WO2023011615A1 - 一种肿瘤评估方法及应用 - Google Patents

一种肿瘤评估方法及应用 Download PDF

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WO2023011615A1
WO2023011615A1 PCT/CN2022/110445 CN2022110445W WO2023011615A1 WO 2023011615 A1 WO2023011615 A1 WO 2023011615A1 CN 2022110445 W CN2022110445 W CN 2022110445W WO 2023011615 A1 WO2023011615 A1 WO 2023011615A1
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region
nucleic acid
dna region
present application
human
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PCT/CN2022/110445
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English (en)
French (fr)
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王辉
杨其昌
苏志熙
马成城
刘蕊
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江苏鹍远生物技术有限公司
上海鹍远生物科技股份有限公司
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Priority claimed from CN202110903691.6A external-priority patent/CN115896277A/zh
Priority claimed from CN202110902676.XA external-priority patent/CN115896275A/zh
Priority claimed from CN202110903841.3A external-priority patent/CN115927606A/zh
Priority claimed from CN202110902703.3A external-priority patent/CN115896276A/zh
Application filed by 江苏鹍远生物技术有限公司, 上海鹍远生物科技股份有限公司 filed Critical 江苏鹍远生物技术有限公司
Priority to EP22852326.2A priority Critical patent/EP4382617A1/en
Publication of WO2023011615A1 publication Critical patent/WO2023011615A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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

  • This application relates to the field of biomedicine, in particular to a tumor assessment method and its application.
  • tumor cell-free DNA detection technology based on liquid biopsy has a higher demand for the discovery of tumor markers.
  • the basic principle is: after the death of tumor cells, free DNA is released into the blood, and by detecting tumor cell-related information such as DNA mutation, DNA methylation, miRNA, and histone modification in the blood, very early tumor signals can be detected. Studies have found that changes in DNA methylation may be earlier than the occurrence of DNA mutations, and are more effective detection markers for early screening of tumors.
  • the method provided in this application can use more efficient methylation markers to confirm or assist in confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease, which can improve the early detection of tumors, such as liver tumors.
  • the efficiency of screening and early diagnosis can solve the problems of low early diagnosis rate of liver cancer and heavy burden of clinical treatment.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, comprising determining the modification status of the DNA region or fragments thereof where the target gene is located in the sample to be tested
  • the presence and/or content of the target gene comprises SEPT9 and IKZF1.
  • the present application also provides a method for assessing the methylation state of a liver tumor-associated DNA region, comprising determining the presence and/or content of the modification state of the DNA region where the target gene is located or a fragment thereof in the sample to be tested, said Target genes include SEPT9 and IKZF1.
  • the present application also provides a method for confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease, including determining the target DNA region in the sample to be tested, or its complementary region, or the above-mentioned
  • the presence and/or content of the modified state of the fragment, the target DNA region comprises the region defined from human chr17:75368651-75370720 and derived from human chr7:50343720-50344547.
  • the present application also provides a method for determining the methylation state of a DNA region, comprising determining the presence and/or content of the modification state of the target DNA region or its complementary region, or the above-mentioned fragments in the sample to be tested,
  • the target DNA region includes regions defined from human chr17:75368651-75370720 and human chr7:50343720-50344547.
  • the present application also provides a nucleic acid comprising the DNA region capable of binding to the target gene as described in the method of the present application, or its complementary region, or the above-mentioned transformed region, or the above-mentioned
  • the sequence of the fragment; or the nucleic acid comprises a sequence capable of binding to the target DNA region as described in the method of the present application, or its complementary region, or the above-mentioned converted region, or the above-mentioned fragment.
  • the present application also provides a method for preparing nucleic acid, said method comprising the DNA region where the target gene is located according to the method of the present application, or its complementary region, or the above-mentioned transformed region, or
  • a method for preparing nucleic acid comprising the DNA region where the target gene is located according to the method of the present application, or its complementary region, or the above-mentioned transformed region, or
  • the modified state of the target DNA region, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment is designed to be able to bind to the target DNA region, or its complementary region, or the above-mentioned transformed Regions, or nucleic acids of fragments of the above.
  • the present application also provides a nucleic acid group, which comprises the DNA region capable of binding to the target gene as described in the method of the present application, or its complementary region, or the above-mentioned transformed region, or The sequence of the above-mentioned fragment; or the nucleic acid group comprises the sequence capable of binding to the target DNA region as described in the method of the present application, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the present application also provides a method for preparing a nucleic acid group, the method comprising the DNA region where the target gene is located, or its complementary region, or the above-mentioned transformed region according to the method of the present application, Or the modified state of the above-mentioned fragments, designing a nucleic acid group that can be combined with the DNA region where the target gene is located, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragments; or the method includes according to the present application
  • the modified state of the target DNA region, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment is designed to be able to combine with the target DNA region, or its complementary region, or the above-mentioned transformed The nucleic acid group of the region, or the above-mentioned fragment.
  • the present application also provides a kit comprising the nucleic acid of the present application and/or the nucleic acid group of the present application.
  • the present application also provides the application of the nucleic acid of the present application, the nucleic acid set of the present application and/or the kit of the present application in the preparation of substances for determining the modification status of DNA regions or fragments thereof.
  • the present application also provides the application of the nucleic acid of the present application, the nucleic acid group of the present application and/or the kit of the present application in the preparation of disease detection products.
  • the present application also provides the nucleic acid of the present application, the nucleic acid group of the present application and/or the kit of the present application, which can be used to confirm the existence of the disease, assess the formation of the disease or the risk of forming the disease, and/or assess the progress of the disease. application of the substance.
  • the present application also provides a nucleic acid, a nucleic acid group and/or a kit for determining the modification state of a DNA region, which is used for confirming the existence of liver tumors, evaluating the formation of liver tumors or the risk of forming liver tumors, and/or assessing the presence of liver tumors.
  • the application of the advanced substance, the DNA region used for determination comprises the DNA region where the target gene is located or a fragment thereof as described in the method of the present application.
  • the present application also provides a nucleic acid, a nucleic acid group and/or a kit for determining the modification state of a DNA region, prepared for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease
  • the DNA region used for determination includes the target DNA region as described in the method of the present application, or its complementary region, or the above-mentioned fragments.
  • the present application also provides the DNA region where the target gene is located in the method of the present application, or its transformed region, or the nucleic acid of the above-mentioned fragments or a combination thereof, which can be used to confirm the presence of liver tumors , Use of a substance for assessing liver neoplasia or the risk of developing and/or assessing the progression of a liver neoplasia.
  • the present application also provides the nucleic acid of the target DNA region, or its complementary region, or the above-mentioned converted region, or the above-mentioned fragments or a combination thereof, as described in the method of the present application, for confirmation during preparation.
  • the presence of a disease the use of a substance in assessing the development or risk of developing a disease and/or assessing the progression of a disease.
  • the present application also provides a storage medium, which records a program capable of running the method of the present application.
  • the present application also provides a device, which includes the storage medium of the present application.
  • Figures 1, 3, 5, and 7 show the comparison of DNA methylation signals in paracancerous tissue, cancer tissue, and leukocyte at the detection site.
  • Figures 2, 4, 6, and 8 show the comparison of DNA methylation signals of detection sites in control plasma and liver cancer plasma.
  • BCAT1 generally refers to a gene or its expression product.
  • the UniProt accession number of BCAT1 (Branched-chain-amino-acid aminotransferase) protein may be P54687.
  • BCAT1 may cover its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • VASH2 generally refers to a gene or its expression product.
  • the UniProt accession number of VASH2 (Tubulinyl-Tyr carboxypeptidase 2) protein may be Q86V25.
  • VASH2 may encompass its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • GPAM generally refers to a gene or its expression product.
  • the UniProt accession number of the GPAM (Glycerol-3-phosphate acyltransferase 1, mitochondrial) protein may be Q9HCL2.
  • GPAM may encompass its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • VAV3 generally refers to a gene or its expression product.
  • the UniProt accession number of VAV3 (Guanine nucleotide exchange factor VAV3) protein may be Q9UKW4.
  • VAV3 may encompass its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • SEPT9 generally refers to a gene or its expression product.
  • the UniProt accession number for SEPT9 (SEPTIN9) protein can be Q9UHD8.
  • SEPT9 may cover its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • SEPT9 in this application may also represent a detection site, such as methylation detection for the specific nucleic acid sequence of the gene.
  • the detection site represented by SEPT9 may be the nucleic acid sequence of one or more regions under the gene.
  • IKZF1 generally refers to a gene or its expression product.
  • the UniProt accession number of IKZF1 (DNA-binding protein Ikaros) protein may be Q13422.
  • IKZF1 may cover its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • IKZF1 may also represent a detection site, such as methylation detection for the specific nucleic acid sequence of the gene.
  • the detection site represented by IKZF1 may be the nucleic acid sequence of one or more regions under the gene.
  • the term "BEST4" generally refers to a gene or its expression product.
  • the UniProt accession number of the BEST4 (Bestrophin-4) protein may be Q8NFU0.
  • BEST4 may cover its unprocessed form, any processed form, its variant or substances comprising its functionally active fragments.
  • BEST4 in this application may also represent a detection site, such as methylation detection for the specific nucleic acid sequence of the gene.
  • the detection site represented by BEST4 may be the nucleic acid sequence of one or more regions under the gene.
  • B4GALNT1 generally refers to a gene or its expression product.
  • the UniProt accession number of the B4GALNT1 (Beta-1,4N-acetylgalactosaminyltransferase 1) protein may be Q00973.
  • B4GALNT1 may cover its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • B4GALNT1 in the present application may also represent a detection site, such as methylation detection for the specific nucleic acid sequence of the gene.
  • the detection site represented by B4GALNT1 may be the nucleic acid sequence of one or more regions under the gene.
  • GRASP GRASP1 (General receptor for phosphoinositides 1)-associated scaffold protein) protein
  • the UniProt accession number of GRASP GRASP1 (General receptor for phosphoinositides 1)-associated scaffold protein) protein
  • GRASP may cover its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • GRASP in the present application may also represent a detection site, such as methylation detection for the specific nucleic acid sequence of the gene.
  • the detection site represented by GRASP can be the nucleic acid sequence of one or more regions under the gene.
  • IRF4 generally refers to a gene or its expression product.
  • the UniProt accession number of IRF4 (Interferon regulatory factor 4) protein may be Q15306.
  • IRF4 may encompass its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • IRF4 may also represent a detection site, such as methylation detection for the specific nucleic acid sequence of the gene.
  • the detection site represented by IRF4 may be the nucleic acid sequence of one or more regions under the gene.
  • BEND4 generally refers to a gene or its expression product.
  • the UniProt accession number of BEND4 (BEN domain-containing protein 4) protein can be Q6ZU67.
  • BEND4 may cover its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • BEND4 may also represent a detection site, for example, methylation detection is performed on a specific nucleic acid sequence of the gene.
  • the detection site represented by BEND4 may be the nucleic acid sequence of one or more regions under the gene.
  • sample to be tested generally refers to a sample that needs to be tested. For example, it can be detected whether one or more gene regions on the sample to be tested are modified.
  • cell-free nucleic acid generally refers to DNA in a sample that is not contained within cells when collected.
  • cell-free nucleic acid may not refer to DNA that is rendered intracellular by in vitro disruption of cells or tissues.
  • cfDNA can include both normal cell and cancer cell-derived DNA.
  • cfDNA can be obtained from blood or plasma ("circulatory system").
  • cfDNA can be released into the circulation through secretion or through cell death processes such as necrosis or apoptosis.
  • complementary nucleic acid generally refers to a nucleotide sequence that is complementary to a reference nucleotide sequence.
  • a complementary nucleic acid can be a nucleic acid molecule that optionally has an opposite orientation.
  • the complementary may refer to having the following complementary associations: guanine and cytosine; adenine and thymine; adenine and uracil.
  • DNA region generally refers to a sequence of two or more covalently bonded naturally occurring or modified deoxyribonucleotides.
  • a DNA region of a gene may refer to the location of a specific sequence of deoxyribonucleotides where the gene is located, eg, the sequence of deoxyribonucleotides encodes the gene.
  • the DNA region of the present application includes the entire length of the DNA region, its complementary region, or a fragment thereof. For example, a sequence of at least about 20 kb upstream and downstream of the detection region provided in the present application can be used as the detection site.
  • At least about 20 kb, at least about 15 kb, at least about 10 kb, at least about 5 kb, at least about 3 kb, at least about 2 kb, at least about 1 kb, or at least about 0.5 kb of the sequence upstream and downstream of the region provided by the application can be used as a detection site.
  • suitable primers and probes can be designed according to the microcomputer to detect the methylation of the sample.
  • the term "modification state” generally refers to the modification state of the gene fragment, nucleotide or its base in the present application.
  • the modification state in the present application may refer to the modification state of cytosine.
  • a gene segment of the present application having a modified state may have altered gene expression activity.
  • the modification status of the present application may refer to the methylation modification of a base.
  • the modified state in this application may refer to the covalent bonding of a methyl group at the 5' carbon position of cytosine in the CpG region of genomic DNA, for example, it may become 5-methylcytosine (5mC).
  • a modification state can refer to the presence or absence of 5-methylcytosine ("5-mCyt") within the DNA sequence.
  • methylation generally refers to the methylation state of a gene fragment, nucleotide or its base in this application.
  • the DNA fragment where the gene in this application is located may have methylation on one strand or multiple strands.
  • the DNA fragment where the gene in this application is located may have methylation at one site or multiple sites.
  • transformation generally refers to the transformation of one or more structures into another structure.
  • the transformations of the present application can be specific.
  • cytosine without methylation modification can be converted into other structures (such as uracil), and cytosine with methylation modification can be substantially unchanged after conversion.
  • cytosine without methylation modification can be cleaved after conversion, and cytosine with methylation modification can be substantially unchanged after conversion.
  • a deamination reagent generally refers to a substance having the ability to remove an amino group.
  • a deamination reagent can deaminate unmodified cytosine.
  • bisulfite generally refers to a reagent that can distinguish between DNA regions that have a modified state and those that do not.
  • bisulfite may include bisulfite, or an analog thereof, or a combination thereof.
  • bisulfite can deaminate the amino group of unmodified cytosine to distinguish it from modified cytosine.
  • analogue generally refers to a substance having a similar structure and/or function.
  • an analog of bisulfite may have a similar structure to bisulfite.
  • an analog of bisulfite may refer to a reagent that can also distinguish between DNA regions that have a modified state and those that do not.
  • methylation-sensitive restriction enzyme generally refers to an enzyme that selectively digests nucleic acid according to the methylation status of its recognition site.
  • a restriction enzyme that specifically cleaves when the recognition site is not methylated may not cleavage, or cleave at a significantly reduced efficiency, when the recognition site is methylated.
  • restriction enzymes that cleave specifically when the recognition site is methylated cleavage may not occur, or be cleaved with significantly reduced efficiency, when the recognition site is not methylated.
  • a methylation-specific restriction enzyme can recognize a sequence containing a CG dinucleotide (eg, cgcg or cccggg).
  • tumor generally refers to cells and/or tissues that exhibit at least partial loss of control in normal growth and/or development.
  • a common tumor or cancer cell may often have lost contact inhibition and may be invasive and/or have the ability to metastasize.
  • the tumors of the present application may be benign or malignant.
  • progression generally refers to a change in disease from a less severe state to a more severe state.
  • tumor progression can include an increase in the number or severity of the tumor, the degree to which cancer cells have metastasized, the rate at which the cancer grows or spreads, and the like.
  • tumor progression can include the progression of the stage of the cancer from a less advanced state to a more advanced state, eg, from stage I to stage II, from stage II to stage III, etc.
  • the term "formation” generally refers to the appearance of a lesion in an individual.
  • the individual can be diagnosed as a tumor patient.
  • fluorescent PCR generally refers to a quantitative or semi-quantitative PCR technique.
  • PCR techniques can be real-time quantitative polymerase chain reaction, quantitative polymerase chain reaction or kinetic polymerase chain reaction.
  • PCR amplification can be used to quantify the initial amount of target nucleic acid with the aid of intercalating fluorescent dyes or sequence-specific probes that can contain fluorescent reporters detectable only by hybridization to the target nucleic acid .
  • PCR amplification generally refers to the polymerase chain amplification reaction.
  • PCR amplification in the present application may include any currently known polymerase chain amplification reaction for DNA amplification.
  • fluorescence Ct value generally refers to a measurement value for quantitative or semi-quantitative evaluation of a target nucleic acid. For example, it may refer to the number of amplification reaction cycles experienced when the fluorescent signal reaches a set threshold value.
  • This application can find a variety of efficient liver tumor-related methylation markers, improve the efficiency of early screening and diagnosis of liver tumors, and solve the problems of low early diagnosis rate of liver cancer and heavy burden of clinical treatment.
  • this application provides multiple sets of efficient liver tumor methylation markers, and lists efficient marker combinations.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region or its fragments where the target gene is located in the sample to be tested
  • the presence and/or content of said target gene may comprise SEPT9 and IKZF1.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the target gene is located or its fragment in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification status of the DNA region where the target gene is located or its fragment in the test sample, assessing whether liver tumor formation is diagnosed.
  • the method of the present application may include, according to the determination results of the existence and/or content of the modification state of the DNA region where the target gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification status of the DNA region where the target gene is located or its fragment in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-associated DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the target gene is located or a fragment thereof in the sample to be tested.
  • Genes may comprise SEPT9 and IKZF1.
  • the methylation status of the liver tumor-related DNA region is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the DNA region of SEPT9 can be derived from human chr17:75276651-75496678.
  • the DNA region of IKZF1 may be derived from human chr7:50343720-50472799.
  • the target gene may further comprise a gene selected from the following group: BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, BEST4 and B4GALNT1.
  • the target gene may comprise SEPT9, IKZF1, BEST4, GRASP and B4GALNT1.
  • the target gene may comprise SEPT9, IKZF1, BEST4, IRF4, B4GALNT1 and BEND4.
  • the target gene may comprise at least two genes.
  • the target gene may include 2 to 7 genes.
  • the target gene of the present application may comprise 2, 3, 4, 5, 6, or 7 target genes provided in the present application.
  • the present application can select one or more DNA regions where the target gene is located.
  • the DNA region of BEST4 can be derived from human chr1:45249257-45253377.
  • the DNA region of B4GALNT1 can be derived from human chr12:58017193-58027138.
  • the DNA region of GRASP can be derived from human chr12:52400724-52409673.
  • the DNA region of IRF4 can be derived from human chr6:391739-411447.
  • the DNA region of BEND4 can be derived from human chr4:42112955-42154895.
  • the target gene may include two genes selected from the following group: SEPT9, IKZF1, BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise SEPT9 and IKZF1.
  • the target gene may comprise SEPT9 and BEST4.
  • the target gene may comprise SEPT9 and B4GALNT1.
  • the target gene may comprise SEPT9 and GRASP.
  • the target gene may comprise SEPT9 and IRF4.
  • the target gene may comprise SEPT9 and BEND4.
  • the target gene may comprise IKZF1 and BEST4.
  • the target gene may comprise IKZF1 and B4GALNT1.
  • the target gene may comprise IKZF1 and GRASP.
  • the target gene may comprise IKZF1 and IRF4.
  • the target gene may comprise IKZF1 and BEND4.
  • the target gene may include BEST4 and B4GALNT1.
  • the target gene may include BEST4 and GRASP.
  • the target gene may comprise BEST4 and IRF4.
  • the target gene may comprise BEST4 and BEND4.
  • the target gene may comprise B4GALNT1 and GRASP.
  • the target gene may comprise B4GALNT1 and IRF4.
  • the target gene may comprise B4GALNT1 and BEND4.
  • the target gene may comprise GRASP and IRF4.
  • the target gene may comprise GRASP and BEND4.
  • the target gene may comprise IRF4 and BEND4.
  • the target gene may include three genes selected from the following group: SEPT9, IKZF1, BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise SEPT9, IKZF1 and BEST4.
  • the target gene may comprise SEPT9, IKZF1 and B4GALNT1.
  • the target gene may comprise SEPT9, IKZF1 and GRASP.
  • the target gene may comprise SEPT9, IKZF1 and IRF4.
  • the target gene may comprise SEPT9, IKZF1 and BEND4.
  • the target gene may comprise SEPT9, BEST4 and B4GALNT1.
  • the target gene may comprise SEPT9, BEST4 and GRASP.
  • the target gene may comprise SEPT9, BEST4 and IRF4.
  • the target gene may comprise SEPT9, BEST4 and BEND4.
  • the target gene may comprise SEPT9, B4GALNT1 and GRASP.
  • the target gene may comprise SEPT9, B4GALNT1 and IRF4.
  • the target gene may comprise SEPT9, B4GALNT1 and BEND4.
  • the target gene may comprise SEPT9, GRASP and IRF4.
  • the target gene may comprise SEPT9, GRASP and BEND4.
  • the target gene may comprise SEPT9, IRF4 and BEND4.
  • the target gene may comprise IKZF1, BEST4 and B4GALNT1.
  • the target gene may comprise IKZF1, BEST4 and GRASP.
  • the target gene may comprise IKZF1, BEST4 and IRF4.
  • the target gene may comprise IKZF1, BEST4 and BEND4.
  • the target gene may comprise IKZF1, B4GALNT1 and GRASP.
  • the target gene may comprise IKZF1, B4GALNT1 and IRF4.
  • the target gene may comprise IKZF1, B4GALNT1 and BEND4.
  • the target gene may comprise IKZF1, GRASP and IRF4.
  • the target gene may comprise IKZF1, GRASP and BEND4.
  • the target gene may comprise IKZF1, IRF4 and BEND4.
  • the target gene may comprise BEST4, B4GALNT1 and GRASP.
  • the target gene may comprise BEST4, B4GALNT1 and IRF4.
  • the target gene may comprise BEST4, B4GALNT1 and BEND4.
  • the target gene may comprise BEST4, GRASP and IRF4.
  • the target gene may comprise BEST4, GRASP and BEND4.
  • the target gene may comprise BEST4, IRF4 and BEND4.
  • the target gene may comprise B4GALNT1, GRASP and IRF4.
  • the target gene may comprise B4GALNT1, GRASP and BEND4.
  • the target gene may comprise B4GALNT1, IRF4 and BEND4.
  • the target gene may comprise GRASP, IRF4 and BEND4.
  • the target gene may include four genes selected from the following group: SEPT9, IKZF1, BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, BEST4 and B4GALNT1.
  • the target gene may comprise SEPT9, IKZF1, BEST4 and GRASP.
  • the target gene may comprise SEPT9, IKZF1, BEST4 and IRF4.
  • the target gene may comprise SEPT9, IKZF1, BEST4 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, B4GALNT1 and GRASP.
  • the target gene may comprise SEPT9, IKZF1, B4GALNT1 and IRF4.
  • the target gene may comprise SEPT9, IKZF1, B4GALNT1 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, GRASP and IRF4.
  • the target gene may comprise SEPT9, IKZF1, GRASP and BEND4.
  • the target gene may comprise SEPT9, IKZF1, IRF4 and BEND4.
  • the target gene may comprise SEPT9, BEST4, B4GALNT1 and GRASP.
  • the target gene may comprise SEPT9, BEST4, B4GALNT1 and IRF4.
  • the target gene may comprise SEPT9, BEST4, B4GALNT1 and BEND4.
  • the target gene may comprise SEPT9, BEST4, GRASP and IRF4.
  • the target gene may comprise SEPT9, BEST4, GRASP and BEND4.
  • the target gene may comprise SEPT9, BEST4, IRF4 and BEND4.
  • the target gene may comprise SEPT9, B4GALNT1, GRASP and IRF4.
  • the target gene may comprise SEPT9, B4GALNT1, GRASP and BEND4.
  • the target gene may comprise SEPT9, B4GALNT1, IRF4 and BEND4.
  • the target gene may comprise SEPT9, GRASP, IRF4 and BEND4.
  • the target gene may comprise IKZF1, BEST4, B4GALNT1 and GRASP.
  • the target gene may comprise IKZF1, BEST4, B4GALNT1 and IRF4.
  • the target gene may comprise IKZF1, BEST4, B4GALNT1 and BEND4.
  • the target gene may comprise IKZF1, BEST4, GRASP and IRF4.
  • the target gene may comprise IKZF1, BEST4, GRASP and BEND4.
  • the target gene may comprise IKZF1, BEST4, IRF4 and BEND4.
  • the target gene may comprise IKZF1, B4GALNT1, GRASP and IRF4.
  • the target gene may comprise IKZF1, B4GALNT1, GRASP and BEND4.
  • the target gene may comprise IKZF1, B4GALNT1, IRF4 and BEND4.
  • the target gene may comprise IKZF1, GRASP, IRF4 and BEND4.
  • the target gene may comprise BEST4, B4GALNT1, GRASP and IRF4.
  • the target gene may comprise BEST4, B4GALNT1, GRASP and BEND4.
  • the target gene may comprise BEST4, B4GALNT1, IRF4 and BEND4.
  • the target gene may comprise B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise 5 genes selected from the following group: SEPT9, IKZF1, BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, B4GALNT1 and GRASP.
  • the target gene may comprise SEPT9, IKZF1, BEST4, B4GALNT1 and IRF4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, B4GALNT1 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, GRASP and IRF4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, GRASP and BEND4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, IRF4 and BEND4.
  • the target gene may comprise SEPT9, BEST4, B4GALNT1, GRASP and IRF4.
  • the target gene may comprise SEPT9, BEST4, B4GALNT1, GRASP and BEND4.
  • the target gene may comprise SEPT9, BEST4, B4GALNT1, IRF4 and BEND4.
  • the target gene may comprise SEPT9, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise IKZF1, BEST4, B4GALNT1, GRASP and IRF4.
  • the target gene may comprise IKZF1, BEST4, B4GALNT1, GRASP and BEND4.
  • the target gene may comprise IKZF1, BEST4, B4GALNT1, IRF4 and BEND4.
  • the target gene may comprise IKZF1, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may include 6 genes selected from the following group: SEPT9, IKZF1, BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, B4GALNT1, GRASP and IRF4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, B4GALNT1, GRASP and BEND4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, B4GALNT1, IRF4 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, GRASP, IRF4 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise SEPT9, BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise IKZF1, BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise seven genes selected from the following group: SEPT9, IKZF1, BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target gene may comprise SEPT9, IKZF1, BEST4, B4GALNT1, GRASP, IRF4 and BEND4.
  • the target DNA region located in the DNA region where the SEPT9 gene is located in the method of the present application may include a region derived from the definition of human chr17:75368651-75370720.
  • the target DNA region can be represented by SEPT(1).
  • the target DNA region located in the DNA region where the IKZF1 gene is located in the method of the present application may include a region defined from human chr7:50343720-50344547.
  • a region defined from human chr7:50343793-50343896 For example, the target DNA region can be represented by IKZF1(1).
  • the target DNA region located in the DNA region where the BEST4 gene is located in the method of the present application may include a region derived from the definition of human chr1:45251728-45252477.
  • the target DNA region can be represented by BEST4(1).
  • the target DNA region located in the DNA region where the B4GALNT1 gene is located in the method of the present application may include a region derived from the definition of human chr12:58020498-58022962. For example derived from the region defined by human chr12:58021586-58021670.
  • the target DNA region can be represented by B4GALNT1(1).
  • the target DNA region located in the DNA region where the GRASP gene is located in the method of the present application may include the region defined in human chr12:52400724-52401698.
  • the target DNA region can be represented by GRASP(1).
  • the target DNA region located in the DNA region where the IRF4 gene is located in the method of the present application may include the region defined in human chr6:391739-394056.
  • the target DNA region can be represented by IRF4(1).
  • the target DNA region located in the DNA region where the BEND4 gene is located in the method of the present application may include a region derived from the definition of human chr4:42152705-42154895.
  • a region derived from the region defined by human chr4:42153816-42153921 derived from the region defined by human chr4:42153816-42153921.
  • the target DNA region can be represented by BEND4(1).
  • the target DNA region located in the DNA region where the SEPT9 gene is located in the method of the present application may include a region derived from the definition of human chr17:75368651-75370720.
  • the target DNA region can be represented by SEPT9(1a).
  • the present application provides a method for confirming the existence of a disease, evaluating the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a specific subregion of the DNA region where the target gene is located in the sample to be tested (such as the target DNA region), or its complementary region, or the presence and/or content of the modified state of the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining the target DNA region in the sample to be tested, or its complementary region, or the above-mentioned fragments
  • the presence and/or content of the modified state of the target DNA region may include regions derived from human chr17:75368651-75370720 and derived from human chr7:50343720-50344547.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the target DNA region or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination results of the presence and/or content of the modification state of the target DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the target DNA region or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the target DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include determining the presence and/or content of the modification state of the target DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, so
  • the target DNA region may include regions defined from human chr17:75368651-75370720 and human chr7:50343720-50344547.
  • the confirmed presence or increased amount of methylation of the target DNA region relative to a reference level can be correlated with the development of a disease.
  • the target DNA region in the present application may refer to one or more specific segments of the target genomic DNA.
  • a target DNA region of the present application can be specified by a gene name or a set of chromosomal coordinates.
  • the target DNA region represented by SEPT9 may be one or more specific segments of the DNA region where the SEPT9 gene is located.
  • the target DNA region represented by SEPT9 may be one or more specific segments of the target DNA region in the SEPT9 gene provided in this application.
  • one or more different specific segments of SEPT9 can be represented separately by distinguishable forms such as SEPT9(1) and SEPT9(1a).
  • a gene can have its sequence and chromosomal position by reference to its name, or its sequence and chromosomal position can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may comprise providing a nucleic acid capable of binding to a DNA region comprising SEQ ID NO: 1, or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the target region may include a region defined from human chr17:75369558-75369622.
  • the target DNA region can be represented by SEPT(1).
  • the method of the present application can comprise providing the nucleic acid shown in SEQ ID NO: 2 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise providing the nucleic acid set shown in SEQ ID NO: 3 and 4 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the probe and/or primer set described above can detect and/or amplify a DNA region derived from the region defined by human chr17:75369558-75369622.
  • the method of the present application may comprise providing a nucleic acid capable of binding to a DNA region comprising SEQ ID NO: 5, or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the target region may include a region defined from human chr7:50343793-50343896.
  • the target DNA region can be represented by IKZF1(1).
  • the method of the present application can comprise providing the nucleic acid shown in SEQ ID NO: 6 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise providing the nucleic acid set shown in SEQ ID NO: 7 and 8 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the aforementioned probe and/or primer sets can detect and/or amplify a region of DNA derived from the region defined by human chr7:50343793-50343896.
  • the target DNA region may also include a region defined by the following group: derived from human chr1: 45251728-45252477, derived from human chr12: 58020498-58022962, derived from human chr12: 52400724-52401698 , from human chr6:391739-394056, and from human chr4:42152705-42154895.
  • the target DNA region may comprise at least two regions.
  • the target DNA region may comprise 2 to 8 regions.
  • the target DNA region may comprise 2, 3, 4, 5, 6, 7, or 8 regions.
  • the target DNA region may comprise human chr17:75368651-75370720, human chr7:50343720-50344547, human chr1:45251728-45252477 and human chr12:58020498-58022962 defined area.
  • the target DNA region may include human chr17:75368651-75370720, human chr7:50343720-50344547, human chr1:45251728-45252477, human chr12:52400724-52401698 and derived from the region defined by human chr12:58020498-58022962.
  • the target DNA region may include human chr6: 391739-394056, human chr7: 50343720-50344547, human chr1: 45251728-45252477, human chr12: 58020498-58022962 , derived from human chr4:42152705-42154895 and derived from the region defined by human chr17:75368651-75370720.
  • the method of the present application may comprise providing a nucleic acid capable of binding to a DNA region comprising SEQ ID NO: 9, or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the target region may include a region defined from human chr1:45252095-45252176.
  • the target DNA region can be represented by BEST4(1).
  • the method of the present application can comprise providing the nucleic acid shown in SEQ ID NO: 10 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise providing the nucleic acid set shown in SEQ ID NO: 11 and 12 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the aforementioned probe and/or primer sets can detect and/or amplify a region of DNA derived from the region defined by human chr1:45252095-45252176.
  • the method of the present application may comprise providing a nucleic acid capable of binding to a DNA region comprising SEQ ID NO: 13, or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the target region may include a region defined from human chr12:58021586-58021670.
