A gene marker combination, kit and detection method

Abstract

The present application relates to the field of biomedicine, and specifically to a gene marker combination, detection method, kit and detection model for urothelial carcinoma. The present application uses a reagent combination for confirming the presence of urothelial carcinoma, assessing the risk of urothelial carcinoma and / or assessing the prognosis / progression of urothelial carcinoma, and the reagent combination comprises a reagent capable of detecting the methylation level of the DNA region or its fragment where the VIM gene and the ONECUT2 gene are located in the sample to be tested, as well as the gene mutations of TERT and FGFR3.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of September 11, 2023, application number 202311160819.X, and invention name “A genetic marker combination, a kit and a detection method”. Technical Field

[0002] The present application relates to the field of biomedicine, and specifically to a detection method, a kit and a detection model for urothelial carcinoma. Background Art

[0003] Among many cancers, the incidence of urinary system tumors is increasing year by year, so the demand for accurate diagnosis and treatment of urinary system tumors is also further expanding. Urothelial carcinoma (UC) includes upper urinary tract urothelial carcinoma (UTUC) and lower urinary tract urothelial carcinoma (mainly bladder cancer, BC), which has the characteristics of high incidence, multiple primary sites, and easy recurrence after surgery. The commonly used clinical bladder cancer examination methods currently include cystoscopy (biopsy of suspected lesions), urine exfoliated cytology, imaging examination, and fluorescence in situ hybridization.

[0004] Clinical diagnosis of UC and post-treatment monitoring mainly rely on imaging, urine testing, endoscopy and biopsy. Traditional imaging examinations are difficult for positioning and qualitative diagnosis of small or atypical lesions. Urine testing is widely used in clinical practice as an important auxiliary diagnostic method, but there are 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.

[0005] In clinical practice, the early diagnosis and monitoring of UC are still not effective. For example, the accuracy of CT examination for muscle-invasive bladder cancer (MIBC) is only 54.9%; urine cytology and fluorescence in situ hybridization (FISH) have low sensitivity and are easily affected by urinary tract infection, cystitis, stones, and hematuria; cystoscopy is the gold standard for the diagnosis of bladder cancer, but due to its invasive disadvantages, patients have psychological resistance, making compliance with this method impossible to guarantee, and carcinoma in situ is not easy to detect under cystoscopy and is easily confused with inflammatory changes in the bladder, requiring biopsy for confirmation.

[0006] Early diagnosis of upper tract urothelial carcinoma (UTUC) is still very difficult. When UTUC is highly suspected but imaging examinations cannot be clear, 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 by microscopic examination. The causes of obstruction include ureteral stones, stenosis of the ureteropelvic junction, 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 probability of exudation and adhesion of surrounding tissues after ureteroscopy is significantly increased, and the difficulty of radical surgery will increase, so it should not be carried out easily.

[0007] Therefore, it is necessary to provide a non-invasive, highly sensitive and specific detection product for accurate early diagnosis and prognosis of urothelial carcinoma. 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 methylated gene fragments as detection indicators for early diagnosis of urothelial carcinoma and determination of prognosis. 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. The sensitivity is 16%: detection kits with simple methylation or mutation markers have the risk of missed detection; and the detection method of the present application uses point mutations and methylation multi-omics together, 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 conducive to patients receiving 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] 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 its fragment where the VIM gene is located in the sample to be tested, the methylation level of the DNA region or its fragment where the ONECUT2 gene is located, and the gene mutation of one or more genes selected from TERT and FGFR3. 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.

[0012] In certain embodiments, 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.

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

[0016] In certain embodiments, the gene mutation of FGFR3 includes one or more selected from R248C and S249C. When the FGFR3 R248C point mutation mentioned in the present application is referred to in the art, it may also be referred to as Chr4:1803564C>T (GRCh37), or "NM_000142 (FGFR3): c.742C>T (p.R248C)", and when the FGFR3 S249C point mutation mentioned in the present application is referred to in the art, it may also be referred to 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 so that bases having methylation and bases not having methylation form different substances after conversion.

