Identification of DNA methylation marker tagme-7 and use thereof in tumor detection

Abstract

Provided are an identification of a DNA methylation marker TAGMe-7 and the use thereof in tumor detection. The tumor marker can be used for molecular diagnosis, early screening, prognosis and efficacy evaluation of clinical tumors, and the significant difference in the methylation level of the tumor marker exists in a wide range of different types of tumors, including solid tumors and non-solid tumors, and thus the tumor marker is a pan-cancer marker.

Description

Identification of the DNA methylation marker TAGMe-7 and its application in tumor detection

[0001] This application claims priority to patent application No. CN 202311288687.9, filed on October 5, 2023; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the field of biotechnology and epigenetic modification. More specifically, the present invention relates to the identification of the DNA methylation marker TAGMe-7 and its use in tumor detection. Background Art

[0003] Changes in DNA sequences, such as mutations, are of interest in cancer research, but abnormal regulatory mechanisms beyond DNA sequence alterations are more common and important in the development and progression of cancer. These heritable regulatory mechanisms, independent of DNA sequence changes, are known as epigenetic mechanisms and include DNA methylation, micoRNA, histone acetylation, and long noncoding RNA. DNA methylation has been extensively studied at the level of individual genes, genome-wide, and in the field of functional regulation, and its application in the diagnosis and treatment of cancer clinical practice has also received significant attention. In tumor cells, some genes that are hypomethylated in normal cells become hypermethylated and transcriptionally inactive. Affected genes include tumor suppressor genes, DNA repair genes, cell cycle control genes, and anti-apoptotic genes, thereby promoting the development and progression of cancer.

[0004] The main approaches to studying DNA methylation include whole-genome and gene-level approaches. At the whole-genome level, these include DNA hybridization-based microarray technologies, such as CHIP-chip, which uses specific proteins to enrich methylated DNA fragments in the genome and then hybridizes them to a microarray, and methylation arrays, which use sulfite-modified genomic DNA before hybridization to a microarray. High-throughput next-generation sequencing technologies also include methods that use specific proteins to enrich methylated DNA fragments in the genome and then sequence them, such as MethylCap-seq and MeDIP-seq, and methods that use sulfite-modified genomic DNA before high-throughput sequencing, such as Genome-wide Bisulfite-seq and Reduced Representation Bisulfite Sequencing (RRBS). It should be noted that only Genome-wide Bisulfite-seq and Reduced Representation Bisulfite Sequencing (RRBS) can detect DNA methylation at single-base resolution; other omics approaches require subsequent verification of methylation status at the single-gene level. Detection methods at the specific gene level include MSP (methylation-specific PCR), qMSP (fluorescence quantitative methylation-specific PCR), and MSRE-qPCR (methylation-sensitive endonuclease-digested quantitative PCR) based on PCR (polymerase chain reaction); and BSP (sulfite-treated PCR product sequencing) and pyrophosphate sequencing based on sequencing.

[0005] The study of DNA methylation can not only help people further understand the occurrence and development mechanism of tumors from a new perspective, but the abnormal methylation pattern of tumor cell DNA has become a new research field of tumor biomarkers. Moreover, due to the reversibility of DNA methylation modification, it also has the potential to become a target for molecular therapy.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide the identification of the DNA methylation marker TAGMe-7 and its use in tumor detection.

[0008] 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-7 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.

[0009] As an embodiment, the SEQ ID NO: 1 also includes sequence variants or homologous sequences thereof.

[0010] 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.

[0011] 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).

[0012] In one embodiment, the detection includes diagnosis, identification, analysis, screening or prognostic assessment.

[0013] As an embodiment, the polynucleotide converted from the isolated polynucleotide is a polynucleotide having a nucleotide sequence shown in SEQ ID NO: 2.

[0014] In one embodiment, the at least one modified CpG site is any one CpG site or a combination thereof selected from Nos. 1 to 71 (such as 2 to 71, more specifically 3, 5, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70) 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. 34 to 41 or 33 to 41, CpG sites 1 to 32 or a combination thereof, or CpG sites 42 to 71 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1.

