Novel target nucleic acid molecules p7 for the identification of multiple cancer types and uses thereof

Abstract

This invention provides a novel target nucleic acid molecule, P7, for the identification of multiple cancer types and its applications. A group of methylated target nucleic acid segments for the diagnosis of multiple pan-cancer diseases were screened and validated. These segments exhibited significant differences in methylation status within tumor samples, which can be used to identify individuals at high risk for cancer. When these target nucleic acid segments are used in combination for detection, they exhibit unexpected improvements in sensitivity and specificity. The tumor target nucleic acid segments of this invention can serve as novel molecules for auxiliary clinical diagnosis or prognosis of cancer.

Description

Technical Field

[0001] This invention belongs to the field of epigenetics, and more specifically, this invention relates to a novel target nucleic acid molecule P7 for the identification of multiple cancer types and its uses. Background Technology

[0002] Early cancer screening methods typically include blood tests, imaging studies, and molecular biological tests. These methods screen for early-stage cancer biomarkers or suspicious lesions. Common screening methods include: 1. Blood tests: These methods include liquid biopsy, detection of circulating tumor DNA (ctDNA), and tumor-associated antigens (such as CA-125, PSA, etc.). Liquid biopsy is an innovative technology that analyzes blood samples to identify cancer-related DNA, RNA, proteins, or other molecular markers, helping to identify cancer at an early stage. 2. Imaging studies: Such as X-rays, CT scans, MRI, ultrasound, and endoscopy, used to detect abnormal tumors or lesions. For example, mammography or ultrasound is commonly used for breast cancer screening, while low-dose CT scans are commonly used for lung cancer screening. 3. Molecular biological tests: Including genomics and transcriptomics technologies, which identify cancer biomarkers through gene mutations, epigenetic alterations, etc., to predict cancer risk at an early stage.

[0003] Epigenetics refers to the regulation or repression of gene expression through chemical modifications (such as DNA methylation and histone modifications) without altering the DNA sequence. The main epigenetic mechanisms include: DNA methylation: Cytosine (C) on DNA molecules is modified with methyl groups, typically occurring on CpG islands. This methylation can suppress gene expression, especially in promoter regions. Histone modifications: Histones are packaging proteins for DNA. Histone modifications such as acetylation, methylation, and phosphorylation can alter chromatin structure, affecting the open or closed state of genes, thereby regulating gene expression. Non-coding RNAs: MicroRNAs and long non-coding RNAs (lncRNAs) play a role in regulating gene expression. They can control gene expression by regulating mRNA stability or the translation process.

[0004] The relationship between cancer and epigenetics is an important area of ​​current tumor biology research. Cancer is a disease caused by mutations in the cell's genome or abnormal gene expression, while epigenetics studies how gene expression is affected by environmental and external factors without altering the DNA sequence itself. In recent years, researchers have discovered that the occurrence and development of cancer are not only related to gene mutations, but epigenetic changes (such as DNA methylation, histone modifications, and the role of non-coding RNA) play a crucial role in the occurrence and progression of cancer.

[0005] Abnormal DNA methylation patterns provide valuable information for early cancer diagnosis, prognostic assessment, and treatment selection. Therefore, researchers have begun exploring the use of DNA methylation as a biomarker to predict cancer. Liquid biopsies (such as the detection of cancer markers in bodily fluids like blood, urine, or saliva) are non-invasive methods for detecting the presence of cancer and monitoring its progression. DNA methylation biomarkers are ideal for liquid biopsies due to their ubiquity and stability in cancer development. Studies have found that circulating cell-free DNA (cfDNA) or exosome DNA in the blood can reflect methylation changes in cancer cells. Methylation of the Septin9 gene has been used for early screening of colorectal cancer and has received FDA approval. Similar research is exploring how to use the detection of methylation markers in the blood for early diagnosis of other types of cancer, such as lung cancer, breast cancer, and stomach cancer.

