New DNA methylation marker TAGMe-5 for tumor identification and use thereof

Abstract

The application provides a novel DNA methylation marker TAGMe-5 for tumor identification and use thereof. The tumor marker TAGMe-5 of the application shows significant DNA methylation difference between cancer adjacent tissue and cancer tissue, and the difference has significant statistical significance, and the difference is shown in numerous tumors such as solid tumors and non-solid tumors. The solid tumors include lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumor, etc. The non-solid tumors include hematological tumors, lymphoma, etc. Therefore, the tumor marker of the application can be used for clinical screening of pan-cancer, molecular diagnosis, prognosis and efficacy evaluation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of oncology and epigenomics, more specifically, the present application relates to a novel DNA methylation marker TAGMe-5 for tumor identification and uses thereof. BACKGROUND

[0002] Malignant tumor is a complex disease that seriously threatens human life and health. Under the action of genetic and carcinogenic factors, it causes damage and mutation of normal cell genetic material DNA, and at the same time, a variety of oncogenes are activated and tumor suppressor genes are inactivated, so that cancer cells grow and proliferate uncontrollably, invade adjacent normal tissues, and metastasize to distant tissues and organs. Invasion and metastasis are the basic biological characteristics of malignant tumors and the main cause of death in tumor patients. Tumor metastasis includes local invasion, penetration into adjacent blood vessels or lymphatic vessels, survival and transportation in the circulatory system, extravasation from the lumen of the circulatory system to the distant tissue, and clonal formation of visible tumors in the distant tissue. Tumor metastasis is a series of complex interactions between tumor cells, host cells and tumor microenvironment, a continuous process of mutual influence, multiple pathways, multiple genes and multiple cytokines are involved in the whole process of invasion and metastasis. At the same time, epigenomics factors are also closely related to the occurrence, development or metastasis of malignant tumors.

[0003] Epigenomics is a discipline that studies the heritable changes in gene function without DNA sequence changes, which ultimately lead to changes in phenotype. Epigenomics mainly includes biochemical processes such as DNA methylation, histone modification, microRNA level change, etc.; DNA methylation is a more in-depth epigenetic mechanism, which has important application prospects in tumor clinical practice including diagnosis and treatment. DNA methylation is a process in which S-adenosyl methionine (SAM) is used as a methyl donor to transfer a methyl group to a specific base under the catalysis of DNA methyltransferase (DMT) in the body. DNA methylation can occur at N-6 of adenine, N-4 of cytosine, N-7 of guanine or C-5 of cytosine. However, in mammals, DNA methylation mainly occurs at C of 5'-CpG-3', generating 5-methylcytosine (5mC).

[0004] More than 98% of CpG dinucleotides in the genome are scattered in repetitive sequences with transcription-dependent transposition potential. In normal cells, these CpGs are in a highly methylated / transcriptionally silenced state, while in tumor cells these CpGs are extensively demethylated, leading to transcription of repetitive sequences, activation of transposons, high genomic instability and enhanced transcription of proto-oncogenes. The remaining 2% of CpGs are densely distributed in smaller regions (CpG islands). About 40-50% of gene promoters or their vicinity contain CpG islands, suggesting that DNA methylation may be involved in the transcriptional regulation mechanism of such genes. In tumor cells, these CpG islands, which are normally in a hypomethylated state in normal cells, are hypermethylated, leading to gene inactivation. The affected genes include DNA repair genes, cell cycle control genes and anti-apoptotic genes, etc. Therefore, this abnormal DNA methylation pattern in tumor cells has become a new research field of tumor biomarkers. After genomic DNA is treated with bisulfite, PCR (methylation-specific PCR, MSP) detection can effectively determine the methylation state of specific sites of the test DNA fragment. As a qualitative analysis method, MSP can detect individual abnormal methylation tumor cells from a complex clinical sample containing 10,000 normal cells, as long as the methylation states of specific regions of the two test DNAs are completely opposite.

[0005] Although DNA methylation-based tumor early screening has gradually gained attention, there are few truly clinical application programs, so tumor screening is still a special examination project. In addition, most existing tumor markers are only for specific tumor types, and there are almost no markers that can be used for multi-cancer screening. Therefore, it is of great significance to find molecular targets that can be used for diagnosis, prognosis and prediction of cancer development for early screening, clinical intervention and guiding patient treatment of cancer. SUMMARY

[0006] The purpose of the present application is to provide a novel DNA methylation marker TAGMe-5 for tumor identification and its use.

[0007] In the first aspect of the present application, the use of an isolated polynucleotide or a polynucleotide derived therefrom in the preparation of a reagent or kit for identifying a tumor is provided; wherein the polynucleotide comprises: (1) a polynucleotide TAGMe-5 represented by the nucleotide sequence of SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein, or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the polynucleotide derived from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), in which the unmodified cytosine is converted to T or U, while the modified cytosine C of the CpG site remains unchanged.

[0008] In another embodiment, the SEQ ID NO: 1 further comprises a sequence variant thereof, or a homologous sequence.

[0009] In another embodiment, the sequence variant or the homologous sequence is a sequence having 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 96% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more sequence identity compared to the sequence shown in SEQ ID NO: 1. Accordingly, also included are polynucleotides resulting from conversion of the sequence variant or the homologous sequence (non-modified cytosine is converted to T or U, while the modified cytosine C at the CpG site is not changed).

[0010] In another embodiment, the identifying comprises diagnosis, detection, screening, or prognosis evaluation.

[0011] In another embodiment, the modification comprises 5-methylation modification (5mC), 5-hydroxymethylation modification (5hmC), 5-formaldehyde methylation modification (5-fC), or 5-carboxymethylation modification (5-caC).

[0012] In another embodiment, the polynucleotide resulting from conversion of the isolated polynucleotide is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 2.

[0013] In another embodiment, the at least 1 modified CpG site is selected from any one of the "CpG" from No. 1 to 22 or a combination thereof (such as 2 to 22, more specifically 3, 5, 8, 10, 11, 12, 15, 20) in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably is selected from any one of the CpG from No. 9 to 16 or a combination thereof, the "CpG" from No. 1 to 8 or a combination thereof, or the CpG from No. 17 to 22 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1.

[0014] In another embodiment, the polynucleotide fragment is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1 from No. 277 to 392 or from No. 247 to 421.

[0015] In another embodiment, the tumor includes (but is not limited to) a tumor of the respiratory system, a tumor of the digestive system, a tumor of the urinary system, a tumor of the gynecological and reproductive system, a tumor of the blood system, a tumor of the nervous system, a tumor of the head and neck, a tumor of the skin system, a tumor of the endocrine system, or a tumor of the skeletal system.

[0016] In another embodiment, the tumor comprises lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial cancer, biliary tract tumor, gastric cancer, breast cancer, esophageal cancer, brain glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal cancer, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma or ovarian cancer.

