Gene marker combination, kit, and detection method

Abstract

The present application relates to the field of biomedicine, and in particular to a gene marker combination, a detection method, a kit and a detection model for urothelial carcinoma. According to the present application, a reagent combination is used for confirming the presence of urothelial carcinoma, assessing the risk of urothelial carcinoma and / or assessing the prognosis / progression of urothelial carcinoma. The reagent combination comprises reagents capable of detecting the methylation level of a DNA region or a fragment thereof where a VIM gene and a ONECUT2 gene are located in a sample under test, and gene mutations of TERT and FGFR3.

Description

Gene marker combination, kit and detection method Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a detection method, kit, and detection model for urothelial carcinoma. Background Art

[0002] Among numerous cancers, the incidence of urinary tract tumors is increasing annually, further expanding the demand for precise diagnosis and treatment of urinary tract tumors. Urothelial carcinoma (UC), which includes upper tract urothelial carcinoma (UTUC) and lower tract urothelial carcinoma (primarily bladder cancer, BC), is characterized by high incidence, multiple primary sites, and high recurrence after surgery. Common clinical screening methods for bladder cancer include cystoscopy (which can biopsy suspected lesions), urine cytology, imaging, and fluorescence in situ hybridization.

[0003] Clinical diagnosis and post-treatment monitoring of UC mainly rely on imaging, urine testing, endoscopy and biopsy. Traditional imaging examinations are difficult for lesions that are small or atypical in morphology, making it difficult to locate and make a qualitative diagnosis. Urine testing is widely used in clinical practice as an important auxiliary diagnostic method, but both have certain shortcomings. For example, urine exfoliative cytology and FISH examinations have the limitations of relatively low sensitivity and specificity, and cannot replace microscopy and biopsy. Endoscopy (ureteroscopy / cystoscopy) combined with biopsy is the gold standard for the diagnosis and follow-up of urothelial tumors, but because it is invasive and relatively costly, patient compliance is poor, making it difficult to detect early tumors and tumor recurrence in some patients, thus missing the opportunity for treatment.

[0004] In clinical practice, early diagnosis and monitoring of UC remain suboptimal. For example, CT scans for muscle-invasive bladder cancer (MIBC) have an accuracy rate of only 54.9%. Urine cytology and fluorescence in situ hybridization (FISH) have low sensitivity and are susceptible to urinary tract infections, cystitis, stones, and hematuria. Cystoscopy, the gold standard for diagnosing bladder cancer, is invasive, leading to patient resistance and poor compliance. Furthermore, carcinoma in situ is difficult to detect under cystoscopy and can be easily confused with inflammatory changes in the bladder, necessitating a biopsy for confirmation.

[0005] Early diagnosis of upper tract urothelial carcinoma (UTUC) remains very difficult. When UTUC is highly suspected but imaging examinations are unclear, ureteroscopy can directly observe the tumor and perform a biopsy. However, microscopic examination is affected by the location and angle of the tumor, resulting in the inability of conventional ureteroscopes to enter the tumor site and diagnose the tumor; ureteral obstruction can also affect the results of microscopic examination, resulting in the inability to distinguish the cause of obstruction. The causes of obstruction include ureteral stones, ureteropelvic junction stenosis, and ureteral malignant tumors. At the same time, ureteroscopy itself also has certain problems. In addition to the trauma and infection risks of the ureteroscope itself, the chance of exudation and adhesion of surrounding tissues after ureteroscopy is significantly increased, which increases the difficulty of radical surgery and should not be carried out easily.

[0006] Therefore, it is necessary to provide a non-invasive, highly sensitive and specific detection product for accurate early diagnosis and prognosis of urothelial carcinoma.

[0007] Summary of the Invention

[0008] In order to improve the sensitivity and specificity of early diagnosis of urothelial carcinoma, so as to achieve accurate early diagnosis of urothelial carcinoma and recurrence monitoring, the present application provides a method for confirming whether urothelial carcinoma exists, assessing the risk of urothelial carcinoma and / or assessing the prognosis / progression of urothelial carcinoma, as well as corresponding detection kits and / or detection models.

[0009] The present application uses specially selected mutation sites and methylation gene fragments as detection indicators for early diagnosis of urothelial carcinoma and prognosis determination. First, the detection method of the present application uses urine gDNA as a template as the initial sample for detection. It is a non-invasive detection method that effectively avoids the occurrence of complications such as urinary and male reproductive system infections, urethral and bladder bleeding, urethral injury and urethral stenosis, and is easy to implement and can be tested at home. Secondly, the sensitivity of conventional urine cytology tests is not enough and there is a risk of false negatives, especially for G1 grade (G1 grade, well-differentiated papillary carcinoma) and low-grade tumors, with a sensitivity of 16%: detection kits for simple methylation or mutation markers have the risk of missed detection; the detection method of the present application uses point mutations and methylation multi-omics, effectively utilizing multiple information within limited samples, which can effectively distinguish especially early tumors, and has the advantages of high sensitivity and specificity; in addition, the detection method of the present application obtains data results quickly, for example, using fluorescent quantitative PCR, which is beneficial for patients to receive follow-up treatment in a timely manner. The method, kit and / or detection model of the present application have the advantages of high sensitivity and specificity

[0010] On the one hand, the present application provides a method for confirming the presence of urothelial carcinoma, assessing the risk of urothelial carcinoma and / or assessing the prognosis / progression of urothelial carcinoma, comprising detecting the methylation level of the DNA region or fragment thereof where the VIM gene is located in the sample to be tested, and genetic mutations of one or more genes selected from TERT and FGFR3.

[0011] In one aspect, the present application provides a method for confirming the presence of urothelial carcinoma, assessing the risk of urothelial carcinoma, and / or assessing the prognosis / progression of urothelial carcinoma, comprising detecting the methylation level of a DNA region or fragment thereof containing the VIM gene, the methylation level of a DNA region or fragment thereof containing the ONECUT2 gene, and genetic mutations in one or more genes selected from TERT and FGFR3 in a test sample. In certain embodiments, the test sample is from a cancer patient, a suspected cancer patient, a population susceptible to cancer, a population at high risk of cancer, or a healthy population.

[0012] In certain embodiments, the tumor patient includes a patient who has not been treated with anti-cancer therapy and / or a patient who has been treated with anti-cancer therapy.

[0013] In certain embodiments, the sample to be tested comprises tissue, cells and / or body fluid.

[0014] In certain embodiments, the test sample comprises urine.

[0015] In certain embodiments, the TERT gene mutation includes one or more selected from C250T and C228T. The TERT C250T point mutation mentioned in this application is located 146 bp upstream of the ATG start site of the TERT gene and may also be referred to in the art as Chr5:1295250C>T (GRCh37) or "c.-146C>T". The TERT C228T point mutation is located 124 bp upstream of the ATG start site of the TERT gene and may also be referred to in the art as Chr5:1295228C>T (GRCh37) or "c.-124C>T".

[0016] In certain embodiments, the FGFR3 gene mutation includes one or more selected from R248C and S249C. The FGFR3 R248C point mutation mentioned in this application may also be referred to in the art as Chr4:1803564C>T (GRCh37), or "NM_000142(FGFR3):c.742C>T(p.R248C)", and the FGFR3 S249C point mutation mentioned in this application may also be referred to in the art as Chr4:1803568C>G (GRCh37), or NM_000142(FGFR3):c.746C>G(p.S249C)".

[0017] In certain embodiments, the DNA region where the VIM gene is located includes chr10:17270951-17271151, chr10:17271330-17271493, chr10:17271498-17271632 and / or chr10:17272405-17272436.

[0018] In certain embodiments, the method includes obtaining nucleic acid from the sample to be tested.

[0019] In certain embodiments, the method comprises converting the nucleic acid such that bases with methylation and bases without methylation form different species after conversion.

[0020] In certain embodiments, the conversion comprises conversion by a deamination agent and / or a methylation-sensitive restriction enzyme.

[0021] In certain embodiments, the deamination reagent comprises bisulfite.

[0022] In certain embodiments, the method comprises contacting the sample to be tested with a reagent that can specifically recognize and / or bind to the DNA region or fragment thereof where the VIM gene is located, and / or the gene mutation.

[0023] In certain embodiments, the reagents include primers and / or probes that can specifically recognize and / or bind to the DNA region or fragment thereof where the VIM gene is located.

[0024] In certain embodiments, the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 13-27.

[0025] In certain embodiments, the method further comprises detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located in the sample to be tested.

[0026] In certain embodiments, the DNA region where the ONECUT2 gene is located includes chr18:55107803-55107828, chr18:55108590-55108672, chr18:55108794-55108826 and / or chr18:55109012-55109014.

[0027] In certain embodiments, the method comprises contacting the sample to be tested with a reagent that can specifically recognize and / or bind to the DNA region or fragment thereof where the ONECUT2 gene is located.

[0028] In certain embodiments, the reagents include primers and / or probes that can specifically recognize and / or bind to the DNA region or fragment thereof where the ONECUT2 gene is located.

[0029] In certain embodiments, the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 1-12.

[0030] In certain embodiments, the method comprises contacting the test sample with a reagent that can specifically recognize and / or bind to a TERT gene mutation.

[0031] In certain embodiments, the reagents include primers and / or probes that can specifically recognize and / or bind to the TERT gene mutation C250T.

[0032] In certain embodiments, the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 29-31.

[0033] In certain embodiments, the reagents include primers and / or probes that can specifically recognize and / or bind to the TERT gene mutation C228T.

[0034] In certain embodiments, the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 28, 30, and 31.

