Human genome t5 series fragments for multi-cancer identification and uses thereof

Abstract

The application provides a human genome T5 series fragment for multi-cancer identification and use thereof. A set of human genome differential methylation regions for multi-cancer identification, referred to as T5 series fragments (T5-1, T5-2, T5-3, T5-4, T5-5, T5-6, T5-7, T5-8) is disclosed. On this basis, the application provides a multi-cancer methylation technology which can cover more than 20 high-incidence cancer types, has high sensitivity (≥90%) and high specificity (≤5% false positive rate), and can realize rapid and micro-sample detection.

Description

Technical Field

[0001] This invention belongs to the fields of oncology, epigenetics, and early cancer diagnosis technology; more specifically, this invention relates to human genome T5 series fragments for multi-cancer identification and their uses. Background Technology

[0002] Unlike other diseases, the five-year survival rate for early-stage cancer (Stage I) is generally over 80%. However, because most early-stage cancers have no specific symptoms, about 60% of patients are already in the middle or late stages when they are diagnosed. After treatment, the five-year survival rate drops sharply to below 20%. Therefore, large-scale screening technology is conducive to early diagnosis and is the core path to reduce cancer mortality.

[0003] Current clinical cancer screening methods still face significant technical bottlenecks, making it difficult to meet the needs of early screening for multiple cancer types: In imaging examinations, while low-dose spiral CT (for lung cancer screening) can identify small lesions in the lungs, it has a high rate of missed diagnoses for early nodules <5mm in diameter, and long-term frequent testing carries a cumulative risk of radiation exposure; breast ultrasound (for initial breast cancer screening) has a generally low detection rate for early cancerous changes in dense breast tissue, requiring verification with other detection methods; among single-cancer serum marker tests, PSA (for prostate cancer) has a high false-positive rate, easily leading to overdiagnosis; CA-125 (for ovarian cancer) has a low positive detection rate in early ovarian cancer and cannot be used as a standalone screening indicator; traditional tissue biopsies require invasive procedures such as endoscopy and puncture to obtain samples, resulting in low patient tolerance (screening compliance in high-risk groups is less than 20%). Furthermore, existing methods can only detect a single suspicious site, making it difficult to achieve simultaneous screening of multiple organs.

[0004] Advances in epigenetics research have provided new directions for early cancer screening. Abnormal DNA methylation has been confirmed as an "early molecular signal" of cancer development. Compared to genomic mutations, changes in CpG island methylation patterns in cancer cells can occur 3-5 years earlier than pathological morphological changes and exhibit significant cancer type specificity. This characteristic gives it an irreplaceable advantage in screening compared to traditional techniques: First, different cancer types have specific methylation markers. For example, the association between RASSF1A methylation and lung cancer, MGMT methylation and glioma, and Septin9 methylation and colorectal cancer has been clinically validated, enabling precise "marker-cancer type" correspondence. Second, the test samples do not rely on tissue; only 10 mL of peripheral blood is needed to extract circulating cell-free DNA (cfDNA), avoiding the risks associated with invasive procedures and increasing patient compliance to over 85%. Third, it allows for dynamic tracking of cfDNA methylation levels after treatment, providing early warning of recurrence 3-6 months earlier than traditional imaging, offering molecular-level evidence for comprehensive cancer management.

[0005] Although DNA methylation screening has made breakthroughs in single-cancer fields (such as the FDA approval of Septin9 testing for colorectal cancer screening), multi-cancer combined screening still has unresolved technical shortcomings: existing multi-cancer methylation detection schemes mostly adopt a "simple superposition of single-cancer biomarkers" model, without considering the cross-interference between different biomarkers, resulting in false positive rates exceeding 15% for some cancer types (such as gastric cancer and liver cancer); the detection process requires batch processing of different targets, with a single sample processing time exceeding 48 hours and consuming ≥5mL of blood sample, which is difficult to meet the clinical needs for rapid, low-volume sample screening; in addition, most schemes only cover 3-5 cancer types, excluding "difficult-to-screen" high-incidence cancers such as pancreatic cancer and esophageal cancer, and cannot achieve a truly "panoramic" screening.

