Novel DNA methylation marker tagme-5 for tumor identification and use thereof

Abstract

Provided are a novel DNA methylation marker TAGMe-5 for tumor identification and a use thereof. The tumor marker TAGMe-5 shows a significant DNA methylation difference in para-carcinoma tissues and carcinoma tissues, the difference has remarkable statistical significance, and the difference is shown in a plurality of tumors such as solid tumors and non-solid tumors. The solid tumors comprise lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, a biliary tract tumor, etc. The non-solid tumors comprise a blood system tumor, lymphoma, etc. Therefore, the tumor marker can be used for screening, molecular diagnosis, prognosis, and therapeutic effect evaluation for clinical multi-tumors (Pan-cancer).

Description

A novel DNA methylation marker TAGMe-5 for tumor identification and its use

[0001] This application claims priority to patent application No. CN 202311288756.6, filed on October 5, 2023; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the fields of oncology and epigenetics, and more specifically, relates to a novel DNA methylation marker TAGMe-5 that can be used for tumor identification and its use. Background Art

[0003] Malignant tumors are complex diseases that pose a serious threat to human health and life. Genetic and carcinogenic factors cause damage and mutations in the genetic material of normal cells, accompanied by the activation of multiple oncogenes and the inactivation of tumor suppressor genes. This leads to uncontrolled growth and proliferation of cancer cells, invasion of adjacent normal tissues, and metastasis to distant tissues and organs. Invasion and metastasis are fundamental biological hallmarks of malignant tumors and the leading cause of death in cancer patients. Tumor metastasis involves local invasion, intravasation into adjacent blood vessels or lymphatic vessels, survival and transport within the circulatory system, extravasation from the circulatory lumen to distant tissues, and colonization and formation of visible tumors in distant tissues. Tumor metastasis is a continuous process characterized by a complex series of interactions and mutual influences between tumor cells, host cells, and the tumor microenvironment. Multiple pathways, multiple genes, and multiple cytokines are involved in the entire process of invasion and metastasis. Furthermore, epigenetic factors are also closely associated with the occurrence, progression, and metastasis of malignant tumors.

[0004] Epigenomics studies the heritable changes in gene function that occur without altering the DNA sequence, ultimately leading to phenotypic changes. Epigenetics primarily encompasses biochemical processes such as DNA methylation, histone modification, and changes in microRNA levels. DNA methylation is a well-studied epigenetic mechanism and holds important application prospects in clinical oncology, including diagnosis and treatment. DNA methylation is the process in vivo where a methyl group is transferred to a specific base using S-adenosylmethionine (SAM) as a methyl donor, catalyzed by DNA methyltransferase (DMT). DNA methylation can occur at the N-6 position of adenine, the N-4 position of cytosine, the N-7 position of guanine, or the C-5 position of cytosine. However, in mammals, DNA methylation primarily occurs at the C of the 5'-CpG-3' position, generating 5-methylcytosine (5mC).

[0005] Over 98% of CpG dinucleotides in the genome are dispersed within repetitive sequences with transcription-dependent transposable potential. In normal cells, these CpGs are hypermethylated and transcriptionally silent, whereas in tumor cells, they undergo extensive demethylation, leading to transcription of repetitive sequences, transposon activation, high genomic instability, and enhanced transcription of oncogenes. The remaining approximately 2% of CpGs are densely distributed within smaller regions (CpG islands). Approximately 40-50% of gene promoters contain CpG islands, suggesting that DNA methylation may be involved in the transcriptional regulation of these genes. In tumor cells, these CpG islands, which are hypomethylated in normal cells, become hypermethylated, leading to transcriptional inactivation of genes. Affected genes include DNA repair genes, cell cycle control genes, and tumor suppressor genes such as anti-apoptosis genes. Therefore, this abnormal DNA methylation pattern in tumor cells has become a new area of ​​research for tumor biomarkers. After the genomic DNA is treated with bisulfite, PCR (methylation-specific PCR, MSP) detection can effectively determine the methylation status of specific sites in the test DNA fragment. As a qualitative analysis method, MSP can detect a single-digit number of abnormally methylated tumor cells from a complex clinical sample containing 10,000 normal cells, as long as the methylation status of the specific regions of the test DNA of the two is completely opposite.

[0006] While early cancer screening based on DNA methylation molecular markers is gaining increasing attention, few clinically applicable protocols exist, relegating cancer screening to specialized testing programs. Furthermore, most existing tumor markers target specific tumor types, and few can be used for multi-cancer screening. Therefore, identifying molecular targets that can be used for diagnosis, prognosis, and prediction of cancer development is crucial for early cancer screening, clinical intervention, and patient treatment guidance.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a novel DNA methylation marker TAGMe-5 that can be used for tumor identification and its use.

[0009] In a first aspect of the present invention, there is provided a use of an isolated polynucleotide or a polynucleotide converted therefrom in the preparation of a reagent or kit for identifying a tumor; wherein the polynucleotide comprises: (1) a polynucleotide TAGMe-5 having a 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 polynucleotide converted from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

[0010] In another embodiment, the SEQ ID NO: 1 also includes sequence variants or homologous sequences thereof.

[0011] In another embodiment, the sequence variant or homologous sequence is a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 92%, greater than 95%, greater than 96%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.8% sequence identity with the sequence shown in SEQ ID NO: 1. Accordingly, polynucleotides derived from the sequence variant or homologous sequence by conversion (conversion of the unmodified cytosine to T or U, while the cytosine C at the modified CpG site remains unchanged) are also included.

[0012] In another embodiment, said identifying comprises diagnosis, detection, screening or prognostic assessment.

[0013] In another embodiment, the modification comprises 5-methylation modification (5mC), 5-hydroxymethylation modification (5hmC), 5-aldehydemethylation modification (5-fC) or 5-carboxymethylation modification (5-caC).

[0014] In another embodiment, the polynucleotide converted from the isolated polynucleotide is a polynucleotide having a nucleotide sequence shown in SEQ ID NO: 2.

