74726227, chr2:74726230, chr2:74726181, chr2:74726179, chr2:74725882, chr2:74726124;
- the 10marker group includes: chr2:74726037, chr2:74726227, chr2:74726230, chr2:74726181, chr2:74726179, chr2:74725882, chr2:74726124, chr2:74726094, chr2:747260 73.chr2:74726006;
- the 20marker group includes: chr2:74726037, chr2:74726227, chr2:74726230, chr2:74726181, chr2:74726179, chr2:74725882, chr2:74726124, chr2:74726094, chr2:747260 73.
- the 34marker group includes: chr2:74725882, chr2:74725887, chr2:74725895, chr2:74725926, chr2:74725934, chr2:74725967, chr2:74725984, chr2:74725992, chr2:747259 94.
- 3marker group includes: chr20:62272362, chr20:62272293, chr20:62272371;
- the 5marker group includes: chr20:62272362, chr20:62272293, chr20:62272371, chr20:62272387, chr20:62272448;
- the 7marker group includes: chr20:62272362, chr20:62272293, chr20:62272371, chr20:62272387, chr20:62272448, chr20:62272382, chr20:62272341;
- the 10marker group includes: chr20:62272362, chr20:62272293, chr20:62272371, chr20:62272387, chr20:62272448, chr20:62272382, chr20:62272341, chr20:62272199, chr 20:62272381, chr20:62272313;
- the 20marker group includes: chr20:62272362, chr20:62272293, chr20:62272371, chr20:62272387, chr20:62272448, chr20:62272382, chr20:62272341, chr20:62272199, chr 20:62272381, chr20:62272313, chr20:62272446, chr20: 62272478 ⁇ chr20:62272434 ⁇ chr20:62272480 ⁇ chr20:62272513 ⁇ chr20:62272453 ⁇ chr20:62272403 ⁇ chr20:62272547 ⁇ chr20:62272388 ⁇ chr20:62272491;
- the 38marker group includes: chr20:62272199, chr20:62272293, chr20:62272309, chr20:62272310, chr20:62272312, chr20:62272313, chr20:62272340, chr20:62272341, chr 20:62272361, chr20:62272362, chr20:62272371, chr20: 62272372 ⁇ chr20:62272381 ⁇ chr20:62272382 ⁇ chr20:62272387 ⁇ chr20:62272388 ⁇ chr20:62272403 ⁇ chr20:62272404 ⁇ chr20:62272433 ⁇ chr20:62272434 ⁇ chr20:62272446, chr20:62272447, chr20:62272448, chr20:62272449, chr20:62272453 ⁇ chr20:
- the 3marker group includes: chr19:48983569, chr19:48983526, chr19:48983663;
- the 5marker group includes: chr19:48983569, chr19:48983526, chr19:48983663, chr19:48983571, chr19:48983638;
- the 7marker group includes: chr19:48983569, chr19:48983526, chr19:48983663, chr19:48983571, chr19:48983638, chr19:48983596, chr19:48983629;
- the 10marker group includes: chr19:48983569, chr19:48983526, chr19:48983663, chr19:48983571, chr19:48983638, chr19:48983596, chr19:48983629, chr19:48983666, chr 19:48983606, chr19:48983509;
- the 18marker group includes: chr19:48983509, chr19:48983526, chr19:48983564, chr19:48983569, chr19:48983571, chr19:48983584, chr19:48983596, chr19:48983601, chr 19:48983606 ⁇ chr19:48983608 ⁇ chr19:48983629 ⁇ chr19: 48983638, chr19:48983653, chr19:48983663, chr19:48983666, chr19:48983671, chr19:48983679, chr19:48983698.
- the 5 marker combinations include: chr2:74726230, chr2:74726227, chr2:74726037, chr2:74726181, chr2:74726179;
- the 10 marker combinations include: chr2:74726230, chr2:74726227, chr2:74726037, chr2:74726181, chr2:74726179, chr20:62272362, chr2:74725882, chr20:62272293, chr2:7472 6094,chr20:62272371;
- the 20marker combinations include: chr2:74726230, chr2:74726227, chr2:74726037, chr2:74726181, chr2:74726179, chr20:62272362, chr2:74725882, chr20:62272293, chr2:7472 6094, chr20:62272371, chr2:74726006, chr2: 74726124, chr2:74726073, chr20:62272341, chr2:74726131, chr2:74726257, chr20:62272381, chr2:74726274, chr20:62272382, chr2:74726083;
- the 50marker combinations include: chr2:74726230, chr2:74726227, chr2:74726037, chr2:74726181, chr2:74726179, chr20:62272362, chr2:74725882, chr20:62272293, chr2:7472 6094, chr20:62272371, chr2:74726006, chr2: 74726124, chr2:74726073, chr20:62272341, chr2:74726131, chr2:74726257, chr20:62272381, chr2:74726274, chr20:62272382, chr2:74726083, chr2:7 4726332 ⁇ chr2:74726276 ⁇ chr2:74726271 ⁇ chr20:62272199 ⁇ chr2:74725967 ⁇
- the 100marker combinations include: chr2:74726230, chr2:74726227, chr2:74726037, chr2:74726181, chr2:74726179, chr20:62272362, chr2:74725882, chr20:62272293, chr2:747 26094, chr20:62272371, chr2:74726006, chr2: 74726124, chr2:74726073, chr20:62272341, chr2:74726131, chr2:74726257, chr20:62272381, chr2:74726274, chr20:62272382, chr2:74726083, chr2:7 4726332 ⁇ chr2:74726276 ⁇ chr2:74726271 ⁇ chr20:62272199 ⁇ chr2:74725967
- the 125marker combination includes: all markers in Appendix 2.
- the 5marker group includes: chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230;
- the 10marker group includes: chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230, chr22:40504031, chr3:197832359, chr2:74726227, chr3:1 97828361,chr7:77687875;
- the 20marker group includes: chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230, chr22:40504031, chr3:197832359, chr2:74726227, chr3:1 97828361, chr7:77687875, chr2:208666424, chr2: 74726181, chr2:74726179, chr1:173375804, chr3:197222254, chr19:4745045, chr6:41251759, chr18:7084620, chr1:166690923, chr19:51017312;
- the 50marker group includes: chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230, chr22:40504031, chr3:197832359, chr2:74726227, chr3:1 97828361, chr7:77687875, chr2:208666424, chr2: 74726181, chr2:74726179, chr1:173375804, chr3:197222254, chr19:4745045, chr6:41251759, chr18:7084620, chr1:166690923, chr19:51017312, chr10 :71338666 ⁇ chr6:161655343 ⁇ chr2:174424852 ⁇ chr16:67427066 ⁇ chr6:42931754 ⁇ chr
- the 100marker group includes: chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230, chr22:40504031, chr3:197832359, chr2:74726227, chr3: 197828361, chr7:77687875, chr2:208666424, chr2: 74726181, chr2:74726179, chr1:173375804, chr3:197222254, chr19:4745045, chr6:41251759, chr18:7084620, chr1:166690923, chr19:51017312, chr10 :71338666 ⁇ chr6:161655343 ⁇ chr2:174424852 ⁇ chr16:67427066 ⁇ chr6:42931754 ⁇ chr1:
- the 200marker group includes: chr20:62272433, chr22:50706553, chr19:35408269, chr2:74726037, chr2:74726230, chr22:40504031, chr3:197832359, chr2:74726227, chr3: 197828361, chr7:77687875, chr2:208666424, chr2: 74726181, chr2:74726179, chr1:173375804, chr3:197222254, chr19:4745045, chr6:41251759, chr18:7084620, chr1:166690923, chr19:51017312, chr10 :71338666 ⁇ chr6:161655343 ⁇ chr2:174424852 ⁇ chr16:67427066 ⁇ chr6:42931754 ⁇ chr1:
- the 341marker group includes all methylation sites in Appendix Table 1.
- Chromosome 1 (chr1) genomic locus Chromosome 1 (chr1) genomic locus:
- Chromosome 10 (chr10) genomic locus Chromosome 10 (chr10) genomic locus:
- Chromosome 11 (chr11) genomic locus Chromosome 11 (chr11) genomic locus:
- Chromosome 12 (chr12) genomic locus Chromosome 12 (chr12) genomic locus:
- Chromosome 13 (chr13) genomic locus Chromosome 13 (chr13) genomic locus:
- Chromosome 14 (chr14) genomic locus Chromosome 14 (chr14) genomic locus:
- Chromosome 15 (chr15) genomic locus Chromosome 15 (chr15) genomic locus:
- Chromosome 16 (chr16) genomic locus Chromosome 16 (chr16) genomic locus:
- Chromosome 17 (chr17) genomic locus Chromosome 17 (chr17) genomic locus:
- Chromosome 18 (chr18) genomic locus Chromosome 18 (chr18) genomic locus:
- Chromosome 19 (chr19) genomic locus Chromosome 19 (chr19) genomic locus:
- Chromosome 2 (chr2) genomic locus Chromosome 2 (chr2) genomic locus:
- Chromosome 20 (chr20) genomic locus Chromosome 20 (chr20) genomic locus:
- Chromosome 21 (chr21) genomic locus Chromosome 21 (chr21) genomic locus:
- Chromosome 22 (chr22) genomic locus Chromosome 22 (chr22) genomic locus:
- Chromosome 3 (chr3) genomic locus Chromosome 3 (chr3) genomic locus:
- Chromosome 4 (chr4) genomic locus
- Chromosome 5 (chr5) genomic locus Chromosome 5 (chr5) genomic locus:
- Chromosome 6 (chr6) genomic locus Chromosome 6 (chr6) genomic locus:
- Chromosome 7 (chr7) genomic locus Chromosome 7 (chr7) genomic locus:
- Chromosome 8 (chr8) genomic locus
- Chromosome 9 (chr9) genomic locus
- Chromosome 13 (chr13) genomic locus Chromosome 13 (chr13) genomic locus:
- Chromosome 19 (chr19) genomic locus Chromosome 19 (chr19) genomic locus:
- Chromosome 2 (chr2) genomic locus Chromosome 2 (chr2) genomic locus:
- Chromosome 20 (chr20) genomic locus Chromosome 20 (chr20) genomic locus:
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Abstract
本发明公开了用于检测结直肠癌淋巴结转移的甲基化生物标记物及其应用。其中，本发明提供了用于结直肠癌淋巴结转移诊断的甲基化生物标记物，所述的甲基化生物标记物包括附表1中提供的甲基化位点中的任一项或其任意组合。进一步地，所述的甲基化生物标记物包括附表2中提供的甲基化位点中的任一项或其任意组合。本发明提供的甲基化生物标记物可以用于诊断患有结直肠癌的受试者是否存在淋巴结转移，具有良好的敏感性、特异性及准确度。
Description
优先权和相关申请
本发明要求2022年5月25日提交的名称为“用于检测结直肠癌淋巴结转移的甲基化生物标记物及其应用”的中国专利申请202210578099.8的优先权，该申请包括附录在内的全部内容作为参考并入本发明。
本发明涉及生物技术和医学诊断领域，具体涉及用于检测结直肠癌淋巴结转移的甲基化生物标记物及其应用。
结直肠癌(Colorectal cancer,CRC)是世界第三大恶性肿瘤，全世界每年结直肠癌死亡率呈上升趋势，据最新统计，目前全球CRC的发病率逐渐升高，死亡率仍然高居不下，并CRC呈现出年轻化趋势。其发病率和死亡率已攀升到第三位和第二位，其中，CRC淋巴结转移是术后复发和死亡率升高的主要原因。在中国，CRC同样是最常见的恶性肿瘤之一，它给国民健康和经济负担都带来沉重的打击。根据2021年世界卫生组织国际癌症研究机构(International Agency for Research on Cancer,IARC)公布的2020年数据显示，中国一年内新发的CRC患者约56万人，死亡近29万人，均呈现上升的趋势。而在我国，CRC具备有三个明显特征，第一是人口特征分布，目前男性CRC患者约是女性CRC患者1.3倍；同时，确诊CRC的平均年龄大概是59岁，也远低于世界平均年龄的69岁；第二，目前CRC的病理特征显示，确诊时是早期CRC患者比例在逐年减少，而确诊时已经是晚期CRC的比例仍在增加；第三，临床诊断方面显示目前计算机断层扫描成像(computed tomography，CT)用于诊断CRC的比例翻倍式升高；磁共振成像(magnetic resonance imaging，MRI)用于诊断CRC也较前增加，而正电子发射计算机断层扫描成像(positron
emission tomography-computed tomography,PET-CT)也明显升高；第四，目前CRC治疗方面仍然以手术为主，手术联合术后放化疗的治疗方案比例升高。
由于CRC淋巴结转移与否决定了患者的重要分期，也是术后复发和预后的重要影响因素，同时也决定了不同的治疗方案，所以一直以来都受到临床上的广泛关注。因此，有必要在CRC治疗前了解患者有无合并淋巴结转移。目前广泛应用于临床的主要有血清学指标和影像学诊断，其中血清学指标主要包括胃肠道相关肿瘤抗原(CA199)、血清癌胚抗原(CEA)，但其检验效能有限，它们尚不能将CEA、CA199用于鉴别淋巴结转移。虽然目前对于CRC淋巴结转移诊断的金标准仍然是通过术后的淋巴结病理活检，但这明显滞后。因此术前的影像学诊断仍被广泛用于对CRC淋巴结转移的判断。但目前关于影像学在CRC淋巴结转移的诊断准确性仍然偏低。有研究指出MRI、CT对CRC淋巴结诊断结果与CRC组织病理学结果的符合率分别只有为57.6％和54.7％，而MRI和CT的敏感性分别为42.6％和25.0％，特异性分别为74.1％和41.3％，且MR和CT评估5年无病生存率和总生存率的符合率分别为56.7％和43.8％。虽然总体MRI诊断性能优于CT，但诊断效能仍十分有限。而近年来被广泛认可的用于肿瘤诊断，尤其是微小病灶具有高度敏感性和特异性的PET-CT，其在关于CRC淋巴结转移的诊断效能也非常有限。有实验证明，PET-CT检测CRC近端淋巴结的灵敏度为66％，特异性为60％，准确度为63％，其特异性及准确性同比高于CT特异性的29％，准确性的59％，但仍到不到70％以上甚至更高的水平。
综上，亟需开发一种新的DNA甲基化标志物，鉴别早期结直肠癌淋巴结转移，辅助临床准确诊断和指导治疗。
发明内容
发明要解决的问题
本发明的目的在于利用DNA甲基化生物标记物来检测结直肠癌淋巴结转移与非转移，本发明提供的甲基化生物标记物可以实现精准检测/诊断的目
的。
用于解决问题的方案
本发明的第一方面提供了一种用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化生物标记物包括附表1中提供的甲基化位点中的任一项或其任意组合。
在一些实施方案中，所述的甲基化生物标记物包括附表2中提供的甲基化位点中的任一项或其任意组合。
在一些具体实施方案中，所述的甲基化位点至少包括chr2:74726037、chr2:74726227和chr2:74726230中的至少一种。
进一步地，所述的甲基化位点还包括chr2:74726181、chr2:74726179中的至少一种。
更进一步地，所述的甲基化位点还包括chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371中的至少一种。
再进一步地，所述的甲基化位点还包括chr2:74726006、chr2:74726124、chr2:74726073、chr20:62272341、chr2:74726131、chr2:74726257、chr20:62272381、chr2:74726274、chr20:62272382、chr2:74726083中的至少一种。