[0015] In one embodiment, the polynucleotide fragment is a polynucleotide having a nucleotide sequence shown at positions 272 to 388 or positions 244 to 418 in SEQ ID NO: 1.

[0016] 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.

[0017] 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.

[0018] As an embodiment, the tumor detection is directed to a tumor (including an early, middle or late stage tumor) or a precancerous lesion thereof.

[0019] As an embodiment, the samples targeted for tumor detection include (but are not limited to): tissue samples, body fluid samples, and blood samples.

[0020] 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.

[0021] In another aspect of the present invention, a method for preparing a reagent for detecting tumors is provided, comprising: (a) providing an isolated polynucleotide TAGMe-7 or a polynucleotide converted therefrom, comprising (1) a polynucleotide 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 a polynucleotide of (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; preferably, the at least one modified CpG site is any one CpG site selected from Nos. 1 to 71 or a combination thereof in the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1; preferably, the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1 In the polynucleotide having the nucleotide sequence shown in NO:1, any CpG site or a combination thereof is selected from CpG sites 34 to 41 or 33 to 41, any CpG site or a combination thereof from 1 to 32, or any CpG site or a combination thereof from 42 to 71; preferably, the polynucleotide fragment is a polynucleotide having the nucleotide sequence shown in SEQ ID NO:1 at positions 272 to 388 or 244 to 418; (b) using the polynucleotide of (a) as a target sequence, designing a detection reagent that specifically detects the modification status of the CpG site of the target sequence.

[0022] As an embodiment, the reagents for detecting tumors include but are not limited to: primers, probes, chips or test strips.

[0023] As an embodiment, one or more sets of reagents may be prepared for the target sequence.

[0024] As an embodiment, the detection reagent is integrated on a chip.

[0025] 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-7 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 272 to 388 or positions 244 to 418 of the nucleotide sequence shown in SEQ ID NO: 1.

[0026] As an embodiment, the reagent or reagent combination is: a primer containing the sequence at positions 29 to 56 in SEQ ID NO: 3 and the sequence at positions 28 to 57 in SEQ ID NO: 4.

[0027] As an embodiment, the reagent or reagent combination is: primers of SEQ ID NO: 3 and SEQ ID NO: 4 sequences.

[0028] 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 29-56 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.

[0029] As an embodiment, the reagent or reagent combination is: primers with sequences shown in SEQ ID NO: 27 and SEQ ID NO: 28.

[0030] As an embodiment, the reagent or reagent combination is: primers with sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30.

[0031] In another embodiment, the reagent or reagent combination further comprises primers with sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0032] In another aspect of the present invention, the use of the reagent or reagent combination is provided 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, glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma or ovarian cancer.

[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-7 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 unmodified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; and the converted polynucleotide corresponds to a polynucleotide of (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.

[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-7 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 34 to 41 or 33 to 41 or a combination thereof, CpG sites 1 to 32 or a combination thereof, or CpG sites 42 to 71 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 272 to 388 or 244 to 418 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.