[0015] In another embodiment, the at least one modified CpG site is any one "CpG" or a combination thereof selected from Nos. 1 to 22 (e.g., 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, it is any one CpG site or a combination thereof selected from Nos. 9 to 16, "CpG" or a combination thereof, or CpG sites 17 to 22 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1.

[0016] In another embodiment, the polynucleotide fragment is a polynucleotide having a nucleotide sequence shown at positions 277 to 392 or positions 247 to 421 in SEQ ID NO: 1.

[0017] In another embodiment, the tumor includes (but is not limited to): respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, blood system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors or skeletal system tumors.

[0018] In another embodiment, the tumor comprises 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 cancer, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma or ovarian cancer.

[0019] In another embodiment, the identifying of a tumor is directed to a tumor (including an early, middle or late stage tumor) or a precancerous lesion thereof.

[0020] In another embodiment, the sample targeted for tumor identification includes (but is not limited to): tissue sample, blood sample, body fluid sample.

[0021] In another embodiment, the samples include (but are not limited to): paraffin-embedded samples, pleural effusion samples and 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 scraping or brush samples, tissue and cell biopsy samples, etc.

[0022] In another aspect of the present invention, a method for preparing a reagent for identifying a tumor is provided, comprising: (a) providing an isolated polynucleotide or a polynucleotide converted therefrom, comprising (1) the 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 corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; preferably, the at least one modified CpG site is any one CpG site selected from Nos. 1 to 22 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, it is any one CpG site selected from 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 shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is SEQ ID NO: 1. (b) using the polynucleotide of (a) as a target sequence, designing a detection reagent that specifically detects the CpG site modification status of the target sequence.

[0023] In another embodiment, the reagents for identifying tumors include but are not limited to primers, probes, chips or test strips.

[0024] In another embodiment, one or more sets of reagents can be prepared for the target sequence.

[0025] In another embodiment, the detection reagent is integrated on a chip.

[0026] In another aspect of the present invention, a reagent or reagent combination for specifically detecting the modification of CpG sites of a target sequence is provided, wherein the target sequence is: (1) a polynucleotide TAGMe-5 having a 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 corresponds to a polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; preferably, the reagent or reagent combination is directed against a gene sequence comprising the target sequence, preferably, the gene sequence comprises a gene panel or a gene group; preferably, the reagent or reagent combination comprises: primers for amplifying positions 277 to 392 or positions 247 to 421 of the nucleotide sequence shown in SEQ ID NO: 1.

[0027] In another embodiment, the reagent or reagent combination is a primer containing the sequence at positions 29 to 58 of SEQ ID NO: 3 and the sequence at positions 28 to 57 of SEQ ID NO: 4.

[0028] In another embodiment, the reagent or reagent combination is primers of SEQ ID NO: 3 and SEQ ID NO: 4.

[0029] In another embodiment, the reagent or reagent combination is: a primer formed by connecting any sequence shown in SEQ ID NO: 7-16 to the sequence at positions 29-58 in SEQ ID NO: 3, and a primer formed by connecting any sequence shown in SEQ ID NO: 17-26 to the sequence at positions 28-57 in SEQ ID NO: 4.

[0030] In another embodiment, the reagent or reagent combination further comprises primers of SEQ ID NO: 5 and SEQ ID NO: 6.

[0031] In another embodiment, the reagent or reagent combination is: primers with sequences shown in SEQ ID NO: 27 and SEQ ID NO: 28.

[0032] In another embodiment, the reagent or reagent combination is: primers with sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30.

[0033] In another aspect of the present invention, there is provided a use of the reagent or reagent combination for preparing a kit for identifying tumors.

[0034] In another aspect of the present invention, a kit for identifying tumors is provided, comprising the reagent or reagent combination.

[0035] In another embodiment, the kit may further include, but is not limited to: DNA purification reagents, DNA extraction reagents, Bisulfite, and PCR amplification reagents.

[0036] In another embodiment, the kit further includes: instructions for indicating the detection operation steps and result judgment criteria.

[0037] In another aspect of the present invention, a method for analyzing the methylation level of a sample to be tested is provided, comprising: (i) obtaining a polynucleotide from the sample to be tested; and (ii) analyzing the CpG site modification of a target sequence or a fragment thereof in the extracted polynucleotide, wherein the target sequence is: (1) a polynucleotide TAGMe-5 having a 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), wherein the unmodified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; and the converted polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1 corresponds to a polynucleotide (1) or (2), wherein the unmodified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

[0038] In another embodiment, the method for detecting the CpG site modification status of the target sequence in the extracted polynucleotide includes: pyrophosphate sequencing, bisulfite conversion sequencing, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion method, methylation chip method, qPCR method, digital PCR method, second-generation sequencing method, third-generation sequencing method, whole-genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, or a combination thereof.

[0039] In another embodiment, the method for analyzing the modification status of the CpG sites of the target sequence in the extracted polynucleotide comprises: (i) treating the extracted polynucleotide to convert the unmodified cytosine therein into uracil; preferably, the modification comprises 5-methylation modification, 5-hydroxymethylation modification, 5-aldehydemethylation modification or 5-carboxymethylation modification; preferably, treating the nucleic acid described in step (i) with Bisulfite; and (ii) analyzing the modification status of the target sequence in the nucleic acid treated in (i).

[0040] In another embodiment, the polynucleotide of step (i) is treated with Bisulfite; and (ii) the modification of the target sequence in the polynucleotide treated in step (i) is analyzed.

[0041] In another embodiment, the abnormal methylation level refers to hypermethylation of C in the CpG of the polynucleotide.

[0042] In another embodiment, other methylation detection methods and methylation detection methods newly developed in the future can also be applied to the present invention.

[0043] In another embodiment, the method of analyzing methylation levels is not a diagnostic method, that is, it is not intended to directly obtain a diagnosis of a disease.

[0044] In another embodiment, the method for detecting the methylation level of a sample is an in vitro method.

[0045] In another embodiment, the methylation-sensitive restriction endonuclease is a restriction endonuclease whose recognition site is sensitive to methylated bases; including but not limited to: HhaI, BmgBI, HaeII, RruI, TaiI, Bsu15I, Hin6I, HpyCH4IV, NarI, etc., or a combination of one or more thereof.