  • the target DNA region can be represented by B4GALNT1(1).
  • the method of the present application can comprise providing the nucleic acid shown in SEQ ID NO: 14 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise providing the nucleic acid set shown in SEQ ID NO: 15 and 16 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the aforementioned probe and/or primer sets can detect and/or amplify a DNA region derived from the region defined by human chr12:58021586-58021670.
  • the method of the present application may comprise providing a nucleic acid capable of binding to a DNA region comprising SEQ ID NO: 17, or a complementary region thereof, or the above-mentioned converted region, or the above-mentioned fragment.
  • the target region may include a region defined from human chr12:52401083-52401169.
  • the target DNA region can be represented by GRASP(1).
  • the method of the present application can comprise providing the nucleic acid shown in SEQ ID NO: 18 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise providing the nucleic acid set shown in SEQ ID NO: 19 and 20 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the aforementioned probe and/or primer sets can detect and/or amplify a DNA region derived from the region defined by human chr12:52401083-52401169.
  • the method of the present application may comprise providing a nucleic acid capable of binding to a DNA region comprising SEQ ID NO: 21, or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the target region may include a region defined from human chr6:392282-392377.
  • the target DNA region can be represented by IRF4(1).
  • the method of the present application can comprise providing the nucleic acid shown in SEQ ID NO: 22 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise providing the nucleic acid set shown in SEQ ID NO: 23 and 24 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the probe and/or primer sets described above can detect and/or amplify a region of DNA derived from the region defined by human chr6:392282-392377.
  • the method of the present application may comprise providing a nucleic acid capable of binding to a DNA region comprising SEQ ID NO: 25, or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the target region may include a region defined from human chr4:42153816-42153921.
  • the target DNA region can be represented by BEND4(1).
  • the method of the present application can comprise providing the nucleic acid shown in SEQ ID NO: 26 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise providing the nucleic acid set shown in SEQ ID NO: 27 and 28 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the aforementioned probe and/or primer sets can detect and/or amplify a region of DNA derived from the region defined by human chr4:42153816-42153921.
  • the method of the present application may comprise providing a nucleic acid capable of binding to a DNA region comprising SEQ ID NO: 1, or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the target region may include a region defined from human chr17:75369603-75369693.
  • the target DNA region can be represented by SEPT9(1a).
  • the method of the present application can comprise providing the nucleic acid shown in SEQ ID NO: 29 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise providing the nucleic acid set shown in SEQ ID NO: 30 and 31 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the aforementioned probe and/or primer sets can detect and/or amplify a region of DNA derived from the region defined by human chr17:75369603-75369693.
  • one or more of the aforementioned target regions can serve as amplification regions and/or detection regions.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region where the BCAT1 gene is located or its fragments in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the BCAT1 gene is located or its fragment in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the BCAT1 gene is located or its fragment in the test sample, assessing whether hepatic neoplasia is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the BCAT1 gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the BCAT1 gene is located or its fragment in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the BCAT1 gene is located or a fragment thereof in the sample to be tested. For example, according to the determination of the presence and/or content of the modification state of the DNA region where the BCAT1 gene is located or its fragment in the sample to be tested, the methylation status of the liver tumor-related DNA region is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr12:24964295-25102393.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease, which may include determining a specific subregion of the DNA region where the BCAT1 gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, evaluating the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following group in the sample to be tested, or a complementary region thereof, or The existence and/or content of the modified state of the above fragments: derived from human chr12:25101630-25102393, derived from human chr12:25078661-25079410 and derived from human chr12:25054781-25056311.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr12:25101630-25102393, derived from human chr12:25078661-25079410 and derived from human chr12:25054781-25056311.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal location by reference to its name, or its sequence and chromosomal location can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation status of a DNA region, which may include: determining the DNA region derived from human chr12:25101630-25102393 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the present application provides a method for determining the methylation status of a DNA region, which may include: determining the DNA region derived from human chr:12:25078661-25079410 in the sample to be tested, or its complementary region, or the above-mentioned fragments The presence and/or content of the modified state.
  • the present application provides a method for determining the methylation status of a DNA region, which may include: determining the DNA region derived from human chr12: 25054781-25056311 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 32, SEQ ID NO: 39 and SEQ ID NO: 43.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr12: 25102016-25102110, derived from human chr12: 25101992-25102093, derived from human chr12: 25079051-25079133 and derived from human chr12: 25056027-25056134 .
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr12: 25102016-25102110, derived from human chr12: 25101992-25102093, derived from human chr12: 25079051-25079133 and derived from human chr12: 25056027-25056134.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr12:25102016-25102110 , derived from human chr12:25101992-25102093, derived from human chr12:25079051-25079133 and derived from human chr12:25056027-25056134.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or complementary nucleic acid thereof, or above-mentioned fragment: SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:40 and SEQ ID NO:44.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application can comprise: providing the nucleic acid shown in SEQ ID NO: 33 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr12:25101630-25102393.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:25102016-25102110.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 36 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr12:25101630-25102393.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:25101992-25102093.
  • the method of the present application can comprise: providing the nucleic acid shown in SEQ ID NO: 40 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr:12:25078661-25079410.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:25079051-25079133.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 44 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr12:25054781-25056311.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:25056027-25056134.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid group or its complementary nucleic acid group, or above-mentioned fragment: SEQ ID NO:34 and 35, SEQ ID NO:37 and 38, SEQ ID NO:41 and 42 and SEQ ID NO: 45 and 46.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 34 and 35 or its complementary nucleic acid group, or the above-mentioned fragments.
  • this nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr12:25101630-25102393.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:25102016-25102110.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 37 and 38 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr12:25101630-25102393.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:25101992-25102093.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 41 and 42 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr:12:25078661-25079410.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:25079051-25079133.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 45 and 46 or its complementary nucleic acid set, or the above-mentioned fragments.
  • this nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:25054781-25056311.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:25056027-25056134.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region or fragments thereof where the IKZF1 gene is located in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the IKZF1 gene is located or its fragment in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination result of the existence and/or content of the modification status of the DNA region where the IKZF1 gene is located or its fragment in the test sample, assessing whether hepatic neoplasia is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the IKZF1 gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of being diagnosed with liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the IKZF1 gene is located or its fragment in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the IKZF1 gene is located or a fragment thereof in the sample to be tested. For example, according to the determination of the presence and/or content of the modification state of the DNA region where the IKZF1 gene is located or its fragment in the sample to be tested, the methylation status of the DNA region associated with liver tumors is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr7:50343720:50472799.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease, which may include determining a specific subregion of the DNA region where the IKZF1 gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, evaluating the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following group in the sample to be tested, or a complementary region thereof, or The presence and/or content of the modified state of the above fragments: derived from human chr7: 50343720-50344547, derived from human chr7: 50450118-50450531 and derived from human chr7: 50467368-50469092.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr7: 50343720-50344547, derived from human chr7: 50450118-50450531 and derived from human chr7: 50467368-50469092.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal location by reference to its name, or its sequence and chromosomal location can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation status of a DNA region, which may include: determining the DNA region derived from human chr7: 50343720-50344547 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the present application provides a method for determining the methylation status of a DNA region, which may include: determining the DNA region derived from human chr7: 50450118-50450531 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr7: 50467368-50469092 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 5, SEQ ID NO: 50 and SEQ ID NO: 54.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above fragments: derived from human chr7: 50343793-50343896, derived from human chr7: 50343867-50343961, derived from human chr7: 50450311-50450411 and derived from human chr7: 50467865-50467980 .
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr7: 50343793-50343896, derived from human chr7: 50343867-50343961, derived from human chr7: 50450311-50450411 and derived from human chr7: 50467865-50467980.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr7: 50343793-50343896 , derived from human chr7:50343867-50343961, derived from human chr7:50450311-50450411 and derived from human chr7:50467865-50467980.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or its complementary nucleic acid, or above-mentioned fragment: SEQ ID NO:6, SEQ ID NO:47, SEQ ID NO:51 and SEQ ID NO:55.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application can comprise: providing the nucleic acid shown in SEQ ID NO: 6 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr7:50343720-50344547.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr7:50343793-50343896.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 47 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr7:50343720-50344547.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr7:50343867-50343961.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 51 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr7:50450118-50450531.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr7:50450311-50450411.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 55 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr7:50467368-50469092.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr7:50467865-50467980.
  • the method of the present application may include: providing a nucleic acid group or its complementary nucleic acid group that may be selected from the following group, or the above-mentioned fragments: SEQ ID NO: 7 and 8, SEQ ID NO: 48 and 49, SEQ ID NO: 52 and 53 and SEQ ID NO: 56 and 57.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 7 and 8 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr7:50343720-50344547.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr7:50343793-50343896.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 48 and 49 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr7:50343720-50344547.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr7:50343867-50343961.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 52 and 53 or its complementary nucleic acid group, or the above-mentioned fragments.
  • this nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr7:50450118-50450531.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr7:50450311-50450411.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 56 and 57 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr7:50467368-50469092.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr7:50467865-50467980.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region where the VAV3 gene is located or its fragments in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the VAV3 gene is located or its fragment in the sample to be tested, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the VAV3 gene is located or its fragment in the test sample, assessing whether hepatic neoplasia is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the VAV3 gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the VAV3 gene is located or its fragment in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the VAV3 gene is located or its fragment in the sample to be tested. For example, according to the determination of the presence and/or content of the modification state of the DNA region where the VAV3 gene is located or its fragment in the sample to be tested, the methylation status of the DNA region associated with liver tumors is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr1:108113782-108507766.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease, which may include determining a specific subregion of the DNA region where the VAV3 gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following, or a complementary region thereof, in the sample to be tested, or Existence and/or content of the modified state of the above fragments: derived from human chr1: 108507215-108507766.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr1: 108507215-108507766.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal location by reference to its name, or its sequence and chromosomal location can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr1: 108507215-108507766 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may comprise: providing a nucleic acid capable of binding to a DNA region selected from the following, or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO:58.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr1: 108507591-108507674 and derived from human chr1: 108507624-108507725.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr1: 108507591-108507674 and derived from human chr1: 108507624-108507725.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr1: 108507591-108507674 and derived from human chr1:108507624-108507725.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or its complementary nucleic acid, or above-mentioned fragment: SEQ ID NO:59 and SEQ ID NO:62.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 59 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr1:108507215-108507766.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:108507591-108507674.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 62 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr1:108507215-108507766.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:108507624-108507725.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid group or its complementary nucleic acid group, or above-mentioned fragment: SEQ ID NO:60 and 61 and SEQ ID NO:63 and 64.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 60 and 61 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:108507215-108507766.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr1:108507591-108507674.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 63 and 64 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:108507215-108507766.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr1:108507624-108507725.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region where the IRF4 gene is located or its fragments in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the IRF4 gene is located or its fragment in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the IRF4 gene is located or its fragment in the test sample, assessing whether hepatic neoplasia is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the IRF4 gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the IRF4 gene is located or its fragment in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the IRF4 gene is located or a fragment thereof in the sample to be tested. For example, according to the determination of the presence and/or content of the modification state of the DNA region where the IRF4 gene is located or its fragment in the sample to be tested, the methylation status of the liver tumor-related DNA region is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr6:391739-411447.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease, which may include determining a specific subregion of the DNA region where the IRF4 gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, evaluating the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following group in the sample to be tested, or a complementary region thereof, or The presence and/or content of the modified state of the above fragments: derived from human chr6:391739-394056 and derived from human chr6:401233-401801.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr6:391739-394056 and derived from human chr6:401233-401801.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal location by reference to its name, or its sequence and chromosomal location can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation status of a DNA region, which may include: determining the DNA region derived from human chr6: 391739-394056 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr6:401233-401801 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 21 and SEQ ID NO: 71.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above fragments: derived from human chr6: 392282-392377, derived from human chr6: 392036-392145, derived from human chr6: 392405-392500 and derived from human chr6: 401641-401752 .
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr6: 392282-392377, derived from human chr6: 392036-392145, derived from human chr6: 392405-392500 and derived from human chr6: 401641-401752.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr6: 392282-392377 , derived from human chr6:392036-392145, derived from human chr6:392405-392500 and derived from human chr6:401641-401752.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application may comprise: providing nucleic acid or its complementary nucleic acid that can be selected from following group, or above-mentioned fragment: SEQ ID NO:22, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:72.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 22 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr6:391739-394056.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr6:392282-392377.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 65 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr6:391739-394056.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr6:392036-392145.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 68 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr6:391739-394056.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr6:392405-392500.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 72 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr6:401233-401801.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr6:401641-401752.
  • the method of the present application may include: providing a nucleic acid group or its complementary nucleic acid group that may be selected from the following group, or the above-mentioned fragments: SEQ ID NO: 23 and 24, SEQ ID NO: 66 and 67, SEQ ID NO: 69 and 70 and SEQ ID NO: 73 and 74.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 23 and 24 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr6:391739-394056.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr6:392282-392377.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 66 and 67 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr6:391739-394056.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr6:392036-392145.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 69 and 70 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr6:391739-394056.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr6:392405-392500.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 73 and 74 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr6:401233-401801.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr6:401641-401752.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation or risk of liver tumors and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region where the B4GALNT1 gene is located or its fragments in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the B4GALNT1 gene is located or its fragment in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the B4GALNT1 gene is located or its fragment in the test sample, assessing whether the diagnosis of liver tumor formation is confirmed.
  • the method of the present application may include, according to the determination of the existence and/or content of the modification state of the DNA region where the B4GALNT1 gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the existence and/or content of the modification state of the DNA region where the B4GALNT1 gene is located or its fragment in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the B4GALNT1 gene is located or a fragment thereof in the sample to be tested. For example, according to the determination of the presence and/or content of the modification state of the DNA region where the B4GALNT1 gene is located or its fragment in the sample to be tested, the methylation status of the DNA region associated with liver tumors is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr12:58017193-58027138.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease, which may include determining a specific subregion of the DNA region where the B4GALNT1 gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, evaluating the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following group in the sample to be tested, or a complementary region thereof, or The presence and/or content of the modified state of the above fragments: derived from human chr12:58020498-58022962 and derived from human chr12:58025539-58027138.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr12:58020498-58022962 and derived from human chr12:58025539-58027138.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal position by reference to its name, or its sequence and chromosomal position can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr12:58020498-58022962 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr12: 58025539-58027138 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 13 and SEQ ID NO: 78.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr12: 58021586-58021670, derived from human chr12: 58021907-58021987 and derived from human chr12: 58026383-58026475.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr12:58021586-58021670, derived from human chr12:58021907-58021987 and derived from human chr12:58026383-58026475.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr12:58021586-58021670 , derived from human chr12:58021907-58021987 and derived from human chr12:58026383-58026475.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or complementary nucleic acid thereof, or above-mentioned fragment: SEQ ID NO:14, SEQ ID NO:75 and SEQ ID NO:79.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 14 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr12:58020498-58022962.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:58021586-58021670.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 75 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr12:58020498-58022962.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:58021907-58021987.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 79 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr12:58025539-58027138.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:58026383-58026475.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid group or its complementary nucleic acid group, or above-mentioned fragment: SEQ ID NO:15 and 16, SEQ ID NO:76 and 77 and SEQ ID NO:80 and 81.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 15 and 16 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr12:58020498-58022962.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:58021586-58021670.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 76 and 77 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr12:58020498-58022962.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:58021907-58021987.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 80 and 81 or its complementary nucleic acid group, or the above-mentioned fragments.
  • this nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr12:58025539-58027138.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr12:58026383-58026475.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region where the GRASP gene is located or its fragments in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the GRASP gene is located or its fragment in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the GRASP gene is located or its fragment in the test sample, assessing whether liver tumor formation is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the GRASP gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the GRASP gene is located or its fragment in the sample to be tested, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the GRASP gene is located or a fragment thereof in the sample to be tested. For example, according to the existence and/or content determination of the modification state of the DNA region where the GRASP gene is located or its fragment in the sample to be tested, the methylation status of the liver tumor-related DNA region is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr12:52400724-52409673.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining the specific subregion of the DNA region where the GRASP gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, evaluating the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following group in the sample to be tested, or a complementary region thereof, or The presence and/or content of the modified state of the above fragments: derived from human chr12:52400724-52401698 and derived from human chr12:52406880-52409127.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may comprise, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr12:52400724-52401698 and derived from human chr12:52406880-52409127.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal position by reference to its name, or its sequence and chromosomal position can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation status of a DNA region, which may include: determining the DNA region derived from human chr12:52400724-52401698 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the present application provides a method for determining the methylation status of a DNA region, which may include: determining the DNA region derived from human chr12:52406880-52409127 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 17 and SEQ ID NO: 85.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr12: 52401083-52401169, derived from human chr12: 52400965-52401055 and derived from human chr12: 52408471-52408566.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr12:52401083-52401169, derived from human chr12:52400965-52401055 and derived from human chr12:52408471-52408566.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr12:52401083-52401169 , derived from human chr12:52400965-52401055 and derived from human chr12:52408471-52408566.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application may comprise: providing nucleic acid or its complementary nucleic acid that can be selected from following group, or above-mentioned fragment: SEQ ID NO: 18, SEQ ID NO: 82 and SEQ ID NO: 86.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application can comprise: providing the nucleic acid shown in SEQ ID NO: 18 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr12:52400724-52401698.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:52401083-52401169.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 82 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr12:52400724-52401698.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:52400965-52401055.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 86 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr12:52406880-52409127.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr12:52408471-52408566.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid group or its complementary nucleic acid group, or above-mentioned fragment: SEQ ID NO:19 and 20, SEQ ID NO:83 and 84 and SEQ ID NO:87 and 88.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 19 and 20 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr12:52400724-52401698.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:52401083-52401169.
  • the method of the present application can comprise: providing the nucleic acid set shown in SEQ ID NO:83 and 84 or its complementary nucleic acid set, or above-mentioned fragment.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr12:52400724-52401698.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:52400965-52401055.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 87 and 88 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr12:52406880-52409127.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr12:52408471-52408566.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region where the BEST4 gene is located or its fragments in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the BEST4 gene is located or its fragment in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination result of the existence and/or content of the modification state of the DNA region where the BEST4 gene is located or its fragment in the test sample, assessing whether hepatic neoplasia is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the BEST4 gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the BEST4 gene is located or its fragment in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the BEST4 gene is located or a fragment thereof in the sample to be tested. For example, according to the determination of the presence and/or content of the modification state of the DNA region where the BEST4 gene is located or its fragment in the sample to be tested, the methylation status of the liver tumor-related DNA region is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr1:45249257-45253377.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a specific subregion of the DNA region where the BEST4 gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, evaluating the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following group in the sample to be tested, or a complementary region thereof, or Existence and/or content of the modified state of the above fragments: derived from human chr1:45251728-45252477 and derived from human chr1:45249853-45250527.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr1: 45251728-45252477 and derived from human chr1: 45249853-45250527.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal location by reference to its name, or its sequence and chromosomal location can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr1: 45251728-45252477 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr1: 45249853-45250527 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may comprise: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 9 and SEQ ID NO: 92.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr1: 45252095-45252176, derived from human chr1: 45252187-45252275 and derived from human chr1: 45249894-45249981.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr1: 45252095-45252176, derived from human chr1: 45252187-45252275 and derived from human chr1: 45249894-45249981.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr1: 45252095-45252176 , derived from human chr1:45252187-45252275 and derived from human chr1:45249894-45249981.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or its complementary nucleic acid, or above-mentioned fragment: SEQ ID NO:10, SEQ ID NO:89 and SEQ ID NO:93.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 10 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr1:45251728-45252477.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:45252095-45252176.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 89 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr1:45251728-45252477.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:45252187-45252275.
  • the method of the present application can comprise: providing the nucleic acid shown in SEQ ID NO: 93 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr1:45249853-45250527.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:45249894-45249981.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid group or its complementary nucleic acid group, or above-mentioned fragment: SEQ ID NO:11 and 12, SEQ ID NO:90 and 91 and SEQ ID NO:94 and 95.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 11 and 12 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:45251728-45252477.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr1:45252095-45252176.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 90 and 91 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:45251728-45252477.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr1:45252187-45252275.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 94 and 95 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:45249853-45250527.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr1:45249894-45249981.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region where the BEND4 gene is located or its fragments in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the BEND4 gene is located or its fragment in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the BEND4 gene is located or its fragment in the test sample, assessing whether hepatic neoplasia is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the BEND4 gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the BEND4 gene is located or its fragment in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the BEND4 gene is located or a fragment thereof in the sample to be tested. For example, according to the existence and/or content determination of the modification state of the DNA region where the BEND4 gene is located or its fragment in the sample to be tested, the methylation status of the liver tumor-related DNA region is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence of methylation in the DNA region or an increase in the amount relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr4:42112955-42154895.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a specific subregion of the DNA region where the BEND4 gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following, or its complementary region, or the above-mentioned The presence and/or content of the modified state of the fragment: derived from human chr4: 42152705-42154895.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and/or modification state of the following DNA regions, or their complementary regions, or the above-mentioned fragments in the sample to be tested Or content: derived from human chr4: 42152705-42154895.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal position by reference to its name, or its sequence and chromosomal position can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr4: 42152705-42154895 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may comprise: providing a nucleic acid capable of binding to a DNA region or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 25.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr4: 42153816-42153921 derived from human chr4: 42153513-42153601.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr4: 42153816-42153921 derived from human chr4: 42153513-42153601.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr4: 42153816-42153921 Derived from human chr4:42153513-42153601.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or complementary nucleic acid thereof, or above-mentioned fragment: SEQ ID NO: 26 and SEQ ID NO: 96.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 26 or its complementary nucleic acid, or the above-mentioned fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr4:42152705-42154895.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr4:42153816-42153921.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 96 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr4:42152705-42154895.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr4:42153513-42153601.
  • the method of the present application may comprise: providing a nucleic acid group or its complementary nucleic acid group that may be selected from the following group, or the above-mentioned fragments: SEQ ID NO: 27 and 28 and SEQ ID NO: 97 and 98.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 27 and 28 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr4:42152705-42154895.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr4:42153816-42153921.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 97 and 98 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr4:42152705-42154895.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr4:42153513-42153601.
  • the present application provides a method for confirming the presence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the DNA region derived from human chr1:145384249:145413094 in the sample to be tested The presence and/or content of the modified state of or fragments thereof.
  • the method of the present application may include, according to the determination result of the existence and/or content of the modification state of the DNA region or its fragment derived from human chr1:145384249:145413094 in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region or fragment thereof derived from human chr1:145384249:145413094 in the test sample, assessing whether hepatic neoplasia is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region or fragment thereof derived from human chr1:145384249:145413094 in the sample to be tested, assessing whether there is a diagnosis of liver tumor formation risk and/or level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region or fragment thereof derived from human chr1:145384249:145413094 in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-associated DNA region, which may include determining the presence and/or the modification state of the DNA region or fragment thereof derived from human chr1:145384249:145413094 in the test sample or content.
  • the methylation status of the DNA region associated with liver tumors is assessed according to the determination of the presence and/or content of the modification state of the DNA region or fragment thereof derived from human chr1:145384249:145413094 in the sample to be tested.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or referred to as "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining the specific DNA region derived from human chr1:145384249:145413094 in the sample to be tested. The existence and/or content of the modified state of the sub-region, or its complementary region, or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, evaluating the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following group in the sample to be tested, or a complementary region thereof, or The existence and/or content of the modified state of the above fragments: derived from human chr1: 145389746-145401075, derived from human chr1: 145384910-145385929 and derived from human chr1: 145406714-145408013.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr1: 145389746-145401075, derived from human chr1: 145384910-145385929 and derived from human chr1: 145406714-145408013.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal position by reference to its name, or its sequence and chromosomal position can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr1: 145389746-145401075 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr1: 145384910-145385929 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr1: 145406714-145408013 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 99, SEQ ID NO: 106 and SEQ ID NO: 110.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr1: 145399249: 145399476, derived from human chr1: 145396880-145396983, derived from human chr1: 145385298-145385376 and derived from human chr1: 145407431-145407518 .
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr1: 145399249:145399476, derived from human chr1: 145396880-145396983, derived from human chr1: 145385298-145385376 and derived from human chr1: 145407431-145407518.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr1:145399249:145399476 , derived from human chr1: 145396880-145396983, derived from human chr1: 145385298-145385376 and derived from human chr1: 145407431-145407518.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or complementary nucleic acid thereof, or above-mentioned fragment: SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:107 and SEQ ID NO:111.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 100 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr1:145389746-145401075.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:145399249:145399476.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 103 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr1:145389746-145401075.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:145396880-145396983.
  • the method of the present application can comprise: providing the nucleic acid shown in SEQ ID NO: 107 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:145384910-145385929.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:145385298-145385376.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 111 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:145406714-145408013.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:145407431-145407518.
  • the method of the present application may include: providing a nucleic acid group or its complementary nucleic acid group that may be selected from the following group, or the above-mentioned fragments: SEQ ID NO: 101 and 102, SEQ ID NO: 104 and 105, SEQ ID NO: 108 and 109 and SEQ ID NO: 112 and 113.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 101 and 102 or its complementary nucleic acid group, or the above-mentioned fragments.
  • this nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:145389746-145401075.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr1:145399249:145399476.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 104 and 105 or its complementary nucleic acid group, or the above-mentioned fragments.
  • this nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:145389746-145401075.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr1:145396880-145396983.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 108 and 109 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:145384910-145385929.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr1:145385298-145385376.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 112 and 113 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:145406714-145408013.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr1:145407431-145407518.
  • the present application provides a method for confirming the presence of liver tumors, assessing the formation or risk of liver tumors and/or assessing the progress of liver tumors, which may include determining the DNA region derived from human chr7:26415938:26416740 in the sample to be tested The presence and/or content of the modified state of or fragments thereof.
  • the method of the present application may include, according to the determination result of the existence and/or content of the modification state of the DNA region or its fragment derived from human chr7:26415938:26416740 in the test sample, confirming whether the liver tumor exists.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region or fragment thereof derived from human chr7:26415938:26416740 in the sample to be tested, assessing whether hepatic neoplasia is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region or fragment thereof derived from human chr7:26415938:26416740 in the test sample, assessing whether there is a diagnosis of liver tumor formation risk and/or level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region or fragment thereof derived from human chr7:26415938:26416740 in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation status of a liver tumor-associated DNA region, which may include determining the presence and/or the modification status of the DNA region or fragment thereof derived from human chr7:26415938:26416740 in the test sample or content.
  • the methylation status of the DNA region associated with liver tumors is evaluated according to the determination of the presence and/or content of the modification state of the DNA region or fragment thereof derived from human chr7:26415938:26416740 in the sample to be tested.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining the specific DNA region derived from human chr7:26415938:26416740 in the sample to be tested. The existence and/or content of the modified state of the sub-region, or its complementary region, or the above-mentioned fragments.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal position by reference to its name, or its sequence and chromosomal position can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • sensitivity may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question;
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr7: 26416257-26416363 derived from human chr7: 26416026-26416126.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr7: 26416257-26416363 derived from human chr7: 26416026-26416126.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr7: 26416257-26416363 Derived from human chr7:26416026-26416126.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or its complementary nucleic acid, or above-mentioned fragment: SEQ ID NO:114 and SEQ ID NO:117.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application can comprise: providing the nucleic acid shown in SEQ ID NO: 114 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr7:26416257-26416363.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 117 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr7:26416026-26416126.
  • the method of the present application may comprise: providing a nucleic acid group or its complementary nucleic acid group selected from the following group, or the fragments mentioned above: SEQ ID NO: 115 and 116 and SEQ ID NO: 118 and 119.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 115 and 116 or its complementary nucleic acid group, or the above fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr7:26416257-26416363.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 118 and 119 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr7:26416026-26416126.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region or fragments thereof where the GPAM gene is located in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the GPAM gene is located or its fragment in the sample to be tested, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the GPAM gene is located or its fragment in the test sample, assessing whether hepatic neoplasia is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the GPAM gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the GPAM gene is located or its fragment in the sample to be tested, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the GPAM gene is located or a fragment thereof in the sample to be tested. For example, according to the determination of the presence and/or content of the modification state of the DNA region where the GPAM gene is located or its fragment in the sample to be tested, the methylation status of the liver tumor-related DNA region is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr10:113909624-113975135.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease, which may include determining the specific subregion of the DNA region where the GPAM gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following, or its complementary region, or the above-mentioned The presence and/or content of the modified state of the fragments: derived from human chr10: 113942657-113943906.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and/or modification state of the following DNA regions, or their complementary regions, or the above-mentioned fragments in the sample to be tested Or content: derived from human chr10:113942657-113943906.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal position by reference to its name, or its sequence and chromosomal position can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the DNA region derived from human chr10: 113942657-113943906 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may comprise: providing a nucleic acid capable of binding to a DNA region selected from the following, or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 120.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr10: 113943540: 113943739 derived from human chr10: 113943511-113943582.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr10: 113943540: 113943739 derived from human chr10: 113943511-113943582.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr10:113943540:113943739 Derived from human chr10:113943511-113943582.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or complementary nucleic acid thereof, or above-mentioned fragment: SEQ ID NO:121 and SEQ ID NO:124.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application can comprise: providing the nucleic acid shown in SEQ ID NO: 121 or its complementary nucleic acid, or above-mentioned fragment.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr10:113942657-113943906.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr10:113943540:113943739.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 124 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr10:113942657-113943906.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr10:113943511-113943582.
  • the method of the present application may comprise: providing a nucleic acid group or its complementary nucleic acid group selected from the following group, or the above-mentioned fragments: SEQ ID NO: 122 and 123 and SEQ ID NO: 125 and 126.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 122 and 123 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr10:113942657-113943906.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr10:113943540:113943739.
  • the method of the present application may comprise: providing the nucleic acid set shown in SEQ ID NO: 125 and 126 or its complementary nucleic acid set, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr10:113942657-113943906.
  • the nucleic acid set can be used as a primer set to amplify a DNA region derived from human chr10:113943511-113943582.
  • the present application provides a method for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include determining the modification status of the DNA region or fragments of the VASH2 gene in the sample to be tested presence and/or content.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the VASH2 gene is located or its fragment in the test sample, confirming whether there is a liver tumor.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the VASH2 gene is located or its fragment in the test sample, assessing whether the liver tumor is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the modification state of the DNA region where the VASH2 gene is located or its fragment in the sample to be tested, assessing whether there is a risk and/or risk of liver tumor formation high and low.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region where the VASH2 gene is located or its fragment in the test sample, assessing the progress of the liver tumor.
  • the present application provides a method for assessing the methylation state of a liver tumor-related DNA region, which may include determining the presence and/or content of the modification state of the DNA region where the VASH2 gene is located or a fragment thereof in the sample to be tested. For example, according to the existence and/or content determination of the modification state of the DNA region where the VASH2 gene is located or its fragment in the sample to be tested, the methylation status of the liver tumor-related DNA region is evaluated.
  • the methylation status of a liver tumor-associated DNA region may refer to the confirmed presence or increase in the amount of methylation of the DNA region relative to a reference level, which may be associated with the occurrence of liver tumors.
  • the DNA region of the present application may be derived from human chr1:213123862-213165379.
  • the genes of the present application can be described by their names and their chromosomal coordinates.
  • the chromosome coordinates may be consistent with the Hg19 version of the Human Genome Database released in February 2009 (or called "Hg19 coordinates").
  • the DNA region of the present application may be derived from a region defined by Hg19 coordinates.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a specific subregion of the DNA region where the VASH2 gene is located in the sample to be tested, or its The existence and/or content of the modified state of the complementary region or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include determining a DNA region selected from the following, or its complementary region, or the above-mentioned The presence and/or content of the modified state of the fragment: derived from human chr1:213123862-213125211.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, confirming whether the disease exists.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modified state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing whether the disease is diagnosed.
  • the method of the present application may include, according to the determination of the presence and/or content of the DNA region or its complementary region, or the modification state of the above-mentioned fragments in the sample to be tested, assessing whether there is a risk of being diagnosed with a disease and and/or the level of risk.
  • the method of the present application may include, according to the determination result of the presence and/or content of the modification state of the DNA region, or its complementary region, or the above-mentioned fragments in the sample to be tested, assessing the progress of the disease.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and/or modification state of the following DNA regions, or their complementary regions, or the above-mentioned fragments in the sample to be tested Or content: derived from human chr1: 213123862-213125211.
