CN117344021A

CN117344021A - Novel tumor diagnosis marker and application thereof

Info

Publication number: CN117344021A
Application number: CN202311472849.4A
Authority: CN
Inventors: 李振艳; 罗怀兵
Original assignee: Shanghai Epiprobe Biotechnology Co Ltd
Current assignee: Shanghai Epiprobe Biotechnology Co Ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2024-01-05
Also published as: CN117248024A; CN117604098A; CN117305461A; CN111020034B; CN111020034A; CN117265121A; CN117265120A; CN117248027A; CN117305463A; CN117248028A; CN117265122A; CN117305462A; CN117265119A; CN117248026A; CN117248025A; CN117512109A

Abstract

The present invention provides a class of DNA apparent modification related disease markers that exhibit significant differences in methylation status in patients with tumors. The tumor marker can be used as a marker for diagnosis, screening, typing, detection and prognosis of clinical tumors, can also be used as a novel molecule for clinical auxiliary diagnosis or prognosis of tumors, or can be used for designing diagnostic reagents and kits.

Description

Novel tumor diagnosis marker and application thereof

This patent application is a divisional application of the inventive patent application with application number CN 201911407889.4.

Technical Field

The invention belongs to the field of disease diagnosis markers, and in particular relates to a novel tumor diagnosis marker and application thereof.

Background

Epigenetics (Epigenomics) is a discipline that studies heritable changes in gene function and ultimately leads to phenotypic changes without DNA sequence alterations. Epigenetic mainly comprises biochemical processes such as DNA methylation (DNA methylation), histone modification (histone modification), microRNA level change and the like. DNA methylation is an epigenetic mechanism with deeper research, and has application prospect in clinical practice of tumor including diagnosis and treatment. DNA methylation refers to the process of transferring a methyl group to a specific base in an organism under the catalysis of DNA methyltransferase (DNA methyltransferase, DMT) using S-adenosylmethionine (SAM) as a methyl donor. DNA methylation may occur at the N-6 position of adenine, the N-4 position of cytosine, the N-7 position of guanine, the C-5 position of cytosine, or the like. In mammals DNA methylation occurs predominantly at the C of 5'-CpG-3', producing 5-methylcytosine (5 mC).

The distribution of more than 98% of the CpG dinucleotides scattered in the genome is located in repetitive sequences with transcription-dependent transposition potential. In normal cells, these cpgs are in a highly methylated/transcriptionally silent state, whereas in tumor cells they undergo extensive demethylation, leading to transcription of repeated sequences, activation of transposons, high genomic instability and protooncogene transcription enhancement. The remaining cpgs, which account for about 2% of the total, are densely distributed in smaller areas (CpG islands). About 40-50% of the gene promoter regions or their vicinity present CpG islands suggesting that DNA methylation may be involved in this type of gene transcriptional regulation mechanism.

In tumor cells, some CpG islands that were otherwise hypomethylated in normal cells are hypermethylated, resulting in transcriptional inactivation of the gene. The affected genes include oncogenes such as DNA repair genes, cell cycle control genes, and anti-apoptotic genes. After bisulphite treatment of genomic DNA, PCR (methylation specific PCR, MSP) detection can be performed to effectively determine the methylation status of a specific site of the test DNA fragment.

Although the whole sequence of human genome is known, the genome sequence is complex, which genes or segments are closely related to diseases, and the important subject of the research in the field still exists. Thus, there is a need to screen for new markers useful for disease diagnosis in combination with epigenetic techniques. In the inventors' prior studies, a part of tumor markers based on methylation modification were found. However, there is still a need to find more novel tumor markers, thereby providing more ways for diagnosis of tumors. Future tumor diagnosis necessarily requires the combined use of multiple markers to improve the accuracy of diagnosis.

Disclosure of Invention

The invention aims to provide a novel tumor diagnosis marker and application thereof.

In a first aspect of the invention there is provided the use of an isolated human nucleic acid or a nucleic acid converted therefrom in the preparation of a reagent or kit for tumour screening, diagnosis, detection or prognostic evaluation; wherein the human nucleic acid comprises (1) a nucleic acid or combination of nucleic acids of any one of the nucleotide sequences shown in SEQ ID NOs 1 to 16, or a nucleic acid or combination of nucleic acid modified CpG sites comprising at least 1 of said sequences, such as comprising 2 to 80 (any number which may be a positive integer of 2 to 80) or 3 to 77 (any number which may be a positive integer of 3 to 77) modified CpG sites; or (2) a nucleic acid or combination of nucleic acids that is complementary in sequence to the nucleic acids of (1); wherein the nucleic acid converted from human nucleic acid is a nucleic acid corresponding to (1) or (2), the unmodified cytosine of which is converted to T or U, and the cytosine C of the modified CpG site of which is unchanged.

In another preferred embodiment, the nucleic acid complementary to the nucleic acid of any one of the nucleotide sequences shown in SEQ ID NOS.1 to 16 is the nucleic acid of the nucleotide sequence shown in SEQ ID NOS.17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, respectively.

In another preferred embodiment, the sample of tumor comprises: tissue samples, paraffin embedded samples, blood samples, pleural effusion samples, alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, stool samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical or brush blade samples, tissue and cell biopsy samples.

In another aspect of the invention, there is provided an isolated human nucleic acid or combination of nucleic acids comprising: (1) Nucleic acid of the nucleotide sequence shown in any one of SEQ ID NO 1-16, or nucleic acid containing at least 1 modified CpG site in the sequence; or (2) a nucleic acid complementary in sequence to the nucleic acid of (1). Preferably, the "nucleic acid comprising at least 1 modified CpG site in the sequence" has a length of about 20-1392 bp, such as a length of 30, 40, 50, 60, 70, 80, 100, 150, 200, 300, 500, 1000bp.

In a preferred embodiment, the modified CpG sites include CpG sites that are modified with 5-aldehyde methylation, 5-hydroxymethyl, 5-methylation, or 5-carboxymethylation.