[0006] Different types of cancer typically exhibit specific DNA methylation patterns. Therefore, by identifying and analyzing characteristic methylation markers of different cancers, researchers can develop highly sensitive diagnostic tools. For example, certain methylation markers may only appear in a specific type of cancer, providing a valuable basis for early cancer detection. Cancers such as breast cancer, lung cancer, stomach cancer, and liver cancer have specific marker genes in their respective DNA methylation patterns. Utilizing the methylation information of these genes can assist in cancer classification and diagnosis.

[0007] Epigenetics research has provided a new perspective for understanding the occurrence and development of cancer. Epigenetic alterations not only play a role in the early stages of cancer but may also have a significant impact on cancer invasion, metastasis, and drug resistance. With the deepening of research in epigenetics and precision medicine, related targeted therapies and early diagnostic methods are expected to play a greater role in cancer treatment. At the same time, epigenetics also offers new directions for cancer prevention. Summary of the Invention

[0008] The purpose of this invention is to provide a novel target nucleic acid molecule P7 for the identification of multiple cancer types and its uses.

[0009] In a first aspect of the invention, the use of a reagent for specifically detecting the level of CpG site methylation modification of a target nucleic acid segment in the preparation of a reagent or kit for detecting tumors is provided; wherein the target nucleic acid segment is a specific segment in the human genome selected from: (1) nucleic acids or combinations thereof with the nucleotide sequences 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 or SEQ ID NO:7; or (2) nucleic acids or combinations thereof that are sequence-complementary to (1) and of the same length.

[0010] In one embodiment, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7 further includes sequence variants or homologous sequences thereof.

[0011] In a preferred embodiment, the sequence variant or homologous sequence is a sequence that has more than 80%, more than 85%, more than 90%, more than 92%, more than 95%, more than 96%, more than 98%, more than 99%, more than 99.5%, or more than 99.8% sequence identity with the sequences 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, or SEQ ID NO:7.

[0012] In one embodiment, the polynucleotide also includes those derived from the sequence variant or homologous sequence transition (unmodified cytosine is converted to T or U, while the cytosine C at the modified CpG site remains unchanged).

[0013] In one implementation, detecting tumors includes diagnosing, screening, detecting, or prognostically assessing tumors.

[0014] In one implementation, the tumor detection includes analyzing the risk of the tumor.

[0015] The detection, screening, diagnosis, 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.

[0016] In one implementation, during detection, a significant increase in the methylation level of CpG sites in the target nucleic acid region indicates a high risk of tumor development.

[0017] In one embodiment, the method for detecting the CpG site methylation modification level of the target nucleic acid segment includes: pyrosequencing, bisulfite conversion sequencing, methylation array method, 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, HPLC, MassArray, or combinations thereof.

[0018] In one embodiment, other methylation detection methods and future newly developed methylation detection methods may also be applied to this invention.

[0019] In one 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.

[0020] In one embodiment, a method for detecting the CpG site methylation modification level of the target nucleic acid segment includes: (1) Extract nucleic acid from the sample to be tested; (2) The extracted nucleic acid is processed to convert the unmodified cytosine into uracil (the unmodified cytosine (C) is converted into T or U, while the cytosine at the modified CpG site remains unchanged); preferably, the nucleic acid described in step (1) is treated with bisulfite. (3) Analyze the sequence segments in the nucleic acid of (2) that correspond to the target nucleic acid segment to obtain the methylation modification level of CpG site.

[0021] In one embodiment, the tumor is pan-cancer, including: solid tumors and non-solid tumors; preferably, the tumor includes: glioma, ovarian cancer, laryngeal cancer, cervical cancer, gastric cancer, colorectal cancer, prostate cancer, osteosarcoma, lung cancer, biliary tract tumor, nasopharyngeal carcinoma, lymphoma, breast cancer, leukemia, melanoma, oral cancer, esophageal cancer, liver cancer, endometrial cancer, renal cell carcinoma, thyroid cancer, pancreatic cancer, urothelial carcinoma, liposarcoma, and bladder cancer.

[0022] In one embodiment, the target nucleic acid segment is a combination of nucleic acids of the nucleotide sequences shown in SEQ ID NO:3 (P7-3) and SEQ ID NO:1 (P7-1).

[0023] In one 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.