  • the confirmed presence or increased amount of methylation of the DNA region relative to a reference level can be associated with the development of a disease.
  • a DNA region in the present application may refer to a specific segment of genomic DNA.
  • a DNA region of the present application may be specified by a gene name or a set of chromosomal coordinates.
  • a gene can have its sequence and chromosomal position by reference to its name, or its sequence and chromosomal position can be determined by reference to its chromosomal coordinates.
  • the present application adopts the methylation status of these specific DNA regions as a series of analysis indicators, which can provide significant improvement in sensitivity and/or specificity, and can simplify the screening process.
  • "sensitivity” may refer to the proportion of positive results correctly identified, i.e., the percentage of individuals correctly identified as having the disease in question
  • specificity may refer to the proportion of negative results correctly identified, i.e., as not having Percentage of individuals with the disease in question.
  • the present application provides a method for determining the methylation status of a DNA region, which may include: determining the DNA region derived from human chr1: 213123862-213125211 in the sample to be tested, or its complementary region, or the modification of the above-mentioned fragments presence and/or content of the state.
  • the DNA regions of the present application may encompass all forms of these molecules and fragments or variants thereof.
  • a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity in common with a DNA region described herein, and a variant may comprise one or more deletions , add, replace, reverse sequence, etc.
  • the modification status of the variants described in this application can achieve the same assessment results.
  • the DNA regions of the present application may contain all forms of any other mutation, polymorphic variation or allelic variation.
  • the method of the present application may comprise: providing a nucleic acid capable of binding to a DNA region or a complementary region thereof, or the above-mentioned transformed region, or the above-mentioned fragment: SEQ ID NO: 127.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include: determining the DNA region or its complementary region in the sample to be tested that may be selected from the following group , or the presence and/or content of the modified state of the above-mentioned fragments: derived from human chr1: 213124569-213124670 derived from human chr1: 213124036-213124162.
  • the present application provides a method for determining the methylation state of a DNA region, which may include: determining the presence and /or content: derived from human chr1: 213124569-213124670 derived from human chr1: 213124036-213124162.
  • the method of the present application may include: providing a nucleic acid capable of binding to a DNA region selected from the following group, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment: derived from human chr1: 213124569-213124670 Derived from human chr1:213124036-213124162.
  • one or more of the aforementioned regions can serve as amplification regions and/or detection regions.
  • the method of the present application can comprise: providing can be selected from following group nucleic acid or complementary nucleic acid thereof, or above-mentioned fragment: SEQ ID NO:128 and SEQ ID NO:131.
  • the nucleic acid can be used to detect a region of interest.
  • the nucleic acid can serve as a probe.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 128 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr1:213123862-213125211.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:213124569-213124670.
  • the method of the present application may comprise: providing the nucleic acid shown in SEQ ID NO: 131 or its complementary nucleic acid, or the above fragments.
  • the nucleic acid can be used as a probe to detect the DNA region derived from human chr1:213123862-213125211.
  • the nucleic acid can be used as a probe to detect a DNA region derived from human chr1:213124036-213124162.
  • the method of the present application may comprise: providing a nucleic acid group or its complementary nucleic acid group that may be selected from the following group, or the above-mentioned fragments: SEQ ID NO: 129 and 130 and SEQ ID NO: 132 and 133.
  • the set of nucleic acids can be used to amplify a region of interest.
  • the set of nucleic acids can serve as a set of primers.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 129 and 130 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:213123862-213125211.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:213124569-213124670.
  • the method of the present application may comprise: providing the nucleic acid group shown in SEQ ID NO: 132 and 133 or its complementary nucleic acid group, or the above-mentioned fragments.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:213123862-213125211.
  • the nucleic acid set can be used as a primer set to amplify the DNA region derived from human chr1:213124036-213124162.
  • the disease may comprise a tumor.
  • the disease may comprise solid tumors.
  • the disease may include arbitrary tumors such as liver tumors.
  • a "primer” may be a natural or synthetic oligonucleotide that, upon duplex formation with a polynucleotide template, is capable of serving as an initiation point for nucleic acid synthesis and extends from its 3' end along the template, thereby forming an extended duplex.
  • the sequence of nucleotides added during extension is determined by the sequence of the template polynucleotide.
  • Primers are typically extendable by a polymerase, such as a nucleic acid polymerase.
  • complementary and substantially complementary in the present application may include between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule, or between an oligonucleotide primer and a single-stranded Hybridization or base pairing or duplex formation between primer binding sites on a nucleic acid.
  • Complementary nucleotides may typically be A and T (or A and U) or C and G.
  • the nucleotides of one strand are optimally aligned and compared and have appropriate nucleotide insertions or deletions, they differ from at least about 80% (usually at least about 90%) of the other strand.
  • two complementary nucleotide sequences are capable of hybridizing and may have less than 25% mismatches, more preferably less than 15% mismatches, may have less than 5% mismatches between the nucleotides in reverse. mismatch, or lack of mismatch.
  • two molecules can hybridize under high stringency conditions.
  • the modification state in the present application may refer to the presence, absence and/or amount thereof at a specific nucleotide or multiple nucleotides within a DNA region.
  • the modification status of the present application may refer to the modification status of each base or each specific base (such as cytosine) in a specific DNA sequence.
  • the modification status of the present application may refer to the base pair combination and/or the modification status of the base combination in a specific DNA sequence.
  • the modification status in this application may refer to information about the density of modification in a specific DNA sequence (including the DNA region where the gene is located or a specific region fragment thereof), but may not provide information on the precise position of where the modification occurs in the sequence.
  • the modification state in the present application may refer to a methylation state or a state similar to methylation.
  • a state of having or having higher methylation can be associated with transcriptional silencing of a particular region.
  • having or having a higher methylation state can be associated with being able to be transformed by a methylation-specific transforming reagent (e.g., a deamination reagent and/or a methylation-sensitive restriction enzyme).
  • converting can mean being transformed into other substances and/or being sheared or digested.
  • methylation state can refer to the presence or absence of 5-methylcytidine at one or more CpG dinucleotides within a DNA sequence.
  • Pyrimidine (“5-mC” or “5-mCyt”).
  • the methylation status at one or more specific CpG methylation sites (each with two CpG dinucleotide sequences) within the DNA sequence includes “unmethylated”, “fully methylated” and " Hemimethylation”.
  • hemimethylation may refer to the methylation state of double-stranded DNA, wherein only one strand of the double-stranded DNA is methylated.
  • hypomethylation may refer to the average methylation state corresponding to the amount of 5-mCyt found at the corresponding CpG dinucleotide in the normal control DNA sample, one of the DNA sequences of the test DNA sample or increased presence of 5-mCyt at multiple CpG dinucleotides.
  • hypermethylation may refer to the average methylation state corresponding to the amount of 5-mCyt found in the DNA sequence of the test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotide in the normal control DNA sample. The presence of 5-mCyt at one or more of the CpG dinucleotides is reduced.
  • the method may further comprise obtaining nucleic acid in the sample to be tested.
  • the nucleic acid can comprise cell-free episomal nucleic acid.
  • the sample to be tested may comprise tissues, cells and/or body fluids.
  • the sample to be tested may comprise plasma.
  • the detection methods of the present application can be performed on any suitable biological sample.
  • the sample to be tested can be any sample of biological material, such as it can be derived from an animal, but is not limited to, cellular material, biological fluid (such as blood), exudate, tissue biopsy specimen, surgical specimen, or has been introduced into the body of an animal And then remove the fluid.
  • the sample to be tested in the present application may include a sample processed in any form after the sample is separated.
  • the method may also comprise transforming the DNA region or a fragment thereof.
  • the bases with the modified state and the bases without the modified state can form different substances after conversion.
  • the base with the modified state is substantially unchanged after conversion, and the base without the modified state may be changed to other bases different from the base after conversion (for example, the The other bases may contain uracil), or be cleaved after conversion.
  • the base may comprise cytosine.
  • the modification state may comprise a methylation modification.
  • the transformation may comprise transformation by a deamination reagent and/or a methylation sensitive restriction enzyme.
  • the deamination reagent may comprise bisulfite or the like. For example, sodium bisulfite or potassium bisulfite.
  • the method may optionally further comprise amplifying the DNA region or fragment thereof in the sample to be tested prior to determining the presence and/or amount of modifications of the DNA region or fragment thereof.
  • the amplification can comprise PCR amplification.
  • the amplification of the present application may include any known amplification system.
  • the amplification step of the present application may be optional.
  • "amplification” can refer to the process of producing multiple copies of a desired sequence.
  • Multiple copies may mean at least two copies.
  • “Copy” may not imply perfect sequence complementarity or identity to the template sequence.
  • copies may include nucleotide analogs such as deoxyinosine, deliberate sequence changes (such as those introduced by primers containing sequences that are hybridizable but not complementary to the template), and/or may occur during amplification. Sequence error.
  • the method for determining the presence and/or content of a modification state may comprise confirming the presence and/or content of a substance formed after the conversion of the base having the modification state.
  • the method for determining the presence and/or content of a modification state may comprise determining the presence and/or content of a DNA region or fragment thereof having the modification state.
  • the presence and/or content of DNA regions or fragments thereof having said modification state can be detected directly.
  • it can be detected in the following manner: DNA regions or fragments thereof having said modification state may have different properties during a reaction (eg amplification reaction) from DNA regions or fragments thereof not having said modification state.
  • a DNA region or a fragment thereof having the modified state may be specifically amplified and emit fluorescence; a DNA region or a fragment thereof not having the modified state may not be substantially amplified, and Almost no fluorescence.
  • alternative methods of determining the presence and/or amount of species formed after said conversion of bases having said modified state may be included within the scope of this application.
  • the presence and/or content of the DNA region or fragment thereof with the modification state can be determined by the fluorescent Ct value detected by the fluorescent PCR method.
  • the presence of a liver tumor, or the formation of a liver tumor can be determined by the presence of a modification state of said DNA region or fragment thereof and/or said DNA region or fragment thereof has a higher content of modification state relative to a reference level or pose risks.
  • the fluorescence Ct value of the sample to be tested is lower relative to the reference fluorescence Ct value
  • the existence of the modification state of the DNA region or its fragment can be determined and/or the modification of the DNA region or its fragment can be determined
  • the abundance of the modified state is higher than that of the modified state in the reference sample.
  • the reference fluorescence Ct value can be determined by detecting a reference sample. For example, when the fluorescence Ct value of the sample to be tested is higher or substantially equal to the reference fluorescence Ct value, the existence of the modified state of the DNA region or its fragment may not be excluded; when the fluorescence of the sample to be tested When the Ct value is higher or substantially equal to the reference fluorescence Ct value, it can be confirmed that the content of the modification state of the DNA region or its fragment is lower than or substantially equal to the content of the modification state in the reference sample.
  • the present application may use the cycle threshold value (ie, Ct value) to indicate the existence and/or content of the modification state of a specific DNA region or its fragment, for example, including the methylation level and the reference level of the sample to be tested.
  • a Ct value can refer to the cycle number at which fluorescence of a PCR product can be detected above background signal.
  • there may be a negative correlation between the Ct value and the initial quantity of the target marker in the sample, that is, the lower the Ct value, the more the number of modification states of the DNA region or its fragments in the sample to be tested.
  • the Ct value of the test sample is the same or lower than its corresponding reference Ct value, it can be confirmed that a specific disease exists, diagnosed as the formation of a specific disease or has the risk of forming a specific disease, or evaluated as a certain progress of a specific disease.
  • the Ct value of the sample to be tested is at least 1 cycle, at least 2 cycles, at least 5 cycles, at least 10 cycles, at least 20 cycles, or at least 50 cycles lower than its corresponding reference Ct value.
  • the presence of a specific disease can be confirmed, diagnosed as developing or at risk of developing a specific disease, or assessed as some progression of a specific disease.
  • the specific disease does not exist, is diagnosed as developing a specific disease, or is at risk of developing a specific disease. Or assessed as a certain progression of a specific disease.
  • the Ct value of a cell sample, tissue sample, or sample derived from a subject is at least 1 cycle, at least 2 cycles, at least 5 cycles, at least 10 cycles, at least 20 cycles higher than its corresponding reference Ct value cycle, or at least 50 cycles, it can be confirmed that the specific disease does not exist, the formation of the specific disease is diagnosed or there is a risk of formation, or it is evaluated as a certain progress of the specific disease.
  • the presence or absence of a particular disease can be confirmed, the development or absence of a particular disease can be diagnosed , has or does not have the risk of forming, or is assessed as a certain progression of a specific disease, and at the same time can give suggestions for further testing.
  • a reference level or control level in the present application may refer to a normal level or a healthy level.
  • said normal level may be the level of modification status of a DNA region in a sample derived from a cell, tissue or individual free of said disease.
  • the normal level when used in the assessment of tumors, the normal level may be the level of modification status of a DNA region in a sample from a tumor-free cell, tissue, or individual.
  • the normal level when used in the assessment of liver tumors, the normal level may be the modification state level of a DNA region in a sample from cells, tissues or individuals without liver tumors.
  • a reference level in this application may refer to a threshold level for confirming the presence or absence of a particular disease in a subject or sample.
  • a reference level may refer to a threshold level at which a subject is diagnosed as having developed or at risk of developing a particular disease.
  • a reference level may refer to a threshold level at which a subject is assessed as having progressed to a particular disease.
  • the reference level may refer to the DNA region of a patient without a specific disease.
  • Modified status can be confirmed as the presence of a particular disease, diagnosed as developing or at risk of developing a particular disease, or assessed as some progression of a particular disease.
  • "substantially equal" between A and B in this application may mean that the difference between A and B is 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or Less or 0.0001% or less.
  • a tissue sample or a sample derived from a subject is at least 1%, at least 5%, at least 10%, at least 20%, at least 50%, at least 1% higher than the corresponding reference level times, at least 2 times, at least 5 times, at least 10 times, or at least 20 times, it can be confirmed that there is a specific disease, diagnosed as the formation of a specific disease or has a risk of formation, or evaluated as a certain progression of a specific disease.
  • the modification status of the DNA region in the cell sample, tissue sample, or sample derived from the subject is at least 1% higher than the corresponding reference level , at least 5%, at least 10%, at least 20%, at least 50%, at least 1 times, at least 2 times, at least 5 times, at least 10 times, or at least 20 times, can be confirmed as the presence of a specific disease, diagnosed as a specific disease The development of or is at risk of developing or is assessed as a certain progression of a specific disease.
  • the reference level here may refer to the modification of a DNA region in a patient with a specific disease A status that can be confirmed as the absence of a specific disease, diagnosed as developing or at risk of developing a specific disease, or assessed as some progression of a specific disease.
  • the modification state of a DNA region in a cell sample, tissue sample or sample derived from a subject is at least 1%, at least 5%, at least 10%, at least 20%, at least 50%, at least 100% lower than the corresponding reference level %, it can be confirmed that there is no specific disease, it can be diagnosed as the development of specific disease or there is a risk of developing specific disease, or it can be evaluated as a certain progress of specific disease.
  • reference levels can be derived from One or more reference samples, where the reference level is obtained from an experiment performed in parallel with the experiment testing the sample of interest.
  • reference levels may also be obtained in a database comprising a collection of data, standards or levels from one or more reference samples or disease reference samples.
  • a collection of data, standards, or levels can be normalized or normalized so that it can be compared with data from one or more samples and used to reduce errors arising under different assay conditions.
  • a reference level may be derived from a database, which may be a reference database, eg, including modification status levels of a marker of interest and/or other laboratory and clinical data from one or more reference samples.
  • a reference database can be established by aggregating reference level data from reference samples obtained from healthy individuals and/or individuals not suffering from the corresponding disease (ie, individuals known to be free of the disease).
  • a reference database can be established by aggregating reference level data obtained from reference samples of individuals with the corresponding disease being treated.
  • a reference database can be established by aggregating data from reference samples obtained from individuals at different stages of the disease. For example, different stages can be evidenced by different modification state levels of markers of interest of the present application, for example.
  • Those skilled in the art can also determine whether an individual suffers from the corresponding disease or has the risk of suffering from the corresponding disease based on various factors, such as age, gender, medical history, family history, symptoms, etc.
  • the method of the present application may comprise the following steps: obtaining the nucleic acid in the sample to be tested; transforming the DNA region or its fragment; confirming the presence and/or content.
  • the method of the present application may comprise the following steps: obtaining the nucleic acid in the sample to be tested; transforming the DNA region or its fragment; amplifying the DNA region or its fragment in the sample to be tested; confirming that the base with the modified state The presence and/or content of substances formed after said conversion.
  • the method of the present application may comprise the following steps: obtaining nucleic acid in the sample to be tested; treating the DNA obtained from the sample to be tested with a reagent capable of distinguishing unmethylated sites and methylated sites in the DNA site, thereby obtaining processed DNA; optionally amplifying said DNA region or a fragment thereof in the test sample; quantitatively, semi-quantitatively or qualitatively analyzing the presence of the methylation state of the processed DNA in the test sample and/or content; comparing the methylation level of the processed DNA in the test sample with the corresponding reference level, when the methylation state of the DNA region in the sample to be tested is higher than or substantially equal to the corresponding reference level, it can be confirmed as A specified disease is present, diagnosed as developing or at risk of developing a specified disease, or assessed as some progression of a specified disease.
  • the present application provides a nucleic acid, which may contain a sequence capable of binding to the DNA region where the target gene of the present application is located, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the nucleic acid can be any of the probes of the present application.
  • the present application provides a method for preparing nucleic acid, which may include designing a DNA region capable of binding The nucleic acid of the DNA region, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the method of preparing the nucleic acid can be any suitable method known in the art.
  • the present application provides a nucleic acid group, which may comprise a sequence capable of binding to the DNA region where the target gene of the present application is located, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment.
  • the nucleic acid set can be any primer set of the present application.
  • the present application provides a method for preparing a nucleic acid group, which may include designing a DNA region capable of A nucleic acid set of said DNA region, or a complementary region thereof, or a transformed region as described above, or a fragment as described above is amplified.
  • the method of preparing the nucleic acids in the set of nucleic acids can be any suitable method known in the art.
  • the methylation status of a target polynucleotide can be assessed using a single probe or primer configured to hybridize to the target polynucleotide.
  • the methylation status of a target polynucleotide can be assessed using a plurality of probes or primers configured to hybridize to the target polynucleotide.
  • the present application provides a nucleic acid comprising the DNA region capable of binding to the target gene in the method described in the present application, or its complementary region, or the above-mentioned transformed region, or the above-mentioned
  • the sequence of the fragment on the other hand, provides a nucleic acid, which may comprise the region capable of binding to the target DNA in the method described in the present application, or its complementary region, or the above-mentioned transformed region, or sequences of fragments described above.
  • the present application provides a method for preparing nucleic acid, which may include the DNA region where the target gene is located in the method as described in the present application, or its complementary region, or the above-mentioned transformed region, Or the modified state of the above-mentioned fragments, designing a nucleic acid that can be combined with the DNA region where the target gene is located, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment; on the other hand, the present application provides a nucleic acid , the method may comprise, according to the modification state of the target DNA region, or its complementary region, or the above-mentioned converted region, or the above-mentioned fragment in the method as described in the present application, designing a target DNA region capable of binding to the target DNA region, or its complementary region, or the above-mentioned transformed region, or the nucleic acid of the above-mentioned fragment.
  • the present application provides a nucleic acid set, which comprises the DNA region capable of binding to the target gene in the method described in the present application, or its complementary region, or the above transformed region, or The sequence of the above-mentioned fragment; on the other hand, the application provides a nucleic acid, and the nucleic acid group may comprise the region capable of binding to the target DNA described in the method described in the application, or its complementary region, or the above transformation region, or the sequence of the above-mentioned fragments.
  • the method of preparing the nucleic acid can be any suitable method known in the art.
  • the present application provides a method for preparing a nucleic acid group, which may include the DNA region where the target gene is located in the method described in the present application, or its complementary region, or the above-mentioned transformed region , or the modified state of the above-mentioned fragments, designing a nucleic acid group that can be combined with the DNA region where the target gene is located, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragments; on the other hand, the application provides a A nucleic acid, the method may comprise, according to the modification state of the target DNA region, or its complementary region, or the above-mentioned converted region, or the above-mentioned fragment in the method as described in the present application, designing the DNA capable of binding to the A target DNA region, or its complementary region, or the above-mentioned transformed region, or a nucleic acid group of the above-mentioned fragments.
  • the methylation status of a target polynucleotide can be assessed using a single probe or primer configured to hybridize to the target polynucleotide.
  • the methylation status of a target polynucleotide can be assessed using a plurality of probes or primers configured to hybridize to the target polynucleotide.
  • the present application provides a kit, which may comprise the nucleic acid of the present application and/or the nucleic acid group of the present application.
  • the kit of the present application may optionally contain a reference sample for a corresponding use or provide a reference level for a corresponding use.
  • the methods of the present application can be used for diagnosis, prognosis, stratification, risk assessment or treatment monitoring of cancer or neoplasia in a subject.
  • a "subject” may refer to an organism or a part or component thereof to which the methods, nucleic acids, sets of nucleic acids, kits, devices, and systems provided herein may be administered or applied.
  • the subject can be a mammal or a cell, tissue, organ or part of the mammal.
  • mammal refers to any kind of mammal, preferably a human (including a human, human subject or human patient).
  • Subjects and mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, and rodents such as mice and rats.
  • the methods described herein can be used to assess cancer or tumor formation in any suitable subject.
  • the methods described herein can be used to assess cancer or tumor formation in a mammal.
  • the mammal may be a non-human mammal such as a pet, farm animal, companion animal or laboratory animal.
  • said mammal is a human.
  • a subject can be a person who needs to be screened for risk of cancer or neoplasia, a person in a high-risk group, a person diagnosed with cancer or neoplasia but needs further stratification or grading, a person diagnosed with cancer or neoplasia A person who has formed and is receiving active treatment, or who has cancer or tumor formation and is in remission.
  • the application provides the application of the nucleic acid of the application, the nucleic acid group of the application and/or the kit of the application in the preparation of substances for determining the modification status of DNA regions or fragments thereof.
  • the present application provides a method for determining the modification state of the DNA region or its fragment, which may include providing the nucleic acid of the present application, such as the nucleic acid set of the present application and/or the kit of the present application.
  • the present application provides the nucleic acid of the present application, the nucleic acid set of the present application and/or the kit of the present application, which can be used to determine the modification state of the DNA region or fragment thereof.
  • the present application provides the application of the nucleic acid of the present application, the nucleic acid group of the present application and/or the kit of the present application in the preparation of disease detection products.
  • the disease may comprise tumors.
  • solid tumors for example, solid tumors.
  • the disease may comprise liver tumors.
  • the present application provides a disease detection method, which may include providing the nucleic acid of the present application, such as the nucleic acid set of the present application and/or the kit of the present application.
  • the disease may comprise tumors.
  • solid tumors for example, in the application of the present application, the disease may comprise liver tumors.
  • the present application provides the nucleic acid of the present application, the nucleic acid set of the present application and/or the kit of the present application, which can be used for disease detection.
  • the disease may comprise tumors.
  • solid tumors for example, solid tumors.
  • the disease may comprise liver tumors.
  • the present application provides the nucleic acid of the present application, the nucleic acid group of the present application and/or the kit of the present application, in the preparation of substances for confirming the existence of the disease, assessing the formation of the disease or the risk of forming the disease, and/or assessing the progress of the disease in the application.
  • the disease may comprise tumors.
  • solid tumors for example, in the application of the present application, the disease may comprise liver tumors.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include providing the nucleic acid of the present application, such as the nucleic acid group of the present application and/or the nucleic acid of the present application.
  • Reagent test kit for example, as used herein, the disease may comprise tumors. For example solid tumors.
  • the disease may comprise liver tumors.
  • the present application provides the nucleic acid of the present application, the nucleic acid group of the present application and/or the kit of the present application, which can be used to confirm the existence of a disease, assess the risk of disease formation or formation and/or assess the progress of a disease .
  • the disease may comprise tumors.
  • solid tumors for example, in the application of the present application, the disease may comprise liver tumors.
  • the present application provides nucleic acids, nucleic acid groups and/or kits for determining the modification status of DNA regions, in preparation for confirming the presence of liver tumors, assessing the formation or risk of liver tumor formation and/or assessing the progression of liver tumors
  • the DNA region used for determination may include the DNA region where the target gene is located or a fragment thereof as described in the method of the present application.
  • the present application provides methods for confirming the presence of a liver tumor, assessing the formation or risk of developing a liver tumor, and/or assessing the progression of a liver tumor, which may comprise providing nucleic acids, nucleic acid groups, and/or reagents for determining the state of modification of a DNA region box.
  • the DNA region used for determination may include the DNA region where the target gene is located or a fragment thereof as described in the method of the present application.
  • the present application provides nucleic acids, nucleic acid sets and/or kits for determining the modification status of DNA regions, which can be used to confirm the presence of liver tumors, assess the formation or risk of liver tumors and/or assess the progression of liver tumors .
  • the DNA region used for determination may include the DNA region where the target gene is located or a fragment thereof as described in the method of the present application.
  • the present application provides nucleic acids, nucleic acid sets and/or kits for determining the modification status of DNA regions, in the manufacture of substances for confirming the presence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progression of a disease
  • the DNA region used for determination may include the target DNA region as described in the method of the present application, or its complementary region, or the above-mentioned fragments.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing a disease, and/or assessing the progress of a disease, which may include providing a nucleic acid, nucleic acid group, and/or kit for determining the modification status of a DNA region.
  • the DNA region used for determination may comprise the target DNA region as described in the method of the present application, or its complementary region, or the fragments mentioned above.
  • the present application provides nucleic acids, sets of nucleic acids and/or kits for determining the modification status of DNA regions, which can be used to confirm the presence of a disease, assess the development or risk of developing a disease, and/or assess the progression of a disease.
  • the DNA region used for determination may comprise the target DNA region as described in the method of the present application, or its complementary region, or the fragments mentioned above.
  • the disease may comprise tumors.
  • solid tumors for example, in the application of the present application, the disease may comprise liver tumors.
  • the modification state may include methylation modification.
  • the present application provides the DNA region where the target gene is located in the method of the present application, or the region transformed therefrom, or the nucleic acid of the above-mentioned fragments, or a combination thereof, which can be used in the preparation for confirming the existence and evaluation of liver tumors Use of substances for liver neoplasia or risk of formation and/or assessment of progression of liver neoplasia.
  • the present application provides methods for confirming the existence of liver tumors, assessing the formation of liver tumors or the risk of forming liver tumors, and/or assessing the progress of liver tumors, which may include providing the DNA region where the target gene in the method of the present application is located, or its transformation The resulting region, or the nucleic acid of the above-mentioned fragments, or a combination thereof.
  • the present application provides the DNA region where the target gene is located in the method of the present application, or the region transformed therefrom, or the nucleic acid of the above-mentioned fragments, or a combination thereof, which can be used to confirm the existence and evaluation of liver tumors Liver neoplasia or risk of development and/or assessment of progression of liver neoplasia.
  • the present application provides the target DNA region as described in the method of the present application, or its complementary region, or the above-mentioned transformed region, or the nucleic acid of the above-mentioned fragment, or a combination thereof, which can be used to confirm the disease Use in substances for the presence, assessment of the development or risk of developing a disease and/or for assessing the progression of a disease.
  • the disease may comprise a tumor.
  • the disease may comprise liver tumors.
  • the present application provides a method for confirming the existence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease, which may include providing the target DNA region as described in the method of the present application, or its complementary region, or the above-mentioned The transformed region, or the nucleic acid of the above-mentioned fragment, or a combination thereof.
  • the disease may comprise tumors.
  • the disease may comprise liver tumors.
  • the present application provides the target DNA region as described in the method of the present application, or its complementary region, or the above-mentioned transformed region, or the nucleic acid of the above-mentioned fragment, or a combination thereof, which can be used to confirm the diagnosis of the disease Presence, assessment of disease development or risk of developing and/or assessment of disease progression.
  • the disease may comprise tumors.
  • the disease may comprise liver tumors.
  • the present application provides a device, which may include the storage medium of the present application.
  • the present application provides a non-volatile computer-readable storage medium, on which a computer program is stored, and the program is executed by a processor to implement any one or more methods described in the present application.
  • the method may comprise a method of using a methylation indicator to classify samples with different likelihoods of being cancerous.
  • Exemplary classification algorithms can be linear discriminant analysis, logistic regression, naive Bayesian classification, perceptual classification, quadratic classification, k-nearest neighbors, boosting methods, decision trees, random forests, neural networks, learning vector quantization, or support vector machines, or a combination thereof.
  • the non-transitory computer readable storage medium may include a floppy disk, a flexible disk, a hard disk, a solid state storage (SSS) (such as a solid state drive (SSD)), a solid state card (SSC), a solid state module (SSM)), an enterprise high-grade flash drives, tape, or any other non-transitory magnetic media, etc.
  • SSD solid state drive
  • SSC solid state card
  • SSM solid state module
  • enterprise high-grade flash drives tape, or any other non-transitory magnetic media, etc.
  • Non-transitory computer readable storage media may also include punched cards, paper tape, cursor sheets (or any other physical media having a pattern of holes or other optically identifiable markings), compact disc read only memory (CD-ROM) , Rewritable Disc (CD-RW), Digital Versatile Disc (DVD), Blu-ray Disc (BD) and/or any other non-transitory optical media.
  • CD-ROM compact disc read only memory
  • CD-RW Rewritable Disc
  • DVD Digital Versatile Disc
  • BD Blu-ray Disc
  • the device of the present application may further include a processor coupled to the storage medium, and the processor is configured to execute based on a program stored in the storage medium to implement the method of the present application.
  • the device may implement various mechanisms to ensure that the methods described herein performed on the database system produce correct results.
  • the device may use a magnetic disk as persistent data storage.
  • the device can provide database storage and processing services for multiple database clients.
  • the appliance may store database data across multiple shared storage devices, and/or may utilize one or more execution platforms with multiple execution nodes.
  • the device can be organized such that storage and computing resources can be effectively infinitely expanded.
  • the invention uses fluorescent PCR detection technology to detect the methylation status of markers related to the buffy coat (most of which are white blood cells), paracancerous tissues and liver cancer tissues. Preliminary confirmation of the potential of this marker for blood detection of liver tumors.
  • An ideal marker for detection of methylation in liver cancer should have the following characteristics: 1. Low level of DNA methylation in buffy coat; 2. High level of DNA methylation in liver cancer tissue.
  • the detection of methylation markers can be carried out by methylation-specific PCR (MSP).
  • MSP methylation-specific PCR
  • the basic principle of MSP is that only methylated sequence templates generate amplification signals during detection, while non-methylated sequence templates do not. Amplifies the signal.
  • the method can be realized by designing methylation-specific sequences for primers or probes, or by simultaneously designing methylation-specific primers and probe pairs.
  • the main steps include: 1. According to the marker sequence, design detection primers and probes in the detection segment suitable for MSP (which may be a CpG-rich region). 2. Perform nucleic acid extraction of white blood cells and tissue samples. 3. Bisulfite treatment is performed on nucleic acid to convert unmethylated cytosine into uracil, while methylated cytosine maintains the sequence. 4. Perform fluorescent PCR detection.
  • the markers satisfying the above-mentioned characteristics through the detection of white blood cells and tissue samples prove that they have the potential to detect liver cancer through blood. Then, these markers were verified in plasma samples, and the test samples included the control group, liver cancer group, and hepatitis B and liver cirrhosis interference population. The reference levels of these markers and marker combination performance were analyzed. Due to the limited free DNA in the plasma of a single sample, the target can be pre-amplified when the fluorescent PCR detection is optional, so that the least amount of DNA can detect as many methylated sites as possible.
  • the main steps include: 1. Nucleic acid extraction of plasma samples. 2. Bisulfite treatment of nucleic acid converts unmethylated cytosine into uracil, while methylated cytosine maintains its sequence. 3. Perform pre-amplification and dilution of the target. 4. Perform fluorescent PCR detection.
  • any combination can be performed on the basis of the above target combination, or the mathematical model of machine learning can be introduced into the data algorithm, such as linear regression, support vector regression, ridge regression, random forest, etc. , to obtain a liver cancer methylation detection marker combination with high detection accuracy.
