CN115572765B

CN115572765B - Tumor detection markers and application thereof

Info

Publication number: CN115572765B
Application number: CN202210321889.8A
Authority: CN
Inventors: 王辉; 苏志熙; 杨其昌; 刘轶颖; 周爽; 马成城; 刘蕊
Original assignee: Jiangsu Huayuan Biotechnology Co ltd; Jiangsu Fuyuan Biotechnology Co ltd
Current assignee: Jiangsu Huayuan Biotechnology Co ltd; Jiangsu Fuyuan Biotechnology Co ltd
Priority date: 2022-03-30
Filing date: 2022-03-30
Publication date: 2024-05-31
Anticipated expiration: 2042-03-30
Also published as: CN115572765A

Abstract

The application relates to a group of tumor detection markers and application thereof, in particular to a method for confirming the existence of liver tumor, evaluating the formation or formation risk of liver tumor and/or evaluating the progress of liver tumor, which comprises the step of evaluating the existence and/or the content of the modified state of a DNA region or a fragment thereof where a group of marker genes are located in a sample to be tested. The application also relates to nucleic acids, nucleic acid sets and/or kits for assessing the modification status of a set of DNA regions, and methods of making the same.

Description

Tumor detection markers and application thereof

Technical Field

The application relates to the field of biological medicine, in particular to a group of tumor detection markers and application thereof.

Background

Finding more efficient tumor markers has been an important direction in tumor-related studies, and liquid biopsy-based plasma free DNA detection techniques have placed a higher demand on the discovery of tumor markers. The basic principle is as follows: after death of tumor cells, free DNA is released into the blood, and extremely early tumor signals can be detected by detecting information related to tumor cells such as DNA mutation, DNA methylation, miRNA, histone modification and the like in the blood. It has been found that DNA methylation changes may occur earlier than DNA mutations, and are more effective detection markers for early screening of tumors.

However, in early cancers, few tumor cells released into the blood are present, and there is an urgent need in the art to find more effective detection markers for liver tumors. Therefore, there is a need to develop a method and/or kit that can efficiently read the epigenetic information from a very limited amount of extracellular free DNA in a biological sample by detecting accurate, stable, effective liver cancer biomarkers or a combination thereof, and preferably that can be easily configured and reliably applied in a hospital clinical laboratory.

Disclosure of Invention

The method provided by the application can be used for confirming or assisting in confirming the existence of diseases, evaluating the formation or formation risk of diseases and/or evaluating the progress of diseases, can improve the early screening and early diagnosis efficiency of tumors, such as liver tumors, and solves the problems of low early diagnosis rate of liver cancer, heavy clinical treatment burden and the like.

In one aspect, the application provides a method of confirming the presence of a liver tumor, assessing liver tumor formation or risk of formation and/or assessing liver tumor progression comprising determining the presence and/or amount of a modified state of a DNA region or fragment thereof of a target gene comprising CHFR and GRASP in a sample to be tested.

In another aspect, the application also provides a method for assessing methylation status of a liver tumor-associated DNA region, comprising determining the presence and/or amount of a modification status of a DNA region or fragment thereof of a target gene in a sample to be tested, said target gene comprising CHFR and GRASP.

In a further aspect, the application provides a method of confirming the presence of a disease, assessing the formation or risk of formation of a disease and/or assessing the progression of a disease comprising determining the presence and/or amount of a modified state of a region of target DNA, or a complementary region thereof, or a fragment thereof, in a sample to be tested, said region of target DNA comprising a region derived from human chr12:133483901-133485740 and a region derived from human chr12: 52400724-52401698.

In another aspect, the application provides a method of determining the methylation status of a DNA region comprising a region derived from human chr12:133483901-133485740 and from human chr12:52400724-52401698, comprising determining the presence and/or amount of a modified status of a target DNA region, or a complementary region or fragment thereof, in a sample to be tested.

In another aspect, the application provides a nucleic acid comprising a sequence capable of binding to a region of DNA in which the target gene is located, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, as in the method of the application; or the nucleic acid comprises a sequence capable of binding to the region of the target DNA, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, as in the method of the application.

In another aspect, the present application provides a method for preparing a nucleic acid, which comprises designing a nucleic acid capable of binding to a DNA region of a target gene, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the DNA region of the target gene, or a complementary region thereof, or the transformed region thereof, or the fragment thereof, as in the method of the present application; or the method comprises designing a nucleic acid capable of binding to the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, as in the method of the present application.

In another aspect, the application provides a nucleic acid set comprising a sequence capable of binding to a region of DNA in which the target gene is located, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, as in the methods of the application; or the nucleic acid set comprises a sequence capable of binding to the region of the target DNA, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, as in the method of the application.

In another aspect, the present application provides a method for preparing a nucleic acid set, the method comprising designing a nucleic acid set capable of binding to a DNA region of a target gene, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the DNA region of the target gene, or the complementary region thereof, or the transformed region thereof, or the fragment thereof, as in the method of the present application; or the method comprises designing a nucleic acid set capable of binding to the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, as in the method of the present application.

In another aspect, the application also provides a kit comprising a nucleic acid according to the application and/or a nucleic acid set according to the application.

In a further aspect, the application also provides the use of a nucleic acid according to the application, a nucleic acid set according to the application and/or a kit according to the application for the preparation of a substance for determining the modification state of a DNA region or a fragment thereof.

In a further aspect, the application also provides the use of a nucleic acid according to the application, a nucleic acid set according to the application and/or a kit according to the application for the preparation of a disease detection product.

In a further aspect, the application also provides the use of a nucleic acid according to the application, a nucleic acid set according to the application and/or a kit according to the application for the preparation of a substance for confirming the presence of a disease, assessing the formation or risk of formation of a disease and/or assessing the progression of a disease.

In a further aspect, the application provides the use of a nucleic acid, set of nucleic acids and/or kit for determining the modification status of a DNA region comprising a DNA region or fragment thereof in which a gene of interest is located as in the method of the application, for the preparation of a substance for determining the presence of a liver tumor, assessing the risk of liver tumor formation or formation and/or assessing the progression of a liver tumor.

In a further aspect, the application provides the use of a nucleic acid, set of nucleic acids and/or kit for determining the modification status of a DNA region comprising a DNA region of interest, or a complementary region thereof, or a fragment thereof, as described in the methods of the application, for the preparation of a substance for confirming the presence of a disease, assessing the risk of disease formation or formation and/or assessing the progression of a disease.

In a further aspect, the application provides the use of a nucleic acid or combination of fragments thereof, in a DNA region of a target gene, or a region transformed therefrom, or a fragment thereof, as described in the methods of the application, for the preparation of a substance for confirming the presence of a liver tumour, assessing liver tumour formation or risk of formation and/or assessing the progression of a liver tumour.

In a further aspect, the application provides the use of a nucleic acid or combination of the above-mentioned regions of target DNA, or complementary regions thereof, or the above-mentioned transformed regions, or the above-mentioned fragments, in the preparation of a substance for use in confirming the presence of a disease, assessing the risk of disease formation or formation and/or assessing the progression of a disease, as in the methods of the application.

In another aspect, the present application also provides a storage medium recording a program that can run the method of the present application.

In another aspect, the application also provides an apparatus comprising the storage medium of the application.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the descriptions in the specification of the present application are intended to be illustrative, and not limiting.

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

Definition of terms

In the present application, the term "CHFR" generally refers to a gene or an expression product thereof. For example, the UniProt accession number for the CHFR (Checkpoint With Forkhead AND RING FINGER Domains) protein may be Q96EP1. In the present application, CHFR may encompass its unprocessed form, any processed form, variants thereof or substances comprising functionally active fragments thereof. For example, CHFR in the context of the present application may also represent a detection site, for example methylation detection of a particular nucleic acid sequence of the gene. For example, the detection site represented by CHFR may be a nucleic acid sequence of one or more regions under the gene.

In the present application, the term "GRASP" generally refers to a gene or its expression product. For example, the UniProt accession number for the GRASP (GRP 1 (General receptor for phosphoinositides) 1) -associated scaffold protein) protein may be K7CHN5. In the present application, GRASP may encompass its unprocessed form, any processed form, variants thereof, or materials comprising functionally active fragments thereof. For example, GRASP in the context of the present application may also represent a detection site, such as methylation detection for a particular nucleic acid sequence of the gene. For example, the detection site represented by GRASP may be a nucleic acid sequence of one or more regions under the gene.

In the present application, the term "IRF4" generally refers to a gene or its expression product. For example, the UniProt accession number for IRF4 (Interferon regulatory factor 4) protein may be Q15306. In the present application IRF4 may encompass its unprocessed form, any processed form, variants thereof or substances comprising functionally active fragments thereof. For example, IRF4 in the present application may also represent a detection site, for example methylation detection of a specific nucleic acid sequence of the gene. For example, the detection site represented as IRF4 may be a nucleic acid sequence of one or more regions under the gene.

In the present application, the term "IKZF1" generally refers to a gene or its expression product. For example, the UniProt accession number for the IKZF1 (DNA-binding protein Ikaros) protein may be Q13422. In the present application IKZF1 may encompass its unprocessed form, any processed form, variants thereof or substances comprising functionally active fragments thereof. For example, IKZF1 in the context of the present application may also represent a detection site, for example methylation detection of a specific nucleic acid sequence of the gene. For example, the detection site represented by IKZF1 may be a nucleic acid sequence of one or more regions under the gene.

In the present application, the term "gene spacer 1" generally refers to a spacer region between genes. For example, gene spacer 1 (chr7: 26415938-26416740) of the present application can represent a detection site, e.g., methylation detection for a particular nucleic acid sequence of that region. For example, the detection site represented by gene spacer 1 (chr 7: 26415938-26416740) may be the nucleic acid sequence of one or more DNA regions within chr7: 26415938-26416740.

In the present application, the term "BCAT1" generally refers to a gene or its expression product. For example, the UniProt accession number for the BCAT1 (Branched-chain-amino-acid aminotransferase) protein may be P54687. In the present application BCAT1 can encompass its unprocessed form, any processed form, variants thereof or substances comprising functionally active fragments thereof. For example, BCAT1 in the present application may also represent a detection site, e.g. methylation detection for a specific nucleic acid sequence of the gene. For example, the detection site represented as BCAT1 may be a nucleic acid sequence of one or more regions under the gene.

In the present application, the term "sample to be tested" generally refers to a sample that is to be tested. For example, the presence or absence of a modification in one or more gene regions on a test sample can be detected.

In the present application, the term "cell-free nucleic acid" or "cfDNA" generally refers to DNA in a sample that is not contained within cells when collected. For example, cell-free nucleic acid may not refer to DNA that is rendered non-intracellular by in vitro disruption of cells or tissues. For example, cfDNA may include both normal cells and DNA derived from cancer cells. For example, cfDNA may be obtained from blood or plasma ("circulatory system"). For example, cfDNA may be released into the circulatory system by secretion or cell death processes, such as cell necrosis or apoptosis.

In the present application, the term "complementary nucleic acid" generally refers to a nucleotide sequence that is complementary to a reference nucleotide sequence. For example, the complementary nucleic acid may be a nucleic acid molecule, optionally with opposite orientation. For example, the complementation may refer to having the following complementation association: guanine and cytosine; adenine and thymine; adenine and uracil.

In the present application, the term "DNA region" generally refers to a sequence of two or more covalently bonded naturally occurring or modified deoxyribonucleotides. For example, a DNA region of a gene may refer to the location of a particular deoxyribonucleotide sequence at which the gene is located, e.g., the deoxyribonucleotide sequence encodes the gene. For example, the DNA region of the present application comprises the full length of the DNA region, its complementary region, or a fragment thereof. For example, a sequence of at least about 20kb upstream and downstream of the detection region provided by the present application may be used as the site of detection. For example, sequences at least about 20kb, at least about 15kb, at least about 10kb, at least about 5kb, at least about 3kb, at least about 2kb, at least about 1kb, or at least about 0.5kb upstream and downstream of the region provided by the present application may serve as the site of detection. For example, methylation detection of a sample can be performed by designing appropriate primers and probes based on the microcomputer.

In the present application, the term "modified state" generally refers to a modified state possessed by a gene fragment, a nucleotide or a base thereof in the present application. For example, the modified state in the present application may refer to a modified state of cytosine. For example, a gene fragment of the application having a modified state may have altered gene expression activity. For example, the modified form of the present application may refer to methylation modification of a base. For example, the modified form of the present application may refer to covalent attachment of a methyl group at the 5' carbon position of cytosine in a CpG region of genomic DNA, e.g., may be 5-methylcytosine (5 mC). For example, the modified state may refer to the presence or absence of 5-methylcytosine ("5-mCyt") within the DNA sequence.

In the present application, the term "methylation" generally refers to the methylation state of a gene fragment, a nucleotide or a base thereof of the present application. For example, a DNA fragment in which a gene of the application is located may have methylation on one or more strands. For example, a DNA fragment in which a gene of the present application is located may have methylation at one site or at multiple sites.

In the present application, the term "transformation" generally refers to the transformation of one or more structures into another structure. For example, the transformation of the application may be specific. For example, a cytosine having no methylation modification may be transformed into another structure (e.g., uracil), and a cytosine having methylation modification may be transformed substantially unchanged. For example, a cytosine having no methylation modification may be cleaved upon conversion, and a cytosine having methylation modification may be substantially unchanged upon conversion.