[0006] In summary, there is an urgent need in this field to develop diagnostic solutions that can achieve large-scale screening and meet the requirements for diagnostic specificity and sensitivity. Summary of the Invention

[0007] The purpose of this invention is to provide a non-invasive multi-cancer screening method based on DNA methylation detection at the T5 gene methylation site. This method can be applied to universal screening of common cancers in healthy individuals and dynamic monitoring of high-risk individuals, providing a highly efficient screening solution for clinical use that "covers multiple cancers with a single test".

[0008] In a first aspect of the invention, the use of a methylation analysis reagent for a human genome CpG site region (differential methylation region) in the preparation of a reagent or kit for diagnosing tumors is provided; said human genome CpG site region is selected from one or more regions of the group consisting of: (a) a region of nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (b) a region that is sequence-complementary to (a) and has the same number of bases.

[0009] In a preferred embodiment, the nucleotide sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 further includes its sequence variants or homologous sequences (including sequences having more than 80%, 85%, 90%, 92%, 95%, 96%, 98%, 99%, 99.5%, or 99.8% sequence identity).

[0010] In another preferred embodiment, the diagnosis of tumor includes analyzing the risk of the tumor.

[0011] In a preferred embodiment, the diagnosis of tumors includes tumor screening, diagnosis, analysis, or prognostic assessment.

[0012] In another preferred embodiment, the diagnosis, detection, screening, testing, or prognostic assessment includes detection, screening, diagnosis, testing, or prognostic assessment of early-stage tumors or precancerous conditions, as well as detection, screening, diagnosis, testing, or prognostic assessment of healthy individuals.

[0013] In another preferred embodiment, the tumor includes solid tumors or non-solid tumors; preferably, the solid tumors include: gastric cancer, colorectal cancer, prostate cancer, osteosarcoma, lung cancer, biliary tract tumors, nasopharyngeal carcinoma, ovarian cancer, breast cancer, melanoma, cervical cancer, oral cancer, esophageal cancer, liver cancer, glioma, endometrial cancer, renal cell carcinoma, thyroid cancer, laryngeal cancer, pancreatic cancer, urothelial carcinoma, liposarcoma, and bladder cancer; the non-solid tumors include: leukemia and lymphoma.

[0014] In another preferred embodiment, human genome CpG site regions such as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 are simultaneously detected to obtain the methylation status of human genome CpG site regions and to diagnose tumors.

[0015] In another preferred embodiment, if the methylation of CpG sites in the human genome is statistically increased during detection, it is judged to have a high tumor risk; preferably, the method for detecting the methylation of CpG sites in the human genome includes the following methods or combinations of methods: bisulfite conversion sequencing, pyrosequencing, methylation array method, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion, qPCR, digital PCR, second-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing technology, HPLC, and MassArray.

[0016] In another preferred embodiment, the methylation-sensitive restriction endonuclease is a restriction endonuclease that is sensitive to methylated bases at its recognition site; including but not limited to one or more of HpaII, AciI, Bsu15I, Hin1I, Hin6I, HpyCH4IV, NarI, etc.

[0017] In another preferred embodiment, the method for detecting the methylation status of CpG sites in the human genome is bisulfite conversion sequencing.

[0018] In another preferred embodiment, the bisulfite conversion sequencing method includes: (i) extracting nucleic acids from the sample to be tested; (ii) processing the extracted nucleic acids to convert unmodified cytosine into uracil (its unmodified C is converted to T or U, while the cytosine at its modified CpG sites remains unchanged); preferably, the nucleic acids in step (i) are treated with bisulfite; (iii) analyzing the sequence regions in the nucleic acids from step (ii) corresponding to the CpG site regions of the human genome to obtain the methylation status of the CpG sites.

[0019] In another preferred embodiment, the reagent or kit is intended for samples including (but not limited to): tissue samples (such as paraffin-embedded samples), blood samples, cell samples (such as cell smear samples), urine samples, pleural effusion samples, bronchoalveolar lavage fluid samples, ascites samples, ascites lavage fluid samples, bile samples, fecal samples, saliva samples, cerebrospinal fluid samples, cervical samples, and uterine cavity samples.

[0020] In another aspect of the invention, the use of a human genome CpG site region or a nucleic acid derived therefrom in the preparation of a reagent or kit for tumor detection is provided; wherein the human genome CpG site region is selected from one or more regions of the group consisting of: (1) a region with a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (2) a region that is sequence-complementary to the region of (1) and has the same number of bases; wherein the nucleic acid derived from the human genome CpG site region is a polynucleotide corresponding to (1) or (2), wherein its unmodified C is converted to T or U, while the cytosine of its modified CpG site remains unchanged; preferably, the nucleotide sequence of the nucleic acid derived from the human genome CpG site region is as shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 ...9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, The region shown in IDNO:15 or SEQ ID NO:16.