[0024] In another aspect of the invention, the use of a target nucleic acid segment or a nucleic acid segment derived therefrom in the preparation of reagents or kits for tumor detection (including screening, diagnosis, detection or prognostic assessment) is provided; wherein the target nucleic acid segment is a specific segment in the human genome selected from: (1) nucleic acids or combinations thereof with the nucleotide sequences 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 or SEQ ID NO:7; or (2) nucleic acids or combinations thereof that are sequence-complementary to (1) and of the same length; the nucleic acid segment derived from the target nucleic acid segment is a polynucleotide corresponding to (1) or (2), wherein its unmodified cytosine (C) is converted to T or U, while the cytosine at its modified CpG site remains unchanged.

[0025] In another aspect of the invention, a method for preparing a reagent for detecting (including screening, diagnosis, detection, or prognostic assessment) tumors is provided, the method comprising: (a) providing a target nucleic acid segment selected from: (1) nucleic acids or combinations thereof with the nucleotide sequences 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, or SEQ ID NO:7; or (2) nucleic acids or combinations thereof that are sequence-complementary to and of the same length as the nucleic acid sequence of (1); and (b) preparing a detection reagent for specifically detecting the level of CpG site methylation modification of the target sequence for the target nucleic acid segment of (a).

[0026] In one embodiment, the detection reagent includes (but is not limited to): primers and probes; preferably, the detection reagent includes: Primers for the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16; Primers for the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18; Primers for the nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20; Primers for the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22; Primers for the nucleotide sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24; Primers for the nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26; and / or, Primers for the nucleotide sequences shown in SEQ ID NO: 27 and SEQ ID NO: 28.

[0027] Preferably, the reagent also includes primers for the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO: 20.

[0028] In one implementation, one or more sets of reagents may be prepared for the target nucleic acid segment.

[0029] In one implementation, the detection reagent is integrated onto a chip or test strip.

[0030] In another aspect of the invention, a reagent for detecting tumors is provided, which specifically detects the level of CpG site methylation modification of a target nucleic acid segment selected from: (1) nucleic acids or combinations thereof with the nucleotide sequences 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 or SEQ ID NO:7; or (2) nucleic acids or combinations thereof that are sequence-complementary to (1) and of the same length.

[0031] In one embodiment, the reagent comprises primers selected from the group consisting of: Primers for the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16; Primers for the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18; Primers for the nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20; Primers for the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22; Primers for the nucleotide sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24; Primers for the nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26; and / or, Primers for the nucleotide sequences shown in SEQ ID NO: 27 and SEQ ID NO: 28.

[0032] In one embodiment, the reagent comprises primers for the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO: 20.

[0033] 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.

[0034] In one embodiment, the kit may also include, but is not limited to: DNA purification reagent, DNA extraction reagent, bisulfite, and PCR amplification reagent.

[0035] In one embodiment, the kit further includes: instructions for specifying the detection procedures and result interpretation criteria.

[0036] In another aspect of the invention, isolated nucleic acids or nucleic acids derived therefrom are provided, wherein the nucleic acids are: (1) nucleic acids or combinations of nucleic acids with nucleotide sequences 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 or SEQ ID NO:7; or (2) nucleic acids or combinations of nucleic acids that are sequence-complementary to the nucleic acids of (1); wherein the nucleic acids derived from the nucleic acids are nucleic acids corresponding to (1) or (2), wherein the unmodified cytosine is converted to T or U, while the cytosine C at the modified CpG site remains unchanged.

[0037] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0038] Figure 1 The results of methylation level analysis targeting P7-1 in 25 different types of tumors were compared with non-tumor tissues as controls.

[0039] Figure 2 The results of methylation level analysis targeting P7-2 in 25 different types of tumors were compared with non-tumor tissues as controls.

[0040] Figure 3 ROC analysis results targeting P7-1 for 25 different types of tumors.