In another preferred embodiment, the isolated human nucleic acid comprises a nucleic acid selected from the group consisting of: nucleic acid of the nucleotide sequence shown in any one of SEQ ID NO 1-16; nucleic acid of 122 th to 175 th nucleotide sequence or complementary sequence in SEQ ID NO. 1; nucleic acid of 35 th to 65 th nucleotide sequence or complementary sequence in SEQ ID NO. 2; nucleic acid of 52 th to 65 th nucleotide sequences or complementary sequences in SEQ ID NO. 3; nucleic acid of 33 th to 70 th nucleotide sequence or complementary sequence in SEQ ID NO. 4; nucleic acid of 273 th to 291 th nucleotide sequence or complementary sequence in SEQ ID NO. 5; nucleic acid of nucleotide sequence 2-43 or complementary sequence in SEQ ID NO. 6; nucleic acid of 112-167 th nucleotide sequence or complementary sequence in SEQ ID NO. 7; nucleic acid of 16 th to 46 th nucleotide sequences or complementary sequences in SEQ ID NO. 8; nucleic acid of nucleotide sequence 1-28 or complementary sequence in SEQ ID NO. 9; nucleic acid of nucleotide sequence 27-40 or complementary sequence in SEQ ID NO. 10; nucleic acid of 63-91 nucleotide sequence or complementary sequence in SEQ ID NO. 11; nucleic acid of nucleotide sequence 1-39 or complementary sequence in SEQ ID NO. 13; nucleic acid of the 21 st to 32 nd nucleotide sequence or the complementary sequence in SEQ ID NO. 14; nucleic acid of 497 to 536 nucleotide sequences or complementary sequences thereof in SEQ ID NO. 15; or the nucleotide sequence from 44 th to 76 th positions in SEQ ID NO. 16 or the complementary sequence thereof.

In another aspect of the invention there is provided a nucleic acid or combination of nucleic acids converted from the aforementioned isolated nucleic acid or combination of nucleic acids, corresponding to the aforementioned isolated nucleic acid, whose unmodified cytosine is converted to T or U, while its modified CpG site has unchanged cytosine C.

In another preferred embodiment, the converted nucleic acid or combination of nucleic acids is converted from a nucleic acid or combination of nucleic acids corresponding to the aforementioned isolated nucleic acids according to SEQ ID NOS 1-16, by bisulfite or bisulfite treatment. Preferably, it includes: a nucleic acid or nucleic acid fragment of a nucleotide sequence as set forth in SEQ ID NOs 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, or a nucleic acid or nucleic acid fragment of a nucleotide sequence as set forth in SEQ ID NOs 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109; the nucleic acid fragment includes at least 1 (e.g., including 2 to 80, any number of positive integers, which may be 2 to 80) modified CpG sites. Preferably, the nucleic acid fragment has a length of about 20 to 1392bp, such as length 30, 40, 50, 60, 70, 80, 100, 150, 200, 300, 500, 1000bp.

In another preferred embodiment, the fragment of the nucleic acid comprises: nucleic acid of nucleotide sequence 122-175 or complementary sequence in SEQ ID NO. 18; nucleic acid of 35 th to 65 th nucleotide sequence or complementary sequence in SEQ ID NO. 24; nucleic acid of 52 th to 65 th nucleotide sequence or complementary sequence in SEQ ID NO. 30; nucleic acid of 33-70 nucleotide sequence or complementary sequence in SEQ ID NO. 36; nucleic acid of 273 th to 291 th nucleotide sequence or complementary sequence in SEQ ID NO. 42; nucleic acid of nucleotide sequence 2-43 or complementary sequence in SEQ ID NO. 48; nucleic acid of 112-167 th nucleotide sequence or complementary sequence in SEQ ID NO. 54; nucleic acid of nucleotide sequence 16-46 or complementary sequence in SEQ ID NO. 60; nucleic acid of nucleotide sequence 1-28 or complementary sequence in SEQ ID NO. 66; nucleic acid of nucleotide sequence 27-40 or complementary sequence in SEQ ID NO. 72; nucleic acid of nucleotide sequence 63-91 or complementary sequence in SEQ ID NO. 78; nucleic acid of nucleotide sequence 1-39 or complementary sequence in SEQ ID NO. 90; nucleic acid of the 21 st to 32 nd nucleotide sequence or the complementary sequence in SEQ ID NO. 96; nucleic acid of 497 to 536 nucleotide sequences or the complementary sequence thereof in SEQ ID NO. 102; or the nucleotide sequence from 44 th to 76 th in SEQ ID NO. 108 or the complementary sequence thereof.

In another aspect of the invention, there is provided a reagent or combination of reagents which specifically detects CpG site modifications of a target sequence, said target sequence being a full length or fragment of the nucleic acid of any one of claims 3 to 8, comprising at least 1 (e.g. comprising 2 to 80, any number which may be a positive integer from 2 to 80) modified CpG site; preferably, the agent or combination of agents is directed against a gene sequence comprising the target sequence, preferably the gene sequence comprises a gene Panel or group of genes; preferably, the reagent or combination of reagents comprises reagents for specifically detecting 2 to 16 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) sequences of SEQ ID NOs 1 to 16.

In another preferred embodiment, the agent or combination of agents is directed against a gene sequence comprising the target sequence (designed based on the gene sequence), preferably the gene sequence comprises a gene Panel or group of genes.

In another aspect of the invention, there is provided a method of preparing a reagent for tumour diagnosis, screening, detection, typing or prognosis evaluation, the method comprising: providing the isolated human nucleic acid, taking the full length or fragment of the nucleic acid as a target sequence, and designing a detection reagent for specifically detecting the CpG site modification condition of the target sequence; wherein the target sequence comprises at least 1 (such as any number comprising 2-80 positive integers which can be 2-80) modified CpG sites; preferably, the detection reagent includes, but is not limited to: primers, probes, chips or strips.

In another preferred embodiment, the detection reagent comprises a reagent for specifically detecting 2 to 16 (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) sequences in SEQ ID NO. 1 to 16; preferably, a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) of said detection reagents are integrated on a chip.

In another aspect of the invention, a kit for performing tumor detection, screening, typing, diagnosis or prognosis evaluation is provided, comprising the reagents or reagent combinations as described above.

In another preferred embodiment, the reagent or combination of reagents comprises a primer.