[0021] In another aspect of the invention, a method for preparing a reagent for diagnosing tumors is provided, comprising: (a) providing a human genome CpG site region, which is one or a combination of regions selected from the group consisting of: (a) a region with a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (b) a region that is sequence-complementary to the region of (a) and has the same number of bases; and (b) preparing a diagnostic reagent specifically for detecting the methylation of CpG sites based on the human genome CpG site region described in (a).

[0022] In another preferred embodiment, the diagnostic reagent comprises (but is not limited to): primers, probes; preferably, the diagnostic reagent comprises: primers with nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18; primers with nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20; primers with nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22; primers with nucleotide sequences as shown in SEQ ID NO: 23 and SEQ ID NO: 24; primers with nucleotide sequences as shown in SEQ ID NO: 25 and SEQ ID NO: 26; primers with nucleotide sequences as shown in SEQ ID NO: 27 and SEQ ID NO: 28; primers with nucleotide sequences as shown in SEQ ID NO: 29 and SEQ ID NO: 30; and / or primers with nucleotide sequences as shown in SEQ ID NO: 31 and SEQ ID NO: 32.

[0023] In another preferred embodiment, the diagnostic reagent also includes primers with nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18, primers with nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20, and primers with nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22.

[0024] In another preferred embodiment, the diagnostic reagent is integrated onto a chip.

[0025] In another aspect of the invention, a reagent for diagnosing tumors is provided, which targets and analyzes the methylation of CpG sites in a human genome CpG site region selected from: (1) regions of nucleotide sequences such as those shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (2) regions that are sequence-complementary to (1) and have the same number of bases.

[0026] In another preferred embodiment, the reagent comprises: primers with nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18; primers with nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20; primers with nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22; primers with nucleotide sequences as shown in SEQ ID NO: 23 and SEQ ID NO: 24; primers with nucleotide sequences as shown in SEQ ID NO: 25 and SEQ ID NO: 26; primers with nucleotide sequences as shown in SEQ ID NO: 27 and SEQ ID NO: 28; primers with nucleotide sequences as shown in SEQ ID NO: 29 and SEQ ID NO: 30; and / or primers with nucleotide sequences as shown in SEQ ID NO: 31 and SEQ ID NO: 32. In another preferred embodiment, the reagent also includes: primers with nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18; primers with nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20; and primers with nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22.

[0027] In another aspect of the invention, a kit is provided for tumor detection (including screening, diagnosis, detection, or prognostic assessment), the kit comprising the aforementioned reagents or combinations of reagents.

[0028] In another preferred embodiment, the kit may also include, but is not limited to: DNA purification reagent, DNA extraction reagent, bisulfite, and PCR amplification reagent.

[0029] In another preferred embodiment, the kit further includes an instruction manual specifying the detection procedure and result determination criteria.

[0030] In another aspect of the invention, isolated nucleic acids or nucleic acids derived therefrom are provided, wherein the nucleic acids are one or more regions selected from the group consisting of: (1) nucleic acids or combinations thereof with nucleotide sequences such as those shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (2) nucleic acids or combinations thereof that are sequence-complementary to (1) and of the same length; wherein the nucleic acid derived from the CpG site region of the human genome is a polynucleotide corresponding to (1) or (2), wherein its unmodified C is converted to T or U, while the cytosine of its modified CpG site remains unchanged.

[0031] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0032] Figure 1 The results of PCR amplification and methylation level analysis were performed on a subset of cancer types (specific cancer types are shown in the figure) using T5-1 as the analysis object.

[0033] Figure 2 The results of PCR amplification and methylation level analysis were performed on a subset of cancer types (specific cancer types are shown in the figure) using T5-1 as the analysis object.

[0034] Figure 3 The results of PROC analysis for the various cancer types shown in the figure.

[0035] Figure 4 The results of PROC analysis for the various cancer types shown in the figure. Detailed Implementation

[0036] Based on in-depth research and screening of multiple cancer types, this invention reveals a set of differentially methylated regions in the human genome for the identification of multiple cancer types, referred to as the T5 series fragments (T5-1, T5-2, T5-3, T5-4, T5-5, T5-6, T5-7, T5-8). Building upon this, this invention provides a multi-cancer methylation technology that can cover more than 20 common cancer types, possesses both high sensitivity (≥90%) and high specificity (≤5% false positive rate), and enables rapid detection of minute samples.