[0035] In certain embodiments, the method comprises contacting the test sample with a reagent that can specifically recognize and / or bind to FGFR3 gene mutations.

[0036] In certain embodiments, the reagents include primers and / or probes that can specifically recognize and / or bind to the FGFR3 gene mutation R248C.

[0037] In certain embodiments, the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 32, 34 and 35.

[0038] In certain embodiments, the reagents include primers and / or probes that can specifically recognize or bind to the FGFR3 gene mutation S249C.

[0039] In certain embodiments, the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 33-35.

[0040] In certain embodiments, the method further comprises contacting the sample to be tested with a reagent that can specifically bind to the DNA region or fragment thereof where the reference gene is located.

[0041] In certain embodiments, the reference gene is the ACTB gene.

[0042] In certain embodiments, the reagent capable of specifically binding to the DNA region or fragment thereof where the reference gene is located comprises a primer and / or a probe, and the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 39-44.

[0043] In certain embodiments, the total concentration of the primers and / or probes is 2-15 μM.

[0044] In certain embodiments, the detection comprises using fluorescent PCR to obtain a fluorescent Ct value. In certain embodiments, if one or more methylation levels in the test sample are detected to be positive, or one or more gene mutations are detected to be positive, the test result is considered positive; if all methylation levels in the test sample are detected to be negative, and all gene mutations are detected to be negative, the test result is considered negative.

[0045] On the other hand, the present application provides a reagent combination for confirming the presence or absence of urothelial carcinoma, assessing the risk of urothelial carcinoma formation and / or assessing the progression of urothelial carcinoma, wherein the reagent combination comprises a reagent capable of detecting the methylation level of the DNA region or fragment thereof where the VIM gene is located in the sample to be tested, and a reagent capable of detecting gene mutations in one or more genes selected from TERT and FGFR3.

[0046] On the other hand, the present application provides a reagent combination for confirming the presence or absence of urothelial carcinoma, assessing the risk of urothelial carcinoma formation and / or assessing the progression of urothelial carcinoma, wherein the reagent combination comprises a reagent capable of detecting the methylation level of the DNA region or fragment thereof where the VIM gene is located in the test sample, a reagent capable of detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and a reagent capable of detecting gene mutations in one or more genes selected from TERT and FGFR3.

[0047] In certain embodiments, the sample to be tested is from a tumor patient, a suspected tumor patient, a tumor-susceptible population, a tumor-high-risk population, or a healthy population.

[0048] In certain embodiments, the tumor patient includes a patient who has not been treated with anti-cancer therapy and / or a patient who has been treated with anti-cancer therapy.

[0049] In certain embodiments, the sample to be tested comprises tissue, cells and / or body fluid.

[0050] In certain embodiments, the test sample comprises urine.

[0051] In certain embodiments, the DNA region where the VIM gene is located includes chr10:17270951-17271151, chr10:17271330-17271493, chr10:17271498-17271632 and / or chr10:17272405-17272436.

[0052] In certain embodiments, the reagent capable of detecting the methylation level of the DNA region or fragment thereof where the VIM gene is located in the test sample comprises primers and / or probes that can specifically recognize and / or bind to the DNA region or fragment thereof where the VIM gene is located.

[0053] In certain embodiments, the primer and / or probe capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the VIM gene is located comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 13-27.

[0054] In certain embodiments, the primers and / or probes capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the VIM gene is located comprise any one of the following groups: (1) primers: SEQ ID NOs. 13-14, probes: SEQ ID NO. 15; (2) primers: SEQ ID NOs. 16-17, probes: SEQ ID NO. 18; (3) primers: SEQ ID NOs. 19-20, probes: SEQ ID NO. 21; (4) primers: SEQ ID NOs. 22-23, probes: SEQ ID NO. 24; and (5) primers: SEQ ID NOs. 25-26, probes: SEQ ID NO. 27.

[0055] In certain embodiments, the TERT gene mutation includes one or more selected from C250T and C228T.

[0056] In certain embodiments, the reagent combination comprises primers and / or probes that can specifically recognize and / or bind to the TERT gene mutation C250T.

[0057] In certain embodiments, the primer and / or probe capable of specifically recognizing and / or binding to the TERT gene mutation C250T comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 29-31.

[0058] In certain embodiments, the reagent combination comprises primers and / or probes that can specifically recognize and / or bind to the TERT gene mutation C228T.

[0059] In certain embodiments, the primer and / or probe capable of specifically recognizing and / or binding to the TERT gene mutation C228T comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 28, 30, and 31.

[0060] In certain embodiments, the FGFR3 gene mutation includes one or more selected from R248C and S249C.

[0061] In certain embodiments, the reagent combination comprises primers and / or probes that can specifically recognize and / or bind to the FGFR3 gene mutation R248C.

[0062] In certain embodiments, the primer and / or probe capable of specifically recognizing and / or binding to the FGFR3 gene mutation R248C comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 32, 34, and 35.

[0063] In certain embodiments, the reagent combination comprises primers and / or probes that can specifically recognize and / or bind to the FGFR3 gene mutation S249C.

[0064] In certain embodiments, the primer and / or probe capable of specifically recognizing and / or binding to the FGFR3 gene mutation S249C comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 33-35.

[0065] In certain embodiments, the reagent combination further comprises detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located in the sample to be tested.

[0066] In certain embodiments, the DNA region where the ONECUT2 gene is located includes chr18:55107803-55107828, chr18:55108590-55108672, chr18:55108794-55108826 and / or chr18:55109012-55109014.

[0067] In certain embodiments, the reagent capable of detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located in the test sample comprises primers and / or probes that can specifically recognize and / or bind to the DNA region or fragment thereof where the ONECUT2 gene is located.

[0068] In certain embodiments, the primer and / or probe capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the ONECUT2 gene is located comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 1-12.

[0069] In certain embodiments, the primers and / or probes capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the ONECUT2 gene is located comprise primers and / or probes selected from any one of the following groups: (1) primers: SEQ ID NO.1-2, probe: SEQ ID NO.3; (2) primers: SEQ ID NO.4-5, probe: SEQ ID NO.6; (3) primers: SEQ ID NO.7-8, probe: SEQ ID NO.9; (4) primers: SEQ ID NO.10-11, probe: SEQ ID NO.12 and primers: SEQ ID NO.36-37, probe: SEQ ID NO.38.

[0070] In certain embodiments, the reagent combination further comprises a reagent capable of specifically binding to the DNA region or fragment thereof where the reference gene is located.

[0071] In certain embodiments, the reference gene is the ACTB gene.

[0072] In certain embodiments, the reagent capable of specifically binding to the DNA region or fragment thereof where the reference gene is located comprises a primer and / or a probe, and the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 39-44.

[0073] In certain embodiments, the total concentration of the primers and / or probes is 2-15 μM.

[0074] In certain embodiments, the reagent combination further comprises a deamination reagent and / or a methylation-sensitive restriction endonuclease.

[0075] In certain embodiments, the deamination reagent comprises bisulfite.

[0076] On the other hand, the present application provides use of the reagent combination in preparing a kit.

[0077] On the other hand, the present application provides a kit comprising the reagent combination.

[0078] On the other hand, the present application provides a model for confirming the presence of urothelial carcinoma, assessing the risk of urothelial carcinoma and / or assessing the prognosis / progression of urothelial carcinoma, wherein the model is implemented based on the kit.

[0079] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the specification of the present application is merely exemplary and not restrictive. DETAILED DESCRIPTION

[0080] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0081] Definition of terms

[0082] In this application, the term "VIM" generally refers to the gene encoding vimentin, also known as CTRCT30 or HEL113. The human VIM gene is located at Chromosome 10:17,270,258-17,279,592, forward strand (GRCh37:CM000672.1). For more information about the human VIM gene, please refer to ensemble number ENSG00000026025.

[0083] In this application, the term "ONECUT2" generally refers to the gene encoding transcription factor onecut family member 2. Human ONECUT2 is located at Chromosome 18:55,102,917-55,158,529 forward strand (GRCh37:CM000680.1). For more information about the human ONECUT2 gene, please refer to ensemble number ENSG00000119547.

[0084] In this application, the term "TERT" generally refers to the gene encoding telomerase reverse transcriptase, which may also be referred to as CMM9, DKCA2, DKCB4, EST2, PFBMFT1, TCS1, TP2, TRT, hEST2, or hTRT. The location of human TERT is at Chromosome 5:1,253,262-1,295,184 on the reverse strand (GRCh37:CM000667.1). For more information about the human TERT gene, please refer to ensemble number ENSG00000164362. The point mutations involving the TERT gene in the present application can be expressed in one or more ways. For example, the two common promoter mutations of the TERT gene in the present application are located 124bp and 146bp upstream of the ATG start site, respectively, including Chr5:1295228C>T (called C228T, which can also be referred to as c.-124C>T in the art) and Chr5:1295250C>T (called C250T, which can also be referred to as c.-146C>T in the art).

[0085] In this application, the term "FGFR3" generally refers to the gene encoding fibroblast growth factor receptor 3, which may also be referred to as ACH, CD333, CEK2, HSFGFR3EX or JTK4. The location of human FGFR3 is at Chromosome 4:1,795,034-1,810,599 forward chain (GRCh37:CM000666.1). For more information about the human FGFR3 gene, reference is made to ensemble number ENSG00000068078. The point mutations involved in the FGFR3 gene in the present application can be expressed in one or more ways. For example, the FGFR3 R248C point mutation mentioned in the present application can also be referred to in the art as Chr4:1803564C>T (GRCh37), or "NM_000142 (FGFR3): c.742C>T (p.R248C)", and the FGFR3 S249C point mutation mentioned in the present application can also be referred to in the art as Chr4:1803568C>G (GRCh37), or NM_000142 (FGFR3): c.746C>G (p.S249C)".

