DNA methylation marker tagme-4 and use thereof in tumor detection

Abstract

Provided is a tumor marker TAGMe-4 related to epigenetic modification. A tumor is detected by means of an abnormal hypermethylation phenomenon at the marker-specific site in the tumor. The TAGMe-4 gene sequence region has a significant methylation difference between cancerous tissue and paracancerous tissue. It can be determined upon detection of an abnormal hypermethylation state in the TAGMe-4 gene sequence region that a subject belongs to a group at high risk of tumors. In addition, such significant difference between tumor tissue and non-tumor tissue presented by the TAGMe-4 is present in a wide range of different types of tumors, including solid tumors and non-solid tumors, and thus the TAGMe-4 can be used as a pan-cancer marker, for example, the TAGMe-4 can be used for large-scale screening of people.

Description

DNA methylation marker TAGMe-4 and its application in tumor detection

[0001] This application claims priority to patent application No. CN 202311284439.7, filed on October 5, 2023; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the fields of pharmacology and gene epigenetic modification. More specifically, the present invention relates to a DNA methylation marker TAGMe-4 and its use in tumor detection. Background Art

[0003] Tumorigenesis is a multifactorial, multi-stage, complex and progressive process. The transformation of normal cells into malignant tumor cells requires multiple steps and stages, including the initiation phase, the promotion phase and the evolution phase. This makes cancer research more complicated.

[0004] The biological research of cancer cells includes a series of contents, such as cancer cell membrane, glycoprotein sugar chain structure, cell transmembrane signal transduction, cell apoptosis and regulation, cell differentiation induction and malignant transformation, information transmission of malignant growth, molecular targets of treatment, the role of oncogenes and tumor suppressor genes, and the mechanism of cancer metastasis.

[0005] In recent years, significant progress has been made in the treatment of tumors. In addition to improvements in traditional tumor treatments such as surgery, radiotherapy, and chemotherapy, the development of fundamental disciplines such as tumor molecular biology and cell biology has led to the continuous emergence of new treatments and approaches. Some explorations have gradually transitioned from laboratory research to clinical studies. However, further in-depth research and clinical observation are needed to truly evaluate their clinical efficacy.

[0006] DNA methylation is a crucial component of epigenetics, playing a crucial role in maintaining normal cell function, embryonic development, disease pathogenesis, and human tumors. The level of DNA methylation, particularly in gene promoter regions, directly influences gene transcriptional activity, controlling gene expression and playing a crucial role in life. Therefore, the study of DNA methylation is a top priority in epigenetics and, indeed, in the entire life sciences.

[0007] There are many methods for detecting DNA methylation. One approach relies on methylation-sensitive restriction endonucleases (MSREs). Methylation-sensitive restriction endonucleases are a class of endonucleases that are sensitive to methylation modifications. These enzymes contain a methylated base within their cleavage site, which blocks DNA cleavage by the endonuclease, allowing detection via Southern blotting or PCR. The most commonly used endonucleases in this approach are HpaII and MspI. HpaII and MspI recognize the same sequence, but HpaII is sensitive to methylation while MspI is not. This approach offers the advantage of simplicity, but the limitation of the endonuclease cleavage sites significantly limits the methylated regions that can be studied. Another approach is based on DNA methylation antibodies. Antibody-based DNA methylation research methods employ antibodies against methylcytosine or methylation-binding proteins to study DNA methylation. The principle and operation are similar to ChIP, where antibody-bound DNA fragments are hybridized or sequenced on a microarray. The advantage is that changes in DNA methylation can be studied genome-wide, but this method lacks precision and cannot determine the methylation status of individual bases. Furthermore, this method is easily affected by the CG content of different regions in the genome, making it insensitive to regions with low CG content in the genome. Another method is based on sodium bisulfite; the most widely used method for detecting DNA methylation is the sodium bisulfite-based method. Its advantage is that it can accurately detect changes in methylation with single-base resolution. The principle of this method is that after DNA is treated with sodium bisulfite, C (cytosine) that has not been methylated can be converted to U (uracil), while C that has been methylated will not change. After PCR amplification and sequencing, and then compared with the original sequence, the methylation modification status of a certain region in the genome can be obtained.