[0037] As used herein, "sample" or "sample" refers to nucleic acid material for DNA methylation testing, which may be derived from an individual (such as a human or animal) or from other sources, such as amplified or unamplified laboratory nucleic acid material, or artificially synthesized test samples. It should be understood that testing of a "sample" or "sample" is not limited to diagnostic purposes but may also involve other non-diagnostic purposes.

[0038] Unless otherwise stated, "sample" or "sample" includes substances obtained from any individual (preferably a human) or isolated tissue, cells, or bodily fluids (such as plasma) suitable for detecting DNA methylation. For example, the sample may include, but is not limited to: tissue samples (such as paraffin-embedded samples), blood samples, cell samples (such as cell smear samples), urine samples, cervical samples, uterine cavity samples, pleural effusion samples, bronchoalveolar lavage fluid samples, ascites samples, ascites lavage fluid samples, bile samples, fecal samples, saliva samples, cerebrospinal fluid samples, cell smear samples, and cell samples.

[0039] This invention is based on a methylation detection method using bisulfite conversion. After DNA samples are treated with a bisulfite solution (such as sodium bisulfite), unmethylated cytosine (C) is converted to uracil (U), while methylated cytosine (mC) remains unchanged. After PCR amplification, U is converted to T, and mC is converted to C, thus transforming the difference between methylation and unmethylation into C / T base polymorphism. By detecting the C / T base status at the target site, it can be determined whether the sample DNA has been methylated.

[0040] In this invention, the methylation status of differentially methylated CpG regions T5-1 (SEQ ID NO:1), T5-2 (SEQ ID NO:2), T5-3 (SEQ ID NO:3), T5-4 (SEQ ID NO:4), T5-5 (SEQ ID NO:5), T5-6 (SEQ ID NO:6), T5-7 (SEQ ID NO:7), and T5-8 (SEQ ID NO:8) in the genome shows significant differences between tumor and non-tumor tissues. When abnormally high methylation is detected in these gene sequence regions, the subject can be considered to have a tumor or belong to a high-risk group for tumors. This difference in methylation status of the (human) genome's T5-1, T5-2, T5-3, T5-4, T5-5, T5-6, T5-7, and T5-8 is very significant in a variety of tumors. The differentially expressed CpG methylation regions can also be the antisense strands (reverse complementary strands) of the T5-1, T5-2, T5-3, T5-4, T5-5, T5-6, T5-7, and T5-8 gene sequences specifically shown in this invention.

[0041] This invention also includes "conservative variant sequences" that are conserved or have high sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or their inverse complementary sequences. "High sequence identity" is defined as, for example, higher than 90%, higher than 92%, higher than 95%, higher than 98%, higher than 99%, etc.

[0042] Gene panels or gene groups containing differentially methylated regions T5-1, T5-2, T5-3, T5-4, T5-5, T5-6, T5-7, T5-, or their complementary sequences are also included in this invention. For the aforementioned gene panels or gene groups, DNA methylation status can also be detected to identify the characteristics of normal and tumor cells.

[0043] A wide variety of techniques for analyzing methylation can be applied in this invention, and there are no particular limitations on such detection techniques. The differentially expressed CpG methylation regions in the (human) genome can serve as key regions for analyzing methylation in the (human) genome, and their methylation can be analyzed using various techniques known in the art, thereby analyzing the occurrence or development of tumors.

[0044] The differentially methylated CpG regions of the genome, or fragments thereof, or their complementary sequences, can be converted from unmethylated cytosine to uracil after bisulfite treatment, while methylated cytosine remains unchanged. This invention also discloses sequences obtained after bisulfite treatment of the aforementioned differentially methylated CpG regions, including nucleotide sequences such as those shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16. These can serve as more direct targets for designing detection reagents or kits.

[0045] The differentially methylated CpG regions of the genome and / or their complementary nucleic acids and / or nucleic acids derived from one or more segments of the present invention (e.g., via bisulfite conversion) can also be integrated into one or more whole units, such as one or more nucleic acid sets, for use by those skilled in the art. For example, one or more CpG methylated regions can be selected from such nucleic acid sets to design targeted analytical reagents. The designed targeted analytical reagents can also be integrated into one or more whole units, such as one or more kits, or one or more chips.