In another preferred embodiment, the primer comprises: the primer comprises a primer selected from the group consisting of: SEQ ID NO. 20 and SEQ ID NO. 21, or SEQ ID NO. 22; SEQ ID NO 26 and SEQ ID NO 27, or SEQ ID NO 28; SEQ ID NO. 32 and SEQ ID NO. 33, or SEQ ID NO. 34; SEQ ID NO 38 and SEQ ID NO 39, or SEQ ID NO 40; SEQ ID NO 44 and SEQ ID NO 45, or SEQ ID NO 46; SEQ ID NO. 50 and SEQ ID NO. 51, or SEQ ID NO. 52; SEQ ID NO. 56 and SEQ ID NO. 57, or SEQ ID NO. 58; SEQ ID NO. 62 and SEQ ID NO. 63, or SEQ ID NO. 64; SEQ ID NO. 68 and SEQ ID NO. 69, or SEQ ID NO. 70; SEQ ID NO 74 and SEQ ID NO 75, or SEQ ID NO 76; 80 and 81 or also includes 82; SEQ ID NO 86 and SEQ ID NO 87, or SEQ ID NO 88; SEQ ID NO. 92 and SEQ ID NO. 93, or SEQ ID NO. 94; 98 and 99, or also SEQ ID NO 100; SEQ ID NO 104 and SEQ ID NO 105, or SEQ ID NO 106; and/or SEQ ID NO 110 and SEQ ID NO 111, or SEQ ID NO 112.

In a preferred embodiment, the kit may further include, but is not limited to: DNA purification reagents, DNA extraction reagents, bisulphite or bisulphite, PCR amplification reagents.

In other preferred embodiments, the kit further comprises: instructions for designating the detection procedure and the result determination criteria.

In another aspect of the invention there is provided the use of said reagent or reagent combination for the preparation of a kit for tumour diagnosis, screening, genotyping or prognostic assessment.

In another aspect of the invention, there is provided a method of detecting the methylation level of a test sample comprising: extracting nucleic acid of a sample to be detected; and detecting CpG site modification of a target sequence in the extracted nucleic acid, wherein the target sequence is any one of the converted nucleic acids.

In another preferred embodiment, the method for detecting CpG site modification of a target sequence in an extracted nucleic acid comprises: pyrosequencing, bisulfite conversion sequencing, methylation chip, qPCR, digital PCR, second generation sequencing, third generation sequencing, whole genome methylation sequencing, DNA enrichment detection, reduced bisulfite sequencing, HPLC, massArray, methylation specific PCR, or a combination thereof; or a combined gene group in vitro detection method of partial or whole methylation sites in the sequence shown in SEQ ID NO. 1 and an in vivo tracing detection method.

In another preferred embodiment, the method for detecting CpG site modification of a target sequence in an extracted nucleic acid comprises: (i) Treating the extracted nucleic acid to convert unmodified cytosine to uracil; preferably, the modification comprises a 5-methylation modification, a 5-hydroxymethylation modification, a 5-aldehyde methylation modification or a 5-carboxymethylation modification; preferably, the nucleic acid of step (i) is treated with bisulphite or bisulphite; (ii) Analyzing the modification of said target sequence in the nucleic acid treated in (i).

In another preferred embodiment, in step (ii), the method of analysis comprises: pyrosequencing, bisulfite conversion sequencing, methylation chip, qPCR, digital PCR, second generation sequencing, third generation sequencing, whole genome methylation sequencing, DNA enrichment detection, reduced bisulfite sequencing, HPLC, massArray, methylation Specific PCR (MSP), or combinations thereof, and combined genome in vitro detection methods and in vivo tracer detection methods for part or all of the methylation sites in the sequence shown in SEQ ID NO. 1. Also, other methylation detection methods and methylation detection methods newly developed in the future may be applied to the present invention.

In another preferred embodiment, the method of methylation of the spectrum is not diagnostic, i.e. it is not aimed at directly obtaining a diagnostic result of the disease.

In another preferred embodiment, the method for detecting the methylation pattern of a sample is an in vitro method.

In another preferred embodiment, step (ii) comprises: (1) Treating the product of (i) to convert unmodified cytosine to uracil; preferably, the modification comprises a 5-methylation modification, a 5-hydroxymethylation modification, a 5-aldehyde methylation modification or a 5-carboxymethylation modification; preferably, the nucleic acid of step (i) is treated with bisulphite or bisulphite; (2) Analyzing the modification of the target sequence in the nucleic acid treated in (1).

In another preferred embodiment, the methylation profile abnormality is a hypermethylation of C in the CpG of the nucleic acid.

According to any of the preceding aspects, the tumor comprises: hematological tumors, digestive system tumors, gynecological tumors, reproductive system tumors, nervous system tumors, urinary system tumors, and other system tumors; preferably, the hematological neoplasm is a leukemia, lymphoma, multiple myeloma; digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer and gallbladder cancer; gynaecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; tumors of the nervous system such as glioma, neuroblastoma, meningioma; head and neck tumors such as oral cavity cancer, tongue cancer, laryngeal cancer and nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer, skin cancer; other systemic tumors such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer; respiratory system tumors such as lung cancer and pleural tumor.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, a graph of the results of analyzing methylation levels of corresponding sites in target genes in tumor tissue or normal tissue based on public database information.

FIG. 2, results of methylation levels of target gene sites in ten tumor cell lines and three control samples of leukocytes.

In the clinical samples of leukemia, the methylation values of the sites in the sequences shown in SEQ ID NOs 1 to 16 are compared in an experimental group and a control group.

In the clinical samples of breast cancer, the methylation values of the sites in the sequences shown in SEQ ID NOs 1-16 are compared in an experimental group and a control group.

In FIG. 5, in the rectal cancer clinical samples, the methylation values of the sites in the sequences shown in SEQ ID NOs 1 to 16 are compared in the experimental group and the control group.

In FIG. 6, esophageal cancer clinical samples, the methylation values of the sites in the sequences shown in SEQ ID NOs 1-16 are compared in an experimental group and a control group.

FIG. 7, methylation value comparison of sites in the sequences shown in SEQ ID NOS.1 to 16 in the experimental group and the control group in the stomach clinical samples.

In the clinical samples of the head and neck tumor, the methylation values of the sites in the sequences shown in SEQ ID NOs 1-16 are compared in an experimental group and a control group.

In FIG. 9, liver cancer clinical samples, methylation values of sites in sequences shown in SEQ ID NOs 1 to 16 in an experimental group and a control group are compared.

In the clinical samples of lung cancer, the methylation values of the sites in the sequences shown in SEQ ID NOs 1 to 16 are compared in an experimental group and a control group.

In FIG. 11, pancreatic cancer clinical samples, the methylation values of the sites in the sequences shown in SEQ ID NOs 1 to 16 were compared in the experimental group and the control group.