[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 the methylation marker TAGMe-7 in lung cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0048] Figure 2. Methylation levels of the methylation marker TAGMe-7 in HCC and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0049] Figure 3. Methylation levels of the methylation marker TAGMe-7 in prostate cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0050] Figure 4. Methylation levels of the methylation marker TAGMe-7 in cervical cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0051] Figure 5. Methylation levels of the methylation marker TAGMe-7 in endometrial cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0052] Figure 6. Methylation levels of the methylation marker TAGMe-7 in urothelial carcinoma and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0053] Figure 7. Methylation levels of the methylation marker TAGMe-7 in biliary tract tumor and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0054] Figure 8. Methylation levels of the methylation marker TAGMe-7 in gastric cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0055] Figure 9. Methylation levels of the methylation marker TAGMe-7 in breast cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0056] Figure 10. Methylation levels of the methylation marker TAGMe-7 in esophageal cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0057] Figure 11. Methylation levels of the methylation marker TAGMe-7 in brain glioma and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0058] Figure 12. Methylation levels of the methylation marker TAGMe-7 in colorectal cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0059] Figure 13. Methylation levels of the methylation marker TAGMe-7 in leukemia and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0060] Figure 14. Methylation levels of the methylation marker TAGMe-7 in pancreatic cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0061] Figure 15. Methylation levels of the methylation marker TAGMe-7 in thyroid cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0062] Figure 16. Methylation levels of the methylation marker TAGMe-7 in melanoma and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0063] Figure 17. Methylation levels of the methylation marker TAGMe-7 in NPC and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0064] Figure 18. Methylation levels of the methylation marker TAGMe-7 in oral cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0065] Figure 19. Methylation levels of the methylation marker TAGMe-7 in laryngeal cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0066] Figure 20. Methylation levels of the methylation marker TAGMe-7 in osteosarcoma and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0067] Figure 21. Methylation levels of the methylation marker TAGMe-7 in lymphoma and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0068] Figure 22. Methylation levels of the methylation marker TAGMe-7 in renal cell carcinoma and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0069] Figure 23. Methylation levels of the methylation marker TAGMe-7 in ovarian cancer and control samples (left panel) and sensitivity and specificity analysis results (right panel).

[0070] FIG24 is a BSP validation of the methylation levels of CpG sites 1-32 in SEQ ID NO: 1 in cancer cells and normal control cells.

[0071] FIG25 . BSP verification of the methylation levels of methylation sites 42-71 in SEQ ID NO: 1 in cancer cells and normal control cells. DETAILED DESCRIPTION

[0072] The inventors are committed to exploring the application of DNA methylation in tumor screening. After in-depth research and screening, they revealed an epigenetic modification-related tumor marker TAGMe-7, which uses the abnormal high methylation phenomenon of marker-specific sites in tumors to detect tumors.

[0073] As a tumor marker, TAGMe-7 exhibits significant, statistically significant differences in DNA methylation between adjacent and cancerous tissues. These differences are observed across a wide range of tumor types, including solid tumors (lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, and biliary tract cancer) and non-solid tumors (hematologic tumors). Therefore, the tumor marker of the present invention can be used for molecular diagnosis, early screening, prognosis, and efficacy assessment of clinical tumors. The significant differences in its methylation levels are present across a broad spectrum of tumor types, including solid and non-solid tumors, making it a pan-cancer marker.

[0074] As used herein, 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.

[0075] 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.

[0076] As used herein, the term "hypermethylation (level)" refers to the presence of hypermethylation, hydroxymethylation, aldehydemethylation, or carboxymethylation modifications at CpG sites within a gene sequence. For example, in methylation-specific PCR (MSP) analysis, a positive PCR result obtained using methylation-specific primers indicates that the DNA (gene) region under investigation is in a hypermethylated state. For example, in real-time quantitative methylation-specific PCR, hypermethylation can be determined by analyzing the relative methylation status of a control sample and determining the statistical difference.

[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 by the present invention is possible, and thus the present invention also encompasses nucleic acid fragments of the nucleotide sequence, which include at least one methylated CpG site. The at least one CpG site may include 1 to 71 of SEQ ID NO: 1 or its reverse complement, more specifically 2, 3, 5, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, or 70. It will be appreciated by those skilled in the art that, after the present invention provides CpG numbering based on a 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.

[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 acids in a sample in vitro 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 basic 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 by Southern blotting. The advantages of this method include: no detailed information about the primary structure of the target DNA is required, and it can provide a direct assessment of the methylation status of the CpG islands, 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 the sequence of a genome, its variation and methylation status known to those skilled in the art can 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, 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.