[0008] Despite this, based on the methodology, this field still urgently needs to find molecular targets that can be used to diagnose, prognose and predict the occurrence and development of cancer in order to provide more and better detection effects.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a DNA methylation marker TAGMe-4 and its use in tumor detection.

[0011] 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 detecting a tumor; wherein the polynucleotide comprises: (1) a polynucleotide TAGMe-4 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.

[0012] In one embodiment, the modification includes 5-methylation modification (5mC), 5-hydroxymethylation modification (5hmC), 5-aldehydemethylation modification (5-fC) or 5-carboxymethylation modification (5-caC).

[0013] As an embodiment, the SEQ ID NO: 1 also includes sequence variants or homologous sequences thereof.

[0014] In one embodiment, the sequence variant or homologous sequence is a sequence having a sequence identity of 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, or 99.8% or more to 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.

[0015] In one embodiment, the identification includes diagnosis, detection, screening or prognostic assessment.

[0016] As an embodiment, the polynucleotide converted from the isolated polynucleotide is a polynucleotide having a nucleotide sequence shown in SEQ ID NO: 2.

[0017] As one embodiment, the at least one modified CpG site is any CpG site or a combination thereof selected from No. 1 to No. 88 (such as 2 to 88, more specifically 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45) in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, it is any CpG site or a combination thereof selected from No. 3 to No. 19, or any CpG site or a combination thereof, in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1.

[0018] In one embodiment, the polynucleotide fragment is a polynucleotide having a nucleotide sequence shown at positions 40 to 157 or positions 10 to 185 in SEQ ID NO: 1.

[0019] As an 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.

[0020] As an embodiment, the tumor includes: 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 cancer, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma or ovarian cancer.

[0021] As an embodiment, the tumor detection is directed to tumors (including early, middle or late stage tumors) and their precancerous lesions.

[0022] As an embodiment, the samples targeted for tumor detection include (but are not limited to): tissue samples, body fluid samples, and blood samples.

[0023] As an 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.

[0024] In another aspect of the present invention, a method for preparing a tumor detection reagent is provided, comprising: (a) providing an isolated polynucleotide or a polynucleotide converted therefrom, comprising (1) the polynucleotide TAGMe-4 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 of CpG sites 1 to 88 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, it is any one of CpG sites 3 to 19 or a combination thereof, or any one of CpG sites 20 to 88 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is any one of CpG sites 40 to 157 or 10 to 157 in 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.

[0025] As an embodiment, one or more sets of reagents may be prepared for the target sequence.

[0026] As an embodiment, the detection reagent includes but is not limited to: primers, probes, chips or test paper.

[0027] As an embodiment, the detection reagent is integrated on a chip.

[0028] 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-4 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: a primer for amplifying positions 40 to 157 or positions 10 to 185 of the nucleotide sequence shown in SEQ ID NO: 1.

[0029] As an embodiment, the reagent or reagent combination is: a primer containing the sequence at positions 29 to 58 in SEQ ID NO: 3 and the sequence at positions 28 to 55 in SEQ ID NO: 4.

[0030] As an embodiment, the reagent or reagent combination is: primers of SEQ ID NO: 3 and SEQ ID NO: 4 sequences.

[0031] As an 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 28-55 in SEQ ID NO: 4.

[0032] In another aspect of the present invention, there is provided a use of the reagent or reagent combination for preparing a kit for detecting tumors.

[0033] In another aspect of the present invention, a kit for detecting tumors is provided, comprising the reagent or reagent combination.

[0034] As an embodiment, the kit may further include but is not limited to: DNA purification reagents, DNA extraction reagents, Bisulfite, and PCR amplification reagents.

[0035] As an embodiment, the kit further includes: instructions for indicating the detection operation steps and result judgment criteria.

[0036] 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-4 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.

[0037] As an 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; or

[0038] As an 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 described in the nucleic acid treated in (i).

[0039] In one 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.

[0040] As an embodiment, abnormal methylation level refers to high methylation of C in the CpG of the polynucleotide.

[0041] As an embodiment, other methylation detection methods and methylation detection methods newly developed in the future can also be applied to the present invention.

[0042] As an embodiment, the method for analyzing methylation levels is not a diagnostic method, that is, it is not intended to directly obtain a diagnosis result of a disease.