  • the content provided by this application 1. Found many liver cancer methylation markers with excellent performance; 2. Developed a combination of liver cancer methylation detection markers with high detection accuracy; 3. The detection performance is better than the existing fluorescent PCR The detection method has been greatly improved; 4. Compared with the second-generation sequencing detection method, it has many advantages such as easy development, easy clinical promotion, and low cost.
  • the application of the markers and marker combinations provided in this application may not be limited to specific detection methods.
  • a method for confirming the existence of liver tumors, assessing the formation or risk of liver tumors and/or evaluating the progress of liver tumors comprising determining BCAT1, IKZF1, VAV3, IRF4, B4GALNT1, GRASP, BEST4, BEND4, The presence and/or content of the modified state of the DNA region where the GPAM and VASH2 genes are located, the DNA region where human chr1:145384249:145413094 is located, and/or the DNA region where human chr7:26415938:26416740 is located or fragments thereof.
  • a method for assessing the methylation status of liver tumor-related DNA regions comprising determining the DNA regions where BCAT1, IKZF1, VAV3, IRF4, B4GALNT1, GRASP, BEST4, BEND4, GPAM, and VASH2 genes are located in samples to be tested, derived from human The presence and/or content of the DNA region where chr1:145384249:145413094 is located, and/or the DNA region or fragment thereof derived from human chr7:26415938:26416740.
  • nucleic acid comprises cell-free nucleic acid.
  • sample to be tested comprises tissues, cells and/or body fluids.
  • the bases having the modified state and the bases not having the modified state form different substances after conversion.
  • the bases having the modified state are substantially unchanged after conversion, and the bases not having the modified state are changed to Other bases that are different from said base, or are cleaved after conversion.
  • the method for determining the presence and/or content of the modified state comprises, confirming that the base with the modified state is formed after the conversion presence and/or content.
  • the method for determining the presence and/or content of a modified state comprises, determining the presence and/or content of a DNA region or fragment thereof having the modified state .
  • the presence and/or content of the DNA region or fragment thereof having the modification state is determined by the fluorescent Ct value detected by the fluorescent PCR method.
  • a method for confirming the presence of a disease, assessing the formation of a disease or the risk of forming a disease, and/or assessing the progress of a disease comprising determining the modification status of a DNA region selected from the following group, or its complementary region, or the above-mentioned fragment in the sample to be tested
  • Presence and/or content derived from human chr12: 25101630-25102393, derived from human chr12: 25078661-25079410 and derived from human chr12: 25054781-25056311, or derived from human chr7: 50343720-50344547, derived from human chr7 :50450118-50450531 and derived from human chr7:50467368-50469092, or derived from human chr1:108507215-108507766, or derived from human chr6:391739-394056 and derived from human chr6:401233-401801, or derived from human chr12:
  • a method for determining the methylation state of a DNA region comprising determining the presence and/or content of the modified state of a DNA region, or its complementary region, or the above-mentioned fragments in a sample to be tested: derived from human chr12 :25101630-25102393, derived from human chr:12:25078661-25079410 and derived from human chr12:25054781-25056311, or derived from human chr7:50343720-50344547, derived from human chr7:50450118-504580531 and derived from human chr7:50450118-504580531 and derived from human 4673:6 -50469092, or from human chr1: 108507215-108507766, or from human chr6: 391739-394056 and from human chr6: 401233-401801, or from human chr12: 58020498-58022962 and from human chr12: 580
  • any one of embodiments 22-23 comprising providing a nucleic acid capable of binding a DNA region comprising a region selected from the group consisting of, or a complementary region thereof, or a transformed region as described above, or a fragment as described above: SEQ ID NO: 32, SEQ ID NO: 39 and SEQ ID NO: 43, or SEQ ID NO: 5, SEQ ID NO: 50 and SEQ ID NO: 54, or SEQ ID NO: 58, or SEQ ID NO: 21 and SEQ ID NO: 71, or SEQ ID NO: 13 and SEQ ID NO: 78, or SEQ ID NO: 17 and SEQ ID NO: 85, or SEQ ID NO: 9 and SEQ ID NO: 92, or SEQ ID NO : 25, or SEQ ID NO: 99, SEQ ID NO: 106 and SEQ ID NO: 110, or SEQ ID NO: 120, or SEQ ID NO: 127.
  • nucleic acid or its complementary nucleic acid, or a fragment thereof selected from the group consisting of: SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 40 and SEQ ID NO: 44, or SEQ ID NO: 6, SEQ ID NO: 47, SEQ ID NO: 51 and SEQ ID NO: 55, or SEQ ID NO: 59 and SEQ ID NO: 62, or SEQ ID NO : 22, SEQ ID NO: 65, SEQ ID NO: 68 and SEQ ID NO: 72, or SEQ ID NO: 14, SEQ ID NO: 75 and SEQ ID NO: 79, or SEQ ID NO: 18, SEQ ID NO : 82 and SEQ ID NO: 86, or SEQ ID NO: 10, SEQ ID NO: 89 and SEQ ID NO: 93, or SEQ ID NO: 26 and SEQ ID NO: 96, or SEQ ID NO: 100, SEQ ID NO: 103, SEQ ID NO:
  • SEQ ID NO: 104 and 105 SEQ ID NO: 108 and 109 and SEQ ID NO: 112 and 113, or SEQ ID NO: 115 and 116 and SEQ ID NO: 118 and 119, or SEQ ID NO: 122 and 123 and SEQ ID NO: 125 and 126, or SEQ ID NO: 129 and 130 and SEQ ID NO: 132 and 133.
  • sample to be tested comprises tissue, cells and/or body fluids.
  • the bases having the modified state are substantially unchanged after conversion, and the bases that do not have the modified state are changed to Other bases that are different from said base, or are cleaved after conversion.
  • the method for determining the presence and/or content of the modified state comprises, confirming that the base with the modified state is formed after the transformation. presence and/or content.
  • the method for determining the presence and/or content of a modification state comprises, determining the presence and/or content of a DNA region or fragment thereof having the modification state .
  • the presence and/or content of the DNA region or fragment thereof having the modification state is determined by the fluorescent Ct value detected by the fluorescent PCR method.
  • a nucleic acid comprising a DNA region capable of binding BCAT1, IKZF1, VAV3, IRF4, B4GALNT1, GRASP, BEST4, BEND4, GPAM, VASH2 genes, a DNA region derived from human chr1:145384249:145413094, and/or Or derived from the DNA region where human chr7:26415938:26416740 is located, or its complementary region, or the above-mentioned transformed region, or the sequence of the above-mentioned fragment.
  • a method for preparing nucleic acid comprising the DNA region where BCAT1, IKZF1, VAV3, IRF4, B4GALNT1, GRASP, BEST4, BEND4, GPAM, VASH2 genes are located, the DNA region where human chr1:145384249:145413094 is located, and/or Derived from the modified state of the DNA region where human chr7:26415938:26416740 is located, or its complementary region, or the above-mentioned transformed region, or the above-mentioned fragment, the design can bind to the DNA region, or its complementary region, or the above-mentioned Nucleic acid of the transformed region or the above-mentioned fragment.
  • a nucleic acid group comprising the DNA region where BCAT1, IKZF1, VAV3, IRF4, B4GALNT1, GRASP, BEST4, BEND4, GPAM, VASH2 genes are located, the DNA region where human chr1:145384249:145413094 is located, And/or derived from the DNA region where human chr7:26415938:26416740 is located, or its complementary region, or the above-mentioned transformed region, or the sequence of the above-mentioned fragment.
  • a method for preparing a nucleic acid group comprising the DNA region where BCAT1, IKZF1, VAV3, IRF4, B4GALNT1, GRASP, BEST4, BEND4, GPAM, VASH2 genes are located, the DNA region where human chr1:145384249:145413094 is located, and/or Or derived from the DNA region where human chr7:26415938:26416740 is located, or its complementary region, or the above-mentioned transformed region, or the modified state of the above-mentioned fragment, the design can amplify the DNA region, or its complementary region, or The nucleic acid group of the above-mentioned transformed region or the above-mentioned fragment.
  • a kit comprising the nucleic acid of embodiment 47 and/or the nucleic acid set of embodiment 49.
  • nucleic acid according to embodiment 47 the nucleic acid set according to embodiment 49 and/or the kit according to embodiment 51 in the preparation of disease detection products.
  • nucleic acid according to embodiment 47 in the preparation of confirmation of the presence of a disease, assessment of disease formation or risk of formation and/or assessment of disease The progress of the application of the substance.
  • nucleic acid according to embodiment 47 Use of the nucleic acid according to embodiment 47, the nucleic acid set according to embodiment 49 and/or the kit according to embodiment 51, in the preparation of a substance for determining the modification state of the DNA region or fragment thereof .
  • the DNA region used for determination includes the DNA region where BCAT1, IKZF1, VAV3, IRF4, B4GALNT1, GRASP, BEST4, BEND4, GPAM, VASH2 genes are located, the DNA region where human chr1:145384249:145413094 is located, and/or source In human chr7:26415938:26416740 DNA region or its fragment.
  • the DNA region comprises a DNA region selected from the following group, or a complementary region thereof, or a fragment thereof: derived from human chr12:25101630-25102393, derived from human chr12:25078661-25079410 and derived from human chr12:25054781-25056311, or From human chr7: 50343720-50344547, from human chr7: 50450118-50450531 and from human chr7: 50467368-50469092, or from human chr1: 108507215-108507766, or from human chr6: 391739-394056 and from human chr6: 401233-401801, or derived from human chr12
  • Nucleic acid selected from the following group of DNA regions, or complementary regions thereof, or the above-mentioned transformed regions, or the above-mentioned fragments, and a combination of the above-mentioned nucleic acids, used in the preparation for confirming the presence of a disease, assessing the formation or formation of a disease
  • a storage medium recording a program capable of performing the method of any one of embodiments 1-46.
  • An apparatus comprising the storage medium of embodiment 59.
  • the device of embodiment 59 further comprising a processor coupled to the storage medium, the processor configured to execute based on a program stored in the storage medium to implement any of embodiments 1-46 any one of the methods described.
  • a processor coupled to the storage medium, the processor configured to execute based on a program stored in the storage medium to implement any of embodiments 1-46 any one of the methods described.
  • DNA samples (10 buffy coat samples and 10 cancer tissues each) were obtained from the buffy coat of healthy people with no abnormal liver and the cancer tissue of liver cancer patients, and the buffy coat DNA was selected as a reference The sample is because most of the plasma free DNA comes from the DNA released after the buffy coat ruptures, and its background background can be a basic background signal of the detection site of the plasma free DNA.
  • Qiagen QIAamp DNA Mini Kit was used to extract buffy coat DNA
  • Qiagen QIAamp DNA FFPE Tissue Kit was used to extract tissue DNA.
  • a 20 ng sample of the DNA obtained in the above steps was treated with bisulfite reagent (D5031, ZYMO RESEARCH) to obtain transformed DNA.
  • the detection fluorescence Ct value of the marker is obtained.
  • the final concentration of each primer is 500 nM, and the final concentration of each detection probe is 200 nM.
  • the PCR reaction system contains: 10 ⁇ L of pre-amplified diluted product, 2.5 ⁇ L of primer and probe premix containing the detection site; 12.5 ⁇ L of PCR reagent ( Universal Probe qPCR Master Mix (NEB).
  • the PCR reaction conditions were as follows: 95° C. for 5 minutes; 95° C. for 30 seconds, 56° C. for 60 seconds (collecting fluorescence), and 50 cycles were performed.
  • Use ABI 7500 Real-Time PCR System to detect different fluorescence in the corresponding fluorescence channel.
  • Cell-free extracellular DNA in the above plasma samples was extracted using commercial Qiagen QIAamp Circulating Nucleic Acid Kit. Use the commercial bisulfite conversion reagent MethylCodeTM Bisulfite Conversion Kit to perform sulfite conversion on the extracted extracellular free DNA to obtain the converted DNA.
  • the above-mentioned transformed DNA is used for pre-amplification, using methylation marker-specific primers and internal reference primers (ACTB) containing the methylation markers shown in Table 1, the forward primer and reverse primer can be as shown in Table 1, respectively.
  • the probe can be a primer pool as shown in Table 2), and the converted DNA is used as a template for PCR amplification, and the final concentration of each primer is 100 nM.
  • the PCR reaction system is 10 ⁇ L of transformed DNA, 2.5 ⁇ L of the master mix containing the above primers; PCR reagent ( Universal Probe qPCR Master Mix (NEB) 12.5 ⁇ L.
  • the PCR reaction conditions are as follows: 95° C. for 5 minutes; 95° C. for 30 seconds, 56° C. for 60 seconds, 15 cycles.
  • the obtained preamplified products were diluted 10 times and used for fluorescent PCR detection.
  • the primers for each methylation marker shown in Table 1, the detection probe sequences shown in Table 2, and the internal reference gene ACTB were used as a control.
  • the final primer concentration was 500nM, and the final probe concentration was 200nM.
  • the PCR reaction system includes: 10 ⁇ L of pre-amplified diluted product, 2.5 ⁇ L of primer and probe premix containing the detection site; PCR reagent ( Universal Probe qPCR Master Mix (NEB) 12.5 ⁇ L.
  • the fluorescent PCR reaction system is the same as the above-mentioned embodiment.
  • the PCR reaction conditions are as follows: 95° C. for 5 minutes; 95° C. for 15 seconds, 56° C. for 40 seconds (collecting fluorescence), 50 cycles.
  • To modify the fluorescence of different gene probes select the corresponding detection fluorescence channel.
  • the target Ct value for which no amplification signal was detected was set at 50. The results show that each target of this application can have the ability to detect liver cancer in blood.
  • results in the above table show that the DNA methylation signals of the detection sites are compared in the control plasma and liver cancer plasma.
  • the selected target markers were demonstrated to have high sensitivity to blood samples from liver cancer patients.
  • the data analysis can adopt the method of setting a positive interpretation threshold for a single target, and when combining targets, if any target is positive, the sample is comprehensively interpreted as positive. Verified this way:
  • the sensitivity of liver cancer detection can be 64.5%, and the specificity of healthy controls is as high as 95.9%.
  • the positive rate of hepatitis B patients is 2.7%, and the positive rate of liver cirrhosis patients is 20.5%.
  • SEPT9(1), IKZF1, BEST4, and B4GALNT1 can make the detection sensitivity of liver cancer 73.1%, and the specificity of healthy controls is 91.0%.
  • the positive rate of hepatitis B patients is 9.1%, and the positive rate of liver cirrhosis patients is 20.5%.
  • DNA samples were obtained from leukocytes from healthy people with no abnormalities in the liver, paracancerous tissues and cancerous tissues from patients with liver cancer (including about 10 white blood cell samples, and 24 paracancerous tissues and cancerous tissues respectively), and selected
  • the white blood cell DNA is used as a reference sample because most of the free plasma DNA is derived from the DNA released after the leukocyte rupture, and its background background can be a basic background signal of the detection site of the free plasma DNA.
  • Qiagen QIAamp DNA Mini Kit was used to extract white blood cell DNA
  • Qiagen QIAamp DNA FFPE Tissue Kit was used to extract tissue DNA.
  • a 20 ng sample of the DNA obtained in the above steps was treated with bisulfite reagent (D5031, ZYMO RESEARCH) to obtain transformed DNA.
  • the primer pool containing the target methylation-specific primers of the application and the internal reference (ACTB) primer pair use the converted DNA as a template to perform PCR amplification, and the final concentration of each primer is 100 nM.
  • the PCR reaction system contains: 10 ⁇ L transformed DNA (2.5 ⁇ L master mix containing the above primers) and 12.5 ⁇ L PCR reagent ( Universal Probe qPCR Master Mix (NEB).
  • the PCR reaction conditions are: 95°C for 5 minutes; 95°C for 30 seconds, 56°C for 60 seconds, and 10 cycles.
  • the positions of lowercase letters in the sequences of the tables of this application may correspond to the positions of natural cytosines on the regions of the genome to which the sequences bind.
  • the bases represented by lowercase letters in the sequence in the application form can be used to pair with converted bases; for example, natural cytosine can be converted to uracil after bisulfite treatment, and the lowercase base a can be paired with The converted uracil is paired, and the lowercase base t can be further paired with the adenine that is paired with the uracil.
  • the primer sequences are shown in Table 2-1.
  • IRF4 reverse primer 1a CGCCTACCCCTCCGCGCG 69
  • IRF4 forward primer 1b AtAAGTGGCGtAGACGCGGG 70
  • IRF4 reverse primer 1b CCTCCGCTCTCCCGaaCCTa 73
  • IRF4 forward primer 2 CGACGGGtTtTATGCGAAAAG 74
  • IRF4 reverse primer 2 aAaCTTaCAaaTCTaaTCTCTCTCC 135 ACTB forward primer GTGATGGAGGAGGTTTAGTAAGTT 136
  • the obtained pre-amplified products were diluted 10 times, and detected by multiplex fluorescent PCR to obtain the detection fluorescence Ct value of the marker.
  • the final concentration of each primer is 500 nM
  • the final concentration of each detection probe is 200 nM.
  • the PCR reaction system contains: 10 ⁇ L of pre-amplified diluted product, 2.5 ⁇ L of primer and probe premix containing the detection site; 12.5 ⁇ L of PCR reagent ( Universal Probe qPCR Master Mix (NEB).
  • the PCR reaction conditions were as follows: 95° C. for 5 minutes; 95° C. for 30 seconds, 56° C. for 60 seconds (collecting fluorescence), and 50 cycles were performed.
  • ABI 7500 Real-Time PCR System to detect different fluorescence in the corresponding fluorescence channel. Calculate and compare the sample Ct values obtained from leukocytes, paracancerous tissues, and cancer tissues, and set the Ct value of the target point where no amplification signal is detected as 50.
  • the primer sequences are shown in Table 2-1, and the probe sequences are shown in Table 2-2.
  • Plasma samples from about 65 individuals with liver cancer and about 76 control individuals were tested using fluorescent PCR assays:
  • Cell-free extracellular DNA in the above plasma samples was extracted using commercial Qiagen QIAamp Circulating Nucleic Acid Kit. Use the commercial bisulfite conversion reagent MethylCodeTM Bisulfite Conversion Kit to perform sulfite conversion on the extracted extracellular free DNA to obtain the converted DNA.
  • the above-mentioned converted DNA is used for pre-amplification, and the primer pool containing the application target methylation-specific primer and internal reference (ACTB) primer pair shown in Table 2-1 is used to convert the transformed DNA DNA was used as a template for PCR amplification, and the final concentration of each primer was 100 nM.
  • the PCR reaction system is 10 ⁇ L of transformed DNA, 2.5 ⁇ L of the master mix containing the above primers; PCR reagent ( Universal Probe qPCR Master Mix (NEB) 12.5 ⁇ L.
  • the PCR reaction conditions are as follows: 95° C. for 5 minutes; 95° C. for 30 seconds, 56° C. for 60 seconds, 15 cycles.
  • the obtained preamplified products were diluted 10 times and used for fluorescent PCR detection. Use the primers shown in Table 2-1, the detection probe sequences shown in Table 2-2, and simultaneously detect the internal reference gene ACTB as a control. The final primer concentration was 500nM, and the final probe concentration was 200nM.
  • the PCR reaction system includes: 10 ⁇ L of pre-amplified diluted product, 2.5 ⁇ L of primer and probe premix containing the detection site; PCR reagent ( Universal Probe qPCR Master Mix (NEB) 12.5 ⁇ L.
  • the fluorescent PCR reaction system is the same as in this example.
  • the PCR reaction conditions are as follows: 95° C. for 5 minutes; 95° C. for 15 seconds, 56° C. for 40 seconds (collecting fluorescence), 50 cycles.
  • To modify the fluorescence of different gene probes select the corresponding detection fluorescence channel.
  • the target Ct value for which no amplification signal was detected was set at 50.
  • DNA samples were obtained from the buffy coat of healthy people with no abnormalities in the liver and the cancer tissue of liver cancer patients (including about 10 samples of buffy coat and about 10 samples of cancer tissue), and the DNA samples of buffy coat were selected. It is used as a reference sample because most of the plasma free DNA comes from the DNA released after the buffy coat ruptures, and its background background can be a basic background signal of the detection site of the plasma free DNA. According to the instructions, Qiagen QIAamp DNA Mini Kit was used to extract buffy coat DNA, and Qiagen QIAamp DNA FFPE Tissue Kit was used to extract tissue DNA. A 20 ng sample of the DNA obtained in the above steps was treated with bisulfite reagent (D5031, ZYMO RESEARCH) to obtain transformed DNA.
  • bisulfite reagent D5031, ZYMO RESEARCH
  • the primer pool containing the target methylation-specific primers of the application and the internal reference (ACTB) primer pair use the converted DNA as a template to perform PCR amplification, and the final concentration of each primer is 100 nM.
  • the PCR reaction system contains: 10 ⁇ L transformed DNA (2.5 ⁇ L master mix containing the above primers) and 12.5 ⁇ L PCR reagent ( Universal Probe qPCR Master Mix (NEB).
  • the PCR reaction conditions are: 95°C for 5 minutes; 95°C for 30 seconds, 56°C for 60 seconds, and 10 cycles.
  • the positions of lowercase letters in the sequences of the tables of this application may correspond to the positions of natural cytosines on the regions of the genome to which the sequences bind.
  • the bases represented by lowercase letters in the sequence in the application form can be used to pair with converted bases; for example, natural cytosine can be converted to uracil after bisulfite treatment, and the lowercase base a can be paired with The converted uracil is paired, and the lowercase base t can be further paired with the adenine that is paired with the uracil.
  • the primer sequences are shown in Table 2-5.
  • the obtained pre-amplified products were diluted 10 times, and detected by multiplex fluorescent PCR to obtain the detection fluorescence Ct value of the marker.
  • the final concentration of each primer is 500 nM
  • the final concentration of each detection probe is 200 nM.
  • the PCR reaction system contains: 10 ⁇ L of pre-amplified diluted product, 2.5 ⁇ L of primer and probe premix containing the detection site; 12.5 ⁇ L of PCR reagent ( Universal Probe qPCR Master Mix (NEB).
  • the PCR reaction conditions were as follows: 95° C. for 5 minutes; 95° C. for 30 seconds, 56° C. for 60 seconds (collecting fluorescence), and 50 cycles were performed.
  • ABI 7500 Real-Time PCR System to detect different fluorescence in the corresponding fluorescence channel. Calculate and compare the sample Ct values obtained from the buffy coat, paracancerous tissue and cancer tissue, and set the target Ct value of no amplification signal to 50.
  • the primer sequences are shown in Table 2-5, and the probe sequences are shown in Table 2-6.
  • Intergenic region 2 represents the human chr1:145384249:145413094 region
  • intergenic region 1 represents the human chr7:26415938:26416740 region.
  • the above-mentioned transformed DNA is used for pre-amplification, and the primer pool containing the target methylation-specific primers of the application shown in Table 2-5 and the internal reference (ACTB) primer pair is used to convert the transformed DNA DNA was used as a template for PCR amplification, and the final concentration of each primer was 100 nM.
  • the PCR reaction system is 10 ⁇ L of transformed DNA, 2.5 ⁇ L of the master mix containing the above primers; PCR reagent ( Universal Probe qPCR Master Mix (NEB) 12.5 ⁇ L.
  • the PCR reaction conditions are as follows: 95° C. for 5 minutes; 95° C. for 30 seconds, 56° C. for 60 seconds, 15 cycles.
  • the obtained preamplified products were diluted 10 times and used for fluorescent PCR detection. Use the primers shown in Table 2-5, the detection probe sequences shown in Table 2-6, and simultaneously detect the internal reference gene ACTB as a control. The final primer concentration was 500nM, and the final probe concentration was 200nM.
  • the PCR reaction system includes: 10 ⁇ L of pre-amplified diluted product, 2.5 ⁇ L of primer and probe premix containing the detection site; PCR reagent ( Universal Probe qPCR Master Mix (NEB) 12.5 ⁇ L.
  • the fluorescent PCR reaction system is the same as in this example.
  • the PCR reaction conditions are as follows: 95° C. for 5 minutes; 95° C. for 15 seconds, 56° C. for 40 seconds (collecting fluorescence), 50 cycles.
  • To modify the fluorescence of different gene probes select the corresponding detection fluorescence channel.
  • the target Ct value for which no amplification signal was detected was set to 50.
  • the results in the above table show the AUC statistics of the markers between the liver cancer group and the healthy control group.
  • the targets of this application can all be used for blood detection of liver cancer.
  • the results showed that the DNA methylation signals of the detection sites were compared in the control plasma and liver cancer plasma.
  • the selected target markers were demonstrated to have high sensitivity to blood samples from liver cancer patients.
  • liver cancer samples were detected for each sub-region of the methylation marker of the present application.
  • the test results are shown in the table below:
  • the numbers after the underline in each detection area number are the sub-area numbers corresponding to the detection area. If the same number after the underline has different letters, it represents a different specific detection area in the same sub-area.
  • the primer pairs and probes used in each detection area are shown in Table 2-1, 2-5 and 2-2 and 2-6.
  • the number after the underline in each detection area number corresponds to the primer pair and probe in the corresponding table.
  • the methylation signal Ct value of cancer tissue reflects the exponential difference in the detection effect of the target.
  • the classification criteria for the detection effect of specific regions of each target point are: A indicates that the methylation signal of cancer tissue is 100 times higher than that of white blood cells, which is the preferred site for liver cancer detection; B indicates that the methylation signal of cancer tissue is between 10 times to 100 times, the site that may be used for liver cancer detection; C indicates that the methylation signal of cancer tissue is less than 10 times that of white blood cells, and the site is likely not to be used for liver cancer detection.
  • DNA samples (10 buffy coat samples and 10 cancer tissues each) were obtained from the buffy coat of healthy people with no abnormal liver and the cancer tissue of liver cancer patients, and the buffy coat DNA was selected as a reference The sample is because most of the plasma free DNA comes from the DNA released after the buffy coat ruptures, and its background background can be a basic background signal of the detection site of the plasma free DNA.
  • Qiagen QIAamp DNA Mini Kit was used to extract buffy coat DNA
  • Qiagen QIAamp DNA FFPE Tissue Kit was used to extract tissue DNA.
  • a 20 ng sample of the DNA obtained in the above steps was treated with bisulfite reagent (D5031, ZYMO RESEARCH) to obtain transformed DNA.
  • the detection fluorescence Ct value of the marker is obtained.
  • the final concentration of each primer is 500 nM, and the final concentration of each detection probe is 200 nM.
  • the PCR reaction system contains: 10 ⁇ L of pre-amplified diluted product, 2.5 ⁇ L of primer and probe premix containing the detection site; 12.5 ⁇ L of PCR reagent ( Universal Probe qPCR Master Mix (NEB).
  • the primer sequences of each methylation marker are shown in Table 4-1, and the probe sequences are shown in Table 4-2.
  • the PCR reaction conditions were as follows: 95° C. for 5 minutes; 95° C. for 30 seconds, 56° C. for 60 seconds (collecting fluorescence), and 50 cycles were performed.