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

[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 as 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 as 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 as 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 the FGFR3 gene mutation.

[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 as 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 as 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 the 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 some embodiments, the detection includes using fluorescent PCR to obtain a fluorescent Ct value. In some embodiments, if one or more methylation levels in the sample to be tested are detected to be positive, or one or more gene mutations are detected to be positive, the test result is considered positive; if the methylation levels in the sample to be tested are all negative, and the gene mutations are all 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, the reagent combination comprising 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 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, the reagent combination comprising 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, 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 patients include patients who have not been treated with anti-cancer therapy and / or patients who have 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 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.

[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 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.

[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 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 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 include primers and / or probes selected from any one of the following groups: (1) primers: SEQ ID NO.1-2, probes: SEQ ID NO.3; (2) primers: SEQ ID NO.4-5, probes: SEQ ID NO.6; (3) primers: SEQ ID NO.7-8, probes: SEQ ID NO.9; (4) primers: SEQ ID NO.10-11, probes: SEQ ID NO.12 and primers: SEQ ID NO.36-37, probes: SEQ ID NO.38.

[0070] In certain embodiments, the reagent combination further includes a reagent that can specifically bind 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 the 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 agent 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 perceive 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 is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art 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, which may also be referred to as CTRCT30 or HEL113. The human VIM gene is located in Chromosome 10:17,270,258-17,279,592 forward chain (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 a gene encoding transcription factor onecut family member 2. The location of human ONECUT2 is in 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 in Chromosome5: 1,253,262-1,295,184 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 in Chromosome 4: 1,795,034-1,810,599 forward chain (GRCh37: CM000666.1). For more information about the human FGFR3 gene, refer to ensemble number ENSG00000068078. The point mutations of 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 the present application, the term "sample to be tested" generally refers to a sample to be tested. For example, one or more gene regions on the sample to be tested may be tested for modification or mutation.

[0087] In this application, the term "healthy population" generally refers to a population in which no tumor is currently detected by tumor detection methods. Healthy population can develop into tumor patients, tumor high-risk population, tumor suspected patients or tumor susceptible population. "Healthy population" is a concept relative to "tumor patients", and "tumor patients" generally refer to people who have been confirmed to have tumors through pathological diagnosis and other detection methods.

[0088] In this application, the term "tumor high-risk population" generally refers to a population with a higher risk of developing tumors. For example, people with a family history of tumors, people carrying tumor susceptibility genes, people with unhealthy lifestyles (such as smokers, alcoholics, people who often stay up late, etc.), and people who are often exposed to toxic and harmful substances in their living or working environment.

[0089] In this application, the term "suspected tumor patients" generally refers to people who have tumor symptoms but have not been confirmed to have a tumor 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 tumors, for example, a population with a family history of tumors, a population carrying tumor-susceptible genes, a population with an unhealthy lifestyle (such as smokers, alcoholics, and those who often stay up late), or a population that is often exposed to toxic and harmful substances in their living or working environment.

[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 who have 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 Chinese herbal medicines.

[0092] In the present 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 may refer to the position of a sequence of a specific deoxyribonucleotide at which the gene is located, such as a sequence of the deoxyribonucleotides encoding the gene. For example, the DNA region of the present application comprises the full length of the DNA region, its complementary region, or the above-mentioned fragment. For example, a sequence of at least about 20 kb upstream and downstream of the detection region provided in the present application can be used as a site for detection. For example, a sequence of 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 the present application can be used as a site for detection. For example, suitable primers and probes can be designed according to the site for methylation detection of a sample. In the present application, when it comes to a DNA region or a base position, if not otherwise specified, it generally refers to the human reference genome version GRCh37, and the UCSC corresponding version number hg19.