[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] In summary, the present invention provides novel pan-cancer markers suitable for detecting cervical exfoliated cells, among other things. Samples can be easily obtained and are non-invasive. Compared to clinical procedures requiring surgical tissue sampling, this approach significantly reduces patient pain and improves patient compliance.

[0101] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0102] Example 1 Determination of methylation detection targets

[0103] 1. Obtain the DNA sequence of TAGMe-7 (SEQ ID NO: 1; human origin);

[0104] In the above sequence (sense strand), each "CG" underlined by a solid line represents a methylated CpG site, numbered from 5' to 3' as CpG sites 1 to 71 (methylated CpG sites 1 to 71). 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.

[0105] 2. Obtain the DNA sequence SEQ ID NO: 2 after bisulfite conversion (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 from 5' to 3' as "YG" 1 to 71 (converted methylated CpG sites 1 to 71). 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] 3. The entire SEQ ID NO: 2 can be used for detection. Considering the length, a partial fragment can be determined as the detection region, and primers can be designed upstream and downstream of the detection region.

[0108] Example 2 Design and synthesis of PCR 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 primers (Table 1);

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

[0112] 2.4 Synthesize primers for the first round of PCR reaction and primers for the second round of PCR reaction with Illumina adapters and indexes.

[0113] Table 1

[0114] In the table, F1 and R1 amplify the sequence segment corresponding to positions 244-418 in SEQ ID NO: 1 or SEQ ID NO: 2 (containing CpG sites 33 to 41), with positions 272 to 388 containing CpG sites 34 to 41. In F1, positions 29-56 are complementary to the target detection sequence segment (upstream and downstream), and positions 22-28 are the tag sequence. In R1, positions 28-57 are complementary to the target detection sequence segment (upstream and downstream), and positions 21-27 are the tag sequence.

[0115] Example 3 PCR reaction system

[0116] Synthesize a pair of primers and perform two rounds of PCR reaction on the positive and negative reference products:

[0117] The first-round PCR reaction system is shown in Table 2.

[0118] Table 2

[0119] The second-round PCR reaction system is shown in Table 3.

[0120] Table 3

[0121] Example 4 Construction of primer pool

[0122] After verifying the efficiency of primer PCR, a primer pool with different barcodes was synthesized, and the primers were diluted and combined. In the first round of PCR, there are M×N (10×10) combinations of F and R primers, which can be used for 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 29-56 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: Methylation differences of TAGMe-7 CpG sites between tumor tissues and non-tumor tissues - NGS sequencing

[0126] 5.1 Obtaining Clinical Samples: Obtain paracancerous / non-cancerous-cancer 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] 5.2 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] 5.3 Bisulfite Treatment: Treat the extracted DNA samples with bisulfite, strictly following the instructions; this experiment used the ZYMO Research EZ DNA Methylation-Gold Kit, Catalog No. D5006 (this kit is not limited to this one).

[0129] 5.4 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] 5.5 After PCR amplification, 2% agarose gel electrophoresis was used to test 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] 5.6 Analyze sequencing results: Extract sample sequencing information based on primer barcode sequences;

[0132] 5.7 Calculation of TAGMe-7 methylation value: NGS sequencing can independently detect the methylation status of individual CpG sites within the target region. The median methylation value of all CpG sites is calculated as the methylation value of TAGMe-7 in the sample.

[0133] 5.8 Analysis of results: Compare the TAGMe-7 methylation values ​​in non-tumor tissues and tumor tissues and determine the cutoff value using the ROC curve.

[0134] Example 6 TAGMe-7: Lung Cancer Clinical Sample Validation - NGS Sequencing

[0135] Forty clinical samples were obtained, including 20 adjacent lung cancer samples as a control group (normal) and 20 lung cancer samples as an experimental group. Two rounds of PCR were performed using the primer combinations described in Example 4 (select any first-round primer pair from Table 4; the second-round primers F2 and R2 from Table 1). An NGS library of clinical lung cancer samples was constructed, and TAGMe-7 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-7 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001). The sensitivity and specificity were both 100%.