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Abstract

提供了一种肿瘤评估方法及应用，具体提供了一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，包含评估待测样本中一组标志物基因所在DNA区域或其片段的修饰状态的存在和/或含量。还涉及用于评估一组DNA区域修饰状态的核酸、核酸组和/或试剂盒，及其制备方法。

Description

一种肿瘤评估方法及应用 技术领域
本申请涉及生物医学领域，具体的涉及一种肿瘤评估方法及应用。
背景技术
寻找更高效的肿瘤标志物一直是肿瘤相关研究中重要的一个方向，基于液体活检的血浆游离DNA检测技术对于肿瘤标志物的发现有了更高的需求。其基本原理为：肿瘤细胞死亡后，释放游离DNA进入血液，通过检测血液中的DNA突变、DNA甲基化、miRNA、组蛋白修饰等肿瘤细胞相关信息，可探测到极早期的肿瘤信号。已有研究发现DNA甲基化变化可能早于DNA突变的发生，是肿瘤早期筛查的更行之有效的检测标志物。
但目前早期癌症中，释放入血的肿瘤细胞极少，并且本领域急需寻找到更多的对于肝肿瘤有效的检测标志物。因此，亟需开发一种方法和/或试剂盒，通过检测准确、稳定、有效的肝癌生物标志物或其组合，且可以从生物样品中数量极为有限的细胞外游离DNA高效地读取到该表观遗传学信息，更优的，该方法应可以在医院检验科里很容易地配置并可靠地应用。
发明内容
本申请提供的方法可以将更多高效的甲基化标志物，用于确认或辅助确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展，可以提高肿瘤，例如肝肿瘤的早筛早诊效率，解决肝癌早诊率低、临床治疗负担重等问题。
一方面，本申请提供了一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，包含确定待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量，所述目标基因包含SEPT9和IKZF1。
另一方面，本申请还提供了一种评估肝肿瘤相关DNA区域甲基化状态的方法，包含确定待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量，所述目标基因包含SEPT9和IKZF1。
另一方面，本申请还提供了一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，包含确定待测样本中目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量，所述目标DNA区域包含来源于人chr17:75368651-75370720和来源于人chr7:50343720-50344547定义的区域。
另一方面，本申请还提供了一种确定DNA区域甲基化状态的方法，包含确定待测样本中目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量，所述目标DNA区域包含来源于人chr17:75368651-75370720和来源于人chr7:50343720-50344547定义的区域。
另一方面，本申请还提供了一种核酸，所述核酸包含能够结合如本申请的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列；或者所述核酸包含能够结合如本申请的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。
另一方面，本申请还提供了一种制备核酸的方法，所述方法包含根据如本申请的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸；或者所述方法包含根据如本申请的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
另一方面，本申请还提供了一种核酸组，所述核酸组包含能够结合如本申请的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列；或者所述核酸组包含能够结合如本申请的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。
另一方面，本申请还提供了一种制备核酸组的方法，所述方法包含根据如本申请的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸组；或者所述方法包含根据如本申请的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸组。
另一方面，本申请还提供了一种试剂盒，包含如本申请的核酸和/或本申请的核酸组。
另一方面，本申请还提供了如本申请的核酸、如本申请的核酸组和/或本申请的试剂盒，在制备确定DNA区域或其片段的修饰状态的物质中的应用。
另一方面，本申请还提供了如本申请的核酸、如本申请的核酸组和/或本申请的试剂盒，在制备疾病检测产品中的应用。
另一方面，本申请还提供了如本申请的核酸、如本申请的核酸组和/或本申请的试剂盒， 在制备确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用。
另一方面，本申请还提供了用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，在制备用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的物质中的应用，所述用于确定的DNA区域包含如本申请的方法中所述目标基因所在DNA区域或其片段。
另一方面，本申请还提供了用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，在制备用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用，所述用于确定的DNA区域包含如本申请的方法中所述目标DNA区域、或其互补区域、或上述的片段。
另一方面，本申请还提供了如本申请的方法中所述目标基因所在DNA区域、或其转化而来的区域、或上述的片段的核酸或其组合，在制备用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的物质中的应用。
另一方面，本申请还提供了如本申请的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸或其组合，在制备用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用。
另一方面，本申请还提供了一种储存介质，其记载可以运行本申请的方法的程序。
另一方面，本申请还提供了一种设备，其包含本申请的储存介质。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1、3、5、7显示的是检测位点癌旁组织、癌组织和白细胞DNA甲基化信号对比。
图2、4、6、8显示的是检测位点在对照血浆和肝癌血浆DNA甲基化信号对比。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书 所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“BCAT1”通常是指一种基因或其表达产物。例如，BCAT1(Branched-chain-amino-acid aminotransferase)蛋白的UniProt登录号可以是P54687。本申请中，BCAT1可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。
在本申请中，术语“VASH2”通常是指一种基因或其表达产物。例如，VASH2(Tubulinyl-Tyr carboxypeptidase 2)蛋白的UniProt登录号可以是Q86V25。本申请中，VASH2可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。
在本申请中，术语“GPAM”通常是指一种基因或其表达产物。例如，GPAM(Glycerol-3-phosphate acyltransferase 1,mitochondrial)蛋白的UniProt登录号可以是Q9HCL2。本申请中，GPAM可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。
在本申请中，术语“VAV3”通常是指一种基因或其表达产物。例如，VAV3(Guanine nucleotide exchange factor VAV3)蛋白的UniProt登录号可以是Q9UKW4。本申请中，VAV3可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。
在本申请中，术语“SEPT9”通常是指一种基因或其表达产物。例如，SEPT9(SEPTIN9)蛋白的UniProt登录号可以是Q9UHD8。本申请中，SEPT9可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。例如，本申请中SEPT9也可以表示检测位点，例如针对该基因特定核酸序列进行甲基化检测。例如，以SEPT9表示的检测位点可以是该基因下的一个或多个区域的核酸序列。
在本申请中，术语“IKZF1”通常是指一种基因或其表达产物。例如，IKZF1(DNA-binding protein Ikaros)蛋白的UniProt登录号可以是Q13422。本申请中，IKZF1可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。例如，本申请中IKZF1也可以表示检测位点，例如针对该基因特定核酸序列进行甲基化检测。例如，以IKZF1表示的检测位点可以是该基因下的一个或多个区域的核酸序列。
在本申请中，术语“BEST4”通常是指一种基因或其表达产物。例如，BEST4(Bestrophin-4)蛋白的UniProt登录号可以是Q8NFU0。本申请中，BEST4可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。例如，本申请中BEST4 也可以表示检测位点，例如针对该基因特定核酸序列进行甲基化检测。例如，以BEST4表示的检测位点可以是该基因下的一个或多个区域的核酸序列。
在本申请中，术语“B4GALNT1”通常是指一种基因或其表达产物。例如，B4GALNT1(Beta-1,4N-acetylgalactosaminyltransferase 1)蛋白的UniProt登录号可以是Q00973。本申请中，B4GALNT1可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。例如，本申请中B4GALNT1也可以表示检测位点，例如针对该基因特定核酸序列进行甲基化检测。例如，以B4GALNT1表示的检测位点可以是该基因下的一个或多个区域的核酸序列。
在本申请中，术语“GRASP”通常是指一种基因或其表达产物。例如，GRASP(GRP1(General receptor for phosphoinositides 1)-associated scaffold protein)蛋白的UniProt登录号可以是K7CHN5。本申请中，GRASP可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。例如，本申请中GRASP也可以表示检测位点，例如针对该基因特定核酸序列进行甲基化检测。例如，以GRASP表示的检测位点可以是该基因下的一个或多个区域的核酸序列。
在本申请中，术语“IRF4”通常是指一种基因或其表达产物。例如，IRF4(Interferon regulatory factor 4)蛋白的UniProt登录号可以是Q15306。本申请中，IRF4可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。例如，本申请中IRF4也可以表示检测位点，例如针对该基因特定核酸序列进行甲基化检测。例如，以IRF4表示的检测位点可以是该基因下的一个或多个区域的核酸序列。
在本申请中，术语“BEND4”通常是指一种基因或其表达产物。例如，BEND4(BEN domain-containing protein 4)蛋白的UniProt登录号可以是Q6ZU67。本申请中，BEND4可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。例如，本申请中BEND4也可以表示检测位点，例如针对该基因特定核酸序列进行甲基化检测。例如，以BEND4表示的检测位点可以是该基因下的一个或多个区域的核酸序列。
在本申请中，术语“待测样本”通常是指需要进行检测的样本。例如，可以检测待测样本上的一个或者多个基因区域是否存在有修饰状态。
在本申请中，术语“无细胞游离核酸”或“cfDNA”通常是指样品中的DNA，当采集时，该DNA没有包含在细胞内。例如，无细胞游离核酸可以不是指通过细胞或组织的体外破裂而使其不在细胞内的DNA。例如，cfDNA可以包括正常细胞和源自癌细胞的DNA两者。例 如，cfDNA可以获自血液或血浆(“循环系统”)。例如，cfDNA可以通过分泌或细胞死亡过程，如细胞坏死或凋亡释放到循环系统中。
在本申请中，术语“互补核酸”通常是指与参考核苷酸序列相比具有互补的核苷酸序列。例如，互补核酸可以为任选地具有相反方向的核酸分子。例如，所述互补可以是指具有下面的互补性关联：鸟嘌呤和胞嘧啶；腺嘌呤和胸腺嘧啶；腺嘌呤和尿嘧啶。
在本申请中，术语“DNA区域”通常是指两个或更多个共价键合的天然存在的或经修饰的脱氧核糖核苷酸的序列。例如，基因的DNA区域可以是指该基因所位于的特定的脱氧核糖核苷酸的序列的位置，例如该脱氧核糖核苷酸的序列编码所述基因。例如，本申请的DNA区域包含DNA区域的全长、其互补区域，或者上述的片段。例如，本申请所提供的检测区域的上下游至少约20kb的序列可以作为检测的位点。例如，本申请所提供的区域的上下游至少约20kb、至少约15kb、至少约10kb、至少约5kb、至少约3kb、至少约2kb、至少约1kb、或至少约0.5kb的序列可以作为检测的位点。例如，可以根据所述微电脑设计合适的引物和探针进行样品的甲基化检测。
在本申请中，术语“修饰状态”通常是指本申请中基因片段、核苷酸或其碱基具有的修饰状态。例如，本申请中的修饰状态可以是指胞嘧啶的修饰状态。例如，本申请的具有修饰状态的基因片段可以具有改变的基因表达活性。例如，本申请的修饰状态可以是指碱基具有的甲基化修饰。例如，本申请的修饰状态可以是指在基因组DNA的CpG区域的胞嘧啶5'碳位共价结合一个甲基基团，例如可以成为5-甲基胞嘧啶(5mC)。例如，修饰状态可以是指DNA序列内存在或不存在5-甲基胞嘧啶(“5-mCyt”)。
在本申请中，术语“甲基化”通常是指本申请中基因片段、核苷酸或其碱基具有的甲基化状态。例如，本申请中基因所在的DNA片段可以在一条链或多条链上具有甲基化。例如，本申请中基因所在的DNA片段可以在一个位点或多个位点上具有甲基化。
在本申请中，术语“转化”通常是指将一种或多种结构转变为另一种结构。例如，本申请的转化可以是具有特异性。例如，不具有甲基化修饰的胞嘧啶经过转化可以变为其它结构(例如尿嘧啶)，且具有甲基化修饰的胞嘧啶经过转化可以基本不发生变化。例如，不具有甲基化修饰的胞嘧啶经过转化可以被剪切，且具有甲基化修饰的胞嘧啶经过转化可以基本不发生变化。
在本申请中，术语“脱氨基试剂”通常是指具有移除氨基能力的物质。例如，脱氨基试剂可以将未修饰的胞嘧啶的氨基脱除。
在本申请中，术语“亚硫酸氢盐”通常是指一种可以区分具有修饰状态和不具有修饰状态的DNA区域的试剂。例如，亚硫酸氢盐可以包括亚硫酸氢盐、或其类似物或上述的组合。例如，亚硫酸氢盐可以使未修饰的胞嘧啶的氨基脱氨基化，以使其与修饰的胞嘧啶区分。在本申请中，术语“类似物”通常是指具有类似结构和/或功能的物质。例如亚硫酸氢盐的类似物可以与亚硫酸氢盐具有类似的结构。例如，亚硫酸氢盐的类似物可以是指一种同样可以区分具有修饰状态和不具有修饰状态的DNA区域的试剂。
在本申请中，术语“甲基化敏感限制酶”通常是指一种根据其识别位点的甲基化状态而选择性消化核酸的酶。例如，对于当识别位点未被甲基化时才特异剪切的限制酶来说，当识别位点被甲基化时，可以不会发生剪切，或以显著降低的效率剪切。对于当识别位点被甲基化时才特异剪切的限制酶来说，当识别位点未被甲基化时，可以不会发生剪切，或以显著降低的效率剪切。例如，甲基化特异的限制酶可以识别含有CG二核苷酸(例如cgcg或cccggg)的序列。
在本申请中，术语“肿瘤”通常是指在正常生长和/或发育中呈现出至少部分失去控制的细胞和/或组织。例如，常见的肿瘤或癌细胞通常可以是失去了接触抑制并可能是入侵性的和/或具有转移的能力。例如，本申请的肿瘤可以是良性的，也可能是恶性的。
在本申请中，术语“进展”通常是指疾病从不太严重状态到较严重状态的变化。例如，肿瘤进展可以包括肿瘤的数量或严重性、癌细胞转移程度、癌症生长或扩散的速度等增大。例如，肿瘤进展可以包括这种癌症从不太严重状态到较严重状态的阶段时期，例如从I期到II期、从II期到III期等的进展。
在本申请中，术语“形成”通常是指个体体内出现病灶。例如，当肿瘤形成时，可以将该个体确诊为肿瘤患者。
在本申请中，术语“荧光PCR”通常是指一种定量或半定量的PCR技术。例如，可以是实时定量聚合酶链反应、定量聚合酶链反应或动力学聚合酶链反应的PCR技术。例如，可以利用PCR扩增并借助嵌入性荧光染料或序列特异性探针定量检测起始的靶核酸量，所述序列特异性探针可以含有仅与靶核酸杂交才可检出的荧光报道分子。
在本申请中，术语“PCR扩增”通常是指聚合酶链扩增反应。例如，本申请中的PCR扩增可以包含目前已知的用于DNA扩增的任意聚合酶链扩增反应。
在本申请中，术语“荧光Ct值”通常是指一种定量或半定量评估靶核酸的测量值。例如，可以是指荧光信号到达设定的域值时所经历的扩增反应循环数。
发明详述
本申请可以找到多种高效的肝脏肿瘤相关甲基化标志物，提高肝肿瘤早筛早诊效率，解决肝癌早诊率低，临床治疗负担重等问题。同时本申请提供多组高效的肝脏肿瘤甲基化标志物，并列举高效的标志物组合。
甲基化标志物组合
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量，所述目标基因可以包含SEPT9和IKZF1。
例如，本申请的方法可以包含，根据待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量，所述目标基因可以包含SEPT9和IKZF1。
例如，根据待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
例如，本申请的方法中，所述SEPT9的DNA区域可以来源于人chr17:75276651-75496678。
例如，本申请的方法中，所述IKZF1的DNA区域可以来源于人chr7:50343720-50472799。
例如，本申请的方法中，所述目标基因还可以包含选自以下组的基因：BEST4、B4GALNT1、GRASP、IRF4和BEND4。
例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4和B4GALNT1。 例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、GRASP和B4GALNT1。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、IRF4、B4GALNT1和BEND4。
例如，本申请的方法中，所述目标基因可以包含至少2种基因。
例如，本申请的方法中，所述目标基因可以包含2种至7种基因。例如，本申请的目标基因可以包含2种、3种、4种、5种、6种、或7种本申请提供的目标基因。例如，对相同的目标基因，本申请可以选择该目标基因所在的一种或更多种的DNA区域。例如，本申请的方法中，所述BEST4的DNA区域可以来源于人chr1:45249257-45253377。例如，本申请的方法中，所述B4GALNT1的DNA区域可以来源于人chr12:58017193-58027138。例如，本申请的方法中，所述GRASP的DNA区域可以来源于人chr12:52400724-52409673。例如，本申请的方法中，所述IRF4的DNA区域可以来源于人chr6:391739-411447。例如，本申请的方法中，所述BEND4的DNA区域可以来源于人chr4:42112955-42154895。
例如，本申请的方法中，所述目标基因可以包含选自以下组的基因中的2种：SEPT9、IKZF1、BEST4、B4GALNT1、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9和IKZF1。例如，本申请的方法中，所述目标基因可以包含SEPT9和BEST4。例如，本申请的方法中，所述目标基因可以包含SEPT9和B4GALNT1。例如，本申请的方法中，所述目标基因可以包含SEPT9和GRASP。例如，本申请的方法中，所述目标基因可以包含SEPT9和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1和BEST4。例如，本申请的方法中，所述目标基因可以包含IKZF1和B4GALNT1。例如，本申请的方法中，所述目标基因可以包含IKZF1和GRASP。例如，本申请的方法中，所述目标基因可以包含IKZF1和IRF4。例如，本申请的方法中，所述目标基因可以包含IKZF1和BEND4。例如，本申请的方法中，所述目标基因可以包含BEST4和B4GALNT1。例如，本申请的方法中，所述目标基因可以包含BEST4和GRASP。例如，本申请的方法中，所述目标基因可以包含BEST4和IRF4。例如，本申请的方法中，所述目标基因可以包含BEST4和BEND4。例如，本申请的方法中，所述目标基因可以包含B4GALNT1和GRASP。例如，本申请的方法中，所述目标基因可以包含B4GALNT1和IRF4。例如，本申请的方法中，所述目标基因可以包含B4GALNT1和BEND4。例如，本申请的方法中，所述目标基因可以包含GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含IRF4和BEND4。
例如，本申请的方法中，所述目标基因可以包含选自以下组的基因中的3种：SEPT9、IKZF1、BEST4、B4GALNT1、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1和BEST4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1和B4GALNT1。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1和GRASP。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4和B4GALNT1。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4和GRASP。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、B4GALNT1和GRASP。例如，本申请的方法中，所述目标基因可以包含SEPT9、B4GALNT1和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、B4GALNT1和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4和B4GALNT1。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4和GRASP。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4和IRF4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、B4GALNT1和GRASP。例如，本申请的方法中，所述目标基因可以包含IKZF1、B4GALNT1和IRF4。例如，本申请的方法中，所述目标基因可以包含IKZF1、B4GALNT1和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含IKZF1、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含BEST4、B4GALNT1和GRASP。例如，本申请的方法中，所述目标基因可以包含BEST4、B4GALNT1和IRF4。例如，本申请的方法中，所述目标基因可以包含BEST4、B4GALNT1和BEND4。例如，本申请的方法中，所述目标基因可以包含BEST4、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含BEST4、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含BEST4、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含B4GALNT1、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含B4GALNT1、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含B4GALNT1、 IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含GRASP、IRF4和BEND4。
例如，本申请的方法中，所述目标基因可以包含选自以下组的基因中的4种：SEPT9、IKZF1、BEST4、B4GALNT1、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4和B4GALNT1。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4和GRASP。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、B4GALNT1和GRASP。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、B4GALNT1和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、B4GALNT1和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、B4GALNT1和GRASP。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、B4GALNT1和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、B4GALNT1和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、B4GALNT1、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、B4GALNT1、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、B4GALNT1、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、B4GALNT1和GRASP。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、B4GALNT1和IRF4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、B4GALNT1和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、B4GALNT1、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含IKZF1、B4GALNT1、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、B4GALNT1、IRF4和BEND4。例 如，本申请的方法中，所述目标基因可以包含IKZF1、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含BEST4、B4GALNT1、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含BEST4、B4GALNT1、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含BEST4、B4GALNT1、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含B4GALNT1、GRASP、IRF4和BEND4。
例如，本申请的方法中，所述目标基因可以包含选自以下组的基因中的5种：SEPT9、IKZF1、BEST4、B4GALNT1、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、B4GALNT1和GRASP。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、B4GALNT1和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、B4GALNT1和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、B4GALNT1、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、B4GALNT1、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、B4GALNT1、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、B4GALNT1、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、B4GALNT1、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、B4GALNT1、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、B4GALNT1、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、B4GALNT1、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含BEST4、B4GALNT1、GRASP、IRF4和BEND4。
例如，本申请的方法中，所述目标基因可以包含选自以下组的基因中的6种：SEPT9、IKZF1、BEST4、B4GALNT1、GRASP和IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、B4GALNT1、GRASP和IRF4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、B4GALNT1、GRASP和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、B4GALNT1、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、B4GALNT1、 GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、BEST4、B4GALNT1、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含IKZF1、BEST4、B4GALNT1、GRASP、IRF4和BEND4。
例如，本申请的方法中，所述目标基因可以包含选自以下组的基因中的7种：SEPT9、IKZF1、BEST4、B4GALNT1、GRASP、IRF4和BEND4。例如，本申请的方法中，所述目标基因可以包含SEPT9、IKZF1、BEST4、B4GALNT1、GRASP、IRF4和BEND4。
例如，本申请的方法中位于SEPT9基因所在DNA区域的目标DNA区域可以包含来源于人chr17:75368651-75370720定义的区域。例如来源于人chr17:75369558-75369622定义的区域。例如，该目标DNA区域可以通过SEPT(1)表示。
例如，本申请的方法中位于IKZF1基因所在DNA区域的目标DNA区域可以包含来源于人chr7:50343720-50344547定义的区域。例如来源于人chr7:50343793-50343896定义的区域。例如，该目标DNA区域可以通过IKZF1(1)表示。
例如，本申请的方法中位于BEST4基因所在DNA区域的目标DNA区域可以包含来源于人chr1:45251728-45252477定义的区域。例如来源于人chr1:45252095-45252176定义的区域。例如，该目标DNA区域可以通过BEST4(1)表示。
例如，本申请的方法中位于B4GALNT1基因所在DNA区域的目标DNA区域可以包含来源于人chr12:58020498-58022962定义的区域。例如来源于人chr12:58021586-58021670定义的区域。例如，该目标DNA区域可以通过B4GALNT1(1)表示。
例如，本申请的方法中位于GRASP基因所在DNA区域的目标DNA区域可以包含来源于人chr12:52400724-52401698定义的区域。例如来源于人chr12:52401083-52401169定义的区域。例如，该目标DNA区域可以通过GRASP(1)表示。
例如，本申请的方法中位于IRF4基因所在DNA区域的目标DNA区域可以包含来源于人chr6:391739-394056定义的区域。例如来源于人chr6:392282-392377定义的区域。例如，该目标DNA区域可以通过IRF4(1)表示。
例如，本申请的方法中位于BEND4基因所在DNA区域的目标DNA区域可以包含来源于人chr4:42152705-42154895定义的区域。例如来源于人chr4:42153816-42153921定义的区域。例如，该目标DNA区域可以通过BEND4(1)表示。
例如，本申请的方法中位于SEPT9基因所在DNA区域的目标DNA区域可以包含来源于人chr17:75368651-75370720定义的区域。例如来源于人chr17:75369603-75369693定义的区域。例如，该目标DNA区域可以通过SEPT9(1a)表示。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中目标基因所在DNA区域的特定的亚区域(例如目标DNA区域)、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量，所述目标DNA区域可以包含来源于人chr17:75368651-75370720和来源于人chr7:50343720-50344547定义的区域。例如，本申请的方法可以包含，根据待测样本中所述目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含确定待测样本中目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量，所述目标DNA区域可以包含来源于人chr17:75368651-75370720和来源于人chr7:50343720-50344547定义的区域。
例如，该目标DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的目标DNA区域可以是指目标基因组DNA的一个或多个特定区段。例如，本申请的目标DNA区域可以通过基因名称或一组染色体坐标来指定。例如，以SEPT9表示的目标DNA区域可以是SEPT9基因所在DNA区域的一个或多个特定区段。例如，以SEPT9表示的目标DNA区域可以是本申请提供的SEPT9基因中目标DNA区域的一个或多个特定区段。例如，SEPT9的一个或多个不同的特定区段可以通过SEPT9(1)和SEPT9(1a)等可以区分的形式分别地表示。
例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含提供能够结合包含SEQ ID NO：1所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
例如，本申请的方法中，所述目标区域可以包含来源于人chr17:75369558-75369622定义的区域。例如，该目标DNA区域可以通过SEPT(1)表示。
例如，本申请的方法可以包含提供SEQ ID NO：2所示的核酸或其互补核酸、或上述的片段。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含提供SEQ ID NO：3与4所示的核酸组或其互补核酸组、或上述的片段。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，上述探针和/或引物组可以对来源于人chr17:75369558-75369622定义的区域的DNA区域进行检测和/或扩增。
例如，本申请的方法可以包含提供能够结合包含SEQ ID NO：5所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
例如，本申请的方法中，所述目标区域可以包含来源于人chr7:50343793-50343896定义的区域。例如，该目标DNA区域可以通过IKZF1(1)表示。
例如，本申请的方法可以包含提供SEQ ID NO：6所示的核酸或其互补核酸、或上述的片段。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含提供SEQ ID NO：7与8所示的核酸组或其互补核酸组、或上述的片段。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，上述探针和/或引物组可以对来源于人chr7:50343793-50343896定义的区域的DNA区域进行检测和/或扩增。
例如，本申请的方法中，所述目标DNA区域还可以包含选自以下组定义的区域：来源于人chr1:45251728-45252477、来源于人chr12:58020498-58022962、来源于人chr12:52400724-52401698、来源于人chr6:391739-394056、和来源于人chr4:42152705-42154895。
例如，本申请的方法中，所述目标DNA区域可以包含至少2个区域。
例如，本申请的方法中，所述目标DNA区域可以包含2个至8个区域。例如，本申请的方法中，所述目标DNA区域可以包含2个、3个、4个、5个、6个、7个、或8个区域。
例如，本申请的方法中，所述目标DNA区域可以包含来源于人chr17:75368651-75370720、来源于人chr7:50343720-50344547、来源于人chr1:45251728-45252477和来源于人chr12:58020498-58022962定义的区域。
例如，本申请的方法中，所述目标DNA区域可以包含来源于人chr17:75368651-75370720、来源于人chr7:50343720-50344547、来源于人chr1:45251728-45252477、来源于人chr12:52400724-52401698和来源于人chr12:58020498-58022962定义的区域。
例如，本申请的方法中，所述目标DNA区域可以包含来源于人chr6:391739-394056、来源于人chr7:50343720-50344547、来源于人chr1:45251728-45252477、来源于人chr12:58020498-58022962、来源于人chr4:42152705-42154895和来源于人chr17:75368651-75370720定义的区域。
例如，本申请的方法可以包含提供能够结合包含SEQ ID NO：9所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
例如，本申请的方法中，所述目标区域可以包含来源于人chr1:45252095-45252176定义的区域。例如，该目标DNA区域可以通过BEST4(1)表示。
例如，本申请的方法可以包含提供SEQ ID NO：10所示的核酸或其互补核酸、或上述的片段。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含提供SEQ ID NO：11与12所示的核酸组或其互补核酸组、或上述的片段。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，上述探针和/或引物组可以对来源于人chr1:45252095-45252176定义的区域的DNA区域进行检测和/或扩增。
例如，本申请的方法可以包含提供能够结合包含SEQ ID NO：13所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
例如，本申请的方法中，所述目标区域可以包含来源于人chr12:58021586-58021670定义的区域。例如，该目标DNA区域可以通过B4GALNT1(1)表示。
例如，本申请的方法可以包含提供SEQ ID NO：14所示的核酸或其互补核酸、或上述的片段。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含提供SEQ ID NO：15与16所示的核酸组或其互补核酸组、或上述的片段。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，上述探针和/或引物组可以对来源于人chr12:58021586-58021670定义的区域的DNA区域进行检测和/或扩增。
例如，本申请的方法可以包含提供能够结合包含SEQ ID NO：17所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
例如，本申请的方法中，所述目标区域可以包含来源于人chr12:52401083-52401169定义的区域。例如，该目标DNA区域可以通过GRASP(1)表示。
例如，本申请的方法可以包含提供SEQ ID NO：18所示的核酸或其互补核酸、或上述的片段。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含提供SEQ ID NO：19与20所示的核酸组或其互补核酸组、或上述的片段。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，上述探针和/或引物组可以对来源于人chr12:52401083-52401169定义的区域的DNA区域进行检测和/或扩增。
例如，本申请的方法可以包含提供能够结合包含SEQ ID NO：21所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
例如，本申请的方法中，所述目标区域可以包含来源于人chr6:392282-392377定义的区域。例如，该目标DNA区域可以通过IRF4(1)表示。
例如，本申请的方法可以包含提供SEQ ID NO：22所示的核酸或其互补核酸、或上述的片段。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含提供SEQ ID NO：23与24所示的核酸组或其互补核酸组、或上述的片段。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，上述探针和/或引物组可以对来源于人chr6:392282-392377定义的区域的DNA区域进行检测和/或扩增。
例如，本申请的方法可以包含提供能够结合包含SEQ ID NO：25所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
例如，本申请的方法中，所述目标区域可以包含来源于人chr4:42153816-42153921定义 的区域。例如，该目标DNA区域可以通过BEND4(1)表示。
例如，本申请的方法可以包含提供SEQ ID NO：26所示的核酸或其互补核酸、或上述的片段。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含提供SEQ ID NO：27与28所示的核酸组或其互补核酸组、或上述的片段。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，上述探针和/或引物组可以对来源于人chr4:42153816-42153921定义的区域的DNA区域进行检测和/或扩增。
例如，本申请的方法可以包含提供能够结合包含SEQ ID NO：1所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
例如，本申请的方法中，所述目标区域可以包含来源于人chr17:75369603-75369693定义的区域。例如，该目标DNA区域可以通过SEPT9(1a)表示。
例如，本申请的方法可以包含提供SEQ ID NO：29所示的核酸或其互补核酸、或上述的片段。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含提供SEQ ID NO：30与31所示的核酸组或其互补核酸组、或上述的片段。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，上述探针和/或引物组可以对来源于人chr17:75369603-75369693定义的区域的DNA区域进行检测和/或扩增。
例如，上述目标区域的一种或多种可以作为扩增区域和/或检测区域。
甲基化标志物
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中BCAT1基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中BCAT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中BCAT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中BCAT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中BCAT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤 的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中BCAT1基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中BCAT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr12:24964295-25102393。例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中BCAT1基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:25101630-25102393、来源于人chr:12:25078661-25079410和来源于人chr12:25054781-25056311。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:25101630-25102393、来源于人chr:12:25078661-25079410和来源于人chr12:25054781-25056311。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特 定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr12:25101630-25102393的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr:12:25078661-25079410的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr12:25054781-25056311的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：32、SEQ ID NO：39和SEQ ID NO：43。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:25102016-25102110、来源于人chr12:25101992-25102093、来源于人chr12:25079051-25079133和来源于人chr12:25056027-25056134。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:25102016-25102110、来源于人chr12:25101992-25102093、来源于人 chr12:25079051-25079133和来源于人chr12:25056027-25056134。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr12:25102016-25102110、来源于人chr12:25101992-25102093、来源于人chr12:25079051-25079133和来源于人chr12:25056027-25056134。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：33、SEQ ID NO：36、SEQ ID NO：40和SEQ ID NO：44。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：33所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr12:25101630-25102393的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:25102016-25102110的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：36所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr12:25101630-25102393的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:25101992-25102093的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：40所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr:12:25078661-25079410的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:25079051-25079133的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：44所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr12:25054781-25056311的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:25056027-25056134的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：34与35、SEQ ID NO：37与38、SEQ ID NO：41与42和SEQ ID NO：45与46。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：34与35所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr12:25101630-25102393 的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:25102016-25102110的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：37与38所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr12:25101630-25102393的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:25101992-25102093的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：41与42所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr:12:25078661-25079410的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:25079051-25079133的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：45与46所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr12:25054781-25056311的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:25056027-25056134的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中IKZF1基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中IKZF1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中IKZF1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中IKZF1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中IKZF1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中IKZF1基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中IKZF1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr7:50343720:50472799。例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中IKZF1基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr7:50343720-50344547、来源于人chr7:50450118-50450531和来源于人chr7:50467368-50469092。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr7:50343720-50344547、来源于人chr7:50450118-50450531和来源于人chr7:50467368-50469092。