[0093] In the present application, the term "methylation" generally refers to the methylation state of a gene fragment, nucleotide or base thereof in the present application. For example, a DNA fragment in which a gene is located in the present application may be methylated on one strand or multiple strands. For example, a DNA fragment in which a gene is located in the present application may be methylated on one site or multiple 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 the present application, the term "conversion" generally refers to converting one or more structures into another structure. For example, the conversion of the present application can be specific. For example, a cytosine without a methylation modification can be converted into another structure (e.g., uracil), and a cytosine with a methylation modification can be substantially unchanged after conversion. For example, a cytosine without a methylation modification can be sheared after conversion, and a cytosine with a methylation modification can be substantially unchanged after conversion.

[0095] In the present 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 the present application, the term "bisulfite" generally refers to a reagent that can distinguish between a DNA region with a modification state and a region without 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 the present 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 a reagent that can also distinguish between a DNA region with a modification state and a region without 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 a sequence containing a CG dinucleotide (e.g., cgcg or cccggg).

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

[0099] In this application, the term "progression" generally refers to the change of 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 cancer cell metastasis, the rate at which the cancer grows or spreads, etc. For example, tumor progression can include the stage period of such cancer from a less severe state to a more severe state, such as the progression 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 the present application, the term "fluorescent PCR" generally refers to a quantitative or semi-quantitative PCR technique. For example, it can be a PCR technique of real-time quantitative polymerase chain reaction, quantitative polymerase chain reaction or kinetic polymerase chain reaction. For example, PCR amplification can be used and the initial target nucleic acid amount can be quantitatively detected by means of an intercalating fluorescent dye or a sequence-specific probe, and the sequence-specific probe can contain a fluorescent reporter molecule that can only be detected 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 the present application, the term "fluorescence Ct value" generally refers to a measurement value for quantitatively or semi-quantitatively evaluating a target nucleic acid. For example, it may refer to the number of amplification reaction cycles experienced when the fluorescence signal reaches a set threshold. DETAILED DESCRIPTION OF THE INVENTION

[0105] Gene marker combination

[0106] The present application provides a combination of gene markers, which can be used to confirm whether urothelial carcinoma exists, assess the risk of urothelial carcinoma, and / or assess the prognosis / progress of urothelial carcinoma. Thus, the present application provides a method for detecting and confirming whether urothelial carcinoma exists, assessing the risk of urothelial carcinoma, and / or assessing the prognosis / progress 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, and a kit comprising 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, it is possible to confirm with high sensitivity and specificity whether urothelial carcinoma exists, assess the risk of urothelial carcinoma occurrence, and / or assess the prognosis / progression of urothelial carcinoma.

[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, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations or more selected from the following table. In certain embodiments, 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 following group: TERT, FGFR and other gene mutations listed in Table 2 below, for example, 1 other mutation, 2 other mutations, 3 other mutations, 4 other mutations or more selected from Table 2 below.

[0109] In the present application, the gene marker combination may include the methylation level of the DNA region where the VIM gene is located or a fragment thereof. 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 about 100bp 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 about 100bp 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 about 100bp 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 about 100bp 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 about 100bp upstream and downstream thereof. The presence of urothelial carcinoma, the risk of urothelial carcinoma, and / or the prognosis / progression of urothelial carcinoma can be confirmed by detecting the methylation level of the DNA region where the VIM gene is located. If the methylation level of the DNA region where the VIM gene is located is positive, it can be considered that urothelial carcinoma exists, the risk of urothelial carcinoma is high, and / or the risk of recurrence of urothelial carcinoma 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 urothelial carcinoma, the risk of urothelial carcinoma, and / or the prognosis / progression of urothelial carcinoma can be confirmed by detecting the presence of a TERT gene mutation. 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 mutations, it can be considered that urothelial carcinoma exists, 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 mutation, for example, FGFR3 gene mutation. FGFR3 gene mutation may include gene mutations that cause R248C and / or S249C amino acid mutations. For example, FGFR3 gene mutation may include point mutations that cause R248C, for example, C742T point mutations. For example, FGFR3 gene mutation may include point mutations that cause S249C, for example, C746G point mutations. It is possible to confirm whether urothelial carcinoma exists, assess the risk of urothelial carcinoma occurrence, and / or assess the prognosis / progress of urothelial carcinoma by detecting whether FGFR3 gene mutation exists. If FGFR3 gene mutation is detected in the sample to be tested, particularly the presence of R248C and / or S249C mutations, it can be considered that urothelial carcinoma exists, the risk of urothelial carcinoma occurrence 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 about 100bp 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 about 100bp 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 about 100bp 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 about 100 bp upstream and downstream thereof. The methylation level of the DNA region where the ONECUT2 gene is located can be detected to confirm whether urothelial carcinoma exists, assess the risk of urothelial carcinoma, and / or assess the prognosis / progression of urothelial carcinoma. If the methylation level of the ONECUT2 region where the VIM gene is located is positive, it can be considered that urothelial carcinoma exists, the risk of urothelial carcinoma is high, and / or the risk of recurrence of urothelial carcinoma 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, TERTC250T, 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 a 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 or fragment thereof where the VIM gene is located and the TERT promoter point mutation.