[0046] As a particularly preferred approach, differentially expressed CpG methylation regions of T5-1 and / or their complementary nucleic acids, and differentially expressed CpG methylation regions of T5-2 and / or their complementary nucleic acids are used in combination for detection, further improving the sensitivity and / or specificity for a subset of tumors.

[0047] Based on the differentially methylated genomic regions proposed in this invention, variations can be made, such as selecting longer sequences that contain the present invention, or selecting sequences that overlap with the sequences of this invention in a region. These technical solutions are also included within the scope of this invention.

[0048] This invention also provides a method for preparing tumor detection reagents, comprising: providing the aforementioned nucleic acid, using the full length or a fragment of the nucleic acid as a target sequence, and designing a detection reagent specifically for detecting the target sequence. The detection reagents may include, but are not limited to, chips, primers, probes, etc.; after obtaining the aforementioned markers, the selection of detection reagents is something that can be done by those skilled in the art.

[0049] Once the sequence of the nucleic acid is known, primer design is known to those skilled in the art. Two primers are positioned flanking a specific sequence of the target gene to be amplified (including the CpG sequence; the primers are complementary to the CpG sequence to target methylated gene regions, and complementary to the CpG sequence to target demethylated gene regions). In a preferred embodiment of the invention, the reagent is a primer, preferably one listed in Table 1. Besides primers, other diagnostic or detection reagents can also be prepared, including but not limited to probes, chips, etc.

[0050] The reagents may also be combinations of reagents, such as primer combinations. For example, the combination may include more than one set of primers, thereby enabling the amplification of the multiple nucleic acids mentioned above.

[0051] The present invention also provides a kit for detecting the methylation level of nucleic acids in an in vitro sample, the kit comprising: a container, and the aforementioned primer pair located in the container.

[0052] The kit may also include various reagents required for DNA extraction, DNA purification, PCR amplification, and other reagents, such as sample processing reagents. Furthermore, the kit may include an instruction manual specifying the detection procedures and result interpretation criteria to facilitate application by those skilled in the art.

[0053] The methods and reagents of this invention exhibit very high accuracy when used to diagnose clinical tumors, as demonstrated in the detection of various clinical tumor samples in the embodiments of this invention. This invention can be applied to fields such as pre-tumor screening, efficacy assessment, auxiliary diagnosis, and prognostic monitoring, or, as mentioned above, situations where the purpose is not to obtain a direct disease diagnosis.

[0054] The differentially expressed CpG methylation regions provided by this invention exhibit very high specificity and sensitivity when used independently. Furthermore, the sensitivity and specificity are further enhanced when two differentially expressed CpG methylation regions are used in combination. Therefore, the differentially expressed CpG methylation regions of this invention have significant application value in fields such as tumor adjuvant diagnosis, efficacy assessment, and prognostic monitoring.

[0055] The differentially expressed CpG methylation regions provided by this invention are universally applicable to the detection of various major cancer types, including lung cancer, breast cancer, colorectal cancer, gastric cancer, and liver cancer. Compared to normal tissues, they all show highly consistent and significant abnormal increases (or decreases), demonstrating an excellent "whole-cancer" recognition ability to distinguish malignant tumors from normal tissues. This discovery lays a crucial foundation for developing novel early cancer screening programs.

[0056] In summary, this invention provides a methylation analysis scheme for early diagnosis, screening, and risk prediction of various cancers, as well as primers and a kit for implementation. The primer combination includes primers for amplifying the T5 gene fragment, and the kit contains the aforementioned primer combination. This invention provides a novel DNA methylation marker, T5, for tumor identification and its applications. The T5 gene sequence region exhibits significant differences in methylation levels among multiple cancerous and adjacent tissues. Detecting high methylation in the T5 gene sequence region is sufficient to determine if a subject belongs to a high-risk group for cancer. Furthermore, this differential methylation pattern of T5 in cancerous and adjacent tissues can be widely applied to various types of cancer.

[0057] The technical solution of this invention can not only fill the technical gap in non-invasive screening for multiple cancers, but also reduce the cost of cancer treatment by improving the early diagnosis rate, which has important clinical value and social significance for promoting the upgrading of the global cancer prevention and control system.