[0041] Figure 4 ROC analysis results for 25 different types of tumors, targeting P7-2. Detailed Implementation

[0042] This invention belongs to the fields of oncology and epigenetics. Based on the analysis and research of a large number of clinical samples, a set of methylated target nucleic acid segments for pan-cancer diagnosis (including early diagnosis), screening, and risk prediction has been screened and demonstrated. These segments exhibit significant differences in methylation status (hypermethylation) in tumor samples, which can be used to identify individuals at high risk for cancer. When these target nucleic acid segments are used in combination for detection, they show an unexpected improvement in sensitivity and specificity.

[0043] The tumor target nucleic acid segment of the present invention can be used as a target nucleic acid segment for clinical tumor diagnosis, screening, classification, detection and prognosis, and can also be used as a novel molecule for auxiliary clinical diagnosis or prognosis of tumors. It can be widely used in a variety of different types of tumors, or can be used to design diagnostic reagents and kits.

[0044] In this invention, the term "sample" or "sample" includes substances obtained from any individual (preferably a human) or isolated tissues, cells, or bodily fluids (such as plasma) suitable for detecting DNA methylation status. 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.

[0045] As used in this invention, "detection" includes screening, diagnosis, testing, or prognostic assessment.

[0046] In this invention, the methylation status of the target nucleic acid segments P7-1 (SEQ ID NO: 1), P7-2 (SEQ ID NO: 2), P7-3 (SEQ ID NO: 3), P7-4 (SEQ ID NO: 4), P7-5 (SEQ ID NO: 5), P7-6 (SEQ ID NO: 6), and P7-7 (SEQ ID NO: 7), or portions thereof, differs significantly between tumor and non-tumor tissues. When an abnormally high methylation status is detected in the gene sequence region, the subject is considered to have a tumor or belong to a high-risk group for tumors. This difference in methylation status among the target nucleic acid segments or portions thereof is very significant in various tumors. The target nucleic acid segments described in this invention can also be the antisense strands of the aforementioned target nucleic acid segments.

[0047] This invention also includes "conserved variant sequences" having conservation or high sequence identity with the nucleotide sequences shown in P7-1 (SEQ ID NO: 1), P7-2 (SEQ ID NO: 2), P7-3 (SEQ ID NO: 3), P7-4 (SEQ ID NO: 4), P7-5 (SEQ ID NO: 5), P7-6 (SEQ ID NO: 6), and P7-7 (SEQ ID NO: 7) or their antisense strands. "High sequence identity" is defined as, for example, higher than 90%, higher than 92%, higher than 95%, higher than 98%, higher than 99%, etc. It should be understood that differences may exist at individual sequence sites between different biological individuals (e.g., some meaningless SNPs may exist), but this does not affect the detection based on the overall scheme of this invention.

[0048] With the information on the specific segments in the human genome provided by the present invention, those skilled in the art can easily obtain and apply the CpG sites. The embodiments of the present invention provide a series of sequence fragments containing CpG sites, which may serve as examples of preferred embodiments. However, it should be understood that variations can be made based on the information provided by the present invention, such as selecting longer sequences that contain the present invention, or selecting sequences that overlap regionally with sequences of the present invention.

[0049] This invention also includes gene panels or gene groups comprising the nucleotide sequences or sequence fragments shown in P7-1 (SEQ ID NO: 1), P7-2 (SEQ ID NO: 2), P7-3 (SEQ ID NO: 3), P7-4 (SEQ ID NO: 4), P7-5 (SEQ ID NO: 5), P7-6 (SEQ ID NO: 6), and P7-7 (SEQ ID NO: 7) or their complementary sequences. For the aforementioned gene panels or gene groups, characteristics of normal cells and tumor cells can also be obtained by detecting DNA methylation status.

[0050] A wide variety of techniques for analyzing methylation status can be applied in this invention, and there are no particular limitations on such detection techniques. The nucleic acids provided by this invention can serve as key regions in the genome for analyzing methylation status, and their methylation status can be analyzed using various techniques known in the art, thereby analyzing the occurrence or development of tumors.