[0017] In another embodiment, the tumor to be identified is a tumor (including early, intermediate or advanced tumor) or a precancerous lesion thereof.

[0018] In another embodiment, the sample to be identified for the tumor comprises, but is not limited to, a tissue sample, a blood sample, a body fluid sample.

[0019] In another embodiment, the sample comprises, but is not limited to, a paraffin-embedded sample, a pleural effusion sample, and an alveolar lavage sample, an ascites and lavage sample, a bile sample, a stool sample, a urine sample, a saliva sample, a sputum sample, a cerebrospinal fluid sample, a cell smear sample, a cervical scraping or brushing sample, a tissue and cell biopsy sample, etc.

[0020] In another aspect of the present application, a method for preparing a reagent for identifying a tumor is provided, comprising: (a) providing an isolated polynucleotide or a polynucleotide transformed therefrom, comprising (1) a polynucleotide TAGMe-5 of the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the transformed polynucleotide is a polynucleotide corresponding to (1) or (2), in which the unmodified cytosine is transformed into T or U, and the modified cytosine C of the CpG site remains unchanged; preferably, the at least one modified CpG site is any one of the CpG sites selected from Nos. 1-22 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, any one of the CpG sites selected from Nos. 9-16 or a combination thereof, Nos. 1-8 or a combination thereof, or Nos. 17-22 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1 at positions 277-392 or 247-421; (b) designing a detection reagent for specifically detecting the modification of the CpG site of the target sequence using the polynucleotide of (a) as the target sequence.

[0021] In another embodiment, the reagent for identifying a tumor comprises, but is not limited to, a primer, a probe, a chip or a test paper.

[0022] In another embodiment, one or more sets of reagents can be prepared for the target sequence.

[0023] In another embodiment, the detection reagent is integrated on a chip.

[0024] In another aspect of the present application, a reagent or a combination of reagents for specifically detecting the modification of a CpG site of a target sequence is provided, wherein the target sequence is: (1) a polynucleotide TAGMe-5 of the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the converted polynucleotide is a polynucleotide corresponding to (1) or (2) in which the unmodified cytosine is converted to T or U, while the modified cytosine C of the CpG site remains unchanged; preferably, the reagent or the combination of reagents is directed to a gene sequence containing the target sequence, preferably, the gene sequence comprises a gene panel or a gene group; preferably, the reagent or the combination of reagents comprises primers amplifying the nucleotide sequence shown in SEQ ID NO: 1 at positions 277-392 or 247-421.

[0025] In another embodiment, the reagent or the combination of reagents is: primers containing the sequence at positions 29-58 of SEQ ID NO: 3 and the sequence at positions 28-57 of SEQ ID NO: 4.

[0026] In another embodiment, the reagent or the combination of reagents is: primers of the sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

[0027] In another embodiment, the reagent or the combination of reagents is: primers formed by connecting the sequence at positions 29-58 of SEQ ID NO: 3 with any one of the sequences shown in SEQ ID NO: 7-16, and connecting the sequence at positions 28-57 of SEQ ID NO: 4 with any one of the sequences shown in SEQ ID NO: 17-26.

[0028] In another embodiment, the reagent or the combination of reagents further comprises: primers of the sequences of SEQ ID NO: 5 and SEQ ID NO: 6.

[0029] In another embodiment, the reagent or the combination of reagents is: primers of the sequences shown in SEQ ID NO: 27 and SEQ ID NO: 28.

[0030] In another embodiment, the reagent or the combination of reagents is: primers of the sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30.

[0031] In another aspect of the present application, there is provided the use of the reagent or reagent combination for preparing a kit for identifying a tumor.

[0032] In another aspect of the present application, there is provided a kit for identifying a tumor, comprising the reagent or reagent combination.

[0033] In another embodiment, the kit can further comprise, but not limited to, DNA purification reagent, DNA extraction reagent, bisulfite, PCR amplification reagent.

[0034] In another embodiment, the kit can further comprise instructions for indicating the detection operation steps and the result determination criteria.

[0035] In another aspect of the present application, there is provided a method for analyzing the methylation level of a sample to be tested, comprising: (i) obtaining a polynucleotide from the sample to be tested; and (ii) analyzing the modification of CpG sites of a target sequence or a fragment thereof in the extracted polynucleotide, the target sequence being: (1) a polynucleotide TAGMe-5 as shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; or (2) a polynucleotide complementary to the polynucleotide or fragment of (1) in sequence; wherein the converted polynucleotide is a polynucleotide corresponding to (1) or (2) in which the unmodified cytosine is converted to T or U, while the modified cytosine C of the CpG site remains unchanged; the converted polynucleotide of the nucleotide sequence as shown in SEQ ID NO: 1, a polynucleotide corresponding to (1) or (2) in which the unmodified cytosine is converted to T or U, while the modified cytosine C of the CpG site remains unchanged.

[0036] In another embodiment, the method for detecting the modification of CpG sites of the target sequence in the extracted polynucleotide comprises pyrosequencing, bisulfite conversion sequencing, methylation-specific PCR, methylation-sensitive restriction enzyme digestion, methylation chip, qPCR, digital PCR, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, reduced bisulfite sequencing, HPLC, MassArray, or a combination thereof.

[0037] In another embodiment, the method for analyzing the modification of CpG sites of a target sequence in the extracted polynucleotide comprises: (i) treating the extracted polynucleotide so that cytosine not modified therein is converted to uracil; preferably, the modification comprises 5-methylation modification, 5-hydroxymethylation modification, 5-formaldehyde methylation modification or 5-carboxymethylation modification; preferably, the nucleic acid of step (i) is treated by Bisulfite; and (ii) analyzing the modification of the target sequence in the nucleic acid treated in (i).

[0038] In another embodiment, the polynucleotide of step (i) is treated by Bisulfite; and (ii) analyzing the modification of the target sequence in the polynucleotide treated in (i).

[0039] In another embodiment, the abnormal methylation level means that the cytosine in the CpG of the polynucleotide is highly methylated.

[0040] In another embodiment, other methylation detection methods and future newly developed methylation detection methods can also be applied in the present application.

[0041] In another embodiment, the method for analyzing the methylation level is not a diagnostic method, i.e. it is not intended to directly obtain a diagnosis of a disease.

[0042] In another embodiment, the method for detecting the methylation level of the sample is an in vitro method.

[0043] In another embodiment, the methylation-sensitive restriction enzyme is a restriction enzyme sensitive to methylated bases in its recognition site; including but not limited to one or more of the following: Hhal, BmgBI, HaeII, RruI, Tali, Bsu15I, Hin6I, HpyCH4IV, NarI, etc.