In the present application, the term "deaminating agent" generally refers to a substance having the ability to remove amino groups. For example, the deaminating agent can deaminate unmodified cytosine.

In the present application, the term "bisulfite" generally refers to an agent that can distinguish between regions of DNA having a modified state and regions of DNA not having a modified state. For example, the bisulphite may comprise bisulphite, or an analogue or combination thereof. For example, bisulphite can deaminate the amino group of an unmodified cytosine to distinguish it from a modified cytosine. In the present application, the term "analogue" generally refers to a substance having a similar structure and/or function. For example, the analogue of bisulphite may have a similar structure to bisulphite. For example, an analog of bisulfite may refer to an agent that can also distinguish between regions of DNA having a modified state and regions of DNA not having a modified state.

In the present application, the term "methylation sensitive restriction enzyme" generally refers to an enzyme that selectively digests nucleic acid according to the methylation state of its recognition site. For example, for a restriction enzyme that specifically cleaves only when the recognition site is unmethylated, cleavage may not occur, or with significantly reduced efficiency, when the recognition site is methylated. For restriction enzymes that cleave specifically when the recognition site is methylated, cleavage may not occur, or with significantly reduced efficiency, when the recognition site is unmethylated. For example, a methylation specific restriction enzyme can recognize a sequence that contains a CG dinucleotide (e.g., cgcg or cccggg).

In the present application, the term "tumor" generally refers to cells and/or tissues that exhibit at least partial loss of control during normal growth and/or development. For example, a common tumor or cancer cell may generally be one that has lost contact inhibition and may be invasive and/or have the ability to metastasize. For example, the neoplasm of the application may be benign or malignant.

In the present application, the term "progression" generally refers to the change of a disease from a less severe state to a more severe state. For example, tumor progression may include an increase in the number or severity of tumors, the extent of metastasis of cancer cells, the rate of growth or spread of cancer, and the like. For example, tumor progression may include the progression of such cancer from a less severe state to a stage of a more severe state, e.g., from stage I to stage II, from stage II to stage III, etc.

In the present application, the term "forming" generally refers to the occurrence of a lesion in an individual. For example, when a tumor forms, the individual may be diagnosed as a tumor patient.

In the present application, the term "fluorescent PCR" generally refers to a quantitative or semi-quantitative PCR technique. For example, PCR techniques that quantify the polymerase chain reaction in real time, quantify the polymerase chain reaction, or kinetic polymerase chain reaction can be used. For example, PCR amplification can be used and the amount of starting target nucleic acid can be quantitatively detected with the aid of an intercalating fluorescent dye or a sequence-specific probe, which can contain a fluorescent reporter that is detectable only upon hybridization with the target nucleic acid.

In the present application, the term "PCR amplification" generally refers to a polymerase chain amplification reaction. For example, the PCR amplification in the present application may comprise any polymerase chain amplification reaction currently known for DNA amplification.

In the present application, the term "fluorescence Ct value" generally refers to a measurement value that quantitatively or semi-quantitatively evaluates a target nucleic acid. For example, the number of cycles of amplification reaction that the fluorescent signal undergoes when reaching a set threshold value may be referred to.

Detailed Description

The application can find out a plurality of high-efficiency methylation markers related to liver tumors, improves the early screening and early diagnosing efficiency of the liver tumors, and solves the problems of low early diagnosing rate of liver cancer, heavy clinical treatment burden and the like. Meanwhile, the application provides a plurality of groups of efficient liver tumor methylation markers, and efficient marker combinations are held in parallel.

In one aspect, the application provides a method for confirming the presence of a liver tumor, assessing liver tumor formation or risk of formation and/or assessing liver tumor progression, which may comprise determining the presence and/or amount of a modified state of a DNA region or fragment thereof in which a target gene, which may comprise CHFR and GRASP, is located in a sample to be tested.

For example, the method of the present application may comprise confirming the presence or absence of a liver tumor according to the result of determining the presence and/or amount of the modified state of the DNA region or the fragment thereof of the target gene in the sample to be tested. For example, the method of the present application may comprise evaluating whether liver tumor formation is confirmed or not based on the determination of the presence and/or amount of the modified state of the DNA region or the fragment thereof of the target gene in the sample to be tested. For example, the method of the present application may comprise evaluating whether there is a risk of diagnosing liver tumor formation and/or the level of risk according to the determination result of the presence and/or the amount of the modified state of the DNA region or the fragment thereof of the target gene in the sample to be tested. For example, the method of the present application may comprise assessing the progression of a liver tumor based on the determination of the presence and/or amount of the modification state of the DNA region or fragment thereof of the target gene in the sample to be tested.

In another aspect, the present application provides a method for assessing methylation status of a liver tumor-associated DNA region, which may comprise determining the presence and/or amount of a modification of a DNA region or fragment thereof of a target gene in a test sample, said target gene may comprise CHFR and GRASP.

For example, the methylation state of the DNA region related to the liver tumor is evaluated according to the determination of the presence and/or the content of the modification state of the DNA region or the fragment thereof of the target gene in the sample to be tested. For example, a methylation state of a liver tumor-associated DNA region can refer to a confirmed presence of methylation of the DNA region or an increase in number relative to a reference level, which can be correlated with the occurrence of a liver tumor.

For example, the genes of the application can be described by their name and their chromosomal coordinates. For example, the chromosome coordinates may be consistent with the Hg19 version of the human genome database published in month 2 2009 (otherwise referred to as "Hg19 coordinates"). For example, the DNA region of the application may be derived from a region defined by Hg19 coordinates.

For example, in the method of the application, the DNA region of the CHFR may be derived from human chr12:133398773-133532890.

For example, in the method of the application, the DNA region of GRASP may be derived from human chr12:52400724-52409673.

For example, in the method of the present application, the target gene may further comprise a gene selected from the group consisting of: IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, GRASP, IRF a 4 and IKZF1. For example, in the methods of the application, the target gene may comprise CHFR, GRASP, IRF4, gene spacer 1 (chr 7: 26415938-26416740), and IKZF1. For example, in the methods of the application, the target gene may comprise CHFR, GRASP, IRF a 4, BCAT1, and IKZF1.

For example, in the method of the present application, the target gene may comprise at least 2 genes.

For example, in the method of the present application, the target gene may comprise 2 to 6 genes. For example, the target gene of the present application may comprise 2, 3, 4, 5, or 6 target genes provided by the present application. For example, for the same target gene, the application may select one or more regions of DNA in which the target gene is located. For example, in the methods of the application, the DNA region of IRF4 may be derived from human chr 6:391739-41447. For example, in the method of the application, the DNA region of IKZF1 may be derived from human chr7:50343720-50472799. For example, in the method of the application, the DNA region of gene spacer 1 (chr 7: 26415938-26416740) may be derived from human chr7:26415938-26416740. For example, in the method of the application, the DNA region of BCAT1 may be derived from human chr12:24964295-25102393.

For example, in the method of the present application, the target gene may comprise 2 kinds of genes selected from the group consisting of: CHFR, GRASP, IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR and GRASP.

For example, in the methods of the application, the target gene may comprise CHFR and IRF4.

For example, in the methods of the application, the target gene may comprise CHFR and IKZF1.

For example, in the methods of the application, the target gene may comprise CHFR and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the method of the present application, the target gene may comprise CHFR and BCAT1.

For example, in the methods of the application, the target gene may comprise GRASP and IRF4.

For example, in the methods of the application, the target gene may comprise GRASP and IKZF1.

For example, in the methods of the application, the target gene may comprise GRASP and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise GRASP and BCAT1.

For example, in the methods of the application, the target gene may comprise IRF4 and IKZF1.

For example, in the methods of the application, the target gene may comprise IRF4 and gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise IRF4 and BCAT1.

For example, in the methods of the application, the target gene may comprise IKZF1 and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise IKZF1 and BCAT1.

For example, in the method of the present application, the target gene may comprise the gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the method of the present application, the target gene may comprise 3 kinds of genes selected from the group consisting of: CHFR, GRASP, IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, GRASP, and IRF4.

For example, in the methods of the application, the target gene may comprise CHFR, GRASP, and IKZF1.

For example, in the methods of the application, the target gene may comprise CHFR, GRASP and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise CHFR, GRASP and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, IRF4, and IKZF1.

For example, in the methods of the application, the target gene may comprise CHFR, IRF4 and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the method of the present application, the target gene may comprise CHFR, IRF4 and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, IKZF1 and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise CHFR, IKZF1, and BCAT1.

For example, in the method of the present application, the target gene may comprise CHFR, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the methods of the application, the target gene may comprise GRASP, IRF4, and IKZF1.

For example, in the methods of the application, the target gene may comprise GRASP, IRF4, and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise GRASP, IRF4, and BCAT1.

For example, in the methods of the application, the target gene may comprise GRASP, IKZF1 and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise GRASP, IKZF1, and BCAT1.

For example, in the methods of the application, the target gene may comprise GRASP, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the methods of the application, the target gene may comprise IRF4, IKZF1 and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise IRF4, IKZF1, and BCAT1.

For example, in the methods of the application, the target gene may comprise IRF4, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the methods of the application, the target gene may comprise IKZF1, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the method of the present application, the target gene may comprise 4 kinds of genes selected from the group consisting of: CHFR, GRASP, IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, GRASP, IRF a4 and IKZF1.

For example, in the method of the application, the target gene may comprise CHFR, GRASP, IRF a 4 and gene spacer 1 (chr 7: 26415938-26416740).

For example, in the method of the present application, the target gene may comprise CHFR, GRASP, IRF a4 and BCAT1.

For example, in the method of the application, the target gene may comprise CHFR, GRASP, IKZF a1 and gene spacer 1 (chr 7: 26415938-26416740).

For example, in the method of the present application, the target gene may comprise CHFR, GRASP, IKZF a1 and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, GRASP, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, IRF4, IKZF1 and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise CHFR, IRF4, IKZF1, and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, IRF4, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, IKZF1, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the methods of the application, the target gene may comprise GRASP, IRF4, IKZF1 and Gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise GRASP, IRF4, IKZF1, and BCAT1.

For example, in the methods of the application, the target gene may comprise GRASP, IRF4, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the methods of the application, the target gene may comprise GRASP, IKZF1, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the methods of the application, the target gene may comprise IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the method of the present application, the target gene may comprise 5 kinds of genes selected from the group consisting of: CHFR, GRASP, IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, GRASP, IRF a 4, IKZF1 and gene spacer 1 (chr 7: 26415938-26416740).

For example, in the methods of the application, the target gene may comprise CHFR, GRASP, IRF a 4, IKZF1, and BCAT1.

For example, in the method of the present application, the target gene may comprise CHFR, GRASP, IRF4, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the methods of the application, the target gene may comprise GRASP, IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

For example, in the method of the present application, the target gene may comprise 6 kinds of genes selected from the group consisting of: CHFR, GRASP, IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, in the methods of the application, the target gene may comprise CHFR, GRASP, IRF a4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740) and BCAT1.

For example, the target DNA region located in the DNA region of the CHFR gene in the method of the application may comprise a region derived from the human chr12:133483901-133485740 definition. For example from the region defined by human chr12: 133485000-133485067. For example, the target DNA region may be represented by CHFR.

For example, the target DNA region located in the DNA region of the GRASP gene in the method of the application may comprise a region derived from the human chr12:52400724-52401698 definition. For example from the region defined by human chr12: 52401083-52401169. For example, the target DNA region may be represented by GRASP.

For example, the target DNA region located in the DNA region of the IRF4 gene in the method of the application may comprise a region derived from the human chr6:391739-394056 definition. For example from the region defined by human chr6:392282-392377. For example, the target DNA region may be represented by IRF 4.

For example, the target DNA region located in the DNA region of the IKZF1 gene in the method of the application may comprise a region derived from the human chr7:50343720-50344547 definition. For example from the region defined by human chr7: 50343793-50343896. For example, the target DNA region may be represented by IKZF 1.

For example, the target DNA region of the method of the application which is located in the DNA region of the gene spacer 1 (chr 7: 26415938-26416740) may comprise a region derived from the human chr7:26415938-26416740 definition. For example from the region defined by human chr7: 26416257-26416363. For example, the target DNA region may be represented by gene spacer 1.

For example, the target DNA region located in the DNA region of the BCAT1 gene in the method of the present application may comprise a region derived from the human chr12:25101630-25102393 definition. For example from the region defined by human chr12: 25102016-25102110. For example, the target DNA region can be represented by BCAT 1.

In another aspect, the application provides a method for confirming the presence of a disease, assessing the risk of disease formation or formation and/or assessing the progression of a disease, which may comprise determining the presence and/or amount of a modification of a specific sub-region of a DNA region of interest (e.g. a region of interest) or a complementary region thereof, or a fragment thereof, in a sample to be tested.