尤其进一步地，所述的甲基化位点还包括chr2:74726332、chr2:74726276、chr2:74726271、chr20:62272199、chr2:74725967、chr2:74726137、chr20:62272313、chr2:74726265、chr20:62272387、chr2:74725994、chr20:62272480、chr20:62272478、chr2:74726106、chr20:62272434、chr2:74726216、chr2:74726058、chr2:74726190、chr2:74725992、chr2:74726312、chr20:62272513、chr20:62272388、chr20:62272453、chr20:62272448、chr20:62272447、chr2:74726293、chr20:62272491、chr20:62272494、chr20:62272403、chr2:74725934、chr20:62272361中的至少一种。
尤其更进一步地，所述的甲基化位点还包括chr20:62272487、chr20:62272547、chr13:20693357、chr20:62272310、chr2:74726320、
chr19:48983569、chr20:62272446、chr2:74725984、chr20:62272404、chr13:20693417、chr2:74725926、chr20:62272449、chr20:62272470、chr13:20693578、chr2:74725887、chr13:20693448、chr13:20693607、chr13:20693414、chr20:62272340、chr13:20693653、chr13:20693415、chr13:20693492、chr20:62272372、chr19:48983571、chr19:48983526、chr19:48983663、chr19:48983596、chr19:48983638、chr13:20693564、chr2:74725895、chr13:20693469、chr13:20693608、chr20:62272309、chr20:62272548、chr13:20693577、chr13:20693565、chr13:20693416、chr13:20693342、chr19:48983606、chr13:20693356、chr19:48983564、chr13:20693460、chr19:48983671、chr13:20693569、chr13:20693636、chr13:20693341、chr19:48983601、chr13:20693459、chr19:48983584、chr13:20693468中的至少一种。
在一些尤其具体的实施方案中，所述的甲基化位点包括chr13:20693341、chr13:20693342、chr13:20693356、chr13:20693357、chr13:20693359、chr13:20693360、chr13:20693384、chr13:20693385、chr13:20693414、chr13:20693415、chr13:20693416、chr13:20693417、chr13:20693447、chr13:20693448、chr13:20693459、chr13:20693460、chr13:20693468、chr13:20693469、chr13:20693491、chr13:20693492、chr13:20693564、chr13:20693565、chr13:20693569、chr13:20693570、chr13:20693571、chr13:20693572、chr13:20693577、chr13:20693578、chr13:20693607、chr13:20693608、chr13:20693631、chr13:20693632、chr13:20693636、chr13:20693637、chr13:20693653、chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679、chr19:48983698、chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、
chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320、chr2:74726332、chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547和chr20:62272548。
在一些实施方案中，所述的甲基化生物标记物包括来自13号染色体基因组区域20693341-20693653的位点、2号染色体基因组区域74725882-74726332的位点、20号染色体基因组区域62272199-62272548的位点和/或19号染色体基因组区域48983509-48983698的位点。
在一些具体的实施方案中，所述的甲基化位点包括来自2号染色体基因组区域74725882-74726332的位点。
在一些任选的实施方案中，所述的甲基化位点包括chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、
chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320和chr2:74726332中的任一项或其任意组合。
在一些更具体的实施方案中，所述的甲基化位点至少包括chr2:74726037、chr2:74726227和chr2:74726230中的至少一种。
进一步地，所述的甲基化位点还包括chr2:74726181、chr2:74726179中的至少一种。
更进一步地，所述的甲基化位点还包括chr2:74725882、chr2:74726124中的至少一种。
再进一步地，所述的甲基化位点还包括chr2:74726094、chr2:74726073、chr2:74726006中的至少一种。
尤其进一步地，所述的甲基化位点还包括chr2:74726332、chr2:74726274、chr2:74726137、chr2:74726257、chr2:74726131、chr2:74726190、chr2:74726271、chr2:74726083、chr2:74725967、chr2:74726106中的至少一种。
在一些尤其具体的实施方案中，所述的甲基化位点包括chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320和chr2:74726332。
在另一些具体的实施方案中，所述的甲基化位点包括来自20号染色体基因组区域62272199-62272548的位点。
在一些任选的实施方案中，所述的甲基化位点包括chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、
chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547和chr20:62272548中的任一项或其任意组合。
在一些更具体的实施方案中，所述的甲基化位点至少包括chr20:62272362、chr20:62272293和chr20:62272371中的至少一种。
进一步地，所述的甲基化位点还包括chr20:62272387、chr20:62272448中的至少一种。
更进一步地，所述的甲基化位点还包括chr20:62272382、chr20:62272341中的至少一种。
再进一步地，所述的甲基化位点还包括chr20:62272199、chr20:62272381、chr20:62272313中的至少一种；
尤其进一步地，所述的甲基化位点还包括chr20:62272446、chr20:62272478、chr20:62272434、chr20:62272480、chr20:62272513、chr20:62272453、chr20:62272403、chr20:62272547、chr20:62272388、chr20:62272491中的至少一种。
在一些尤其具体的实施方案中，所述的甲基化位点包括chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、
chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547和chr20:62272548。
在另一些具体的实施方案中，所述的甲基化位点包括来自19号染色体基因组区域48983509-48983698的位点。
在一些任选的实施方案中，所述的甲基化位点包括chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679和chr19:48983698中的任一项或其任意组合。
在一些更具体的实施方案中，所述的甲基化位点至少包括chr19:48983569、chr19:48983526和chr19:48983663中的至少一种。
进一步地，所述的甲基化位点还包括chr19:48983571、chr19:48983638中的至少一种。
更进一步地，所述的甲基化位点还包括chr19:48983596、chr19:48983629中的至少一种。
再进一步地，所述的甲基化位点还包括chr19:48983666、chr19:48983606、chr19:48983509中的至少一种。
在一些尤其具体的实施方案中，所述的甲基化位点包括chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679和chr19:48983698。
在另一些具体的实施方案中，所述的甲基化位点包括来自13号染色体基
因组区域20693341-20693653的位点。
在一些任选的实施方案中，所述的甲基化位点包括chr13:20693341、chr13:20693342、chr13:20693356、chr13:20693357、chr13:20693359、chr13:20693360、chr13:20693384、chr13:20693385、chr13:20693414、chr13:20693415、chr13:20693416、chr13:20693417、chr13:20693447、chr13:20693448、chr13:20693459、chr13:20693460、chr13:20693468、chr13:20693469、chr13:20693491、chr13:20693492、chr13:20693564、chr13:20693565、chr13:20693569、chr13:20693570、chr13:20693571、chr13:20693572、chr13:20693577、chr13:20693578、chr13:20693607、chr13:20693608、chr13:20693631、chr13:20693632、chr13:20693636、chr13:20693637和chr13:20693653中的任一项或其任意组合。
在另一些实施方案中，所述的甲基化生物标记物至少包括chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037和chr2:74726230中的至少一种。
进一步地，所述的甲基化生物标记物还包括chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875中的至少一种；
更进一步地，所述的甲基化生物标记物还包括chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312中的至少一种；
再进一步地，所述的甲基化生物标记物还包括chr10:71338666、chr6:161655343、chr2:174424852、chr16:67427066、chr6:42931754、chr1:68295979、chr15:28381565、chr11:44820675、chr9:140157146、chr15:101097210、chr4:4039187、chr14:44168216、chr15:41264369、chr13:19174394、chr21:15497019、chr15:70574296、chr2:74726094、chr2:74726006、chr13:97991180、chr15:101098460、chr19:13944958、
chr4:55379617、chr2:74726131、chr9:37949852、chr8:10825518、chr11:119355687、chr16:5237372、chr2:74726073、chr2:74726271、chr8:10924461中的至少一种；
尤其进一步地，所述的甲基化生物标记物还包括chr7:129752313、chr2:74726332、chr1:16553114、chr2:74726124、chr19:33863663、chr2:74726257、chr8:7004715、chr2:74726137、chr22:45363303、chr2:74726276、chr2:74725882、chr10:71338651、chr2:74726083、chr1:161450901、chr2:74726274、chr2:74725967、chr7:77687820、chr7:77687882、chr12:80462103、chr19:43778881、chr6:71980418、chr8:136263479、chr17:3548648、chr11:128590262、chr4:174462424、chr8:2480920、chr20:62272362、chr15:95662730、chr19:54172983、chr3:197832357、chr2:176624742、chr19:35408270、chr2:74726265、chr10:102648751、chr12:12766094、chr10:106093878、chr13:19174407、chr9:79138639、chr2:242755966、chr16:77147779、chr2:74725994、chr2:74726106、chr3:36174109、chr2:74726058、chr19:38983253、chr12:1932190、chr3:197222253、chr6:144881470、chr1:161450899、chr2:74726216中的至少一种；
尤其更进一步地，所述的甲基化生物标记物还包括chr20:24899315、chr6:144881495、chr4:190963293、chr9:118501681、chr2:28700693、chr10:108412225、chr19:49891736、chr20:62272293、chr5:77165020、chr4:909786、chr16:2076342、chr6:144881587、chr2:32205788、chr13:19174421、chr1:54121906、chr3:197830661、chr17:62690049、chr3:197217049、chr6:144881549、chr2:36716536、chr2:74726293、chr5:476865、chr17:77047294、chr8:106967372、chr2:74726190、chr8:28157735、chr2:174853968、chr19:51483067、chr1:219944199、chr3:97973710、chr16:27431703、chr21:46238778、chr20:62272371、chr15:101096974、chr11:45743460、chr10:64613369、chr2:74725992、chr20:62272341、chr17:77181458、chr18:60020618、chr2:74725934、chr2:191625107、chr4:48945163、
chr6:144881603、chr11:133800611、chr17:79855640、chr2:74726312、chr15:101097067、chr4:7128051、chr5:138861157、chr6:121069941、chr13:19174425、chr20:62272478、chr5:61065238、chr20:62272382、chr15:26543473、chr21:44197355、chr20:62272480、chr19:8211090、chr3:97982831、chr8:11062433、chr8:10825314、chr1:147827054、chr20:42427883、chr3:3225323、chr21:15497020、chr2:96990550、chr8:95627967、chr3:38618469、chr21:46238680、chr8:89712289、chr20:62272313、chr10:3870830、chr19:1545977、chr4:31261294、chr8:2181379、chr1:9327169、chr10:71338662、chr16:88619484、chr5:138861242、chr20:62272434、chr2:74726320、chr5:2749278、chr12:9856328、chr11:133800801、chr4:41299758、chr6:35892438、chr2:242789465、chr15:91305688、chr2:74725984、chr14:74439495、chr6:170338877、chr4:1404633、chr20:62272381、chr10:3870880、chr22:24253568、chr7:18901757、chr5:103114389、chr12:53216896、chr2:54952741中的至少一种；
在一些尤其具体的实施方案中，所述的甲基化生物标记物包括chr1:147827054、chr1:161450899、chr1:161450901、chr1:16553114、chr1:166690923、chr1:173375804、chr1:219944199、chr1:54121906、chr1:68295979、chr1:9327169、chr10:102648751、chr10:106093878、chr10:108412225、chr10:131724514、chr10:3870825、chr10:3870830、chr10:3870833、chr10:3870842、chr10:3870873、chr10:3870880、chr10:62452853、chr10:64613369、chr10:71338651、chr10:71338662、chr10:71338666、chr11:119355687、chr11:128590262、chr11:133800611、chr11:133800801、chr11:44820675、chr11:45743460、chr12:11423121、chr12:12766094、chr12:1932190、chr12:53216896、chr12:80462103、chr12:9856328、chr13:113373213、chr13:19174394、chr13:19174407、chr13:19174421、chr13:19174425、chr13:20693341、chr13:20693342、chr13:20693356、chr13:20693357、chr13:20693359、chr13:20693360、chr13:20693384、
chr13:20693385、chr13:20693414、chr13:20693415、chr13:20693416、chr13:20693417、chr13:20693447、chr13:20693448、chr13:20693459、chr13:20693460、chr13:20693468、chr13:20693469、chr13:20693491、chr13:20693492、chr13:20693564、chr13:20693565、chr13:20693569、chr13:20693570、chr13:20693571、chr13:20693572、chr13:20693577、chr13:20693578、chr13:20693607、chr13:20693608、chr13:20693631、chr13:20693632、chr13:20693636、chr13:20693637、chr13:20693653、chr13:97991180、chr14:44168216、chr14:74439495、chr15:101096974、chr15:101097067、chr15:101097210、chr15:101098460、chr15:26543473、chr15:28381565、chr15:41264369、chr15:70574296、chr15:91305688、chr15:95662730、chr16:2076342、chr16:27431703、chr16:34521898、chr16:5237372、chr16:67427066、chr16:71260701、chr16:77147779、chr16:88619484、chr17:10101011、chr17:3548648、chr17:58637740、chr17:62690049、chr17:77047294、chr17:77181458、chr17:79855640、chr17:8658715、chr18:112353、chr18:54240802、chr18:60020618、chr18:67901956、chr18:67901984、chr18:67902044、chr18:67902075、chr18:7084620、chr19:13944958、chr19:1545977、chr19:33863663、chr19:35408269、chr19:35408270、chr19:38983253、chr19:43778881、chr19:4745045、chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679、chr19:48983698、chr19:49891736、chr19:51017312、chr19:51483067、chr19:54172983、chr19:8211090、chr2:174424852、chr2:174853968、chr2:176624742、chr2:191625107、chr2:208666424、chr2:242755966、chr2:242789465、chr2:28700693、chr2:32205788、chr2:36716536、chr2:54952741、chr2:74725882、chr2:74725887、
chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320、chr2:74726332、chr2:96990550、chr20:24899315、chr20:42427883、chr20:60740447、chr20:60740448、chr20:60745017、chr20:60745018、chr20:60745185、chr20:60745224、chr20:60745246、chr20:60750111、chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547、chr20:62272548、chr21:15497019、chr21:15497020、chr21:30527898、chr21:30527912、chr21:30527927、chr21:30527936、chr21:30527966、chr21:30527984、chr21:44197355、chr21:46238680、chr21:46238778、chr22:24253568、chr22:38123993、chr22:40504031、chr22:45363303、chr22:50706553、chr3:157614707、chr3:197217049、chr3:197222253、chr3:197222254、chr3:197828361、chr3:197830661、chr3:197832357、chr3:197832359、chr3:20317639、chr3:3225323、chr3:36174109、chr3:38618469、chr3:97973710、chr3:97982831、chr4:1404633、chr4:174462424、chr4:190963243、chr4:190963254、