[0086] In this application, the term "test sample" generally refers to a sample that needs to be tested. For example, one or more gene regions in the test sample can be tested for modification or mutation.

[0087] In this application, the term "healthy population" generally refers to a population in which no tumors are currently detected by tumor detection methods. A healthy population can progress to include cancer patients, those at high risk for cancer, suspected cancer patients, or those susceptible to cancer. "Healthy population" is a concept relative to "cancer patients," which generally refers to a population confirmed to have a tumor through pathological diagnosis or other testing methods.

[0088] In this application, the term "high-risk population for cancer" generally refers to people who are at a higher risk of developing cancer. For example, people with a family history of cancer, people who carry cancer-susceptibility genes, people who have unhealthy lifestyles (such as smoking, alcoholism, and staying up late), and people who are frequently exposed to toxic and hazardous substances in their living or working environments.

[0089] In this application, the term "suspected tumor patients" generally refers to people who have tumor symptoms but have not been confirmed to have tumors through pathological diagnosis or other detection methods.

[0090] In this application, the term "tumor-susceptible population" generally refers to a population that is prone to developing tumors. For example, this includes people with a family history of tumors, people who carry tumor-susceptibility genes, people who have unhealthy lifestyles (such as smoking, alcoholism, and staying up late), and people who are frequently exposed to toxic and hazardous substances in their living or working environments.

[0091] For urothelial cancer, for example, bladder cancer, the tumor-susceptible population or high-risk population may include but is not limited to the following populations: patients with unknown clinical diagnosis, high-risk populations for urothelial cancer (age > 50 years old; long-term smokers; people with long-term exposure to carcinogens such as industrial chemical products; people with a family history of bladder cancer; and people who have taken aristolochic acid-containing Chinese herbal medicines.

[0092] In this application, the term "DNA region" generally refers to a sequence of two or more covalently bonded naturally occurring or modified deoxyribonucleotides. For example, the DNA region of a gene can refer to the location of a specific deoxyribonucleotide sequence at which the gene is located, such as the deoxyribonucleotide sequence that encodes the gene. For example, the DNA region of this application includes the full length of the DNA region, its complementary region, or a fragment thereof. For example, a sequence at least about 20 kb upstream and downstream of the detection region provided in this application can be used as a detection site. For example, a sequence at least about 20 kb, at least about 15 kb, at least about 10 kb, at least about 5 kb, at least about 3 kb, at least about 2 kb, at least about 1 kb, or at least about 0.5 kb upstream and downstream of the detection region provided in this application can be used as a detection site. For example, suitable primers and probes can be designed based on the described sites to perform methylation detection in a sample. In this application, when referring to a DNA region or a base position, unless otherwise specified, it generally refers to the human reference genome version GRCh37, corresponding to the UCSC version number hg19.

[0093] In this application, the term "methylation" generally refers to the methylation state of a gene fragment, nucleotide, or base thereof in this application. For example, a DNA fragment in which a gene is located in this application may be methylated on one or more chains. For example, a DNA fragment in which a gene is located in this application may be methylated on one or more sites. For example, methylation may refer to the conversion of the 5'-end cytosine of a dinucleotide of a CpG island on a DNA sequence to a 5'methylcytosine (5mC), and may also include the formation of N6-methylpurine (N6-mA) and 7-methylguanine (7-mG).

[0094] In this application, the term "conversion" generally refers to the conversion of one or more structures into another structure. For example, the conversion of the present application can be specific. For example, a cytosine that does not have a methylation modification can be converted into another structure (e.g., uracil), and a cytosine that has a methylation modification can remain substantially unchanged after conversion. For example, a cytosine that does not have a methylation modification can be sheared after conversion, and a cytosine that has a methylation modification can remain substantially unchanged after conversion.

[0095] In this application, the term "deaminating agent" generally refers to a substance capable of removing an amino group. For example, a deaminating agent can remove the amino group of unmodified cytosine.

[0096] In this application, the term "bisulfite" generally refers to an agent that can distinguish between regions of DNA that have a modification state and regions that do not have a modification state. For example, bisulfite can include bisulfite, or an analog thereof, or a combination thereof. For example, bisulfite can deaminize the amino group of unmodified cytosine to distinguish it from modified cytosine. In this application, the term "analog" generally refers to a substance with a similar structure and / or function. For example, an analog of bisulfite can have a similar structure to bisulfite. For example, an analog of bisulfite can refer to an agent that can also distinguish between regions of DNA that have a modification state and regions that do not have a modification state.

[0097] In the present application, the term "methylation-sensitive restriction endonuclease" generally refers to an enzyme that selectively digests nucleic acids based on the methylation state of its recognition site. For example, for a restriction endonuclease that specifically shears when the recognition site is not methylated, shearing may not occur, or shearing may be performed with significantly reduced efficiency when the recognition site is methylated. For a restriction endonuclease that specifically shears when the recognition site is methylated, shearing may not occur, or shearing may be performed with significantly reduced efficiency when the recognition site is not methylated. For example, a methylation-specific restriction endonuclease can recognize sequences containing CG dinucleotides (e.g., cgcg or cccggg).

[0098] In this application, the term "urothelial carcinoma" generally refers to a cancer associated with the malignant proliferation of urothelial cells. Urothelial carcinoma can include upper tract urothelial carcinoma (UTUC, e.g., renal pelvis cancer and ureter cancer) and lower tract urothelial carcinoma (e.g., bladder cancer). Urothelial carcinoma can occur in the renal pelvis, ureter, or bladder.

[0099] As used herein, the term "progression" generally refers to a change in a disease from a less severe state to a more severe state. For example, tumor progression can include an increase in the number or severity of tumors, the extent of metastasis, the rate at which the cancer grows or spreads, etc. For example, tumor progression can include the stage of such cancer from a less severe state to a more severe state, such as from stage I to stage II, from stage II to stage III, etc.

[0100] In this application, the term "occurrence" generally refers to the occurrence of a lesion in an individual. For example, when a tumor occurs, the individual can be diagnosed as a tumor patient.

[0101] In this application, the term "fluorescent PCR" generally refers to a quantitative or semi-quantitative PCR technique. For example, the PCR technique may be a real-time quantitative polymerase chain reaction, a quantitative polymerase chain reaction, or a kinetic polymerase chain reaction. For example, PCR amplification can be used and the amount of the initial target nucleic acid can be quantitatively detected using an intercalating fluorescent dye or a sequence-specific probe. The sequence-specific probe may contain a fluorescent reporter molecule that is detectable only by hybridization with the target nucleic acid.

[0102] In this application, the term "PCR amplification" generally refers to a polymerase chain amplification reaction. For example, the PCR amplification in this application can include any polymerase chain amplification reaction currently known for DNA amplification.

[0103] In this application, the term "fluorescence Ct value" generally refers to a measurement value for quantitatively or semi-quantitatively evaluating a target nucleic acid, for example, the number of amplification reaction cycles required for the fluorescence signal to reach a set threshold.

[0104] Detailed Description of the Invention

[0105] Gene marker combination

[0106] The present application provides a gene marker combination that can be used to confirm the presence of urothelial carcinoma, assess the risk of developing urothelial carcinoma, and / or assess the prognosis / progression of urothelial carcinoma. Thus, the present application provides a method for detecting and confirming the presence of urothelial carcinoma, assessing the risk of developing urothelial carcinoma, and / or assessing the prognosis / progression of urothelial carcinoma. The method can be used for early diagnosis and / or recurrence monitoring of urothelial carcinoma. The present application also provides a reagent combination for detecting the gene marker combination, as well as a kit containing the reagent combination.

[0107] The inventors of the present application have discovered that by detecting the methylation level of the VIM gene and the point mutation of TERT in the test sample, the presence of urothelial carcinoma can be confirmed with high sensitivity and specificity, the risk of urothelial carcinoma occurrence and / or the prognosis / progression of urothelial carcinoma can be assessed.

[0108] In the present application, the gene marker combination comprises the methylation level of the DNA region or fragment thereof where the VIM gene is located, and gene mutations of one or more genes selected from Table 2 below, for example, one mutation, two mutations, three mutations, four mutations, five mutations or more selected from the table below. In certain embodiments, the combination comprises the methylation level of the DNA region or fragment thereof where the VIM gene is located, and gene mutations of one or more genes selected from the group consisting of TERT, FGFR, and other gene mutations listed in Table 2 below, for example, one other mutation, two other mutations, three other mutations, four other mutations or more selected from the table below.

[0109] [Corrected 20.05.2024 in accordance with Rule 26] In the present application, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located. In the present application, the DNA region where the VIM gene is located may be chr10:17,270,258-17,279,592. For example, the DNA region where the VIM gene is located may be chr10:17,271,345-17,271,493, or a region extending approximately 100 bp upstream and downstream thereof. For example, the DNA region where the VIM gene is located may be chr10:17,271,051-17,271,051, or a region extending approximately 100 bp upstream and downstream thereof. For example, the DNA region where the VIM gene is located may be chr10:17,270,951-17,271,151, or a region extending approximately 100 bp upstream and downstream thereof. For example, the DNA region where the VIM gene is located can be chr10:17,272,405-17,272,436, or a region extending approximately 100 bp upstream and downstream thereof. For example, the DNA region where the VIM gene is located can be chr10:17,271,498-17,271,632, or a region extending approximately 100 bp upstream and downstream thereof. The methylation level of the DNA region where the VIM gene is located can be tested to confirm the presence of urothelial cancer, assess the risk of developing urothelial cancer, and / or assess the prognosis / progression of urothelial cancer. If the methylation level of the DNA region where the VIM gene is located is positive, it can be considered that urothelial cancer is present, the risk of developing urothelial cancer is high, and / or the risk of recurrence of urothelial cancer is high.