[0044] In another aspect of the present application, there is provided an isolated polynucleotide or a polynucleotide derived therefrom, which comprises: (1) a polynucleotide TAGMe-5 of the nucleotide sequence of SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site; preferably, the at least one modified CpG site is any one of the CpG sites selected from the group consisting of Nos. 9 to 16, or a combination thereof, Nos. 1 to 8, or a combination thereof, or Nos. 17 to 22, or a combination thereof, in the polynucleotide of the nucleotide sequence of SEQ ID NO: 1; preferably, the polynucleotide fragment is a polynucleotide of the nucleotide sequence of SEQ ID NO: 1 from the 277th to 392nd or from the 247th to 421st; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the polynucleotide derived from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2) in which the unmodified cytosine is converted to T or U, while the modified cytosine C of the CpG site remains unchanged.

[0045] Other aspects of the present application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 , Comparison of methylation values of TAGMe-5 in lung cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0047] Figure 2 , Comparison of methylation values of TAGMe-5 in liver cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0048] Figure 3 , Comparison of methylation values of TAGMe-5 in prostate cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0049] Figure 4 , Comparison of methylation values of TAGMe-5 in cervical cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0050] Figure 5 , Comparison of methylation values of TAGMe-5 in endometrial cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0051] Figure 6 , Comparison of methylation values of TAGMe-5 in urothelial cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0052] Figure 7, Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in bile duct tumor and control tissues.

[0053] Figure 8 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in gastric cancer and control tissues.

[0054] Figure 9 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in breast cancer and control tissues.

[0055] Figure 10 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in esophageal cancer and control tissues.

[0056] Figure 11 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in brain glioma and control tissues.

[0057] Figure 12 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in colorectal cancer and control tissues.

[0058] Figure 13 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in leukemia and control tissues.

[0059] Figure 14 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in pancreatic cancer and control tissues.

[0060] Figure 15 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in thyroid cancer and control tissues.

[0061] Figure 16 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in melanoma and control tissues.

[0062] Figure 17 , Results of methylation value comparison (left panel), sensitivity, specificity analysis (right panel) of TAGMe-5 in nasopharyngeal carcinoma and control tissues.

[0063] Figure 18Comparison of TAGMe-5 methylation values ​​in oral cancer and control tissues (left figure), and results of sensitivity and specificity analysis (right figure).

[0064] Figure 19 Comparison of TAGMe-5 methylation values ​​in laryngeal cancer and control tissues (left figure), and results of sensitivity and specificity analysis (right figure).

[0065] Figure 20 Comparison of TAGMe-5 methylation values ​​in osteosarcoma and control tissues (left figure), and results of sensitivity and specificity analysis (right figure).

[0066] Figure 21 Comparison of TAGMe-5 methylation values ​​in lymphoma and control tissues (left figure), and results of sensitivity and specificity analysis (right figure).

[0067] Figure 22 Comparison of TAGMe-5 methylation values ​​in renal cell carcinoma and control tissues (left figure), and results of sensitivity and specificity analysis (right figure).

[0068] Figure 23 Comparison of TAGMe-5 methylation values ​​in ovarian cancer and control tissues (left figure), and results of sensitivity and specificity analysis (right figure).

[0069] Figure 24 The methylation level of methylation sites 1-8 (CpG) in SEQ ID NO:1 in cancer cells was analyzed by sequencing after bisulfite treatment, with normal control cells as a control.

[0070] Figure 25 The methylation level of methylation sites (CpG) 17-22 in SEQ ID NO:1 in cancer cells was analyzed by sequencing after bisulfite treatment, with normal control cells as a control. Detailed Implementation

[0071] In view of the technical problem of lack of DNA methylation and tumor markers in the art and lack of clinical diagnostic significance, the inventors have conducted extensive research and screening, and on the basis of clinical research, disclosed a DNA methylation tumor marker TAGMe-5 which can be used for multi-cancer screening. The tumor marker shows significant DNA methylation difference in cancer-adjacent tissues and cancer tissues, and the difference has significant statistical significance, and the difference is shown in many tumors such as solid tumors and non-solid tumors. The solid tumors include lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumor, etc. The non-solid tumors include hematological tumors, lymphoma, etc. Therefore, the tumor marker of the present application can be used for clinical multi-tumor (pan-cancer) screening (including early screening), molecular diagnosis, prognosis and efficacy evaluation.

[0072] As used herein, "sample" or "specimen" includes material obtained from any individual (preferably a human) or isolated tissue, cells or body fluids (such as plasma) suitable for DNA extraction and can be used for methylation detection. For example, the sample can include but is not limited to: tissue samples, paraffin-embedded samples, blood samples, pleural effusion samples, and alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, fecal samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brushing samples, tissue and cell biopsy samples.

[0073] As used herein, the term "high (degree) methylation (level)" refers to the presence of high methylation, hydroxymethylation, aldehyde methylation or carboxymethylation modification of CpG in a gene sequence. For example, in terms of methylation-specific PCR (MSP) analysis, a PCR reaction performed with methylation-specific primers can obtain a positive PCR result, which can be considered as a high methylation state of the DNA (gene) region of the subject. For example, in terms of real-time quantitative methylation-specific PCR, the determination of high methylation state can be based on the relative value analysis of the methylation state of the control sample and the statistical difference.

[0074] In the present application, the term "tumor" refers to a relatively broad tumor (Pan-cancer) which has a high methylation state in the SEQ ID NO: 1 segment in the genome as described in the present application, which can be a solid tumor or a non-solid tumor, and can include (but not limited to): respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, hematological tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors or skeletal system tumors.

[0075] In the present application, the methylation state of the nucleotide sequence shown in SEQ ID NO: 1 or a partial region (fragment) thereof is significantly different between tumor tissue and non-tumor tissue. When an abnormally high methylation state of the sequence region is detected, it can be considered that the subject suffers from tumor or belongs to a high-risk group of tumor. The significant difference in the methylation state of the sequence region shown in SEQ ID NO: 1 or a partial region thereof is very significant in a variety of tumors (including early stages).

[0076] In the present application, a "conservative variant sequence" of SEQ ID NO: 1 (or a nucleotide sequence shown in the reverse complement sequence thereof) having a high sequence identity or a high sequence identity is also included. The "high sequence identity" is, for example, higher than 90%, higher than 92%, higher than 95%, higher than 98%, higher than 99%, and the like. It should be understood that there can be differences between different biological individuals at individual sequence sites (for example, there can be some meaningless SNPs), but this does not affect the detection based on the overall scheme of the present application.

[0077] According to the above, the present application provides a nucleic acid derived from a specific region of the human genome, which has a sequence region shown in SEQ ID NO: 1 or a partial region thereof, and also includes an antisense strand thereof. In tumor cells, 5-methylcytosine (5mC) or other similar epigenetic modifications are generated at the C position of 5'-CpG-3' in the nucleic acid sequence.