In another aspect, the application provides a method of confirming the presence of a disease, assessing the formation or risk of developing a disease and/or assessing the progression of a disease, which may comprise determining the presence and/or amount of a modified state of a region of target DNA, or a complementary region thereof, or a fragment thereof, in a test sample, which may comprise a region derived from human chr12:133483901-133485740 and a region derived from human chr12: 52400724-52401698. For example, the method of the present application may comprise confirming the presence or absence of a disease based on the determination of the presence and/or amount of the modified state of the target DNA region, or a complementary region thereof, or a fragment thereof in the sample to be tested. For example, the method of the present application may comprise evaluating whether or not the diagnosis of disease formation is established based on the determination of the presence and/or amount of the modified state of the target DNA region, or the complementary region thereof, or the fragment thereof in the sample to be tested. For example, the method of the present application may comprise evaluating whether there is a risk of diagnosing a disease and/or the level of risk according to the determination result of the presence and/or the amount of the modified state of the target DNA region, or the complementary region thereof, or the fragment thereof in the sample to be tested. For example, the method of the present application may comprise assessing the progression of the disease based on the determination of the presence and/or amount of the modified state of the target DNA region, or a complementary region thereof, or a fragment thereof, in the sample to be tested.

In another aspect, the application provides a method of determining the methylation status of a DNA region, which may comprise a region derived from human chr12:133483901-133485740 and a region derived from human chr12:52400724-52401698, comprising determining the presence and/or amount of a modified status of a target DNA region, or a complementary region or fragment thereof, in a sample to be tested.

For example, the presence of a confirmed methylation of the target DNA region or an increased number relative to a reference level can be correlated with the occurrence of a disease. For example, a region of a target DNA of the present application may refer to one or more specific segments of the target genomic DNA. For example, the target DNA region of the present application may be specified by a gene name or a set of chromosome coordinates. For example, the target DNA region represented by CHFR may be one or more specific segments of the DNA region in which the CHFR gene is located. For example, the target DNA region represented by CHFR may be one or more specific segments of the target DNA region in the CHFR gene provided herein. For example, one or more different specific segments of the CHFR may be represented separately by distinguishable forms of CHFR (1) and CHFR (1 a) or the like.

For example, a gene may obtain its sequence and chromosomal location by reference to its name, or determine its sequence and chromosomal location by reference to its chromosomal coordinates. The present application uses these specific DNA region methylation states as a series of analytical indicators, can provide significant improvements in sensitivity and/or specificity, and can simplify the screening process. For example, "sensitivity" may refer to the proportion of positive results that are correctly identified, i.e., the percentage of individuals who are correctly identified as having the disease in question; "specificity" may refer to the proportion of negative results that are correctly identified, i.e., the percentage of individuals that are correctly identified as not having the disease in question.

The DNA regions of the application may comprise all forms of these molecules, as well as fragments or variants thereof. For example, a variant may comprise at least 80%, at least 85%, at least 90%, 95%, 98%, or 99% sequence identity relative to a DNA region described herein, and a variant may comprise one or more deletions, additions, substitutions, inverted sequences, and the like. For example, the modified status of the variants of the application may achieve the same evaluation result. The DNA region of the application may comprise all forms of any other mutation, polymorphic variation or allelic variation.

For example, the methods of the application may comprise providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:1, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

For example, in the method of the application, the target region may comprise a region derived from the human chr12:133485000-133485067 definition. For example, the target DNA region may be represented by CHFR.

For example, the methods of the application may comprise providing SEQ ID NO:2 or a complementary nucleic acid thereof, or a fragment thereof. For example, the nucleic acid may be used to detect a target region. For example, the nucleic acid may be used as a probe.

For example, the methods of the application may comprise providing SEQ ID NO:3 and 4, or a complementary nucleic acid set thereof, or a fragment thereof. For example, the nucleic acid set may be used to amplify a target region. For example, the nucleic acid set may be a primer set.

For example, the above-described probe and/or primer set may detect and/or amplify a region of DNA derived from a region defined by human chr12: 133485000-133485067.

For example, the methods of the application may comprise providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:5, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

For example, in the method of the application, the target region may comprise a region derived from the human chr12:52401083-52401169 definition. For example, the target DNA region may be represented by GRASP.

For example, the methods of the application may comprise providing SEQ ID NO:6 or a complementary nucleic acid thereof, or a fragment thereof. For example, the nucleic acid may be used to detect a target region. For example, the nucleic acid may be used as a probe.

For example, the methods of the application may comprise providing SEQ ID NO:7 and 8 or a complementary nucleic acid set thereof, or a fragment thereof. For example, the nucleic acid set may be used to amplify a target region. For example, the nucleic acid set may be a primer set.

For example, the above-described probe and/or primer set may detect and/or amplify a region of DNA derived from a region defined by human chr12: 52401083-52401169.

For example, in the method of the application, the target DNA region may further comprise a region defined from the group consisting of: derived from human chr6:391739-394056, derived from human chr7:50343720-50344547, derived from human chr7:26415938-26416740, and derived from human chr12:25101630-25102393.

For example, in the method of the application, the target DNA region may comprise at least 2 regions.

For example, in the method of the present application, the target DNA region may comprise 2 to 6 regions. For example, in the methods of the application, the target DNA region may comprise 2,3, 4, 5, or 6 regions.

For example, in the method of the application, the target DNA region may comprise a region defined by human chr12:133483901-133485740, human chr12:52400724-52401698, human chr6:391739-394056 and human chr7: 50343720-50344547.

For example, in the method of the application, the target DNA region may comprise a region defined by human chr12:133483901-133485740, human chr12:52400724-52401698, human chr6:391739-394056, human chr7:26415938-26416740 and human chr7: 50343720-50344547.

For example, in the method of the application, the target DNA region may comprise a region defined by human chr12:25101630-25102393, human chr12:52400724-52401698, human chr6:391739-394056, human chr7:50343720-50344547, and human chr12: 133483901-133485740.

For example, the methods of the application may comprise providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:9, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

For example, in the method of the application, the target region may comprise a region derived from the human chr6:392282-392377 definition. For example, the target DNA region may be represented by IRF 4.

For example, the methods of the application may comprise providing SEQ ID NO:10 or a complementary nucleic acid thereof, or a fragment thereof. For example, the nucleic acid may be used to detect a target region. For example, the nucleic acid may be used as a probe.

For example, the methods of the application may comprise providing SEQ ID NO:11 and 12 or a complementary nucleic acid set thereof, or a fragment thereof. For example, the nucleic acid set may be used to amplify a target region. For example, the nucleic acid set may be a primer set.

For example, the above-described probe and/or primer set may detect and/or amplify a DNA region derived from a region defined by human chr6:392282-392377.

For example, the methods of the application may comprise providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:13, or a complementary region thereof, or a region obtained by transformation as described above, or a fragment thereof.

For example, in the method of the application, the target region may comprise a region derived from the human chr7:50343793-50343896 definition. For example, the target DNA region may be represented by IKZF 1.

For example, the methods of the application may comprise providing SEQ ID NO:14 or a complementary nucleic acid thereof, or a fragment thereof. For example, the nucleic acid may be used to detect a target region. For example, the nucleic acid may be used as a probe.

For example, the methods of the application may comprise providing SEQ ID NO:15 and 16 or a complementary nucleic acid set thereof, or a fragment thereof. For example, the nucleic acid set may be used to amplify a target region. For example, the nucleic acid set may be a primer set.

For example, the above-described probe and/or primer set may detect and/or amplify a region of DNA derived from a region defined by human chr7: 50343793-50343896.

For example, the methods of the application may comprise providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:17, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

For example, in the method of the application, the target region may comprise a region derived from the human chr7:26416257-26416363 definition. For example, the target DNA region may be represented by gene spacer 1.

For example, the methods of the application may comprise providing SEQ ID NO:18 or a complementary nucleic acid thereof, or a fragment thereof. For example, the nucleic acid may be used to detect a target region. For example, the nucleic acid may be used as a probe.

For example, the methods of the application may comprise providing SEQ ID NO:19 and 20 or a complementary nucleic acid set thereof, or a fragment thereof. For example, the nucleic acid set may be used to amplify a target region. For example, the nucleic acid set may be a primer set.

For example, the above-described probe and/or primer set may detect and/or amplify a region of DNA derived from a region defined by human chr7: 26416257-26416363.

For example, the methods of the application may comprise providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:21, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

For example, in the method of the application, the target region may comprise a region derived from the human chr12:25102016-25102110 definition. For example, the target DNA region can be represented by BCAT 1.

For example, the methods of the application may comprise providing SEQ ID NO:22 or a complementary nucleic acid thereof, or a fragment thereof. For example, the nucleic acid may be used to detect a target region. For example, the nucleic acid may be used as a probe.

For example, the methods of the application may comprise providing SEQ ID NO:23 and 24 or a complementary nucleic acid set thereof, or a fragment thereof. For example, the nucleic acid set may be used to amplify a target region. For example, the nucleic acid set may be a primer set.

For example, the above-described probe and/or primer set may detect and/or amplify a region of DNA derived from a region defined by human chr12: 25102016-25102110.

For example, one or more of the above-described target regions may serve as an amplification region and/or a detection region.

For example, the disease may comprise a tumor. For example, the disease may comprise a solid tumor. For example, the disease may include any tumor such as a liver tumor.

For example, a "primer" may be a natural or synthetic oligonucleotide that, upon formation of a duplex with a polynucleotide template, is capable of acting as a point of initiation of nucleic acid synthesis and extending along the template from its 3' end, thereby forming an extended duplex. The sequence of the nucleotides added during extension is determined by the sequence of the template polynucleotide. Primers can typically be extended by a polymerase such as a nucleic acid polymerase.

For example, "complementary" and "substantially complementary" of the present application may include hybridization or base pairing or duplex formation between nucleotides or nucleic acids, e.g., between two strands of a double-stranded DNA molecule, or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid. The complementary nucleotides may generally be A and T (or A and U) or C and G. For two single stranded RNA or DNA molecules, one strand of nucleotides can be considered substantially complementary when they are paired with at least about 80% (typically at least about 90% to about 95%, even about 98% to about 100%) of the other strand when optimally aligned and compared and having the appropriate nucleotide insertions or deletions. In one aspect, two complementary nucleotide sequences are capable of hybridizing and may have less than 25% mismatches between the opposite nucleotides, more may have less than 15% mismatches, may have less than 5% mismatches, or no mismatches. For example, two molecules may hybridize under high stringency conditions.

For example, a modified state of the application may refer to the presence, absence and/or amount of the modified state at a particular nucleotide or nucleotides within a DNA region. For example, the modified state of the present application may refer to the modified state of each base or each specific base (e.g., cytosine) in a specific DNA sequence. For example, the modified state of the present application may refer to a base pair combination and/or a modified state of a base combination in a particular DNA sequence. For example, the modification status of the present application may refer to information about the density of region modifications in a particular DNA sequence (including the DNA region in which the gene is located or a particular region fragment thereof), and may not provide precise positional information of where in the sequence the modification occurs.

For example, the modified state of the application may be a nail methylation state or a state similar to methylation. For example, a state with or with higher methylation may be associated with transcriptional silencing of a particular region. For example, a state with or with higher methylation may be associated with being able to be converted by a methylation specific conversion reagent (e.g., deamination reagent and/or methylation sensitive restriction enzyme). For example, conversion may refer to being converted to another substance and/or being sheared or digested.

For example, "methylation state (methylation state)" or "methylation state (methylation status)" may refer to the presence or absence of 5-methylcytosine ("5-mC" or "5-mCyt") at one or more CpG dinucleotides within a DNA sequence. Methylation states at one or more specific CpG methylation sites (each having two CpG dinucleotide sequences) within the DNA sequence include "unmethylated", "fully methylated" and "semi-methylated". The term "hemimethylation" may refer to the methylation state of double stranded DNA, wherein only one strand of the double stranded DNA is methylated. The term "hypermethylation" may refer to an average methylation state corresponding to: the presence of 5-mCyt at one or more CpG dinucleotides in the DNA sequence of the test DNA sample is increased relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides in the normal control DNA sample. The term "hypomethylation" may refer to an average methylation state corresponding to: the presence of 5-mCyt at one or more CpG dinucleotides in the DNA sequence of the test DNA sample is reduced relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides in the normal control DNA sample.

For example, the method may further comprise obtaining nucleic acid in the sample to be tested. For example, the nucleic acid may comprise cell-free nucleic acid. For example, the sample to be tested may comprise tissue, cells and/or body fluids. For example, the sample to be tested may comprise plasma. For example, the detection methods of the present application may be performed on any suitable biological sample. For example, the sample to be tested may be any sample of biological material, e.g., it may be derived from an animal, but is not limited to cellular material, biological fluids (e.g., blood), effluents, tissue biopsy specimens, surgical specimens, or fluids that have been introduced into the animal's body and subsequently removed. For example, the sample to be tested of the present application may comprise a sample that has been processed in any form after separation of the sample.

For example, the method may further comprise transforming the DNA region or fragment thereof. For example, by the conversion step of the present application, a base having the modified state and a base not having the modified state can be formed into different substances after conversion. For example, a base having the modified state does not substantially change after conversion, and the base not having the modified state may change to another base different from the base after conversion (e.g., the other base may comprise uracil), or be sheared after conversion. For example, the base may comprise cytosine. For example, the modification state may comprise a methylation modification. For example, the transformation may comprise transformation by deamination reagents and/or methylation sensitive restriction enzymes. For example, the deaminating agent may comprise bisulphite or an analogue thereof. For example sodium or potassium hydrogen sulphite.