[0117] 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 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 a 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 or fragment thereof where the VIM gene is located, 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 a 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 a 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 a 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 a 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 a 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 a fragment thereof where the VIM gene is located, the methylation level of the DNA region or a 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 its fragment where the VIM gene is located, the methylation level of the DNA region or its fragment 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 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.

[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 its fragment where the VIM gene is located, the methylation level of the DNA region or its fragment 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 its fragment where the VIM gene is located, the methylation level of the DNA region or its fragment 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 its fragment where the VIM gene is located, the methylation level of the DNA region or its fragment 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 have 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, it is possible to confirm the presence of urothelial carcinoma, assess the risk of urothelial carcinoma, and / or assess the prognosis / progression of urothelial carcinoma with high sensitivity and specificity.

[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 FGFR3R248C point mutation, and a reagent for detecting the FGFR3 S249C point mutation.

[0140] Gene marker detection method

[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 method, 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. Primers and fluorescent dyes can also be used for detection.

[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 NO.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 NO.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 is 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 mentioned above may include the nucleic acid sequences shown in SEQ ID NO.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 mentioned above may include the nucleic acid sequences shown in SEQ ID NO.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 mentioned above may include the nucleic acid sequences shown in SEQ ID NO.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 above VIM may include the nucleic acid sequences shown in SEQ ID NO.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 above 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, 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 gene mutations, 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 may 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 include the nucleic acid sequences shown in SEQ ID NO.29-30, and the probe may include 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 include the nucleic acid sequences shown in SEQ ID NOs. 32 and 34, and the probe may include the nucleic acid sequence shown in SEQ ID NO. 35.

[0160] For example, the primers for detecting FGFR3 point mutations may include the nucleic acid sequences shown in SEQ ID NO.33-34, and the probe may include 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) in 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 result. 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 concentration 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 the gene marker in the combination may be about 2-15 μM. For another example, the concentration of the primer and / or probe mixture for the gene marker in the combination may be about 6-12 μM. For another example, the concentration of the primer and / or probe mixture for the gene marker in the combination may be about 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 combination of gene markers, 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 according to the ΔCt value can be that when the ΔCt value of the methylated 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 the site has a mutation, otherwise the site has no mutation. 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] Gene △Ct Genetic testing results ONECUT2 ≤8 Positive VIM ≤8 Positive FGFR3-R248C ≤12 Positive TERT-C250T ≤12 Positive FGFR3-S249C ≤11 Positive TERT-C228T ≤11 Positive

[0166] In the present application, the scoring value of the sample to be tested, such as the UC-Score value, can also be calculated by a scoring model to determine whether the sample to be tested is negative or positive. If the sample UC-Score>0, the sample to be tested is judged to be positive; if UC-Score≦0, the sample to be tested is judged to be negative. Those skilled in the art can obtain a suitable UC-Score scoring model by analyzing the sample library data.

[0167] 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.

[0168] On the other hand, the present application provides the use of the 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 introducing the method of use. In the kit, the primers and / or probes for detecting each of the gene markers, and other required reagents may be placed in the same container or in different containers.

[0169] In another aspect, the present application provides a program capable of executing the method.