[0058] The invention will be better understood from the following examples. However, those skilled in the art will understand that the specific methods and results are merely for illustrating the invention and not for limiting it. Experimental methods in the following examples that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0059] Example 1: Determination of methylation detection targets

[0060] 1.1 Obtain the (human) T5-1, T5-2, T5-3, T5-4, T5-5, T5-6, T5-7, and T5-8 gene sequences.

[0061] In the above left-hand column sequence (positive strand), each "CG" marked with a solid underline represents one CpG site. The dashed underlines correspond to the upstream and downstream primer design regions and detection target regions in some schemes of the embodiments.

[0062] In the sequence (justice chain) in the right column above, each "YG" marked by a solid underline represents a CpG site that has been transformed and methylated. The black underline corresponds to the upstream and downstream primer design regions in some of the schemes of the embodiments.

[0063] In the DNA sequence after Bisulfite transformation, Y represents C or U; Example 2: Design and Synthesis of Diagnostic Reagents 2.1 The designed multiplex PCR primers are 20-35 bp in length (Table 1), methylated PCR specific primers with appropriate CG content, and the amplification length is ensured to be 150-300 bp; Table 1

[0064] Example 3, Verification of the Reaction Reagents

[0065] First round of PCR: The experimental conditions for targeted methylation-specific multiplex PCR are shown in Table 2.

[0066] Table 2

[0067] The PCR procedure is shown in Table 3.

[0068] Table 3

[0069] To verify the specificity of PCR primers for targeted sequencing, 2.5% agarose gel electrophoresis was used to detect the specificity of the target primers.

[0070] Example 4, Construction of multiplex PCR libraries

[0071] The multiplex PCR amplification products were purified, and the purified PCR products were subjected to a second round of PCR (sequencing adapter ligation). The sequencing adapters were designed and synthesized using barcode sequences provided by Illumina.

[0072] The experimental conditions for the second round of PCR, methylation-targeted sequencing library preparation PCR, are shown in Table 4.

[0073] Table 4

[0074] The PCR procedure is shown in Table 5.

[0075] Table 5

[0076] Example 5: Acquisition and Processing of Clinical Samples

[0077] 1. Obtaining clinical samples: Obtain adjacent / non-cancerous to cancerous tissue samples from clinical settings. The adjacent / non-cancerous samples serve as the control group, while the cancerous tissue samples serve as the tumor detection experimental group.

[0078] 2. DNA extraction: DNA was extracted from the experimental group and the control group respectively; the adsorption column method was used for extraction in this experiment.

[0079] 3. Bisulfite treatment: The extracted DNA sample was treated with bisulfite, and the procedure was strictly followed. In this experiment, the EZ DNA Methylation-Gold Kit of ZYMO Research, catalog number D5006, was used (this method is not limited to this one).

[0080] 4. Using primers from the multiplex PCR primer pool (first-round PCR primers) for targeted amplification and universal sequencing primers from the Illumina system (second-round PCR primers), two rounds of PCR amplification were performed using conventional methods to construct an NGS library. After library construction, the purified targeted methylation sequencing library was sent to a sequencing company for sequencing, and the sequencing data was analyzed.

[0081] 5. After PCR amplification, the PCR fragment specificity was detected by 2.5% agarose gel electrophoresis. 5 μL of PCR product was taken from each sample, mixed, purified, and the target fragment library was recovered for NGS sequencing.

[0082] 6. Sequencing results analysis: Samples were distinguished based on the different index sequences added in the second round of PCR, and the methylation values ​​of the target regions in the cancer group samples and the negative control group samples were analyzed separately.

[0083] 7. Calculation of T5-1, T5-2, T5-3, T5-4, T5-5, T5-6, T5-7, and T5-8 methylation values: 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 T5-1, T5-2, T5-3, T5-4, T5-5, T5-6, T5-7, and T5-8 in the sample.

[0084] Example 6: Validation using clinical samples from multiple cancer types

[0085] Approximately 20 samples were obtained from clinical practice for each type of cancer, including: gastric cancer, colorectal cancer, prostate cancer, osteosarcoma, lung cancer, biliary tract tumors, nasopharyngeal carcinoma, ovarian cancer, lymphoma, breast cancer, leukemia, melanoma, cervical cancer, oral cancer, esophageal cancer, liver cancer, glioma, endometrial cancer, renal cell carcinoma, thyroid cancer, laryngeal cancer, pancreatic cancer, urothelial carcinoma, liposarcoma, bladder cancer, and other cancers.