chr4:190963293、chr4:190963359、chr4:31261294、chr4:4039187、chr4:41299758、chr4:48945163、chr4:55379617、chr4:6574370、chr4:7128051、chr4:909786、chr5:103114389、chr5:122429706、chr5:138861107、chr5:138861157、chr5:138861242、chr5:138861258、chr5:170349468、chr5:2749278、chr5:476865、chr5:61065238、chr5:77165020、chr5:93019223、chr6:121069941、chr6:130005127、chr6:144881470、chr6:144881495、chr6:144881549、chr6:144881587、chr6:144881603、chr6:161655343、chr6:170338877、chr6:35892438、chr6:41251759、chr6:42931754、chr6:48950482、chr6:71980418、chr7:121081132、chr7:121081133、chr7:121081142、chr7:121081143、chr7:121081187、chr7:121081188、chr7:129752313、chr7:18901757、chr7:21742092、chr7:77687820、chr7:77687875、chr7:77687882、chr7:90895650、chr8:106967372、chr8:10825314、chr8:10825518、chr8:10924461、chr8:11062433、chr8:136263479、chr8:2181379、chr8:2480920、chr8:28157735、chr8:56013334、chr8:7004715、chr8:89712289、chr8:95627967、chr9:117064345、chr9:118501681、chr9:140157146、chr9:37949852、chr9:69260268和chr9:79138639。
在一些实施方案中，所述诊断是区分患有结直肠癌的受试者是否存在淋巴结转移。
在一些任选的实施方案中，所述结直肠癌选自TNM分期中T分期的T1期、T2期、T3期或T4期的结直肠癌。
本发明的第二方面提供了如本发明第一方面中所述的甲基化生物标记物在制备用于诊断患有结直肠癌的受试者是否存在淋巴结转移的试剂或试剂盒中的用途。
本发明的第三方面提供了一种用于结直肠癌淋巴结转移诊断的试剂盒，其中，所述的试剂盒包含用于检测待测样本中如本发明第一方面中所述的甲基化生物标记物的甲基化水平的试剂。
在一些实施方案中，所述的试剂为选自以下的检测甲基化水平的方法中所使用的试剂：荧光定量PCR、甲基化特异性PCR、数字PCR、DNA甲基
化芯片、靶向DNA甲基化测序、全基因组甲基化测序和DNA甲基化质谱中的一种或多种。
在一些实施方案中，所述的待测样本选自组织、血液、血浆、唾液、血清、尿液、尿液脱落细胞、尿沉渣、尿液上清中的一种或多种。
本发明的第四方面提供了一种用于结直肠癌淋巴结转移诊断的系统，其中，所述系统包括检测装置、计算装置和输出装置；
所述检测装置包括进样器和检测器，所述进样器用于采集来自患有结直肠癌的受试者的样本，所述检测器用于检测所述样本中如本发明第一方面中所述的甲基化生物标记物的甲基化水平；
所述计算装置包括存储器和处理器，所述存储器中存储有计算机程序，所述处理器被配置为执行所述存储器中存储的计算机程序，以实现如下判别：
所述样本中所述的甲基化生物标记物的甲基化水平不同于不存在淋巴结转移的患有结直肠癌的受试者中测定的甲基化生物标记物的甲基化水平，则判别所述样本对应的受试者的存在结直肠癌淋巴结转移。
发明的效果
本发明提供的甲基化生物标记物可以用于诊断患有结直肠癌的受试者是否存在淋巴结转移，具有良好的敏感性、特异性及准确度。
图1为341个标记物在19例结直肠癌淋巴结转移的癌和11例结直肠癌非淋巴结转移的癌的热图。
图2为125个标记物的热图。
以下，针对本发明的内容进行详细说明。以下所记载的技术特征的说明基于本发明的代表性的实施方案、具体例子而进行，但本发明不限定于这些实施方案、具体例子。需要说明的是：
本说明书中，使用“数值A～数值B”表示的数值范围是指包含端点数值A、B的范围。
本说明书中，使用“基本上”或“实质上”表示与理论模型或理论数据的标准偏差在5％、优选为3％、更优选为1％范围以内。
本说明书中，使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。
本说明书中，“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生，并且该描述包括该事件发生的情况和该事件不发生的情况。
本说明书中，所提及的“一些具体/优选的实施方案”、“另一些具体/优选的实施方案”、“实施方案”等是指所描述的与该实施方案有关的特定要素(例如，特征、结构、性质和/或特性)包括在此处所述的至少一种实施方案中，并且可存在于其它实施方案中或者可不存在于其它实施方案中。另外，应理解，所述要素可以任何合适的方式组合在各种实施方案中。
本发明的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤的过程、方法、装置、产品或设备没有限定于已列出的步骤或模块，而是可选地还包括没有列出的步骤，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤。
在本发明中提及的“多个”是指两个或两个以上。“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。字符“/”一般表示前后关联对象是一种“或”的关系。
本说明书中，术语“结直肠”指的是结肠、直肠和/或阑尾，即整个大肠。
本说明书中，术语“癌症”(也称为癌)通常指任何类型的恶性新生物，即与未受影响的(健康)野生型对照细胞相比显示或具有发生癌特征倾向的靶细胞的任何形态学和/或生理学改变(基于遗传重编程(genetic re-programming))。这种改变的例子可涉及细胞大小和形状(变大或变小)、
细胞增殖(细胞数增加)、细胞分化(生理学状态变化)、凋亡(程序性细胞死亡)或细胞存活。因此，术语“结直肠癌”指的是结肠、直肠和阑尾的癌性生长。
最常见的结直肠癌细胞类型是腺癌，大约占95％。其他类型的CRC包括尤其是淋巴瘤和鳞癌。
本说明书中，TNM(Tumor Node Metastasis)是肿瘤学中对肿瘤的一种分期形式，其中T(Tumor)指肿瘤原发灶的情况，随着肿瘤体积的增加和邻近组织受累范围的增加，依次用T1～T4来表示，其中，T4期还包括T4a期和T4b期，其中，T4a期为肿瘤穿透脏层腹膜(包括通过肿瘤的肠穿孔和通过内脏腹膜表面的炎症区域的连续侵入)；T4b期为肿瘤直接侵入或者黏附于邻近器官和结构；N(Node)指区域淋巴结(regional lymph node)受累情况。淋巴结未受累时，用N0表示。随着淋巴结受累程度和范围的增加，依次用N1～N3表示；M(Metastasis)指远处转移(通常是血道转移)，没有远处转移者用M0表示，有远处转移者用M1表示。在此基础上，用TNM三个指标的组合划出特定的分期。
本说明书中，术语“样本”是指可能包含需要进行分析的靶分子的任何物质，包括生物样本。如本文所用，“样本”或“生物样本”是指从活的或病毒性(或朊病毒的)来源或其他大分子和生物分子来源获得的任何样本，并且包括可以从之获得核酸、蛋白质和/或其他大分子的受试者的任何细胞类型或组织。样本或生物样本可以是直接从生物来源获得的样本或者是被处理的样本。样本或生物样本包括，但不限于，体液(例如血液、血浆、血清、脑脊髓液、滑液、尿液、汗液、精液、粪便、痰、眼泪、粘液、羊水等)、渗出液、骨髓样本、腹水、骨盆冲洗液、胸膜液、脊髓液、淋巴液、眼液、鼻、喉或生殖器拭子的提取物、消化组织的细胞悬浮液、或粪类物质的提取物、以及来自人、动物(例如非人哺乳动物)和植物的组织和器官样本，以及由此衍生出的加工样本。
本说明书中，术语“受试者”可以是哺乳动物或所述哺乳动物的细胞、
组织、器官或一部分。在本发明中，哺乳动物是指任何种类的哺乳动物，优选人(包括人、人受试者或人患者)。受试者和哺乳动物包括，但不限于，农场动物、运动动物、宠物、灵长类动物、马、狗、猫和啮齿类动物如小鼠和大鼠。
本说明书中，诊断包括受试者疾病状态或病症的检测或鉴定、确定受试者将患给定疾病或病症的可能性、确定患有疾病或病症的受试者将对治疗有反应的可能性、确定患有疾病或病症的受试者的预后(或其可能的进展或消退)以及确定治疗对患有疾病或病症的受试者的效果。
在本发明的一些具体实施方案中，诊断还意指区分患有结直肠癌的受试者是否存在淋巴结转移。
术语“互补”和“互补性”是指与碱基配对规则相关的核苷酸(例如，1个核苷酸)或多核苷酸(例如核苷酸的序列)。例如，序列5′-A-G-T-3′与序列3′-T-C-A-5′互补。互补可以是“部分的”，其中仅一些核酸碱基根据碱基配对规则进行匹配。或者，核酸之间可能存在“完全”或“总”互补。核酸链之间的互补程度影响核酸链之间杂交的效率和强度。这在扩增反应和依赖核酸之间的结合的检测方法中尤其重要。
术语“聚合酶链式反应”用于扩增靶序列，该方法由以下步骤组成：将大量过量的两种寡核苷酸引物引入到含有期望靶序列的DNA混合物中，随后在DNA聚合酶存在下进行精确的热循环顺序。两种引物与双链靶序列的相应链互补。为了进行扩增，将混合物变性，然后引物与靶分子内的其互补序列退火。退火后，用聚合酶扩增引物，形成一对新的互补链。变性、引物退火和聚合酶延伸的步骤可以重复多次(即，变性、退火和延伸构成一个“循环”；可以有许多“循环”)以获得高浓度的期望靶序列的扩增片段。期望靶序列的扩增片段的长度由引物相对于彼此的相对位置确定，因此该长度是可控参数。由于该方法的重复方面，该方法被称为“聚合酶链式反应”(“PCR”)。由于靶序列的期望扩增片段成为混合物中的主要序列(以浓度计)，所以称其被“PCR扩增”，是“PCR产物”或“扩增子”。
本说明书中，术语“可扩增核酸”是指可以通过任何扩增方法扩增的核酸。预期“可扩增核酸”通常将包含“样本模板”。
本说明书中，术语“样本模板”是指来源于样本的用于分析“靶”的存在的核酸。相比之下，“背景模板”用于指样本模板以外的核酸，其可能存在或可能不存在于样本中。背景模板通常是无意的。这可能是遗留的结果，或者可能是由于试图从样本中纯化走的核酸污染物的存在。例如，来自生物体的待检测核酸以外的核酸可以作为测试样本的背景存在。
本说明书中，术语“引物”是指在纯化的限制性消化物中天然存在的或合成产生的寡核苷酸，当处于其中诱导与核酸链互补的引物延伸产物合成的条件下(例如，在核苷酸和诱导剂如DNA聚合酶的存在下并且在合适的温度和pH下)时，其能够作为合成的起点。引物优选是单链的，用于扩增的最大效率，但也可以是双链的。如果是双链，则在用于制备延伸产物之前首先处理引物以分离其链。优选地，引物是寡脱氧核糖核苷酸。引物必须足够长以在诱导剂的存在下引发延伸产物的合成。引物的确切长度将取决于许多因素，包括温度、引物来源以及方法的使用。
本说明书中，术语“探针”是指在纯化的限制性消化物中天然存在的或者合成、重组或通过PCR扩增产生的寡核苷酸(例如，核苷酸序列)，其能够与另一种目标寡核苷酸杂交。探针可以是单链或双链的。探针可用于特定基因序列的检测、鉴定和分离(例如，“捕获探针”)。预期在一些实施方案中，本发明中使用的任何探针可以用任何“报道分子”进行标记，使得在任何检测系统中可检测。
本说明书中，“扩增”通常是指产生所需序列的多个拷贝的过程。“多个拷贝”是指至少两个拷贝。“拷贝”并不一定意味着与模板序列具有完美的序列互补性或同一性。例如，拷贝可以包括核苷酸类似物如脱氧肌苷，有意的序列改变(例如通过包含与模板可杂交但不互补的序列的引物引入的序列改变)，和/或在扩增过程中发生的序列错误。
本说明书中，“序列确定”等包括确定与核酸的核苷酸碱基序列有关的
信息。这样的信息可以包括对核酸的部分或全部序列信息的鉴定或确定。可以以不同程度的统计可靠性或置信度来确定序列信息。在一个方面，所述术语包括确定核酸中多个连续核苷酸的身份和顺序。
本说明书中，术语“测序”、“高通量测序”或“下一代测序”包括使用这样的方法进行序列确定：所述方法以本质上平行的方式确定许多(通常数千至数十亿)个核酸序列，即在这种方法中，制备DNA模板并不是用于每次测序一个，而是以批量过程进行，并且在这种方法中许多序列优选地被并行读取，或者使用本身可以并行化的超高通量串行过程读取。此类方法包括但不限于焦磷酸测序(例如，如454Life Sciences,Inc.,Branford,CT所商业化的)；通过连接进行测序(例如，如SOLiDTM技术，Life Technologies,Inc.,Carlsbad,CA所商业化的)；使用修饰的核苷酸通过合成进行测序(例如，如Illumina,Inc.,San Diego,CA所商业化的TruSeqTM和HiSeqTM技术，Helicos Biosciences Corporation,Cambridge,MA所商业化的HeliScopeTM；和Pacific Biosciences of California,Inc.,Menlo Park,CA所商业化的PacBio RS)，通过离子检测技术进行测序(例如，Ion TorrentTM技术,Life Technologies,Carlsbad,CA)；DNA纳米球测序(Complete Genomics,Inc.,Mountain View,CA)；基于纳米孔的测序技术(例如，由Oxford Nanopore Technologies,LTD,Oxford,UK所开发的)等高度并行的测序方法。
本说明书中，“DNA甲基化”或“甲基化”是指生物体内在DNA甲基转移酶的催化下，以S-腺苷甲硫氨酸(SAM)为甲基供体，将甲基转移到特定的碱基上的过程。在哺乳动物体内，主要是核苷酸胞嘧啶残基的5’C端的甲基化。在人类基因组上，大量的DNA甲基化发生在CpG双核苷酸中的胞嘧啶上，C是胞嘧啶，G是鸟嘌呤，p是磷酸基团。DNA甲基化也可以发生在CHG和CHH等核苷酸序列的胞嘧啶中，其中H是腺嘌呤、胞嘧啶或胸腺嘧啶。DNA甲基化还可以发生在非胞嘧啶上，例如N6-甲基腺嘌呤。此外，DNA甲基化还可以是5-羟甲基胞嘧啶等形式。在大部分情况下，DNA甲基化为对侧DNA链或DNA碱基附近其它的CpG位点上的甲基化修饰所诱导产生。
在本说明书中，“DNA甲基化位点”、“甲基化位点”是指可能发生DNA甲基化修饰的单个或多个碱基位置。例如CpG位点、CHG位点或CHH位点。在一些情况下，DNA甲基化位点等同于CpG位点。
本说明书中，“甲基化核苷酸”或“甲基化核苷酸碱基”是指在核苷酸碱基上存在甲基部分，其中甲基部分不存在于公认的典型核苷酸碱基中。例如，胞嘧啶在其嘧啶环上不包含甲基部分，但是5-甲基胞嘧啶在其嘧啶环的5位包含甲基部分。因此，胞嘧啶不是甲基化核苷酸，5-甲基胞嘧啶是甲基化核苷酸。在另一个实例中，胸腺嘧啶在其嘧啶环的5位含有甲基部分。
本说明书中，核酸分子的“甲基化水平”、“甲基化状态”、“甲基化谱”和“甲基化状况”是指在核酸分子中存在或不存在一个或多个甲基化核苷酸碱基。例如，包含甲基化胞嘧啶的核酸分子被视为甲基化的(例如，核酸分子的甲基化状态为甲基化的)。不含任何甲基化核苷酸的核酸分子被视为未甲基化的。
本说明书中，甲基化状态可任选地由“甲基化值”表示或指示(例如，表示甲基化频率、分数、比例、百分比等)。甲基化值可以例如在用甲基化依赖性限制酶限制性消化之后定量存在的完整核酸的量，或者通过比较亚硫酸氢盐反应后的扩增谱，或者通过比较亚硫酸氢盐处理和未处理的核酸的序列来产生。因此，诸如甲基化值的值代表甲基化状态，因此可用作基因座的多个拷贝中甲基化状态的定量指标。
如本文所用，“甲基化频率”或“甲基化百分比(％)”是指分子或基因座为甲基化的实例数相对于分子或基因座为未甲基化的实例数。例如，在一些实施方案中，甲基化百分比是指甲基化胞嘧啶的百分比，以β值表示，即β＝携带甲基化胞嘧啶数量/(携带甲基化胞嘧啶数量+未携带甲基化胞嘧啶的数量)。
因此，甲基化状态描述核酸(例如，基因组序列)的甲基化的状态。此外，甲基化状态是指核酸片段在特定的基因组基因座处与甲基化相关的特性。此类特性包括但不限于：此DNA序列内的任何胞嘧啶(C)残基是否为甲基化
的，甲基化C残基的位置，遍及核酸的任何特定区的甲基化C的频率或百分比，以及因例如等位基因来源中的差异而导致的甲基化中的等位基因差异。术语“甲基化状态”、“甲基化谱”和“甲基化状况”还指遍及生物样品中核酸的任何特定区的甲基化C或未甲基化C的相对、绝对浓度或模式。例如，如果使核酸序列内的胞嘧啶(C)残基甲基化，则其可称为“高甲基化”或具有“增加的甲基化”，而如果DNA序列内的胞嘧啶(C)残基未甲基化，则其可称为“低甲基化”或具有“降低的甲基化”。同样，如果核酸序列内的胞嘧啶(C)残基与另一个核酸序列(例如，来自不同的区或来自不同的个体等)相比甲基化，则该序列被视为与另一个核酸序列相比高甲基化或具有增加的甲基化。或者，如果DNA序列内的胞嘧啶(C)残基与另一个核酸序列(例如，来自不同的区或来自不同的个体等)相比未甲基化，则该序列被视为与另一个核酸序列相比低甲基化或具有降低的甲基化。另外，如本文所用的术语“甲基化模式”是指在核酸的某个区上甲基化和未甲基化核苷酸的集体位点。两个核苷酸可具有相同的或相似的甲基化频率或甲基化百分比，但当甲基化和未甲基化核苷酸的数量在整个区中相同或相似但甲基化和未甲基化核苷酸的位置不同时具有不同的甲基化模式。当序列在甲基化的程度(例如，一个相对于另一个具有增加或降低的甲基化)、频率或模式不同时，将所述序列称为“差异甲基化的”或称为具有“甲基化差异”或具有“不同的甲基化状态”。术语“差异甲基化”是指在癌症阳性(或癌症转移阳性，例如结直肠癌淋巴结转移)样品中的核酸甲基化水平或模式与在癌症阴性样品(或癌症转移阴性，例如结直肠癌未淋巴结转移)中的核酸甲基化水平或模式相比的差异。其还可以指在手术后癌症复发的患者与未复发的患者之间的水平或模式的差异。差异甲基化以及DNA甲基化的特定水平或模式是诊断和预测性生物标记物，例如，一旦定义正确的截止值或预测特性后。
如本文所用，术语“差异甲基化区域”(Differentially Methylated Region,DMR)是指包含一个或多个差异甲基化位点的DNA区域。在选定的感兴趣的条件下，例如癌症状态(或癌症转移状态，例如结直肠癌淋巴结转移)，
包括更多数量或频率的甲基化位点的DMR可以被称为高甲基化DMR。在选定的感兴趣的条件下，例如癌症状态(或癌症转移状态，例如结直肠癌淋巴结转移)，包括较少数量或频率的甲基化位点的DMR可以被称为低甲基化DMR。作为结直肠癌甲基化生物标志物的DMR可称为结肠直肠癌DMR。作为结直肠癌淋巴结转移的甲基化生物标志物的DMR可称为结直肠癌淋巴结转移DMR。在一些情况下，DMR可以是单个核苷酸，该单个核苷酸是甲基化位点。
本说明书中，术语“亚硫酸氢盐试剂”是指在一些实施方案中包含亚硫酸氢盐(bisulfite)、亚硫酸氢盐(disulfite)、亚硫酸氢盐(hydrogen sulfite)或其组合的试剂，经过亚硫酸氢盐试剂处理的DNA，其未经过甲基化的胞嘧啶核苷酸将转化为尿嘧啶，而甲基化的胞嘧啶及其他碱基维持不变，因此可以区分例如CpG二核苷酸序列中的甲基化和未甲基化胞苷。
以下对于本发明的技术方案进行具体说明。
<甲基化生物标记物>
本发明的一些方面，提供了一种用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化生物标记物包括后文附表1中提供的甲基化位点中的任一项或其任意组合。
在本发明的一些实施方案中，所述的甲基化生物标记物包括来自13号染色体基因组区域20693341-20693653的位点、2号染色体基因组区域74725882-74726332的位点、20号染色体基因组区域62272199-62272548的位点和/或19号染色体基因组区域48983509-48983698的位点。
在本发明的一些实施方案中，所述的甲基化生物标记物包括后文附表2中提供的甲基化位点中的任一项或其任意组合。
在一些实施方案中，所述的甲基化位点至少包括chr2:74726037、chr2:74726227和chr2:74726230中的任一种。
进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr2:74726181、chr2:74726179中的至少一种。例如，在一些具体的实施方案
中，所述的甲基化位点至少包括chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181和chr2:74726179。
更进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371中的至少一种。
再进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr2:74726006、chr2:74726124、chr2:74726073、chr20:62272341、chr2:74726131、chr2:74726257、chr20:62272381、chr2:74726274、chr20:62272382、chr2:74726083中的至少一种。
尤其进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr2:74726332、chr2:74726276、chr2:74726271、chr20:62272199、chr2:74725967、chr2:74726137、chr20:62272313、chr2:74726265、chr20:62272387、chr2:74725994、chr20:62272480、chr20:62272478、chr2:74726106、chr20:62272434、chr2:74726216、chr2:74726058、chr2:74726190、chr2:74725992、chr2:74726312、chr20:62272513、chr20:62272388、chr20:62272453、chr20:62272448、chr20:62272447、chr2:74726293、chr20:62272491、chr20:62272494、chr20:62272403、chr2:74725934、chr20:62272361中的至少一种；
尤其更进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr20:62272487、chr20:62272547、chr13:20693357、chr20:62272310、chr2:74726320、chr19:48983569、chr20:62272446、chr2:74725984、chr20:62272404、chr13:20693417、chr2:74725926、chr20:62272449、chr20:62272470、chr13:20693578、chr2:74725887、chr13:20693448、chr13:20693607、chr13:20693414、chr20:62272340、chr13:20693653、chr13:20693415、chr13:20693492、chr20:62272372、chr19:48983571、chr19:48983526、chr19:48983663、chr19:48983596、chr19:48983638、chr13:20693564、chr2:74725895、chr13:20693469、chr13:20693608、