[0078] It is possible to detect one or more CpGs provided by the present application, and therefore the present application also includes a fragment of the nucleic acid of the nucleotide sequence, and at least one methylated CpG site is included therein. The at least one can include 1-22, more specifically 2, 3, 5, 8, 10, 11, 12, 15, 20 of SEQ ID NO: 1 or the reverse complement sequence thereof. Those skilled in the art can understand that after the CpG number based on a DNA strand is provided by the present application, the corresponding CpG sites in the complementary DNA strand to the sense strand are easily obtained according to the content provided by the present application.

[0079] After the information of the specific fragment in the human genome provided by the present application is obtained, those skilled in the art can easily obtain the CpG sites and apply them. A series of sequence fragments containing CpG sites are provided in the embodiments of the present application, which can be used as some examples of preferred embodiments, but it should be understood that according to the information provided by the present application, changes can be made, for example, longer sequences containing the sequences of the present application are selected, or sequences overlapping with the sequences of the present application in the region are selected.

[0080] The present application also includes a gene panel or gene group of the nucleotide sequence shown in SEQ ID NO: 1 or a sequence fragment thereof or a complementary sequence thereof. The characteristics of normal cells and tumor cells can also be obtained by detecting the DNA methylation state for the gene panel or gene group.

[0081] It should be understood that a variety of techniques for analyzing the methylation state can be applied in the present application, and the present application does not have a particular limitation on such detection techniques. The nucleic acids provided by the present application can be the key regions in the genome for analyzing the methylation state, and their methylation state can be analyzed by various techniques known in the art, thereby analyzing the occurrence or development of tumors.

[0082] The nucleic acid of SEQ ID NO: 1 or a fragment thereof or a complementary sequence thereof of the present application can be converted after being subjected to bisulfite treatment, in which the unmethylated cytosine is converted into uracil, and the methylated cytosine remains unchanged. Therefore, the present application also provides the nucleic acid obtained after the bisulfite treatment of the above-mentioned nucleic acid (including the complementary strand (antisense strand)), including: a nucleic acid or a nucleic acid fragment of the nucleotide sequence shown in SEQ ID NO: 2. These nucleic acids can be more direct targets for designing detection reagents or detection kits.

[0083] The nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1 and / or its complementary nucleic acid and / or one or more fragments thereof of the present application can be integrated into one or several units, such as one or several nucleic acid collections, for use by those skilled in the art, such as selecting one or more nucleic acids or nucleic acid fragments from the nucleic acid collection to design targeted analysis reagents. The designed targeted analysis reagents can also be integrated into one or several units, such as one or several kits.

[0084] The nucleic acid converted (e.g., converted by bisulfite) from the nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1 and / or its complementary nucleic acid and / or one or more fragments thereof of the present application can also be integrated into one or several units, such as one or several nucleic acid collections, for use by those skilled in the art, such as selecting one or more nucleic acids or nucleic acid fragments from the nucleic acid collection to design targeted analysis reagents. The designed targeted analysis reagents can also be integrated into one or several units, such as one or several kits, or one or several chips.

[0085] On the basis of the target genes and their epigenetic features provided by the present application, these techniques known in the art and some techniques to be developed can be applied in the present application to implement the detection of methylation level. The determination of the methylation pattern of nucleic acid can be performed by the existing techniques (such as methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight) or other techniques still in development and to be developed. For example, the method of quantitative methylation-specific PCR (QMSP) can be used for detecting the methylation level, which is based on the continuous optical monitoring of fluorescent PCR and is more sensitive than the MSP method. The method has high throughput and avoids the analysis of the results by electrophoresis method. In addition, other available techniques include the qPCR (Me-qPCR) method, the second-generation sequencing method, the pyrosequencing method, the Sanger sequencing method, the bisulfite conversion sequencing method, the whole-genome methylation sequencing method, the DNA enrichment detection method, the simplified bisulfite sequencing technology or the HPLC method and the combined gene group detection method and other conventional methods in the field. Although some preferred methods are provided in the embodiments of the present application, the overall scheme of the present application is not limited thereto.

[0086] As a preferred method of the present application, a method for detecting the methylation pattern of nucleic acid in a sample in vitro is also provided. The principle of the method is that the bisulfite can convert the unmethylated cytosine into uracil, which is converted into thymine in the subsequent PCR amplification process, while the methylated cytosine remains unchanged; thus, after the nucleic acid is treated with bisulfite, the methylated site produces a nucleic acid polymorphism (SNP) similar to a C / T. Based on the above principle, the methylation pattern of nucleic acid in the detection sample can be identified, which can effectively distinguish the methylated and unmethylated cytosine.

[0087] The method of the present application comprises: first providing a sample, extracting genomic DNA; second, treating the genomic DNA in step (a) with bisulfite, so that the unmethylated cytosine in the genomic DNA is converted into uracil; third, analyzing whether the methylation pattern of the genomic DNA treated in step (b) is abnormal.

[0088] The method of the present application can be used for detecting the sample of a subject to evaluate whether the subject has a tumor or for distinguishing a high-risk population of tumor. The method can be used in cases without the purpose of obtaining a direct disease diagnosis result, such as cases without the purpose of judging the final result of the disease, regional analysis of population, scientific research, population census, etc.

[0089] In the preferred embodiments of the present application, the DNA methylation is detected by PCR amplification and pyrosequencing. However, the present application is not limited to this method, and other methods known in the art or being developed for detecting DNA methylation can also be used. In the PCR amplification, the primers used are not limited to those provided in the embodiments, and primers that are different from those provided in the embodiments in sequence but are still directed to the nucleic acid or the corresponding CpG site indicated in the present application can also be used.

[0090] As a preferred method of the present application, a method for detecting the methylation status of a nucleic acid in a sample in vitro is also provided, which is the methylation sensitive restriction endonuclease (MSRE) method. The methylation sensitive restriction endonuclease cannot cut DNA when a methylated base is contained in its cutting site. The MSRE method is based on the principle that the methylation sensitive type II restriction endonuclease cannot cut a fragment containing one or more methylated cutting site sequences. The fragment containing one or more methylated CpG sequences is cut with the methylation sensitive type II endonuclease and its isozyme (not sensitive to methylation), and then analyzed by Southern blotting. The advantages of this method include that detailed information of the primary structure of the target DNA is not required, and a direct evaluation of the methylation status of the CpG island can be provided, including obtaining quantitative analysis information of the methylation of the detected gene.

[0091] Other methods and reagents known to those skilled in the art for determining the sequence, variations and methylation status of a genome around the marker nucleic acid provided in the present application can also be included in the present application.