Detailed Description

Through intensive researches and analyses, the inventor separates and obtains a kind of DNA apparent modification related disease markers, the markers show significant methylation state differences in patients suffering from tumors, the statistical significance of the differences is significant, and the differences can be shown in solid tumors, non-solid tumors and the like, such as blood tumors, and the solid tumors such as liver cancer, lung cancer and the like. Therefore, the tumor marker can be used as a marker for diagnosis, screening, parting, detection and prognosis of clinical tumors, can also be used as a novel molecule for clinical auxiliary diagnosis or prognosis of tumors, or can be used for designing diagnostic reagents and kits.

In the present invention, the term "sample" or "specimen" includes substances obtained from any individual (preferably a human) or isolated tissue, cells or body fluids (such as plasma) suitable for detection of the methylation state of DNA. For example, the samples shown may include, but are not limited to: blood samples, tissue samples, paraffin embedded samples, pleural effusion samples, alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, stool samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, cervical or brush samples, tissue and cell biopsy samples.

In the present invention, the term "hypermethylation" refers to the presence of hypermethylation, methylolation, aldehyde methylation or carboxymethylation modifications of CpG in a gene sequence. For example, in the case of Methylation Specific PCR (MSP) analysis, a positive PCR result can be obtained by a PCR reaction with methylation specific primers, and the DNA (gene) region to be tested can be considered to be in a hypermethylated state. For example, in the case of real-time quantitative methylation-specific PCR, the determination of hypermethylation status can be based on the analysis of statistical differences in relative values of methylation status of its control samples.

In the invention, the methylation state of any one of the gene sequences shown in SEQ ID NO 1-16 or a part of the gene sequence has a remarkable difference between tumor tissues and non-tumor tissues, and the subject can be judged to suffer from tumor or belong to a tumor high risk group as long as the abnormal hypermethylation state of the gene sequence region is detected. Furthermore, the significant differences between tumor tissue and non-tumor tissue represented by any one of the gene sequences shown in SEQ ID NOs 1 to 16 or a partial region thereof are widely present in different kinds of tumors, including solid tumors such as intestinal cancer, lung cancer and the like, and also include non-solid tumors. The results of studies on solid tumors as well as non-solid tumors are confirmed in clinical studies, and the details can be seen in the specific description of the examples.

According to the above, the present invention provides a nucleic acid derived from a specific region of the human genome, which has a gene sequence shown in any one of SEQ ID NOS 1 to 16 or a partial region thereof, and also includes an antisense strand thereof. In tumor cells, 5-methylcytosine (5 mC) is produced at the base C position of the 5'-CpG-3' at multiple positions in the nucleic acid sequence.

Detection of one or more CpG's provided herein is possible, and thus the invention also encompasses fragments of nucleic acids of the nucleotide sequences, including at least 1 methylated CpG site. The at least one may comprise any number from 2 to 80, which may be a positive integer from 2 to 80, more particularly 3,5,8, 10, 12, 15, 18, 20, 30, 40, 50, 60, 70.

The CpG sites can be conveniently obtained and used by a person skilled in the art after the information of the specific fragment in the human genome is provided by the invention. In the examples of the present invention a series of fragments of sequences containing CpG sites are provided, as examples of preferred embodiments, but it will be appreciated that one could make variations and select other fragments based on the information provided by the present invention.

The invention also includes a gene Panel or a gene group of the nucleotide sequence or the sequence fragment shown in any one of SEQ ID NOs 1 to 16 or the complementary sequence thereof. For the gene Panel or gene group, the characteristics of normal cells and tumor cells can also be obtained through DNA methylation state detection.

It will be appreciated that a wide variety of techniques that can be used to analyze methylation status can be used in the present invention, and that the present invention is not particularly limited to such detection techniques. The nucleic acid provided by the invention can be used as a key region for analyzing methylation state in genome, and can be used for analyzing methylation state by various techniques known in the art so as to analyze occurrence or development of tumors.

The nucleic acid of any one of SEQ ID NOS 1 to 16 or a fragment thereof or a sequence complementary thereto according to the present invention may be such that, after bisulfite or bisulfite treatment, unmethylated cytosines are converted to uracil while methylated cytosines remain unchanged. Thus, the present invention also provides a nucleic acid obtained by subjecting the above-mentioned nucleic acid including its complementary strand (antisense strand) to bisulfite or bisulfite treatment, comprising: a nucleic acid or nucleic acid fragment of the nucleotide sequence shown in SEQ ID NO. 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108 or a nucleic acid or nucleic acid fragment of the nucleotide sequence shown in SEQ ID NO. 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109. These nucleic acids can be used as more direct targets for designing detection reagents or detection kits.

The invention also encompasses fragments of the above nucleic acids or nucleic acids obtained by bisulfite or bisulfite treatment of the antisense strand thereof and which comprise at least 1 methylated CpG site. The at least one may comprise any number from 2 to 80, which may be a positive integer from 2 to 80, more particularly 3,5,8, 10, 12, 15, 18, 20, 30, 40, 50, 60, 70. It will be appreciated by those skilled in the art that after the present invention provides a CpG numbering based on one DNA strand, the numbering of the individual CpG sites in the complementary DNA strand corresponding to the sense strand is readily available in accordance with the teachings provided herein.

The nucleic acid of the nucleotide sequence shown in any one of SEQ ID NOs 1 to 16 and/or the complementary nucleic acid thereof can be integrated into one or a plurality of integers, such as one or a plurality of nucleic acid sets, for the application of the technical personnel, such as selecting one or a plurality of nucleic acids or fragments of the nucleic acids from the nucleic acid sets, so as to design a target analysis reagent. The designed targeted assay reagents may also be integrated into one or several entities, such as one or several kits. This integration is advantageous for achieving high throughput analysis.

The nucleic acids according to the invention, which are converted from the nucleic acids of the nucleotide sequences indicated in any one of SEQ ID NOS.1 to 16 and/or the complementary nucleic acids thereof, can also be integrated in one or several entities, for example one or several nucleic acid sets, from which one or more nucleic acids or fragments of nucleic acids are selected for use by a person skilled in the art, for example for designing targeted analysis reagents. The designed targeted assay reagents may also be integrated into one or several entities, such as one or several kits, or one or several chips. This integration is advantageous for achieving high throughput analysis.