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

[0138] Forty clinical samples were obtained, including 20 liver cancer adjacent to liver cancer samples as a control group and 20 liver cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (select any first-round primer pair from Table 4; the second-round primers F2 and R2 from Table 1). An NGS library of clinical liver cancer samples was constructed, and TAGMe-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 80% and a specificity of 90%.

[0140] Example 8 TAGMe-7: Prostate cancer clinical sample validation - NGS sequencing method

[0141] Twenty-four clinical samples were obtained, including 12 prostate cancer adjacent paracancerous samples as a control group and 12 prostate cancer samples as an experimental group. Two rounds of PCR were performed using the primer combinations described in Example 4 (select any first-round primer pair from Table 4; the second-round primers F2 and R2 from Table 1). NGS libraries of prostate cancer clinical samples were constructed, and TAGMe-7 methylation levels were analyzed according to the NGS sequencing protocol.

[0142] The results are shown in Figure 3, showing that in prostate cancer clinical samples, the methylation value of TAGMe-7 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001). The sensitivity and specificity were 91.67%.

[0143] Example 9 TAGMe-7: Validation of cervical cancer clinical samples - NGS sequencing

[0144] 32 clinical samples were obtained, including 16 adjacent cervical adenocarcinoma samples as a control group and 16 cervical cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (select any one pair of first-round primers from Table 4, and the second-round primers F2 and R2 from Table 1). NGS libraries of cervical cancer clinical samples were constructed, and TAGMe-7 methylation levels were analyzed according to the NGS sequencing protocol.

[0145] The results are shown in Figure 4, showing that in cervical cancer clinical samples, the methylation value of TAGMe-7 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001), with a sensitivity of 87.50% and a specificity of 93.75%.

[0146] Example 10 TAGMe-7: 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 was selected, and the second-round primers were F2 and R2 in Table 1). An NGS library of endometrial cancer clinical samples was constructed, and TAGMe-7 methylation levels were analyzed according to the NGS sequencing protocol.

[0148] The results are shown in Figure 5, showing that in endometrial cancer clinical samples, the methylation value of TAGMe-7 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001), with a sensitivity of 88.89% and a specificity of 100%.

[0149] Example 11 TAGMe-7: Validation of Urothelial Carcinoma Clinical Samples - NGS Sequencing

[0150] Thirty-eight clinical samples were obtained, including 19 urothelial carcinoma paracancerous tissue samples as a control group and 19 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-7 methylation levels were analyzed according to the NGS sequencing protocol.

[0151] The results are shown in Figure 6, which show that in clinical samples of urothelial carcinoma, the methylation value of TAGMe-7 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 80% and a specificity of 90%.

[0152] Example 12 TAGMe-7: Validation of Biliary Tract Tumor Clinical Samples - NGS Sequencing

[0153] Thirty-eight clinical samples were obtained, including 19 paracancerous biliary tract tumor samples as a control group and 19 biliary tract tumor samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (select any first-round primer pair from Table 4; the second-round primers F2 and R2 from Table 1). An NGS library of clinical biliary tract tumor samples was constructed, and TAGMe-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 90% and a specificity of 80%.

[0155] Example 13 TAGMe-7: Validation of Gastric Cancer Clinical Samples - NGS Sequencing

[0156] 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-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 80% and a specificity of 100%.

[0158] Example 14 TAGMe-7: 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). An NGS library of clinical breast cancer samples was constructed, and TAGMe-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 100% and a specificity of 80%.

[0161] Example 15 TAGMe-7: Validation of Esophageal Cancer Clinical Samples - NGS Sequencing

[0162] Ten clinical samples were obtained, including five esophageal cancer paracancerous samples as a control group and five 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). An NGS library of clinical esophageal cancer samples was constructed, and TAGMe-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 80% and a specificity of 80%.