[0046] In another aspect of the present invention, an isolated polynucleotide or a polynucleotide converted therefrom is provided, wherein the polynucleotide comprises: (1) the polynucleotide TAGMe-5 having the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; preferably, the at least one modified CpG site is selected from any one of CpG sites 9 to 16 or a combination thereof, CpG sites 1 to 8 or a combination thereof, or CpG sites 17 to 22 or a combination thereof in the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is a polynucleotide having the nucleotide sequence shown in positions 277 to 392 or positions 247 to 421 in SEQ ID NO: 1; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the polynucleotide converted from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), wherein the non-modified cytosine is converted to T or U, and the cytosine C of the modified CpG site remains unchanged.

[0047] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1. Comparison of TAGMe-5 methylation values ​​in lung cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0049] Figure 2. Comparison of TAGMe-5 methylation values ​​in liver cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0050] Figure 3. Comparison of TAGMe-5 methylation values ​​in prostate cancer and control tissues (left panel), and the results of sensitivity and specificity analysis (right panel).

[0051] Figure 4. Comparison of TAGMe-5 methylation values ​​in cervical cancer and control tissues (left panel), and the results of sensitivity and specificity analysis (right panel).

[0052] Figure 5. Comparison of TAGMe-5 methylation values ​​in endometrial cancer and control tissues (left panel), and the results of sensitivity and specificity analysis (right panel).

[0053] Figure 6. Comparison of TAGMe-5 methylation values ​​in urothelial carcinoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0054] Figure 7. Comparison of TAGMe-5 methylation values ​​in biliary tract tumors and control tissues (left panel), and the results of sensitivity and specificity analysis (right panel).

[0055] Figure 8. Comparison of TAGMe-5 methylation values ​​in gastric cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0056] Figure 9. Comparison of TAGMe-5 methylation values ​​in breast cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0057] Figure 10. Comparison of TAGMe-5 methylation values ​​in esophageal cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0058] Figure 11. Comparison of TAGMe-5 methylation values ​​in brain glioma and control tissues (left panel), and the results of sensitivity and specificity analysis (right panel).

[0059] Figure 12. Comparison of TAGMe-5 methylation values ​​in colorectal cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0060] Figure 13. Comparison of TAGMe-5 methylation values ​​in leukemia and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0061] Figure 14. Comparison of TAGMe-5 methylation values ​​in pancreatic cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0062] Figure 15. Comparison of TAGMe-5 methylation values ​​in thyroid cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0063] FIG16 . Comparison of TAGMe-5 methylation values ​​in melanoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0064] Figure 17. Comparison of TAGMe-5 methylation values ​​in nasopharyngeal carcinoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0065] FIG18 . Comparison of TAGMe-5 methylation values ​​in oral cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0066] FIG19 . Comparison of TAGMe-5 methylation values ​​in laryngeal cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0067] FIG20 . Comparison of TAGMe-5 methylation values ​​in osteosarcoma and control tissues (left panel), and the results of sensitivity and specificity analysis (right panel).

[0068] FIG21 . Comparison of TAGMe-5 methylation values ​​in lymphoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0069] FIG22 . Comparison of TAGMe-5 methylation values ​​in renal cell carcinoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0070] FIG23 . Comparison of TAGMe-5 methylation values ​​in ovarian cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0071] Figure 24 shows the methylation levels of CpG sites 1-8 in SEQ ID NO: 1 in cancer cells analyzed by sequencing after bisulfite treatment, with normal control cells used as a control.

[0072] Figure 25 shows the methylation levels of CpG sites 17-22 in SEQ ID NO: 1 in cancer cells analyzed by bisulfite sequencing after treatment, with normal control cells used as a control. DETAILED DESCRIPTION

[0073] In view of the technical problems of the lack of DNA methylation and tumor markers in the field and the lack of clinical diagnostic significance, the inventors conducted extensive research and screening, and based on clinical research, revealed a DNA methylation tumor marker TAGMe-5 that can be used for screening of multiple cancer types. This tumor marker shows significant DNA methylation differences in paracancerous tissues and cancerous tissues. This difference has significant statistical significance and is shown in many tumors, including solid tumors and non-solid tumors. Solid tumors include lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumors, etc. Non-solid tumors include hematological tumors, lymphomas, etc. Therefore, the tumor marker of the present invention can be used for clinical multi-tumor (pan-cancer) screening (including early screening), molecular diagnosis, prognosis and efficacy evaluation.

[0074] As used herein, "sample" or "specimen" includes any material obtained from any individual (preferably a human) or isolated tissue, cell, or body fluid (such as plasma), suitable for DNA extraction and useful for methylation detection. For example, the sample may 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, stool samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brush samples, and tissue and cell biopsy samples.

[0075] As used herein, the term "hypermethylation (level)" refers to the presence of high methylation, hydroxymethylation, aldehyde methylation, or carboxymethylation modifications at CpG sites in a gene sequence. For example, in the context of methylation-specific PCR (MSP) analysis, a positive PCR result obtained in a PCR reaction using methylation-specific primers indicates that the DNA (gene) region under test is in a hypermethylated state. For example, in the context of real-time quantitative methylation-specific PCR, the determination of a hypermethylated state can be based on the statistical difference in the relative value of the methylation state of a control sample.

[0076] In the present invention, the term "tumor" refers to a relatively broad range of tumors (Pan-cancer), in which the SEQ ID NO: 1 segment in the genome presents a high methylation state as described in the present invention, which can be a solid tumor or a non-solid tumor, and can include (but is not limited to): respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, blood system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors or skeletal system tumors.

[0077] In the present invention, the methylation status of the nucleotide sequence represented by SEQ ID NO: 1 or a partial region (fragment) thereof significantly differs between tumor tissue and non-tumor tissue. When abnormally high methylation is detected in the gene sequence region, the subject can be considered to have a tumor or be at high risk for cancer. This significant difference in methylation status of the gene sequence represented by SEQ ID NO: 1 or a partial region thereof is very prominent in a variety of tumors (including those at early stages).