On the basis of the present invention that provides a target gene, these techniques, as well as techniques that are well known in the art and that are to be developed, can be applied to the present invention to conduct the detection of methylation level. The determination of the methylation profile of a nucleic acid can be performed by existing techniques such as Methylation Specific PCR (MSP) or real-time quantitative methylation specific PCR (methyl) or other techniques that are still under development and that will be developed. For example, quantitative methylation-specific PCR (QMSP) methods, which are based on the continuous optical monitoring of a fluorescent PCR, are more sensitive than MSP methods, can be used in detecting methylation levels. The throughput is high and the analysis of the results by electrophoresis is avoided. In addition, other available techniques are: qPCR method, second generation sequencing method, pyrosequencing method, bisulfite conversion sequencing method, whole genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technique or HPLC method, and combined genome detection method. While certain preferred forms are provided in the embodiments of the invention, the general inventive concept is not so limited.

As a preferred mode of the present invention, there is also provided a method for detecting the methylation profile of a nucleic acid in a sample in vitro. The method is based on the following principle: bisulphite or bisulphite can convert unmethylated cytosines to uracil, which in the subsequent PCR amplification process is converted to thymine, while methylated cytosines remain unchanged; thus, after bisulfite or bisulfite treatment of nucleic acids, methylated sites produce nucleic acid polymorphisms (SNPs) resembling a C/T. By identifying the methylation pattern of the nucleic acid in the test sample based on the above principle, methylated and unmethylated cytosines can be effectively distinguished.

The method of the invention comprises the following steps: comprising the following steps: firstly, providing a sample and extracting genome DNA; second, treating the genomic DNA of step (a) with bisulfite or bisulfite, whereby unmethylated cytosines in the genomic DNA are converted to uracil; again, the genomic DNA treated in step (b) was analyzed for the presence of methylation pattern abnormalities.

The method of the invention can be used for: detecting a sample of the subject, and assessing whether the subject has a tumor; or for distinguishing high risk groups of tumors. The method may be a situation that is not aimed at obtaining a direct disease diagnosis, such as a situation that is not aimed at judging the final disease outcome, a regional analysis study of the population, a scientific study, a census, etc.

In the preferred embodiment of the present invention, DNA methylation is detected by PCR amplification and pyrosequencing, and the practice is not limited to this method, and other DNA methylation detection methods known in the art or being modified may be used. In performing PCR amplification, the primers used are not limited to those provided in the examples, but primers differing in sequence from those provided in the examples of the present invention, but still directed to the nucleic acid or corresponding CpG sites indicated in the present invention, can be obtained.

Other methods and reagents known to those skilled in the art for determining genomic sequences, variations thereof, and methylation status of marker nucleic acids provided herein are included in the present invention.

The invention provides a method for preparing a tumor detection reagent, which comprises the following steps: providing the nucleic acid, taking the whole length or the fragment of the nucleic acid as a target sequence, and designing a detection reagent for specifically detecting the target sequence; wherein the target sequence comprises at least 1 methylation CpG site. The detection reagent may include, but is not limited to: chips, primers, probes, etc.; after the label is obtained, the selection of the detection reagent is within the skill of the art.

After knowledge of the sequence of the nucleic acid, it is known to the person skilled in the art to design primers which flank the specific sequence of the target gene to be amplified (including CpG sequences, gene regions in which CpG is complementary to the gene for the original methylation and gene regions in which TpG is complementary to the gene for the original demethylation). In a preferred embodiment of the invention, the reagents are primers, preferably those listed in tables 2 to 17. In addition to primers, other diagnostic or detection reagents may be prepared, including but not limited to probes, chips, and the like.

The reagent may also be a combination of reagents, such as a primer combination. For example, the combination may include more than one set of primers, such that the plurality of nucleic acids may be amplified separately.

The invention also provides a kit for detecting methylation profile of nucleic acid in a sample in vitro, the kit comprising: a container, and the primer pair located in the container.

The kit may further include various reagents required for DNA extraction, DNA purification, PCR amplification, etc., and other reagents such as sample processing reagents. In addition, instructions for use may be included in the kit, wherein the assay procedures and result determination criteria are indicated for use by those skilled in the art.

The method and the reagent provided by the invention have very high accuracy when being used for diagnosing clinical tumors, and are embodied in detection of various tumor clinical samples in the embodiment of the invention. The invention can be applied to the fields of pre-tumor screening, curative effect judgment, auxiliary diagnosis, prognosis monitoring and the like, or the situation that the aim of obtaining a direct disease diagnosis result is not achieved as described above.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 screening and obtaining of tumor-associated markers

The occurrence or progression of human diseases has a close correlation with genes. The human genome has huge and complex genetic information, which hides the genes closely related to the occurrence and development of diseases, and may have some genes or fragments capable of distinguishing diseases from non-diseases. The present inventors have made intensive studies on tumor diagnosis, and have conducted extensive analyses of human genome in order to obtain genes closely related to tumors, have searched for molecules having significant differences in methylation in tumors as well as in non-tumors, and have determined a series of gene segments, including gene segments selected from SEQ ID NO:1 to SEQ ID NO:16, as specific sequences are listed in Table 1, on the basis of extensive analyses and experimental demonstration.

The bisulfite treated sequences of SEQ ID NO. 1-16 are SEQ ID NO. 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, respectively.

Meanwhile, the inventors have also obtained the reverse complements of SEQ ID NO. 1 to SEQ ID NO. 16, which are SEQ ID NO. 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, respectively. The bisulfite treated sequences for the reverse complement are SEQ ID NOs 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, respectively.

The inventors grouped each target gene segment, each target segment, its reverse sequence, and their bisulfite treated sequences into 16 groups as shown in Table 1.

Table 1 x

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* In the table, the base marked by the term "is taken as a methylated CpG site or a corresponding site after being treated by bisulfite, and the corresponding number of the site (lower part) is CpG number; the underlined position is the detection target position region in the subsequent partial embodiment.

Example 2 analysis of methylation level of corresponding target Gene based on public database information

The inventors have summarized a DNA methylation dataset of tumor tissue samples in a common database (the common database comprising TCGA, roadmap, ENCODE). The tumor tissue samples included the following 17 total: bladder cancer, breast cancer, cervical cancer, gall bladder cancer, colon cancer, rectal cancer, head and neck tumor, esophageal cancer, kidney cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, stomach cancer, thyroid cancer, endometrial cancer); normal tissue samples included the following 17: bladder tissue, breast tissue, cervical tissue, gall bladder tissue, colon tissue, head and neck tissue, esophageal tissue, kidney tissue, liver tissue, lung tissue, pancreatic tissue, prostate tissue, skin tissue, stomach tissue, thyroid tissue, endometrial tissue. After obtaining the DNA methylation dataset of each sample, the average methylation levels of the sequences shown in SEQ ID NOs 1 to 16 in all tumors as well as in the control samples were calculated.