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样 本中来源于人chr7:50343720-50344547的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr7:50450118-50450531的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr7:50467368-50469092的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：5、SEQ ID NO：50和SEQ ID NO：54。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr7:50343793-50343896、来源于人chr7:50343867-50343961、来源于人chr7:50450311-50450411和来源于人chr7:50467865-50467980。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr7:50343793-50343896、来源于人chr7:50343867-50343961、来源于人chr7:50450311-50450411和来源于人chr7:50467865-50467980。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr7:50343793-50343896、来源于人chr7:50343867-50343961、来源于人chr7:50450311-50450411和来源于人chr7:50467865-50467980。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片 段：SEQ ID NO：6、SEQ ID NO：47、SEQ ID NO：51和SEQ ID NO：55。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：6所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr7:50343720-50344547的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr7:50343793-50343896的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：47所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr7:50343720-50344547的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr7:50343867-50343961的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：51所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr7:50450118-50450531的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr7:50450311-50450411的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：55所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr7:50467368-50469092的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr7:50467865-50467980的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：7与8、SEQ ID NO：48与49、SEQ ID NO：52与53和SEQ ID NO：56与57。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：7与8所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr7:50343720-50344547的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr7:50343793-50343896的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：48与49所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr7:50343720-50344547的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr7:50343867-50343961的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：52与53所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr7:50450118-50450531 的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr7:50450311-50450411的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：56与57所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr7:50467368-50469092的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr7:50467865-50467980的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中VAV3基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中VAV3基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中VAV3基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中VAV3基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中VAV3基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中VAV3基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中VAV3基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr1:108113782-108507766。例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中VAV3基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病 的进展的方法，可以包含确定待测样本中选自以下的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:108507215-108507766。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:108507215-108507766。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr1:108507215-108507766的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：58。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:108507591-108507674和来源于人chr1:108507624-108507725。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:108507591-108507674和来源于人chr1:108507624-108507725。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr1:108507591-108507674和来源于人chr1:108507624-108507725。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：59和SEQ ID NO：62。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：59所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:108507215-108507766的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:108507591-108507674的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：62所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:108507215-108507766的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:108507624-108507725的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：60与61和SEQ ID NO：63与64。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：60与61所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:108507215-108507766的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:108507591-108507674的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：63与64所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:108507215- 108507766的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:108507624-108507725的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中IRF4基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中IRF4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中IRF4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中IRF4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中IRF4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中IRF4基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中IRF4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr6:391739-411447。例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中IRF4基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr6:391739-394056和来源于人chr6:401233-401801。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状 态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr6:391739-394056和来源于人chr6:401233-401801。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr6:391739-394056的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr6:401233-401801的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：21和SEQ ID NO：71。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病 的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr6:392282-392377、来源于人chr6:392036-392145、来源于人chr6:392405-392500和来源于人chr6:401641-401752。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr6:392282-392377、来源于人chr6:392036-392145、来源于人chr6:392405-392500和来源于人chr6:401641-401752。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr6:392282-392377、来源于人chr6:392036-392145、来源于人chr6:392405-392500和来源于人chr6:401641-401752。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：22、SEQ ID NO：65、SEQ ID NO：68和SEQ ID NO：72。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：22所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr6:391739-394056的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr6:392282-392377的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：65所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr6:391739-394056的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr6:392036-392145的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：68所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr6:391739-394056的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr6:392405-392500的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：72所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr6:401233-401801的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr6:401641-401752的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：23与24、SEQ ID NO：66与67、SEQ ID NO：69与70和SEQ ID NO：73与74。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：23与24所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr6:391739-394056的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr6:392282-392377的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：66与67所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr6:391739-394056的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr6:392036-392145的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：69与70所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr6:391739-394056的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr6:392405-392500的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：73与74所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr6:401233-401801的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr6:401641-401752的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中B4GALNT1基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中B4GALNT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中B4GALNT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中B4GALNT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中B4GALNT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中B4GALNT1基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中B4GALNT1基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr12:58017193-58027138。例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中B4GALNT1基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:58020498-58022962和来源于人chr12:58025539-58027138。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:58020498-58022962和来源于人chr12:58025539-58027138。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定 DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr12:58020498-58022962的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr12:58025539-58027138的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：13和SEQ ID NO：78。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:58021586-58021670、来源于人chr12:58021907-58021987和来源于人chr12:58026383-58026475。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:58021586-58021670、来源于人chr12:58021907-58021987和来源于人chr12:58026383-58026475。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr12:58021586-58021670、来源于人chr12:58021907-58021987和来源于人chr12:58026383-58026475。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片 段：SEQ ID NO：14、SEQ ID NO：75和SEQ ID NO：79。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：14所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr12:58020498-58022962的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:58021586-58021670的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：75所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr12:58020498-58022962的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:58021907-58021987的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：79所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr12:58025539-58027138的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:58026383-58026475的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：15与16、SEQ ID NO：76与77和SEQ ID NO：80与81。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：15与16所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr12:58020498-58022962的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:58021586-58021670的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：76与77所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr12:58020498-58022962的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:58021907-58021987的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：80与81所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr12:58025539-58027138的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:58026383-58026475的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中GRASP基因所在DNA区域或其片段的修饰状态 的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中GRASP基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中GRASP基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中GRASP基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中GRASP基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中GRASP基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中GRASP基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr12:52400724-52409673。例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中GRASP基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:52400724-52401698和来源于人chr12:52406880-52409127。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例 如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:52400724-52401698和来源于人chr12:52406880-52409127。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr12:52400724-52401698的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr12:52406880-52409127的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：17和SEQ ID NO：85。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:52401083-52401169、来源于人chr12:52400965-52401055和来源于人chr12:52408471-52408566。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:52401083-52401169、来源于人chr12:52400965-52401055和来源于人chr12:52408471-52408566。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr12:52401083-52401169、来源于人chr12:52400965-52401055和来源于人chr12:52408471-52408566。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：18、SEQ ID NO：82和SEQ ID NO：86。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：18所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr12:52400724-52401698的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:52401083-52401169的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：82所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr12:52400724-52401698的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:52400965-52401055的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：86所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr12:52406880-52409127的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr12:52408471-52408566的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：19与20、SEQ ID NO：83与84和SEQ ID NO：87与88。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：19与20所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr12:52400724-52401698的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:52401083-52401169的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：83与84所示的核酸组或其互补核酸 组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr12:52400724-52401698的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:52400965-52401055的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：87与88所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr12:52406880-52409127的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr12:52408471-52408566的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中BEST4基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中BEST4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中BEST4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中BEST4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中BEST4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中BEST4基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中BEST4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr1:45249257-45253377。例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中BEST4基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:45251728-45252477和来源于人chr1:45249853-45250527。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:45251728-45252477和来源于人chr1:45249853-45250527。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr1:45251728-45252477的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr1:45249853-45250527的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请 所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：9和SEQ ID NO：92。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:45252095-45252176、来源于人chr1:45252187-45252275和来源于人chr1:45249894-45249981。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:45252095-45252176、来源于人chr1:45252187-45252275和来源于人chr1:45249894-45249981。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr1:45252095-45252176、来源于人chr1:45252187-45252275和来源于人chr1:45249894-45249981。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：10、SEQ ID NO：89和SEQ ID NO：93。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：10所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:45251728-45252477的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:45252095-45252176的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：89所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:45251728-45252477的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:45252187-45252275的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：93所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:45249853-45250527的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:45249894-45249981的DNA区 域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：11与12、SEQ ID NO：90与91和SEQ ID NO：94与95。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：11与12所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:45251728-45252477的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:45252095-45252176的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：90与91所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:45251728-45252477的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:45252187-45252275的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：94与95所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:45249853-45250527的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:45249894-45249981的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中BEND4基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中BEND4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中BEND4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中BEND4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中BEND4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中BEND4基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中BEND4基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以 是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr4:42112955-42154895。例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中BEND4基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr4:42152705-42154895。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr4:42152705-42154895。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr4:42152705-42154895的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：25。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr4:42153816-42153921来源于人chr4:42153513-42153601。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr4:42153816-42153921来源于人chr4:42153513-42153601。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr4:42153816-42153921来源于人chr4:42153513-42153601。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：26和SEQ ID NO：96。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：26所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr4:42152705-42154895的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr4:42153816-42153921的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：96所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr4:42152705-42154895的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr4:42153513-42153601的DNA区 域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：27与28和SEQ ID NO：97与98。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：27与28所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr4:42152705-42154895的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr4:42153816-42153921的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：97与98所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr4:42152705-42154895的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr4:42153513-42153601的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中来源于人chr1:145384249:145413094所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中来源于人chr1:145384249:145413094所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中来源于人chr1:145384249:145413094所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中来源于人chr1:145384249:145413094所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中来源于人chr1:145384249:145413094所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中来源于人chr1:145384249:145413094所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中来源于人chr1:145384249:145413094所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐 标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中来源于人chr1:145384249:145413094所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:145389746-145401075、来源于人chr1:145384910-145385929和来源于人chr1:145406714-145408013。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:145389746-145401075、来源于人chr1:145384910-145385929和来源于人chr1:145406714-145408013。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr1:145389746-145401075的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr1:145384910-145385929的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr1:145406714-145408013的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：99、SEQ ID NO：106和SEQ ID NO：110。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:145399249:145399476、来源于人chr1:145396880-145396983、来源于人chr1:145385298-145385376和来源于人chr1:145407431-145407518。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:145399249:145399476、来源于人chr1:145396880-145396983、来源于人chr1:145385298-145385376和来源于人chr1:145407431-145407518。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr1:145399249:145399476、来源于人chr1:145396880-145396983、来源于人chr1:145385298-145385376和来源于人chr1:145407431-145407518。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：100、SEQ ID NO：103、SEQ ID NO：107和SEQ ID NO：111。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：100所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:145389746-145401075的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:145399249:145399476的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：103所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:145389746-145401075的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:145396880-145396983的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：107所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:145384910-145385929的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:145385298-145385376的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：111所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:145406714-145408013的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:145407431-145407518的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：101与102、SEQ ID NO：104与105、SEQ ID NO：108与109和SEQ ID NO：112与113。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：101与102所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:145389746-145401075的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:145399249:145399476的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：104与105所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:145389746-145401075的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:145396880-145396983的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：108与109所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:145384910-145385929的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人 chr1:145385298-145385376的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：112与113所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:145406714-145408013的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:145407431-145407518的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中来源于人chr7:26415938:26416740所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中来源于人chr7:26415938:26416740所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中来源于人chr7:26415938:26416740所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中来源于人chr7:26415938:26416740所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中来源于人chr7:26415938:26416740所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中来源于人chr7:26415938:26416740所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中来源于人chr7:26415938:26416740所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中来源于人chr7:26415938:26416740所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方 法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr7:26416257-26416363来源于人chr7:26416026-26416126。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr7:26416257-26416363来源于人chr7:26416026-26416126。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr7:26416257-26416363来源于人chr7:26416026-26416126。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片 段：SEQ ID NO：114和SEQ ID NO：117。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：114所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr7:26416257-26416363的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：117所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr7:26416026-26416126的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：115与116和SEQ ID NO：118与119。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：115与116所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr7:26416257-26416363的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：118与119所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr7:26416026-26416126的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中GPAM基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中GPAM基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中GPAM基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中GPAM基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中GPAM基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中GPAM基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中GPAM基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情 况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr10:113909624-113975135。例如，本申请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中GPAM基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr10:113942657-113943906。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr10:113942657-113943906。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百 分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr10:113942657-113943906的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：120。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr10:113943540:113943739来源于人chr10:113943511-113943582。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr10:113943540:113943739来源于人chr10:113943511-113943582。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr10:113943540:113943739来源于人chr10:113943511-113943582。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：121和SEQ ID NO：124。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：121所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr10:113942657-113943906的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr10:113943540:113943739的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：124所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr10:113942657-113943906的DNA区 域进行检测。例如，该核酸作为探针，可以针对来源于人chr10:113943511-113943582的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：122与123和SEQ ID NO：125与126。例如，所述核酸组可以用于扩增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：122与123所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr10:113942657-113943906的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr10:113943540:113943739的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：125与126所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr10:113942657-113943906的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr10:113943511-113943582的DNA区域进行扩增。