[0170] On the other hand, the present application provides the following implementation modes:

[0171] 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 a fragment thereof where a VIM gene is located in a sample to be tested, and detecting gene mutations of one or more genes selected from TERT and FGFR3.

[0172] 2. According to the method described in embodiment 1, 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.

[0173] 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.

[0174] 4. The method according to any one of embodiments 1-3, wherein the sample to be tested comprises tissue, cells and / or body fluid.

[0175] 5. The method according to any one of embodiments 1-4, wherein the sample to be tested comprises urine.

[0176] 6. The method according to any one of embodiments 1-5, wherein the gene mutation of TERT comprises a mutation selected from C250T and

[0177] One or more of C228T.

[0178] 7. The method according to any one of embodiments 1-6, wherein the gene mutation of FGFR3 comprises a mutation selected from R248C and

[0179] One or more of S249C.

[0180] 8. The method according to any one of embodiments 1-7, wherein the DNA region where the VIM gene is located comprises

[0181] chr10:17270951-17271151, chr10:17271330-17271493, chr10:17271498-17271632 and / or

[0182] chr10:17272405-17272436.

[0183] 9. A method according to any one of embodiments 1-8, wherein the method comprises obtaining nucleic acid in the sample to be tested.

[0184] 10. The method according to embodiment 9, wherein the method comprises converting the nucleic acid so that bases with methylation and bases without methylation form different substances after conversion.

[0185] 11. A method according to embodiment 10, wherein the conversion comprises conversion by a deamination agent and / or a methylation-sensitive restriction endonuclease.

[0186] 12. The method of embodiment 11, wherein the deamination reagent comprises bisulfite.

[0187] 13. A method according to any one of embodiments 1 to 12, wherein 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.

[0188] 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 a fragment thereof where the VIM gene is located.

[0189] 15. The method according to embodiment 14, wherein the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NO.13-27.

[0190] 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 sample to be tested.

[0191] 17. The method according to embodiment 16, wherein the DNA region where the ONECUT2 gene is located comprises

[0192] chr18:55107803-55107828, chr18:55108590-55108672, chr18:55108794-55108826 and / or

[0193] chr18:55109012-55109014.

[0194] 18. The method according to any one of embodiments 16-17, wherein 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.

[0195] 19. The method according to embodiment 18, wherein the reagent comprises a

[0196] Primers and / or probes for the DNA region or fragment thereof where the ONECUT2 gene is located.

[0197] 20. The method according to embodiment 19, wherein the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NO.1-12.

[0198] 21. The method according to any one of embodiments 1-20, wherein the method comprises contacting the sample to be tested with a reagent that can specifically recognize and / or bind to a TERT gene mutation.

[0199] 22. A method according to embodiment 21, wherein the reagent comprises primers and / or probes capable of specifically recognizing and / or binding to the TERT gene mutation C250T.

[0200] 23. A method according to embodiment 22, wherein the primers and / or probes comprise a nucleic acid sequence shown in any one of SEQ ID NOs. 29-31.

[0201] 24. A 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.

[0202] 25. A method according to embodiment 24, wherein the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 28, 30 and 31.

[0203] 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.

[0204] 27. A method according to embodiment 26, wherein the reagent comprises primers and / or probes capable of specifically recognizing and / or binding to the FGFR3 gene mutation R248C.

[0205] 28. A method according to embodiment 27, wherein the primer and / or probe comprises a nucleic acid sequence shown in any one of SEQ ID NOs. 32, 34 and 35.

[0206] 29. A method according to embodiment 26, wherein the reagents include primers and / or probes that can specifically recognize or bind to the FGFR3 gene mutation S249C.

[0207] 30. A method according to embodiment 29, wherein the primers and / or probes comprise a nucleic acid sequence as shown in any one of SEQ ID NOs. 33-35.

[0208] 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.

[0209] 32. A method according to embodiment 31, wherein the reference gene is the ACTB gene.

[0210] 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.

[0211] 34. A method according to any one of embodiments 1-33, wherein the detection includes using fluorescent PCR to obtain a fluorescent Ct value.