[0043] As an embodiment, the method for detecting the methylation level of a sample is an in vitro method.

[0044] As an 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., one or more combinations thereof.

[0045] 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-4 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 any one of CpG sites 1 to 88 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, it is any one of CpG sites 3 to 19 or a combination thereof, or any one of CpG sites 20 to 88 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is any one of CpG sites 40 to 157 or 10 to 157 in SEQ ID NO: 1; (1) a polynucleotide having the nucleotide sequence shown at position 85; or (2) a polynucleotide having a sequence complementary 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.

[0046] 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

[0047] Figure 1. Methylation levels of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in lung cancer clinical samples, with adjacent adjacent tissues as controls.

[0048] Figure 2. Methylation levels of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in clinical samples of liver cancer, with adjacent adjacent tissues as controls.

[0049] Figure 3. Methylation levels of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in prostate cancer clinical samples, with adjacent adjacent tissues as controls.

[0050] Figure 4. Methylation levels of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in cervical cancer clinical samples, with adjacent adjacent tissues as controls.

[0051] Figure 5. Methylation levels of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in endometrial cancer clinical samples, with adjacent adjacent tissues as controls.

[0052] Figure 6. Methylation levels of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in clinical samples of urothelial carcinoma, with adjacent adjacent tissues as controls.

[0053] Figure 7. Methylation levels of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in clinical samples of biliary tract tumors, with adjacent adjacent tissues as controls.

[0054] Figure 8. Methylation level of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in gastric cancer clinical samples, with adjacent adjacent tissues as controls.

[0055] Figure 9. Methylation levels of TAGMe-4 in breast cancer tissue (left panel) and sensitivity and specificity (right panel) in clinical breast cancer samples, with adjacent adjacent tissues as controls.

[0056] Figure 10. Methylation level of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in esophageal cancer clinical samples, with adjacent adjacent tissues as controls.

[0057] Figure 11. Methylation levels of TAGMe-4 in cancerous tissues (left panel) and sensitivity and specificity (right panel) in clinical glioma samples, with adjacent adjacent tissues as controls.

[0058] Figure 12. Methylation levels of TAGMe-4 in cancer tissues (left panel) and sensitivity and specificity (right panel) in colorectal cancer clinical samples, with adjacent adjacent tissues as controls.

[0059] Figure 13. Methylation levels of TAGMe-4 in cancer tissues (left panel) and sensitivity and specificity (right panel) in leukemia clinical samples, with non-leukemia bone marrow smear samples as controls.

[0060] Figure 14. Methylation levels of TAGMe-4 in pancreatic cancer tissue (left panel) and sensitivity and specificity (right panel) in pancreatic cancer clinical samples, with adjacent adjacent tissues as controls.

[0061] Figure 15. Methylation levels of TAGMe-4 in cancer tissues (left panel) and sensitivity and specificity (right panel) in thyroid cancer clinical samples, with adjacent adjacent tissues as controls.

[0062] Figure 16. Methylation levels of TAGMe-4 in cancerous tissue (left panel) and sensitivity and specificity (right panel) in melanoma clinical samples, with adjacent adjacent tissues as controls and normal skin tissue samples as controls.

[0063] Figure 17. Methylation levels of TAGMe-4 in cancer tissues (left panel) and sensitivity and specificity (right panel) in nasopharyngeal carcinoma clinical samples, with adjacent adjacent tissues as controls.

[0064] Figure 18. Methylation levels of TAGMe-4 in oral cancer tissues (left panel) and sensitivity and specificity (right panel) in clinical oral cancer samples, with adjacent adjacent tissues as controls.

[0065] Figure 19. Methylation levels of TAGMe-4 in cancerous tissues (left panel) and sensitivity and specificity (right panel) in laryngeal cancer clinical samples, with adjacent adjacent tissues as controls.

[0066] Figure 20. Methylation levels of TAGMe-4 in cancerous tissues (left panel) and sensitivity and specificity (right panel) in osteosarcoma clinical samples, with adjacent adjacent tissues as controls.

[0067] Figure 21. Methylation levels of TAGMe-4 in cancer tissues (left panel) and sensitivity and specificity (right panel) in lymphoma clinical samples, with normal lymphoid samples as controls.