For example, the method may optionally further comprise amplifying the DNA region or fragment thereof in the test sample prior to determining the presence and/or amount of modification of the DNA region or fragment thereof. For example, the amplification may comprise PCR amplification. For example, the amplification of the present application may comprise any known amplification system. For example, the amplification step of the application may be optional. For example, "amplification" may refer to the process of producing multiple copies of a desired sequence. "multiple copies" may refer to at least two copies. "copy" may not mean perfect sequence complementarity or identity with the template sequence. For example, copies may include nucleotide analogs such as deoxyinosine, intentional sequence alterations (e.g., introduced by primers that include sequences that hybridize to the template but are not complementary), and/or sequence errors may occur during amplification.

For example, the method of determining the presence and/or amount of a modification state may comprise confirming the presence and/or amount of a substance formed after the conversion of a base having the modification state. For example, the method of determining the presence and/or amount of a modification state may comprise determining the presence and/or amount of a DNA region or fragment thereof having the modification state. For example, the presence and/or amount of a DNA region or fragment thereof having the modified state may be directly detected. For example, detection may be by: the DNA region or fragment thereof having the modified state may have different properties during a reaction (e.g., an amplification reaction) than the DNA region or fragment thereof not having the modified state. For example, in a fluorescent PCR method, a DNA region or a fragment thereof having the modified state may be specifically amplified and fluoresced; the DNA region or fragment thereof not having the modified state may be substantially unamplified and substantially non-fluorescent. For example, alternative methods of determining the presence and/or amount of a substance formed after the conversion of a base having the modified state may be included within the scope of the present application.

For example, the presence and/or amount of a DNA region or fragment thereof having the modified state can be determined by the fluorescence Ct value detected by the fluorescence PCR method. For example, the presence of, or the risk of liver tumor formation or formation may be determined by the presence of a modified state of said DNA region or fragment thereof and/or the content of said DNA region or fragment thereof having a higher modified state relative to a reference level. For example, when the fluorescence Ct value of the test sample is lower relative to the reference fluorescence Ct value, the presence of the modified state of the DNA region or fragment thereof may be determined and/or the content of the modified state of the DNA region or fragment thereof may be determined to be higher than the content of the modified state in the reference sample. For example, the reference fluorescence Ct value may be determined by detecting a reference sample. For example, the presence of a modified state of the DNA region or fragment thereof may not be excluded when the fluorescence Ct value of the test sample is higher or substantially equivalent relative to a reference fluorescence Ct value; when the fluorescence Ct value of the test sample is higher or substantially equivalent relative to the reference fluorescence Ct value, it can be confirmed that the modified state content of the DNA region or fragment thereof is lower than or substantially equal to the modified state content in the reference sample.

For example, the present application may indicate the presence and/or amount of a modification state of a particular DNA region or fragment thereof by a cycle threshold (i.e., ct value), e.g., including the methylation level and reference level of a test sample. For example, the Ct value may refer to the number of cycles over which fluorescence of the PCR product can be detected above the background signal. For example, the Ct value may be inversely related to the initial amount of the target marker in the sample, i.e., the lower the Ct value, the greater the amount of modification of the DNA region or fragment thereof in the sample to be tested.

For example, a test sample may be identified as having a particular disease, diagnosed as forming a particular disease, or having a risk of forming a particular disease, or assessed as having some progression of a particular disease when the Ct value of the test sample is the same or lower relative to its corresponding reference Ct value. For example, a particular disease is identified as present, diagnosed as forming of a particular disease, or at risk of forming, or assessed as some progression of a particular disease, when the Ct value of a test sample 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.

For example, when the Ct value of a cell sample, tissue sample, or sample derived from a subject is the same or higher relative to its corresponding reference Ct value, it may be confirmed that a particular disease is not present, diagnosed as forming of a particular disease, or has a risk of forming, or is assessed as some progression of a particular disease. For example, when 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, or at least 50 cycles higher relative to its corresponding reference Ct value, it may be confirmed that a particular disease is not present, diagnosed with or at risk of developing, or assessed as a certain progression of a particular disease. For example, when the Ct values of a cell sample, a tissue sample, or a sample derived from a subject are disease-identical with respect to their respective reference Ct values, the presence or absence of a particular disease, the diagnosis of the formation or absence of a particular disease, the presence or absence of a risk of formation, or the assessment of some progression of a particular disease may be confirmed, and at the same time advice may be given that further detection is required.

For example, a reference level or control level of the application may refer to a normal level or a healthy level. For example, the normal level may be a modified status level of a sample DNA region derived from a cell, tissue or individual without the disease. For example, when used in the assessment of a tumor, the normal level may be the level of modification of a sample DNA region derived from a cell, tissue or individual that is tumor-free. For example, when used in the assessment of liver tumors, the normal level may be the modified status level of a sample DNA region derived from cells, tissues or individuals without liver tumors.

For example, a reference level in the present application may refer to a threshold level that identifies a subject or sample as being in the presence or absence of a particular disease. For example, a reference level in the present application may refer to a threshold level that diagnoses a subject as developing or at risk of developing a particular disease. For example, a reference level in the present application may refer to a threshold level of progression of a subject to a particular disease. For example, when the modification state of a DNA region in a cell sample, tissue sample or sample derived from a subject is higher than or substantially equal to a corresponding reference level, for example, a reference level herein may refer to a modification state of a DNA region of a patient not having a particular disease, may be identified as having a particular disease, diagnosed as forming a particular disease, or having a risk of forming, or assessed as having a certain progression of a particular disease. For example, a and B in the present application "substantially equal to" 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. For example, a particular disease is identified as present, diagnosed as forming of the particular disease or having a risk of forming or being assessed as a certain progression of the particular disease when the modified 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 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold higher than a corresponding reference level. For example, a cell sample, tissue sample, or sample derived from a subject may be identified as having, diagnosed with, or at risk of developing, or assessed as having a certain progression of a particular disease when the modified status of the DNA region in at least one, at least two, or at least three of the multiple assays is at least 1%, at least 5%, at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold higher than the corresponding reference level.

For example, when the modification state of a DNA region in a cell sample, tissue sample or sample derived from a subject is lower than or substantially equal to a corresponding reference level, for example, a reference level herein may refer to a modification state of a DNA region of a patient with a particular disease, may be confirmed as absent, diagnosed as forming of a particular disease, or having a risk of forming, or assessed as some progression of a particular disease. For example, a cell sample, tissue sample, or sample derived from a subject may be identified as being absent, diagnosed with, or at risk of developing, or assessed as having a certain progression of a particular disease when the modified status of the DNA region in the sample is at least 1%, at least 5%, at least 10%, at least 20%, at least 50%, at least 100% below the corresponding reference level.

The reference level can be selected by one skilled in the art depending on the desired sensitivity and specificity. For example, the reference level in each case in the present application may be readily identifiable by a person skilled in the art, such as by a limited number of attempts to identify a suitable reference level and/or a suitable means to obtain a reference level, e.g. the reference level may be derived from one or more reference samples, wherein the reference level is obtained from an experiment performed in parallel with the experiment to detect the sample of interest. Alternatively, the reference level may be obtained in a database comprising a collection of data, standards or levels from one or more reference samples or disease reference samples. In some embodiments, the set of data, criteria, or levels may be normalized or normalized so as to be available for comparison with data from one or more samples, thereby to reduce errors generated under different detection conditions.

For example, the reference level may be derived from a database, which may be a reference database, e.g., including the level of modification status of target markers and/or other laboratory and clinical data from one or more reference samples. For example, a reference database may be established by aggregating reference level data obtained from reference samples of healthy individuals and/or individuals other than the respective disease patient (i.e., individuals known to be free of the disease). For example, a reference database may be established by aggregating reference level data obtained from reference samples of individuals suffering from the respective disease who are undergoing treatment. For example, a reference database may be established by aggregating data obtained from reference samples of individuals at different stages of the disease. For example, the different phases may be evidenced by different levels of modification states of the target markers of the application. One skilled in the art can also determine whether an individual is suffering from or at risk of suffering from a corresponding disease based on various factors, such as age, sex, medical history, family history, symptoms, and the like.

For example, the method of the present application may comprise the steps of: obtaining nucleic acid in a sample to be detected; transforming the DNA region or fragment thereof; confirming the presence and/or amount of a substance formed after the conversion of the base having the modified state.

For example, the method of the present application may comprise the steps of: obtaining nucleic acid in a sample to be detected; transforming the DNA region or fragment thereof; amplifying the DNA region or the fragment thereof in the sample to be detected; confirming the presence and/or amount of a substance formed after the conversion of the base having the modified state.

For example, the method of the present application may comprise the steps of: obtaining nucleic acid in a sample to be detected; treating DNA obtained from a sample to be tested with a reagent capable of distinguishing between unmethylated and methylated sites in the DNA, thereby obtaining treated DNA; optionally amplifying the DNA region or fragment thereof in the sample to be tested; quantitatively, semi-quantitatively or qualitatively analyzing the presence and/or amount of methylation status of the treated DNA in the test sample; comparing the methylation level of the treated DNA in the test sample with a corresponding reference level, when the methylation state of the DNA region in the test sample is higher than or substantially equal to the corresponding reference level, it can be confirmed that a specific disease is present, diagnosed as forming a specific disease or is at risk of forming or is assessed as some progression of a specific disease.

In another aspect, the application provides a nucleic acid which may comprise a sequence capable of binding to the region of DNA in which the gene of interest of the application is located, or to the region complementary thereto, or to the region transformed therefrom, or to a fragment thereof. For example, the nucleic acid may be any of the probes of the present application. In another aspect, the present application provides a method for preparing a nucleic acid, which may comprise designing a nucleic acid capable of binding to a DNA region of the target gene of the present application, or a complementary region thereof, or a transformed region thereof, or a modified state of a fragment thereof according to the present application. For example, the method of preparing the nucleic acid may be any suitable method known in the art.

In another aspect, the application provides a nucleic acid set comprising a sequence capable of binding to a region of DNA in which a gene of interest of the application is located, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof. For example, the nucleic acid set may be any of the primer sets of the present application. In another aspect, the present application provides a method for preparing a nucleic acid set, which comprises designing a nucleic acid set capable of amplifying a DNA region, a complementary region thereof, a transformed region thereof, or a fragment thereof according to the modified state of the DNA region, the complementary region thereof, the transformed region thereof, or the fragment thereof, in which the target gene is located. For example, the method of preparing a nucleic acid in a nucleic acid set may be any suitable method known in the art. For example, the methylation state of a target polynucleotide can be assessed using a single probe or primer configured to hybridize to the target polynucleotide. For example, the methylation state of a target polynucleotide can be assessed using a plurality of probes or primers configured to hybridize to the target polynucleotide.

In another aspect, the application provides a nucleic acid comprising a sequence capable of binding to the region of DNA in which the target gene is located, or the complement thereof, or the transformed region described above, or a fragment of the above, in a method according to the application; in another aspect, the application provides a nucleic acid which may comprise a sequence capable of binding to the region of the target DNA, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, in a method according to the application.

In another aspect, the present application provides a method for preparing a nucleic acid, which comprises designing a nucleic acid capable of binding to a DNA region of a target gene, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the DNA region of the target gene, or the complementary region thereof, or the transformed region thereof, or the fragment thereof, in the method of the present application; in another aspect, the present application provides a nucleic acid which may comprise a nucleic acid designed to bind to the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, in the method of the present application.

In another aspect, the application provides a nucleic acid set comprising a sequence capable of binding to the region of DNA in which the target gene is located, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, in a method according to the application; in another aspect, the application provides a nucleic acid which may comprise a sequence capable of binding to the region of the target DNA, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, in a method according to the application. For example, the method of preparing the nucleic acid may be any suitable method known in the art.

In another aspect, the present application provides a method for preparing a nucleic acid set, which comprises designing a nucleic acid set capable of binding to a DNA region of a target gene, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the DNA region of the target gene, or the complementary region thereof, or the transformed region thereof, or the fragment thereof, in the method of the present application; in another aspect, the present application provides a nucleic acid, which may comprise designing a nucleic acid set capable of binding to the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, in the method of the present application. For example, the methylation state of a target polynucleotide can be assessed using a single probe or primer configured to hybridize to the target polynucleotide. For example, the methylation state of a target polynucleotide can be assessed using a plurality of probes or primers configured to hybridize to the target polynucleotide.

In another aspect, the application provides a kit, which may comprise a nucleic acid of the application and/or a nucleic acid set of the application. For example, the kits of the application may optionally comprise a reference sample for a corresponding use or provide a reference level for a corresponding use.

Diagnostic method, preparation and use

For example, the methods of the application can be used for diagnosis, prognosis, stratification, risk assessment or therapy monitoring of cancer or neoplasia in a subject. For example, a "subject" may refer to an organism or a portion or component of an organism to which the methods, nucleic acids, nucleic acid sets, kits, devices and systems provided herein may be administered or applied. For example, the subject may be a mammal or a cell, tissue, organ or portion of the mammal. As used herein, "mammal" refers to any kind of mammal, preferably a human (including a human, a human subject, or a human patient). Subjects and mammals include, but are not limited to, farm animals, sports animals, pets, primates, horses, dogs, cats, and rodents such as mice and rats.