[0086] Ten clinically positive tissue samples and adjacent normal tissue samples were obtained as positive and negative control groups, respectively. Multiplex targeted PCR amplification was performed according to the multiplex PCR primer combination method described in Examples 2-4 above (the primers in the table are all primers in the targeted multiplex PCR primer pool). The NGS library of the samples was constructed using the targeted amplification products, and the T5-1 methylation level was analyzed according to the NGS sequencing procedure.

[0087] Using T5-1 as the analysis target, the results of PCR amplification and methylation level analysis for the aforementioned cancer type are as follows: Figure 1 and Figure 2 As shown.

[0088] Sensitivity and specificity analyses were performed on the aforementioned cancer types using the PROC package. The results are as follows: Figure 3 and Figure 4 As shown.

[0089] The inventors conducted the above experiments on the other seven differentially methylated regions of the gene, analyzed the sensitivity and specificity of each fragment, and summarized the results of T5-1 (fragment 1), T5-2 (fragment 2), T5-3 (fragment 3), T5-4 (fragment 4), T5-5 (fragment 5), T5-6 (fragment 6), T5-7 (fragment 7), and T5-8 (fragment 8) in Table 6.

[0090] Table 6 Diagnostic efficacy (%) of T5-1 to T8 in different cancers

[0091] Example 7: Combined application to further enhance diagnostic efficacy

[0092] Furthermore, to further improve the accuracy of the method, the inventors chose to evaluate and combine the detection efficacy of different fragments. Ultimately, the three fragments T5-1, T5-2, and T5-3 (fragments 1, 2, and 3) were used in combination. If the sample to be tested is methylated positive in at least one of the fragments T5-1, T5-2, or T5-3, then the sample to be tested is a cancer-positive sample; if all three fragments of the sample to be tested are methylated negative, then the sample to be tested is a negative sample.

[0093] The detection performance of T5-1, T5-2 and T5-3 combined is shown in Table 7.

[0094] Table 7. Detection efficacy of T5-1, T5-2 and T5-3 combined (%)

[0095] As shown in the table above, for various tumors, including glioma, leukemia, melanoma, oral cancer, pancreatic cancer, renal cell carcinoma, thyroid cancer, esophageal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, liver cancer, biliary tract cancer, cervical cancer, laryngeal cancer, lung cancer, gastric cancer, osteosarcoma, and nasopharyngeal carcinoma, the combination of T5-1, T5-2, and T5-3 can significantly improve sensitivity when used for analysis.

[0096] Example 8: Diagnostic performance comparison with existing target tumors (taking ovarian cancer and urothelial carcinoma as examples)

[0097] The inventors compared the diagnostic performance of the TAGMe-1 ​​target in the previously published patent CN 115725591 A with the three targets T5-1, T5-2 and T5-3 in this invention in ovarian cancer, as well as the diagnostic performance of the TAGMe-1 ​​target and the three targets T5-1, T5-2 and T5-3 in this invention in urothelial carcinoma.

[0098] Experiments and comparisons were conducted using the experimental methods in Example 5 and the samples in Example 6.

[0099] Comparative analysis of diagnostic performance is shown in Table 8 (ovarian cancer) and Table 9 (urothelial carcinoma).

[0100] Table 8. Comparison of diagnostic performance of different biomarkers for ovarian cancer

[0101] Table 9. Comparison of diagnostic performance of different biomarkers for urothelial carcinoma

[0102] As can be seen from the above, the three targets T5-1, T5-2 and T5-3 of the present invention are significantly superior to the targets in patent CN 115725591 A in the diagnosis of ovarian cancer and urothelial carcinoma.

[0103] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Use of a methylation assay reagent for human genome CpG site regions in the preparation of reagents or kits for diagnosing tumors; wherein the human genome CpG site regions are selected from one or more regions of the group consisting of: (a) regions of nucleotide sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (b) regions that are complementary to (a) in sequence and have the same number of bases.