[0068] Figure 22. Methylation levels of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in renal cell carcinoma clinical samples, with adjacent adjacent tissues as controls.

[0069] Figure 23. Methylation level of TAGMe-4 in cancer tissue (left panel) and sensitivity and specificity (right panel) in ovarian cancer clinical samples, with adjacent adjacent tissues as controls.

[0070] FIG24 is a BSP validation of the methylation levels of CpG sites 20-88 in SEQ ID NO: 1 in cancer cells and normal control cells. DETAILED DESCRIPTION

[0071] The present invention provides a tumor marker TAGMe-4 related to epigenetic modification, which uses the abnormal hypermethylation phenomenon of marker-specific sites in tumors to detect tumors. The TAGMe-4 gene sequence region has significant methylation differences between cancerous tissue and adjacent tissue. As long as the abnormal hypermethylation state of the TAGMe-4 gene sequence region is detected, the subject can be determined to be a high-risk group for tumors. Moreover, the significant differences between tumor tissue and non-tumor tissue presented by TAGMe-4 are widely present in different types of tumors, covering solid tumors and non-solid tumors, and can thus be used as a pan-cancer marker, for example, for screening of the population.

[0072] 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 "higher sequence identity" is, for example, greater than 90%, greater than 92%, greater than 95%, greater than 98%, greater 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.

[0073] 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.

[0074] Detection of one or more CpG sites provided by the present invention is possible, and thus the present invention also encompasses nucleic acid fragments of the nucleotide sequence described herein, which include at least one methylated CpG site. The at least one CpG site can include 1 to 88, more specifically 2, 3, 5, 10, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, or 85, of SEQ ID NO: 1 or its reverse complement. Obviously, after 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 can be readily obtained based on the information provided by the present invention.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] Based on the target gene and its epigenetic characteristics provided in the present invention, these technologies 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 level of nucleic acids can be measured 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 method, and combined gene group detection methods and other 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.

[0082] As a preferred embodiment of the present invention, a method for detecting the methylation level of nucleic acids in a sample in vitro is also provided. The method is based on the principle that bisulfite can convert unmethylated cytosine into uracil, which is then converted into 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. Based on this principle, the methylation level of nucleic acids in the test sample can be identified and effectively distinguished between methylated and unmethylated cytosines.

[0083] 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 abnormal methylation level in the genomic DNA treated in step (b).

[0084] 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.

[0085] 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.

[0086] 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.

[0087] Regarding the marker nucleic acids provided by the present invention, other detection methods and reagents for determining the sequence of a genome, its variation and methylation status known to those skilled in the art can be included in the present invention.

[0088] 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, a chip, primers, probes, etc. After obtaining the marker, the selection of the detection reagent is within the skill of those skilled in the art.

[0089] 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.

[0090] 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.

[0091] The present invention also provides a kit for detecting the methylation level of nucleic acid in a sample in vitro. The kit comprises: a container and the above primer pair located in the container.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] The following further describes the specific embodiments of the present invention in conjunction with the accompanying drawings and examples. The examples are only used to more clearly illustrate the technical solutions of the present invention, and the scope of protection of the present invention is based on the claims.

[0096] Example 1 Determination of methylation detection targets

[0097] 1. Obtain human DNA sequence SEQ ID NO: 1, which is the TAGMe-4 gene sequence;

[0098] In the above sequence (sense strand), each underlined "CG" represents a methylated CpG site, numbered 1 to 88 (methylated CpG sites 1 to 88) 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.

[0099] 2. Obtain the DNA sequence SEQ ID NO: 2 after bisulfite conversion, wherein Y represents C or U (T);

[0100] In the above sequence (sense strand), each "YG" underlined in a solid line represents a converted methylated CpG site, numbered from 5' to 3' as "YG" 1 to 88 (converted methylated CpG sites 1 to 88). 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.

[0101] 3. Determine the detection region and design primers upstream and downstream of the detection region.

[0102] Example 2 Primer design and synthesis

[0103] 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;

[0104] 2. Add a barcode (Sample-ID) to the end of the first round primers (Table 1);

[0105] 3. Add a tag sequence to the end of the first round of primers for library construction;

[0106] 4. Synthesize primers for the first-round PCR reaction and primers for the second-round PCR reaction with Illumina adapters and indexes (Table 1).