[0110] In the present application, the gene marker combination may include a TERT gene mutation, for example, a TERT promoter mutation. The TERT promoter mutation may include a point mutation located at C228 and / or C250. For example, the TERT gene mutation may include a C228T point mutation. For another example, the TERT gene mutation may include a C250T point mutation. The presence of a TERT gene mutation can be used to confirm the presence of urothelial carcinoma, assess the risk of urothelial carcinoma, and / or assess the prognosis / progression of urothelial carcinoma. If a TERT gene mutation is detected in the sample to be tested, in particular the presence of TERT C228T and / or TERT C250T point mutation, it can be considered that urothelial carcinoma is present, the risk of urothelial carcinoma is high, and / or the risk of recurrence of urothelial carcinoma is high.

[0111] In the present application, the gene marker combination may include FGFR3 gene mutations, for example, FGFR3 gene mutations. FGFR3 gene mutations may include gene mutations that cause R248C and / or S249C amino acid mutations. For example, FGFR3 gene mutations may include point mutations that cause R248C, for example, C742T point mutations. For example, FGFR3 gene mutations may include point mutations that cause S249C, for example, C746G point mutations. The presence of urothelial carcinoma can be confirmed by detecting the presence of FGFR3 gene mutations, assessing the risk of urothelial carcinoma, and / or assessing the prognosis / progression of urothelial carcinoma. If the presence of FGFR3 gene mutations, particularly the presence of R248C and / or S249C mutations, is detected in the sample to be tested, it can be considered that urothelial carcinoma is present, the risk of urothelial carcinoma is high, and / or the risk of recurrence of urothelial carcinoma is high.

[0112] In the present application, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located. In the present application, the DNA region where the ONECUT2 gene is located may be chr 18:55,102,917-55,158,529. For example, the DNA region where the ONECUT2 gene is located may be chr18:55,107,803-55,107,828, or a region extending approximately 100 bp upstream and downstream thereof. For example, the DNA region where the ONECUT2 gene is located may be chr18:55,108,590-55,108,672, or a region extending approximately 100 bp upstream and downstream thereof. For example, the DNA region where the ONECUT2 gene is located may be chr18:55,109,012-55,109,014, or a region extending approximately 100 bp upstream and downstream thereof. For example, the DNA region where the ONECUT2 gene is located can be chr18:55,108,794-55,108,826, or a region extending approximately 100 bp upstream and downstream thereof. The methylation level of the DNA region where the ONECUT2 gene is located can be detected to confirm the presence of urothelial cancer, assess the risk of urothelial cancer, and / or assess the prognosis / progression of urothelial cancer. If the methylation level of the ONECUT2 region where the VIM gene is located is positive, it can be considered that urothelial cancer is present, the risk of urothelial cancer is high, and / or the risk of recurrence of urothelial cancer is high.

[0113] The present application confirms the presence of urothelial carcinoma, assesses the risk of urothelial carcinoma and / or assesses the prognosis / progression of urothelial carcinoma by detecting a combination of the above-mentioned gene markers.

[0114] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, and one or more point mutations selected from the group consisting of TERT C228T, TERT C250T, FGFR3 R248C and FGFR3 S249C, for example, two, three or four point mutations.

[0115] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, and any one point mutation selected from the following group: TERT C228T, TERT C250T, FGFR3 R248C and FGFR3 S249C.

[0116] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region where the VIM gene is located or a fragment thereof and a TERT promoter point mutation.

[0117] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region where the VIM gene is located or a fragment thereof and the TERT C228T point mutation.

[0118] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located and the TERT C250T point mutation.

[0119] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located and the FGFR3 R248C point mutation.

[0120] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located and the FGFR3 S249C point mutation.

[0121] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region where the VIM gene is located or a fragment thereof, the FGFR3 point mutation, and the FGFR3 point mutation.

[0122] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, and any two point mutations selected from the following group: TERT C228T, TERT C250T, FGFR3 R248C, and FGFR3 S249C.

[0123] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the TERT C228T point mutation, and the FGFR3 S248C point mutation.

[0124] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the TERT C250T point mutation, and the FGFR3 R249C point mutation.

[0125] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, and any three point mutations selected from the following group: TERT C228T, TERT C250T, FGFR3 R248C and FGFR3 S249C.

[0126] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, and the following four point mutations: TERT C228T, TERT C250T, FGFR3 R248C, and FGFR3 S249C.

[0127] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and any one or more of the following point mutations: TERT C228T, TERT C250T, FGFR3 R248C and FGFR3 S249C, for example, two, three or four point mutations.

[0128] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and any one of the following point mutations: TERT C228T, TERT C250T, FGFR3 R248C, and FGFR3 S249C.

[0129] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and TERT C228T.

[0130] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region where the VIM gene is located or a fragment thereof, the methylation level of the DNA region where the ONECUT2 gene is located or a fragment thereof, and TERT C250T.

[0131] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and FGFR3 R248C.

[0132] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and FGFR3 S249C.

[0133] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and any two of the following point mutations: TERT C228T, TERT C250T, FGFR3 R248C, and FGFR3 S249C.

[0134] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and TERT C228T and FGFR3 R248C.

[0135] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and TERT C250T and FGFR3 S249C.

[0136] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and any three of the following point mutations: TERT C228T, TERT C250T, FGFR3 R248C, and FGFR3 S249C.

[0137] In a specific embodiment, the gene marker combination may include the methylation level of the DNA region or fragment thereof where the VIM gene is located, the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located, and the following four point mutations: TERT C228T, TERT C250T, FGFR3 R248C and FGFR3 S249C.

[0138] The inventors of the present application discovered that by detecting the methylation level of the VIM gene, the methylation level of the ONECUT2 gene, the point mutations C250T and C228T of TERT, and the point mutations R248C and S249C of FGFR3 in the test sample, the presence of urothelial carcinoma can be confirmed with high sensitivity and specificity, the risk of occurrence of urothelial carcinoma can be assessed, and the prognosis / progression of urothelial carcinoma can be assessed.

[0139] In one embodiment, the reagent combination of the present application may include a reagent for detecting the methylation level of the DNA region chr10:17271330-17271493 of VIM, a reagent for detecting the methylation level of the DNA region chr18:55107803-55107828 of ONECUT2, a reagent for detecting the TERT C228T point mutation, a reagent for detecting the TERT C250T point mutation, a reagent for detecting the FGFR3 R248C point mutation, and a reagent for detecting the FGFR3 S249C point mutation.

[0140] Gene marker detection methods

[0141] Methods for detecting the methylation level of a DNA region or a DNA region fragment where a gene is located are well known to those skilled in the art, including but not limited to: methylation-specific PCR (MS-PCR), bisulfite conversion sequencing (BS-Seq), restriction endonuclease analysis combined with sodium bisulfite, fluorescence quantitative analysis, methylation-sensitive high-resolution melting curve analysis, pyrophosphate sequencing, chip-based methylation profiling, high-throughput sequencing, and flight mass spectrometry.

[0142] In a specific embodiment, a fluorescence quantitative method can be used to design specific primers and probe sequences for methylation of the gene to be tested based on the CpG island information of the gene to be tested (e.g., VIM or ONECUT2) in combination with fluorescent probe technology to detect the methylation level. Detection can also be performed using primers and fluorescent dyes.

[0143] For example, the methylation level of one or more ONECUT2 DNA regions selected from the group consisting of chr18:55107803-55107828, chr18:55108794-55108826, chr18:55108590-55108672, and chr18:55109012-55109014 can be detected. For example, the methylation level of at least one of the above DNA regions can be detected.

[0144] For example, the primers for detecting the methylation level of the DNA region of ONECUT2 may include the nucleic acid sequence shown in SEQ ID NO. 1-2, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 3.

[0145] For example, the primers for detecting the methylation level of the DNA region of ONECUT2 may include the nucleic acid sequences shown in SEQ ID NOs. 4-5, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 6.

[0146] For example, the primers for detecting the methylation level of the DNA region of ONECUT2 may include the nucleic acid sequences shown in SEQ ID NOs. 7-8, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 9.

[0147] For example, the primers for detecting the methylation level of the DNA region of ONECUT2 may include the nucleic acid sequences shown in SEQ ID NOs. 10-11, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 12.

[0148] For example, the methylation level of one or more VIM DNA regions selected from the group consisting of chr10:17270951-17271151, chr10:17271330-17271493, chr10:17271051-17271051, chr10:17272405-17272436, and chr10:17271498-17271632 can be detected. For example, the methylation level of at least one of the above DNA regions can be detected.

[0149] For example, the primers for detecting the methylation level of the DNA region of the VIM may include the nucleic acid sequences shown in SEQ ID NOs. 13-14, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 15.

[0150] For example, the primers for detecting the methylation level of the DNA region of the VIM may include the nucleic acid sequences shown in SEQ ID NOs. 16-17, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 18.

[0151] For example, the primers for detecting the methylation level of the DNA region of the VIM may include the nucleic acid sequences shown in SEQ ID NOs. 19-20, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 21.

[0152] For example, the primers for detecting the methylation level of the DNA region of the VIM may include the nucleic acid sequences shown in SEQ ID NOs. 22-23, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 24.