[0078] The present invention also includes "conservative variant sequences" of SEQ ID NO: 1 (or its reverse complementary sequence) that are conservative or have a higher sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 (or its reverse complementary sequence). The "high sequence identity" is, for example, higher than 90%, higher than 92%, higher than 95%, higher than 98%, higher than 99%, etc. It should be understood that different biological individuals may have differences at individual sequence sites (for example, there may be some meaningless SNPs), but this does not affect the detection based on the overall scheme of the present invention.

[0079] Based on the above, the present invention provides a nucleic acid derived from a specific region of the human genome, comprising the gene sequence set forth in SEQ ID NO: 1 or a portion thereof, and also including its antisense strand. In tumor cells, 5-methylcytosine (5mC) or other similar epigenetic modifications are generated at multiple 5'-CpG-3' C positions within the nucleic acid sequence.

[0080] Detection of one or more CpG sites provided herein is possible, and thus the present invention also encompasses nucleic acid fragments of the nucleotide sequence described herein, including at least one methylated CpG site. The at least one CpG site described herein may include 1 to 22, more specifically 2, 3, 5, 8, 10, 11, 12, 15, or 20, of SEQ ID NO: 1 or its reverse complement. Those skilled in the art will appreciate that, while the present invention provides CpG numbering based on one DNA strand, the numbering of each CpG site in the complementary DNA strand corresponding to the sense strand is readily identifiable based on the information provided herein.

[0081] Based on the information provided herein regarding the specific fragments in the human genome, those skilled in the art can readily obtain and utilize the CpG sites. The embodiments of the present invention provide a series of sequence fragments containing CpG sites, which serve as examples of preferred embodiments. However, it should be understood that variations can be made based on the information provided herein, such as selecting longer sequences containing the sequences of the present invention, or selecting sequences that overlap with the sequences of the present invention in certain regions.

[0082] The present invention also includes a gene panel or gene group having the nucleotide sequence or sequence fragment shown in SEQ ID NO: 1 or its complementary sequence. For the gene panel or gene group, the characteristics of normal cells and tumor cells can also be obtained by detecting the DNA methylation status.

[0083] It should be understood that a variety of techniques for analyzing methylation status can be applied to the present invention, and the present invention is not particularly limited to such detection techniques. The nucleic acids provided by the present invention can serve as key regions for analyzing methylation status in the genome, and their methylation status can be analyzed using various techniques known in the art, thereby analyzing the occurrence or progression of tumors.

[0084] The nucleic acid described in SEQ ID NO: 1 of the present invention, or a fragment thereof, or a sequence complementary thereto, can be treated with bisulfite, wherein unmethylated cytosine is converted to uracil, while methylated cytosine remains unchanged. Therefore, the present invention also provides nucleic acids obtained by bisulfite treatment of the aforementioned nucleic acid (including its complementary strand (antisense strand)), including nucleic acids or nucleic acid fragments having the nucleotide sequence of SEQ ID NO: 2. These nucleic acids can serve as more direct targets for designing detection reagents or kits.

[0085] The nucleic acid of the nucleotide sequence set forth in SEQ ID NO: 1 and / or its complementary nucleic acid and / or one or more fragments thereof of the present invention can be incorporated into one or more entities, such as one or more 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 analytical reagents. The designed targeted analytical reagents can also be incorporated into one or more entities, such as one or more kits.

[0086] The nucleic acids of the present invention, derived from the nucleic acid of SEQ ID NO: 1 and / or its complementary nucleic acid and / or one or more fragments thereof (e.g., by bisulfite conversion), can also be incorporated into one or more entities, such as one or more 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 analytical reagents. The designed targeted analytical reagents can also be incorporated into one or more entities, such as one or more kits, or one or more chips.

[0087] Based on the target gene and its epigenetic characteristics provided in the present invention, these techniques known in the art and some technologies to be developed can all be applied to the present invention to implement the detection of methylation levels. The methylation profile of nucleic acids can be determined using existing technologies (such as methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight), or other technologies that are still under development or to be developed. For example, quantitative methylation-specific PCR (QMSP) can be used to detect methylation levels. It is based on the continuous optical monitoring of a fluorescent PCR and is more sensitive than the MSP method. It has a high throughput and avoids the use of electrophoresis to analyze its results. In addition, other available technologies include: qPCR (Me-qPCR), second-generation sequencing, pyrosequencing, Sanger sequencing, bisulfite conversion sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing technology or HPLC, and combined gene group detection methods, etc. Conventional methods in this field. Although some preferred methods are provided in the embodiments of the present invention, the overall scheme of the present invention is not limited thereto.

[0088] As a preferred embodiment of the present invention, a method for detecting the methylation pattern of nucleic acids in a sample in vitro is also provided. This method is based on the principle that bisulfite can convert unmethylated cytosine into uracil, which is then converted to thymine during the subsequent PCR amplification process, while methylated cytosine remains unchanged. Therefore, after bisulfite treatment of nucleic acids, the methylated sites produce a single nucleotide polymorphism (SNP) similar to a C / T. Identifying the methylation pattern of nucleic acids in a test sample based on this principle can effectively distinguish between methylated and unmethylated cytosines.

[0089] The method of the present invention comprises: first providing a sample and extracting genomic DNA; secondly, treating the genomic DNA described in step (a) with bisulfite, thereby converting unmethylated cytosine in the genomic DNA into uracil; and thirdly, analyzing whether there is any abnormal methylation spectrum in the genomic DNA treated in step (b).

[0090] The methods of the present invention can be used to: test samples from subjects to assess whether they have a tumor; or to identify high-risk populations for tumors. These methods can be used in situations where the goal is not to obtain a direct disease diagnosis, such as situations where the goal is not to determine the final outcome of a disease, regional population analysis studies, scientific research, or censuses.

[0091] In a preferred embodiment of the present invention, DNA methylation is detected by PCR amplification and pyrosequencing. However, the present invention is not limited to this method in practical applications, and other DNA methylation detection methods known in the art or being improved may also be used. In performing PCR amplification, the primers used are not limited to those provided in the embodiments. Primers that differ in sequence from the primers provided in the embodiments of the present invention but are still specific to the nucleic acids or corresponding CpG sites indicated by the present invention may also be obtained.