[0107] Table 1

[0108] Among them, F1 and R1 amplify the sequence corresponding to positions 10 to 185 in SEQ ID NO: 1 or SEQ ID NO: 2 (including the primer complementary region, containing CpG sites 3 to 20), wherein positions 40 to 157 contain CpG sites 3 to 19.

[0109] Example 3 Primer Verification

[0110] Synthesize a pair of primers and perform two rounds of PCR reaction on the positive and negative reference products:

[0111] The first-round PCR reaction system is shown in Table 2.

[0112] Table 2

[0113] The second-round PCR reaction system is shown in Table 3.

[0114] Table 3

[0115] Example 4 Construction of primer pool

[0116] 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 28-55 in SEQ ID NO: 4). The second round primers are F2 and R2 in Table 1.

[0117] Table 4

[0118] Here, R represents A or G.

[0119] Example 5: Methylation differences of TAGMe-4 CpG sites between tumor tissues and non-tumor tissues - NGS sequencing

[0120] 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.

[0121] 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);

[0122] 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).

[0123] 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.

[0124] 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.

[0125] 6. Analyze sequencing results: Extract sample sequencing information based on primer barcode sequences;

[0126] 7. Calculation of TAGMe-4 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-4 in the sample;

[0127] 8. Result analysis: Compare the TAGMe-4 methylation values ​​in non-tumor tissues and tumor tissues and determine the cutoff value using the ROC curve.

[0128] Example 6 TAGMe-4: Lung Cancer Clinical Sample Validation - NGS Sequencing

[0129] Forty clinical samples were obtained, including 20 adjacent normal lung cancer samples as a control group and 20 lung 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 lung cancer samples. TAGMe-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0130] The results are shown in Figure 1, showing that in lung cancer clinical samples, the methylation value of TAGMe-4 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001). The sensitivity was 90% and the specificity was 100%.

[0131] Example 7 TAGMe-4: Liver cancer clinical sample verification - NGS sequencing method

[0132] Thirty-four clinical samples were obtained, including 17 liver cancer adjacent to liver cancer samples as a control group and 17 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 analyzed for TAGMe-4 methylation levels according to the NGS sequencing protocol.

[0133] The results are shown in Figure 2, which shows that in clinical liver cancer samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 70.59% and a specificity of 94.12%.

[0134] Example 8 TAGMe-4: Prostate cancer clinical sample validation - NGS sequencing method

[0135] Forty clinical samples were obtained, including 20 prostate cancer adjacent to prostate cancer samples as a control group and 20 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0136] The results are shown in Figure 3, which show that in prostate cancer clinical samples, the methylation value of TAGMe-4 in cancer tissue was significantly higher than that in adjacent tissue (***P<0.001), with a sensitivity of 75% and a specificity of 80%.

[0137] Example 9 TAGMe-4: Cervical cancer clinical sample verification - NGS sequencing method

[0138] 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0139] The results are shown in Figure 4, which shows that in cervical cancer clinical samples, the methylation value of TAGMe-4 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001). The sensitivity was 90% and the specificity was 90%.

[0140] Example 10 TAGMe-4: Endometrial cancer clinical sample validation - NGS sequencing method

[0141] 36 clinical samples were obtained, including 18 endometrial cancer para-cancer samples as a control group and 18 endometrial 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 endometrial cancer clinical samples were constructed, and TAGMe-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0142] The results are shown in Figure 5, showing that in endometrial cancer clinical samples, the methylation value of TAGMe-4 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001), with a sensitivity of 66.67% and a specificity of 94.44%.

[0143] Example 11 TAGMe-4: Validation of clinical samples of urothelial carcinoma - NGS sequencing

[0144] Forty clinical samples were obtained, including 20 urothelial carcinoma paracancerous tissue samples as a control group and 20 urothelial carcinoma 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 urothelial carcinoma clinical samples were constructed, and TAGMe-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0145] The results are shown in Figure 6, which shows that in clinical samples of urothelial carcinoma, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (****P<0.0001). The sensitivity was 100% and the specificity was 80%.