chr20:62272309、chr20:62272548、chr13:20693577、chr13:20693565、chr13:20693416、chr13:20693342、chr19:48983606、chr13:20693356、chr19:48983564、chr13:20693460、chr19:48983671、chr13:20693569、chr13:20693636、chr13:20693341、chr19:48983601、chr13:20693459、chr19:48983584、chr13:20693468中的至少一种。
尤其再进一步地，在一些任选的实施方案中，所述的甲基化位点包括后文附表2中提供的甲基化位点。
在一些更具体实施方案中，所述的甲基化位点包括选自以下的位点组合中的任一组或其任意组合：
5marker组：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179；
10marker组：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179、chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371；
20marker组：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179、chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371、chr2:74726006、chr2:74726124、chr2:74726073、chr20:62272341、chr2:74726131、chr2:74726257、chr20:62272381、chr2:74726274、chr20:62272382、chr2:74726083；
50marker组：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179、chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371、chr2:74726006、chr2:74726124、chr2:74726073、chr20:62272341、chr2:74726131、chr2:74726257、chr20:62272381、chr2:74726274、chr20:62272382、chr2:74726083、chr2:74726332、chr2:74726276、chr2:74726271、chr20:62272199、chr2:74725967、chr2:74726137、chr20:62272313、chr2:74726265、chr20:62272387、chr2:74725994、chr20:62272480、chr20:62272478、chr2:74726106、
chr20:62272434、chr2:74726216、chr2:74726058、chr2:74726190、chr2:74725992、chr2:74726312、chr20:62272513、chr20:62272388、chr20:62272453、chr20:62272448、chr20:62272447、chr2:74726293、chr20:62272491、chr20:62272494、chr20:62272403、chr2:74725934、chr20:62272361；
100marker组：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179、chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371、chr2:74726006、chr2:74726124、chr2:74726073、chr20:62272341、chr2:74726131、chr2:74726257、chr20:62272381、chr2:74726274、chr20:62272382、chr2:74726083、chr2:74726332、chr2:74726276、chr2:74726271、chr20:62272199、chr2:74725967、chr2:74726137、chr20:62272313、chr2:74726265、chr20:62272387、chr2:74725994、chr20:62272480、chr20:62272478、chr2:74726106、chr20:62272434、chr2:74726216、chr2:74726058、chr2:74726190、chr2:74725992、chr2:74726312、chr20:62272513、chr20:62272388、chr20:62272453、chr20:62272448、chr20:62272447、chr2:74726293、chr20:62272491、chr20:62272494、chr20:62272403、chr2:74725934、chr20:62272361、chr20:62272487、chr20:62272547、chr13:20693357、chr20:62272310、chr2:74726320、chr19:48983569、chr20:62272446、chr2:74725984、chr20:62272404、chr13:20693417、chr2:74725926、chr20:62272449、chr20:62272470、chr13:20693578、chr2:74725887、chr13:20693448、chr13:20693607、chr13:20693414、chr20:62272340、chr13:20693653、chr13:20693415、chr13:20693492、chr20:62272372、chr19:48983571、chr19:48983526、chr19:48983663、chr19:48983596、chr19:48983638、chr13:20693564、chr2:74725895、chr13:20693469、chr13:20693608、chr20:62272309、chr20:62272548、chr13:20693577、chr13:20693565、chr13:20693416、chr13:20693342、chr19:48983606、chr13:20693356、chr19:48983564、chr13:20693460、chr19:48983671、chr13:20693569、
chr13:20693636、chr13:20693341、chr19:48983601、chr13:20693459、chr19:48983584、chr13:20693468；
125marker组：包括附表2中全部甲基化位点。
在一些实施方案中，所述的甲基化位点包括来自2号染色体基因组区域74725882-74726332的位点。具体地，所述的甲基化位点包括chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320和chr2:74726332中的任一项或其任意组合。
在一些任选的实施方案中，所述的甲基化位点至少包括chr2:74726037、chr2:74726227和chr2:74726230中的至少一种。在一些具体实施方案中，所述的甲基化位点至少包括chr2:74726037、chr2:74726227和chr2:74726230。
进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr2:74726181、chr2:74726179中的至少一种。
更进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr2:74725882、chr2:74726124中的至少一种。
再进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr2:74726094、chr2:74726073、chr2:74726006中的至少一种。
尤其进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr2:74726332、chr2:74726274、chr2:74726137、chr2:74726257、chr2:74726131、chr2:74726190、chr2:74726271、chr2:74726083、chr2:74725967、chr2:74726106中的至少一种。
尤其更进一步地，在一些任选的实施方案中，所述的甲基化位点包括chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、
chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320和chr2:74726332。
在一些更具体实施方案中，所述的甲基化位点包括选自以下的位点组合中的任一组或其任意组合：
3marker组：chr2:74726037、chr2:74726227、chr2:74726230；
5marker组：chr2:74726037、chr2:74726227、chr2:74726230、chr2:74726181、chr2:74726179；
7marker组：chr2:74726037、chr2:74726227、chr2:74726230、chr2:74726181、chr2:74726179、chr2:74725882、chr2:74726124；
10marker组：chr2:74726037、chr2:74726227、chr2:74726230、chr2:74726181、chr2:74726179、chr2:74725882、chr2:74726124、chr2:74726094、chr2:74726073、chr2:74726006；
20marker组：chr2:74726037、chr2:74726227、chr2:74726230、chr2:74726181、chr2:74726179、chr2:74725882、chr2:74726124、chr2:74726094、chr2:74726073、chr2:74726006、chr2:74726332、chr2:74726274、chr2:74726137、chr2:74726257、chr2:74726131、chr2:74726190、chr2:74726271、chr2:74726083、chr2:74725967、chr2:74726106；
34marker组：chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、
chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320、chr2:74726332。
在本发明的一些实施方案中，所述的甲基化位点包括来自20号染色体基因组区域62272199-62272548的位点；具体地，所述的甲基化位点包括chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547和chr20:62272548中的任一项或其任意组合。
在一些任选的实施方案中，所述的甲基化位点至少包括chr20:62272362、chr20:62272293和chr20:62272371中的至少一种。在一些具体实施方案中，所述的甲基化位点至少包括chr20:62272362、chr20:62272293和chr20:62272371。
进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr20:62272387、chr20:62272448中的至少一种。
更进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr20:62272382、chr20:62272341中的至少一种。
再进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr20:62272199、chr20:62272381、chr20:62272313中的至少一种。
尤其进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr20:62272446、chr20:62272478、chr20:62272434、chr20:62272480、chr20:62272513、chr20:62272453、chr20:62272403、chr20:62272547、chr20:62272388、chr20:62272491中的至少一种。
尤其更进一步地，在一些任选的实施方案中，所述的甲基化位点包括chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547和chr20:62272548。
在一些更具体实施方案中，所述的甲基化位点包括选自以下的位点组合中的任一组或其任意组合：
3marker组：chr20:62272362、chr20:62272293、chr20:62272371；
5marker组：chr20:62272362、chr20:62272293、chr20:62272371、chr20:62272387、chr20:62272448；
7marker组：chr20:62272362、chr20:62272293、chr20:62272371、chr20:62272387、chr20:62272448、chr20:62272382、chr20:62272341；
10marker组：chr20:62272362、chr20:62272293、chr20:62272371、chr20:62272387、chr20:62272448、chr20:62272382、chr20:62272341、chr20:62272199、chr20:62272381、chr20:62272313；
20marker组：chr20:62272362、chr20:62272293、chr20:62272371、chr20:62272387、chr20:62272448、chr20:62272382、chr20:62272341、chr20:62272199、chr20:62272381、chr20:62272313、chr20:62272446、chr20:62272478、chr20:62272434、chr20:62272480、chr20:62272513、chr20:62272453、chr20:62272403、chr20:62272547、chr20:62272388、chr20:62272491。
38marker组：chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547、chr20:62272548
在一些实施方案中，所述的甲基化位点包括来自19号染色体基因组区域48983509-48983698的位点；具体地，所述的甲基化位点包括chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679和chr19:48983698中的任一项或其任意组合。
进一步地，在一些任选的实施方案中，所述的甲基化位点至少包括chr19:48983569、chr19:48983526和chr19:48983663中的至少一种。在一些具体实施方案中，所述的甲基化位点至少包括chr19:48983569、chr19:48983526和chr19:48983663。
更进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr19:48983571、chr19:48983638中的至少一种。
再进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr19:48983596、chr19:48983629中的至少一种。
尤其进一步地，在一些任选的实施方案中，所述的甲基化位点还包括chr19:48983666、chr19:48983606、chr19:48983509中的至少一种。
尤其进一步地，在一些任选的实施方案中，所述的甲基化位点包括chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679和chr19:48983698。
在一些更具体实施方案中，所述的甲基化位点包括选自以下的位点组合中的任一组或其任意组合：
3marker组：chr19:48983569、chr19:48983526、chr19:48983663；
5marker组：chr19:48983569、chr19:48983526、chr19:48983663、chr19:48983571、chr19:48983638；
7marker组：chr19:48983569、chr19:48983526、chr19:48983663、chr19:48983571、chr19:48983638、chr19:48983596、chr19:48983629；
10marker组：chr19:48983569、chr19:48983526、chr19:48983663、chr19:48983571、chr19:48983638、chr19:48983596、chr19:48983629、chr19:48983666、chr19:48983606、chr19:48983509；
18marker组：chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679、chr19:4898369。
在一些具体实施方案中，所述的甲基化位点来自13号染色体基因组区域20693341-20693653；具体地，所述的甲基化位点包括chr13:20693341、chr13:20693342、chr13:20693356、chr13:20693357、chr13:20693359、chr13:20693360、chr13:20693384、chr13:20693385、chr13:20693414、chr13:20693415、chr13:20693416、chr13:20693417、chr13:20693447、chr13:20693448、chr13:20693459、chr13:20693460、chr13:20693468、
chr13:20693469、chr13:20693491、chr13:20693492、chr13:20693564、chr13:20693565、chr13:20693569、chr13:20693570、chr13:20693571、chr13:20693572、chr13:20693577、chr13:20693578、chr13:20693607、chr13:20693608、chr13:20693631、chr13:20693632、chr13:20693636、chr13:20693637和chr13:20693653中的任一项或其任意组合。
在本发明中，不限于将来自同一染色体基因组区域的甲基化位点组合使用，实际上，来自不同染色体基因组区域的甲基化位点组合使用，也可以获得相似的诊断结直肠癌淋巴结转移的作用及效果，正如后文实施例中所证实的。因此，在一些任选的实施方案中，所述的甲基化生物标记物至少包括chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037和chr2:74726230中的至少一种。在本发明的一些具体实施方案中，所述的甲基化生物标记物可以至少包括chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037和chr2:74726230。
进一步地，在一些任选的实施方案中，所述的甲基化生物标记物还包括chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875中的至少一种。
更进一步地，在一些任选的实施方案中，所述的甲基化生物标记物还包括chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312中的至少一种。
再进一步地，在一些任选的实施方案中，所述的甲基化生物标记物还包括chr10:71338666、chr6:161655343、chr2:174424852、chr16:67427066、chr6:42931754、chr1:68295979、chr15:28381565、chr11:44820675、chr9:140157146、chr15:101097210、chr4:4039187、chr14:44168216、chr15:41264369、chr13:19174394、chr21:15497019、chr15:70574296、chr2:74726094、chr2:74726006、chr13:97991180、chr15:101098460、chr19:13944958、chr4:55379617、chr2:74726131、chr9:37949852、chr8:10825518、
chr11:119355687、chr16:5237372、chr2:74726073、chr2:74726271、chr8:10924461中的至少一种。
尤其进一步地，在一些任选的实施方案中，所述的甲基化生物标记物还包括chr7:129752313、chr2:74726332、chr1:16553114、chr2:74726124、chr19:33863663、chr2:74726257、chr8:7004715、chr2:74726137、chr22:45363303、chr2:74726276、chr2:74725882、chr10:71338651、chr2:74726083、chr1:161450901、chr2:74726274、chr2:74725967、chr7:77687820、chr7:77687882、chr12:80462103、chr19:43778881、chr6:71980418、chr8:136263479、chr17:3548648、chr11:128590262、chr4:174462424、chr8:2480920、chr20:62272362、chr15:95662730、chr19:54172983、chr3:197832357、chr2:176624742、chr19:35408270、chr2:74726265、chr10:102648751、chr12:12766094、chr10:106093878、chr13:19174407、chr9:79138639、chr2:242755966、chr16:77147779、chr2:74725994、chr2:74726106、chr3:36174109、chr2:74726058、chr19:38983253、chr12:1932190、chr3:197222253、chr6:144881470、chr1:161450899、chr2:74726216中的至少一种。
尤其更进一步地，在一些任选的实施方案中，所述的甲基化生物标记物还包括chr20:24899315、chr6:144881495、chr4:190963293、chr9:118501681、chr2:28700693、chr10:108412225、chr19:49891736、chr20:62272293、chr5:77165020、chr4:909786、chr16:2076342、chr6:144881587、chr2:32205788、chr13:19174421、chr1:54121906、chr3:197830661、chr17:62690049、chr3:197217049、chr6:144881549、chr2:36716536、chr2:74726293、chr5:476865、chr17:77047294、chr8:106967372、chr2:74726190、chr8:28157735、chr2:174853968、chr19:51483067、chr1:219944199、chr3:97973710、chr16:27431703、chr21:46238778、chr20:62272371、chr15:101096974、chr11:45743460、chr10:64613369、chr2:74725992、chr20:62272341、chr17:77181458、chr18:60020618、chr2:74725934、chr2:191625107、