[0092] As a preferred embodiment of the present invention, a method for detecting the methylation of nucleic acid in an in vitro detection sample is also provided, which is a methylation sensitive restriction endonuclease (MSRE) method. When a methylated base is contained in its cleavage site, the methylation sensitive restriction endonuclease cannot cut DNA. The MSRE method is based on the fundamental principle that methylation sensitive type II restriction endonucleases cannot cut sequences containing one or more methylated cleavage sites. Fragments containing one or more methylated CpG sequences are cut using methylation sensitive type II endonucleases and their isozymes (insensitive to methylation) and then analyzed using Southern blotting. The advantages of this method include: no need to know the detailed information of the target DNA primary structure, and direct evaluation of the methylation status of the CpG island can be provided, including obtaining some quantitative analysis information on the methylation of the gene being tested.

[0093] Regarding the marker nucleic acids provided by the present invention, other detection methods and reagents for determining genomic sequences, their variations and methylation status known to those skilled in the art may be included in the present invention.

[0094] The present invention provides a method for preparing a tumor detection reagent, comprising: providing a nucleic acid, using the full-length or fragmented nucleic acid as a target sequence, and designing a detection reagent that specifically detects the target sequence; wherein the target sequence includes at least one methylated CpG site. The detection reagent may include, but is not limited to, chips, primers, probes, etc. After obtaining the marker, the selection of the detection reagent is within the skill of those skilled in the art.

[0095] After knowing the sequence of the nucleic acid, it is known to those skilled in the art to design primers, with two primers flanking the specific sequence of the target gene to be amplified (including CpG sequences, complementary to CpG sequences for previously methylated gene regions, and complementary to TpG sequences for previously demethylated gene regions). In preferred embodiments of the present invention, the reagents are primers, preferably those listed in the Examples. In addition to primers, other diagnostic or detection reagents can also be prepared, including but not limited to probes, chips, etc.

[0096] The reagent may also be a reagent combination, such as a primer combination. For example, the combination includes more than one set of primers, thereby being able to amplify the above-mentioned multiple nucleic acids separately.

[0097] The present invention also provides a kit for detecting the methylation profile of nucleic acid in a sample in vitro. The kit comprises: a container and the above primer pair located in the container.

[0098] The kit may also include various reagents required for DNA extraction, DNA purification, PCR amplification, and other reagents, such as sample processing reagents. In addition, the kit may also include instructions for use, which indicate the detection steps and result judgment criteria for use by those skilled in the art.

[0099] The methods and reagents of the present invention demonstrate high accuracy in clinical tumor diagnosis, as demonstrated in the examples of the present invention, using a variety of clinical tumor samples. The present invention can be applied to areas such as early-stage tumor screening, therapeutic efficacy assessment, auxiliary diagnosis, and prognosis monitoring, as well as in situations where obtaining a direct disease diagnosis is not the primary objective, as previously mentioned.

[0100] The present invention provides multi-cancer markers suitable for detecting cervical exfoliated cells, among other things. Obtaining test samples is easy and non-invasive. Compared to surgical tissue sampling in clinical settings, this significantly reduces patient pain, improves compliance, and makes the procedure easier for clinicians. Clearly, this represents a significant advancement.

[0101] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in Molecular Cloning Laboratory Manual (ed. by J. Sambrook et al., Science Press), or according to the conditions recommended by the manufacturer.

[0102] Example 1 Determination of methylation detection targets

[0103] 1.1 Obtain the TAGMe-5 gene (human) sequence SEQ ID NO: 1;

[0104] In the above sequence (sense strand), each underlined "CG" represents a methylated CpG site, numbered 1 to 22 (methylated CpG sites 1 to 22) from 5' to 3'. The underlined dashed lines correspond to the upstream and downstream primer design regions in some embodiments; the italicized bold areas correspond to the detection target regions in some embodiments.

[0105] 1.2 Obtain the DNA sequence after Bisulfite conversion: SEQ ID NO: 2, wherein Y represents C or U (T);

[0106] In the above sequence (sense strand), each "YG" underlined in a solid line represents a converted methylated CpG site, numbered 1 to 22 from 5' to 3' ("YG"). The dotted underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold region corresponds to the detection target region in some embodiments.

[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 first-round PCR primers with a length of 25-35 bp and a moderate CG content, and amplify the length of 100-300 bp;

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

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

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

[0113] Table 1

[0114] In the table, F1 and R1 amplify the sequence segment corresponding to positions 277 to 392 (containing CpGs 9 to 16) in SEQ ID NO: 1 or SEQ ID NO: 2, and the sequence containing the primer complementary segment is the sequence segment 247 to 421 (containing CpGs 8 to 16).

[0115] In the sequence of Table F1 above, positions 29-58 (underlined) are bases complementary to the target detection sequence segment (upstream and downstream), and positions 22-28 are the tag sequence; in the sequence of R1, positions 28-57 are bases complementary to the target detection sequence segment (upstream and downstream), and positions 21-27 are the tag sequence.

[0116] Example 3 Validation of detection reagents

[0117] Synthesize a pair of primers to perform two rounds of PCR reaction on the positive and negative reference products:

[0118] The first-round PCR system and reaction conditions are shown in Table 2.

[0119] Table 2

[0120] The second-round PCR system and reaction conditions are shown in Table 3.

[0121] Table 3

[0122] After verifying the efficiency of primer PCR, a primer pool with different barcodes was synthesized, and the primers were diluted and combined (Table 4). For the first round of PCR, there are M×N (10×10) combinations of F and R primers, enabling simultaneous detection and localization of multiple samples. In Table 4, the lowercase bases in the F1 primer correspond to positions 1-21 (Illumina adapter) in the upstream primer F1 in Table 1, and the uppercase bases are the sequencing tag. The tag is followed by bases complementary to the target detection sequence segment (in subsequent examples, it is connected to positions 29-58 in SEQ ID NO: 3); the lowercase bases in the R1 primer correspond to positions 1-20 in the upstream primer R1 in Table 1, and the uppercase bases are the sequencing tag. The tag is followed by bases complementary to the target detection sequence segment (in subsequent examples, it is connected to positions 28-57 in SEQ ID NO: 4). The second round primers are F2 and R2 in Table 1.