[0153] For example, the primers for detecting the methylation level of the DNA region of the VIM may include the nucleic acid sequences shown in SEQ ID NO. 25-26, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 27.

[0154] Methods for detecting target gene mutations or point mutations are well known to those skilled in the art, for example, detection methods based on PCR technology, including but not limited to: PCR-SSCP, heteroduplex analysis (HA), mutant enrichment PCR, deformed gradient gel electrophoresis (DGGE), chemical cleavage mismatch method (CCM), allele-specific oligonucleotide analysis (ASO), DNA chip ligase reaction (LCR), allele-specific amplification, RNase A cleavage method, chromosome in situ hybridization, fluorescence in situ hybridization technology, DNA sequence analysis, fluorescence quantitative PCR, digital PCR and / or high-throughput sequencing. For example, primers, such as multiple specific primers, can be involved in the gene mutation and fluorescence quantitative PCR can be used for detection. Primers and fluorescently labeled probes can also be used for detection.

[0155] For example, the TERT C228T point mutation and / or the TERT C250T point mutation can be detected.

[0156] For example, the primers for detecting TERT point mutations may include the nucleic acid sequences shown in SEQ ID NOs. 28 and 30, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 21.

[0157] For example, the primers for detecting TERT point mutations may comprise the nucleic acid sequences shown in SEQ ID NOs. 29-30, and the probe may comprise the nucleic acid sequence shown in SEQ ID NO. 21.

[0158] For example, the FGFR3 R248C point mutation and / or the FGFR3 S249C point mutation can be detected.

[0159] For example, the primers for detecting FGFR3 point mutations may comprise the nucleic acid sequences shown in SEQ ID NOs. 32 and 34, and the probe may comprise the nucleic acid sequence shown in SEQ ID NO. 35.

[0160] For example, the primers for detecting FGFR3 point mutations may comprise the nucleic acid sequences shown in SEQ ID NOs. 33-34, and the probe may comprise the nucleic acid sequence shown in SEQ ID NO. 35.

[0161] In the present application, the method further includes one or more of the following steps: (1) obtaining nucleic acid (e.g., gDNA) from the sample to be tested, (2) converting the nucleic acid using a deamination reagent (e.g., bisulfite), (3) adding the primer and / or probe combination described in the present application, as well as the primer and / or probe combination of the reference gene, (4) performing real-time fluorescence quantitative PCR, and (5) obtaining a ΔCt value and analyzing the results. The ΔCt value can be: the ΔCt value of the methylated fragment = the Ct value of the methylated fragment - the Ct value of the reference gene, the ΔCt value of the point mutation = the Ct value of the point mutation - the Ct value of the reference gene. The reference gene (also referred to as an internal reference gene) can include the ACTB gene (β-actin), GAPDH, 18S rRNA, 28S rRNA, β2-MG, and PBGD. For example, the reference gene can be the ACTB gene.

[0162] In one embodiment, the concentrations of primers and / or probes for each gene marker in the combination may be the same or different. For example, the concentration of the primer and / or probe mixture for each gene marker in the combination may be approximately 2-15 μM. For another example, the concentration of the primer and / or probe mixture for each gene marker in the combination may be approximately 6-12 μM. For another example, the concentration of the primer and / or probe mixture for each gene marker in the combination may be approximately 9 μM.

[0163] The present application includes detecting the above-mentioned combination of gene markers in the sample to be tested to confirm whether urothelial carcinoma exists, assess the risk of urothelial carcinoma and / or assess the prognosis / progression of urothelial carcinoma. For example, according to the ΔCt value of the point mutation of each gene marker, if there is one or more positives in the above-mentioned gene marker combination, the sample to be tested can be considered to be positive, and it can be considered that the subject from which the sample to be tested has urothelial carcinoma, a high risk of urothelial carcinoma, or a high risk of recurrence of urothelial carcinoma. For example, the method for judging whether each gene marker is positive based on the ΔCt value can be that when the ΔCt value of the methylation fragment is less than a certain value, the test result is considered to be positive and it is judged that the target gene is highly methylated, otherwise the target gene is hypomethylated, or, when the ΔCt value of the point mutation is less than a certain value, the test result is considered to be positive and it is judged that there is a mutation at the site, otherwise there is no mutation at the site. The numerical value compared with ΔCt can refer to Table 1 below. Those skilled in the art can also select a suitable judgment value according to actual needs.

[0164] Table 1

[0165] In the present application, a scoring model can also be used to calculate a score value for the test sample, such as a UC-Score value, to determine whether the test sample is negative or positive. If the sample UC-Score is greater than 0, the test sample is judged to be positive; if the UC-Score is less than 0, the test sample is judged to be negative. Those skilled in the art can obtain an appropriate UC-Score scoring model by analyzing sample library data.

[0166] In another aspect, the present application provides one or more reagents or a combination of reagents for detecting the gene marker combination. The one or more reagents may include primers and / or probes.

[0167] In another aspect, the present application provides the use of one or more reagents or reagent combinations in preparing a kit. The kit may include primers and / or probes for detecting the gene marker combination, and optionally, a pharmaceutically acceptable solvent, carrier, and / or instructions for use. In the kit, the primers and / or probes for detecting each of the gene markers, as well as other required reagents, may be placed in the same container or in separate containers.

[0168] In another aspect, the present application provides a program capable of executing the method.

[0169] On the other hand, the present application provides the following embodiments:

[0170] 1. A method for confirming the presence of urothelial carcinoma, assessing the risk of urothelial carcinoma, and / or assessing the prognosis / progression of urothelial carcinoma, comprising detecting the methylation level of a DNA region or fragment thereof in which the VIM gene is located, and detecting gene mutations in one or more genes selected from TERT and FGFR3 in a test sample.

[0171] 2. The method according to embodiment 1, wherein the sample to be tested is from a tumor patient, a suspected tumor patient, a tumor-susceptible population, a tumor-high-risk population, or a healthy population.

[0172] 3. The method according to embodiment 2, wherein the tumor patients include patients who have not been treated with anti-cancer therapy and / or patients who have been treated with anti-cancer therapy.

[0173] 4. The method according to any one of embodiments 1-3, wherein the sample to be tested comprises tissue, cells and / or body fluid.

[0174] 5. The method according to any one of embodiments 1-4, wherein the test sample comprises urine.

[0175] 6. The method according to any one of embodiments 1-5, wherein the genetic mutation of TERT comprises one or more selected from C250T and C228T.

[0176] 7. The method according to any one of embodiments 1-6, wherein the genetic mutation of FGFR3 comprises one or more selected from R248C and S249C.

[0177] 8. The method according to any one of embodiments 1-7, wherein the DNA region where the VIM gene is located comprises chr10:17270951-17271151, chr10:17271330-17271493, chr10:17271498-17271632 and / or chr10:17272405-17272436.

[0178] 9. The method according to any one of embodiments 1-8, wherein the method comprises obtaining nucleic acid in the sample to be tested.

[0179] 10. The method according to embodiment 9, wherein the method comprises converting the nucleic acid so that the bases having methylation and the bases not having methylation form different substances after conversion.

[0180] 11. A method according to embodiment 10, wherein the conversion comprises conversion by a deamination agent and / or a methylation-sensitive restriction endonuclease.

[0181] 12. The method of embodiment 11, wherein the deamination reagent comprises bisulfite.

[0182] 13. The method according to any one of embodiments 1 to 12, wherein the method comprises contacting the test sample with a reagent that can specifically recognize and / or bind to the DNA region or fragment thereof where the VIM gene is located, and / or the gene mutation.

[0183] 14. The method according to embodiment 13, wherein the reagent comprises a primer and / or a probe capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the VIM gene is located.

[0184] 15. The method according to embodiment 14, wherein the primers and / or probes comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 13-27.

[0185] 16. The method according to any one of embodiments 1-13, further comprising detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located in the test sample.

[0186] 17. The method according to embodiment 16, wherein the DNA region where the ONECUT2 gene is located comprises chr18:55107803-55107828, chr18:55108590-55108672, chr18:55108794-55108826 and / or chr18:55109012-55109014.

[0187] 18. The method according to any one of embodiments 16-17, wherein the method comprises contacting the test sample with a reagent that can specifically recognize and / or bind to the DNA region or fragment thereof where the ONECUT2 gene is located.

[0188] 19. The method according to embodiment 18, wherein the reagent comprises a primer and / or a probe that can specifically recognize and / or bind to the DNA region or fragment thereof where the ONECUT2 gene is located.

[0189] 20. The method according to embodiment 19, wherein the primers and / or probes comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 1-12.

[0190] 21. The method according to any one of embodiments 1-20, wherein the method comprises contacting the test sample with a reagent that can specifically recognize and / or bind to the TERT gene mutation.

[0191] 22. The method according to embodiment 21, wherein the reagent comprises a primer and / or a probe capable of specifically recognizing and / or binding to the TERT gene mutation C250T.

[0192] 23. The method according to embodiment 22, wherein the primers and / or probes comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 29-31.

[0193] 24. The method according to embodiment 21, wherein the reagent comprises a primer and / or a probe capable of specifically recognizing and / or binding to the TERT gene mutation C228T.

[0194] 25. The method according to embodiment 24, wherein the primers and / or probes comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 28, 30 and 31.

[0195] 26. The method according to any one of embodiments 1-25, wherein the method comprises contacting the test sample with a reagent that can specifically recognize and / or bind to FGFR3 gene mutation.

[0196] 27. The method according to embodiment 26, wherein the reagent comprises a primer and / or a probe capable of specifically recognizing and / or binding to the FGFR3 gene mutation R248C.