一方面，本申请提供一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含确定待测样本中VASH2基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，本申请的方法可以包含，根据待测样本中VASH2基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，确认肝肿瘤是否存在。例如，本申请的方法可以包含，根据待测样本中VASH2基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为肝肿瘤形成。例如，本申请的方法可以包含，根据待测样本中VASH2基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为肝肿瘤形成的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中VASH2基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定结果，评估肝肿瘤的进展情况。
另一方面，本申请提供一种评估肝肿瘤相关DNA区域甲基化状态的方法，可以包含确定待测样本中VASH2基因所在DNA区域或其片段的修饰状态的存在和/或含量。例如，根据待测样本中VASH2基因所在DNA区域或其片段的修饰状态的存在和/或含量的确定情况，评估肝肿瘤相关DNA区域甲基化状态。例如，肝肿瘤相关DNA区域甲基化状态可以是指该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与肝肿瘤的发生有关联。
例如，本申请的所述DNA区域可以来源于人chr1:213123862-213165379。例如，本申 请的基因可以通过它们的名称和它们的染色体坐标来描述。例如，染色体坐标可以与2009年2月发布的人类基因组数据库Hg19版(或称作“Hg19坐标”)一致。例如，本申请的DNA区域可以是来源于由Hg19坐标限定的区域。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中VASH2基因所在DNA区域的特定的亚区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含确定待测样本中选自以下DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:213123862-213125211。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，确认疾病是否存在。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否确诊为疾病形成。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估是否有确诊为疾病的风险和/或风险的高低。例如，本申请的方法可以包含，根据待测样本中所述DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量的确定结果，评估疾病的进展情况。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:213123862-213125211。例如，该DNA区域的甲基化的确认存在或相对于参考水平的数量提高，可以与疾病的发生有关联。例如，本申请的DNA区域可以是指基因组DNA的特定区段。例如，本申请的DNA区域可以通过基因名称或一组染色体坐标来指定。例如，一个基因可以通过参考其名称获得其序列和染色体位置，或通过参考其染色体坐标确定其序列和染色体位置。本申请采用这些特定DNA区域甲基化状态作为一个系列分析指标，可以在灵敏度和/或特异性方面提供显著的改进，并且可以简化筛查过程。例如，“灵敏度”可以指正确鉴定的阳性结果的比例，即，正确鉴定为具有所讨论疾病的个体的百分数；“特异性”可以指正确鉴定的阴性结果的比例，即，正确鉴定为不具有所讨论疾病的个体的百分数。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中来源于人chr1:213123862-213125211的DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量。
本申请的DNA区域可以包含这些分子的全部形式及其片段或变体。例如，变体可以包含相对于本申请所述的DNA区域共有至少80％、至少85％、至少90％、95％、98％、或99％序列同一性，变体可以包含一个或多个缺失、添加、置换、倒转序列等。例如，本申请所述变体的修饰状态可以实现相同的评估结果。本申请的DNA区域可以包含全部形式的任何其他的突变、多态性变异或等位变异。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：127。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:213124569-213124670来源于人chr1:213124036-213124162。
另一方面，本申请提供一种确定DNA区域甲基化状态的方法，可以包含：确定待测样本中可以选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr1:213124569-213124670来源于人chr1:213124036-213124162。
例如，本申请的方法可以包含：提供能够结合可以包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr1:213124569-213124670来源于人chr1:213124036-213124162。
例如，上述区域的一种或多种可以作为扩增区域和/或检测区域。
例如，本申请的方法可以包含：提供可以选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：128和SEQ ID NO：131。例如，所述核酸可以用于检测目标区域。例如，所述核酸可以作为探针。
例如，本申请的方法可以包含：提供SEQ ID NO：128所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:213123862-213125211的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:213124569-213124670的DNA区域进行检测。
例如，本申请的方法可以包含：提供SEQ ID NO：131所示的核酸或其互补核酸、或上述的片段。例如该核酸作为探针，可以针对来源于人chr1:213123862-213125211的DNA区域进行检测。例如，该核酸作为探针，可以针对来源于人chr1:213124036-213124162的DNA区域进行检测。
例如，本申请的方法可以包含：提供可以选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：129与130和SEQ ID NO：132与133。例如，所述核酸组可以用于扩 增目标区域。例如，所述核酸组可以作为引物组。
例如，本申请的方法可以包含：提供SEQ ID NO：129与130所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:213123862-213125211的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:213124569-213124670的DNA区域进行扩增。
例如，本申请的方法可以包含：提供SEQ ID NO：132与133所示的核酸组或其互补核酸组、或上述的片段。例如该核酸组作为引物组，可以针对来源于人chr1:213123862-213125211的DNA区域进行扩增。例如，该核酸组作为引物组，可以针对来源于人chr1:213124036-213124162的DNA区域进行扩增。
例如，所述疾病可以包含肿瘤。例如，所述疾病可以包含实体瘤。例如，所述疾病可以包含肝肿瘤等任意的肿瘤。
例如，“引物”可以是天然的或合成的寡核苷酸，其在与多核苷酸模板形成双链体后能够充当核酸合成的起始点并从其3'端沿模板延伸，从而形成延伸的双链体。在延伸过程中添加的核苷酸的序列由模板多核苷酸的序列决定。引物通常可以通过聚合酶如核酸聚合酶延伸。
例如，本申请的“互补的”和“基本上互补的”可以包括在核苷酸或核酸之间，例如在双链DNA分子的两条链之间，或在寡核苷酸引物和单链核酸上的引物结合位点之间的杂交或碱基配对或双链体的形成。互补的核苷酸可以通常是A和T(或A和U)或C和G。对于两个单链RNA或DNA分子，当一条链的核苷酸在进行最佳比对和比较并且具有适当的核苷酸插入或缺失时与另一条链的至少约80％(通常至少约90％至约95％，甚至约98％至约100％)成对时，可以认为它们是基本互补的。在一个方面，两个互补的核苷酸序列能够杂交，并且可以在反向的核苷酸之间有小于25％的错配，更可以以小于15％的错配，可以以小于5％的错配，或不具有错配。例如，两个分子可以在高严格条件下杂交。
例如，本申请的修饰状态可以是指该修饰状态在DNA区域内部一个特定核苷酸或多个核苷酸处的存在、不存在和/或其含量。例如，本申请的修饰状态可以是指特定DNA序列中每个碱基或每个特定碱基(例如胞嘧啶)的修饰状态。例如，本申请的修饰状态可以是指特定DNA序列中碱基对组合和/或碱基组合的修饰状态。例如，本申请的修饰状态可以是指特定DNA序列(包括基因所在DNA区域或其特定区域片段)中关于区域修饰密度的信息，而可以不提供该序列中何处发生修饰的精确位置信息。
例如，本申请的修饰状态可以是指甲基化状态或与甲基化类似的状态。例如，具有或具有较高的甲基化的状态可以是与特定区域的转录沉默相关的。例如，具有或具有较高的甲基 化的状态可以是与能够被甲基化特异性转化试剂(例如脱氨基试剂和/或甲基化敏感限制酶)转化相关的。例如，转化可以是指被转变为其它物质和/或被剪切或消化。
例如，“甲基化状态(methylation state)”或“甲基化状态(methylation status)”可以是指在DNA序列内的一个或多个CpG二核苷酸处存在或不存在5-甲基胞嘧啶(“5-mC”或“5-mCyt”)。DNA序列内一个或多个特定CpG甲基化位点(每个都有两个CpG二核苷酸序列)处的甲基化状态包括“未甲基化”、“完全甲基化”和“半甲基化”。术语“半甲基化”可以是指双链DNA的甲基化状态，其中所述双链DNA仅有一条链被甲基化。术语“高甲基化”可以是指与以下情况相对应的平均甲基化状态：相对于正常对照DNA样品中相应CpG二核苷酸处发现的5-mCyt量，测试DNA样品的DNA序列中的一个或多个CpG二核苷酸处5-mCyt的存在增加。术语“低甲基化”可以是指与以下情况相对应的平均甲基化状态：相对于正常对照DNA样品中相应CpG二核苷酸处发现的5-mCyt量，测试DNA样品的DNA序列中的一个或多个CpG二核苷酸处5-mCyt的存在降低。
例如，所述方法还可以包含获取待测样本中的核酸。例如，所述核酸可以包含无细胞游离核酸。例如，所述待测样本可以包含组织、细胞和/或体液。例如，所述待测样本可以包含血浆。例如，本申请的检测方法可以对任何适合的生物样品进行。例如，待测样本可以为生物材料的任何样品，例如其可以源自动物，但不限于细胞材料、生物流体(例如血液)、排出物、组织活组织检查标本、手术标本或已经导入动物身体中并且随后取出的流体。例如，本申请的待测样本可以包含在所述样本分离后经任何形式处理的样本。
例如，所述方法还可以包含转化所述DNA区域或其片段。例如，通过本申请的转化步骤，具有所述修饰状态的碱基以及不具有所述修饰状态的所述碱基，在转化后可以形成不同的物质。例如，具有所述修饰状态的碱基在转化后基本不发生改变，且不具有所述修饰状态的所述碱基在转化后可以改变为与所述碱基不同的其它碱基(例如，所述其它碱基可以包含尿嘧啶)、或在转化后被剪切。例如，所述碱基可以包含胞嘧啶。例如，所述修饰状态可以包含甲基化修饰。例如，所述转化可以包含通过脱氨基试剂和/或甲基化敏感限制酶转化。例如，所述脱氨基试剂可以包含亚硫酸氢盐或其类似物。例如，亚硫酸氢钠或亚硫酸氢钾。
例如，所述方法还可以任选地包含在确定所述DNA区域或其片段的修饰的存在和/或含量之前，扩增待测样本中所述DNA区域或其片段。例如，所述扩增可以包含PCR扩增。例如，本申请的扩增可以包含已知的任意一种扩增系统。例如，本申请的扩增步骤可以是任选地。例如，“扩增”可以是指产生所需序列的多个拷贝的过程。“多个拷贝”可以是指至少两个拷贝。“拷贝”可以不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/或在扩增过程中可以发生序列错误。
例如，所述确定修饰状态的存在和/或含量的方法可以包含，确认具有所述修饰状态的碱基在所述转化后形成的物质的存在和/或含量。例如，所述确定修饰状态的存在和/或含量的方法可以包含，确定具有所述修饰状态的DNA区域或其片段的存在和/或含量。例如，可以直接检测具有所述修饰状态的DNA区域或其片段的存在和/或含量。例如，可以通过以下方式检测：具有所述修饰状态的DNA区域或其片段可以在反应(例如扩增反应)的过程中可以与不具有所述修饰状态的DNA区域或其片段具有不同的特性。例如，在荧光PCR方法中，具有所述修饰状态的DNA区域或其片段可以被特异性扩增，并发出荧光；不具有所述修饰状态的DNA区域或其片段可以基本不被扩增，并基本不发出荧光。例如，确定具有所述修饰状态的碱基在所述转化后形成的物质的存在和/或含量的替代方法，可以包含在本申请的范围之内。
例如，可以通过所述荧光PCR方法检测的荧光Ct值，确定具有所述修饰状态的DNA区域或其片段的存在和/或含量。例如，可以通过所述DNA区域或其片段的修饰状态的存在和/或所述DNA区域或其片段相对于参考水平具有更高的修饰状态的含量，确定肝肿瘤的存在、或者有肝肿瘤形成或形成的风险。例如，当所述待测样本的荧光Ct值相对于参考荧光Ct值更低时，可以确定所述DNA区域或其片段的修饰状态的存在和/或可以确定所述DNA区域或其片段的修饰状态的含量高于参考样本中的修饰状态的含量。例如，可以通过检测参考样本确定所述参考荧光Ct值。例如，当所述待测样本的荧光Ct值相对于参考荧光Ct值更高或基本相当时，也可以不排除所述DNA区域或其片段的修饰状态的存在；当所述待测样本的荧光Ct值相对于参考荧光Ct值更高或基本相当时，可以确认所述DNA区域或其片段的修饰状态的含量低于或基本等于参考样本中的修饰状态的含量。
例如，本申请可以通过循环阈值(即Ct值)来表示特定DNA区域或其片段的修饰状态的存在和/或含量，例如包括待测样本的甲基化水平和参考水平。例如，Ct值可以是指在背景信号以上可以检测到PCR产物的荧光的循环数。例如，Ct值与样品中目标标记物的起始数量可以成负相关关系，即Ct值越低，待测样品中DNA区域或其片段的修饰状态的数量越多。
例如，当待测样品的Ct值相对于其相应的参考Ct值相同或更低可以确认为存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。例如，当待测样品的Ct值相对于其相应的参考Ct值低至少1个循环、至少2个循环、至少5个循环、至少10个循环、至少20个循环、或至少50个循环时，可以确认为存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。
例如，当细胞样本、组织样本或来源于受试者的样本的Ct值相对于其相应的参考Ct值相同或更高，可以确认为不存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。例如，当细胞样本、组织样本或来源于受试者的样本的Ct值相对于其相应的参考Ct值高至少1个循环、至少2个循环、至少5个循环、至少10个循环、至少20个循环、或至少50个循环时，可以确认为不存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。例如，当细胞样本、组织样本或来源于受试者的样本的Ct值相对于其相应的参考Ct值疾病相同时，可以确认为存在或不存在特定疾病、诊断为特定疾病的形成或未形成、具有或不具有形成风险或者评估为特定疾病的某种进展，并同时可以给出需要进一步检测的建议。
例如，本申请的参考水平或对照水平可以是指是正常水平或健康水平。例如，所述正常水平可以是来源于无所述疾病的细胞、组织或个体的样本DNA区域的修饰状态水平。例如，当用于肿瘤的评估，所述正常水平可以是来源于无肿瘤的细胞、组织或个体的样本DNA区域的修饰状态水平。例如，当用于肝肿瘤的评估，所述正常水平可以是来源于无肝肿瘤的细胞、组织或个体的样本DNA区域的修饰状态水平。
例如，在本申请中参考水平可以是指将受试者或样本确认为存在或不存在特定疾病的阈值水平。例如，在本申请中参考水平可以是指将受试者诊断为特定疾病的形成或具有形成风险的阈值水平。例如，在本申请中参考水平可以是指将受试者评估为特定疾病的某种进展的阈值水平。例如，当细胞样本、组织样本或来源于受试者的样本中的DNA区域的修饰状态高于或基本等于相应参考水平时，例如此处参考水平可以是指不具有特定疾病患者的DNA区域的修饰状态，可以确认为存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。例如，本申请中的A与B“基本等于”可以是指A与B的差值为1％或更少、0.5％或更少、0.1％或更少、0.01％或更少、0.001％或更少或0.0001％或更少。例如，当细胞样本、组织样本或来源于受试者的样本中的DNA区域的修饰状态高于相应参考水平至少1％、至少5％、至少10％、至少20％、至少50％、至少1倍、至少2倍、至少5倍、至少10倍、或至少20倍时，可以确认为存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。例如，当多次检测中的至少一次、至少两次、或至少三次的检测中，细胞样本、组织样本或来源于受试者的样本中的DNA区域的修饰状态高于相应参考水平至少1％、至少5％、至少10％、至少20％、至少50％、至少1倍、至少2倍、至少5倍、至少10倍、或至少20倍时，可以确认为存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。
例如，当细胞样本、组织样本或来源于受试者的样本中的DNA区域的修饰状态低于或基本等于相应参考水平时，例如此处参考水平可以是指具有特定疾病患者的DNA区域的修饰状态，可以确认为不存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。例如，当细胞样本、组织样本或来源于受试者的样本中的DNA区域的修饰状态低于相应参考水平至少1％、至少5％、至少10％、至少20％、至少50％、至少100％时，可以确认为不存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。
本领域技术人员可以根据期望的灵敏度和特异性来选择参考水平。例如，在本申请中各种情况下的参考水平可以是本领域人员容易确认的，如根据有限次尝试确认合适的参考水平和/或合适的获取参考水平的手段，例如，参考水平可以源自一个或多个参考样品，其中参考水平获自与检测目的样品的实验平行进行的实验。或者，也可以在数据库中获得参考水平，该数据库包括来自一个或多个参考样品或疾病参考样品的数据、标准或水平的集合。在一些实施方式中，数据、标准或水平的集合可以被标准化或归一化，以便可用于与来自一个或多个样品的数据进行比较，从而用于减少不同检测条件下产生的误差。
例如，参考水平可以来源于数据库，该数据库可以是参考数据库，例如包括来自一个或多个参考样品的目标标记物和/或其他实验室和临床数据的修饰状态水平。例如，可以通过汇总获自健康个体和/或非相应疾病患者个体(即已知没有该疾病的个体)的参考样品的参考水平数据来建立参考数据库。例如，可以通过汇总获自正在接受治疗的患有相应疾病个体的参考样品的参考水平数据来建立参考数据库。例如，可以通过汇总获自疾病不同阶段的个体的参考样品的数据来建立参考数据库。例如，例如不同阶段可以是通过本申请目标标记物的不同的修饰状态水平来证明的。本领域技术人员还可以基于各种因素，例如年龄、性别、病史、家族史、症状等，来确定个体是否患相应疾病或具有患相应疾病的风险。
例如，本申请的方法可以包含以下步骤：获取待测样本中的核酸；转化所述DNA区域或其片段；确认具有所述修饰状态的碱基在所述转化后形成的物质的存在和/或含量。
例如，本申请的方法可以包含以下步骤：获取待测样本中的核酸；转化所述DNA区域或其片段；扩增待测样本中所述DNA区域或其片段；确认具有所述修饰状态的碱基在所述转化后形成的物质的存在和/或含量。
例如，本申请的方法可以包含以下步骤：获取待测样本中的核酸；用试剂处理从待测样品中获得的DNA,所述试剂能够区分所述DNA中的未甲基化位点和甲基化位点，从而获得经处理的DNA；可选地扩增待测样本中所述DNA区域或其片段；定量、半定量或定性分析 待测样本中经处理的DNA的甲基化状态的存在和/或含量；比较测样本中经处理的DNA的甲基化水平以及相应的参考水平，当待测样本中的DNA区域的甲基化状态高于或基本等于相应参考水平时，可以确认为存在特定疾病、诊断为特定疾病的形成或具有形成风险或者评估为特定疾病的某种进展。
另一方面，本申请提供一种核酸，所述核酸可以包含能够结合本申请目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。例如，所述核酸可以是本申请的任一种探针。另一方面，本申请提供一种制备核酸的方法，可以包含根据本申请目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。例如，制备核酸的方法可以是本领域已知的任意合适的方法。
另一方面，本申请提供一种核酸组，所述核酸组可以包含能够结合本申请目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。例如，所述核酸组可以是本申请的任一种引物组。另一方面，本申请提供一种制备核酸组的方法，可以包含根据本申请目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够扩增所述DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸组。例如，制备核酸组中的核酸的方法可以是本领域已知的任意合适的方法。例如，可以使用单个探针或引物评估靶多核苷酸的甲基化状态，所述单个探针或引物被配置成与所述靶多核苷酸杂交。例如，可以使用多个探针或引物评估靶多核苷酸的甲基化状态，所述多个探针或引物被配置成与所述靶多核苷酸杂交。
另一方面，本申请提供一种核酸，所述核酸包含能够结合如本申请所述的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列；另一方面，本申请提供一种核酸，所述核酸可以包含能够结合如本申请所述的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。
另一方面，本申请提供一种制备核酸的方法，所述方法可以包含根据如本申请所述的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸；另一方面，本申请提供一种核酸，所述方法可以包含根据如本申请所述的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
另一方面，本申请提供一种核酸组，所述核酸组包含能够结合如本申请所述的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列；另一方面，本申请提供一种核酸，所述核酸组可以包含能够结合如本申请所述的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。例如，制备核酸的方法可以是本领域已知的任意合适的方法。
另一方面，本申请提供一种制备核酸组的方法，所述方法可以包含根据如本申请所述的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸组；另一方面，本申请提供一种核酸，所述方法可以包含根据如本申请所述的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸组。例如，可以使用单个探针或引物评估靶多核苷酸的甲基化状态，所述单个探针或引物被配置成与所述靶多核苷酸杂交。例如，可以使用多个探针或引物评估靶多核苷酸的甲基化状态，所述多个探针或引物被配置成与所述靶多核苷酸杂交。
另一方面，本申请提供一种试剂盒，可以包含如本申请的核酸和/或本申请的核酸组。例如，本申请的试剂盒可以可选地包含相应用途的参考样本或提供相应用途的参考水平。
诊断方法、制备用途
例如，本申请的方法可以用于受试者中的癌症或肿瘤形成的诊断、预后、分层、风险评估或治疗监测。例如，“受试者”可以是指可以对之施用或施加本申请提供的方法、核酸、核酸组、试剂盒、装置和系统的生物体或所述生物体的一部分或组分。例如，所述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。如本文所用，“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。
另一方面，本申请所述方法可用于评估任何合适的受试者中的癌症或肿瘤形成。例如，本申请所述方法可用于评估哺乳动物中的癌症或肿瘤形成。所述哺乳动物可以是非人哺乳动物，例如宠物，农场动物，伴侣动物或实验动物。优选地，所述哺乳动物是人。例如，受试者可以是需要进行癌症或肿瘤形成风险筛查的人，高危人群中的人，被诊断为患有癌症或肿瘤形成但需要进一步分层或分级的人，被诊断为患有癌症或肿瘤形成且正在接受积极治疗的人，或患有癌症或肿瘤形成并正在缓解的人。
另一方面，本申请提供如本申请的核酸、如本申请的核酸组和/或本申请的试剂盒，在制 备确定DNA区域或其片段的修饰状态的物质中的应用。
另一方面，本申请提供一种确定所述DNA区域或其片段的修饰状态的方法，可以包括提供本申请的核酸、如本申请的核酸组和/或本申请的试剂盒。
另一方面，本申请提供如本申请的核酸、如本申请的核酸组和/或本申请的试剂盒，其可以用于确定所述DNA区域或其片段的修饰状态。
另一方面，本申请提供如本申请的核酸、如本申请的核酸组和/或本申请的试剂盒，在制备疾病检测产品中的应用。例如，本申请应用中，所述疾病可以包含肿瘤。例如实体瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。
另一方面，本申请提供一种疾病检测方法，可以包括提供本申请的核酸、如本申请的核酸组和/或本申请的试剂盒。例如，本申请应用中，所述疾病可以包含肿瘤。例如实体瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。
另一方面，本申请提供如本申请的核酸、如本申请的核酸组和/或本申请的试剂盒，其可以用于进行疾病检测。例如，本申请应用中，所述疾病可以包含肿瘤。例如实体瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。
另一方面，本申请提供如本申请的核酸、如本申请的核酸组和/或本申请的试剂盒，在制备确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用。例如，本申请应用中，所述疾病可以包含肿瘤。例如实体瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。
另一方面，本申请提供一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包括提供本申请的核酸、如本申请的核酸组和/或本申请的试剂盒。例如，本申请应用中，所述疾病可以包含肿瘤。例如实体瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。
另一方面，本申请提供如本申请的核酸、如本申请的核酸组和/或本申请的试剂盒，其可以用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展。例如，本申请应用中，所述疾病可以包含肿瘤。例如实体瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。
另一方面，本申请提供用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，在制备用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的物质中的应用，所述用于确定的DNA区域可以包含如本申请的方法中所述目标基因所在DNA区域或其片段。
另一方面，本申请提供确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤 的进展的方法，可以包含提供用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒。所述用于确定的DNA区域可以包含如本申请的方法中所述目标基因所在DNA区域或其片段。
另一方面，本申请提供用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，其可以用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展。所述用于确定的DNA区域可以包含如本申请的方法中所述目标基因所在DNA区域或其片段。
另一方面，本申请提供用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，在制备用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用，所述用于确定的DNA区域可以包含如本申请的方法中所述目标DNA区域、或其互补区域、或上述的片段。
另一方面，本申请提供确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含提供用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒。所述用于确定的DNA区域可以包含如本申请的方法中所述目标DNA区域、或其互补区域、或上述的片段。
另一方面，本申请提供用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，其可以用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展。所述用于确定的DNA区域可以包含如本申请的方法中所述目标DNA区域、或其互补区域、或上述的片段。
例如，本申请应用中，所述疾病可以包含肿瘤。例如实体瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。例如，本申请应用中，所述修饰状态可以包含甲基化修饰。
另一方面，本申请提供如本申请的方法中所述目标基因所在DNA区域、或其转化而来的区域、或上述的片段的核酸或其组合，在制备用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的物质中的应用。
另一方面，本申请提供确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，可以包含提供本申请的方法中所述目标基因所在DNA区域、或其转化而来的区域、或上述的片段的核酸或其组合。
另一方面，本申请提供如本申请的方法中所述目标基因所在DNA区域、或其转化而来的区域、或上述的片段的核酸或其组合，其可以用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展。
另一方面，本申请提供如本申请的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸或其组合，在制备用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用。例如，本申请应用中，所述疾病可以包 含肿瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。
另一方面，本申请提供确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，可以包含提供如本申请的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸或其组合。例如，本申请应用中，所述疾病可以包含肿瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。
另一方面，本申请提供如本申请的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸或其组合，其可以用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展。例如，本申请应用中，所述疾病可以包含肿瘤。例如，本申请应用中，所述疾病可以包含肝肿瘤。
另一方面，本申请提供一种设备，其可以包含本申请的储存介质。另一方面，本申请提供了一种非易失性计算机可读存储介质，其上存储有计算机程序，该程序被处理器执行以实现本申请所述的任一种或多种的方法。例如，所述方法可以含有使用甲基化指标对具有不同癌性可能性的样品进行分类的方法。示例性分类算法可以是线性判别分析、逻辑回归、朴素贝叶斯分类、感知分类、二次分类、k近邻法、提升方法、决策树、随机森林、神经网络、学习向量量化或支持向量机、或其组合。例如，所述非易失性计算机可读存储介质可以包括软盘、柔性盘、硬盘、固态存储(SSS)(例如固态驱动(SSD))、固态卡(SSC)、固态模块(SSM))、企业级闪存驱动、磁带或任何其他非临时性磁介质等。非易失性计算机可读存储介质还可以包括打孔卡、纸带、光标片(或任何其他具有孔型图案或其他光学可识别标记的物理介质)、压缩盘只读存储器(CD-ROM)、可重写式光盘(CD-RW)、数字通用光盘(DVD)、蓝光光盘(BD)和/或任何其他非临时性光学介质。
例如，本申请的设备还可以包含耦接至所述储存介质的处理器，所述处理器被配置为基于存储在所述储存介质中的程序执行以实现本申请的方法。例如，所述设备可以实现各种机制以便确保在数据库系统上执行的本申请所述的方法产生正确的结果。在本申请中，所述设备可以使用磁盘作为永久性数据存储器。在本申请中，所述设备可以为多个数据库客户端提供数据库存储和处理服务。所述设备可以跨多个共享存储设备存储数据库数据，和/或可以利用具有多个执行节点的一个或更多个执行平台。所述设备可以被组织成使得存储和计算资源可以被有效地无限扩展。
本发明利用荧光PCR检测技术，检测白膜层(其中大多数为白细胞)、癌旁组织和肝癌组织相关标志物的甲基化情况。初步确认该标志物用于血液检测肝肿瘤的潜能。理想的肝癌甲基化检测标志物应该具备以下特征：1.白膜层DNA甲基化水平低；2.肝癌组织DNA甲基 化水平高。
甲基化标志物检测可以采用甲基化特异PCR(MSP)来进行，MSP的基本原理是在检测中，只有甲基化的序列模板产生扩增信号，而非甲基化的序列模板不产生扩增信号。该方法可通过引物或者探针设计甲基化特异序列来实现，也可以同时设计甲基化特异的引物、探针对来实现。其主要步骤包括：1.根据标志物序列，在适合进行MSP的检测区段(可以为CpG富集区域)，设计检测引物和探针。2.进行白细胞和组织样本的核酸提取。3.对核酸进行亚硫酸氢盐处理，使未甲基化的胞嘧啶转化成尿嘧啶，而甲基化的胞嘧啶维持序列不变。4.进行荧光PCR检测。
经白细胞和组织样本检测，满足上述特征的标志物，证明其存在通过血液来检测肝癌的潜能。然后在血浆样本中，对这些标志物进行验证，检测样本包括对照组、肝癌组以及乙肝、肝硬化干扰人群。分析这些标志物的参考水平及标志物组合性能。因单个样本血浆游离DNA有限，可选地进行荧光PCR检测时，可以对靶点进行了预扩增，以使最少量的DNA能够检测尽可能多的甲基化位点。其主要步骤包括：1.血浆样本的核酸提取。2.对核酸进行亚硫酸氢盐处理，使未甲基化的胞嘧啶转化成尿嘧啶，而甲基化的胞嘧啶维持序列不变。3.进行靶点的预扩增及稀释。4.进行荧光PCR检测。
在靶点组合分析时，也可在上述靶点组合基础上，再进行任意组合形式，或者在数据算法上引入机器学习的数学模型，如线性回归、支持向量回归、脊回归、随机森林等等，得到检测准确性高的肝癌甲基化检测标志物组合。
本申提供的内容：1.找到了众多具有优异性能的肝癌甲基化标志物；2.开发了检测准确性高的肝癌甲基化检测标志物组合；3.检测性能较已有的荧光PCR检测方法有大幅提升；4.可以较二代测序检测方法相比，具备易开展、易临床推广、成本低等众多优势。本申请提供的标志物以及标志物的组合，其应用方式可以不限于特定的检测方式。
实施方案
1.一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，包含确定待测样本中BCAT1、IKZF1、VAV3、IRF4、B4GALNT1、GRASP、BEST4、BEND4、GPAM、VASH2基因所在DNA区域、来源于人chr1:145384249:145413094所在DNA区域、和/或来源于人chr7:26415938:26416740所在DNA区域或其片段的修饰状态的存在和/或含量。
2.一种评估肝肿瘤相关DNA区域甲基化状态的方法，包含确定待测样本中BCAT1、IKZF1、VAV3、IRF4、B4GALNT1、GRASP、BEST4、BEND4、GPAM、VASH2基因 所在DNA区域、来源于人chr1:145384249:145413094所在DNA区域、和/或来源于人chr7:26415938:26416740所在DNA区域或其片段的修饰状态的存在和/或含量。
3.如实施方案1-2中任一项所述的方法，所述DNA区域来源于人chr12:24964295-25102393、来源于人chr7:50343720:50472799、来源于人chr1:108113782-108507766、来源于人chr6:391739-411447、来源于人chr12:58017193-58027138、来源于人chr12:52400724-52409673、来源于人chr1:45249257-45253377、来源于人chr4:42112955-42154895、来源于人chr1:145384249:145413094、来源于人chr7:26415938:26416740、来源于人chr10:113909624-113975135、和/或来源于人chr1:213123862-213165379。
4.如实施方案1-3中任一项所述的方法，所述方法还包含获取待测样本中的核酸。
5.如实施方案4所述的方法，所述核酸包含无细胞游离核酸。
6.如实施方案1-5中任一项所述的方法，所述待测样本包含组织、细胞和/或体液。
7.如实施方案1-6中任一项所述的方法，所述待测样本包含血浆。
8.如实施方案1-7中任一项所述的方法，所述方法还包含转化所述DNA区域或其片段。
9.如实施方案8所述的方法，具有所述修饰状态的碱基以及不具有所述修饰状态的所述碱基，在转化后形成不同的物质。
10.如实施方案8-9中任一项所述的方法，具有所述修饰状态的碱基在转化后基本不发生改变，且不具有所述修饰状态的所述碱基在转化后改变为与所述碱基不同的其它碱基、或在转化后被剪切。
11.如实施方案9-10中任一项所述的方法，所述碱基包含胞嘧啶。
12.如实施方案1-11中任一项所述的方法，所述修饰状态包含甲基化修饰。
13.如实施方案10-12中任一项所述的方法，所述其它碱基包含尿嘧啶。
14.如实施方案8-13中任一项所述的方法，所述转化包含通过脱氨基试剂和/或甲基化敏感限制酶转化。
15.如实施方案14中任一项所述的方法，所述脱氨基试剂包含亚硫酸氢盐或其类似物。
16.如实施方案1-15中任一项所述的方法，所述确定修饰状态的存在和/或含量的方法包含，确认具有所述修饰状态的碱基在所述转化后形成的物质的存在和/或含量。
17.如实施方案1-16中任一项所述的方法，所述确定修饰状态的存在和/或含量的方法包含，确定具有所述修饰状态的DNA区域或其片段的存在和/或含量。
18.如实施方案1-17中任一项所述的方法，通过所述荧光PCR方法检测的荧光Ct值确定具有所述修饰状态的DNA区域或其片段的存在和/或含量。
19.如实施方案1-18中任一项所述的方法，通过确认所述DNA区域或其片段的修饰状态的 存在和/或所述DNA区域或其片段相对于参考水平具有更高的修饰状态的含量，确定肝肿瘤的存在、或者有肝肿瘤形成或形成的风险。
20.如实施方案1-19中任一项所述的方法，所述方法还包含在确定所述DNA区域或其片段的修饰的存在和/或含量之前，扩增待测样本中所述DNA区域或其片段。
21.如实施方案20所述的方法，所述扩增包含PCR扩增。
22.一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，包含确定待测样本中选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:25101630-25102393、来源于人chr:12:25078661-25079410和来源于人chr12:25054781-25056311、或者来源于人chr7:50343720-50344547、来源于人chr7:50450118-50450531和来源于人chr7:50467368-50469092、或者来源于人chr1:108507215-108507766、或者来源于人chr6:391739-394056和来源于人chr6:401233-401801、或者来源于人chr12:58020498-58022962和来源于人chr12:58025539-58027138、或者来源于人chr12:52400724-52401698和来源于人chr12:52406880-52409127、或者来源于人chr1:45251728-45252477和来源于人chr1:45249853-45250527、或者来源于人chr4:42152705-42154895、或者来源于人chr1:145389746-145401075、来源于人chr1:145384910-145385929和来源于人chr1:145406714-145408013、或者来源于人chr7:26416257-26416363和来源于人chr7:26416026-26416126、或者来源于人chr10:113942657-113943906、或者来源于人chr1:213123862-213125211。
23.一种确定DNA区域甲基化状态的方法，包含确定待测样本中选自以下组DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量：来源于人chr12:25101630-25102393、来源于人chr:12:25078661-25079410和来源于人chr12:25054781-25056311、或者来源于人chr7:50343720-50344547、来源于人chr7:50450118-50450531和来源于人chr7:50467368-50469092、或者来源于人chr1:108507215-108507766、或者来源于人chr6:391739-394056和来源于人chr6:401233-401801、或者来源于人chr12:58020498-58022962和来源于人chr12:58025539-58027138、或者来源于人chr1:45251728-45252477和来源于人chr1:45249853-45250527、或者来源于人chr4:42152705-42154895、或者来源于人chr1:145389746-145401075、来源于人chr1:145384910-145385929和来源于人chr1:145406714-145408013、或者来源于人chr7:26416257-26416363和来源于人chr7:26416026-26416126、或者来源于人chr10:113942657-113943906、或者来源于人chr1:213123862-213125211。
24.如实施方案22-23中任一项所述的方法，包含提供能够结合包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：SEQ ID NO：32、SEQ ID NO：39和SEQ ID NO：43、或者SEQ ID NO：5、SEQ ID NO：50和SEQ ID NO：54、或者SEQ ID NO：58、或者SEQ ID NO：21和SEQ ID NO：71、或者SEQ ID NO：13和SEQ ID NO：78、或者SEQ ID NO：17和SEQ ID NO：85、或者SEQ ID NO：9和SEQ ID NO：92、或者SEQ ID NO：25、或者SEQ ID NO：99、SEQ ID NO：106和SEQ ID NO：110、或者SEQ ID NO：120、或者SEQ ID NO：127。
25.如实施方案22-24中任一项所述的方法，包含提供能够结合包含选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸：来源于人chr12:25102016-25102110、来源于人chr12:25101992-25102093、来源于人chr12:25079051-25079133和来源于人chr12:25056027-25056134、或者来源于人chr7:50343793-50343896、来源于人chr7:50343867-50343961、来源于人chr7:50450311-50450411和来源于人chr7:50467865-50467980、或者来源于人chr1:108507591-108507674和来源于人chr1:108507624-108507725、或者来源于人chr6:392282-392377、来源于人chr6:392036-392145、来源于人chr6:392405-392500和来源于人chr6:401641-401752、或者来源于人chr12:58021586-58021670、来源于人chr12:58021907-58021987和来源于人chr12:58026383-58026475、或者来源于人chr12:52401083-52401169、来源于人chr12:52400965-52401055和来源于人chr12:52408471-52408566、或者来源于人chr1:45252095-45252176、来源于人chr1:45252187-45252275和来源于人chr1:45249894-45249981、或者来源于人chr4:42153816-42153921和来源于人chr4:42153513-42153601、或者来源于人chr1:145399249:145399476、来源于人chr1:145396880-145396983、来源于人chr1:145385298-145385376和来源于人chr1:145407431-145407518、或者来源于人chr10:113943540:113943739和来源于人chr10:113943511-113943582、或者来源于人chr1:213124569-213124670和来源于人chr1:213124036-213124162。
26.如实施方案22-25中任一项所述的方法，包含提供选自以下组核酸或其互补核酸、或上述的片段：SEQ ID NO：33、SEQ ID NO：36、SEQ ID NO：40和SEQ ID NO：44、或者SEQ ID NO：6、SEQ ID NO：47、SEQ ID NO：51和SEQ ID NO：55、或者SEQ ID NO：59和SEQ ID NO：62、或者SEQ ID NO：22、SEQ ID NO：65、SEQ ID NO：68和SEQ ID NO：72、或者SEQ ID NO：14、SEQ ID NO：75和SEQ ID NO：79、或者SEQ ID NO：18、SEQ ID NO：82和SEQ ID NO：86、或者SEQ ID NO：10、SEQ  ID NO：89和SEQ ID NO：93、或者SEQ ID NO：26和SEQ ID NO：96、或者SEQ ID NO：100、SEQ ID NO：103、SEQ ID NO：107和SEQ ID NO：111、或者SEQ ID NO：114和SEQ ID NO：117、或者SEQ ID NO：121和SEQ ID NO：124、或者SEQ ID NO：128和SEQ ID NO：131。
27.如实施方案22-26中任一项所述的方法，包含提供选自以下组核酸组或其互补核酸组、或上述的片段：SEQ ID NO：34与35、SEQ ID NO：37与38、SEQ ID NO：41与42和SEQ ID NO：45与46、或者SEQ ID NO：7与8、SEQ ID NO：48与49、SEQ ID NO：52与53和SEQ ID NO：56与57、或者SEQ ID NO：60与61和SEQ ID NO：63与64、或者SEQ ID NO：23与24、SEQ ID NO：66与67、SEQ ID NO：69与70和SEQ ID NO：73与74、或者SEQ ID NO：15与16、SEQ ID NO：76与77和SEQ ID NO：80与81、或者SEQ ID NO：19与20、SEQ ID NO：83与84和SEQ ID NO：87与88、或者SEQ ID NO：11与12、SEQ ID NO：90与91和SEQ ID NO：94与95、或者SEQ ID NO：27与28和SEQ ID NO：97与98、或者SEQ ID NO：101与102、SEQ ID NO：104与105、SEQ ID NO：108与109和SEQ ID NO：112与113、或者SEQ ID NO：115与116和SEQ ID NO：118与119、或者SEQ ID NO：122与123和SEQ ID NO：125与126、或者SEQ ID NO：129与130和SEQ ID NO：132与133。
28.如实施方案22-27中任一项所述的方法，所述疾病包含肿瘤。
29.如实施方案22-28中任一项所述的方法，所述方法还包含获取待测样本中的核酸。
30.如实施方案29所述的方法，所述核酸包含无细胞游离核酸。
31.如实施方案22-30中任一项所述的方法，所述待测样本包含组织、细胞和/或体液。
32.如实施方案22-31中任一项所述的方法，所述待测样本包含血浆。
33.如实施方案22-32中任一项所述的方法，所述方法还包含转化所述DNA区域或其片段。
34.如实施方案33中任一项所述的方法，具有所述修饰状态的碱基以及不具有所述修饰状态的所述碱基，在转化后形成不同的物质。
35.如实施方案33-34中任一项所述的方法，具有所述修饰状态的碱基在转化后基本不发生改变，且不具有所述修饰状态的所述碱基在转化后改变为与所述碱基不同的其它碱基、或在转化后被剪切。
36.如实施方案34-35中任一项所述的方法，所述碱基包含胞嘧啶。
37.如实施方案22-36中任一项所述的方法，所述修饰状态包含甲基化修饰。
38.如实施方案35-37中任一项所述的方法，所述其它碱基包含尿嘧啶。
39.如实施方案33-38中任一项所述的方法，所述转化包含通过脱氨基试剂和/或甲基化敏感限制酶转化。
40.如实施方案39所述的方法，所述脱氨基试剂包含亚硫酸氢盐或其类似物。
41.如实施方案22-40中任一项所述的方法，所述确定修饰状态的存在和/或含量的方法包含，确认具有所述修饰状态的碱基在所述转化后形成的物质的存在和/或含量。