[0212] 35. According to the method described in any one of embodiments 1-34, it also 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.

[0213] 36. 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, the reagent combination comprising 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 in one or more genes selected from TERT and FGFR3.

[0214] 37. A 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.

[0215] 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.

[0216] 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.

[0217] 40. The reagent combination according to any one of embodiments 36-39, wherein the sample to be tested comprises urine.

[0218] 41. The reagent combination according to any one of embodiments 36-40, wherein the DNA region where the VIM gene is located includes chr10:17270951-17271151, chr10:17271330-17271493, chr10:17271498-17271632 and / or chr10:17272405-17272436.

[0219] 42. A 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 sample to be tested 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.

[0220] 43. The reagent combination according to embodiment 42, 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 a nucleic acid sequence shown in any one of SEQ ID NOs. 13-27.

[0221] 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:

[0222] (1) Primers: SEQ ID NO.13-14, probe: SEQ ID NO.15;

[0223] (2) Primers: SEQ ID NO.16-17, probe: SEQ ID NO.18;

[0224] (3) Primers: SEQ ID NO.19-20, probe: SEQ ID NO.21;

[0225] (4) Primers: SEQ ID NO. 22-23, probe: SEQ ID NO. 24; and

[0226] (5) Primers: SEQ ID NO.25-26, probe: SEQ ID NO.27.

[0227] 45. The reagent combination according to any one of embodiments 36-44, wherein the genetic mutation of TERT includes one or more selected from C250T and C228T.

[0228] 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.

[0229] 47. A 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.

[0230] 48. A 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.

[0231] 49. A 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.

[0232] 50. The agent combination according to any one of embodiments 36-49, wherein the genetic mutation of FGFR3 includes one or more selected from R248C and S249C.

[0233] 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.

[0234] 52. A 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 NOs. 32, 34 and 35.

[0235] 53. A 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.

[0236] 54. A 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.

[0237] 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 sample to be tested.

[0238] 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.

[0239] 57. A 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 sample to be tested 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.

[0240] 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.

[0241] 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:

[0242] (1) Primer: SEQ ID NO.1-2, probe: SEQ ID NO.3;

[0243] (2) Primers: SEQ ID NO. 4-5, probe: SEQ ID NO. 6;

[0244] (3) Primers: SEQ ID NO. 7-8, probe: SEQ ID NO. 9; and

[0245] (4) Primers: SEQ ID NO.10-11, probe: SEQ ID NO.12.

[0246] 60. A reagent combination according to any one of embodiments 36-59, which also includes a reagent that can specifically bind to the DNA region or a fragment thereof where the reference gene is located.

[0247] 61. A reagent combination according to embodiment 60, wherein the reference gene is the ACTB gene.

[0248] 62. A reagent combination according to any one of embodiments 60-61, wherein the reagent capable of specifically binding to the DNA region or a 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 SEQID NO.39-44.

[0249] 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.

[0250] 64. A reagent combination according to embodiment 63, wherein the deamination reagent comprises bisulfite.

[0251] 65. Use of the reagent combination described in any one of embodiments 37-64 in preparing a kit.

[0252] 66. A kit comprising the reagent combination described in any one of embodiments 37-64.

[0253] 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.

[0254] Table 2

[0255]

[0256]

[0257]

[0258]

[0259] 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 use of the present application, and are not intended to limit the scope of the invention of the present application.

[0260] Example

[0261] The primer and probe sequences used in the examples of this application are shown in Table 3 below:

[0262] Table 3

[0263]

[0264]

[0265]

[0266] In the sequence, Y represents C or T, and R represents A or G.