In another aspect, the methods of the application can be used to assess cancer or tumor formation in any suitable subject. For example, the methods of the application 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. Preferably, the mammal is a human. For example, the subject may be a person in need of cancer or neoplasia risk screening, a person in a high risk group, a person diagnosed with cancer or neoplasia but in need of further stratification or stratification, a person diagnosed with cancer or neoplasia and undergoing active treatment, or a person with cancer or neoplasia and alleviating.

In a further aspect, the application provides the use of a nucleic acid according to the application, a nucleic acid set according to the application and/or a kit according to the application for the preparation of a substance for determining the modification state of a DNA region or a fragment thereof.

In another aspect, the application provides a method of determining the modification status of a region of said DNA or fragment thereof, which may comprise providing a nucleic acid of the application, e.g. a nucleic acid set of the application and/or a kit of the application.

In another aspect, the application provides a nucleic acid as in the application, a nucleic acid set as in the application and/or a kit as in the application, which can be used to determine the modified status of the DNA region or fragment thereof.

In a further aspect, the application provides the use of a nucleic acid according to the application, a nucleic acid set according to the application and/or a kit according to the application for the preparation of a disease detection product. For example, in the application of the present application, the disease may comprise a tumor. Such as a solid tumor. For example, in the application of the present application, the disease may comprise a liver tumor.

In another aspect, the application provides a method of detecting a disease, which may comprise providing a nucleic acid of the application, e.g. a nucleic acid set of the application and/or a kit of the application. For example, in the application of the present application, the disease may comprise a tumor. Such as a solid tumor. For example, in the application of the present application, the disease may comprise a liver tumor.

In another aspect, the application provides a nucleic acid as in the application, a set of nucleic acids as in the application and/or a kit as in the application, which can be used for disease detection. For example, in the application of the present application, the disease may comprise a tumor. Such as a solid tumor. For example, in the application of the present application, the disease may comprise a liver tumor.

In a further aspect, the application provides the use of a nucleic acid according to the application, a nucleic acid set according to the application and/or a kit according to the application for the preparation of a substance for confirming the presence of a disease, assessing the formation or risk of formation of a disease and/or assessing the progression of a disease. For example, in the application of the present application, the disease may comprise a tumor. Such as a solid tumor. For example, in the application of the present application, the disease may comprise a liver tumor.

In another aspect, the application provides a method of confirming the presence of a disease, assessing the formation or risk of formation of a disease and/or assessing the progression of a disease, which may comprise providing a nucleic acid of the application, e.g. a nucleic acid set of the application and/or a kit of the application. For example, in the application of the present application, the disease may comprise a tumor. Such as a solid tumor. For example, in the application of the present application, the disease may comprise a liver tumor.

In another aspect, the application provides a nucleic acid as in the application, a nucleic acid set as in the application and/or a kit as in the application, which can be used to confirm the presence of a disease, assess the risk of disease formation or formation and/or assess the progression of a disease. For example, in the application of the present application, the disease may comprise a tumor. Such as a solid tumor. For example, in the application of the present application, the disease may comprise a liver tumor.

In a further aspect, the present application provides the use of a nucleic acid, set of nucleic acids and/or kit for determining the modification status of a DNA region, which may comprise the DNA region or fragment thereof in which the gene of interest is located as in the method of the application, for the preparation of a substance for determining the presence of a liver tumor, assessing the risk of liver tumor formation or formation and/or assessing the progression of a liver tumor.

In another aspect, the application provides methods of confirming the presence of a liver tumor, assessing liver tumor formation or risk of formation, and/or assessing progression of a liver tumor, which may comprise providing a nucleic acid, set of nucleic acids, and/or kit for determining the modified status of a DNA region. The DNA region for determination may comprise a DNA region in which the target gene is located as in the method of the present application or a fragment thereof.

In another aspect, the application provides nucleic acids, nucleic acid sets and/or kits for determining the modified status of a DNA region, which may be used to confirm the presence of a liver tumor, assess the risk of liver tumor formation or formation, and/or assess the progression of a liver tumor. The DNA region for determination may comprise a DNA region in which the target gene is located as in the method of the present application or a fragment thereof.

In a further aspect, the present application provides the use of a nucleic acid, set of nucleic acids and/or kit for determining the modification status of a DNA region, which may comprise a DNA region of interest, or a complementary region thereof, or a fragment thereof, as described in the methods of the application, for the preparation of a substance for confirming the presence of a disease, assessing the risk of disease formation or formation and/or assessing the progression of a disease.

In another aspect, the application provides methods of confirming the presence of a disease, assessing disease formation or risk of developing a disease, and/or assessing the progression of a disease, which may comprise providing a nucleic acid, set of nucleic acids, and/or kit for determining the status of modification of a DNA region. The DNA region for determination may comprise the target DNA region, or a complementary region thereof, or a fragment thereof, as described in the methods of the present application.

In another aspect, the application provides nucleic acids, nucleic acid sets and/or kits for determining the modified status of a DNA region, which may be used to confirm the presence of a disease, assess the risk of disease formation or formation, and/or assess the progression of a disease. The DNA region for determination may comprise the target DNA region, or a complementary region thereof, or a fragment thereof, as described in the methods of the present application.

For example, in the application of the present application, the disease may comprise a tumor. Such as a solid tumor. For example, in the application of the present application, the disease may comprise a liver tumor. For example, in the practice of the application, the modification state may comprise a methylation modification.

In a further aspect, the application provides the use of a nucleic acid or combination of the above fragments or a region of DNA in which the gene of interest is located, or a region transformed therefrom, as in the method of the application, for the preparation of a substance for use in the confirmation of the presence of a liver tumour, in the assessment of liver tumour formation or risk of formation and/or in the assessment of the progression of a liver tumour.

In another aspect, the application provides a method of confirming the presence of a liver tumor, assessing liver tumor formation or risk of formation and/or assessing liver tumor progression, which may comprise providing a nucleic acid or a combination thereof of the DNA region of the target gene or the transformed region thereof, or a fragment thereof, in the method of the application.

In another aspect, the application provides a nucleic acid or combination of the above fragments or a region of DNA in which the target gene is located, or a region transformed therefrom, as in the methods of the application, which can be used to confirm the presence of a liver tumor, assess liver tumor formation or risk of formation, and/or assess the progression of a liver tumor.

In a further aspect, the application provides the use of a nucleic acid of the target DNA region, or a complementary region thereof, or a transformed region as described above, or a fragment as described above, or a combination thereof, in the preparation of a substance for use in confirming the presence of a disease, assessing the formation or risk of formation of a disease and/or assessing the progression of a disease, as in the methods of the application. For example, in the application of the present application, the disease may comprise a tumor. For example, in the application of the present application, the disease may comprise a liver tumor.

In another aspect, the application provides a method of confirming the presence of a disease, assessing the formation or risk of formation of a disease and/or assessing the progression of a disease, which may comprise providing a nucleic acid or a combination thereof of a region of a target DNA, or a complementary region thereof, or a transformed region as described above, or a fragment of the above, as described in the methods of the application. For example, in the application of the present application, the disease may comprise a tumor. For example, in the application of the present application, the disease may comprise a liver tumor.

In another aspect, the application provides a nucleic acid of the target DNA region, or a complementary region thereof, or a transformed region as described above, or a fragment as described above, or a combination thereof, as in the methods of the application, which can be used to confirm the presence of a disease, assess the risk of disease formation or formation, and/or assess the progression of a disease. For example, in the application of the present application, the disease may comprise a tumor. For example, in the application of the present application, the disease may comprise a liver tumor.

In another aspect, the present application provides an apparatus, which may comprise a storage medium of the present application. In another aspect, the application provides a non-transitory computer readable storage medium having stored thereon a computer program for execution by a processor to perform any one or more of the methods of the application. For example, the method may comprise a method of classifying samples having different likelihood of cancer using methylation indicators. Exemplary classification algorithms may be linear discriminant analysis, logistic regression, naive bayes classification, perceptual classification, quadratic classification, k-nearest neighbor, lifting method, decision tree, random forest, neural network, learning vector quantization or support vector machine, or a combination thereof. For example, the non-volatile computer-readable storage medium may include a floppy disk, a flexible disk, a hard disk, a Solid State Storage (SSS) (e.g., solid State Drive (SSD)), a Solid State Card (SSC), a Solid State Module (SSM)), an enterprise-level flash drive, a tape, or any other non-transitory magnetic medium, etc. The non-volatile computer-readable storage medium may also include punch cards, paper tape, optical discs (or any other physical medium having a hole pattern or other optically recognizable indicia), compact disc read-only memory (CD-ROM), rewritable optical discs (CD-RW), digital Versatile Discs (DVD), blu-ray discs (BD), and/or any other non-transitory optical medium.

For example, the apparatus of the present application may further comprise a processor coupled to the storage medium, the processor being configured to execute based on a program stored in the storage medium to implement the method of the present application. For example, the device may implement various mechanisms to ensure that the methods of the present application executing on the database system produce the correct results. In the present application, the device may use a disk as the persistent data storage. In the present application, the device may provide database storage and processing services for a plurality of database clients. The device may store database data across multiple shared storage devices and/or may utilize one or more execution platforms having multiple execution nodes. The devices may be organized such that storage and computing resources may be effectively infinitely extended.

The invention uses fluorescent PCR detection technology to detect methylation of related markers of leucoderma (most of which are leucocytes), paracancerous tissues and liver cancer tissues. The potential of the marker for blood detection of liver tumors was initially confirmed. An ideal liver cancer methylation detection marker should possess the following characteristics: 1. the DNA methylation level of the tunica albuginea layer is low; 2. liver cancer tissue has high DNA methylation level.

Methylation marker detection can be performed using Methylation Specific PCR (MSP) based on the principle that in detection, only methylated sequence templates produce an amplified signal, while non-methylated sequence templates do not. The method can be realized by designing a methylation specific sequence through a primer or a probe, and can also be realized by designing a methylation specific primer and a probe pair at the same time. The method mainly comprises the following steps: 1. based on the marker sequences, detection primers and probes are designed in a detection zone (which may be a CpG-rich region) suitable for MSP. 2. Nucleic acid extraction of white blood cells and tissue samples is performed. 3. The nucleic acid is bisulphite treated to convert unmethylated cytosines to uracil, while methylated cytosines maintain the sequence unchanged. 4. Fluorescence PCR detection was performed.

Through the detection of white blood cells and tissue samples, the markers meeting the characteristics prove that the potential of detecting liver cancer through blood exists. And then verifying the markers in a plasma sample, wherein the detection samples comprise a control group, a liver cancer group and a hepatitis B and cirrhosis interference population. The reference levels of these markers and the marker combination properties were analyzed. Because of the limited free DNA in the plasma of a single sample, optionally with fluorescent PCR detection, targets can be pre-amplified to allow the least amount of DNA to detect as many methylation sites as possible. The method mainly comprises the following steps: 1. nucleic acid extraction of plasma samples. 2. The nucleic acid is bisulphite treated to convert unmethylated cytosines to uracil, while methylated cytosines maintain the sequence unchanged. 3. And (5) pre-amplifying and diluting the target spots. 4. Fluorescence PCR detection was performed.

In the target spot combination analysis, any combination form can be performed on the basis of the target spot combination, or a mathematical model of machine learning, such as linear regression, support vector regression, ridge regression, random forest and the like, is introduced into a data algorithm, so that the liver cancer methylation detection marker combination with high detection accuracy is obtained.

The present application provides the following: 1. numerous liver cancer methylation markers with excellent performance are found; 2. developing a liver cancer methylation detection marker combination with high detection accuracy; 3. compared with the existing fluorescence PCR detection method, the detection performance is greatly improved; 4. compared with the second generation sequencing detection method, the method has the advantages of easy development, easy clinical popularization, low cost and the like. The application mode of the marker and the combination of the markers provided by the application can be not limited to a specific detection mode.

Description of the embodiments

1. A method of confirming the presence of a liver tumor, assessing liver tumor formation or risk of formation and/or assessing liver tumor progression comprising determining the presence and/or amount of a modified state of a DNA region or fragment thereof of a target gene in a test sample, said target gene comprising CHFR and GRASP.

2. A method of assessing methylation status of a liver tumor-associated DNA region comprising determining the presence and/or amount of a modification of the DNA region or fragment thereof of a target gene in a test sample, said target gene comprising CHFR and GRASP.

3. The method of any one of embodiments 1-2, wherein the DNA region of CHFR is derived from human chr12:133398773-133532890.

4. The method of any one of embodiments 1-3, wherein the DNA region of GRASP is derived from human chr12:52400724-52409673.

5. The method of any one of embodiments 1-4, wherein the target gene further comprises a gene selected from the group consisting of: IRF4, IKZF1, gene spacer 1 (chr 7: 26415938-26416740), and BCAT1.

6. The method of any one of embodiments 1-5, wherein the target gene comprises at least 2 genes.

7. The method of any one of embodiments 1-6, wherein the target gene comprises 2 to 6 genes.

8. The method of any one of embodiments 1-7, wherein the target gene comprises CHFR, GRASP, IRF a4 and IKZF1.

9. The method of any one of embodiments 1-8, wherein the target gene comprises CHFR, GRASP, IRF a4, gene spacer 1 (chr 7: 26415938-26416740), and IKZF1.