[0197] 28. The method according to embodiment 27, wherein the primers and / or probes comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 32, 34 and 35.

[0198] 29. The method according to embodiment 26, wherein the reagent comprises a primer and / or a probe capable of specifically recognizing or binding to the FGFR3 gene mutation S249C.

[0199] 30. The method according to embodiment 29, wherein the primers and / or probes comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 33-35.

[0200] 31. The method according to any one of embodiments 1-30 further comprises contacting the sample to be tested with a reagent that can specifically bind to the DNA region or fragment thereof where the reference gene is located.

[0201] 32. The method of embodiment 31, wherein the reference gene is the ACTB gene.

[0202] 33. A method according to any one of embodiments 31-32, wherein the reagent capable of specifically binding to the DNA region or fragment thereof where the reference gene is located includes primers and / or probes, and the primers and / or probes include the nucleic acid sequence shown in any one of SEQ ID NO.39-44.

[0203] 34. A method according to any one of embodiments 1-33, wherein the detection includes using fluorescent PCR to obtain a fluorescent Ct value.

[0204] 35. The method according to any one of embodiments 1-34 further includes: if one or more methylation levels in the test sample are detected to be positive, or one or more gene mutations are detected to be positive, the test result is considered to be positive; if the methylation levels in the test sample are detected to be negative, and the gene mutations are detected to be negative, the test result is considered to be negative.

[0205] 36. A reagent combination for confirming the presence or absence of urothelial carcinoma, assessing the risk of urothelial carcinoma development and / or assessing the progression of urothelial carcinoma, the reagent combination comprising a reagent capable of detecting the methylation level of a DNA region or fragment thereof where the VIM gene is located in a test sample, and a reagent capable of detecting gene mutations in one or more genes selected from TERT and FGFR3.

[0206] 37. The reagent combination according to embodiment 36, wherein the sample to be tested is from a tumor patient, a suspected tumor patient, a tumor-susceptible population, a tumor-high-risk population, or a healthy population.

[0207] 38. The reagent combination according to embodiment 37, wherein the tumor patients include patients who have not been treated with anti-cancer therapy and / or patients who have been treated with anti-cancer therapy.

[0208] 39. The reagent combination according to any one of embodiments 36-38, wherein the sample to be tested comprises tissue, cells and / or body fluid.

[0209] 40. The reagent combination according to any one of embodiments 36-39, wherein the test sample comprises urine.

[0210] 41. The reagent combination according to any one of embodiments 36-40, wherein the DNA region where the VIM gene is located comprises chr10:17270951-17271151, chr10:17271330-17271493, chr10:17271498-17271632 and / or chr10:17272405-17272436.

[0211] 42. The reagent combination according to any one of embodiments 36-41, wherein the reagent capable of detecting the methylation level of the DNA region or fragment thereof where the VIM gene is located in the test sample comprises primers and / or probes that can specifically recognize and / or bind to the DNA region or fragment thereof where the VIM gene is located.

[0212] 43. The reagent combination according to embodiment 42, wherein the primers and / or probes capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the VIM gene is located comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 13-27.

[0213] 44. The reagent combination according to any one of embodiments 42-43, wherein the primers and / or probes capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the VIM gene is located comprise nucleic acid sequences selected from any one of the following groups:

[0214] (1) Primers: SEQ ID NO. 13-14, probe: SEQ ID NO. 15;

[0215] (2) Primers: SEQ ID NO. 16-17, probe: SEQ ID NO. 18;

[0216] (3) Primers: SEQ ID NO. 19-20, probe: SEQ ID NO. 21;

[0217] (4) Primers: SEQ ID NO. 22-23, probe: SEQ ID NO. 24; and

[0218] (5) Primers: SEQ ID NO. 25-26, probe: SEQ ID NO. 27.

[0219] 45. The reagent combination according to any one of embodiments 36-44, wherein the genetic mutation of TERT comprises one or more selected from C250T and C228T.

[0220] 46. ​​The reagent combination according to embodiment 45 comprises primers and / or probes capable of specifically recognizing and / or binding to the TERT gene mutation C250T.

[0221] 47. The reagent combination according to embodiment 46, wherein the primers and / or probes capable of specifically recognizing and / or binding to the TERT gene mutation C250T comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 29-31.

[0222] 48. The reagent combination according to any one of embodiments 36-47, comprising primers and / or probes capable of specifically recognizing and / or binding to the TERT gene mutation C228T.

[0223] 49. The reagent combination according to embodiment 48, wherein the primers and / or probes capable of specifically recognizing and / or binding to the TERT gene mutation C228T comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 28, 30 and 31.

[0224] 50. The agent combination according to any one of embodiments 36-49, wherein the genetic mutation of FGFR3 comprises one or more selected from R248C and S249C.

[0225] 51. The reagent combination according to embodiment 50 comprises primers and / or probes capable of specifically recognizing and / or binding to the FGFR3 gene mutation R248C.

[0226] 52. The reagent combination according to embodiment 51, wherein the primers and / or probes capable of specifically recognizing and / or binding to the FGFR3 gene mutation R248C comprise the nucleic acid sequence shown in any one of SEQ ID NO. 32, 34 and 35.

[0227] 53. The reagent combination according to any one of embodiment 50, comprising primers and / or probes capable of specifically recognizing and / or binding to the FGFR3 gene mutation S249C.

[0228] 54. The reagent combination according to embodiment 53, wherein the primers and / or probes capable of specifically recognizing and / or binding to the FGFR3 gene mutation S249C comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 33-35.

[0229] 55. The reagent combination according to any one of embodiments 36-54, further comprising detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located in the test sample.

[0230] 56. The reagent according to embodiment 55, wherein the DNA region where the ONECUT2 gene is located comprises chr18:55107803-55107828, chr18:55108590-55108672, chr18:55108794-55108826 and / or chr18:55109012-55109014.

[0231] 57. The reagent combination according to any one of embodiments 55-56, wherein the reagent capable of detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located in the test sample comprises primers and / or probes that can specifically recognize and / or bind to the DNA region or fragment thereof where the ONECUT2 gene is located.

[0232] 58. The reagent combination according to embodiment 57, wherein the primers and / or probes capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the ONECUT2 gene is located comprise the nucleic acid sequence shown in any one of SEQ ID NOs. 1-12.

[0233] 59. The reagent combination according to any one of embodiments 55-58, wherein the primer and / or probe capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the ONECUT2 gene is located comprises a nucleic acid sequence selected from any one of the following groups:

[0234] (1) Primer: SEQ ID NO. 1-2, probe: SEQ ID NO. 3;

[0235] (2) Primers: SEQ ID NO. 4-5, probe: SEQ ID NO. 6;

[0236] (3) Primers: SEQ ID NO. 7-8, probe: SEQ ID NO. 9; and

[0237] (4) Primers: SEQ ID NO. 10-11, probe: SEQ ID NO. 12.

[0238] 60. The reagent combination according to any one of embodiments 36-59, further comprising a reagent capable of specifically binding to a DNA region or a fragment thereof where a reference gene is located.

[0239] 61. The reagent combination according to embodiment 60, wherein the reference gene is the ACTB gene.

[0240] 62. A reagent combination according to any one of embodiments 60-61, wherein the reagent capable of specifically binding to the DNA region or fragment thereof where the reference gene is located includes primers and / or probes, and the primers and / or probes include the nucleic acid sequence shown in any one of SEQ ID NO.39-44.

[0241] 63. A reagent combination according to any one of embodiments 36-62, which also includes a deamination reagent and / or a methylation-sensitive restriction endonuclease.

[0242] 64. The reagent combination of embodiment 63, wherein the deamination reagent comprises bisulfite.

[0243] 65. Use of the reagent combination described in any one of embodiments 37-64 in preparing a kit.

[0244] 66. A kit comprising the reagent combination of any one of embodiments 37-64.

[0245] 67. A model for confirming the presence or absence of urothelial carcinoma, assessing the risk of developing urothelial carcinoma and / or assessing the prognosis / progression of urothelial carcinoma, wherein the model is implemented based on the kit described in embodiment 66.

[0246] Table 2

[0247] Without intending to be bound by any theory, the following examples are merely intended to illustrate the gene marker combination, reagent combination, kit, detection method and uses of the present application, and are not intended to limit the scope of the invention of the present application.

[0248] Example

[0249] The primer and probe sequences used in the examples of this application are shown in Table 3 below:

[0250] Table 3

[0251] In the sequence, Y represents C or T, and R represents A or G.