[0164] Example 16 TAGMe-7: 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 was selected, and the second-round primers were F2 and R2 in Table 1). An NGS library of clinical glioma samples was constructed, and TAGMe-7 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 brain glioma, the methylation value of TAGMe-7 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 80% and a specificity of 100%.

[0167] Example 17 TAGMe-7: Validation of Colorectal Cancer Clinical Samples - NGS Sequencing

[0168] Ten clinical samples were obtained, including five colorectal cancer adjacent paracancerous samples as a control group and five 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). An NGS library of colorectal cancer clinical samples was constructed, and TAGMe-7 methylation levels were analyzed according to the NGS sequencing protocol.

[0169] The results are shown in Figure 12, which show that in colorectal cancer clinical samples, the methylation value of TAGMe-7 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 18 TAGMe-7: Leukemia Clinical Sample Validation - NGS Sequencing

[0171] 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 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 leukemia clinical samples were constructed, and TAGMe-7 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-7 in leukemia bone marrow smears was significantly higher than that in non-leukemia bone marrow smears (*P<0.05), with a sensitivity of 100% and a specificity of 100%.

[0173] Example 19 TAGMe-7: Pancreatic cancer clinical sample validation - NGS sequencing method

[0174] Sixteen clinical samples were obtained, including eight pancreatic cancer adjacent tissue samples as a control group and eight 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-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 87.5% and a specificity of 87.5%.

[0176] Example 20 TAGMe-7: Validation of thyroid cancer clinical samples - NGS sequencing

[0177] Sixteen clinical samples were obtained, including eight thyroid cancer paracancerous tissue samples as a control group and eight 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). An NGS library of thyroid tumor clinical samples was constructed, and TAGMe-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 75% and a specificity of 62.5%.

[0179] Example 21 TAGMe-7: Melanoma Clinical Sample Validation - NGS Sequencing

[0180] Sixteen clinical samples were obtained, including eight normal skin tissue samples as a control group and eight 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-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 100% and a specificity of 100%.

[0182] Example 22 TAGMe-7: Validation of Nasopharyngeal Carcinoma Clinical Samples - NGS Sequencing

[0183] Sixteen clinical samples were obtained, including eight nasopharyngeal carcinoma paracancerous samples as a control group and eight 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-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 100% and a specificity of 87.5%.

[0185] Example 23 TAGMe-7: Validation of Oral Cancer Clinical Samples - NGS Sequencing

[0186] Sixteen clinical samples were obtained, including eight oral cancer paracancerous samples as a control group and eight oral cancer samples as an experimental group. Two rounds of PCR were performed using the primer combinations 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-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 87.5% and a specificity of 100%.

[0188] Example 24 TAGMe-7: Laryngeal cancer clinical sample validation - NGS sequencing method

[0189] Sixteen clinical samples were obtained, including eight paracancerous laryngeal cancer samples as a control group and eight 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 was selected, and the second-round primers were F2 and R2 in Table 1). An NGS library of laryngeal cancer clinical samples was constructed, and TAGMe-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 100% and a specificity of 100%.

[0191] Example 25 TAGMe-7: Validation of Osteosarcoma Clinical Samples - NGS Sequencing

[0192] Sixteen clinical samples were obtained, including eight adjacent osteosarcoma samples as a control group and eight 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 was selected, and the second-round primers were F2 and R2 in Table 1). An NGS library of osteosarcoma clinical samples was constructed, and TAGMe-7 methylation levels were analyzed according to the NGS sequencing protocol.

[0193] The results are shown in Figure 20, which show that in osteosarcoma clinical samples, the methylation value of TAGMe-7 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 87.5% and a specificity of 62.5%.

[0194] Example 26 TAGMe-7: Lymphoma Clinical Sample Validation - NGS Sequencing

[0195] Sixteen clinical samples were obtained, including eight normal lymphoid samples as a control group and eight lymphoma 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 the lymphoma clinical samples. TAGMe-7 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-7 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 87.5% and a specificity of 75%.