As a result, as shown in FIG. 1, it was found that the methylation levels of the sequences shown by SEQ ID NOS.1 to 16 were generally over 70% in tumor samples, and generally lower than 10% in control normal tissue samples. Thus, the methylation level of the sequences shown in SEQ ID NOS 1-16 in the tumor sample is significantly higher than that of the control sample.

Example 3 preparation of diagnostic reagents and detection of methylation level of tumor cells

1. Design of diagnostic reagents

1. Group 1

Primer design was performed for the target gene positions of group 1. Wherein, the CpG sites detected by pyrophosphate are CpG sites corresponding to 013-022 in SEQ ID NO. 1 (namely CpG contained in the corresponding position of the underlined partial sequence in the table 1). The primer sequences are specifically shown in Table 2.

TABLE 2

2. Group 2

Primer design was performed for the target gene positions of the groups. Wherein, the CpG sites detected by pyrophosphate are CpG sites corresponding to the 05 to 11 numbers of SEQ ID NO. 2 (namely CpG contained in the corresponding position of the underlined partial sequence in the table 1). The primer sequences are specifically shown in Table 3.

TABLE 3 Table 3

3. Group 3

Primer design was performed for the target gene positions of the groups. Wherein, the CpG sites detected by pyrophosphate are CpG sites corresponding to the numbers 03 to 06 in SEQ ID NO. 3 (namely CpG contained in the corresponding position of the underlined partial sequence in the table 1). The primer sequences are specifically shown in Table 4.

TABLE 4 Table 4

4. Group 4

Primer design was performed for the target gene positions of the groups. Wherein the pyrophosphate detection CpG sites are CpG sites corresponding to the numbers 04 to 12 in SEQ ID NO. 4 (namely CpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 5.

TABLE 5

5. Group 5

Primer design was performed for the target gene positions of the groups. Wherein the pyrophosphate detection CpG sites are CpG sites corresponding to the numbers 13 to 17 in SEQ ID NO. 5 (i.e., cpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 6.

TABLE 6

6. Group 6

Primer design was performed for the target gene positions of the groups. Wherein the CpG sites detected by pyrophosphate are CpG sites corresponding to the numbers 01 to 04 in SEQ ID NO. 6 (namely CpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 7.

TABLE 7

7. Group 7

Primer design was performed for the target gene positions of the groups. Wherein the pyrophosphate detection CpG sites are CpG sites corresponding to the numbers 08 to 18 in SEQ ID NO. 7 (i.e., cpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 8.

TABLE 8

8. Group 8

Primer design was performed for the target gene positions of the groups. Wherein the pyrophosphate detection CpG sites are CpG sites corresponding to the numbers 02 to 07 in SEQ ID NO. 8 (i.e., cpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 9.

TABLE 9

9. Group 9

Primer design was performed for the target gene positions of the groups. Wherein the CpG sites detected by pyrophosphate are CpG sites corresponding to the numbers 01 to 05 in SEQ ID NO. 9 (namely CpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 10.

Table 10

10. Group 10

Primer design was performed for the target gene positions of the groups. Wherein the CpG sites detected by pyrophosphate are CpG sites corresponding to the numbers 05 to 09 in SEQ ID NO. 10 (i.e., cpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 11.

TABLE 11

11. Group 11

Primer design was performed for the target gene positions of the groups. Wherein the pyrophosphate detection CpG sites are CpG sites corresponding to the numbers 04 to 07 in SEQ ID NO. 11 (i.e., cpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 12.

Table 12

12. Group 12

Primer design was performed for the target gene positions of the groups. Wherein the CpG sites detected by pyrophosphate are CpG sites corresponding to the numbers 01 to 05 in SEQ ID NO. 12 (namely CpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 13.

TABLE 13

13. Group 13

Primer design was performed for the target gene positions of the groups. Wherein the CpG sites detected by pyrophosphate are CpG sites corresponding to the numbers 01 to 07 in SEQ ID NO. 13 (i.e., cpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 14.

TABLE 14

14. Group 14

Primer design was performed for the target gene positions of the groups. Wherein the pyrophosphate detection CpG sites are CpG sites corresponding to the numbers 02-05 in SEQ ID NO. 14 (i.e., cpG sites are included in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 15.

TABLE 15

15. Group 15

Primer design was performed for the target gene positions of the groups. Wherein the CpG sites detected by pyrophosphate are CpG sites corresponding to the numbers 26 to 31 in SEQ ID NO. 15 (i.e., cpG sites contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 16.

Table 16

16. Group 16

Primer design was performed for the target gene positions of the groups. Wherein the pyrophosphate detection CpG sites are CpG sites corresponding to numbers 07 to 12 in SEQ ID NO. 16 (i.e., cpG sites are contained in the corresponding positions of the underlined partial sequences in Table 1). The primer sequences are specifically shown in Table 17.

TABLE 17

2. Methylation detection against tumor cells

The inventors collected ten tumor cells, obtained their genomes, and examined the methylation levels of the sequences shown in SEQ ID NOs 1 to 16. The tumor cells are A549 (lung cancer), HCT116 (colorectal cancer), K562 (chronic myelogenous leukemia), 97L (liver cancer), hela (cervical cancer), HUCCT1 (cholangiocarcinoma), SW1990 (pancreatic cancer), GBC-SD (gallbladder cancer), MCF7 (breast cancer) and 746T (gastric cancer). Meanwhile, the present inventors collected three cases of white blood cells (WBCs 1 to 3) as control samples.