chr4:48945163、chr6:144881603、chr11:133800611、chr17:79855640、chr2:74726312、chr15:101097067、chr4:7128051、chr5:138861157、chr6:121069941、chr13:19174425、chr20:62272478、chr5:61065238、chr20:62272382、chr15:26543473、chr21:44197355、chr20:62272480、chr19:8211090、chr3:97982831、chr8:11062433、chr8:10825314、chr1:147827054、chr20:42427883、chr3:3225323、chr21:15497020、chr2:96990550、chr8:95627967、chr3:38618469、chr21:46238680、chr8:89712289、chr20:62272313、chr10:3870830、chr19:1545977、chr4:31261294、chr8:2181379、chr1:9327169、chr10:71338662、chr16:88619484、chr5:138861242、chr20:62272434、chr2:74726320、chr5:2749278、chr12:9856328、chr11:133800801、chr4:41299758、chr6:35892438、chr2:242789465、chr15:91305688、chr2:74725984、chr14:74439495、chr6:170338877、chr4:1404633、chr20:62272381、chr10:3870880、chr22:24253568、chr7:18901757、chr5:103114389、chr12:53216896、chr2:54952741中的至少一种。
尤其再进一步地，在一些任选的实施方案中，所述的甲基化生物标记物包括附表1中全部甲基化位点。
在本发明的一些具体实施方案中，所述的甲基化生物标记物可以包括以下位点组合中的任一组或其任意组合：
5marker组：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230；
10marker组：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875；
20marker组：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875、chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、
chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312；
50marker组：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875、chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312、chr10:71338666、chr6:161655343、chr2:174424852、chr16:67427066、chr6:42931754、chr1:68295979、chr15:28381565、chr11:44820675、chr9:140157146、chr15:101097210、chr4:4039187、chr14:44168216、chr15:41264369、chr13:19174394、chr21:15497019、chr15:70574296、chr2:74726094、chr2:74726006、chr13:97991180、chr15:101098460、chr19:13944958、chr4:55379617、chr2:74726131、chr9:37949852、chr8:10825518、chr11:119355687、chr16:5237372、chr2:74726073、chr2:74726271、chr8:10924461；
100marker组：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875、chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312、chr10:71338666、chr6:161655343、chr2:174424852、chr16:67427066、chr6:42931754、chr1:68295979、chr15:28381565、chr11:44820675、chr9:140157146、chr15:101097210、chr4:4039187、chr14:44168216、chr15:41264369、chr13:19174394、chr21:15497019、chr15:70574296、chr2:74726094、chr2:74726006、chr13:97991180、chr15:101098460、chr19:13944958、chr4:55379617、chr2:74726131、chr9:37949852、chr8:10825518、chr11:119355687、chr16:5237372、
chr2:74726073、chr2:74726271、chr8:10924461、chr7:129752313、chr2:74726332、chr1:16553114、chr2:74726124、chr19:33863663、chr2:74726257、chr8:7004715、chr2:74726137、chr22:45363303、chr2:74726276、chr2:74725882、chr10:71338651、chr2:74726083、chr1:161450901、chr2:74726274、chr2:74725967、chr7:77687820、chr7:77687882、chr12:80462103、chr19:43778881、chr6:71980418、chr8:136263479、chr17:3548648、chr11:128590262、chr4:174462424、chr8:2480920、chr20:62272362、chr15:95662730、chr19:54172983、chr3:197832357、chr2:176624742、chr19:35408270、chr2:74726265、chr10:102648751、chr12:12766094、chr10:106093878、chr13:19174407、chr9:79138639、chr2:242755966、chr16:77147779、chr2:74725994、chr2:74726106、chr3:36174109、chr2:74726058、chr19:38983253、chr12:1932190、chr3:197222253、chr6:144881470、chr1:161450899、chr2:74726216；
200marker组：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875、chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312、chr10:71338666、chr6:161655343、chr2:174424852、chr16:67427066、chr6:42931754、chr1:68295979、chr15:28381565、chr11:44820675、chr9:140157146、chr15:101097210、chr4:4039187、chr14:44168216、chr15:41264369、chr13:19174394、chr21:15497019、chr15:70574296、chr2:74726094、chr2:74726006、chr13:97991180、chr15:101098460、chr19:13944958、chr4:55379617、chr2:74726131、chr9:37949852、chr8:10825518、chr11:119355687、chr16:5237372、chr2:74726073、chr2:74726271、chr8:10924461、chr7:129752313、chr2:74726332、chr1:16553114、chr2:74726124、chr19:33863663、chr2:74726257、chr8:7004715、
chr2:74726137、chr22:45363303、chr2:74726276、chr2:74725882、chr10:71338651、chr2:74726083、chr1:161450901、chr2:74726274、chr2:74725967、chr7:77687820、chr7:77687882、chr12:80462103、chr19:43778881、chr6:71980418、chr8:136263479、chr17:3548648、chr11:128590262、chr4:174462424、chr8:2480920、chr20:62272362、chr15:95662730、chr19:54172983、chr3:197832357、chr2:176624742、chr19:35408270、chr2:74726265、chr10:102648751、chr12:12766094、chr10:106093878、chr13:19174407、chr9:79138639、chr2:242755966、chr16:77147779、chr2:74725994、chr2:74726106、chr3:36174109、chr2:74726058、chr19:38983253、chr12:1932190、chr3:197222253、chr6:144881470、chr1:161450899、chr2:74726216、chr20:24899315、chr6:144881495、chr4:190963293、chr9:118501681、chr2:28700693、chr10:108412225、chr19:49891736、chr20:62272293、chr5:77165020、chr4:909786、chr16:2076342、chr6:144881587、chr2:32205788、chr13:19174421、chr1:54121906、chr3:197830661、chr17:62690049、chr3:197217049、chr6:144881549、chr2:36716536、chr2:74726293、chr5:476865、chr17:77047294、chr8:106967372、chr2:74726190、chr8:28157735、chr2:174853968、chr19:51483067、chr1:219944199、chr3:97973710、chr16:27431703、chr21:46238778、chr20:62272371、chr15:101096974、chr11:45743460、chr10:64613369、chr2:74725992、chr20:62272341、chr17:77181458、chr18:60020618、chr2:74725934、chr2:191625107、chr4:48945163、chr6:144881603、chr11:133800611、chr17:79855640、chr2:74726312、chr15:101097067、chr4:7128051、chr5:138861157、chr6:121069941、chr13:19174425、chr20:62272478、chr5:61065238、chr20:62272382、chr15:26543473、chr21:44197355、chr20:62272480、chr19:8211090、chr3:97982831、chr8:11062433、chr8:10825314、chr1:147827054、chr20:42427883、chr3:3225323、chr21:15497020、chr2:96990550、chr8:95627967、chr3:38618469、
chr21:46238680、chr8:89712289、chr20:62272313、chr10:3870830、chr19:1545977、chr4:31261294、chr8:2181379、chr1:9327169、chr10:71338662、chr16:88619484、chr5:138861242、chr20:62272434、chr2:74726320、chr5:2749278、chr12:9856328、chr11:133800801、chr4:41299758、chr6:35892438、chr2:242789465、chr15:91305688、chr2:74725984、chr14:74439495、chr6:170338877、chr4:1404633、chr20:62272381、chr10:3870880、chr22:24253568、chr7:18901757、chr5:103114389、chr12:53216896、chr2:54952741；
341marker组：包括附表1中全部甲基化位点。
在本发明的一些实施方案中，所述诊断是区分患有结直肠癌的受试者是否存在淋巴结转移。
在本发明的一些具体实施方案中，所述的受试者是哺乳动物；优选地，所述的哺乳动物是人；进一步优选地，所述的受试者是结直肠癌患者。
在本发明的一些具体实施方案中，所述结直肠癌选自TNM分期中T分期的T1期、T2期、T3期或T4期的结直肠癌。
<甲基化生物标记物的用途>
在本发明的一些方面中，提供了上述的甲基化生物标记物在制备用于诊断患有结直肠癌的受试者是否存在淋巴结转移的试剂或试剂盒中的用途。
在另一些实施方案中，提供了检测上述的甲基化生物标记物的甲基化水平的试剂在制备用于诊断患有结直肠癌的受试者是否存在淋巴结转移的试剂或试剂盒中的用途。
<结直肠癌淋巴结转移诊断的试剂盒>
在本发明的一些方面中，提供了用于结直肠癌淋巴结转移诊断的试剂盒，其中，所述的试剂盒包含用于检测待测样本中上述的甲基化生物标记物的甲基化水平的试剂。
在本发明的一些具体的实施方案中，所述的试剂为选自以下的检测甲基化水平的方法中所使用的试剂：荧光定量PCR(qPCR)、甲基化特异性PCR(MSP)、数字PCR(ddPCR)、DNA甲基化芯片、靶向DNA甲基化测序、全基因组甲基化测序(Whole Genome Bisulfite Sequencing,WGBS)、DNA甲基化质谱(MassArray)中的一种或多种。
在本发明的一些具体的实施方案中，所述的待测样本选自组织、血液、血浆、唾液、血清、尿液、尿液脱落细胞、尿沉渣、尿液上清中的一种或多种。在本发明的一些优选实施方案中，所述的待测样本为组织，例如结直肠癌组织。
<结直肠癌淋巴结转移诊断系统>
本发明的一些方面提供了一种用于结直肠癌淋巴结转移诊断的系统，其中，所述系统包括检测装置、计算装置和输出装置；
所述检测装置包括进样器和检测器，所述进样器用于采集来自患有结直肠癌的受试者的样本，所述检测器用于检测所述样本中上述的甲基化生物标记物的甲基化水平；
所述计算装置包括存储器和处理器，所述存储器中存储有计算机程序，所述处理器被配置为执行所述存储器中存储的计算机程序，以实现如下判别：
所述样本中如上述的甲基化生物标记物的甲基化水平水平不同于不存在淋巴结转移的患有结直肠癌的受试者中测定的甲基化生物标记物的甲基化水平，则判别所述样本对应的受试者的存在结直肠癌淋巴结转移。
在一些具体的实施方案中，所述输出装置用于输出所述检测装置的检测结果和/或所述计算装置的判别结果，所述输出装置包括显示器、打印机和音频输出装置中的至少一种；所述计算装置包括电脑主机、中央处理器和网络服务器中的至少一种。
<结直肠癌淋巴结转移诊断方法>
在本发明的一些方面，提供了一种结直肠癌淋巴结转移诊断方法，其包括以下步骤。
获得受试者的样本；
提取所述样本的基因组DNA和/或游离DNA；
检测所述DNA中，上述的甲基化生物标记物的甲基化水平；
判别受试者是否存在结直肠癌淋巴结转移，即患有结直肠癌的受试者是否存在淋巴结转移。
以下通过实施例与测试例进一步说明本发明，但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是，应当理解的是，这些仅仅是示例性的，并不意图限制本发明，与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例与测试例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例与测试例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例
1、样本来源：
样本包括19例结直肠癌组织并且发生了淋巴结转移(CRC LN+)的新鲜冰冻组织样本和11例结直肠癌组织但是未发生淋巴结转移(CRC LN-)新鲜冰冻组织样本，所述样本信息如下：
2、实验、数据分析及标记物筛选及验证：
将上述样本，对新鲜冰冻组织进行建库，流程和步骤如下：
2.1、组织DNA提取及甲基化建库
2.1.1、组织DNA的提取
上述结直肠癌组织样本DNA的提取步骤按照QIAGEN公司的DNeasy
Blood & Tissue Kit操作说明进行；
2.1.2、转化
将提取的组织DNA(50ng)进行亚硫酸氢盐转化，使DNA中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶，而甲基化的胞嘧啶保持不变，得到亚硫酸氢盐转化后的DNA，转化具体操作按照Zymo Research的EZ DNA Methylation-Lightning Kit说明书进行。
2.1.3、末端修复
将上述转化后的17μL样本加入以下试剂进行反应(按照Zymo Research公司的试剂盒进行操作，所用试剂均来自Zymo Research公司)：
置于PCR仪中按照以下程序进行反应：
当PCR反应第二步(95℃)达到5min时，立即将样本从PCR仪中取出，直接插入冰中，放置2min以上再进行下一步操作。
2.1.4、连接I
配制如下反应液(按照Zymo Research公司的试剂盒进行操作，所用试剂均来自Zymo Research公司)：
置于PCR仪中按照以下程序进行反应：
2.1.5、扩增I
配制如下反应液(按照Zymo Research公司的试剂盒进行操作，所用试剂均来自Zymo Research公司)：
置于PCR仪中按照以下程序进行反应：
2.1.6、纯化I：
加入166μL 1:6倍稀释的Beckman Agencourt AM Pure Beads(需提前于室温平衡半小时)的对扩增I反应后的产物进行纯化，用21μL EB进行洗脱，纯化具体步骤如下(按照Beckman公司的试剂盒进行操作)：
取上一步反应产物并进行离心，每个样本加入166μL 1:6倍稀释的Agencourt AM Pure Beads，用移液器吹打混匀。室温孵育5min。离心，置于磁力架上静置5min。吸去上清。加入200μL 80％乙醇(EtOH)，静置30s，吸走乙醇，重复一次后，离心，将PCR管置于磁力架上，吸走剩余乙醇，开盖干燥磁珠2-3min，注意不要过干。加入21μL EB进行洗脱，用移液器充分吹打混匀，室温静置3min。离心，将PCR管置于磁力架上，静置3min。吸取
20μL上清于新的PCR管中。
2.1.7、连接II
配制如下反应液：
置于PCR仪中按照以下程序进行反应：
2.1.8、Indexing PCR(扩增产物文库构建)：
配制如下反应液(KAPA HiFi Hot Start Ready Mix试剂盒进行，试剂来自该试剂盒)：
置于PCR仪中按照以下程序进行反应
2.1.9、纯化II
加入Agencourt AM Pure Beads(需提前于室温平衡半小时)对Indexing
PCR反应后的产物进行纯化，用41μL EB进行洗脱，纯化具体步骤如下：
取上一步反应产物并进行离心，每个样本加入71μL未稀释的Agencourt AM Pure Beads，用移液器吹打混匀。室温孵育5min。离心，置于磁力架上静置5min。吸去上清。加入200μl 80％EtOH，静置30s，吸走乙醇，重复步骤一次后，离心，将PCR管置于磁力架上，吸走剩余乙醇。开盖干燥磁珠2-3min，注意不要过干。加入41μL EB进行洗脱，用移液器充分吹打混匀，室温静置3min。离心，将PCR管置于磁力架上，静置3min。吸取20μl上清于新的PCR管中。Qubit定量：取1μL用Qubit dsDNA HS Assay Kit对文库进行定量。
2.2、通过对已建库后的样本进行寡核苷酸探针捕获富集，得到特定区域的上机终文库。杂交捕获试剂盒为IDT公司的xGen Lockdown Reagents，具体按照说明书进行操作。
2.3、采用Illumina公司的测序仪对杂交捕获后的样本进行测序，得到测序结果。
2.4、下机数据的分析：
对测序仪的下机原始数据，进行常规的生物信息学分析处理，先通过fastp过滤低质量(QC低，长度短、太多N等)的读长(reads)，然后去除reads双端的衔接子(adapter)、共有序列、PolyA/T，得到理想的插入片段序列(靶(target)区间)，使用bismark(v0.17.0)软件将这些reads比对hg19对应的位置后，根据UMI对reads进行去重，得到每份样本被探针捕获得到的真实reads数据(bam file)，对bam文件进行统计和分析，得到甲基化数据，用于后续的数据再分析。
2.5、对原始测序数据进行了相关清理及处理分析，并基于对reads读数来确定每一个甲基化位点甲基化胞嘧啶的百分比(β值)；利用DSS软件(R package，Version 2.38.0)进行差异分析，按照p值(p-value)<0.05，错误发现率(false discovery rate,FDR)<0.05,|diff|>0.1进行筛选，其中，diff表示，结直肠癌淋巴结转移受试者一甲基化位点的β值的平均
值(平均甲基化水平)与肠癌淋巴非结转移受试改甲基化位点的β值的平均值(平均甲基化水平)的差值，|diff|表示该差值的绝对值。
对两组样本进行比较分析，筛选到341个标记物，这341个标记物在19例结直肠癌淋巴结转移的癌和11例结直肠癌非淋巴结转移的癌的热图(heatmap)如图1所示，可以看出这些标记物在结直肠癌淋巴结转移和非转移之间存在显著性的甲基化水平上的差异。
2.6、通过对这341个标记物的基因注释信息，以及在19例结直肠癌淋巴结转移的癌和11例结直肠癌淋巴非结转移的癌每个标记物的AUC如下表1所示，其中单标记物AUC大于0.6的有341个，大于0.7有338个，大于0.8有266个，大于0.9有28个，显示这些标记物均具有良好的区分结直肠癌淋巴结转移与非转移的能力。
在下表1和表2中，根据2009年2月human genome assembly GRCh37/hg19(参见例如Rosenbloom等(2012)“ENCODE whole-genome data in the UCSC Genome Browser：update 2012”Nucleic Acids Research 40：D912-D917)对碱基编号。此外，表1和表2中，类型为基因间(intergenic)的基因一般不具有基因名称，因此“基因”一栏为空白。
表1：341个标记物基因注释信息及AUC
2.7、我们通过19例结直肠癌淋巴结转移的癌与11例结直肠癌非淋巴结转移的癌按照甲基化水平进行差异甲基化区域分析，将相邻的甲基化位点进行合并成为区域，这些区域涵盖341个标记物中的125个标记物，这125个标记