[0146] Example 12 TAGMe-4: Biliary Tract Tumor Clinical Sample Validation - NGS Sequencing

[0147] Thirty-two clinical samples were obtained, including 16 adjacent biliary tract tumor samples as a control group and 16 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0148] The results are shown in Figure 7, which shows that in clinical samples of biliary tract tumors, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (****P<0.0001). The sensitivity was 75% and the specificity was 93.75%.

[0149] Example 13 TAGMe-4: Gastric Cancer Clinical Sample Validation - NGS Sequencing

[0150] Ten clinical samples were obtained, including five gastric cancer paracancerous samples as a control group and five 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 and second-round primers F2 and R2 in Table 1) to construct an NGS library of gastric cancer clinical samples. TAGMe-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0151] The results are shown in Figure 8, which show that in gastric cancer clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 80% and a specificity of 100%.

[0152] Example 14 TAGMe-4: Breast cancer clinical sample validation - NGS sequencing method

[0153] Sixteen clinical samples were obtained, including eight adjacent breast cancer samples as a control group and eight 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 second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical breast cancer samples. TAGMe-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0154] The results are shown in Figure 9, which show that in breast cancer clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 75% and a specificity of 87.5%.

[0155] Example 15 TAGMe-4: Esophageal cancer clinical sample validation - NGS sequencing method

[0156] Twelve clinical samples were obtained, including six esophageal cancer adjacent normal 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 and second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical esophageal cancer samples. TAGMe-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0157] The results are shown in Figure 10, which show that in esophageal cancer clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 83.3%.

[0158] Example 16 TAGMe-4: Validation of Clinical Samples of Brain Glioma - NGS Sequencing

[0159] Fourteen clinical samples were obtained, including seven adjacent glioma samples as a control group and seven 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 second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical glioma samples. TAGMe-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0160] The results are shown in Figure 11, which show that in clinical samples of glioma, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 85.71% and a specificity of 71.43%.

[0161] Example 17 TAGMe-4: Validation of colorectal cancer clinical samples - NGS sequencing

[0162] 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0163] The results are shown in Figure 12, which shows that in colorectal cancer clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.33% and a specificity of 100%.

[0164] Example 18 TAGMe-4: Leukemia Clinical Sample Verification - NGS Sequencing

[0165] Ten clinical samples were obtained, including five non-leukemia bone marrow smear samples as a control group and five 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 the second-round primers F2 and R2 in Table 1) to construct an NGS library of leukemia clinical samples. TAGMe-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0166] The results are shown in Figure 13, which show that in leukemia clinical samples, the methylation value of TAGMe-4 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%.

[0167] Example 19 TAGMe-4: Pancreatic cancer clinical sample validation - NGS sequencing method

[0168] Ten clinical samples were obtained, including five pancreatic cancer adjacent tissue samples as a control group and five 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0169] The results are shown in Figure 14, which show that in pancreatic cancer clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 80% and a specificity of 100%.

[0170] Example 20 TAGMe-4: Validation of thyroid cancer clinical samples - NGS sequencing

[0171] Twelve clinical samples were obtained, including six thyroid cancer paracancerous 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0172] The results are shown in Figure 15, which show that in thyroid cancer clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0173] Example 21 TAGMe-4: Melanoma Clinical Sample Validation - NGS Sequencing

[0174] 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0175] The results are shown in Figure 16, which show that in melanoma clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.33% and a specificity of 80%.

[0176] Example 22 TAGMe-4: Validation of nasopharyngeal carcinoma clinical samples - NGS sequencing

[0177] 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 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of nasopharyngeal carcinoma clinical samples were constructed, and TAGMe-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0178] The results are shown in Figure 17, which show that in NPC clinical samples, the methylation value of TAGMe-4 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%.

[0179] Example 23 TAGMe-4: Oral Cancer Clinical Sample Validation - NGS Sequencing

[0180] 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0181] The results are shown in Figure 18, which show that in oral cancer clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (**P < 0.01). The sensitivity was 83.33% and the specificity was 100%.