[0092] The present application provides a method for preparing a tumor detection reagent, comprising: providing the nucleic acid, using the full length or fragment of the nucleic acid as a target sequence, and designing a detection reagent that specifically detects the target sequence; wherein the target sequence contains at least one methylated CpG site. The detection reagent can include but is not limited to a chip, a primer, a probe, etc.; after the marker is obtained, the selection of the detection reagent can be achieved by those skilled in the art.

[0093] After the sequence of the nucleic acid is known, the design of primers is known to those skilled in the art, and the two primers are on both sides of the specific sequence of the target gene to be amplified (including the CpG sequence, and the TpG complementary to the CpG is directed to the gene region that was originally methylated, and the TpG complementary to the CpG is directed to the gene region that was originally demethylated). In the preferred embodiments of the present application, the reagent is a primer, and the primer is preferably those listed in the embodiments. In addition to primers, other diagnostic or detection reagents can also be prepared, including but not limited to probes, chips, etc.

[0094] The reagent can also be a reagent combination, such as a primer combination. For example, the combination includes more than one set of primers, so that the above-mentioned multiple nucleic acids can be amplified respectively.

[0095] The present application also provides a kit for detecting the methylation pattern of nucleic acid in a sample in vitro, which comprises a container and the above-mentioned primer pair in the container.

[0096] The kit can also include various reagents required for DNA extraction, DNA purification, PCR amplification, and other reagents, such as sample processing reagents. In addition, the kit can also include an instruction manual indicating the detection operation steps and result determination criteria for the convenience of the skilled in the art.

[0097] The method and reagent of the present application have very high accuracy when used for diagnosing clinical tumors, which is embodied in the detection of various tumor clinical samples in the embodiments of the present application. The present application can be applied to the fields of pre-tumor screening, efficacy determination, auxiliary diagnosis, prognosis monitoring, etc., or the cases as mentioned above which are not aimed at obtaining direct disease diagnosis results.

[0098] The present application provides a pan-cancer marker, which can be applied to the detection of cervical exfoliated cells and the like. The sample to be detected is easy to obtain and is non-invasive. Compared with the situation that tissue samples need to be obtained by surgery in the clinic, the pain of the subject is greatly reduced, the compliance is good, and the operation of the clinician is also easier. Obviously, this presents a very significant progress.

[0099] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, and the conditions are generally according to the conventional conditions, such as the conditions described in J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Third Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0100] Example 1 Determination of methylation detection target

[0101] 1.1 Obtain the TAGMe-5 gene (human) sequence SEQ ID NO: 1;

[0102]

[0103] In the above sequence (sense strand), each "CG" marked by solid underlined line represents one methylation-modified CpG site, numbered as No. 1-22 "CG" (No. 1-22 methylation-modified CpG sites) from 5' to 3'. The region under dotted underlined line corresponds to the upstream primer and downstream primer design region in the partial scheme of the example; the italicized and blackened region corresponds to the detection target region in the partial scheme of the example.

[0104] 1.2 Obtain the sequence of bisulfite-converted DNA SEQ ID NO: 2, wherein Y represents C or U (T);

[0105]

[0106] In the above sequence (sense strand), each "YG" marked by solid underlined line represents one converted methylation-modified CpG site, numbered as No. 1-22 "YG" (No. 1-22 converted methylation-modified CpG sites) from 5' to 3'. The region under dotted underlined line corresponds to the upstream primer and downstream primer design region in the partial scheme of the example; the italicized and blackened region corresponds to the detection target region in the partial scheme of the example.

[0107] 1.3 Determine the detection region, and design primers upstream and downstream of the detection region.

[0108] Example 2 Design and synthesis of detection reagents

[0109] 2.1 Design the first round of PCR primers with a length of 25-35 bp and a moderate CG content, and make the amplification length 100-300 bp;

[0110] 2.2 Add barcode (Sample-ID) to the end of the first round of primers;

[0111] 2.3 Add tag sequence to the end of the first round of primers for library construction;

[0112] 2.4 Synthesize the primers for the first round of PCR reaction and the primers for the second round of PCR reaction with Illumina adapters and indexes, and the primer sequences are shown in Table 1.

[0113] Table 1

[0114]

[0115] In the table, F1 and R1 amplify the sequence region corresponding to positions 277-392 (containing No. 9-16 CpG) in SEQ ID NO: 1 or SEQ ID NO: 2, and the sequence containing the primer complementary segment is the sequence region at positions 247-421 (containing No. 8-16 CpG).

[0116] In the sequence of Table F1, the bases from 29th to 58th (underlined) are complementary to the target detection sequence segment (upstream and downstream), and the bases from 22nd to 28th are the tag sequence; in the sequence of R1, the bases from 28th to 57th are complementary to the target detection sequence segment (upstream and downstream), and the bases from 21st to 27th are the tag sequence.

[0117] Verification of the detection reagent in Example 3

[0118] A pair of primers was synthesized to perform two rounds of PCR reaction on the positive and negative reference samples:

[0119] The first round of PCR system and reaction conditions are shown in Table 2.

[0120] Table 2

[0121]

[0122] The second round of PCR system and reaction conditions are shown in Table 3.

[0123] Table 3

[0124]

[0125] After verifying the PCR efficiency of the primers, a primer pool with different barcodes was synthesized, and the primers were diluted and combined (Table 4). The F primers and the R primers in the first round of PCR have M x N (10 x 10) combinations, which can be used for simultaneous detection and positioning of multiple samples. In Table 4, the lowercase bases in the F1 primer correspond to the 1st to 21st bases (Illumina adapter) in the upstream primer F1 in Table 1, and the uppercase bases are the sequencing tag. The bases complementary to the target detection sequence segment are connected after the tag (the 29th to 58th bases in SEQ ID NO: 3 in the subsequent examples). The lowercase bases in the R1 primer correspond to the 1st to 20th bases in the upstream primer R1 in Table 1, and the uppercase bases are the sequencing tag. The bases complementary to the target detection sequence segment are connected after the tag (the 28th to 57th bases in SEQ ID NO: 4 in the subsequent examples). The second round of primers are F2 and R2 in Table 1.

[0126] Table 4

[0127]

[0128]

[0129] wherein R represents A or G.

[0130] Example 5 Difference in methylation of TAGMe-5 CpG sites between tumor tissue and non-tumor tissue-NGS sequencing method

[0131] 1.Obtain clinical samples: Obtain paracancerous / non-cancer-cancer tissue samples from the clinic, paracancerous / non-cancer samples as control group, cancer tissue samples as tumor detection experimental group;

[0132] 2. DNA extraction: Extract DNA from the experimental group and the control group, respectively; the adsorption column method is used in this experiment (which can not be limited to this method);

[0133] 3. Bisulfite treatment: Bisulfite treatment of extracted DNA samples, strictly according to the steps; ZYMO Research Company's EZ DNA Methylation-Gold Kit, item number D5006 (which can not be limited to this method) is used in this experiment;

[0134] 4. Use primers in primer pool (first round of PCR primers) and Illumina system universal sequencing primers (second round of PCR primers) to perform two rounds of PCR amplification to construct NGS library by conventional method.