物在合并后，主要在基因间区chr13:20693341-20693653有35个CpG位点，CYTH2基因(chr19:48983509-48983698)有18个CpG位点，LBX2基因(chr2:74725882-74726332)有34个CpG位点，STMN3基因(chr20:62272199-62272548)有38个CpG位点，这125个标记物在19例淋巴结转移和11例淋巴结非转移受试者中的平均甲基化水平如下表2所示，如前所述，平均甲基化水平为每个甲基化位点分别在19例转移和11例非转移受试者，在该位点处的β值的平均值，利用DSS软件进行差异分析得到p-value值，显示出这些区域及区域内的CpG位点均具有良好的区分转移和非转移的能力。
如图2所示为上述125个标记物的热图，下表2是这125个标记物在19个淋巴结转移和11个淋巴结非转移受试者中平均甲基化水平，显示出在转移和非转移之间这些标记物的diff均在±0.1以上，p-value均小于0.05。
表2：125个标记物平均甲基化水平
2.8、取LBX2基因(chr2:74725882-74726332)内34个CpG位点任意CpG位点及组合，将前面所使用的30个样本按照5:5的比例切分100次，利用随机森林(Random Forest)建模，每一次切分里利用随机森林在训练(train)集合里面建模，用该模型在测试(test)集合里面计算每个样本的风险得分，风险得分对比标准诊断得到该甲基化区域组合的判别灵敏度、特异性、AUC、阴性预测值(Negative predictive value,NPV)以及阳性预测值(Positive predictive value,PPV)等。如下表3所示；显示在不同的生物标记物(marker)数的情况下，AUC均在0.86以上，说明这些生物标记物(marker)或者生物标记物的组合均具有良好的区分结直肠癌淋巴结转移和非转移的能力。
表3：
其中：3marker组包括：chr2:74726037、chr2:74726227、chr2:74726230；
5marker组包括：chr2:74726037、chr2:74726227、chr2:74726230、chr2:74726181、chr2:74726179；
7marker组包括：chr2:74726037、chr2:74726227、chr2:74726230、chr2:74726181、chr2:74726179、chr2:74725882、chr2:74726124；
10marker组包括：chr2:74726037、chr2:74726227、chr2:74726230、chr2:74726181、chr2:74726179、chr2:74725882、chr2:74726124、chr2:74726094、chr2:74726073、chr2:74726006；
20marker组包括：chr2:74726037、chr2:74726227、chr2:74726230、chr2:74726181、chr2:74726179、chr2:74725882、chr2:74726124、chr2:74726094、chr2:74726073、chr2:74726006、chr2:74726332、chr2:74726274、chr2:74726137、chr2:74726257、chr2:74726131、chr2:74726190、chr2:74726271、chr2:74726083、chr2:74725967、chr2:74726106；
34marker组包括：chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320、chr2:74726332。
2.9、取STMN3基因(chr20:62272199-62272548)内38个CpG位点任意CpG位点及组合，将30个样本按照5:5的比例切分100次，利用随机森林建模，每一次切分里利用随机森林在train集合里面建模，用该模型在test集合里面计算每个样本的风险得分，风险得分对比标准诊断得到该甲基化区域组合的判别灵敏度、特异性、AUC、NPV以及PPV等。如下表4所示；显示在不同的生物标记物数的情况下，AUC均在0.78以上，说明这些生物标记物(marker)或者生物标记物的组合均具有良好的区分结直肠癌淋巴结转移和非转移的能力。
表4：
其中：3marker组包括：chr20:62272362、chr20:62272293、chr20:62272371；
5marker组包括：chr20:62272362、chr20:62272293、chr20:62272371、chr20:62272387、chr20:62272448；
7marker组包括：chr20:62272362、chr20:62272293、chr20:62272371、chr20:62272387、chr20:62272448、chr20:62272382、chr20:62272341；
10marker组包括：chr20:62272362、chr20:62272293、chr20:62272371、chr20:62272387、chr20:62272448、chr20:62272382、chr20:62272341、chr20:62272199、chr20:62272381、chr20:62272313；
20marker组包括：chr20:62272362、chr20:62272293、chr20:62272371、chr20:62272387、chr20:62272448、chr20:62272382、chr20:62272341、chr20:62272199、chr20:62272381、chr20:62272313、chr20:62272446、chr20:62272478、chr20:62272434、chr20:62272480、chr20:62272513、chr20:62272453、chr20:62272403、chr20:62272547、chr20:62272388、
chr20:62272491；
38marker组包括：chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547、chr20:62272548。
2.10、取CYTH2基因(chr19:48983509-48983698)内18个CpG位点任意CpG位点及组合，将30个样本按照5:5的比例切分100次，利用随机森林建模，每一次切分里利用随机森林在train集合里面建模，用该模型在test集合里面计算每个样本的风险得分，风险得分对比标准诊断得到该甲基化区域组合的判别灵敏度、特异性、AUC、NPV以及PPV等。如下表5所示；显示在不同的生物标记物数的情况下，AUC均在0.7以上，说明这些生物标记物或者生物标记物的组合均具有良好的区分结直肠癌淋巴结转移和非转移的能力。
表5：
其中，3marker组包括：chr19:48983569、chr19:48983526、chr19:48983663；
5marker组包括：chr19:48983569、chr19:48983526、chr19:48983663、chr19:48983571、chr19:48983638；
7marker组包括：chr19:48983569、chr19:48983526、chr19:48983663、
chr19:48983571、chr19:48983638、chr19:48983596、chr19:48983629；
10marker组包括：chr19:48983569、chr19:48983526、chr19:48983663、chr19:48983571、chr19:48983638、chr19:48983596、chr19:48983629、chr19:48983666、chr19:48983606、chr19:48983509；
18marker组包括：chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679、chr19:48983698。
2.11、利用125个CpG位点(附表2)任意CpG位点及组合，将30个样本按照5:5的比例切分100次，利用随机森林建模，每一次切分里利用随机森林在train集合里面建模，用该模型在test集合里面计算每个样本的风险得分，风险得分对比标准诊断得到该甲基化区域组合的判别灵敏度、特异性、AUC、NPV以及PPV等。如下表6所示；显示在不同的生物标记物数的情况下，AUC均在0.85以上，说明这些生物标记物或者生物标记物的组合均具有良好的区分结直肠癌淋巴结转移和非转移的能力。
表6：
其中，5marker组合包括：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179；
10marker组合包括：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179、chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371；
20marker组合包括：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179、chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371、chr2:74726006、chr2:74726124、chr2:74726073、chr20:62272341、chr2:74726131、chr2:74726257、chr20:62272381、chr2:74726274、chr20:62272382、chr2:74726083；
50marker组合包括：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179、chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371、chr2:74726006、chr2:74726124、chr2:74726073、chr20:62272341、chr2:74726131、chr2:74726257、chr20:62272381、chr2:74726274、chr20:62272382、chr2:74726083、chr2:74726332、chr2:74726276、chr2:74726271、chr20:62272199、chr2:74725967、chr2:74726137、chr20:62272313、chr2:74726265、chr20:62272387、chr2:74725994、chr20:62272480、chr20:62272478、chr2:74726106、chr20:62272434、chr2:74726216、chr2:74726058、chr2:74726190、chr2:74725992、chr2:74726312、chr20:62272513、chr20:62272388、chr20:62272453、chr20:62272448、chr20:62272447、chr2:74726293、chr20:62272491、chr20:62272494、chr20:62272403、chr2:74725934、chr20:62272361；
100marker组合包括：chr2:74726230、chr2:74726227、chr2:74726037、chr2:74726181、chr2:74726179、chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371、chr2:74726006、chr2:74726124、chr2:74726073、chr20:62272341、chr2:74726131、chr2:74726257、chr20:62272381、chr2:74726274、chr20:62272382、chr2:74726083、chr2:74726332、chr2:74726276、chr2:74726271、chr20:62272199、chr2:74725967、chr2:74726137、chr20:62272313、chr2:74726265、chr20:62272387、chr2:74725994、chr20:62272480、chr20:62272478、chr2:74726106、chr20:62272434、chr2:74726216、chr2:74726058、chr2:74726190、chr2:74725992、chr2:74726312、chr20:62272513、chr20:62272388、chr20:62272453、
chr20:62272448、chr20:62272447、chr2:74726293、chr20:62272491、chr20:62272494、chr20:62272403、chr2:74725934、chr20:62272361、chr20:62272487、chr20:62272547、chr13:20693357、chr20:62272310、chr2:74726320、chr19:48983569、chr20:62272446、chr2:74725984、chr20:62272404、chr13:20693417、chr2:74725926、chr20:62272449、chr20:62272470、chr13:20693578、chr2:74725887、chr13:20693448、chr13:20693607、chr13:20693414、chr20:62272340、chr13:20693653、chr13:20693415、chr13:20693492、chr20:62272372、chr19:48983571、chr19:48983526、chr19:48983663、chr19:48983596、chr19:48983638、chr13:20693564、chr2:74725895、chr13:20693469、chr13:20693608、chr20:62272309、chr20:62272548、chr13:20693577、chr13:20693565、chr13:20693416、chr13:20693342、chr19:48983606、chr13:20693356、chr19:48983564、chr13:20693460、chr19:48983671、chr13:20693569、chr13:20693636、chr13:20693341、chr19:48983601、chr13:20693459、chr19:48983584、chr13:20693468；
125marker组合包括：附表2中全部标记物。
2.12、利用341个CpG位点(附表1)任意CpG位点及组合，将30个样本按照5:5的比例切分100次，利用随机森林建模，每一次切分里利用随机森林在train集合里面建模，用该模型在test集合里面计算每个样本的风险得分，风险得分对比标准诊断得到该甲基化区域组合的判别灵敏度、特异性、AUC、NPV以及PPV等。如下表7所示；显示在不同的生物标记物数的情况下，AUC均在0.88以上，说明这些生物标记物或者生物标记物的组合均具有良好的区分结直肠癌淋巴结转移和非转移的能力。
表7：
其中，5marker组包括：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230；
10marker组包括：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875；
20marker组包括：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875、chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312；
50marker组包括：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875、chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312、chr10:71338666、chr6:161655343、chr2:174424852、chr16:67427066、chr6:42931754、chr1:68295979、chr15:28381565、chr11:44820675、chr9:140157146、chr15:101097210、chr4:4039187、chr14:44168216、chr15:41264369、chr13:19174394、chr21:15497019、
chr15:70574296、chr2:74726094、chr2:74726006、chr13:97991180、chr15:101098460、chr19:13944958、chr4:55379617、chr2:74726131、chr9:37949852、chr8:10825518、chr11:119355687、chr16:5237372、chr2:74726073、chr2:74726271、chr8:10924461；
100marker组包括：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875、chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312、chr10:71338666、chr6:161655343、chr2:174424852、chr16:67427066、chr6:42931754、chr1:68295979、chr15:28381565、chr11:44820675、chr9:140157146、chr15:101097210、chr4:4039187、chr14:44168216、chr15:41264369、chr13:19174394、chr21:15497019、chr15:70574296、chr2:74726094、chr2:74726006、chr13:97991180、chr15:101098460、chr19:13944958、chr4:55379617、chr2:74726131、chr9:37949852、chr8:10825518、chr11:119355687、chr16:5237372、chr2:74726073、chr2:74726271、chr8:10924461、chr7:129752313、chr2:74726332、chr1:16553114、chr2:74726124、chr19:33863663、chr2:74726257、chr8:7004715、chr2:74726137、chr22:45363303、chr2:74726276、chr2:74725882、chr10:71338651、chr2:74726083、chr1:161450901、chr2:74726274、chr2:74725967、chr7:77687820、chr7:77687882、chr12:80462103、chr19:43778881、chr6:71980418、chr8:136263479、chr17:3548648、chr11:128590262、chr4:174462424、chr8:2480920、chr20:62272362、chr15:95662730、chr19:54172983、chr3:197832357、chr2:176624742、chr19:35408270、chr2:74726265、chr10:102648751、chr12:12766094、chr10:106093878、chr13:19174407、chr9:79138639、chr2:242755966、chr16:77147779、chr2:74725994、chr2:74726106、chr3:36174109、chr2:74726058、
chr19:38983253、chr12:1932190、chr3:197222253、chr6:144881470、chr1:161450899、chr2:74726216；
200marker组包括：chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037、chr2:74726230、chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875、chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312、chr10:71338666、chr6:161655343、chr2:174424852、chr16:67427066、chr6:42931754、chr1:68295979、chr15:28381565、chr11:44820675、chr9:140157146、chr15:101097210、chr4:4039187、chr14:44168216、chr15:41264369、chr13:19174394、chr21:15497019、chr15:70574296、chr2:74726094、chr2:74726006、chr13:97991180、chr15:101098460、chr19:13944958、chr4:55379617、chr2:74726131、chr9:37949852、chr8:10825518、chr11:119355687、chr16:5237372、chr2:74726073、chr2:74726271、chr8:10924461、chr7:129752313、chr2:74726332、chr1:16553114、chr2:74726124、chr19:33863663、chr2:74726257、chr8:7004715、chr2:74726137、chr22:45363303、chr2:74726276、chr2:74725882、chr10:71338651、chr2:74726083、chr1:161450901、chr2:74726274、chr2:74725967、chr7:77687820、chr7:77687882、chr12:80462103、chr19:43778881、chr6:71980418、chr8:136263479、chr17:3548648、chr11:128590262、chr4:174462424、chr8:2480920、chr20:62272362、chr15:95662730、chr19:54172983、chr3:197832357、chr2:176624742、chr19:35408270、chr2:74726265、chr10:102648751、chr12:12766094、chr10:106093878、chr13:19174407、chr9:79138639、chr2:242755966、chr16:77147779、chr2:74725994、chr2:74726106、chr3:36174109、chr2:74726058、chr19:38983253、chr12:1932190、chr3:197222253、chr6:144881470、chr1:161450899、chr2:74726216、chr20:24899315、chr6:144881495、