[0123] Table 4

[0124] Here, R represents A or G.

[0125] Example 5: Differences in methylation of TAGMe-5 CpG sites between tumor tissues and non-tumor tissues - NGS sequencing

[0126] 1. Obtain clinical samples: Obtain paracancerous / non-cancerous tissue samples from the clinic. Paracancerous / non-cancerous samples serve as the control group, and cancerous tissue samples serve as the tumor detection experimental group.

[0127] 2. DNA extraction: DNA was extracted from the experimental group and the control group respectively; this experiment used the adsorption column method (but it is not limited to this method);

[0128] 3. Bisulfite Treatment: Treat the extracted DNA sample with bisulfite, strictly following the instructions; this experiment used the ZYMO Research EZ DNA Methylation-Gold Kit, Catalog No. D5006 (this method is not limited to this method).

[0129] 4. Use primers in the primer pool (first-round PCR primers) and Illumina system universal sequencing primers (second-round PCR primers) to perform two rounds of PCR amplification using conventional methods to construct the NGS library.

[0130] 5. After PCR amplification, 2% agarose gel electrophoresis was used to detect the specificity of the PCR fragments. 5 μl of PCR product was collected from each sample, mixed, and purified to recover the target fragment library for NGS sequencing.

[0131] 6. Analyze sequencing results: Extract sample sequencing information based on primer barcode sequences;

[0132] 7. Calculation of TAGMe-5 methylation value: NGS sequencing can independently detect the methylation status of individual CpG sites within the target region and calculate the median methylation value of all CpG sites as the methylation value of TAGMe-5 in the sample;

[0133] 8. Result analysis: Compare the TAGMe-5 methylation values ​​in non-tumor tissues and tumor tissues and determine the cutoff value using the ROC curve.

[0134] Example 6 TAGMe-5: Lung Cancer Clinical Sample Validation - NGS Sequencing

[0135] 38 clinical samples were obtained, including 19 normal lung cancer samples as a control group and 19 lung cancer samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical lung cancer samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0136] The results are shown in Figure 1, showing that in lung cancer clinical samples, the methylation value of TAGMe-5 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001). The sensitivity was 63.16% and the specificity was 89.47%.

[0137] Example 7 TAGMe-5: Validation of Liver Cancer Clinical Samples - NGS Sequencing

[0138] Thirty clinical samples were obtained, including 15 liver cancer adjacent to the tumor samples as a control group and 15 liver cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of the liver cancer clinical samples were constructed and TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0139] The results are shown in Figure 2, which shows that in clinical samples of liver cancer, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 73.33% and a specificity of 93.33%.

[0140] Example 8 TAGMe-5: Prostate cancer clinical sample validation - NGS sequencing method

[0141] Thirty clinical samples were obtained, including 15 prostate cancer adjacent paracancerous samples as a control group and 15 prostate cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of prostate cancer clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0142] The results are shown in Figure 3, which shows that in prostate cancer clinical samples, the methylation value of TAGMe-5 in cancer tissue was significantly higher than that in adjacent tissue (***P<0.001), with a sensitivity of 86.67% and a specificity of 93.33%.

[0143] Example 9 TAGMe-5: Validation of Cervical Cancer Clinical Samples - NGS Sequencing

[0144] 40 clinical samples were obtained, including 20 adjacent cervical adenocarcinoma samples as a control group and 20 cervical cancer samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of cervical cancer clinical samples were constructed, and TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0145] The results are shown in Figure 4, which show that in cervical cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 70% and a specificity of 90%.

[0146] Example 10 TAGMe-5: Endometrial cancer clinical sample validation - NGS sequencing method

[0147] Eighteen clinical samples were obtained, including nine endometrial cancer para-cancer samples as a control group and nine endometrial cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of endometrial cancer clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0148] The results are shown in Figure 5, which shows that in endometrial cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (****P<0.0001). The sensitivity was 100% and the specificity was 100%.

[0149] Example 11 TAGMe-5: Validation of clinical samples of urothelial carcinoma - NGS sequencing

[0150] Twenty clinical samples were obtained, including 10 urothelial carcinoma adjacent tissue samples as a control group and 10 urothelial carcinoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of urothelial carcinoma clinical samples were constructed, and TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0151] The results are shown in Figure 6, which shows that in clinical samples of urothelial carcinoma, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (****P<0.0001). The sensitivity was 100% and the specificity was 100%.

[0152] Example 12 TAGMe-5: Validation of Biliary Tract Tumor Clinical Samples - NGS Sequencing

[0153] Twenty clinical samples were obtained, including 10 adjacent biliary tract tumor samples as a control group and 10 biliary tract tumor samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical biliary tract tumor samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0154] The results are shown in Figure 7, which show that in clinical samples of biliary tract tumors, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 80% and a specificity of 80%.

[0155] Example 13 TAGMe-5: Gastric Cancer Clinical Sample Validation - NGS Sequencing

[0156] Twelve clinical samples were obtained, including six gastric cancer adjacent to the cancer sample as a control group and six gastric cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of gastric cancer clinical samples were constructed and TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0157] The results are shown in Figure 8, which show that in gastric cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.33% and a specificity of 83.33%.

[0158] Example 14 TAGMe-5: Breast Cancer Clinical Sample Verification - NGS Sequencing

[0159] Ten clinical samples were obtained, including five adjacent breast cancer samples as a control group and five breast cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and the second-round primers F2 and R2 in Table 1). NGS libraries of clinical breast cancer samples were constructed and TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0160] The results are shown in Figure 9, which show that in breast cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 80% and a specificity of 100%.

[0161] Example 15 TAGMe-5: Validation of Esophageal Cancer Clinical Samples - NGS Sequencing

[0162] Twelve clinical samples were obtained, including six esophageal cancer adjacent paracancerous samples as a control group and six esophageal cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of clinical esophageal cancer samples were constructed, and TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0163] The results are shown in Figure 10, which show that in esophageal cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.3% and a specificity of 100%.