[0135] 5. After PCR amplification, 2% agarose gel electrophoresis is used to detect the specificity of PCR fragments, 5ul of PCR product is taken from each sample, mixed, and the target fragment library is purified and recovered for NGS sequencing;

[0136] 6. Analyze sequencing results: Extract the sequencing information of the sample according to the primer barcode sequence;

[0137] 7. TAGMe-5 methylation value calculation: NGS sequencing can independently detect the methylation status of individual CpG sites in the target region, and the median value of all CpG site methylation is calculated as the methylation value of TAGMe-5 in the sample;

[0138] 8. Result analysis: Compare the TAGMe-5 methylation values in non-tumor tissue and tumor tissue and determine the cutoff value through ROC curve.

[0139] Example 6 TAGMe-5: Lung cancer clinical sample verification-NGS sequencing method

[0140] Thirty-eight clinical samples were obtained, including 19 paracancerous samples of lung cancer (normal) as the control group and 19 lung cancer samples as the experimental group. Two-round PCR reaction was performed according to the primer combination method described in Example 4 above (Table 4, optional first-round primer 1 pair, second-round primer F2 and R2 in Table 1), to construct NGS library of lung cancer clinical samples, and the methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps;

[0141] The results are as follows Figure 1, shows that in lung cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues is significantly higher than that in paracancer tissues (****P<0.0001). The sensitivity is 63.16%, and the specificity is 89.47%.

[0142] Example 7 TAGMe-5: Liver cancer clinical sample verification-NGS sequencing method

[0143] 30 clinical samples were obtained, including 15 liver cancer paracancer samples as a control group and 15 liver cancer samples as an experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (optionally primer pair 1 in Table 4 for the first round, and primer F2 and R2 in Table 1 for the second round), to construct NGS libraries of liver cancer clinical samples. The methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0144] The results are shown in Figure 2 , which shows that in liver cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues is significantly higher than that in paracancer tissues (***P<0.001). The sensitivity is 73.33%, and the specificity is 93.33%.

[0145] Example 8 TAGMe-5: Prostate cancer clinical sample verification-NGS sequencing method

[0146] 30 clinical samples were obtained, including 15 prostate cancer paracancer samples as a control group and 15 prostate cancer samples as an experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (optionally primer pair 1 in Table 4 for the first round, and primer F2 and R2 in Table 1 for the second round), to construct NGS libraries of prostate cancer clinical samples. The methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0147] The results are shown in Figure 3 , which shows that in prostate cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues is significantly higher than that in paracancer tissues (***P<0.001). The sensitivity is 86.67%, and the specificity is 93.33%.

[0148] Example 9 TAGMe-5: Cervical cancer clinical sample verification-NGS sequencing method

[0149] 40 clinical samples were obtained, including 20 cervical adenocarcinoma paracancer samples as a control group and 20 cervical cancer samples as an experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (optionally primer pair 1 in Table 4 for the first round, and primer F2 and R2 in Table 1 for the second round), to construct NGS libraries of cervical cancer clinical samples. The methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0150] The results are shown inFigure 4 , shows that in endometrial carcinoma clinical samples, the methylation value of TAGMe-5 in cancer tissues is significantly higher than that in para-cancer tissues (****P<0.0001). The sensitivity is 100%, and the specificity is 100%.

[0151] Example 10 TAGMe-5: Endometrial carcinoma clinical sample verification-NGS sequencing method

[0152] 18 samples were obtained clinically, of which 9 endometrial carcinoma para-cancer samples were used as the control group, and 9 endometrial carcinoma samples were used as the experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally primer pair 1 in Table 4 for the first round, and primer F2 and R2 in Table 1 for the second round), to construct the NGS library of endometrial carcinoma clinical samples, and the methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0153] The results are shown in Table 6. Figure 5 , shows that in endometrial carcinoma clinical samples, the methylation value of TAGMe-5 in cancer tissues is significantly higher than that in para-cancer tissues (****P<0.0001). The sensitivity is 100%, and the specificity is 100%.

[0154] Example 11 TAGMe-5: Urothelial carcinoma clinical sample verification-NGS sequencing method

[0155] 20 samples were obtained clinically, of which 10 urothelial carcinoma para-cancer tissue samples were used as the control group, and 10 urothelial carcinoma samples were used as the experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally primer pair 1 in Table 4 for the first round, and primer F2 and R2 in Table 1 for the second round), to construct the NGS library of urothelial carcinoma clinical samples, and the methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0156] The results are shown in Table 7. Figure 6 , shows that in endometrial carcinoma clinical samples, the methylation value of TAGMe-5 in cancer tissues is significantly higher than that in para-cancer tissues (****P<0.0001). The sensitivity is 100%, and the specificity is 100%.

[0157] Example 12 TAGMe-5: Biliary tract tumor clinical sample verification-NGS sequencing method

[0158] 20 samples were obtained clinically, of which 10 biliary tract tumor para-cancer samples were used as the control group, and 10 biliary tract tumor samples were used as the experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally primer pair 1 in Table 4 for the first round, and primer F2 and R2 in Table 1 for the second round), to construct the NGS library of biliary tract tumor clinical samples, and the methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0159] The results are as follows Figure 7 The results showed that in clinical samples of biliary tract tumors, the methylation value of TAGMe-5 in cancerous tissue was significantly higher than that in adjacent normal tissue (**P<0.01). The sensitivity and specificity were 80%.

[0160] Example 13 TAGMe-5: Validation of Gastric Cancer Clinical Samples - NGS Sequencing

[0161] Twelve clinical samples were obtained, including six gastric cancer adjacent normal samples as the control group and six gastric cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of gastric cancer clinical samples. The TAGMe-5 methylation level was analyzed according to the NGS sequencing procedure.

[0162] The results are as follows Figure 8 The results showed that in clinical samples of gastric cancer, the methylation value of TAGMe-5 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 83.33%, and the specificity was 83.33%.

[0163] Example 14 TAGMe-5: Clinical Sample Validation for Breast Cancer - NGS Sequencing

[0164] Ten clinical samples were obtained, including five breast cancer adjacent normal samples as the control group and five breast cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers in the first round from Table 4, and F2 and R2 in Table 1 for the second round of primers) to construct NGS libraries of breast cancer clinical samples. The TAGMe-5 methylation level was analyzed according to the NGS sequencing steps.

[0165] The results are as follows Figure 9 The results showed that in clinical samples of breast cancer, the methylation value of TAGMe-5 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 80% and the specificity was 100%.