10. The method of any one of embodiments 1-9, wherein the target gene comprises CHFR, GRASP, IRF a 4, BCAT1, and IKZF1.

11. The method of any one of embodiments 5-10, wherein the DNA region of IRF4 is derived from human chr6:391739-41447.

12. The method of any one of embodiments 5-11 wherein the DNA region of IKZF1 is derived from human chr7:50343720-50472799.

13. The method of any one of embodiments 5-12, wherein the DNA region of gene spacer 1 (chr 7: 26415938-26416740) is derived from human chr7:26415938-26416740.

14. The method of any one of embodiments 5-13, wherein the DNA region of BCAT1 is derived from human chr12:24964295-25102393.

15. A method of confirming the presence of a disease, assessing the formation or risk of developing a disease and/or assessing the progression of a disease comprising determining the presence and/or amount of a modified state of a region of target DNA, or a complementary region thereof, or a fragment thereof, in a test sample, said region of target DNA comprising a region derived from human chr12:133483901-133485740 and a region derived from human chr12: 52400724-52401698.

16. A method of determining the methylation status of a DNA region comprising determining the presence and/or amount of a modified status of a target DNA region, or a complementary region thereof, or a fragment thereof, in a test sample, said target DNA region comprising a region derived from human chr12:133483901-133485740 and derived from human chr12: 52400724-52401698.

17. The method of any one of embodiments 1-16, comprising providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:1, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

18. The method of any one of embodiments 15-17, wherein the target region comprises a region derived from human chr12: 133485000-133485067.

19. The method of any one of embodiments 1-18, comprising providing the sequence of SEQ ID NO:2 or a complementary nucleic acid thereof, or a fragment thereof.

20. The method of any one of embodiments 1-19, comprising providing the sequence of SEQ ID NO:3 and 4, or a complementary nucleic acid set thereof, or a fragment thereof.

21. The method of any one of embodiments 1-20, comprising providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:5, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

22. The method of any one of embodiments 15-21, wherein the region of interest comprises a region derived from human chr12: 52401083-52401169.

23. The method of any one of embodiments 1-22, comprising providing SEQ ID NO:6 or a complementary nucleic acid thereof, or a fragment thereof.

24. The method of any one of embodiments 1-23, comprising providing the sequence of SEQ ID NO:7 and 8 or a complementary nucleic acid set thereof, or a fragment thereof.

25. The method of any one of embodiments 15-24, wherein the region of target DNA further comprises a region selected from the group consisting of: derived from human chr6:391739-394056, derived from human chr7:50343720-50344547, derived from human chr7:26415938-26416740, and derived from human chr12:25101630-25102393.

26. The method of any one of embodiments 15-25, wherein the region of target DNA comprises at least 2 regions.

27. The method of any one of embodiments 15-26, wherein the region of target DNA comprises 2 to 6 regions.

28. The method of any one of embodiments 15-27, wherein the region of target DNA comprises a region defined by human chr12:133483901-133485740, human chr12:52400724-52401698, human chr6:391739-394056, and human chr7: 50343720-50344547.

29. The method of any one of embodiments 15-28, wherein the region of target DNA comprises a region defined by human chr12:133483901-133485740, human chr12:52400724-52401698, human chr6:391739-394056, human chr7:26415938-26416740, and human chr7: 50343720-50344547.

30. The method of any one of embodiments 15-29, wherein the region of target DNA comprises a region defined by human chr12:25101630-25102393, human chr12:52400724-52401698, human chr6:391739-394056, human chr7:50343720-50344547, and human chr12: 133483901-133485740.

31. The method of any one of embodiments 1-30, comprising providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:9, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

32. The method of any one of embodiments 15-31, wherein the region of interest comprises a region derived from human chr6:392282-392377.

33. The method of any one of embodiments 1-32, comprising providing SEQ ID NO:10 or a complementary nucleic acid thereof, or a fragment thereof.

34. The method of any one of embodiments 1-33, comprising providing SEQ ID NO:11 and 12 or a complementary nucleic acid set thereof, or a fragment thereof.

35. The method of any one of embodiments 1-34, comprising providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:13, or a complementary region thereof, or a region obtained by transformation as described above, or a fragment thereof.

36. The method of any one of embodiments 15-35, wherein the region of interest comprises a region derived from human chr7: 50343793-50343896.

37. The method of any one of embodiments 1-36, comprising providing SEQ ID NO:14 or a complementary nucleic acid thereof, or a fragment thereof.

38. The method of any one of embodiments 1-37, comprising providing SEQ ID NO:15 and 16 or a complementary nucleic acid set thereof, or a fragment thereof.

39. The method of any one of embodiments 1-38, comprising providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:17, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

40. The method of any one of embodiments 15-39, wherein the target region comprises a region derived from the human chr7:26416257-26416363 definition.

41. The method of any one of embodiments 1-40, comprising providing SEQ ID NO:18 or a complementary nucleic acid thereof, or a fragment thereof.

42. The method of any one of embodiments 1-41, comprising providing SEQ ID NO:19 and 20 or a complementary nucleic acid set thereof, or a fragment thereof.

43. The method of any one of embodiments 1-42, comprising providing a polypeptide capable of binding a polypeptide comprising SEQ ID NO:21, or a complementary region thereof, or a region obtained by transformation of the above, or a fragment thereof.

44. The method of any one of embodiments 15-43, wherein the region of interest comprises a region derived from human chr12: 25102016-25102110.

45. The method of any one of embodiments 1-44, comprising providing SEQ ID NO:22 or a complementary nucleic acid thereof, or a fragment thereof.

46. The method of any one of embodiments 1-45, comprising providing SEQ ID NO:23 and 24 or a complementary nucleic acid set thereof, or a fragment thereof.

47. The method of any one of embodiments 15-46, wherein the disease comprises a tumor.

48. The method of any one of embodiments 15-47, wherein the disease comprises a liver tumor.

49. The method of any one of embodiments 1-48, further comprising obtaining nucleic acid in the sample to be tested.

50. The method of embodiment 49, wherein the nucleic acid comprises cell-free nucleic acid.

51. The method of any one of embodiments 1-50, wherein the sample to be tested comprises a tissue, a cell, and/or a body fluid.

52. The method of any one of embodiments 1-51, wherein the sample to be tested comprises plasma.

53. The method of any one of embodiments 1-52, further comprising transforming the DNA region or fragment thereof.

54. The method of embodiment 53, wherein the base having the modified state and the base not having the modified state form different substances after conversion.

55. The method of any one of embodiments 1-54, wherein the base with the modified state does not substantially change after conversion, and wherein the base without the modified state changes to another base different from the base after conversion, or is sheared after conversion.

56. The method of any one of embodiments 54-55, wherein the base comprises a cytosine.

57. The method of any one of embodiments 1-56, wherein the modified state comprises a methylation modification.

58. The method of any one of embodiments 55-57, wherein the additional base comprises uracil.

59. The method of any of embodiments 53-58, wherein the converting comprises converting by a deamination reagent and/or a methylation sensitive restriction enzyme.

60. The method of embodiment 59, wherein the deaminating agent comprises bisulfite or an analog thereof.

61. The method of any one of embodiments 1-60, the method of determining the presence and/or amount of a modified state comprising confirming the presence and/or amount of a substance formed after the conversion of a base having the modified state.

62. The method of any one of embodiments 1-61, wherein the method of determining the presence and/or amount of a modification state comprises determining the presence and/or amount of a DNA region or fragment thereof having the modification state.

63. The method of any one of embodiments 1-62, wherein the presence and/or amount of a DNA region or fragment thereof having the modified state is determined by a fluorescent Ct value detected by the fluorescent PCR method.

64. The method of any one of embodiments 1-63, determining the presence of, or the risk of liver tumor formation or formation by confirming the presence of a modified state of the DNA region or fragment thereof and/or the content of the DNA region or fragment thereof having a higher modified state relative to a reference level.

65. The method of any one of embodiments 1-64, further comprising amplifying the DNA region or fragment thereof in the test sample prior to determining the presence and/or amount of modification of the DNA region or fragment thereof.

66. The method of embodiment 65, wherein said amplifying comprises PCR amplifying.

67. A nucleic acid comprising a sequence capable of binding to the DNA region of the target gene, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, according to the method of any one of embodiments 1-14 and 49-66; or the nucleic acid comprises a sequence capable of binding to the region of the target DNA, or a region complementary thereto, or a transformed region as described above, or a fragment thereof, according to the method of any one of embodiments 15-66.

68. A method for producing a nucleic acid, the method comprising designing a nucleic acid capable of binding to a DNA region of the target gene, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the DNA region of the target gene, or a complementary region thereof, or the transformed region thereof, or the fragment thereof, in the method according to any one of embodiments 1 to 14 and 49 to 66; or the method comprising designing a nucleic acid capable of binding to the region of the target DNA, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the region of the target DNA, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, as described in any one of embodiments 15-66.

69. A nucleic acid set comprising a sequence capable of binding to a DNA region in which the target gene is located, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, according to the method of any one of embodiments 1-14 and 49-66; or the nucleic acid set comprises a sequence capable of binding to the region of the target DNA, or a region complementary thereto, or a region transformed therefrom, or a fragment thereof, according to the method of any one of embodiments 15-66.

70. A method for producing a nucleic acid set, the method comprising designing a nucleic acid set capable of binding to a DNA region of the target gene, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the DNA region of the target gene, or a complementary region thereof, or the transformed region thereof, or the fragment thereof, in the method according to any one of embodiments 1 to 14 and 49 to 66; or the method comprising designing a nucleic acid set capable of binding to the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, according to the modified state of the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, as described in the method of any one of embodiments 15 to 66.

71. A kit comprising a nucleic acid according to embodiment 67 and/or a nucleic acid set according to embodiment 69.

72. The nucleic acid of embodiment 67, the set of nucleic acids of embodiment 69 and/or the kit of embodiment 71 for use in the preparation of a substance for determining the modification status of a DNA region or fragment thereof.

73. The use of the nucleic acid of embodiment 67, the set of nucleic acids of embodiment 69 and/or the kit of embodiment 71 in the preparation of a disease detection product.

74. Use of the nucleic acid of embodiment 67, the set of nucleic acids of embodiment 69 and/or the kit of embodiment 71 for the preparation of a substance for confirming the presence of a disease, assessing the formation or risk of formation of a disease and/or assessing the progression of a disease.

75. The use of any one of embodiments 73-74, wherein the disease comprises a tumor.

76. The use of any one of embodiments 73-75, wherein the disease comprises a liver tumor.

77. Use of a nucleic acid, set of nucleic acids and/or kit for determining the modification status of a DNA region comprising a DNA region or fragment thereof in which the gene of interest is located in a method according to any one of embodiments 1-14 and 49-66, for the preparation of a substance for determining the presence of a liver tumor, assessing liver tumor formation or risk of formation and/or assessing the progression of a liver tumor.

78. Use of a nucleic acid, set of nucleic acids and/or kit for determining the modification status of a DNA region comprising the target DNA region, or a complementary region thereof, or a fragment thereof, according to the method of any one of embodiments 15-66, in the preparation of a substance for confirming the presence of a disease, assessing the formation or risk of formation of a disease and/or assessing the progression of a disease.

79. The use of embodiment 78, wherein the disease comprises a tumor.

80. The use of any one of embodiments 78-79, wherein the disease comprises a liver tumor.

81. The use of any one of embodiments 72 and 77-80, wherein the modified state comprises a methylation modification.

82. Use of a nucleic acid or combination of fragments thereof of any of embodiments 1-14 and 49-66 of the DNA region of the target gene, or a transformed region thereof, or a fragment thereof, in the preparation of a substance for confirming the presence of a liver tumor, assessing liver tumor formation or risk of formation and/or assessing the progression of a liver tumor.

83. The use of a nucleic acid or combination of the target DNA region, or a complementary region thereof, or a transformed region thereof, or a fragment thereof, in the method of any one of embodiments 15-66, for the preparation of a substance for confirming the presence of a disease, assessing the risk of disease formation or formation, and/or assessing the progression of a disease.

84. The use of embodiment 83, wherein the disease comprises a tumor.

85. The use according to any one of embodiments 83-84, wherein the disease comprises a liver tumor.

86. A storage medium recording a program capable of executing the method of any one of embodiments 1 to 66.

87. An apparatus comprising the storage medium of embodiment 86.

88. The apparatus of embodiment 87, further comprising a processor coupled to the storage medium, the processor configured to execute to implement the method of any of embodiments 1-66 based on a program stored in the storage medium.

Without intending to be limited by any theory, the following examples are meant to illustrate the methods and uses of the present application and the like and are not intended to limit the scope of the application.

Examples

Example 1 comparison of methylation abundance of liver cancer, paracancestral tissue and leucoma DNA samples

DNA samples were obtained from the buffy coat of healthy people without abnormality in the liver, cancer tissues from liver cancer patients (10 buffy coat samples, 10 cancer tissues each), respectively, and buffy coat DNA was selected as the reference sample because most of plasma free DNA was derived from DNA released after rupture of buffy coat, and the background thereof could be a basic background signal of the detection site of plasma free DNA. The tunica albuginea DNA was extracted with QIAGEN QIAAMP DNA MINI KIT and the Tissue DNA was extracted with the QIAGEN QIAAMP DNA FFPE Tissue Kit according to the requirements of the specification.