[0164] Example 16 TAGMe-5: Validation of Clinical Samples of Brain Glioma - NGS Sequencing

[0165] Ten clinical samples were obtained, including five adjacent glioma samples as a control group and five glioma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and the second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical glioma samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0166] The results are shown in Figure 11, which show that in clinical samples of glioma, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0167] Example 17 TAGMe-5: Validation of Colorectal Cancer Clinical Samples - NGS Sequencing

[0168] Twelve clinical samples were obtained, including six colorectal cancer adjacent to the cancer sample as a control group and six colorectal cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of colorectal cancer clinical samples were constructed, and TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0169] The results are shown in Figure 12, which shows that in colorectal cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 83.33% and a specificity of 100%.

[0170] Example 18 TAGMe-5: Leukemia Clinical Sample Validation - NGS Sequencing

[0171] Twelve clinical samples were obtained, including 6 non-leukemia bone marrow smear samples as a control group and 6 leukemia bone marrow smear samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of leukemia clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0172] The results are shown in Figure 13, which show that in leukemia clinical samples, the methylation value of TAGMe-5 in leukemia bone marrow smears was significantly higher than that in non-leukemia bone marrow smears (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0173] Example 19 TAGMe-5: Pancreatic cancer clinical sample validation - NGS sequencing method

[0174] Twelve clinical samples were obtained, including six pancreatic cancer adjacent tissue samples as a control group and six pancreatic cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of liposarcoma clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0175] The results are shown in Figure 14, which show that in pancreatic cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 83.33% and a specificity of 100%.

[0176] Example 20 TAGMe-5: Validation of thyroid cancer clinical samples - NGS sequencing

[0177] Twelve clinical samples were obtained, including six thyroid cancer adjacent tissue samples as a control group and six thyroid cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of thyroid tumor clinical samples were constructed, and TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0178] The results are shown in Figure 15, which show that in thyroid cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 83.33% and a specificity of 83.33%.

[0179] Example 21 TAGMe-5: Melanoma Clinical Sample Validation - NGS Sequencing

[0180] Twelve clinical samples were obtained, including six normal skin tissue samples as a control group and six skin melanoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of melanoma clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0181] The results are shown in Figure 16, which show that in melanoma clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0182] Example 22 TAGMe-5: Validation of Nasopharyngeal Carcinoma Clinical Samples - NGS Sequencing

[0183] Twelve clinical samples were obtained, including six nasopharyngeal carcinoma paracancerous samples as a control group and six nasopharyngeal carcinoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of nasopharyngeal carcinoma clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0184] The results are shown in Figure 17, which show that in NPC clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.33% and a specificity of 66.67%.

[0185] Example 23 TAGMe-5: Oral Cancer Clinical Sample Validation - NGS Sequencing

[0186] Twelve clinical samples were obtained, including six oral cancer paracancerous samples as a control group and six oral cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of oral cancer clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0187] The results are shown in Figure 18, which show that in oral cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 100% and a specificity of 83.33%.

[0188] Example 24 TAGMe-5: Laryngeal cancer clinical sample validation - NGS sequencing method

[0189] Ten clinical samples were obtained, including five paracancerous laryngeal cancer samples as a control group and five laryngeal cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of laryngeal cancer clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0190] The results are shown in Figure 19, which show that in laryngeal cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0191] Example 25 TAGMe-5: Validation of Osteosarcoma Clinical Samples - NGS Sequencing

[0192] Twelve clinical samples were obtained, including 6 adjacent osteosarcoma samples as a control group and 6 osteosarcoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and the second-round primers F2 and R2 in Table 1) to construct an NGS library of osteosarcoma clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0193] The results are shown in Figure 20, which shows that in clinical osteosarcoma samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0194] Example 26 TAGMe-5: Lymphoma Clinical Sample Validation - NGS Sequencing

[0195] Twelve clinical samples were obtained, including 6 normal lymphoid samples as a control group and 6 lymphoma samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of the lymphoma clinical samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0196] The results are shown in Figure 21, which show that in lymphoma clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01). The sensitivity was 100% and the specificity was 100%.

[0197] Example 27 TAGMe-5: Validation of Renal Cell Carcinoma Clinical Samples - NGS Sequencing

[0198] Twelve clinical samples were obtained, including 6 adjacent renal cell carcinoma samples as a control group and 6 renal cell carcinoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of renal cell carcinoma clinical samples were constructed, and TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0199] The results are shown in Figure 22, which show that in clinical samples of renal cell carcinoma, the methylation value of TAGMe-5 in cancer tissue was significantly higher than that in adjacent tissue (*P<0.05), with a sensitivity of 83.33% and a specificity of 83.33%.

[0200] Example 28 TAGMe-5: Validation of ovarian cancer clinical samples - NGS sequencing

[0201] Twelve clinical samples were obtained, including six adjacent ovarian cancer samples as a control group and six ovarian cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical ovarian cancer samples. TAGMe-5 methylation levels were analyzed according to the NGS sequencing protocol.

[0202] The results are shown in Figure 23, which show that in ovarian cancer clinical samples, the methylation value of TAGMe-5 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 83.33% and a specificity of 100%.

[0203] Example 29 Detection Performance Analysis of Single CpG Sites

[0204] Using the clinical samples obtained in the previous examples, we analyzed CpG sites 9 through 16 (CpG sites within positions 277 to 392 in SEQ ID NO: 1) as single CpG sites for cancer detection. Methylation at these single CpG sites was analyzed using NGS sequencing.

[0205] The results are shown in Tables 5 to 9.

[0206] Table 5

[0207] Table 6

[0208] Table 7

[0209] Table 8

[0210] Table 9

[0211] The results in Tables 5 to 9 indicate that a single CpG site has high sensitivity and / or specificity, and a single CpG site can also be used as a target for methylation modification analysis to guide clinical auxiliary diagnosis analysis of cancer.