[0252] Example 1 Non-diagnostic detection method for urothelial carcinoma

[0253] S1 extracts gDNA from urine samples and performs bisulfite conversion

[0254] 1. Extraction of gDNA from urine sediment

[0255] Extract gDNA from urine precipitate using a cell extraction kit purchased from Zymo, product model Quick-DNA Urine Kit (D3061). Specific steps are as follows:

[0256] Urine pretreatment: Centrifuge the collected 50 ml urine sample at 1600 g for 10 minutes. After centrifugation, discard the supernatant, add 1 ml of PBS buffer, mix thoroughly by pipetting, and transfer to a 1.5 ml centrifuge tube. Centrifuge the 1.5 ml tube containing the urine pellet at 12,000 g for 1 minute, and discard the waste liquid. Rinse the pellet obtained in 1.2 once with 1 ml of PBS buffer, centrifuge at 12,000 g for 1 minute, and discard the waste liquid. Repeat the washing process and discard the waste liquid. Add 200 μl of GA buffer (used for cell suspension) directly to the 1.5 ml centrifuge tube containing the pellet and mix thoroughly by pipetting. Add 20 μl of proteinase K and vortex to mix thoroughly. Add 200 μl of GB buffer (used for cell lysis and nucleic acid and protein separation), mix thoroughly by vortexing, and briefly centrifuge. Incubate at 70°C for 10 minutes. After incubation, add 200 μl of anhydrous ethanol and mix thoroughly by vortexing for 15 seconds. Apply the solution to the CB3 column and centrifuge at 12,000 g for 1 minute. Discard the liquid. Then, add 500 μl of GD rinse buffer (used to remove protein impurities) to the CB3 column and centrifuge at 12,000 rpm for 30 seconds. Discard the liquid. Then, add 600 μl of PW (used to remove salts) to the CB3 column and centrifuge at 12,000 rpm for 1 minute. Repeat this process. Centrifuge at 12,000 rpm for 2 minutes, discard the liquid, and aspirate the column. Then, incubate the CB3 column at room temperature for 5 minutes and air dry. Transfer the CB3 column to a clean 1.5 ml tube and add 53 μl of TE (used to dissolve DNA) to the column membrane. Incubate at room temperature for 5 minutes. Centrifuge at 12,000 g for 2 minutes and discard the liquid.

[0257] 2. Bisulfite conversion of urine precipitated gDNA

[0258] Urine precipitated gDNA was sulfite-converted using the zymo kit (EZ DNA Methylation-Lightning Kit (D3051) purchased from Tianmo Biotechnology). The specific steps are as follows:

[0259] Take 80ng of gDNA obtained in step 1 and add nuclease-free water (NFW) to 20ul, add 130ul of Lightning Conversion Reagent, shake and mix, and react under the following reaction conditions: 98℃ for 8min, then 54℃ for 60min, and maintain at 4℃. TM Add 600 μl of binding buffer (M-Binding Buffer) to the IC column, and transfer all the incubation solutions to the Zymo-Spin TM IC, invert 10 times to mix, centrifuge at 10000g for 1min, and discard the waste liquid. TM Add 100ul of M-Wash Buffer to the IC column, centrifuge at 10000g for 1min, and discard the waste liquid. TM Add 200ul of L-Desulphonation Buffer to the IC column, let it stand at room temperature for 15min, then centrifuge at 10000g for 30s and discard the waste liquid. TM Add 200ul of M-Wash Buffer to the IC column, centrifuge at 10000g for 1min, discard the waste liquid, and repeat once. TM The IC was placed in a new 1.5 ml centrifuge tube, 10 μl of M-Elution Buffer was added, and then centrifuged at 10000 g for 30 seconds. The sample was collected and used Qubit TM ssDNA detection kit for quantitative detection.

[0260] S2. Using the sulfite-converted gDNA as a template, the mixture is mixed with the primer probe set for the methylated gene fragment, the primer probe set for the point mutation site, and the primer probe set for the control gene to perform real-time fluorescence quantitative PCR.

[0261] Prepare a reaction mixture on ice using 12 μl of detection reaction mix, 9 μl of probe and primer mix, and 30 ng of template. (To ensure accurate reaction mixture preparation, prepare the detection reaction mix and probe and primer mix for each reaction (number of reactions + 2), then aliquot each mixture into individual reaction tubes. Finally, add the converted DNA sample.) Simultaneously with the test sample, add the positive control, negative control, and negative sample to determine if the experiment is qualified. Reaction conditions are shown in Table 4.

[0262] Table 4 PCR reaction conditions

[0263] S3. Analyze the data results.

[0264] After the reaction, the data were read and analyzed using the software provided with the Real-Time PCR Instrument 7500. The ΔCt values ​​for each methylated gene fragment and mutation site were obtained.

[0265] The reference method for reading the ΔCt value is:

[0266] I. Reading of PCR reaction results of methylation probe and primer mixture:

[0267] ① Baseline Setting: Manually set the baseline. The baseline's Start Cycle is typically set at 3, and the End Cycle is typically set at 15. ② Threshold Setting: Manually set the threshold, typically during the exponential phase of PCR amplification, at the inflection point of the amplification curve. ③ Ct Value Determination: After setting the baseline and fluorescence threshold, click the "Analyze" button to obtain the Ct value for each sample.

[0268] ΔCt value of methylated fragment = Ct value of methylated fragment - Ct value of reference gene.

[0269] According to Table 1 above, when the ΔCt value of the methylated fragment is ≤8, it is judged as positive, indicating that the target gene is hypermethylated; otherwise, the target gene is hypomethylated.

[0270] II. Reading the results of the PCR reaction mixture of point mutation probes and primers: ① Baseline Setup: Manually set the baseline. The ACTB-M baseline (Start Cycle) is generally set at 3, and the End Cycle is generally set at 15. ② Fluorescence Threshold Setup: Manually set the threshold, generally set during the exponential phase of PCR amplification, at the inflection point of the amplification curve. ③ Ct Value Determination: After setting the baseline and fluorescence threshold, click the "Analyze" button to obtain the Ct value for each sample.

[0271] ΔCt value of point mutation = Ct value of point mutation - Ct value of reference gene.

[0272] According to Table 1 above, when the ΔCt value of the point mutation is ≤11 or 12, it is determined that the site has a mutation, otherwise the site has no mutation.

[0273] Example 2 Detection results of two gene markers in early diagnosis of urothelial carcinoma

[0274] 252 suspected patients with urothelial carcinoma were collected (urine samples were collected from patients with no history of malignancy in the past five years and with microscopic or macroscopic hematuria. All patients in the pathology-positive group were pathologically confirmed by endoscopy, abdominal ultrasound, CT scan or abdominal and pelvic MRI. For patients in the gold standard-negative group, after endoscopic examination and imaging evaluation, the patients were excluded from the diagnosis of urothelial tumors), and the detection process of Example 1 was used for detection. After the experiment, the software of the instrument was used for analysis, and the threshold was adjusted to above the baseline fluorescence value so that the negative control Ct value did not show any numerical value, and the sample Ct value calculated by the automatic analysis of the instrument was recorded. A Ct with a test result of no detection or higher than 45 was considered to be below the detection limit, which was converted to 45, and then ΔCt was calculated (ΔCt = Ct of the detection gene - Ct of the internal reference gene ACTB).

[0275] Judgment Criteria: Gene marker combinations include gene mutations and methylation. If either or both of these are positive, the patient is considered to have a positive test result. For gene marker combinations that include mutations at more than one locus, if one or two loci are positive, the gene marker combination is considered mutation-positive.

[0276] Comparison with the gold standard cystoscopic pathology results revealed 131 positive cases and 121 negative results. The gene marker combinations used are shown in Table 5, and the test results are shown in Tables 6-1 to 6-5 and Table 7. The results showed that the experimental group had higher sensitivity and specificity than the control group, exceeding 80%.

[0277] Table 5 Two-gene marker combination

[0278] Table 6-1 Test results of experimental group 1

[0279] Table 6-2 Test results of experimental group 2

[0280] Table 6-3 Test results of experimental group 3

[0281] Table 6-4 Test results of control group 1

[0282] Table 6-5 Test results of control group 2

[0283] Table 7 Summary of the test results of two-gene combination for early diagnosis

[0284] Example 3 Detection results of two gene markers in monitoring recurrence of urothelial carcinoma

[0285] Forty-seven patients with suspected recurrence of urothelial carcinoma were collected and tested using the method of Example 2. The results were compared with the gold standard cystoscopic pathological examination. The gene marker combination used is shown in Table 5, and the test results are shown in Tables 8-1, 8-2, 8-3, 8-4, and 9. The results showed that compared with the control group, the test group had higher sensitivity and specificity, exceeding 80%.

[0286] Table 8-1 Test results of experimental group 1

[0287] Table 8-2 Test results of experimental group 2

[0288] Table 8-3 Test results of control group 1

[0289] Table 8-4 Test results of control group 2

[0290] Table 9 Summary of the results of the two-gene combination test for recurrence monitoring

[0291] Example 4 Detection results of three gene markers in early diagnosis of urothelial carcinoma

[0292] 252 suspected patients with urothelial carcinoma were collected (urine samples were collected from patients with no history of malignancy in the past five years and with microscopic or macroscopic hematuria. All patients in the pathology-positive group underwent endoscopy, abdominal ultrasound, CT scan or abdominal and pelvic MRI examination, and pathological confirmation was obtained. For patients in the gold standard-negative group, after endoscopic examination and imaging evaluation, the patients were excluded from the diagnosis of urothelial tumors). The detection process of Example 1 was used for detection. After the experiment, the software of the instrument was used for analysis, and the threshold was adjusted to above the baseline fluorescence value so that the negative control Ct value did not show any numerical value. The sample Ct value calculated by the automatic analysis of the instrument was recorded. A Ct with a test result of no detection or higher than 45 was considered to be below the detection limit, converted to 45, and then ΔCt was calculated (ΔCt = Ct of the detection gene - Ct of the internal reference gene ACTB).

[0293] Judgment Criteria: Gene marker combinations include gene mutations and methylation. If either or both of these are positive, the patient is considered to have a positive test result. For gene marker combinations that include mutations at more than one locus, if one or two loci are positive, the gene marker combination is considered mutation-positive.

[0294] Compared with the gold standard cystoscopic pathology results, cystoscopic pathology results were positive in 131 cases and negative in 121. The gene marker combinations used are shown in Table 10, and the test results are shown in Tables 11-1, 11-2, and 12. The results showed that the detection sensitivity and specificity of experimental groups 1 and 2 were both high, exceeding 70%, and that of experimental group 1 was above 85%.