The DNA obtained in the above step was sampled by 20ng and treated with a bisulfite reagent (D5031, ZYMO RESEARCH) to obtain a converted DNA.

The fluorescence Ct value of the marker is obtained through fluorescence PCR detection. In the fluorescent PCR reaction system, the final concentration of each primer was 500nM and the final concentration of each detection probe was 200nM. The PCR reaction system comprises: 10. Mu.L of pre-amplified dilution product, 2.5. Mu.L of primer and probe premix containing the detection sites; 12.5 mu L of PCR reagentUniversal Probe QPCR MASTER Mix (NEB). Wherein the primer sequences of the methylation markers are shown in Table 1, and the probe sequences are shown in Table 2. The PCR conditions were as follows: 95 ℃ for 5 minutes; 50 cycles were performed at 95℃for 30 seconds, 56℃for 60 seconds (fluorescence was collected). Different fluorescence was detected using ABI7500 Real-TIME PCR SYSTEM in the corresponding fluorescence channel. Sample Ct values obtained from the buffy coat, the paracancerous tissue and the cancerous tissue are calculated and compared, and the target Ct value at which no amplification signal is detected is set to 50.

TABLE 1 primer sequences

TABLE 2 detection probe sequences

Table 3 summary of sample test results

The results of the above table show that the average Ct value for cancer tissue detection is small, representing a stronger methylation signal. The detection rate of methylation signals in cancer tissues can be far higher than that of a white membrane layer, and the methylation signals are also strong. Most samples of the buffy coat failed to detect target methylation signals. These targets can all have the potential for blood detection of liver cancer. The selected target markers proved to be viable and specific for tumor tissue.

Example 2 comparison of plasma sample methylation Signal in liver cancer patients and people with no liver abnormality

Samples of 170 liver-unseen abnormal healthy control plasma, 321 liver cancer patient preoperative plasma (with phase I accounting for 86%), 36 hepatitis b patient plasma, and 20 liver cirrhosis plasma were selected.

Extracellular free DNA in the above plasma samples was extracted using commercial QIAGEN QIAAMP Circulating Nucleic Acid Kit. The extracted extracellular free DNA was subjected to sulfite conversion treatment using commercial bisulfite conversion reagent MethylCodeTM Bisulfite Conversion Kit to obtain converted DNA.

Alternatively, the above-mentioned converted DNA was used for pre-amplification, and PCR amplification was performed using a primer pool containing methylation marker specific primers shown in Table 1 and internal reference primers (ACTB, forward primer and reverse primer may be shown in Table 1, respectively, and probe may be shown in Table 2) using the converted DNA as a template, each primer having a final concentration of 100nM. The PCR reaction system was 10. Mu.L of the transformed DNA, and 2.5. Mu.L of the premix containing the above primers; PCR reagent [ ]Universal Probe QPCR MASTER Mix (NEB) 12.5. Mu.L. The PCR conditions were as follows: 95 ℃ for 5 minutes; 95℃for 30 seconds, 56℃for 60 seconds, 15 cycles.

The obtained pre-amplified product was diluted 10 times and used for fluorescent PCR detection. Primers for each methylation marker shown in Table 1, and the detection probe sequences shown in Table 2 were used, and the reference gene ACTB was detected at the same time, as a control. The final primer concentration was 500nM and the final probe concentration was 200nM. The PCR reaction system comprises: 10. Mu.L of pre-amplified dilution containing 2.5. Mu.L of primer and probe premix for the detection sites; PCR reagent [ ]Universal Probe qPCR Master Mix(NEB)12.5μL。

The fluorescent PCR reaction system was the same as in the above examples. The PCR conditions were as follows: 95 ℃ for 5 minutes; 95℃for 15 seconds, 56℃for 40 seconds (fluorescence is collected), 50 cycles. And aiming at the modified fluorescence of different gene probes, selecting a corresponding detection fluorescence channel. The Ct value of the target spot at which no amplification signal was detected was set to 50. The results show that each target point of the application can have the capability of detecting liver cancer by blood.

The detection sensitivity statistics for the detection sites at about 90% specificity are shown in table 4:

TABLE 4 detection sensitivity of detection sites

Site(s)	Sensitivity%	Specificity%
			CHFR	69.16	89.81
GRASP	53.89	93.2
			IRF4	40.0	90.3
IKZF1	36.14	98.06
			Gene spacer 1	19.94	99.03
BCAT1	19.63	96.6

The results of the above table show that the detection sites are compared with the DNA methylation signals of the control plasma and the liver cancer plasma. The selected target marker is proved to have higher sensitivity to blood samples of liver cancer patients.

When the combined analysis is carried out on the targets, the data analysis can adopt a mode of setting a positive interpretation threshold value for a single target, and when the targets are combined, the positive mode of any target, namely the integrated interpretation of the sample, is balanced sensitivity and specificity, and different positive interpretation threshold values can be used for each site in different combined modes. In this way, it was verified that:

The combination of CHFR and GRASP can lead the liver cancer detection sensitivity to be 72.6 percent, the specificity to be 90.0 percent, and the positive rate of a patient with hepatitis B to be 11.1 percent and the positive rate of a patient with cirrhosis to be 20 percent; by combining CHFR, GRASP, IRF < 4 > and IKZF1, the liver cancer detection sensitivity is 75.4%, the healthy contrast specificity is 91.2%, meanwhile, the positive rate of a patient with hepatitis B is 8.3%, and the positive rate of a patient with cirrhosis is 20%; by combining CHFR, GRASP, IRF4, IKZF1, gene spacer 1 and BCAT1, the liver cancer detection sensitivity is 79.4%, the healthy contrast specificity is 90.0%, meanwhile, the positive rate of a patient with hepatitis B is 13.9%, and the positive rate of a patient with cirrhosis is 20%; the target combinations all prove that the performance of distinguishing healthy control from liver cancer is superior, and the specificity is good in patients with hepatitis B and liver cirrhosis.

The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Numerous variations of the presently illustrated embodiments of the application will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and equivalents thereof.

Sequence listing

<110> Jiangsu Kunyuan Biotechnology Co., Ltd.; Jiangsu Kunyuan Biotechnology Co., Ltd.

<120> A set of tumor detection markers and uses thereof

<130> 0266-PA-037

<160> 27

<170> PatentIn version 3.5

<210> 1

<211> 1840

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> CHFR subregion

<400> 1

tcgcacctcc aaagcccacg tccacacgtt ccgagcccgc cgtcccatcg caaacatccg 60

ggggccgcca ctgcgcctaa cgaccgccca tctctgacgc ctcgggtcgg cgtttgattt 120

ttaacccaag aaaagagtaa aagtcaagcg gaaatcaacg cagaagcggt gctgcagcct 180

ctgagcttcc tgacggccgt gtccagagcc gggctcctcc tccggcggca gccgccgggc 240

tcacttcagt gcgctcagct tctcgcgggg acaagcgtct gggcggggac ggcccgaggc 300

gggggtgtgg cgaggctgga gaacgctgaa agctggtggg cttgggaaga caaacgacgc 360

tctgaaattt ggctcccagg taatacgttc cggcacttcg cggcggctca gtcagcgctg 420

ctgggttcca cccctcctat gcgttgctca gggacaggtt tcttagtgac aggcccctca 480

ctattcccat tggccaggac gcccaaggcc cacgcagccg ttggctaaaa ttgttgtctg 540

tttcagacac gttggcgaat tccccgcctc tacgttgccg ggagacagca tgttgcctag 600

cagcgtgcgg ctcccattgc ctttgggcgc ggctctccgc cccgacccgc ccctctggga 660

cccatccggg tctgcgcctc agctagacag ggcgggctgc ggcgcgcagt cttaggcgtg 720

cgcttcgggg aaggggccct cgcgtgggag gagcgcgtct gcgagggatg cgccggcgca 780

ggcgagaccc ggagccgcgt ggtctctgcg cagtgcgtcg tgtggggcgg ccgaccccgg 840

cccgggtttc cctgctcgtt ttcgtccggg cgggcgtctt ggttcccgtt ccaggccagc 900

cccatcctag gtcgctgctt catccagcgc ttggggcatt tgtgtcgttg gcgttgtttt 960

cctcttaact acaaacttta aatgtgttca tctactcgtg acggcgagtt tagcagactt 1020

tcattcccag aagacgtgtc acacgatctg tatatttttc atacagaaat tgacggacgt 1080

cgttacttct aaatttccac aggggctggt tcatcccgcg gctttaccta gagaaaaacg 1140

tctgaggccg ggcgcggcgg ctcccgcctg taatcccaac actttgggag gccgaggcgg 1200

gcgaatcacg aggtcaggag ttcgagacca gactggccaa catggtgaaa cctcgtctct 1260

actaaaaata caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaatagccg ggcgtggcgg 1320