2. The use as described in claim 1, characterized in that, The tumors include solid tumors or non-solid tumors; preferably, the solid tumors include: gastric cancer, colorectal cancer, prostate cancer, osteosarcoma, lung cancer, biliary tract tumors, nasopharyngeal carcinoma, ovarian cancer, breast cancer, melanoma, cervical cancer, oral cancer, esophageal cancer, liver cancer, glioma, endometrial cancer, renal cell carcinoma, thyroid cancer, laryngeal cancer, pancreatic cancer, urothelial carcinoma, liposarcoma, and bladder cancer; the non-solid tumors include: leukemia and lymphoma.

3. The use as described in claim 1, characterized in that, Nucleotide sequences such as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 of the human genome CpG site regions were simultaneously detected to obtain the methylation status of the human genome CpG site regions and to diagnose tumors.

4. The use as described in any one of claims 1, 2, or 3, characterized in that, During detection, if the methylation of CpG sites in the human genome is statistically increased, it is judged to have a high risk of tumors. Preferably, the method for detecting the methylation of CpG sites in the human genome includes the following methods or combinations of methods: bisulfite conversion sequencing, pyrosequencing, methylation microarray, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion, qPCR, digital PCR, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing technology, HPLC, and MassArray.

5. The use as described in claim 4, characterized in that, The method for detecting the methylation status of CpG sites in the human genome is bisulfite conversion sequencing. Preferably, the bisulfite conversion sequencing method includes: (i) Extract nucleic acid from the sample to be tested; (ii) The extracted nucleic acid is processed to convert the unmodified cytosine into uracil; preferably, the nucleic acid described in step (i) is treated with bisulfite; (iii) Analyze the sequence regions in the nucleic acids of (ii) that correspond to the CpG site regions of the human genome to obtain the methylation status of the CpG sites.

6. The use of human genome CpG site regions or nucleic acids derived from them in the preparation of reagents or kits for tumor detection; among which, The human genome CpG site region is selected from one or more regions of the following group: (1) a region with a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (2) a region that is complementary to the region of (1) in sequence and has the same number of bases; wherein the nucleic acid derived from the human genome CpG site region is a polynucleotide corresponding to (1) or (2), wherein its unmodified C is converted to T or U, while the cytosine of its modified CpG site remains unchanged; preferably, the nucleotide sequence of the nucleic acid derived from the human genome CpG site region is a region shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:

16.

7. A method for preparing a reagent for diagnosing tumors, comprising: (a) Provides a CpG site region of the human genome, which is one or a combination of regions selected from the group consisting of: (a) a region with a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (b) a region that is complementary to the region of (a) in sequence and has the same number of bases; (b) Prepare diagnostic reagents that specifically detect the methylation status of CpG sites in the human genome, based on the CpG site regions described in (a).

8. The method as described in claim 7, characterized in that, The diagnostic reagent includes: primers and probes; preferably, the diagnostic reagent includes: Primers with nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18; Primers with nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20; Primers with nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22; Primers with nucleotide sequences as shown in SEQ ID NO: 23 and SEQ ID NO: 24; Primers with nucleotide sequences as shown in SEQ ID NO: 25 and SEQ ID NO: 26; Primers with nucleotide sequences as shown in SEQ ID NO: 27 and SEQ ID NO: 28; Primers with nucleotide sequences as shown in SEQ ID NO: 29 and SEQ ID NO: 30; and / or, Primers with nucleotide sequences as shown in SEQ ID NO: 31 and SEQ ID NO:

32.

9. The method as described in claim 8, characterized in that, The diagnostic reagent also includes primers with nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18, primers with nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20, and primers with nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO:

22.

10. A reagent for diagnosing tumors, which targets and analyzes the methylation of CpG sites in a region of the human genome, wherein the CpG site region of the human genome is selected from one or more regions of the group consisting of: (1) a region of nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (2) a region that is complementary to the region of (1) in sequence and has the same number of bases; Preferably, the reagent comprises: Primers with nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18; Primers with nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20; primers with nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22; primers with nucleotide sequences as shown in SEQ ID NO: 23 and SEQ ID NO: 24; primers with nucleotide sequences as shown in SEQ ID NO: 25 and SEQ ID NO: 26; primers with nucleotide sequences as shown in SEQ ID NO: 27 and SEQ ID NO: 28; primers with nucleotide sequences as shown in SEQ ID NO: 29 and SEQ ID NO: 30; And / or primers with nucleotide sequences as shown in SEQ ID NO: 31 and SEQ ID NO: 32; More preferably, the reagent also includes primers with nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18; Primers with nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20; primers with nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22.

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