[0212] Example 30: Differences in methylation of TAGMe-5 CpG sites between tumor cells and non-tumor cells

[0213] Bisulfite Sequencing PCR (BSP) was used. The steps of this sequencing method are as follows:

[0214] 1. Extract genomic DNA from hematological tumor cell lines (myeloid leukemia cell line K562), colorectal cancer cell line (HCT116), pancreatic cancer cell line (SW1990), human renal clear cell adenocarcinoma cell line (786-O), gastric cancer cell line (BGC-823), breast cancer cell line (BT-549), and cervical cancer cell line (HeLa) and their corresponding normal cell genomic DNA;

[0215] 2. Treat the extracted genomic DNA of cancer cell lines and normal cell lines with bisulfite and use them as templates for subsequent PCR amplification;

[0216] 3. Design amplification primers according to the sequence of SEQ ID NO: 2, as shown in Table 10, and perform amplification.

[0217] 4. After PCR amplification, the specificity of the PCR fragments was tested by 2% agarose gel electrophoresis. The target fragments were recovered by gel excision, inserted into the T vector, transformed into competent E. coli, and plated. The next day, clones were selected for sequencing. More than 10 clones were selected for each fragment for Sanger sequencing.

[0218] Table 10, BSP primers

[0219] BSP verification of the methylation levels of cancer cells and normal control cells at methylation sites 1-8 in the SEQ ID NO: 1 region is shown in Figure 24, which shows that the methylation level of TAGMe-5 in cancer cells is significantly higher than that in normal cells.

[0220] BSP verification of the methylation levels of cancer cells and normal control cells at methylation sites 17-22 in the SEQ ID NO: 1 region is shown in Figure 25, which shows that the methylation level of TAGMe-5 in cancer cells is significantly higher than that in normal cells.

[0221] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of an isolated polynucleotide or a polynucleotide converted therefrom in the preparation of a reagent or kit for identifying a tumor; wherein: The polynucleotide comprises: (1) a polynucleotide TAGMe-5 having a nucleotide sequence as 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); The polynucleotide converted from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

2. The use according to claim 1, characterized in that The polynucleotide converted from the isolated polynucleotide is a polynucleotide with a nucleotide sequence shown in SEQ ID NO: 2; or The at least one modified CpG site is any one of CpG sites 1 to 22 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably any one of CpG sites 9 to 16 or a combination thereof, CpG sites 1 to 8 or a combination thereof, or CpG sites 17 to 22 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; or The polynucleotide fragment is a polynucleotide having a nucleotide sequence shown at positions 277 to 392 or positions 247 to 421 in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that The tumors include: respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, blood system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors or bone system tumors; Preferably, the tumor includes: 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.

4. The use according to claim 1, characterized in that The samples targeted for tumor identification include: tissue samples, blood samples, and body fluid samples.

5. A method for preparing a reagent for identifying a tumor, comprising: (a) providing an isolated polynucleotide or a polynucleotide converted therefrom, comprising (1) a polynucleotide TAGMe-5 having a nucleotide sequence as 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 corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; preferably, the at least one modified CpG site is any one of CpG sites 1 to 22 or a combination thereof selected from the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1; preferably, any one of CpG sites 9 to 16 or a combination thereof, CpG sites 1 to 8 or a combination thereof, or CpG sites 17 to 22 or a combination thereof selected from the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is any one of CpG sites 9 to 16 or a combination thereof, CpG sites 1 to 8 or a combination thereof, or CpG sites 17 to 22 or a combination thereof selected from the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1; A polynucleotide having the nucleotide sequence shown at positions 277 to 392 or 247 to 421 in NO: 1; (b) Using the polynucleotide of (a) as the target sequence, a detection reagent is designed to specifically detect the CpG site modification status of the target sequence.

6. A reagent or reagent combination for specifically detecting the modification of a CpG site of a target sequence, wherein the target sequence is: (1) a polynucleotide TAGMe-5 having a nucleotide sequence as 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 corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; preferably, the reagent or reagent combination is directed to a gene sequence comprising the target sequence, preferably, the gene sequence comprises a gene panel or a gene group; preferably, the reagent or reagent combination comprises: Primers for amplifying the nucleotide sequence at positions 277 to 392 or 247 to 421 in SEQ ID NO:

1.

7. Use of the reagent or reagent combination according to claim 6 for preparing a kit for identifying a tumor; preferably, a respiratory system tumor, a digestive system tumor, a urinary system tumor, a gynecological and reproductive system tumor, a blood system tumor, a nervous system tumor, a head and neck tumor, a skin system tumor, an endocrine system tumor or a skeletal system tumor; more preferably, the tumor comprises: Lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumors, 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.

8. A kit for identifying a tumor, comprising the reagent or reagent combination according to claim 6.

9. A method for analyzing the methylation level of a sample to be tested, comprising: (i) a polynucleotide obtained from a sample to be tested; and (ii) analyzing the CpG site modification of the target sequence or its fragment in the extracted polynucleotide, wherein the target sequence is: (1) the 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 corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; the converted polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1 corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

10. The method according to claim 9, characterized in that Methods for detecting the modification of CpG sites in the target sequence of the extracted polynucleotide include: pyrophosphate sequencing, bisulfite conversion sequencing, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion, methylation chip method, qPCR method, digital PCR method, second-generation sequencing method, third-generation sequencing method, whole-genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, or a combination thereof; or The method for analyzing the modification of the CpG site of the target sequence in the extracted polynucleotide comprises: (i) treating the extracted polynucleotide to convert the unmodified cytosine therein into uracil; preferably, the modification comprises 5-methylation modification, 5-hydroxymethylation modification, 5-aldehyde methylation modification or 5-carboxymethylation modification; preferably, treating the nucleic acid described in step (i) with Bisulfite; and (ii) analyzing the modification of the target sequence described in the nucleic acid treated by (i).

11. An isolated polynucleotide or a polynucleotide converted therefrom, the polynucleotide comprising: (1) The 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; preferably, the at least one modified CpG site is selected from any one of CpG sites 9 to 16 or a combination thereof, CpG sites 1 to 8 or a combination thereof, or CpG sites 17 to 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 positions 277 to 392 or positions 247 to 421 of SEQ ID NO: 1; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); The polynucleotide converted from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

Images