[0051] The nucleic acids or fragments thereof, or complementary sequences thereof, described in P7-1 (SEQ ID NO: 1), P7-2 (SEQ ID NO: 2), P7-3 (SEQ ID NO: 3), P7-4 (SEQ ID NO: 4), P7-5 (SEQ ID NO: 5), P7-6 (SEQ ID NO: 6), and P7-7 (SEQ ID NO: 7) of the present invention can be converted to uracil after bisulfite treatment, while the methylated cytosine remains unchanged. Therefore, the present invention also provides nucleic acids obtained by treating the above-mentioned nucleic acids (including their complementary strands (antisense strands)) with bisulfite, comprising: nucleic acids or nucleic acid fragments of the nucleotide sequences shown in SEQ ID NO: 8-SEQ ID NO: 14. These nucleic acids can serve as more direct targets for designing detection reagents or detection kits.

[0052] The nucleic acids and / or their complementary nucleic acids and / or one or more fragments thereof derived from the nucleotide sequences shown in P7-1 (SEQ ID NO: 1), P7-2 (SEQ ID NO: 2), P7-3 (SEQ ID NO: 3), P7-4 (SEQ ID NO: 4), P7-5 (SEQ ID NO: 5), P7-6 (SEQ ID NO: 6), and P7-7 (SEQ ID NO: 7) of the present invention can also be integrated into one or more units, such as one or more sets of nucleic acids, for use by those skilled in the art, such as selecting one or more nucleic acids or fragments of nucleic acids from the set of nucleic acids to design targeted analytical reagents. The designed targeted analytical reagents can also be integrated into one or more units, such as one or more kits, or one or more chips.

[0053] In the most preferred embodiment of the present invention, the nucleic acid and / or its complementary nucleic acid of the nucleotide sequence shown in SEQ ID NO: 3 are used in combination with the nucleic acid and / or its complementary nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1 for detection.

[0054] Based on the target nucleic acid segments and their epigenetic modification characteristics provided in this invention, these techniques known in the art, as well as some techniques that are about to be developed, can all be applied to this invention to detect methylation modification levels. The determination of nucleic acid methylation profiles can be performed using existing techniques (such as methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight), or other techniques that are still under development or will be developed. For example, quantitative methylation-specific PCR (QMSP) can be used to detect methylation modification levels. Based on continuous optical monitoring of fluorescent PCR, it is more sensitive than the MSP method. It has high throughput and avoids the need for electrophoresis analysis. In addition, other available techniques include: qPCR (Me-qPCR), next-generation sequencing, pyrosequencing, Sanger sequencing, bisulfite conversion sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing, HPLC, and combinatorial gene group detection, etc., which are conventional methods in this field. Although some preferred methods are provided in the embodiments of this invention, the overall scheme of this invention is not limited thereto.

[0055] As a preferred embodiment of the present invention, a method for in vitro detection of the methylation profile of nucleic acids in a sample is also provided. The method is based on the principle that bisulfite can convert unmethylated cytosine into uracil, which is then converted into thymine during subsequent PCR amplification, while methylated cytosine remains unchanged. Therefore, after nucleic acid treatment with bisulfite, the methylated sites produce a nucleic acid polymorphism (SNP) similar to a C / T ratio. Identifying the methylation profile of nucleic acids in a sample based on this principle can effectively distinguish between methylated and unmethylated cytosine.

[0056] The method described in this invention includes: first, providing a sample and extracting genomic DNA; second, treating the genomic DNA obtained in step (a) with bisulfite, thereby converting unmethylated cytosine in the genomic DNA into uracil; and third, analyzing whether there are abnormal methylation patterns in the genomic DNA treated in step (b).

[0057] The method of this invention can be used to: test subject samples to assess whether the subject has a tumor; or to identify high-risk groups for tumors. The method 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 the disease, population geographic analysis studies, scientific research, population censuses, etc.

[0058] In a preferred embodiment of the present invention, DNA methylation is detected by PCR amplification and next-generation sequencing. However, this method is not limited to practical applications; other DNA methylation detection methods known in the art or currently being improved may also be used. In the PCR amplification, the primers used are not limited to those provided in the embodiments; primers that differ in sequence from those provided in the embodiments of the present invention, but still target the nucleic acid or corresponding CpG site indicated by the present invention, may also be obtained.