[0166] Example 15 TAGMe-5: Validation of Clinical Samples from Esophageal Cancer - NGS Sequencing

[0167] Twelve clinical samples were obtained, including six esophageal cancer adjacent normal samples as the control group and six esophageal cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of esophageal cancer clinical samples. The TAGMe-5 methylation level was analyzed according to the NGS sequencing procedure.

[0168] The results are as follows Figure 10The results showed that in esophageal cancer clinical samples, the methylation value of TAGMe-5 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 83.3% and the specificity was 100%.

[0169] Example 16 TAGMe-5: Clinical Sample Validation of Glioma - NGS Sequencing

[0170] Ten clinical samples were obtained, including five glioma adjacent normal samples as the control group and five glioma samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of clinical glioma samples. The TAGMe-5 methylation level was analyzed according to the NGS sequencing procedure.

[0171] The results are as follows Figure 11 The results showed that in clinical samples of glioma, the methylation value of TAGMe-5 in cancerous tissue was significantly higher than that in adjacent normal tissue (**P<0.01). Sensitivity and specificity were 100%.

[0172] Example 17 TAGMe-5: Clinical Sample Validation for Colorectal Cancer - NGS Sequencing

[0173] Twelve clinical samples were obtained, including six adjacent normal colorectal cancer samples as the control group and six colorectal cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (one pair of primers was selected from Table 4 for the first round, and F2 and R2 from Table 1 for the second round). NGS libraries of colorectal cancer clinical samples were constructed, and TAGMe-5 methylation levels were analyzed according to the NGS sequencing procedure.

[0174] The results, shown in Figure 12, indicate that in clinical samples of colorectal cancer, the methylation value of TAGMe-5 in cancerous tissue was significantly higher than that in adjacent normal tissue (**P<0.01). The sensitivity was 83.33%, and the specificity was 100%.

[0175] Example 18 TAGMe-5: Validation of Clinical Samples from Leukemia - NGS Sequencing

[0176] Twelve clinical samples were obtained, including six non-leukemia bone marrow smear samples as the control group and six leukemia bone marrow smear samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (one pair of primers was selected from Table 4 for the first round, and F2 and R2 from Table 1 for the second round). NGS libraries of leukemia clinical samples were constructed, and TAGMe-5 methylation levels were analyzed according to the NGS sequencing steps.

[0177] The results are as follows Figure 13, showed that in the clinical samples of leukemia, the methylation value of TAGMe-5 in the bone marrow smears of leukemia was significantly higher than that in the bone marrow smears of non-leukemia (**P<0.01). The sensitivity was 100%, and the specificity was 100%.

[0178] Example 19 TAGMe-5: Pancreatic cancer clinical sample verification-NGS sequencing method

[0179] Twelve samples were clinically obtained, of which six samples of pancreatic cancer paracancerous tissue were used as a control group, and six samples of pancreatic cancer were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally, the first round of primer 1 pair in Table 4, and the second round of primer F2 and R2 in Table 1), to construct the NGS library of the clinical sample of liposarcoma, and the methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0180] The results are shown in Figure 14 , which showed that in the clinical samples of pancreatic cancer, the methylation value of TAGMe-5 in the cancer tissue was significantly higher than that in the paracancerous tissue (**P<0.01). The sensitivity was 83.33%, and the specificity was 100%.

[0181] Example 20 TAGMe-5: Thyroid cancer clinical sample verification-NGS sequencing method

[0182] Twelve samples were clinically obtained, of which six samples of thyroid cancer paracancerous tissue were used as a control group, and six samples of thyroid cancer were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally, the first round of primer 1 pair in Table 4, and the second round of primer F2 and R2 in Table 1), to construct the NGS library of the clinical sample of thyroid tumor, and the methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0183] The results are shown in Figure 15 , which showed that in the clinical samples of thyroid cancer, the methylation value of TAGMe-5 in the cancer tissue was significantly higher than that in the paracancerous tissue (**P<0.01). The sensitivity was 83.33%, and the specificity was 83.33%.

[0184] Example 21 TAGMe-5: Melanoma clinical sample verification-NGS sequencing method

[0185] Twelve samples were clinically obtained, of which six samples of normal skin tissue were used as a control group, and six samples of skin melanoma were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally, the first round of primer 1 pair in Table 4, and the second round of primer F2 and R2 in Table 1), to construct the NGS library of the clinical sample of melanoma, and the methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0186] The results are shown inFigure 16 , showed that in the melanoma clinical samples, the methylation value of TAGMe-5 in the cancer tissue was significantly higher than that in the paracancerous tissue (**P<0.01). The sensitivity was 100%, and the specificity was 100%.

[0187] Example 22 TAGMe-5: nasopharyngeal carcinoma clinical sample verification - NGS sequencing method

[0188] Twelve samples were clinically obtained, of which six nasopharyngeal carcinoma paracancer samples were used as a control group, and six nasopharyngeal carcinoma samples were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally the first round of primer 1 pair in Table 4, and the second round of primer F2 and R2 in Table 1), to construct the NGS library of nasopharyngeal carcinoma clinical samples. The methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0189] The results are shown in Table 6. Figure 17 , showed that in the nasopharyngeal carcinoma clinical samples, the methylation value of TAGMe-5 in the cancer tissue was significantly higher than that in the paracancerous tissue (*P<0.05). The sensitivity was 83.33%, and the specificity was 66.67%.

[0190] Example 23 TAGMe-5: oral cancer clinical sample verification - NGS sequencing method

[0191] Twelve samples were clinically obtained, of which six oral cancer paracancer samples were used as a control group, and six oral cancer samples were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally the first round of primer 1 pair in Table 4, and the second round of primer F2 and R2 in Table 1), to construct the NGS library of oral cancer clinical samples. The methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0192] The results are shown in Table 7. Figure 18 , showed that in the oral cancer clinical samples, the methylation value of TAGMe-5 in the cancer tissue was significantly higher than that in the paracancerous tissue (*P<0.05). The sensitivity was 100%, and the specificity was 83.33%.

[0193] Example 24 TAGMe-5: laryngeal carcinoma clinical sample verification - NGS sequencing method

[0194] Ten samples were clinically obtained, of which five laryngeal carcinoma paracancer samples were used as a control group, and five laryngeal carcinoma samples were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally the first round of primer 1 pair in Table 4, and the second round of primer F2 and R2 in Table 1), to construct the NGS library of laryngeal carcinoma clinical samples. The methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0195] The results are shown in Table 8. Figure 19, shows that in the clinical samples of laryngeal cancer, the methylation value of TAGMe-5 in cancer tissues is significantly higher than that in paracancer tissues (**P<0.01). The sensitivity is 100%, and the specificity is 100%.