cgcatgcctg taatcccagc tactcaggag gctgaggcag gagaatcgct tgaacccgag 1380

atggtgccac tgcactccag catgggcgat agagcgagac tccgtctcaa aaaataataa 1440

taaaataaaa accatggaca atgggaggag aacatcacac accggggcct gtcgggggtg 1500

gggggccagg ggagggatag catcaggaga aatacgtaat gtagataact ggttgatggg 1560

tgcagcaaac caccatggca cgtgtatacc tatgtaacaa acctgcaggt tctgcacatg 1620

taccccagaa cttaaagtat aattttaaaa agagagagag aagcgtctga gcgtttctca 1680

gggacggcgg gtggcagcca ggacgggctg gggcggtgaa gtgcggggcg ggaagcgacg 1740

cgtgcgcctc ctgcgctggc ttctgtgaaa ggagggactg attggagatc actgctgaaa 1800

agtggactac gcatcagtga atagttagtg aaatatgagc 1840

<210> 2

<211> 19

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> CHFR Probe

<400> 2

gcggcgcgta gttttaggc 19

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> CHFR Forward primer

<400> 3

gcgttttagt tagatagggc gg 22

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> CHFR reverse primer

<400> 4

gaaaacccct tccccgaaac 20

<210> 5

<211> 975

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> GRASP subregion

<400> 5

ccggctggca tcccccagcc gccgccagcc ccgccgaggg gagccagcgc cgtctctgag 60

gggcgtccgg cgccggagcc atgaccctcc gccgactcag gaagctgcag cagaaggagg 120

aggcggcggc caccccggac cccgccgccc ggactcccga ctcggaagtc gcgcccgccg 180

ctccggtccc gaccccggga ccccctgccg cagccgccac ccctgggccc ccagcggacg 240

agctgtacgc ggcgctggag gactatcacc ctgccgagct gtaccgcgcg ctcgccgtgt 300

ccgggggcac cctgccccgc cgaaaggtgc gtcccccgcc cgccttcagg atctgctcag 360

cccctctccg actccctaca gggcctgctg actccgcagt gccctctcct cggcgtccgc 420

ggagtccccc accttcttcc ccggcccgct gggtgcctcg actccccgcg ttccccgctg 480

ctgcgaaggc cgtggccctc gcctgcacac cgcgcccagg ctcggtggct cttaactccg 540

cgccccatgc acgccccctc tctccctcct tgactcctcc cagcaccccc cttctcctac 600

ccgctccatc tggctttctg ccccccatgc cccgcctccc cgtggccagg tgtcctgggt 660

ccccaggagc ccctcgcccg agggacagag acagccccag gcaagttgaa ggtccgagag 720

cccccggtgg gagaagcggg ccggtggctg cgccgcgtgc gttctcactc tgaggaagtg 780

cgtggggagc cgctgactcc ggatagcaca cccttccgag gggactcccc gattcctggg 840

ctgggggcct gccgcctggc cccacgtctg acgtacgggg cgcgagggcc actgctccct 900

ggacttctgt cggaaccgga cgcagtggga ggggtcgcag ggcgcccgcg gggcaggaag 960

gatgcgggcc gcgcc 975

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> GRASP Probe

<400> 6

tgtttttttt tcggcgttcg cg 22

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> GRASP Forward primer

<400> 7

gttttttttc gattttttat aggg 24

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> GRASP reverse primer

<400> 8

aaccgaaaaa aaaaataaaa aactc 25

<210> 9

<211> 2318

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> IRF4 subdomain

<400> 9

acctcgcact ctcagtttca ccgctcgatc ttgggaccca ccgctgccct cagctccgag 60

tccagggcga ggtaagggct ggagtcgggc aggaggaggg gtgtgaggct gataccagag 120

aggacccgga gcgcgaacca gaggttcgac ctccagggca gcgcagggta ccccggcttc 180

ggagcgggaa gggagcgcgc cccgtcctgg agctccgact cccaccccat ctgcgctgag 240

ccggaggcgc tggtttgggc tccaaggccc gcctccttgg ctctgcccga gcctccccgc 300

ctgccctccg cgctcctgcg acggggtcgc cacaagctgg acgggatgag ctaaccggac 360

tgtcggggcc ccaggagtgg ctgaggcggg gccgtccaag gcacccacac aagacggcac 420

aactgcctgc gagaaacagg cccggccctg tggaccccaa tccgaggctc cttcccctgc 480

tcttcgttcc taaggggccc aagctcacgg cggcctccgg cgcggtgctc acccgctggc 540

gcaggaggag gaggagctcc acatttgggt cgctccgagc cttgcgtgcg gtggcctagc 600

cggcctggcg cggtccctgc ctcccaggct ccgcagctgt cgtcgccctc tcccgcgccc 660

tccccgcctc cgctctcccg ggcctgctcc ggggtccggc ggacgctctg cgcgcggaat 720

cccccgtact ggggctgcag cccccgcgtc tgcgccactt gtcgtttgca gagcccactt 780

agtgcgcgct agctgggcag ggataggggt cctattcggg gcgaagggtc tggatgcgag 840

cagagaaagc ggagggtgga ggaacccggg gctgcgcccc tggaacgccc ggccgcaggc 900

gaggtcctcc gcgcgtggag gccgccaggg gagtggaaac tgacagagtc gcggggaagg 960

ggcgagaagc gggttgggag tgagcgaagg caagcgagag ctgcgagtga gtgcggaagg 1020

agggccagga ggggtggcgg ctgggtgggg agagagggtg caagacgagc ggcgcgtgtc 1080

gggagccttt gggctgcggg tgcgttacag gagagcaggc gggtaggagc cttcgcgggg 1140

gccgagctcg gaaggcggac ggctgtgccc gcccagggga tgcgcccggg ccggccgcga 1200

aggtgccttc ttccgggggc ccggacgacc ctgacacggc acgcgcgcgc ttcgcagcct 1260

caaagactcc ggggcctcgt ggtcactggc gcaggggatc ggggcggggt gcccggagtg 1320

cggtgcctcg tggctgaagg gcagctcttc tccccgcagt gcagagcaga gcgggcggag 1380

gaccccgggc gcgggcgcgg acggcacgcg gggcatgaac ctggagggcg gcggccgagg 1440

cggagagttc ggcatgagcg cggtgagctg cggcaacggg aagctccgcc agtggctgat 1500

cgaccagatc gacagcggca agtaccccgg gctggtgtgg gagaacgagg agaagagcat 1560

cttccgcatc ccctggaagc acgcgggcaa gcaggactac aaccgcgagg aggacgccgc 1620

gctcttcaag gtctccggcc tcgggagccg gcgggggcgc gccggggagg gcccagagac 1680

agagcccggg gtccccggcg ccgcctccga ggcgagccca ggggaccgcg cggggcggac 1740

gggcgggcgg cggaggcatc aggtggcgtc gccggagccg caggaggagg aaaggaggcc 1800

tcggctctca gcgggaccgc gggggccggg agccgggtcc tgggcgcgtg gaggctgcag 1860

ggaaaccgct gaaggcccgg ccgggcccgg ggaagggcgg ccaaaggctt gaggggtttt 1920

gcgcgttcgt ccgtgcgttc tcgtttccac gcaagcctcc cgcccttcct ccgggctccc 1980

gtctgccgcc tccgtccgtg ggtccccctc gccctctccg tgcgtccgcg cctgtgccgg 2040

cggctgtttt cgtctctcac cgcgtctctg tttctctttt cgctgctttt ctctctgagt 2100

ctctctctct ccatgttttt cctgaggtca gcctctcttc tcgctcctgc tagctctctg 2160

cgggtactcc cacctctgtc tttctctttg tgtgtctctg tctctctctt tcccccatcg 2220

cagtggaact cagggcctct gtctagagct gtctcccttg ccctttgcgc gagtgcacac 2280

acgtgtgtcg ttgttacgat tgttctcccc taaggcag 2318

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> IRF4 Probe

<400> 10

atcgtacgta aggttcggag cga 23

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> IRF4 Forward primer

<400> 11

aaaaaaaaaa aaactccaca ttt 23

<210> 12

<211> 17

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> IRF4 reverse primer

<220>

<221> misc_feature

<222> (7)..(7)

<223> n = A, T, G or C

<400> 12

tagttgngga gtttggg 17

<210> 13

<211> 828

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> IKZF1 subdomain

<400> 13

gcacccgccg ccgccccggc ggcctttggg ggctgagccg gagcccggcg cgattgcaaa 60

gttttcgtgc gcggcccctc tggcccggag ttgcggctga gacgcgcgcc gcgcgagccg 120

ggggactcgg cgacggggcg gggacgggac gacgcaccct ctccgtgtcc cgctctgcgc 180

ccttctgcgc gccccgctcc ctgtaccgga gcagcgatcc gggaggcggc cgagaggtgc 240

gcgcggggcc gagccggctg cggggcaggt cgagcaggga ccgccagcgt gcgtcacccc 300

aaagtttgcg gggtggcagg gcgcgcgctc tggccacccg ccgctctggg cggcagctgg 360

tggcaacgca agggcgcggc gggggcggcc ggcgcggagg gggccaggta cggggcccgc 420

gggcggcgct gtgcgcgcgg ggcagccggt cggccgggag cgcgaaagcc tggtctgagc 480

cggctggggg cggggagtgt ggcggagaaa tggggaacaa tgcgagtgag caacttcagg 540

aagtcattgt gaaagaaagc tgggaagagc tccgcggcca agttagcagg acactctaac 600

aagtgactgc gcggcccgcg cccggggcgg tgactgcggc aagccccctg ggtccccgcg 660

cggcgcatcc cagcctgggc gggacgctcg gccgcggcga ggcgggcaag cctggcaggg 720

cagagggagc cccggctccg aggttgctct tcgcacccga ggatcagtct tggccccaaa 780

gcgcgacgca caaatccacg tgagtgtttt caaattgaat ttcaatag 828

<210> 14

<211> 16

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> IKZF1 Probe

<400> 14

cgccccgtcg ccgaat 16

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> IKZF1 Forward primer

<400> 15

gtttttttgg ttcggagttg 20

<210> 16

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> IKZF1 reverse primer

<400> 16

caaaacgaaa cacgaaaaaa ata 23

<210> 17

<211> 803

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> Gene spacer 1 subdomain

<400> 17

cgccctggta acccatggca actgcgcggc ggaaagaaaa aaaagaaccg tggtggggcg 60

gggggcggcg ggcggtgaat gggaacaacg accgcaaaga gacagcgatt taccgcgcgg 120

gcctgcggct cccggccgca cctgctgcag acggcccgcg aggccccttc ccgcgctacg 180

gtgacgattc tggctgctgg ggaaaagagc aataagcaga aagccctcgt gcagagggga 240

tccgggcggt gcacttggtg tgggaggctg ccttaaacgc cgatgaccct cccggcctcc 300

gcgtgtcccg gcttccaggg ccccgccagg ctggagagcg cgcgtggaga gggccttgcc 360

gcctgggggt tggttgaggg gcggccgcgc cggggccggg gctggactcc aggctttgtt 420

ctgcagacgc tcgcgcccgg ccggaggagg ggctacaccg tgctgccccg gccccatggg 480

gcccggcccc cgagggtccc cgcgagcgga cgcggtgggg ccgggcaaac tctacgtgcc 540

ctaaattttg tcatctgcac acgcatcgca cacattagtt aacccctttc ccttctaggc 600

ccccgagaac ctaacctgcc cggcgggcgc gcggctgcgt taacctggcc ccttccgtct 660

ggagagggag gggacggtcg cgcctggtgg taggactggg ctgtgtgtgt gtgtgtgtgt 720

gtgtgtgtgt gtgtgtgtgt gtgtgtgtag gggggtgata caagttttcc acccgcttta 780

ggctgaggcc ctgtaagcgg gac 803

<210> 18

<211> 15

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> Gene spacer 1 Probe

<400> 18

ttgaggggcg gtcgc 15

<210> 19

<211> 19

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> Gene spacer 1 Forward primer

<400> 19

gtttcgttag gttggagag 19

<210> 20

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> Gene spacer 1 reverse primer

<400> 20

ctacaaaaca aaacctaaaa tcc 23

<210> 21

<211> 764

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> BCAT1 subdomain

<400> 21

agtagggagg tgggcaggag ccagtgatga cggaatggca atcacatttg acctctgatc 60

tgtttatttc ctcctccttg acgtctccat ataaatgtta cacgggcatc cccacactcg 120

gatacgcacc cacagtggct gattcggggg taaccgtgtc atttgcttgc aacactggca 180

cctctgccct gcaccccggg agtgagcagt gagtgaggct cgggtctggg cgctggctcc 240

gaatcttcgg gctgggagag actccaccat ctgggggcgg cctgggggag cagccttagt 300

gtcttcctgc tgatgcaatc cgctaggtcg cgagtctccg ccgcgagagg gccggtctgc 360

aatccagccc gccacgtgta ctcgccgccg cctcgggcac tgccccaggt cttgctgcag 420

ccgggaccgc gctctgcagc cgcagacccg gtccacacgg ccaggggcta cgacccttgg 480

gatctgccct ccgctcagct cgagcttccc tcgtggccga cggaacaatg aaggtaacta 540

cttatggttt tgtccgtgtt ttacaaaaat gtgtgcgtga atcgaaccgg cgatttctcc 600

aagaaacata gttggcaggg aggggaggaa ggcgagacaa ccatggctta tatcccccgc 660

aaacgtctca gtatcttctt tatcaatcgt agtttgcggg gaccgtgcat tctgttcaga 720

tttcggttta acctccactc gcaggacgtg ccttctcgga cttt 764

<210> 22

<211> 17

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> BCAT1 Probe

<400> 22

tcggtttttt cgcggcg 17

<210> 23

<211> 15

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> BCAT1 Forward primer

<400> 23

tacgtggcgg gttgg 15

<210> 24

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> BCAT1 reverse primer

<400> 24

aaaaaaacaa ccttaatatc ttc 23

<210> 25

<211> 30

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> ACTB Probe

<400> 25

accaccaccc aacacacaat aacaaacaca 30

<210> 26

<211> 24

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> ACTB Forward primer

<400> 26

gtgatggagg aggtttagta agtt 24

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<223> ACTB reverse primer

<400> 27

ccaataaaac ctactcctcc cttaa 25

Claims

1. Use of a nucleic acid set comprising a sequence capable of determining the methylation status of a DNA region of a target gene, a region complementary thereto, or a region transformed therefrom in a sample to be tested, wherein the target gene comprises CHFR and GRASP, the DNA region of CHFR is the nucleic acid sequence of SEQ ID No. 1, the DNA region of GRASP is the nucleic acid sequence of SEQ ID No. 5, and after said transformation unmethylated cytosines are converted to uracils, and methylated cytosines are unchanged, in the preparation of a liver tumor detection product.

2. The use of claim 1, wherein the nucleic acid set comprises a nucleic acid capable of binding to the DNA region in which the CHFR is located.

3. The use according to claim 1, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO: 2.

4. The use according to claim 1, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO:3 and 4.

5. The use of claim 1, wherein the nucleic acid set comprises a nucleic acid capable of binding to the DNA region in which the GRASP is located.

6. The use according to claim 1, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO: shown at 6.

7. The use according to claim 1, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO:7 and 8.

8. The use according to claim 1, wherein the target gene further comprises a gene selected from the group consisting of: IRF4, IKZF1, gene interval region 1 and BCAT1, wherein, the DNA region where IRF4 is located is the nucleic acid sequence shown in SEQ ID NO. 9, the DNA region where IKZF1 is located is the nucleic acid sequence shown in SEQ ID NO. 13, the DNA region where gene interval region 1 is located is the nucleic acid sequence shown in SEQ ID NO. 17, and the DNA region where BCAT1 is located is the nucleic acid sequence shown in SEQ ID NO. 21.

9. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid capable of binding to the region of DNA in which the IRF4 is located.

10. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO: shown at 10.

11. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO:11 and 12.

12. The use according to claim 8 wherein the nucleic acid set comprises a nucleic acid capable of binding to the region of DNA in which the IKZF1 is located.

13. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO: 14.

14. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO:15 and 16.

15. The use according to claim 8, wherein said nucleic acid set comprises a nucleic acid capable of binding to the DNA region in which said gene spacer 1 is located.

16. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO: shown at 18.

17. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO:19 and 20.

18. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid capable of binding to the DNA region in which the BCAT1 is located.

19. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO: shown at 22.

20. The use according to claim 8, wherein the nucleic acid set comprises a nucleic acid having a sequence as set forth in SEQ ID NO:23 and 24.

21. The use according to claim 8 wherein the gene of interest comprises CHFR, GRASP, IRF a 4 and IKZF1.

22. The use of claim 21, wherein the nucleic acid set comprises a nucleic acid having a sequence set forth in SEQ ID NO: 2. 3-4, 6, 7-8, 10, 11-12 and 14, 15-16.

23. The use according to claim 8, wherein the gene of interest comprises CHFR, GRASP, IRF a4, IKZF1, gene spacer 1 and BCAT1.

24. The use of claim 23, wherein the nucleic acid set comprises a nucleic acid having a sequence set forth in SEQ ID NO: 2. 3-4, 6, 7-8, 10, 11-12, 14, 15-16, 18, 19-20 and 22, 23-24.

25. The use according to claim 1, wherein the sample to be tested is plasma.

26. An application of a nucleic acid set in preparing a liver tumor detection kit, wherein the nucleic acid set comprises a sequence capable of determining the methylation state of a DNA region where a target gene is located, the target gene comprises CHFR and GRASP, the DNA region where CHFR is located is a nucleic acid sequence shown as SEQ ID NO. 1, and the DNA region where GRASP is located is a nucleic acid sequence shown as SEQ ID NO. 5.