chr4:190963293、chr9:118501681、chr2:28700693、chr10:108412225、chr19:49891736、chr20:62272293、chr5:77165020、chr4:909786、chr16:2076342、chr6:144881587、chr2:32205788、chr13:19174421、chr1:54121906、chr3:197830661、chr17:62690049、chr3:197217049、chr6:144881549、chr2:36716536、chr2:74726293、chr5:476865、chr17:77047294、chr8:106967372、chr2:74726190、chr8:28157735、chr2:174853968、chr19:51483067、chr1:219944199、chr3:97973710、chr16:27431703、chr21:46238778、chr20:62272371、chr15:101096974、chr11:45743460、chr10:64613369、chr2:74725992、chr20:62272341、chr17:77181458、chr18:60020618、chr2:74725934、chr2:191625107、chr4:48945163、chr6:144881603、chr11:133800611、chr17:79855640、chr2:74726312、chr15:101097067、chr4:7128051、chr5:138861157、chr6:121069941、chr13:19174425、chr20:62272478、chr5:61065238、chr20:62272382、chr15:26543473、chr21:44197355、chr20:62272480、chr19:8211090、chr3:97982831、chr8:11062433、chr8:10825314、chr1:147827054、chr20:42427883、chr3:3225323、chr21:15497020、chr2:96990550、chr8:95627967、chr3:38618469、chr21:46238680、chr8:89712289、chr20:62272313、chr10:3870830、chr19:1545977、chr4:31261294、chr8:2181379、chr1:9327169、chr10:71338662、chr16:88619484、chr5:138861242、chr20:62272434、chr2:74726320、chr5:2749278、chr12:9856328、chr11:133800801、chr4:41299758、chr6:35892438、chr2:242789465、chr15:91305688、chr2:74725984、chr14:74439495、chr6:170338877、chr4:1404633、chr20:62272381、chr10:3870880、chr22:24253568、chr7:18901757、chr5:103114389、chr12:53216896、chr2:54952741；
341marker组包括附表1中全部甲基化位点。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未
对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围，因此，本发明专利的保护范围应以所附权利要求为准。
附表1(与表1中位点对应)：
1号染色体(chr1)基因组位点：
chr1:147827054、chr1:161450899、chr1:161450901、chr1:16553114、chr1:166690923、chr1:173375804、chr1:219944199、chr1:54121906、chr1:68295979、chr1:9327169；
10号染色体(chr10)基因组位点：
chr10:102648751、chr10:106093878、chr10:108412225、chr10:131724514、chr10:3870825、chr10:3870830、chr10:3870833、chr10:3870842、chr10:3870873、chr10:3870880、chr10:62452853、chr10:64613369、chr10:71338651、chr10:71338662、chr10:71338666；
11号染色体(chr11)基因组位点：
chr11:119355687、chr11:128590262、chr11:133800611、chr11:133800801、chr11:44820675、chr11:45743460；
12号染色体(chr12)基因组位点：
chr12:11423121、chr12:12766094、chr12:1932190、chr12:53216896、chr12:80462103、chr12:9856328；
13号染色体(chr13)基因组位点：
chr13:113373213、chr13:19174394、chr13:19174407、chr13:19174421、chr13:19174425、chr13:20693341、chr13:20693342、chr13:20693356、
chr13:20693357、chr13:20693359、chr13:20693360、chr13:20693384、chr13:20693385、chr13:20693414、chr13:20693415、chr13:20693416、chr13:20693417、chr13:20693447、chr13:20693448、chr13:20693459、chr13:20693460、chr13:20693468、chr13:20693469、chr13:20693491、chr13:20693492、chr13:20693564、chr13:20693565、chr13:20693569、chr13:20693570、chr13:20693571、chr13:20693572、chr13:20693577、chr13:20693578、chr13:20693607、chr13:20693608、chr13:20693631、chr13:20693632、chr13:20693636、chr13:20693637、chr13:20693653、chr13:97991180；
14号染色体(chr14)基因组位点：
chr14:44168216、chr14:74439495；
15号染色体(chr15)基因组位点：
chr15:101096974、chr15:101097067、chr15:101097210、chr15:101098460、chr15:26543473、chr15:28381565、chr15:41264369、chr15:70574296、chr15:91305688、chr15:95662730；
16号染色体(chr16)基因组位点：
chr16:2076342、chr16:27431703、chr16:34521898、chr16:5237372、chr16:67427066、chr16:71260701、chr16:77147779、chr16:88619484；
17号染色体(chr17)基因组位点：
chr17:10101011、chr17:3548648、chr17:58637740、chr17:62690049、chr17:77047294、chr17:77181458、chr17:79855640、chr17:8658715；
18号染色体(chr18)基因组位点：
chr18:112353、chr18:54240802、chr18:60020618、chr18:67901956、chr18:67901984、chr18:67902044、chr18:67902075、chr18:7084620；
19号染色体(chr19)基因组位点：
chr19:13944958、chr19:1545977、chr19:33863663、chr19:35408269、chr19:35408270、chr19:38983253、chr19:43778881、chr19:4745045、
chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679、chr19:48983698、chr19:49891736、chr19:51017312、chr19:51483067、chr19:54172983、chr19:8211090；
2号染色体(chr2)基因组位点：
chr2:174424852、chr2:174853968、chr2:176624742、chr2:191625107、chr2:208666424、chr2:242755966、chr2:242789465、chr2:28700693、chr2:32205788、chr2:36716536、chr2:54952741、chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320、chr2:74726332、chr2:96990550；
20号染色体(chr20)基因组位点：
chr20:24899315、chr20:42427883、chr20:60740447、chr20:60740448、chr20:60745017、chr20:60745018、chr20:60745185、chr20:60745224、chr20:60745246、chr20:60750111、chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、
chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547、chr20:62272548；
21号染色体(chr21)基因组位点：
chr21:15497019、chr21:15497020、chr21:30527898、chr21:30527912、chr21:30527927、chr21:30527936、chr21:30527966、chr21:30527984、chr21:44197355、chr21:46238680、chr21:46238778；
22号染色体(chr22)基因组位点：
chr22:24253568、chr22:38123993、chr22:40504031、chr22:45363303、chr22:50706553；
3号染色体(chr3)基因组位点：
chr3:157614707、chr3:197217049、chr3:197222253、chr3:197222254、chr3:197828361、chr3:197830661、chr3:197832357、chr3:197832359、chr3:20317639、chr3:3225323、chr3:36174109、chr3:38618469、chr3:97973710、chr3:97982831；
4号染色体(chr4)基因组位点：
chr4:1404633、chr4:174462424、chr4:190963243、chr4:190963254、chr4:190963293、chr4:190963359、chr4:31261294、chr4:4039187、chr4:41299758、chr4:48945163、chr4:55379617、chr4:6574370、chr4:7128051、chr4:909786；
5号染色体(chr5)基因组位点：
chr5:103114389、chr5:122429706、chr5:138861107、chr5:138861157、chr5:138861242、chr5:138861258、chr5:170349468、chr5:2749278、chr5:476865、chr5:61065238、chr5:77165020、chr5:93019223；
6号染色体(chr6)基因组位点：
chr6:121069941、chr6:130005127、chr6:144881470、chr6:144881495、chr6:144881549、chr6:144881587、chr6:144881603、chr6:161655343、chr6:170338877、chr6:35892438、chr6:41251759、chr6:42931754、chr6:48950482、
chr6:71980418；
7号染色体(chr7)基因组位点：
chr7:121081132、chr7:121081133、chr7:121081142、chr7:121081143、chr7:121081187、chr7:121081188、chr7:129752313、chr7:18901757、chr7:21742092、chr7:77687820、chr7:77687875、chr7:77687882、chr7:90895650；
8号染色体(chr8)基因组位点：
chr8:106967372、chr8:10825314、chr8:10825518、chr8:10924461、chr8:11062433、chr8:136263479、chr8:2181379、chr8:2480920、chr8:28157735、chr8:56013334、chr8:7004715、chr8:89712289、chr8:95627967；
9号染色体(chr9)基因组位点：
chr9:117064345、chr9:118501681、chr9:140157146、chr9:37949852、chr9:69260268、chr9:79138639。
附表2(与表2中位点对应)：
13号染色体(chr13)基因组位点：
chr13:20693341、chr13:20693342、chr13:20693356、chr13:20693357、chr13:20693359、chr13:20693360、chr13:20693384、chr13:20693385、chr13:20693414、chr13:20693415、chr13:20693416、chr13:20693417、chr13:20693447、chr13:20693448、chr13:20693459、chr13:20693460、chr13:20693468、chr13:20693469、chr13:20693491、chr13:20693492、chr13:20693564、chr13:20693565、chr13:20693569、chr13:20693570、chr13:20693571、chr13:20693572、chr13:20693577、chr13:20693578、chr13:20693607、chr13:20693608、chr13:20693631、chr13:20693632、chr13:20693636、chr13:20693637、chr13:20693653；
19号染色体(chr19)基因组位点：
chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、
chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679、chr19:48983698；
2号染色体(chr2)基因组位点：
chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320、chr2:74726332；
20号染色体(chr20)基因组位点：
chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547、chr20:62272548。
Claims (18)
- 一种用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化生物标记物包括附表1中提供的甲基化位点中的任一项或其任意组合。
- 根据权利要求1所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化生物标记物包括附表2中提供的甲基化位点中的任一项或其任意组合。
- 根据权利要求1或2所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化位点至少包括chr2:74726037、chr2:74726227和chr2:74726230中的至少一种；任选地，所述的甲基化位点还包括chr2:74726181、chr2:74726179中的至少一种；任选地，所述的甲基化位点还包括chr20:62272362、chr2:74725882、chr20:62272293、chr2:74726094、chr20:62272371中的至少一种；任选地，所述的甲基化位点还包括chr2:74726006、chr2:74726124、chr2:74726073、chr20:62272341、chr2:74726131、chr2:74726257、chr20:62272381、chr2:74726274、chr20:62272382、chr2:74726083中的至少一种；任选地，所述的甲基化位点还包括chr2:74726332、chr2:74726276、chr2:74726271、chr20:62272199、chr2:74725967、chr2:74726137、chr20:62272313、chr2:74726265、chr20:62272387、chr2:74725994、chr20:62272480、chr20:62272478、chr2:74726106、chr20:62272434、chr2:74726216、chr2:74726058、chr2:74726190、chr2:74725992、chr2:74726312、chr20:62272513、chr20:62272388、chr20:62272453、chr20:62272448、chr20:62272447、chr2:74726293、chr20:62272491、chr20:62272494、chr20:62272403、chr2:74725934、chr20:62272361中的至少一种；任选地，所述的甲基化位点还包括chr20:62272487、chr20:62272547、chr13:20693357、chr20:62272310、chr2:74726320、chr19:48983569、 chr20:62272446、chr2:74725984、chr20:62272404、chr13:20693417、chr2:74725926、chr20:62272449、chr20:62272470、chr13:20693578、chr2:74725887、chr13:20693448、chr13:20693607、chr13:20693414、chr20:62272340、chr13:20693653、chr13:20693415、chr13:20693492、chr20:62272372、chr19:48983571、chr19:48983526、chr19:48983663、chr19:48983596、chr19:48983638、chr13:20693564、chr2:74725895、chr13:20693469、chr13:20693608、chr20:62272309、chr20:62272548、chr13:20693577、chr13:20693565、chr13:20693416、chr13:20693342、chr19:48983606、chr13:20693356、chr19:48983564、chr13:20693460、chr19:48983671、chr13:20693569、chr13:20693636、chr13:20693341、chr19:48983601、chr13:20693459、chr19:48983584、chr13:20693468中的至少一种；进一步地，所述的甲基化位点包括chr13:20693341、chr13:20693342、chr13:20693356、chr13:20693357、chr13:20693359、chr13:20693360、chr13:20693384、chr13:20693385、chr13:20693414、chr13:20693415、chr13:20693416、chr13:20693417、chr13:20693447、chr13:20693448、chr13:20693459、chr13:20693460、chr13:20693468、chr13:20693469、chr13:20693491、chr13:20693492、chr13:20693564、chr13:20693565、chr13:20693569、chr13:20693570、chr13:20693571、chr13:20693572、chr13:20693577、chr13:20693578、chr13:20693607、chr13:20693608、chr13:20693631、chr13:20693632、chr13:20693636、chr13:20693637、chr13:20693653、chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679、chr19:48983698、chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、 chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320、chr2:74726332、chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547和chr20:62272548。
- 根据权利要求1～3中任一项所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化生物标记物包括来自13号染色体基因组区域20693341-20693653的位点、2号染色体基因组区域74725882-74726332的位点、20号染色体基因组区域62272199-62272548的位点和/或19号染色体基因组区域48983509-48983698的位点。
- 根据权利要求1～4中任一项所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化位点包括来自2号染色体基因组区域74725882-74726332的位点；任选地，所述的甲基化位点包括chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、 chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320和chr2:74726332中的任一项或其任意组合。
- 根据权利要求5所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化位点至少包括chr2:74726037、chr2:74726227和chr2:74726230中的至少一种；任选地，所述的甲基化位点还包括chr2:74726181、chr2:74726179中的至少一种；任选地，所述的甲基化位点还包括chr2:74725882、chr2:74726124中的至少一种；任选地，所述的甲基化位点还包括chr2:74726094、chr2:74726073、chr2:74726006中的至少一种；任选地，所述的甲基化位点还包括chr2:74726332、chr2:74726274、chr2:74726137、chr2:74726257、chr2:74726131、chr2:74726190、chr2:74726271、chr2:74726083、chr2:74725967、chr2:74726106中的至少一种；进一步地，所述的甲基化位点包括chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320和chr2:74726332。
- 根据权利要求1～6中任一项所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化位点包括来自20号染色体基因组区域 62272199-62272548的位点；任选地，所述的甲基化位点包括chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547和chr20:62272548中的任一项或其任意组合。