[0059] In relation to the marker nucleic acid provided by this invention, other methods and reagents known to those skilled in the art for determining the sequence of a genome, its variations, and methylation status may also be included in this invention.

[0060] This invention provides a method for preparing a tumor detection reagent, 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; wherein the target sequence includes at least one methylation CpG site. The detection reagent may include, but is not limited to, primers, probes, etc.; after obtaining the aforementioned marker, the selection of the detection reagent is a matter that can be accomplished by those skilled in the art.

[0061] Once the sequence of the nucleic acid is known, it is known to those skilled in the art to design primers. Two primers are positioned flanking a specific sequence of the target gene to be amplified (including the CpG sequence, with the primer complementary to the CpG to target a previously methylated gene region, and with the primer complementary to the CpG to target a previously demethylated gene region). 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.

[0062] 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.

[0063] The present invention also provides a kit for in vitro detection of methylation profiles of nucleic acids in samples, the kit comprising: a container, and the aforementioned primer pair located in the container.

[0064] 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.

[0065] 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.

[0066] The target nucleic acid segments provided by this invention exhibit very high specificity and sensitivity when used independently. Furthermore, the sensitivity and specificity are further enhanced when two target nucleic acid segments are used in combination. Therefore, the target nucleic acid segments of this invention have significant application value in fields such as tumor adjuvant diagnosis, efficacy assessment, and prognostic monitoring.

[0067] The target nucleic acid segment provided by this invention has universality, high sensitivity and specificity across tissue sources, and is applicable to a single detection platform, thus providing a technical approach to achieve true "one-time detection, multiple cancer early warning".

[0068] Example 1: Determination of methylation detection targets

[0069] 1.1 Obtain the human sequences of genes P7-1, 2, 3, 4, 5, 6, and 7.

[0070] The sequence is shown in Table 1.

[0071] Table 1

[0072] In the above sequence (positive chain), each "" is indicated by a solid underline. CG "" represents one CpG site. The dashed and underlined lines correspond to the upstream and downstream primer design regions and detection target regions in some schemes of the embodiments.

[0073] 1.2 Obtain the DNA sequence after Bisulfite transformation, where Y represents C or U.

[0074] In the above sequence (justice chain), each "YG" marked by a solid underline represents a transformed methylated CpG site, and the black underline corresponds to the upstream and downstream primer design regions in some schemes of the embodiments.

[0075] Example 2: Design and Synthesis of Detection Reagents

[0076] The designed multiplex PCR primers with a length of 25-35 bp (Table 2) are preferred, and the methylated PCR specific primers with appropriate CG content are ensured to have an amplification length of 150-300 bp.

[0077] Table 2

[0078] Example 3: Verification of the reaction reagents

[0079] First round of PCR: The experimental conditions for targeted methylation-specific multiplex PCR are shown in Tables 3A and 3B.

[0080] Table 3A

[0081] The PCR procedure is shown in Table 3B.

[0082] Table 3B

[0083] 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.

[0084] Example 4: Construction of Multiplex PCR Libraries

[0085] 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.

[0086] The experimental conditions for the second round of PCR, methylation-targeted sequencing library preparation PCR, are shown in Table 4.

[0087] Table 4

[0088] The PCR procedure is shown in Table 5.

[0089] Table 5

[0090] Example 5: CpG site methylation analysis

[0091] 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. 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 (Shanghai Yipu Jinding Biotechnology Co., Ltd., A01 nucleic acid extraction kit); 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). 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.

[0092] 5. After PCR amplification, 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. 6. Sequencing results analysis: Samples were differentiated based on the different index sequences added in the second round of PCR, and the methylation values ​​of the target regions in cancer group samples and negative control group samples were analyzed separately; 7. Calculation of P7-1, 2, 3, 4, 5, 6, 7 methylation values: NGS sequencing can independently detect the methylation status of individual CpG sites within the target region. The median value of methylation at all CpG sites is calculated as the methylation of P7-1, 2, 3, 4, 5, 6, 7 in the sample.

[0093] Example 6, P7-1 and P7-2: Validation of multiple cancer types in clinical samples

[0094] Approximately 30 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, and bladder cancer.