The pyrosequencing method (pyrosequencing) is adopted during detection, and the main steps are as follows:

methylation differences between tumor and non-tumor samples are detected by pyrosequencing, which comprises the following steps:

(1) Obtaining a sample: selecting a tumor cell line-leucocyte control or clinically obtaining a cancer side/non-cancer side/cancer tissue sample, wherein leucocytes and the cancer side/non-cancer sample are used as a control group, and the tumor cell line and the cancer tissue sample are used as a tumor detection experimental group;

(2) DNA extraction: extracting DNA of an experimental group and DNA of a control group respectively; phenol chloroform extraction method is used in the experiment;

(3) Bisulfite treatment: treating the extracted DNA sample with bisulfite, and performing the steps; EZ DNA Methylation-Gold Kit from ZYMO Research, cat# D5006;

(4) Primer design: designing PCR amplification primers and pyrophosphoric acid sequencing primers according to the target gene sequence, as shown in the subsequent corresponding list;

(5) PCR amplification and agarose gel electrophoresis: taking the sample treated by the bisulfite as a template of PCR, performing PCR amplification, and identifying the specificity of the PCR amplification of the amplified product by agarose gel electrophoresis;

(6) Pyrosequencing: detecting by a Pyro Mark Q96 ID pyrosequencer of QIAGEN company, and operating according to the specification steps;

(7) Methylation value calculation: the methylation condition of single CpG sites in the target area can be independently detected by pyrosequencing, and the methylation average value of all CpG sites is calculated and used as the methylation value in the sample;

(8) Analysis of results: methylation values of target gene sequences in the control and experimental groups were compared.

As a result, as shown in FIG. 2 (each column corresponds to the top-down order from left to right, and the arrows indicate the control), the methylation level (about 50-99%) of the sequences shown in SEQ ID NOS 1-16 was significantly higher than that of the white blood cell control samples (about 20% or less), both in ten tumor cell lines and in three white blood cell control samples.

Example 4 clinical detection of leukemia

The inventors obtained 12 non-leukemia bone marrow smear samples clinically as a control group and obtained 12 leukemia bone marrow smear samples simultaneously as an experimental group, and compared methylation levels of sequences shown in SEQ ID NOs 1 to 16 of the control group and the experimental group according to the pyrophosphoric acid test procedure of example 3.

As a result, as shown in FIG. 3, in the leukemia clinical samples, the methylation values of the sequences shown in SEQ ID NOs 1 to 16 are significantly higher in the experimental group than in the control group, and the methylation level is generally increased by about 2 to 8 times.

Therefore, the methylation level detection of any one sequence shown in SEQ ID NO 1-16 can be used for clinical diagnosis of leukemia.

Example 5 clinical detection of Breast cancer

The inventors obtained 5 cases of tissue samples beside breast cancer as a control group and 5 cases of tissue samples beside breast cancer as an experimental group clinically, and compared methylation levels of sequences shown in SEQ ID NOs 1 to 16 of the control group and the experimental group according to the pyrophosphoric acid test procedure of example 3.

As a result, as shown in FIG. 4, in the clinical sample of breast cancer, the methylation values of the sequences shown in SEQ ID NOs 1 to 16 are significantly higher in the experimental group than in the control group (the columns indicated by the arrows), and the increase of the methylation level is generally about 2 to 8 times.

Therefore, the methylation level detection of any one sequence shown in SEQ ID NO. 1-16 can be used for clinical diagnosis of breast cancer.

Example 6 clinical detection of colorectal cancer

The inventors obtained 7 cases of colorectal cancer-side tissue samples clinically as a control group and 7 cases of colorectal cancer tissue samples simultaneously as an experimental group, and compared methylation levels of sequences shown in SEQ ID NOs 1 to 16 of the control group with those of the experimental group according to the pyrophosphoric acid test procedure of example 3.

As a result, as shown in FIG. 5, in the colorectal cancer clinical samples, the methylation values of the sequences shown in SEQ ID NOs 1 to 16 were significantly higher in the experimental group than in the control group (the columns indicated by the arrows), and the increase in methylation level was generally about 2 to 8 times.

Therefore, the methylation level detection of any one of the sequences shown in SEQ ID NO 1-16 can be used for the clinical diagnosis of rectal cancer.

Example 7 clinical detection of esophageal cancer

The inventors obtained 10 cases of esophageal cancer tissue samples clinically as a control group and 10 cases of esophageal cancer tissue samples as an experimental group, and compared methylation levels of sequences shown in SEQ ID NOs 1 to 16 of the control group and the experimental group according to the pyrophosphoric acid test procedure of example 3 above.

As a result, as shown in FIG. 6, in the clinical esophageal cancer sample, the methylation values of the sequences shown in SEQ ID NOs 1 to 16 were significantly higher in the experimental group than in the control group (the columns indicated by the arrows), and the increase in methylation level was generally about 2 to 7 times.

Therefore, the methylation level detection of any one sequence shown in SEQ ID NO 1-16 can be used for clinical diagnosis of esophageal cancer.

Example 8 clinical detection of gastric cancer

The inventors obtained 5 cases of tissue samples of patients suffering from stomach inflammation from clinic as a control group, and obtained 5 cases of stomach cancer tissue samples as an experimental group, and compared methylation levels of sequences shown in SEQ ID NOs 1 to 16 of the control group and the experimental group according to the above pyrophosphoric acid test procedure of example 3.

As shown in FIG. 7, the methylation values of the sequences shown in SEQ ID NOs 1 to 16 in the clinical samples of gastric cancer are significantly higher than those of the control group (the columns indicated by arrows), and the methylation level is generally increased by about 2 to 4 times.

Therefore, the methylation level detection of any one sequence shown in SEQ ID NO 1-16 can be used for clinical diagnosis of gastric cancer.

Example 9 clinical detection of head and neck tumor

The inventors obtained 10 cases of head and neck tumor tissue samples from clinic as a control group and 10 cases of head and neck tumor tissue samples as an experimental group, and compared methylation levels of sequences shown in SEQ ID NOs 1 to 16 of the control group and the experimental group according to the pyrophosphoric acid test procedure of example 3 above.

As shown in FIG. 8, in clinical samples of head and neck tumor, the methylation values of the sequences shown in SEQ ID NOs 1 to 16 are significantly higher in the experimental group than in the control group (the columns indicated by arrows), and the methylation level is generally increased by about 2 to 4 times.

Therefore, the methylation level detection of any one sequence shown in SEQ ID NO 1-16 can be used for clinical diagnosis of head and neck tumor.

Example 10 clinical detection of liver cancer

The inventors obtained 9 cases of tissue samples beside liver cancer as a control group and 9 cases of tissue samples of liver cancer as an experimental group, and compared methylation levels of sequences shown in SEQ ID NO 1 to 16 of the control group and the experimental group according to the pyrophosphoric acid test procedure of example 3 above.

As shown in FIG. 9, in the clinical liver cancer samples, the methylation values of the sequences shown in SEQ ID NOs 1 to 16 are significantly higher than those of the control group (the columns indicated by arrows), and the methylation level is generally increased by about 2 to 4 times.