42.如实施方案22-41中任一项所述的方法，所述确定修饰状态的存在和/或含量的方法包含，确定具有所述修饰状态的DNA区域或其片段的存在和/或含量。
43.如实施方案22-42中任一项所述的方法，通过所述荧光PCR方法检测的荧光Ct值确定具有所述修饰状态的DNA区域或其片段的存在和/或含量。
44.如实施方案22-43中任一项所述的方法，通过确认所述DNA区域或其片段的修饰状态的存在和/或所述DNA区域或其片段相对于参考水平具有更高的修饰状态的含量，确定肝肿瘤的存在、或者有肝肿瘤形成或形成的风险。
45.如实施方案22-44中任一项所述的方法，所述方法还包含在确定所述DNA区域或其片段的修饰的存在和/或含量之前，扩增待测样本中所述DNA区域或其片段。
46.如实施方案45所述的方法，所述扩增包含PCR扩增。
47.一种核酸，所述核酸包含能够结合BCAT1、IKZF1、VAV3、IRF4、B4GALNT1、GRASP、BEST4、BEND4、GPAM、VASH2基因所在DNA区域、来源于人chr1:145384249:145413094所在DNA区域、和/或来源于人chr7:26415938:26416740所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。
48.一种制备核酸的方法，包含根据BCAT1、IKZF1、VAV3、IRF4、B4GALNT1、GRASP、BEST4、BEND4、GPAM、VASH2基因所在DNA区域、来源于人chr1:145384249:145413094所在DNA区域、和/或来源于人chr7:26415938:26416740所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
49.一种核酸组，所述核酸组包含能够结合BCAT1、IKZF1、VAV3、IRF4、B4GALNT1、GRASP、BEST4、BEND4、GPAM、VASH2基因所在DNA区域、来源于人chr1:145384249:145413094所在DNA区域、和/或来源于人chr7:26415938:26416740所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。
50.一种制备核酸组的方法，包含根据BCAT1、IKZF1、VAV3、IRF4、B4GALNT1、GRASP、BEST4、BEND4、GPAM、VASH2基因所在DNA区域、来源于人 chr1:145384249:145413094所在DNA区域、和/或来源于人chr7:26415938:26416740所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够扩增所述DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸组。
51.一种试剂盒，包含如实施方案47所述的核酸和/或实施方案49所述的核酸组。
52.如实施方案47所述的核酸、如实施方案49所述的核酸组和/或实施方案51所述的试剂盒，在制备疾病检测产品中的应用。
53.如实施方案47所述的核酸、如实施方案49所述的核酸组和/或实施方案51所述的试剂盒，在制备确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用。
54.如实施方案47所述的核酸、如实施方案49所述的核酸组和/或实施方案51所述的试剂盒，在制备确定所述DNA区域或其片段的修饰状态的物质中的应用。
55.用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，在制备用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的物质中的应用，所述用于确定的DNA区域包含BCAT1、IKZF1、VAV3、IRF4、B4GALNT1、GRASP、BEST4、BEND4、GPAM、VASH2基因所在DNA区域、来源于人chr1:145384249:145413094所在DNA区域、和/或来源于人chr7:26415938:26416740所在DNA区域或其片段。
56.用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，在制备用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用，所述DNA区域包含选自以下组DNA区域、或其互补区域、或上述的片段：来源于人chr12:25101630-25102393、来源于人chr:12:25078661-25079410和来源于人chr12:25054781-25056311、或者来源于人chr7:50343720-50344547、来源于人chr7:50450118-50450531和来源于人chr7:50467368-50469092、或者来源于人chr1:108507215-108507766、或者来源于人chr6:391739-394056和来源于人chr6:401233-401801、或者来源于人chr12:58020498-58022962和来源于人chr12:58025539-58027138、或者来源于人chr12:52400724-52401698和来源于人chr12:52406880-52409127、或者来源于人chr1:45251728-45252477和来源于人chr1:45249853-45250527、或者来源于人chr4:42152705-42154895、或者来源于人chr1:145389746-145401075、来源于人chr1:145384910-145385929和来源于人chr1:145406714-145408013、或者来源于人chr7:26416257-26416363和来源于人chr7:26416026-26416126、或者来源于人chr10:113942657-113943906、或者来源于人chr1:213123862-213125211、。
57.BCAT1、IKZF1、VAV3、IRF4、B4GALNT1、GRASP、BEST4、BEND4、GPAM、VASH2基因所在DNA区域、来源于人chr1:145384249:145413094所在DNA区域、和/或来源于人chr7:26415938:26416740所在DNA区域、或其转化而来的区域、或上述的片段的核酸，以及上述核酸的组合，在制备用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的物质中的应用。
58.选自以下组DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸，以及上述核酸的组合，在制备用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用：来源于人chr12:25101630-25102393、来源于人chr:12:25078661-25079410和来源于人chr12:25054781-25056311、或者来源于人chr7:50343720-50344547、来源于人chr7:50450118-50450531和来源于人chr7:50467368-50469092、或者来源于人chr1:108507215-108507766、或者来源于人chr6:391739-394056和来源于人chr6:401233-401801、或者来源于人chr12:58020498-58022962和来源于人chr12:58025539-58027138、或者来源于人chr12:52400724-52401698和来源于人chr12:52406880-52409127、或者来源于人chr1:45251728-45252477和来源于人chr1:45249853-45250527、或者来源于人chr4:42152705-42154895、或者来源于人chr1:145389746-145401075、来源于人chr1:145384910-145385929和来源于人chr1:145406714-145408013、或者来源于人chr7:26416257-26416363和来源于人chr7:26416026-26416126、或者来源于人chr10:113942657-113943906、或者来源于人chr1:213123862-213125211。
59.一种储存介质，其记载可以运行实施方案1-46中任一项所述的方法的程序。
60.一种设备，其包含实施方案59所述的储存介质。
61.如实施方案59所述的设备，还包含耦接至所述储存介质的处理器，所述处理器被配置为基于存储在所述储存介质中的程序执行以实现实施方案1-46中任一项所述的方法。不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1
比较肝癌、癌旁组织及白膜层DNA样本甲基化丰度
分别从来源于肝脏未见异常的健康人群的白膜层、来源于肝癌患者的癌组织中获得DNA样品(其中白膜层样品10个，癌组织各10个)，选择白膜层DNA作为参考样本是因为血浆 游离DNA大多数来源于白膜层破裂后释放的DNA，其本底背景可以是血浆游离DNA该检测位点的一个基础背景信号。按照说明书的要求，用Qiagen QIAamp DNA Mini Kit提取白膜层DNA，用Qiagen QIAamp DNA FFPE Tissue Kit提取组织DNA。
将上述步骤中获得的DNA取20ng样品用亚硫酸氢盐试剂(D5031，ZYMO RESEARCH)处理，以获得转化的DNA。
通过荧光PCR检测，获得标记物的检测荧光Ct值。在荧光PCR反应体系中，每个引物的终浓度为500nM，每个检测探针的终浓度为200nM。PCR反应体系包含：10μL的预扩增稀释产物，2.5μL包含检测位点的引物和探针预混液；12.5μL PCR试剂(
Figure PCTCN2022110445-appb-000001
Universal Probe qPCR Master Mix(NEB)。PCR反应条件如下：95℃5分钟；95℃30秒，56℃60秒(采集荧光)，进行50个循环。使用ABI 7500Real-Time PCR System在相应的荧光通道检测不同的荧光。计算并比较从白膜层、癌旁组织和癌组织获得的样品Ct值，未检测到扩增信号的靶点Ct值被设定为50。其中各个甲基化标志物的引物序列见表1-1，探针序列见表1-2。
表1-1引物序列
Figure PCTCN2022110445-appb-000002
Figure PCTCN2022110445-appb-000003
表1-2检测探针序列
SEQ ID NO. 名称 序列
2 SEPT9(1)探针 TTAACCGCGAAATCCGAC
6 IKZF1探针 CGCCCCGTCGCCGAAT
10 BEST4探针 CGGCGTATTTGCGTTTATTACGT
14 B4GALNT1探针 AGATTTCGCGTATCGCGTTTT
18 GRASP探针 TGTTTTTTTTTCGGCGTTCGCG
22 IRF4探针 ATCGTACGTAAGGTTCGGAGCGA
26 BEND4探针 TAGGACGGCGACGACGA
29 SEPT9(1a)探针 TTGTTGCGGTCGCGGACG
32 ACTB探针 ACCACCACCCAACACACAATAACAAACACA
表1-3样本检测结果汇总
Figure PCTCN2022110445-appb-000004
上表结果显示，癌组织检测的平均Ct值小，代表具有更强的甲基化信号。癌组织中甲基化信号检出率可以远高于白膜层，也代表甲基化信号强。白膜层大多数样本不能检出靶点甲基化信号。这些靶点都可以具备用于血液检测肝癌的潜能。证明所选目标标记物对肿瘤组织具有可行性和特异性。
比较肝癌患者、肝脏未见异常人群血浆样本甲基化信号
选取266个肝脏未见异常健康对照血浆、372个肝癌患者术前血浆(其中I期占比为86％)、37例乙肝患者血浆和39例肝硬化血浆样本进行检测。
使用商业化Qiagen QIAamp Circulating Nucleic Acid Kit抽提上述血浆样本中的细胞外游离DNA。使用商业化亚硫酸氢盐转化试剂MethylCodeTM Bisulfite Conversion Kit对抽提出的细胞外游离DNA进行亚硫酸盐转化处理，得到转化后的DNA。
可选地，将上述转化后的DNA用于预扩增，用含有表1所示的甲基化标志物特异性引物和内参引物(ACTB，正向引物和反向引物可以分别如表1所示，探针可以如表2所示)的引物池，以转化后的DNA为模板，进行PCR扩增，每条引物的终浓度为100nM。PCR反应体系为10μL转化的DNA，包含上述引物的预混液2.5μL；PCR试剂(
Figure PCTCN2022110445-appb-000005
Universal Probe qPCR Master Mix(NEB)12.5μL。PCR反应条件如下：95℃5分钟；95℃30秒，56℃60秒，15个循环。
将获得的预扩增产物稀释10倍后用于荧光PCR检测。使用如表1所示的各个甲基化标志物的引物、表2所示的检测探针序列，并且同时对内参基因ACTB进行检测，作为对照。引物终浓度为500nM，探针终浓度为200nM。PCR反应体系包含：10μL的预扩增稀释产物，包含检测位点的引物和探针预混液2.5μL；PCR试剂(
Figure PCTCN2022110445-appb-000006
Universal Probe qPCR Master Mix(NEB)12.5μL。
荧光PCR反应体系与上述实施例相同。PCR反应条件如下：95℃5分钟；95℃15秒，56℃40秒(采集荧光)，50个循环。针对不同基因探针修饰荧光，选择相应检测荧光通道。未检测到扩增信号的靶点Ct值被设定为50。结果显示，本申请的各个靶点都可以具备用于血液检测肝癌的能力。
在约90％特异性的情况下，检测位点的检测灵敏度统计如表5所示：
表1-4检测位点的检测灵敏度
位点 特异性 灵敏度
BEST4 94.9％ 48.8％
GRASP 91.8％ 40.2％
B4GALNT1 92.9％ 40.2％
IRF4 90.3％ 40.0％
SEPT9(1a) 91.8％ 40.0％
SEPT9(1) 92.3％ 40.0％
IKZF1 98.3％ 35.1％
BEND4 99.1％ 17.8％
上表结果显示，检测位点在对照血浆和肝癌血浆DNA甲基化信号对比。证明所选目标标记物对肝癌患者血液样本具有较高的灵敏度。
在对靶点进行组合分析时，数据分析可采用对单一靶点设定阳性判读阈值，联合靶点时，任一靶点阳性即样本综合判读为阳性的方式。以此方式验证了：
联合SEPT9(1)、IKZF1，可使肝癌检测灵敏性64.5％，健康对照特异性高达95.9％，同时乙肝患者的阳性率为2.7％，肝硬化患者阳性率为20.5％；联合SEPT9(1)、IKZF1、BEST4、B4GALNT1，可使肝癌检测灵敏性73.1％，健康对照特异性为91.0％，同时乙肝患者的阳性率为9.1％，肝硬化患者阳性率为20.5％；联合SEPT9(1)、SEPT9(1a)、IKZF1、BEST4、GRASP、B4GALNT1，可使肝癌检测灵敏性75.8％，健康对照特异性为90.2％，同时乙肝患者的阳性率为16.2％，肝硬化患者阳性率为20.5％；联合IRF4、IKZF1、BEST4、B4GALNT1、BEND4、SEPT9(1)时，可使肝癌检测灵敏性76.9％，健康对照特异性为91.0％，同时乙肝患者的阳性率为9.1％，肝硬化患者阳性率为20.5％；以上靶点组合均证实区分健康对照与肝癌的性能优越，且在乙肝和肝硬化患者中特异性良好。
实施例2
比较肝癌、癌旁组织及白细胞DNA样本甲基化丰度
分别从来源于肝脏未见异常的健康人群的白细胞、来源于肝癌患者的癌旁组织和癌组织中获得DNA样品(其中白细胞样品约10个，癌旁组织和癌组织各约24个)，选择白细胞DNA作为参考样本是因为血浆游离DNA大多数来源于白细胞破裂后释放的DNA，其本底背景可以是血浆游离DNA该检测位点的一个基础背景信号。按照说明书的要求，用Qiagen QIAamp DNA Mini Kit提取白细胞DNA，用Qiagen QIAamp DNA FFPE Tissue Kit提取组织DNA。将上述步骤中获得的DNA取20ng样品用亚硫酸氢盐试剂(D5031，ZYMO RESEARCH)处理，以获得转化的DNA。
可选地，用含有本申请靶点甲基化特异性引物和内参(ACTB)引物对的引物池，以转化后的DNA为模板，进行PCR扩增，每条引物的终浓度为100nM。PCR反应体系包含：10μL转化的DNA(包含2.5μL上述引物的预混液)和12.5μL PCR试剂(
Figure PCTCN2022110445-appb-000007
Universal Probe qPCR Master Mix(NEB)。PCR反应条件为：95℃5分钟；95℃30秒，56℃60秒，进行10个循环。例如，本申请表格序列中的小写字母所在的位置可以对应该序列所结合的基因组的 区域上天然胞嘧啶的位点。例如，本申请表格中序列的小写字母表示的碱基，可以用于与转化后的碱基配对；如天然的胞嘧啶在亚硫酸氢盐处理后可以转化尿嘧啶，小写的碱基a可以与该转化后的尿嘧啶配对，小写的碱基t可以进一步与该尿嘧啶配对的腺嘌呤配对。其中引物序列如表2-1所示。
表2-1本申请靶点特异性引物
SEQ ID NO. 名称 序列
34 BCAT1正向引物1 TACGTGGCGGGTTGG
35 BCAT1反向引物1 AAAAAAACAACCTTAATATCTTC
37 BCAT1正向引物1a GTTTTTTTGTTGATGTAATTCGTTAGGTC
38 BCAT1反向引物1a CAATACCCGAAACGACGACG
41 BCAT1正向引物2 tAGGGtAGAGGCGtTttttAtAT
42 BCAT1反向引物2 CCGaCTaCCATCCCGTCTAa
45 BCAT1正向引物3 tTGGGACGAGACGGTTGGAG
46 BCAT1反向引物3 AaaaCGCTaaaTACCACGACCTa
7 IKZF1正向引物1 GTTTTTTTGGTTCGGAGTTG
8 IKZF1反向引物1 CAAAACGAAACACGAAAAAAATA
48 IKZF1正向引物1a GACGACGTATTTTTTTCGTGTTTC
49 IKZF1反向引物1a GCGCACCTCTCGACCG
52 IKZF1正向引物2 GtTGtATTtCGGGGAGAAGtt
53 IKZF1反向引物2 ACCTACCGaAaTaCGTCCTC
56 IKZF1正向引物3 AtAGCGtCGTGGAGAAttTGt
57 IKZF1反向引物3 CTCGTTaTTaCTCTCGaTaTCCG
60 VAV3正向引物1 CGGAGTCGAGTTTAG
61 VAV3反向引物1 ACCGCCGACCCTTT
63 VAV3正向引物1a CGCGGGATTCGTTGTAGC
64 VAV3反向引物1a AACAAAAACCGCGACTAACGA
23 IRF4正向引物1 AAAAAAAAAAAAACTCCACATTT
24 IRF4反向引物1 TAGTTGNGGAGTTTGGG
66 IRF4正向引物1a TGGGTGTTTTGGACGGTTTC
67 IRF4反向引物1a CGCCTACCCTCCGCG
69 IRF4正向引物1b AtAAGTGGCGtAGACGCGGG
70 IRF4反向引物1b CCTCCGCTCTCCCGaaCCTa
73 IRF4正向引物2 CGACGGGtTtTATGCGAAAAG
74 IRF4反向引物2 aAaCTTaCAaaTCTaaTCTCTCTCC
135 ACTB正向引物 GTGATGGAGGAGGTTTAGTAAGTT
136 ACTB反向引物 CCAATAAAACCTACTCCTCCCTTAA
将获得的预扩增产物稀释10倍，通过多重荧光PCR检测，获得标记物的检测荧光Ct值。在荧光PCR反应体系中，每个引物的终浓度为500nM，每个检测探针的终浓度为200nM。PCR反应体系包含：10μL的预扩增稀释产物，2.5μL包含检测位点的引物和探针预混液；12.5μL PCR试剂(
Figure PCTCN2022110445-appb-000008
Universal Probe qPCR Master Mix(NEB)。PCR反应条件如下：95℃5分钟；95℃30秒，56℃60秒(采集荧光)，进行50个循环。使用ABI 7500Real-Time PCR System在相应的荧光通道检测不同的荧光。计算并比较从白细胞、癌旁组织和癌组织获得的样品Ct值，未检测到扩增信号的靶点Ct值被设定为50。其中引物序列见表2-1，探针序列见表2-2。
表2-2本申请靶点检测探针序列
SEQ ID NO. 名称 序列
33 BCAT1探针1 TCGGTTTTTTCGCGGCG
36 BCAT1探针1a TTCGTCGCGAGAGGGTCGGTT
40 BCAT1探针2 TtAGACGATGGGCGGtCG
44 BCAT1探针3 TGCGTCGTAtttTGCGttTG
6 IKZF1探针1 CGCCCCGTCGCCGAAT
47 IKZF1探针1a TTTGTATCGGAGTAGCGATTCGGGAGG
51 IKZF1探针2 TACGttTGtCGtCGGAGGG
55 IKZF1探针3 TGtttTCGGAGCGCGAGGC
59 VAV3探针1 TTTCGATTTCGCGCGGGG
62 VAV3探针1a CGCGGCGTTCGCGATTCGTT
22 IRF4探针1 ATCGTACGTAAGGTTCGGAGCGA
65 IRF4探针1a TCGTTTAGTTTGTGGCGATTTCGTCG
68 IRF4探针1b TACGGGGGATTtCGCGCGtA
72 IRF4探针2 TGGCGtTGTGtAACGAtCGGt
134 ACTB探针 ACCACCACCCAACACACAATAACAAACACA
结果如图1、3、5、7所示，可见这些检测位点甲基化在白细胞DNA中本底信号均很低，组织信号强于白细胞DNA信号，癌组织信号强于癌旁组织信号。证明所选目标标记物对肿瘤组织具有可行性和特异性。
比较肝癌患者、肝脏未见异常人群血浆样本甲基化信号
使用荧光PCR检测方法对约65例肝癌个体的血浆样本和约76例对照个体的血浆样本进行检测：
使用商业化Qiagen QIAamp Circulating Nucleic Acid Kit抽提上述血浆样本中的细胞外游离DNA。使用商业化亚硫酸氢盐转化试剂MethylCodeTM Bisulfite Conversion Kit对抽提出的细胞外游离DNA进行亚硫酸盐转化处理，得到转化后的DNA。
可选地，将上述转化后的DNA用于预扩增，用含有表2-1所示的本申请靶点甲基化特异性引物和内参(ACTB)引物对的引物池，以转化后的DNA为模板，进行PCR扩增，每条引物的终浓度为100nM。PCR反应体系为10μL转化的DNA，包含上述引物的预混液2.5μL；PCR试剂(
Figure PCTCN2022110445-appb-000009
Universal Probe qPCR Master Mix(NEB)12.5μL。PCR反应条件如下：95℃5分钟；95℃30秒，56℃60秒，15个循环。
将获得的预扩增产物稀释10倍后用于荧光PCR检测。使用如表2-1所示的引物、表2-2所示的检测探针序列，并且同时对内参基因ACTB进行检测，作为对照。引物终浓度为500nM，探针终浓度为200nM。PCR反应体系包含：10μL的预扩增稀释产物，包含检测位点的引物和探针预混液2.5μL；PCR试剂(
Figure PCTCN2022110445-appb-000010
Universal Probe qPCR Master Mix(NEB)12.5μL。
荧光PCR反应体系与本实施例相同。PCR反应条件如下：95℃5分钟；95℃15秒，56℃40秒(采集荧光)，50个循环。针对不同基因探针修饰荧光，选择相应检测荧光通道。未检测到扩增信号的靶点Ct值被设定为50。
结果如图2、4、6、8所示，检测位点在对照血浆和肝癌血浆DNA甲基化信号对比。证明所选目标标记物对肿瘤组织具有较高的灵敏度。
在大于90％特异性的情况下，检测位点的检测灵敏度统计如下表所示：
表2-3检测位点的检测灵敏度
位点 灵敏度
BCAT1 34％
IKZF1 55％
VAV3 35％
IRF4 51％
将5个标记物组(Septin9、IKZF1、IRF4、VAV3和BCAT1)综合考量，在对照组特异性为90.8％的情况下，肝癌的检测灵敏度可以实现90.8％，具体信息如下表所示：
表2-4检测多个位点的检测准确性
Figure PCTCN2022110445-appb-000011
比较肝癌、癌旁组织及白膜层DNA样本甲基化丰度
分别从来源于肝脏未见异常的健康人群的白膜层、来源于肝癌患者的癌组织中获得DNA样品(其中白膜层样品约10个，癌组织各约10个)，选择白膜层DNA作为参考样本是因为血浆游离DNA大多数来源于白膜层破裂后释放的DNA，其本底背景可以是血浆游离DNA该检测位点的一个基础背景信号。按照说明书的要求，用Qiagen QIAamp DNA Mini Kit提取白膜层DNA，用Qiagen QIAamp DNA FFPE Tissue Kit提取组织DNA。将上述步骤中获得的DNA取20ng样品用亚硫酸氢盐试剂(D5031，ZYMO RESEARCH)处理，以获得转化的DNA。
可选地，用含有本申请靶点甲基化特异性引物和内参(ACTB)引物对的引物池，以转化后的DNA为模板，进行PCR扩增，每条引物的终浓度为100nM。PCR反应体系包含：10μL转化的DNA(包含2.5μL上述引物的预混液)和12.5μL PCR试剂(
Figure PCTCN2022110445-appb-000012
Universal Probe qPCR Master Mix(NEB)。PCR反应条件为：95℃5分钟；95℃30秒，56℃60秒，进行10个循环。例如，本申请表格序列中的小写字母所在的位置可以对应该序列所结合的基因组的区域上天然胞嘧啶的位点。例如，本申请表格中序列的小写字母表示的碱基，可以用于与转化后的碱基配对；如天然的胞嘧啶在亚硫酸氢盐处理后可以转化尿嘧啶，小写的碱基a可以与该转化后的尿嘧啶配对，小写的碱基t可以进一步与该尿嘧啶配对的腺嘌呤配对。其中引物序列如表2-5所示。
将获得的预扩增产物稀释10倍，通过多重荧光PCR检测，获得标记物的检测荧光Ct值。在荧光PCR反应体系中，每个引物的终浓度为500nM，每个检测探针的终浓度为200nM。PCR反应体系包含：10μL的预扩增稀释产物，2.5μL包含检测位点的引物和探针预混液； 12.5μL PCR试剂(
Figure PCTCN2022110445-appb-000013
Universal Probe qPCR Master Mix(NEB)。PCR反应条件如下：95℃5分钟；95℃30秒，56℃60秒(采集荧光)，进行50个循环。使用ABI 7500Real-Time PCR System在相应的荧光通道检测不同的荧光。计算并比较从白膜层、癌旁组织和癌组织获得的样品Ct值，未检测到扩增信号的靶点Ct值被设定为50。其中引物序列见表2-5，探针序列见表2-6。基因间隔区2表示人chr1:145384249:145413094区域，基因间隔区1表示人chr7:26415938:26416740区域。
表2-5引物序列
SEQ ID NO. 名称 序列
15 B4GALNT1正向引物1 AGtAGtTGtCgATAAGTGGT
16 B4GALNT1反向引物1 GCCTaaAaaCAACCTCCCT
76 B4GALNT1正向引物1a GGTttAGCGGCGTtCGtTttAG
77 B4GALNT1反向引物1a aaaTaaACGACGACTTCGTCTTC
80 B4GALNT1正向引物2 GAATtATGttCGTGtTGGCGGG
81 B4GALNT1反向引物2 GGAtAGAGTGGAtCGGGAAGCG
19 GRASP正向引物1 GttttTtTtCgAtTtttTAtAGGG
20 GRASP反向引物1 aaCcgaaaAAaAAaaTaaaaaACTC
83 GRASP正向引物1a GtTGTACGCGGCGtTGGAGG
84 GRASP反向引物1a ACGCACCTTTCGaCGaaaCA
87 GRASP正向引物2 TGCGtAGGttTGGTGGTGAAG
88 GRASP反向引物2 CAaCGAaCCCGaaAaCAaaCCC
11 BEST4正向引物1 TGTGGGtCgGAtttttAGAG
12 BEST4反向引物1 TaCTTAAacGCTTCCCCAC
90 BEST4正向引物1a GAtCGAGCGtAGtAttAGtACGG
91 BEST4反向引物1a TaCACGaCGTaaACCAaCGaa
94 BEST4正向引物2 GGttTCGTtttCGGAtTtCGt
95 BEST4反向引物2 TCTaCTaCGCTTCCGaaCGaA
27 BEND4正向引物1 AGTttTtAAGTGGtttTGGGAT
28 BEND4反向引物1 CCAaacgCAaAaCTCCTAC
97 BEND4正向引物1a TtTCGAAGTTTtCGGGTGCG
98 BEND4反向引物1a CGaaaaaCCGCCGACACTTA
101 基因间隔区2正向引物1 TTAtTtTTTtttTGAtCgGGAAT
102 基因间隔区2反向引物1 ATAaCTaCAACcGaaAAaaaTTA
104 基因间隔区2正向引物1a tTGtCGTGAtTCGGATTCGAA
105 基因间隔区2反向引物1a CAaCTaTAaCGCGCCGCCTa
108 基因间隔区2正向引物2 ACGCGGTGAGGAtAttACGG
109 基因间隔区2反向引物2 GttCGGCGGAAATGAtTGTGAG
112 基因间隔区2正向引物3 GAtAAGAGAtCGGGCGCGGT
113 基因间隔区2反向引物3 CTCGATCTCCTaACCTCGTaATCC
115 基因间隔区1正向引物1 GtttCgttAGGtTGGAGAG
116 基因间隔区1反向引物1 CTaCAaAACAAAaCCTaaAaTCC
118 基因间隔区1正向引物1a CGAtCGtAAAGAGAtAGCGATTT
119 基因间隔区1反向引物1a AATCGTCACCGTAaCGCGaa
122 GPAM正向引物1 gGttTGcgAGttTtAGGG
123 GPAM反向引物1 CAaacgCTCACACACTATC
125 GPAM正向引物1a GtTGtAGTGGCGtAtttTGATGAC
126 GPAM反向引物1a CGaCGCTaaCTAaCCGaCGa
129 VASH2正向引物1 TtAGCgTGtAGGGGGGA
130 VASH2反向引物1 cGaAaACACAaCTaACACCA
132 VASH2正向引物1a ttCGtCGttCGGAGAGGtTt
133 VASH2反向引物1a TTCCAaCGCCTATCACCGaaA
135 ACTB正向引物 GTGATGGAGGAGGTTTAGTAAGTT
136 ACTB反向引物 CCAATAAAACCTACTCCTCCCTTAA
表2-6检测探针序列
Figure PCTCN2022110445-appb-000014
Figure PCTCN2022110445-appb-000015
表2-7样本检测结果汇总
Figure PCTCN2022110445-appb-000016
Figure PCTCN2022110445-appb-000017
结果显示，癌组织中甲基化信号检出率可以远高于白膜层，也代表甲基化信号强。白膜层大多数样本不能检出靶点甲基化信号。这些靶点都可以具备用于血液检测肝癌的潜能。证明所选目标标记物对肿瘤组织具有可行性和特异性。
比较肝癌患者、肝脏未见异常人群血浆样本甲基化信号
选取约170个肝脏未见异常健康对照血浆、约321个肝癌患者术前血浆(其中I期占比为86％)、约36例乙肝患者血浆和20例肝硬化血浆样本进行检测：使用商业化Qiagen QIAamp Circulating Nucleic Acid Kit抽提上述血浆样本中的细胞外游离DNA。使用商业化亚硫酸氢盐转化试剂MethylCodeTM Bisulfite Conversion Kit对抽提出的细胞外游离DNA进行亚硫酸盐转化处理，得到转化后的DNA。
可选地，将上述转化后的DNA用于预扩增，用含有表2-5所示的本申请靶点甲基化特异性引物和内参(ACTB)引物对的引物池，以转化后的DNA为模板，进行PCR扩增，每条引物的终浓度为100nM。PCR反应体系为10μL转化的DNA，包含上述引物的预混液2.5μL；PCR试剂(
Figure PCTCN2022110445-appb-000018
Universal Probe qPCR Master Mix(NEB)12.5μL。PCR反应条件如下：95℃5分钟；95℃30秒，56℃60秒，15个循环。
将获得的预扩增产物稀释10倍后用于荧光PCR检测。使用如表2-5所示的引物、表2-6所示的检测探针序列，并且同时对内参基因ACTB进行检测，作为对照。引物终浓度为500nM，探针终浓度为200nM。PCR反应体系包含：10μL的预扩增稀释产物，包含检测位点的引物和探针预混液2.5μL；PCR试剂(
Figure PCTCN2022110445-appb-000019
Universal Probe qPCR Master Mix(NEB)12.5μL。
荧光PCR反应体系与本实施例相同。PCR反应条件如下：95℃5分钟；95℃15秒，56℃40秒(采集荧光)，50个循环。针对不同基因探针修饰荧光，选择相应检测荧光通道。未检 测到扩增信号的靶点Ct值被设定为50。
表2-8样本检测结果汇总
  AUC p值
B4GALNT1 0.743 7.12E-19
GRASP 0.748 1.67E-19
BEST4 0.787 1.05E-25
BEND4 0.586 1.64E-03
基因间隔区2 0.777 4.97E-24
基因间隔区1 0.597 4.14E-04
GPAM 0.755 1.24E-20
VASH2 0.592 8.00E-04
上表结果显示标志物在肝癌组和健康对照组之间的AUC统计，本申请的靶点都可以具备用于血液检测肝癌。
在大于90％特异性的情况下，检测位点的检测灵敏度统计如下表所示：
表2-9检测位点的检测灵敏度
位点 灵敏度 特异性
B4GALNT1 52.02％ 93.69％
GRASP 53.89％ 93.2％
BEST4 57.01％ 89.81％
BEND4 17.76％ 99.51％
基因间隔区2 54.21％ 86.41％
基因间隔区1 19.94％ 99.03％
GPAM 56.7％ 89.81％
VASH2 18.38％ 100％
结果显示，检测位点在对照血浆和肝癌血浆DNA甲基化信号对比。证明所选目标标记物对肝癌患者血液样本具有较高的灵敏度。
实施例3
本申请甲基化标志物各个亚区域的样本检测能力
参考以上实施例，对于本申请的甲基化标志物的各个亚区域进行肝癌样本的检测。检测 结果如下表所示：
表3样本检测结果汇总
Figure PCTCN2022110445-appb-000020
Figure PCTCN2022110445-appb-000021
其中各个检测区域编号中下划线之后的数字编号为该检测区域对应的亚区域编号，下划线之后的相同数字编号如有不同字母，代表相同亚区域中的不同的特定检测区域。各个检测区域用到的引物对和探针如表2-1、2-5以及2-2和2-6所示，各个检测区域编号中下划线之后的编号，与对应使用的表格中引物对和探针名称结尾的编号对应。
癌组织甲基化信号Ct值相较于白细胞甲基化信号Ct值，体现了靶点检测效果的指数级差异。各个靶点特定区域检测效果等级划分标准为：A表示癌组织甲基化信号相较于白细胞大于100倍，是肝癌检测的优选位点；B表示癌组织甲基化信号相较于白细胞介于10倍到100倍，有可能用于肝癌检测的位点；C表示癌组织甲基化信号相较于白细胞小于10倍，大概率不可用于肝癌检测的位点。
实施例4
分别从来源于肝脏未见异常的健康人群的白膜层、来源于肝癌患者的癌组织中获得DNA样品(其中白膜层样品10个，癌组织各10个)，选择白膜层DNA作为参考样本是因为血浆游离DNA大多数来源于白膜层破裂后释放的DNA，其本底背景可以是血浆游离DNA该检测位点的一个基础背景信号。按照说明书的要求，用Qiagen QIAamp DNA Mini Kit提取白膜层DNA，用Qiagen QIAamp DNA FFPE Tissue Kit提取组织DNA。
将上述步骤中获得的DNA取20ng样品用亚硫酸氢盐试剂(D5031，ZYMO RESEARCH)处理，以获得转化的DNA。
通过荧光PCR检测，获得标记物的检测荧光Ct值。在荧光PCR反应体系中，每个引物的终浓度为500nM，每个检测探针的终浓度为200nM。PCR反应体系包含：10μL的预扩增稀释产物，2.5μL包含检测位点的引物和探针预混液；12.5μL PCR试剂(
Figure PCTCN2022110445-appb-000022
Universal Probe qPCR Master Mix(NEB)。其中各个甲基化标志物的引物序列见表4-1，探针序列见表4-2。PCR反应条件如下：95℃5分钟；95℃30秒，56℃60秒(采集荧光)，进行50个循环。使用ABI 7500 Real-Time PCR System在相应的荧光通道检测不同的荧光。计算并比较从白膜层、癌旁组织和癌组织获得的样品Ct值，未检测到扩增信号的靶点Ct值被设定为50。
表4-1引物序列
Figure PCTCN2022110445-appb-000023
表4-2检测探针序列
SEQ ID NO. 名称 序列
137 SDC2探针 TTCGGGGCGTAGTTGCGGGCGG
表4-3样本检测结果汇总
Figure PCTCN2022110445-appb-000024
上表结果显示，肝癌组织检测的平均Ct值相较于白膜层大，代表肝癌组织样本SDC2甲基化信号比白膜层弱。例如并不是所有本领域已知的甲基化靶点都可用于肝癌血液样本检测。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (97)

  1. 一种确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的方法，包含确定待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量，所述目标基因包含SEPT9和IKZF1。
  2. 一种评估肝肿瘤相关DNA区域甲基化状态的方法，包含确定待测样本中目标基因所在DNA区域或其片段的修饰状态的存在和/或含量，所述目标基因包含SEPT9和IKZF1。
  3. 如权利要求1-2中任一项所述的方法，所述SEPT9的DNA区域来源于人chr17:75276651-75496678。
  4. 如权利要求1-3中任一项所述的方法，所述IKZF1的DNA区域来源于人chr7:50343720-50472799。
  5. 如权利要求1-4中任一项所述的方法，所述目标基因还包含选自以下组的基因：BEST4、B4GALNT1、GRASP、IRF4和BEND4。
  6. 如权利要求1-5中任一项所述的方法，所述目标基因包含至少2种基因。
  7. 如权利要求1-6中任一项所述的方法，所述目标基因包含2种至7种基因。
  8. 如权利要求1-7中任一项所述的方法，所述目标基因包含SEPT9、IKZF1、BEST4和B4GALNT1。
  9. 如权利要求1-8中任一项所述的方法，所述目标基因包含SEPT9、IKZF1、BEST4、GRASP和B4GALNT1。
  10. 如权利要求1-9中任一项所述的方法，所述目标基因包含SEPT9、IKZF1、BEST4、IRF4、B4GALNT1和BEND4。
  11. 如权利要求5-10中任一项所述的方法，所述BEST4的DNA区域来源于人chr1:45249257-45253377。
  12. 如权利要求5-11中任一项所述的方法，所述B4GALNT1的DNA区域来源于人chr12:58017193-58027138。
  13. 如权利要求5-12中任一项所述的方法，所述GRASP的DNA区域来源于人chr12:52400724-52409673。
  14. 如权利要求5-13中任一项所述的方法，所述IRF4的DNA区域来源于人chr6:391739-411447。
  15. 如权利要求5-14中任一项所述的方法，所述BEND4的DNA区域来源于人chr4:42112955-42154895。
  16. 一种确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的方法，包含确定待测样本中目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含 量，所述目标DNA区域包含来源于人chr17:75368651-75370720和来源于人chr7:50343720-50344547定义的区域。
  17. 一种确定DNA区域甲基化状态的方法，包含确定待测样本中目标DNA区域、或其互补区域、或上述的片段的修饰状态的存在和/或含量，所述目标DNA区域包含来源于人chr17:75368651-75370720和来源于人chr7:50343720-50344547定义的区域。
  18. 如权利要求1-17中任一项所述的方法，包含提供能够结合包含SEQ ID NO：1所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
  19. 如权利要求16-18中任一项所述的方法，所述目标区域包含来源于人chr17:75369558-75369622定义的区域。
  20. 如权利要求1-19中任一项所述的方法，包含提供SEQ ID NO：2所示的核酸或其互补核酸、或上述的片段。
  21. 如权利要求1-20中任一项所述的方法，包含提供SEQ ID NO：3与4所示的核酸组或其互补核酸组、或上述的片段。
  22. 如权利要求1-21中任一项所述的方法，包含提供能够结合包含SEQ ID NO：5所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
  23. 如权利要求16-22中任一项所述的方法，所述目标区域包含来源于人chr7:50343793-50343896定义的区域。
  24. 如权利要求1-23中任一项所述的方法，包含提供SEQ ID NO：6所示的核酸或其互补核酸、或上述的片段。
  25. 如权利要求1-24中任一项所述的方法，包含提供SEQ ID NO：7与8所示的核酸组或其互补核酸组、或上述的片段。
  26. 如权利要求16-25中任一项所述的方法，所述目标DNA区域还包含选自以下组定义的区域：来源于人chr1:45251728-45252477、来源于人chr12:58020498-58022962、来源于人chr12:52400724-52401698、来源于人chr6:391739-394056、和来源于人chr4:42152705-42154895。
  27. 如权利要求16-26中任一项所述的方法，所述目标DNA区域包含至少2个区域。
  28. 如权利要求16-27中任一项所述的方法，所述目标DNA区域包含2个至8个区域。
  29. 如权利要求16-28中任一项所述的方法，所述目标DNA区域包含来源于人chr17:75368651-75370720、来源于人chr7:50343720-50344547、来源于人chr1:45251728-45252477和来源于人chr12:58020498-58022962定义的区域。
  30. 如权利要求16-29中任一项所述的方法，所述目标DNA区域包含来源于人chr17:75368651-75370720、来源于人chr7:50343720-50344547、来源于人chr1:45251728-45252477、来源于人chr12:52400724-52401698和来源于人chr12:58020498-58022962定义的区域。
  31. 如权利要求16-30中任一项所述的方法，所述目标DNA区域包含来源于人chr6:391739-394056、来源于人chr7:50343720-50344547、来源于人chr1:45251728-45252477、来源于人chr12:58020498-58022962、来源于人chr4:42152705-42154895和来源于人chr17:75368651-75370720定义的区域。
  32. 如权利要求1-31中任一项所述的方法，包含提供能够结合包含SEQ ID NO：9所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
  33. 如权利要求16-32中任一项所述的方法，所述目标区域包含来源于人chr1:45252095-45252176定义的区域。
  34. 如权利要求1-33中任一项所述的方法，包含提供SEQ ID NO：10所示的核酸或其互补核酸、或上述的片段。
  35. 如权利要求1-34中任一项所述的方法，包含提供SEQ ID NO：11与12所示的核酸组或其互补核酸组、或上述的片段。
  36. 如权利要求1-35中任一项所述的方法，包含提供能够结合包含SEQ ID NO：13所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
  37. 如权利要求16-36中任一项所述的方法，所述目标区域包含来源于人chr12:58021586-58021670定义的区域。
  38. 如权利要求1-37中任一项所述的方法，包含提供SEQ ID NO：14所示的核酸或其互补核酸、或上述的片段。
  39. 如权利要求1-38中任一项所述的方法，包含提供SEQ ID NO：15与16所示的核酸组或其互补核酸组、或上述的片段。
  40. 如权利要求1-39中任一项所述的方法，包含提供能够结合包含SEQ ID NO：17所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
  41. 如权利要求16-40中任一项所述的方法，所述目标区域包含来源于人chr12:52401083-52401169定义的区域。
  42. 如权利要求1-41中任一项所述的方法，包含提供SEQ ID NO：18所示的核酸或其互补核酸、或上述的片段。
  43. 如权利要求1-42中任一项所述的方法，包含提供SEQ ID NO：19与20所示的核酸组或其互补核酸组、或上述的片段。
  44. 如权利要求1-43中任一项所述的方法，包含提供能够结合包含SEQ ID NO：21所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
  45. 如权利要求16-44中任一项所述的方法，所述目标区域包含来源于人chr6:392282-392377定义的区域。
  46. 如权利要求1-45中任一项所述的方法，包含提供SEQ ID NO：22所示的核酸或其互补核酸、或上述的片段。
  47. 如权利要求1-46中任一项所述的方法，包含提供SEQ ID NO：23与24所示的核酸组或其互补核酸组、或上述的片段。
  48. 如权利要求1-47中任一项所述的方法，包含提供能够结合包含SEQ ID NO：25所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
  49. 如权利要求16-48中任一项所述的方法，所述目标区域包含来源于人chr4:42153816-42153921定义的区域。
  50. 如权利要求1-49中任一项所述的方法，包含提供SEQ ID NO：26所示的核酸或其互补核酸、或上述的片段。
  51. 如权利要求1-50中任一项所述的方法，包含提供SEQ ID NO：27与28所示的核酸组或其互补核酸组、或上述的片段。
  52. 如权利要求1-51中任一项所述的方法，包含提供能够结合包含SEQ ID NO：1所示的DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
  53. 如权利要求16-52中任一项所述的方法，所述目标区域包含来源于人chr17:75369603-75369693定义的区域。
  54. 如权利要求1-53中任一项所述的方法，包含提供SEQ ID NO：29所示的核酸或其互补核酸、或上述的片段。
  55. 如权利要求1-54中任一项所述的方法，包含提供SEQ ID NO：30与31所示的核酸组或其互补核酸组、或上述的片段。
  56. 如权利要求16-55中任一项所述的方法，所述疾病包含肿瘤。
  57. 如权利要求16-56中任一项所述的方法，所述疾病包含肝肿瘤。
  58. 如权利要求1-57中任一项所述的方法，所述方法还包含获取待测样本中的核酸。
  59. 如权利要求58所述的方法，所述核酸包含无细胞游离核酸。
  60. 如权利要求1-59中任一项所述的方法，所述待测样本包含组织、细胞和/或体液。
  61. 如权利要求1-60中任一项所述的方法，所述待测样本包含血浆。
  62. 如权利要求1-61中任一项所述的方法，所述方法还包含转化所述DNA区域或其片段。
  63. 如权利要求62所述的方法，具有所述修饰状态的碱基以及不具有所述修饰状态的所述碱基，在转化后形成不同的物质。
  64. 如权利要求1-63中任一项所述的方法，具有所述修饰状态的碱基在转化后基本不发生改变，且不具有所述修饰状态的所述碱基在转化后改变为与所述碱基不同的其它碱基、或在转化后被剪切。
  65. 如权利要求63-64中任一项所述的方法，所述碱基包含胞嘧啶。
  66. 如权利要求1-65中任一项所述的方法，所述修饰状态包含甲基化修饰。
  67. 如权利要求64-66中任一项所述的方法，所述其它碱基包含尿嘧啶。
  68. 如权利要求62-67中任一项所述的方法，所述转化包含通过脱氨基试剂和/或甲基化敏感限制酶转化。
  69. 如权利要求68所述的方法，所述脱氨基试剂包含亚硫酸氢盐或其类似物。
  70. 如权利要求1-69中任一项所述的方法，所述确定修饰状态的存在和/或含量的方法包含，确认具有所述修饰状态的碱基在所述转化后形成的物质的存在和/或含量。
  71. 如权利要求1-70中任一项所述的方法，所述确定修饰状态的存在和/或含量的方法包含，确定具有所述修饰状态的DNA区域或其片段的存在和/或含量。
  72. 如权利要求1-71中任一项所述的方法，通过所述荧光PCR方法检测的荧光Ct值确定具有所述修饰状态的DNA区域或其片段的存在和/或含量。
  73. 如权利要求1-72中任一项所述的方法，通过确认所述DNA区域或其片段的修饰状态的存在和/或所述DNA区域或其片段相对于参考水平具有更高的修饰状态的含量，确定肝肿瘤的存在、或者有肝肿瘤形成或形成的风险。
  74. 如权利要求1-73中任一项所述的方法，所述方法还包含在确定所述DNA区域或其片段的修饰的存在和/或含量之前，扩增待测样本中所述DNA区域或其片段。
  75. 如权利要求74所述的方法，所述扩增包含PCR扩增。
  76. 一种核酸，所述核酸包含能够结合如权利要求1-15和58-75中任一项所述的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列；或者所述核酸包含能够结合如权利要求16-75中任一项所述的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。
  77. 一种制备核酸的方法，所述方法包含根据如权利要求1-15和58-75中任一项所述的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的 片段的修饰状态，设计能够结合所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸；或者所述方法包含根据如权利要求16-75中任一项所述的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸。
  78. 一种核酸组，所述核酸组包含能够结合如权利要求1-15和58-75中任一项所述的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列；或者所述核酸组包含能够结合如权利要求16-75中任一项所述的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的序列。
  79. 一种制备核酸组的方法，所述方法包含根据如权利要求1-15和58-75中任一项所述的方法中所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标基因所在DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸组；或者所述方法包含根据如权利要求16-75中任一项所述的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的修饰状态，设计能够结合所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸组。
  80. 一种试剂盒，包含如权利要求76所述的核酸和/或权利要求78所述的核酸组。
  81. 如权利要求76所述的核酸、如权利要求78所述的核酸组和/或权利要求80所述的试剂盒，在制备确定DNA区域或其片段的修饰状态的物质中的应用。
  82. 如权利要求76所述的核酸、如权利要求78所述的核酸组和/或权利要求80所述的试剂盒，在制备疾病检测产品中的应用。
  83. 如权利要求76所述的核酸、如权利要求78所述的核酸组和/或权利要求80所述的试剂盒，在制备确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用。
  84. 如权利要求82-83中任一项所述的应用，所述疾病包含肿瘤。
  85. 如权利要求82-84中任一项所述的应用，所述疾病包含肝肿瘤。
  86. 用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，在制备用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的物质中的应用，所述用于确定的DNA区域包含如权利要求1-15和58-75中任一项所述的方法中所述目标基因所在DNA区域或其片段。
  87. 用于确定DNA区域修饰状态的核酸、核酸组和/或试剂盒，在制备用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用，所述用于确定的DNA区域包含如权利要求16-75中任一项所述的方法中所述目标DNA区域、或其互补区域、或上述的片段。
  88. 如权利要求87所述的应用，所述疾病包含肿瘤。
  89. 如权利要求87-88中任一项所述的应用，所述疾病包含肝肿瘤。
  90. 如权利要求81和86-89中任一项所述的应用，所述修饰状态包含甲基化修饰。
  91. 如权利要求1-15和58-75中任一项所述的方法中所述目标基因所在DNA区域、或其转化而来的区域、或上述的片段的核酸或其组合，在制备用于确认肝肿瘤的存在、评估肝肿瘤形成或形成风险和/或评估肝肿瘤的进展的物质中的应用。
  92. 如权利要求16-75中任一项所述的方法中所述目标DNA区域、或其互补区域、或上述的转化而来的区域、或上述的片段的核酸或其组合，在制备用于确认疾病的存在、评估疾病形成或形成风险和/或评估疾病的进展的物质中的应用。
  93. 如权利要求92所述的应用，所述疾病包含肿瘤。
  94. 如权利要求92-93中任一项所述的应用，所述疾病包含肝肿瘤。
  95. 一种储存介质，其记载可以运行权利要求1-75中任一项所述的方法的程序。
  96. 一种设备，其包含权利要求95所述的储存介质。
  97. 如权利要求96所述的设备，还包含耦接至所述储存介质的处理器，所述处理器被配置为基于存储在所述储存介质中的程序执行以实现权利要求1-75中任一项所述的方法。
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