[0095] Fifteen clinically positive tissue samples and adjacent normal tissue samples were obtained as the positive group and negative control group, respectively, and multiplex targeted PCR amplification was performed according to the multiplex PCR primer combination methods described in Examples 2, 3, and 4 above (the primers in the table are all primers in the targeted multiplex PCR primer pool).

[0096] NGS libraries of gastric cancer samples were constructed using targeted amplification products, and the methylation levels of P7-1 and P7-2 were analyzed according to the NGS sequencing procedure.

[0097] The cancer samples were analyzed separately, with P7-1 as the analytical target, using the method described in Example 5 for CpG site methylation analysis. The results of the differential analysis of methylation modification levels for P7-1 and P7-2 are as follows: Figure 1 and Figure 2 As shown.

[0098] Simultaneously, ROC analysis was performed on the aforementioned cancer types: statistical analysis and plotting were all completed using the R language environment (version 4.4.2). For continuous variables, normality was first tested, and data conforming to a normal distribution were compared between groups using the independent samples t-test (Student's t-test). Statistical significance was graded using asterisks. This means P < 0.05. This means P < 0.01. This means P < 0.001. This represents P < 0.0001. To assess the discriminative power of the indicator, the pROC package was used to plot the receiver operating characteristic (ROC) curve, and the area under the curve (AUC) and confidence interval were calculated. The optimal cutoff value was determined based on the Youden Index. The ROC analysis results are as follows: Figure 3 and Figure 4 As shown.

[0099] Furthermore, the inventors conducted the above experiments on other P7 differentially methylated regions and analyzed the sensitivity and specificity of each fragment. The results for P7-1, 2, 3, 4, 5, 6, and 7 are statistically presented in Table 6 (unit: %) and Table 7 (unit: %).

[0100] Table 6, P7-1, 2, 3, 4: Detection performance data

[0101] Table 7, P7-5, 6, 7: Detection performance data

[0102] Example 7: Further Improvement Scheme for Diagnostic Performance

[0103] To further improve the accuracy of multi-tumor diagnosis, the inventors, through in-depth analysis, obtained a series of dual-fragment diagnostic target combinations. The number of positive and negative samples was consistent, with 15 negative and 15 positive samples each for 18 common cancers, including breast cancer, bladder cancer, gastric cancer, glioma, nasopharyngeal carcinoma, prostate cancer, laryngeal cancer, pancreatic cancer, liposarcoma, thyroid cancer, urothelial carcinoma, colorectal cancer, biliary tract cancer, cervical cancer, liver cancer, osteosarcoma, ovarian cancer, and renal cell carcinoma. Leukemia had 23 negative and 23 positive samples, esophageal cancer had 26 negative and 26 positive samples, while lung cancer, melanoma, oral cancer, lymphoma, and endometrial cancer each had 20 negative and 20 positive samples. If at least one fragment of the combination of fragment P7-3 and fragment P7-1 is methylated positive, then the sample to be tested is a cancer-positive sample; if both fragments of the sample to be tested are methylated negative, then the sample to be tested is a cancer-negative sample. This combination can better distinguish between the control group and the cancer group in multiple cancer types. The sensitivity and specificity of this fragment combination in different cancer types are shown in Table 8.

[0104] Table 8. Diagnostic performance of the parallel combination of fragment P7-1 and fragment P7-3

[0105] As shown in Table 5, when fragments P7-1 and P7-3 are combined in parallel, they exhibit significant improvements in sensitivity or specificity across various cancer types.

[0106] Example 7: Clinical analysis of the target of the present invention in glioma or ovarian cancer.

[0107] In this embodiment, the diagnostic performance of the STAMP-EP2 target in CN 108866191 B and the two targets P7-1 and P7-3 of this invention in glioma and ovarian cancer is compared.

[0108] The analysis was performed using the experimental methods in Example 5 and the samples (glioma and ovarian cancer) in Example 6. The diagnostic performance results are shown in Tables 9 and 10.