[0295] Table 10 Three-gene marker combination

[0296] Table 11-1 Test results of experimental group 1

[0297] Table 11-2 Test results of experimental group 2

[0298] Table 12 Summary of the results of the three-gene combination test for early diagnosis

[0299] Example 5 Detection results of six gene markers in early diagnosis of urothelial carcinoma

[0300] 252 suspected patients with urothelial carcinoma were collected (urine samples were collected from patients with no history of malignancy in the past five years and with microscopic or macroscopic hematuria. All patients in the pathology-positive group underwent endoscopy, abdominal ultrasound, CT scan or abdominal and pelvic MRI examination, and pathological confirmation was obtained. For patients in the gold standard-negative group, after endoscopic examination and imaging evaluation, the patients were excluded from the diagnosis of urothelial tumors). The detection process of Example 1 was used for detection. After the experiment, the software of the instrument was used for analysis, and the threshold was adjusted to above the baseline fluorescence value so that the negative control Ct value did not show any numerical value. The sample Ct value calculated by the automatic analysis of the instrument was recorded. A Ct with a test result of no detection or higher than 45 was considered to be below the detection limit, converted to 45, and then ΔCt was calculated (ΔCt = Ct of the detection gene - Ct of the internal reference gene ACTB).

[0301] Judgment Criteria: Gene marker combinations include gene mutations and methylation. If either or both of these are positive, the patient is considered to have a positive test result. For gene marker combinations that include mutations at more than one locus, if one or two loci are positive, the gene marker combination is considered mutation-positive.

[0302] Comparison with the gold standard cystoscopic pathology results revealed 131 positive cases and 121 negative results. The gene marker combinations used are shown in Table 13, and the test results are shown in Tables 14-1, 14-2, and 15. The results showed that compared with the control group, the sensitivity and specificity of experimental group 1 were both higher, exceeding 90%, while the sensitivity and specificity of the control group were both below 70%.

[0303] Table 13 Six-gene marker combination

[0304] Table 14-1 Test results of experimental group 1

[0305] Table 14-2 Test results of control group 1

[0306] Table 15 Summary of the test results of the six-gene combination for early diagnosis

[0307] Example 6 Detection Results of Six Gene Markers in Monitoring Recurrence of Urothelial Carcinoma

[0308] Forty-seven patients with suspected recurrence of urothelial carcinoma were collected and tested using the method of Example 5. The results were compared with the gold standard cystoscopic pathological examination. The gene marker combination used is shown in Table 13, and the test results are shown in Tables 16-1, 16-2, and 17. The results showed that compared with the control group, the test group had higher sensitivity and specificity, exceeding 90%, while the control group had less than 60%.

[0309] Table 16-1 Test results of experimental group 1

[0310] Table 16-2 Test results of control group 1

[0311] Table 17 Summary of the test results of the six-gene combination for early diagnosis

[0312] Example 7 Detection results of different primer / probe sequences in six gene combinations

[0313] Based on the multiple methylation regions of VIM or ONECUT2, different primer and probe combinations were designed. Urine samples from 252 suspected urothelial carcinoma patients and 47 recurrent patients were tested according to the methods of Example 5 or Example 6. The primer and probe combinations for the six-gene marker panel were the same as in Example 5, except that the primer probes for VIM or ONECUT2 methylation were replaced with the primer probes listed in Table 18. The test results are shown in Table 19. The results showed that the six-gene detection sensitivity and specificity were both high, exceeding 85%, for different methylation regions.

[0314] Table 18

[0315] Table 19

[0316] Example 8 Detection Results of Six Gene Markers in Early Diagnosis of Urothelial Carcinoma

[0317] A total of 529 suspected patients with urothelial carcinoma (296 of whom were pathologically diagnosed as positive and 233 of whom were pathologically diagnosed as negative) were collected and tested using the method of Example 5. The gene marker combinations used are shown in Table 13, and the test results are shown in Table 20. The results showed that the six-gene test experimental group had high sensitivity and specificity, exceeding 85%.

[0318] Table 20 Test results of experimental group

[0319] The sensitivity and specificity of the detection results of this application are: sensitivity = 258 / 296 = 87.2%, specificity = 200 / 233 = 85.8%.

Claims

1. Use of a reagent combination in preparing a kit, wherein: The reagent combination comprises a reagent capable of detecting the methylation level of a DNA region or a fragment thereof where the VIM gene is located in a sample to be tested, and a reagent capable of detecting gene mutations of one or more genes selected from TERT and FGFR3. The kit is used to confirm the presence or absence of urothelial carcinoma, assess the risk of urothelial carcinoma formation and / or assess the progression of urothelial carcinoma, and the DNA region where the VIM gene is located comprises any one of the following groups: chr10: 17270851-17271251, chr10: 17271245-17271593, chr10: 17271398-17271732 and chr10: 17272305-17272536.

2. The use according to claim 1, wherein the sample to be tested is from a tumor patient, a suspected tumor patient, a tumor-susceptible population, a tumor-high-risk population or a healthy population.

3. The use according to claim 2, wherein the tumor patients include patients who have not been treated with anti-cancer therapy and / or patients who have been treated with anti-cancer therapy.

4. The use according to any one of claims 1 to 3, wherein the sample to be tested comprises tissue, cells and / or body fluid.

5. The use according to any one of claims 1 to 4, wherein the sample to be tested comprises urine.

6. The use according to any one of claims 1 to 5, wherein the reagent capable of detecting the methylation level of the DNA region or fragment thereof where the VIM gene is located in the sample to be tested comprises primers and / or probes capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the VIM gene is located.

7. The use according to claim 6, wherein the primer and / or probe capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the VIM gene is located comprises the nucleic acid sequence shown in any one of SEQ ID NOs. 13-27.

8. The use according to any one of claims 6-7, wherein the primers and / or probes capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the VIM gene is located comprise any one of the following nucleic acid sequences: (1) Primers: SEQ ID NO.13-14, probe: SEQ ID NO.15; (2) Primers: SEQ ID NO.16-17, probe: SEQ ID NO.18; (3) Primers: SEQ ID NO.19-20, probe: SEQ ID NO.21; (4) Primers: SEQ ID NO. 22-23, probe: SEQ ID NO. 24; and (5) Primers: SEQ ID NO.25-26, probe: SEQ ID NO.

27.

9. The use according to any one of claims 1 to 8, wherein the gene mutation of TERT comprises one or more selected from C250T and C228T.

10. The use according to claim 9, wherein the reagent combination comprises primers and / or probes capable of specifically recognizing and / or binding to the TERT gene mutation C250T.

11. The use according to claim 10, wherein the primer and / or probe capable of specifically recognizing and / or binding to the TERT gene mutation C250T comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 29-31.

12. The use according to any one of claims 1 to 11, wherein the reagent combination comprises primers and / or probes that can specifically recognize and / or bind to the TERT gene mutation C228T.

13. The use according to claim 12, wherein the primer and / or probe capable of specifically recognizing and / or binding to the TERT gene mutation C228T comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 28, 30 and 31.

14. The use according to any one of claims 1 to 13, wherein the gene mutation of FGFR3 comprises one or more selected from R248C and S249C.

15. The use according to claim 14, wherein the reagent combination comprises primers and / or probes capable of specifically recognizing and / or binding to the FGFR3 gene mutation R248C.

16. The use according to claim 15, wherein the primer and / or probe capable of specifically recognizing and / or binding to the FGFR3 gene mutation R248C comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 32, 34 and 35.

17. The use according to any one of claims 1 to 16, wherein the reagent combination comprises primers and / or probes that can specifically recognize and / or bind to the FGFR3 gene mutation S249C.

18. The use according to claim 17, wherein the primer and / or probe capable of specifically recognizing and / or binding to the FGFR3 gene mutation S249C comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 33-35.

19. The use according to any one of claims 1 to 18, wherein the reagent combination further comprises detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located in the sample to be tested.

20. The use according to claim 19, wherein the DNA region where the ONECUT2 gene is located comprises any one of the following groups: chr18: 55107703-55107928, chr18: 55108490-55108772, chr18: 55108694-55108926 and / or chr18: 55108912-55109114.

21. The use according to any one of claims 19-20, wherein the reagent capable of detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located in the sample to be tested comprises a primer and / or a probe capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the ONECUT2 gene is located.

22. The use according to claim 21, wherein the primer and / or probe capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the ONECUT2 gene is located comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 1-12.

23. The use according to any one of claims 19 to 22, wherein the primer and / or probe capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the ONECUT2 gene is located comprises a nucleic acid sequence selected from any one of the following groups: (1) Primer: SEQ ID NO.1-2, probe: SEQ ID NO.3; (2) Primers: SEQ ID NO. 4-5, probe: SEQ ID NO. 6; (3) Primers: SEQ ID NO. 7-8, probe: SEQ ID NO. 9; and (4) Primers: SEQ ID NO.10-11, probe: SEQ ID NO.

12.

24. The use according to any one of claims 1 to 23, further comprising a reagent capable of specifically binding to the DNA region or fragment thereof where the reference gene is located. The use according to claim 24 , wherein the reference gene is the ACTB gene.

26. The use according to any one of claims 24-25, wherein the reagent capable of specifically binding to the DNA region or fragment thereof where the reference gene is located comprises a primer and / or a probe, and the primer and / or the probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 39-44.

27. The method according to any one of claims 1 to 26, further comprising a deaminating agent and / or a methylation-sensitive restriction endonuclease.

28. Use according to claim 27, wherein the deamination agent comprises bisulfite.

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