Therefore, the methylation level detection of any sequence shown in SEQ ID NO 1-16 can be used for clinical diagnosis of liver cancer.

Example 11 clinical detection of Lung cancer

The inventors obtained 8 lung cancer tissue samples clinically as a control group and 8 lung cancer tissue samples as an experimental group, and compared methylation levels of sequences shown in SEQ ID NOs 1 to 16 of the control group and the experimental group according to the pyrophosphoric acid test procedure of example 3 above.

As a result, as shown in FIG. 10, in the clinical samples of lung cancer, the methylation values of the sequences shown in SEQ ID NOs 1 to 16 were significantly higher in the experimental group than in the control group (the columns indicated by the arrows), and the increase in methylation level was generally about 2 to 5 times.

Therefore, the methylation level detection of any one sequence shown in SEQ ID NO 1-16 can be used for clinical diagnosis of lung cancer.

Example 12 clinical detection of pancreatic cancer

The inventors obtained 6 pancreatic cancer tissue samples from clinic as a control group and 6 pancreatic cancer tissue samples as an experimental group, and compared methylation levels of sequences shown in SEQ ID NOs 1 to 16 of the control group and the experimental group according to the pyrophosphoric acid test procedure of example 3 above.

As a result, as shown in FIG. 11, in the pancreatic cancer clinical samples, the methylation values of the sequences shown in SEQ ID NOs 1 to 16 were significantly higher in the experimental group than in the control group (columns indicated by arrows), and the increase in methylation level was generally about 2 to 6 times.

Therefore, the methylation level detection of any one of the sequences shown in SEQ ID NO 1-16 can be used for pancreatic cancer clinical diagnosis.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. Use of an isolated human nucleic acid or a nucleic acid converted therefrom in the preparation of a reagent or kit for tumor screening, diagnosis, detection or prognostic evaluation;

wherein said human nucleic acid comprises (1) a nucleic acid of the nucleotide sequence shown in SEQ ID NO. 13 or positions 1 to 39 thereof, or a nucleic acid comprising at least 1 modified CpG site in said sequence; or (2) a nucleic acid complementary in sequence to the nucleic acid of (1);

Wherein the nucleic acid converted from human nucleic acid is a nucleic acid corresponding to (1) or (2), the unmodified cytosine of which is converted to T or U, and the cytosine C of the modified CpG site of which is unchanged.

2. The use of claim 1, wherein the sample of a tumor comprises: tissue samples, paraffin embedded samples, blood samples, pleural effusion samples, alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, stool samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical or brush blade samples, tissue and cell biopsy samples.

3. The use of any one of claims 1-2, wherein the modified CpG site comprises a CpG site that is subject to a 5-aldehyde methylation modification, a 5-hydroxymethylation modification, a 5-methylation modification or a 5-carboxymethylation modification.

4. The use according to claim 1, wherein the nucleic acid converted from human nucleic acid is a nucleic acid or a nucleic acid fragment of the nucleotide sequence shown in SEQ ID NO. 90 or a nucleic acid fragment of the nucleotide sequence shown in SEQ ID NO. 91; the nucleic acid fragment comprises at least 1 modified CpG site; preferably, the nucleotide sequence of SEQ ID NO. 90 at positions 1 to 39 or the sequence complementary thereto.

5. A reagent or combination of reagents which specifically detects CpG site modification of a target sequence which is a full length or fragment of any one of the nucleic acids defined in claim 1 or 4; preferably, the agent or combination of agents is directed against a gene sequence comprising the target sequence, preferably the gene sequence comprises a gene Panel or group of genes.

6. Use of the reagent or reagent combination of claim 5 for the preparation of a kit for tumor diagnosis, screening, detection typing or prognosis evaluation.

7. A kit for tumor detection, screening, typing, diagnosis or prognosis evaluation, comprising: the agent or combination of agents of claim 5.

8. The reagent or reagent combination of claim 5, the use of claim 6 or the kit of claim 7, wherein the reagent or reagent combination comprises a primer; preferably, the primer comprises: SEQ ID NO. 92 and SEQ ID NO. 93, or SEQ ID NO. 94.

9. A method of preparing a reagent for tumor diagnosis, screening, detection, typing or prognosis evaluation, comprising: providing any one of the nucleic acids defined in claim 1 or 4, taking the full length or fragment of the nucleic acid as a target sequence, and designing a detection reagent for specifically detecting the CpG site modification condition of the target sequence; wherein the target sequence comprises at least 1 modified CpG site; preferably, the detection reagent comprises: primers, probes, chips or strips.

10. A method for detecting the methylation level of a sample to be tested, comprising: extracting nucleic acid of a sample to be detected; and detecting CpG site modification of a target sequence in the extracted nucleic acid, said target sequence being any one of the nucleic acids as defined in claim 1 or 4; preferably, the method for detecting CpG site modification of a target sequence in an extracted nucleic acid comprises: pyrosequencing, bisulfite conversion sequencing, methylation chip, qPCR, digital PCR, second generation sequencing, third generation sequencing, whole genome methylation sequencing, DNA enrichment detection, reduced bisulfite sequencing, HPLC, massArray, methylation specific PCR, or a combination thereof; or a combined gene group in vitro detection method of partial or all methylation sites in the sequence shown in SEQ ID NO. 13 and an in vivo tracing detection method;

more preferably, the method for detecting CpG site modification of a target sequence in an extracted nucleic acid comprises:

(i) Treating the extracted nucleic acid to convert unmodified cytosine to uracil; preferably, the modification comprises a 5-methylation modification, a 5-hydroxymethylation modification, a 5-aldehyde methylation modification or a 5-carboxymethylation modification; preferably, the nucleic acid of step (i) is treated with bisulphite or bisulphite;

(ii) Analyzing the modification of said target sequence in the nucleic acid treated in (i).

11. The method of any one of claims 1, 6, 7 or 9, wherein the tumor comprises: hematological tumors, digestive system tumors, gynecological tumors, reproductive system tumors, nervous system tumors, urinary system tumors, and other system tumors; preferably, the hematological neoplasm is a leukemia, lymphoma, multiple myeloma; digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer and gallbladder cancer; gynaecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; tumors of the nervous system such as glioma, neuroblastoma, meningioma; head and neck tumors such as oral cavity cancer, tongue cancer, laryngeal cancer and nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer, skin cancer; other systemic tumors such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer; respiratory system tumors such as lung cancer and pleural tumor.