[0182] Example 24 TAGMe-4: Laryngeal cancer clinical sample verification - NGS sequencing method

[0183] Twelve clinical samples were obtained, including six paracancerous laryngeal cancer samples as a control group and six 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0184] The results are shown in Figure 19, which show that in laryngeal cancer clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0185] Example 25 TAGMe-4: Validation of Osteosarcoma Clinical Samples - NGS Sequencing

[0186] 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 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). The NGS library of the osteosarcoma clinical samples was constructed and analyzed according to the NGS sequencing steps. e -4 methylation level;

[0187] The results are shown in Figure 20, which shows that in osteosarcoma clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0188] Example 26 TAGMe-4: Lymphoma Clinical Sample Validation - NGS Sequencing

[0189] 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0190] The results are shown in Figure 21, which show that in lymphoma clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 66.67% and a specificity of 83.33%.

[0191] Example 27 TAGMe-4: Validation of Renal Cell Carcinoma Clinical Samples - NGS Sequencing

[0192] 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-4 methylation levels were analyzed according to the NGS sequencing protocol.

[0193] The results are shown in Figure 22, which show that in clinical samples of renal cell carcinoma, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.33% and a specificity of 100%.

[0194] Example 28 TAGMe-4: Validation of ovarian cancer clinical samples - NGS sequencing

[0195] Twelve clinical samples were obtained, including 6 ovarian cancer paracancerous samples as a control group and 6 ovarian 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). The NGS library of the clinical ovarian cancer samples was constructed and analyzed according to the NGS sequencing steps. e -4 methylation level;

[0196] The results are shown in Figure 23, which show that in ovarian cancer clinical samples, the methylation value of TAGMe-4 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 83.33%.

[0197] Example 29: Detection Performance Analysis of a Single CG Site

[0198] Using the clinical samples obtained in the previous examples, we analyzed the feasibility of using CG sites 9 to 19 (CG sites within the 10-185 segment of SEQ ID NO: 1) as single CG sites for cancer detection. Methylation modifications at the single CG sites were analyzed using NGS sequencing.

[0199] As shown in Tables 5-9, a single CG site can already exhibit relatively high sensitivity and specificity, and can also be used as a target for methylation modification analysis, enabling analysis and detection of cancer.

[0200] Table 5

[0201] Table 6

[0202] Table 7

[0203] Table 8

[0204] Table 9

[0205] Example 30: Methylation Differences of TAGMe-4 CpG Sites between Tumor Cells and Non-Tumor Cells—Bisulfite Sequencing PCR (BSP)

[0206] The steps of the bisulfite treatment and sequencing method are as follows:

[0207] 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;

[0208] 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;

[0209] 3. Design amplification primers according to the sequence of SEQ ID NO: 2, as shown in Table 10, and perform amplification.

[0210] 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.

[0211] Table 10. BSP primers

[0212] BSP verification of the methylation levels of cancer cells and normal control cells at methylation sites 20-88 in the SEQ ID NO: 1 region is shown in Figure 24. The results show that the methylation level of TAGMe-4 in cancer cells is significantly higher than that in normal cells.

[0213] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. Use of an isolated polynucleotide or a polynucleotide converted therefrom in the preparation of a reagent or kit for detecting a tumor; wherein: The polynucleotide comprises: (1) a polynucleotide TAGMe-4 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 88 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably any one of CpG sites 3 to 19 or a combination thereof, any one of CpG sites 20 to 88 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 40 to 157 or positions 10 to 185 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 for tumor detection include: tissue samples, body fluid samples, and blood samples.

5. A method for preparing a tumor detection reagent, comprising: (a) providing an isolated polynucleotide or a polynucleotide converted therefrom, comprising (1) a polynucleotide TAGMe-4 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 88 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 3 to 19 or a combination thereof, or any one of CpG sites 20 to 88 or a combination thereof selected from the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is a polynucleotide having a nucleotide sequence as shown in positions 40 to 157 or positions 10 to 185 of SEQ ID 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-4 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 40th to 157th or 10th to 185th nucleotide sequence of SEQ ID NO:

1.

7. Use of the reagent or reagent combination according to claim 6 for preparing a kit for detecting tumors; preferably, 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; more preferably, the tumors include: 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 detecting 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-4 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), 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-4 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 any one CpG site 1 to 88 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, it is any one CpG site 3 to 19 or a combination thereof, or any one CpG site 20 to 88 or a combination thereof selected from 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 40 to 157 or positions 10 to 185 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.

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