[0109] Table 9. Comparison of diagnostic performance of gliomas in different target segments

[0110] Table 10. Comparison of diagnostic performance of different target regions for ovarian cancer

[0111] As can be seen from the comparison results in Tables 9 and 10, the performance of the P7-1 and P7-3 targets of the present invention is significantly better than that of the STAMP-EP2 target in CN 108866191 B in clinical analysis of glioma or ovarian cancer.

[0112] 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. The application of a reagent for specifically detecting the CpG site methylation modification level of a target nucleic acid segment in the preparation of a reagent or kit for detecting tumors; wherein the target nucleic acid segment is a specific segment in the human genome, selected from: (1) nucleic acids or combinations thereof with the nucleotide sequences 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 or SEQ ID NO:7; or (2) nucleic acids or combinations thereof that are complementary to the nucleic acid sequences of (1) in sequence and have the same length.

2. The application as described in claim 1, characterized in that, During detection, a significant increase in the methylation level of CpG sites in the target nucleic acid region indicates a high risk of tumor development. Preferably, the methods for detecting the methylation level of CpG sites in the target nucleic acid region include: pyrosequencing, bisulfite conversion sequencing, 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, HPLC, MassArray, or combinations thereof.

3. The application as described in claim 2, characterized in that, The method for detecting the CpG site methylation modification level of the target nucleic acid segment includes: (1) Extract nucleic acid from the sample to be tested; (2) The extracted nucleic acid is processed to convert the unmodified cytosine into uracil; preferably, the nucleic acid described in step (1) is treated with bisulfite. (3) Analyze the sequence segments in the nucleic acid of (2) that correspond to the target nucleic acid segment to obtain the methylation modification level of CpG site.

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

5. The application as described in claim 1, characterized in that, The target nucleic acid segment is a combination of nucleic acids with nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:

1.

6. The application as described in claim 1, characterized in that, The reagents or kits are designed for the following types of samples: tissue samples, blood samples, cell 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.

7. The application of target nucleic acid segments or nucleic acid segments derived from them in the preparation of reagents or kits for tumor detection; among which, The target nucleic acid segment is a specific segment in the human genome, selected from: (1) nucleic acids or combinations thereof with the nucleotide sequences 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 or SEQ ID NO:7; or (2) nucleic acids or combinations thereof that are complementary to (1) in sequence and have the same length; the nucleic acid segment derived from the target nucleic acid segment is a polynucleotide corresponding to (1) or (2), wherein its unmodified cytosine (C) is converted to T or U, while the cytosine at its modified CpG site remains unchanged.

8. A method for preparing a reagent for detecting tumors, characterized in that, The method includes: (a) Provide a target nucleic acid segment, said target nucleic acid segment being selected from: (1) nucleic acids or combinations thereof with the nucleotide sequences 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 or SEQ ID NO:7; or (2) nucleic acids or combinations thereof that are sequence-complementary to (1) and of the same length; (b) For the target nucleic acid segment described in (a), prepare a detection reagent specifically for detecting the level of methylation modification at the CpG site of the target sequence.

9. The method as described in claim 8, characterized in that, The detection reagents include: primers and probes; preferably, the detection reagents include: Primers selected from the following group: Primers for the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16; Primers for the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18; Primers for the nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20; Primers for the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22; Primers for the nucleotide sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24; Primers for the nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26; and / or, Primers for the nucleotide sequences shown in SEQ ID NO: 27 and SEQ ID NO:

28. Preferably, the reagent also includes primers for the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO:

20.

10. A reagent for detecting tumors, specifically detecting the level of CpG site methylation modification of a target nucleic acid segment, said target nucleic acid segment being selected from: (1) nucleic acids or combinations thereof with the nucleotide sequences 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 or SEQ ID NO:7; or (2) nucleic acids or combinations thereof that are sequence-complementary to (1) and of the same length; Preferably, the reagent comprises primers selected from the group consisting of: Primers for the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16; Primers for the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18; Primers for the nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20; Primers for the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22; Primers for the nucleotide sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24; Primers for the nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26; and / or, Primers for the nucleotide sequences shown in SEQ ID NO: 27 and SEQ ID NO:

28. Preferably, the reagent also includes primers for the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO: 20.

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