- 根据权利要求7所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化位点至少包括chr20:62272362、chr20:62272293和chr20:62272371中的至少一种；任选地，所述的甲基化位点还包括chr20:62272387、chr20:62272448中的至少一种；任选地，所述的甲基化位点还包括chr20:62272382、chr20:62272341中的至少一种；任选地，所述的甲基化位点还包括chr20:62272199、chr20:62272381、chr20:62272313中的至少一种；任选地，所述的甲基化位点还包括chr20:62272446、chr20:62272478、chr20:62272434、chr20:62272480、chr20:62272513、chr20:62272453、chr20:62272403、chr20:62272547、chr20:62272388、chr20:62272491中的至少一种；进一步地，所述的甲基化位点包括chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、 chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547和chr20:62272548。
- 根据权利要求1～8中任一项所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化位点包括来自19号染色体基因组区域48983509-48983698的位点；任选地，所述的甲基化位点包括chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679和chr19:48983698中的任一项或其任意组合。
- 根据权利要求9所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化位点至少包括chr19:48983569、chr19:48983526和chr19:48983663中的至少一种；任选地，所述的甲基化位点还包括chr19:48983571、chr19:48983638中的至少一种；任选地，所述的甲基化位点还包括chr19:48983596、chr19:48983629中的至少一种；任选地，所述的甲基化位点还包括chr19:48983666、chr19:48983606、chr19:48983509中的至少一种；进一步地，所述的甲基化位点包括chr19:48983509、chr19:48983526、 chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679和chr19:48983698。
- 根据权利要求1～10中任一项所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化位点包括来自13号染色体基因组区域20693341-20693653的位点；任选地，所述的甲基化位点包括chr13:20693341、chr13:20693342、chr13:20693356、chr13:20693357、chr13:20693359、chr13:20693360、chr13:20693384、chr13:20693385、chr13:20693414、chr13:20693415、chr13:20693416、chr13:20693417、chr13:20693447、chr13:20693448、chr13:20693459、chr13:20693460、chr13:20693468、chr13:20693469、chr13:20693491、chr13:20693492、chr13:20693564、chr13:20693565、chr13:20693569、chr13:20693570、chr13:20693571、chr13:20693572、chr13:20693577、chr13:20693578、chr13:20693607、chr13:20693608、chr13:20693631、chr13:20693632、chr13:20693636、chr13:20693637和chr13:20693653中的任一项或其任意组合。
- 根据权利要求1所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述的甲基化生物标记物至少包括chr20:62272433、chr22:50706553、chr19:35408269、chr2:74726037和chr2:74726230中的至少一种；任选地，所述的甲基化生物标记物还包括chr22:40504031、chr3:197832359、chr2:74726227、chr3:197828361、chr7:77687875中的至少一种；任选地，所述的甲基化生物标记物还包括chr2:208666424、chr2:74726181、chr2:74726179、chr1:173375804、chr3:197222254、chr19:4745045、chr6:41251759、chr18:7084620、chr1:166690923、chr19:51017312中的至少一 种；任选地，所述的甲基化生物标记物还包括chr10:71338666、chr6:161655343、chr2:174424852、chr16:67427066、chr6:42931754、chr1:68295979、chr15:28381565、chr11:44820675、chr9:140157146、chr15:101097210、chr4:4039187、chr14:44168216、chr15:41264369、chr13:19174394、chr21:15497019、chr15:70574296、chr2:74726094、chr2:74726006、chr13:97991180、chr15:101098460、chr19:13944958、chr4:55379617、chr2:74726131、chr9:37949852、chr8:10825518、chr11:119355687、chr16:5237372、chr2:74726073、chr2:74726271、chr8:10924461中的至少一种；任选地，所述的甲基化生物标记物还包括chr7:129752313、chr2:74726332、chr1:16553114、chr2:74726124、chr19:33863663、chr2:74726257、chr8:7004715、chr2:74726137、chr22:45363303、chr2:74726276、chr2:74725882、chr10:71338651、chr2:74726083、chr1:161450901、chr2:74726274、chr2:74725967、chr7:77687820、chr7:77687882、chr12:80462103、chr19:43778881、chr6:71980418、chr8:136263479、chr17:3548648、chr11:128590262、chr4:174462424、chr8:2480920、chr20:62272362、chr15:95662730、chr19:54172983、chr3:197832357、chr2:176624742、chr19:35408270、chr2:74726265、chr10:102648751、chr12:12766094、chr10:106093878、chr13:19174407、chr9:79138639、chr2:242755966、chr16:77147779、chr2:74725994、chr2:74726106、chr3:36174109、chr2:74726058、chr19:38983253、chr12:1932190、chr3:197222253、chr6:144881470、chr1:161450899、chr2:74726216中的至少一种；任选地，所述的甲基化生物标记物还包括chr20:24899315、chr6:144881495、chr4:190963293、chr9:118501681、chr2:28700693、chr10:108412225、chr19:49891736、chr20:62272293、chr5:77165020、chr4:909786、chr16:2076342、chr6:144881587、chr2:32205788、chr13:19174421、 chr1:54121906、chr3:197830661、chr17:62690049、chr3:197217049、chr6:144881549、chr2:36716536、chr2:74726293、chr5:476865、chr17:77047294、chr8:106967372、chr2:74726190、chr8:28157735、chr2:174853968、chr19:51483067、chr1:219944199、chr3:97973710、chr16:27431703、chr21:46238778、chr20:62272371、chr15:101096974、chr11:45743460、chr10:64613369、chr2:74725992、chr20:62272341、chr17:77181458、chr18:60020618、chr2:74725934、chr2:191625107、chr4:48945163、chr6:144881603、chr11:133800611、chr17:79855640、chr2:74726312、chr15:101097067、chr4:7128051、chr5:138861157、chr6:121069941、chr13:19174425、chr20:62272478、chr5:61065238、chr20:62272382、chr15:26543473、chr21:44197355、chr20:62272480、chr19:8211090、chr3:97982831、chr8:11062433、chr8:10825314、chr1:147827054、chr20:42427883、chr3:3225323、chr21:15497020、chr2:96990550、chr8:95627967、chr3:38618469、chr21:46238680、chr8:89712289、chr20:62272313、chr10:3870830、chr19:1545977、chr4:31261294、chr8:2181379、chr1:9327169、chr10:71338662、chr16:88619484、chr5:138861242、chr20:62272434、chr2:74726320、chr5:2749278、chr12:9856328、chr11:133800801、chr4:41299758、chr6:35892438、chr2:242789465、chr15:91305688、chr2:74725984、chr14:74439495、chr6:170338877、chr4:1404633、chr20:62272381、chr10:3870880、chr22:24253568、chr7:18901757、chr5:103114389、chr12:53216896、chr2:54952741中的至少一种；进一步地，所述的甲基化生物标记物包括chr1:147827054、chr1:161450899、chr1:161450901、chr1:16553114、chr1:166690923、chr1:173375804、chr1:219944199、chr1:54121906、chr1:68295979、chr1:9327169、chr10:102648751、chr10:106093878、chr10:108412225、chr10:131724514、chr10:3870825、chr10:3870830、chr10:3870833、chr10:3870842、chr10:3870873、chr10:3870880、chr10:62452853、chr10:64613369、chr10:71338651、 chr10:71338662、chr10:71338666、chr11:119355687、chr11:128590262、chr11:133800611、chr11:133800801、chr11:44820675、chr11:45743460、chr12:11423121、chr12:12766094、chr12:1932190、chr12:53216896、chr12:80462103、chr12:9856328、chr13:113373213、chr13:19174394、chr13:19174407、chr13:19174421、chr13:19174425、chr13:20693341、chr13:20693342、chr13:20693356、chr13:20693357、chr13:20693359、chr13:20693360、chr13:20693384、chr13:20693385、chr13:20693414、chr13:20693415、chr13:20693416、chr13:20693417、chr13:20693447、chr13:20693448、chr13:20693459、chr13:20693460、chr13:20693468、chr13:20693469、chr13:20693491、chr13:20693492、chr13:20693564、chr13:20693565、chr13:20693569、chr13:20693570、chr13:20693571、chr13:20693572、chr13:20693577、chr13:20693578、chr13:20693607、chr13:20693608、chr13:20693631、chr13:20693632、chr13:20693636、chr13:20693637、chr13:20693653、chr13:97991180、chr14:44168216、chr14:74439495、chr15:101096974、chr15:101097067、chr15:101097210、chr15:101098460、chr15:26543473、chr15:28381565、chr15:41264369、chr15:70574296、chr15:91305688、chr15:95662730、chr16:2076342、chr16:27431703、chr16:34521898、chr16:5237372、chr16:67427066、chr16:71260701、chr16:77147779、chr16:88619484、chr17:10101011、chr17:3548648、chr17:58637740、chr17:62690049、chr17:77047294、chr17:77181458、chr17:79855640、chr17:8658715、chr18:112353、chr18:54240802、chr18:60020618、chr18:67901956、chr18:67901984、chr18:67902044、chr18:67902075、chr18:7084620、chr19:13944958、chr19:1545977、chr19:33863663、chr19:35408269、chr19:35408270、chr19:38983253、chr19:43778881、chr19:4745045、chr19:48983509、chr19:48983526、chr19:48983564、chr19:48983569、chr19:48983571、chr19:48983584、chr19:48983596、chr19:48983601、chr19:48983606、 chr19:48983608、chr19:48983629、chr19:48983638、chr19:48983653、chr19:48983663、chr19:48983666、chr19:48983671、chr19:48983679、chr19:48983698、chr19:49891736、chr19:51017312、chr19:51483067、chr19:54172983、chr19:8211090、chr2:174424852、chr2:174853968、chr2:176624742、chr2:191625107、chr2:208666424、chr2:242755966、chr2:242789465、chr2:28700693、chr2:32205788、chr2:36716536、chr2:54952741、chr2:74725882、chr2:74725887、chr2:74725895、chr2:74725926、chr2:74725934、chr2:74725967、chr2:74725984、chr2:74725992、chr2:74725994、chr2:74726006、chr2:74726037、chr2:74726058、chr2:74726073、chr2:74726083、chr2:74726094、chr2:74726106、chr2:74726124、chr2:74726131、chr2:74726137、chr2:74726179、chr2:74726181、chr2:74726190、chr2:74726216、chr2:74726227、chr2:74726230、chr2:74726257、chr2:74726265、chr2:74726271、chr2:74726274、chr2:74726276、chr2:74726293、chr2:74726312、chr2:74726320、chr2:74726332、chr2:96990550、chr20:24899315、chr20:42427883、chr20:60740447、chr20:60740448、chr20:60745017、chr20:60745018、chr20:60745185、chr20:60745224、chr20:60745246、chr20:60750111、chr20:62272199、chr20:62272293、chr20:62272309、chr20:62272310、chr20:62272312、chr20:62272313、chr20:62272340、chr20:62272341、chr20:62272361、chr20:62272362、chr20:62272371、chr20:62272372、chr20:62272381、chr20:62272382、chr20:62272387、chr20:62272388、chr20:62272403、chr20:62272404、chr20:62272433、chr20:62272434、chr20:62272446、chr20:62272447、chr20:62272448、chr20:62272449、chr20:62272453、chr20:62272470、chr20:62272478、chr20:62272480、chr20:62272487、chr20:62272491、chr20:62272494、chr20:62272513、chr20:62272521、chr20:62272522、chr20:62272531、chr20:62272532、chr20:62272547、chr20:62272548、chr21:15497019、chr21:15497020、chr21:30527898、chr21:30527912、chr21:30527927、chr21:30527936、chr21:30527966、chr21:30527984、 chr21:44197355、chr21:46238680、chr21:46238778、chr22:24253568、chr22:38123993、chr22:40504031、chr22:45363303、chr22:50706553、chr3:157614707、chr3:197217049、chr3:197222253、chr3:197222254、chr3:197828361、chr3:197830661、chr3:197832357、chr3:197832359、chr3:20317639、chr3:3225323、chr3:36174109、chr3:38618469、chr3:97973710、chr3:97982831、chr4:1404633、chr4:174462424、chr4:190963243、chr4:190963254、chr4:190963293、chr4:190963359、chr4:31261294、chr4:4039187、chr4:41299758、chr4:48945163、chr4:55379617、chr4:6574370、chr4:7128051、chr4:909786、chr5:103114389、chr5:122429706、chr5:138861107、chr5:138861157、chr5:138861242、chr5:138861258、chr5:170349468、chr5:2749278、chr5:476865、chr5:61065238、chr5:77165020、chr5:93019223、chr6:121069941、chr6:130005127、chr6:144881470、chr6:144881495、chr6:144881549、chr6:144881587、chr6:144881603、chr6:161655343、chr6:170338877、chr6:35892438、chr6:41251759、chr6:42931754、chr6:48950482、chr6:71980418、chr7:121081132、chr7:121081133、chr7:121081142、chr7:121081143、chr7:121081187、chr7:121081188、chr7:129752313、chr7:18901757、chr7:21742092、chr7:77687820、chr7:77687875、chr7:77687882、chr7:90895650、chr8:106967372、chr8:10825314、chr8:10825518、chr8:10924461、chr8:11062433、chr8:136263479、chr8:2181379、chr8:2480920、chr8:28157735、chr8:56013334、chr8:7004715、chr8:89712289、chr8:95627967、chr9:117064345、chr9:118501681、chr9:140157146、chr9:37949852、chr9:69260268和chr9:79138639。
- 根据权利要求1～12中任一项所述的用于结直肠癌淋巴结转移诊断的甲基化生物标记物，其中，所述诊断是区分患有结直肠癌的受试者是否存在淋巴结转移；任选地，所述结直肠癌选自TNM分期中T分期的T1期、T2期、T3期或T4期的结直肠癌。
- 权利要求1～13中任一项所述的甲基化生物标记物在制备用于诊断患有结直肠癌的受试者是否存在淋巴结转移的试剂或试剂盒中的用途。
- 一种用于结直肠癌淋巴结转移诊断的试剂盒，其中，所述的试剂盒包含用于检测待测样本中权利要求1～13中任一项所述的甲基化生物标记物的甲基化水平的试剂。
- 根据权利要求15所述的用于结直肠癌淋巴结转移诊断的试剂盒，其中，所述的试剂为选自以下的检测甲基化水平的方法中所使用的试剂：荧光定量PCR、甲基化特异性PCR、数字PCR、DNA甲基化芯片、靶向DNA甲基化测序、全基因组甲基化测序和DNA甲基化质谱中的一种或多种。
- 根据权利要求15或16中所述的用于结直肠癌淋巴结转移诊断的试剂盒，其中，所述的待测样本选自组织、血液、血浆、唾液、血清、尿液、尿液脱落细胞、尿沉渣、尿液上清中的一种或多种。
- 一种用于结直肠癌淋巴结转移诊断的系统，其中，所述系统包括检测装置、计算装置和输出装置；所述检测装置包括进样器和检测器，所述进样器用于采集来自患有结直肠癌的受试者的样本，所述检测器用于检测所述样本中权利要求1～13中任一项所述的甲基化生物标记物的甲基化水平；所述计算装置包括存储器和处理器，所述存储器中存储有计算机程序，所述处理器被配置为执行所述存储器中存储的计算机程序，以实现如下判别：所述样本中所述的甲基化生物标记物的甲基化水平不同于不存在淋巴结转移的患有结直肠癌的受试者中测定的甲基化生物标记物的甲基化水平，则判别所述样本对应的受试者的存在结直肠癌淋巴结转移。
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