[0197] Example 27 TAGMe-7: Validation of Renal Cell Carcinoma Clinical Samples - NGS Sequencing

[0198] Sixteen clinical samples were obtained, including eight adjacent renal cell carcinoma samples as a control group and eight 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-7 methylation levels were analyzed according to the NGS sequencing protocol.

[0199] The results are shown in Figure 22, which show that in renal cell carcinoma clinical samples, the methylation value of TAGMe-7 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 62.5% and a specificity of 87.5%.

[0200] Example 28 TAGMe-7: Validation of ovarian cancer clinical samples - NGS sequencing

[0201] Sixteen clinical samples were obtained, including eight adjacent ovarian cancer samples as a control group and eight ovarian cancer samples as an experimental group. Two rounds of PCR were performed using the primer combinations 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 ovarian cancer samples were constructed, and TAGMe-7 methylation levels were analyzed according to the NGS sequencing protocol.

[0202] The results are shown in Figure 23, which shows that in ovarian cancer clinical samples, the methylation value of TAGMe-7 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 100% and a specificity of 87.5%.

[0203] Example 29: Methylation differences of TAGMe-7 CpG sites between tumor cells and non-tumor cells - sequencing after bisulfite treatment

[0204] Bisulfite Sequencing PCR (BSP) was used, and the steps were as follows:

[0205] 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;

[0206] 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;

[0207] 3. Design amplification BSP primers according to the sequence of SEQ ID NO: 2, as shown in Table 5, and perform amplification.

[0208] 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.

[0209] Table 5

[0210] BSP verification of the methylation levels of cancer cells and normal control cells at methylation sites 1-32 in the SEQ ID NO: 1 region is shown in Figure 24. The results show that the methylation level of TAGMe-7 in cancer cells is significantly higher than that in normal cells.

[0211] BSP verification of the methylation levels of cancer cells and normal control cells at methylation sites 42-71 in the SEQ ID NO: 1 region is shown in Figure 25. The results show that the methylation level of TAGMe-7 in cancer cells is significantly higher than that in normal cells.

[0212] Example 30 Feasibility Analysis of a Single CpG Site as a Target for Methylation Detection

[0213] Using the clinical samples obtained in the previous examples, the feasibility of analyzing CpG sites 34 to 41 (CpG sites within the segment 272 to 388 in SEQ ID NO: 1) as single CpG sites for cancer detection was assessed. Methylation at the single CpG sites was analyzed using NGS sequencing.

[0214] The results are shown in Tables 6 to 10. It can be seen that a single CpG site has relatively ideal sensitivity and specificity, and can also be used as a target for methylation modification analysis, which is meaningful for the diagnosis of cancer.

[0215] Table 6

[0216] Table 7

[0217] Table 8

[0218] Table 9

[0219] Table 10

[0220] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

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-7 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 CpG site or a combination thereof selected from CpG sites 1 to 71 in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably any one CpG site or a combination thereof selected from CpG sites 34 to 41 or 33 to 41, CpG sites 1 to 32 or a combination thereof, or CpG sites 42 to 71 or a combination thereof in 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 272 to 388 or positions 244 to 418 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 reagent for detecting a tumor, comprising: (a) providing an isolated polynucleotide TAGMe-7 or a polynucleotide converted therefrom, comprising (1) a polynucleotide 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 71 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, any one of CpG sites 34 to 41 or 33 to 41 or a combination thereof, any one of CpG sites 1 to 32 or a combination thereof, or any one of CpG sites 42 to 71 or a combination thereof selected from the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is SEQ ID NO: A polynucleotide having the nucleotide sequence shown at positions 272 to 388 or 244 to 418 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-7 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 272 to 388 or 244 to 418 in 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) obtaining polynucleotides 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-7 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-7 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 CpG site 34 to 41 or 33 to 41 or a combination thereof, CpG site 1 to 32 or a combination thereof, or CpG site 42 to 71 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 272 to 388 or positions 244 to 418 in 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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