[0196] Example 25 TAGMe-5: osteosarcoma clinical sample verification-NGS sequencing method

[0197] Twelve samples were clinically obtained, of which six osteosarcoma paracancer samples were used as a control group, and six osteosarcoma samples were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally primer pair 1 in Table 4 for the first round, and F2 and R2 in Table 1 for the second round), to construct the NGS library of osteosarcoma clinical samples. The methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0198] The results are shown in Table 6. Figure 20 , shows that in the clinical samples of laryngeal cancer, the methylation value of TAGMe-5 in cancer tissues is significantly higher than that in paracancer tissues (**P<0.01). The sensitivity is 100%, and the specificity is 100%.

[0199] Example 26 TAGMe-5: lymphoma clinical sample verification-NGS sequencing method

[0200] Twelve samples were clinically obtained, of which six normal lymph samples were used as a control group, and six lymphoma samples were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally primer pair 1 in Table 4 for the first round, and F2 and R2 in Table 1 for the second round), to construct the NGS library of lymphoma clinical samples. The methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0201] The results are shown in Table 6. Figure 21 , shows that in the clinical samples of laryngeal cancer, the methylation value of TAGMe-5 in cancer tissues is significantly higher than that in paracancer tissues (**P<0.01). The sensitivity is 100%, and the specificity is 100%.

[0202] Example 27 TAGMe-5: renal cell carcinoma clinical sample verification-NGS sequencing method

[0203] Twelve samples were clinically obtained, of which six renal cell carcinoma paracancer samples were used as a control group, and six renal cell carcinoma samples were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally primer pair 1 in Table 4 for the first round, and F2 and R2 in Table 1 for the second round), to construct the NGS library of renal cell carcinoma clinical samples. The methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0204] The results are shown in Table 6. Figure 22, showed that in the clinical samples of renal cell carcinoma, the methylation value of TAGMe-5 in the cancer tissue was significantly higher than that in the paracancerous tissue (*P<0.05). The sensitivity was 83.33%, and the specificity was 83.33%.

[0205] Example 28 TAGMe-5: ovarian cancer clinical sample verification-NGS sequencing method

[0206] Twelve samples were obtained clinically, of which six ovarian cancer paracancer samples were used as a control group, and six ovarian cancer samples were used as an experimental group. Two rounds of PCR reactions were performed according to the primer combination mode described in Example 4 above (optionally the first round of primer 1 pair in Table 4, and the second round of primer F2 and R2 in Table 1). The NGS library of the ovarian cancer clinical sample was constructed, and the methylation level of TAGMe-5 was analyzed according to the NGS sequencing steps.

[0207] The results are shown in Table 6. Figure 23 , showed that in the clinical samples of renal cell carcinoma, the methylation value of TAGMe-5 in the cancer tissue was significantly higher than that in the paracancerous tissue (*P<0.05). The sensitivity was 83.33%, and the specificity was 83.33%.

[0208] Example 29 Analysis of detection performance of single CpG site

[0209] Using the clinical samples obtained in the foregoing examples, the feasibility of each of the 9th to 16th CpG sites (CpG sites in the 277th to 392nd segment in SEQ ID NO: 1) as a single CpG site for detecting cancer was analyzed. The methylation modification of the single CpG site was analyzed by NGS sequencing method.

[0210] The results are shown in Tables 5 to 9.

[0211] Table 5

[0212]

[0213] Table 6

[0214]

[0215]

[0216] Table 7

[0217]

[0218] Table 8

[0219]

[0220] Table 9

[0221]

[0222] The results of Tables 5 to 9 show that a single CpG site has high sensitivity and / or specificity, and the single CpG site can also be used as a target for methylation modification analysis to guide clinical auxiliary diagnosis analysis of cancer.

[0223] Example 30 Methylation difference of TAGMe-5 CpG site between tumor cells and non-tumor cells

[0224] Bisulfite Sequencing PCR (BSP) was used after bisulfite treatment, and the sequencing method was as follows:

[0225] 1. Extract genomic DNA of hematological tumor cell lines (myeloid leukemia cell line K562), colorectal cancer cell lines (HCT116), pancreatic cancer cell lines (SW1990), human renal clear cell adenocarcinoma cells (786-O), gastric cancer cell lines (BGC-823), breast cancer cell lines (BT-549) and cervical cancer cell lines (HeLa) and their corresponding normal cell genomic DNA;

[0226] 2. Bisulfite was used to treat the extracted genomic DNA of cancer cell lines and normal cell lines, respectively, as a template for subsequent PCR amplification;

[0227] 3. The amplification primers were designed according to the sequence of SEQ ID NO: 2, as shown in Table 10, for amplification.

[0228] 4. After PCR amplification, 2% agarose gel electrophoresis was used to detect the specificity of the PCR fragments, the target fragments were recovered by cutting the gel, inserted into T vector, transformed into competent E. coli, plated, and the next day the clones were picked for sequencing. More than 10 clones were picked for Sanger sequencing for each fragment.

[0229] Table 10, BSP primers

[0230]

[0231] BSP verification of methylation levels of cancer cells and normal control cells in 1-8 methylation sites in SEQ ID NO: 1 region as shown in Figure 24 , showing that the methylation level of cancer cells TAGMe-5 is significantly higher than that of normal cells.

[0232] BSP verification of methylation levels of cancer cells and normal control cells in 17-22 methylation sites in SEQ ID NO: 1 region as shown in Figure 25 , showing that the methylation level of cancer cells TAGMe-5 is significantly higher than that of normal cells.

[0233] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that embodiments can be practiced without the specific details that are set forth herein. Further, it should be noted that, in this application, "exemplary" or "for example" is used on a purely illustrative basis to aid the reader in understanding the application. Any process steps, or sequences of steps, or examples, or embodiments, or materials, or components, or compounds, or elements described herein are understood to be illustrative only and not limiting.

Claims

1. The use of a detection reagent in the preparation of a reagent for identifying tumors, comprising: (a) Provides an isolated polynucleotide having the nucleotide sequence shown in positions 277–392 of SEQ ID NO: 1; (b) Using the polynucleotide in (a) as the target sequence, design a detection reagent that specifically detects the CpG site modification status of the target sequence; The tumor referred to is pan-cancer, selected from lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumor, gastric cancer, breast cancer, esophageal cancer, glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma, or ovarian cancer.

2. The use of a reagent or combination of reagents for specifically detecting CpG site modifications of a target sequence in the preparation of a kit for identifying tumors; wherein the tumor is pan-cancer, selected from lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumors, gastric cancer, breast cancer, esophageal cancer, glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma, or ovarian cancer; wherein the target sequence is a polynucleotide of the nucleotide sequence shown at positions 277-392 of SEQ ID NO:

1.

3. The use as described in claim 2, characterized in that, The samples used for tumor identification include tissue samples and blood samples.

4. The use as described in claim 2, characterized in that, The samples used for tumor identification include bodily fluid samples.

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