[0267] Example 1 Non-diagnostic detection method for urothelial carcinoma

[0268] S1 Extraction and bisulfite conversion of gDNA from urine samples

[0269] 1. Extraction of gDNA from urine sediment

[0270] The gDNA from urine precipitation was extracted using a cell extraction kit purchased from zymo, product model Quick-DNA Urine Kit (D3061). The specific steps are as follows:

[0271] Urine pretreatment: 50 ml of urine sample was collected and centrifuged at 1600 g for 10 min. After centrifugation, the supernatant was discarded, 1 ml of PBS buffer was added, and the mixture was mixed and transferred to a 1.5 ml centrifuge tube. The 1.5 ml tube containing the urine precipitate was centrifuged at 12000 g for 1 min, and the waste liquid was discarded. 1 ml of PBS buffer was added to rinse the precipitate obtained in 1.2 once, and the precipitate was centrifuged at 12000 g for 1 min, and the waste liquid was discarded. The precipitate was washed again and the waste liquid was discarded. 200 ul of GA buffer (GA buffer, used to suspend cells) was directly added to the 1.5 ml centrifuge tube containing the precipitate, and the mixture was mixed by blowing; then 20 ul of proteinase k was added, and the mixture was mixed by shaking; then 200 ul of GB buffer (GB buffer, used for cell lysis and separation of nucleic acid and protein) was added, and the mixture was mixed by shaking, and the mixture was centrifuged instantly; then incubated at 70 ° C for 10 min. After incubation, add 200ul of anhydrous ethanol and shake for 15s; add the solution to the CB3 column, centrifuge at 12000g for 1min, and discard the waste liquid. Then add 500μl of GD rinse solution (rinse solution for removing protein impurities) to the CB3 column, centrifuge at 12000rpm for 30s, and discard the waste liquid. Then add 600μl of PW (rinse solution for removing salt) to the CB3 column, centrifuge at 12000rpm for 1min, discard the waste liquid, and repeat once. Centrifuge at 12000rpm for 2min, discard the waste liquid, and aspirate the column, then place the CB3 column at room temperature for 5min and dry it. Transfer the above CB3 column to a clean 1.5ml tube, add 53μl of TE (for dissolving DNA) to the column membrane, place it at room temperature for 5min, centrifuge at 12000g for 2min, and discard the waste liquid.

[0272] 2. Urine precipitation gDNA sulfite conversion

[0273] The urine precipitated gDNA was converted to sulfite using the zymo kit, which was purchased from Tianmo Biotechnology, product model EZ DNA Methylation-Lightning Kit (D3051). The specific steps are as follows:

[0274] Take 80 ng of the gDNA obtained in step 1, add nuclease-free water (NFW) to 20 ul, add 130 ul of Lightning Conversion Reagent, shake and mix, and react under the following reaction conditions: 98°C for 8 min, then 54°C for 60 min, and keep at 4°C. TM Add 600 μl of M-Binding Buffer to the IC column and transfer all the incubation solution to the Zymo-Spin TMIC, invert 10 times to mix, centrifuge at 10000g for 1 min, 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 s. The sample was collected and eluted using Qubit TM ssDNA detection kit for quantitative detection.

[0275] S2. Using the sulfite-converted gDNA as a template, and mixing it with the primer probe set of the methylated gene fragment, the primer probe set of the point mutation site, and the primer probe set of the control gene, real-time fluorescence quantitative PCR is performed.

[0276] Prepare the reaction mixture on ice according to the configuration scheme of detection reaction mix 12μl, probe and primer mix 9μl, and template 30ng (in order to ensure the accuracy of the reaction solution preparation, when performing each reaction, the detection reaction mix and probe and primer mix should be prepared according to the amount of reaction number + 2, and then divided into each reaction tube, and finally the transformed DNA sample is added). When adding the sample to be tested, add the positive quality control product, negative quality control product, and negative sample to determine whether the experiment is qualified. The reaction conditions are shown in Table 4.

[0277] Table 4 PCR reaction conditions

[0278]

[0279] S3. Analyze the data results.

[0280] After the reaction, the data were read and analyzed using the software of the Real-Time PCR Instrument 7500, and the ΔCt values ​​of each methylated gene fragment and mutation site were obtained.

[0281] The reference method for reading ΔCt value is:

[0282] I. Reading of PCR reaction results of methylation probe and primer mixture:

[0283] ① Baseline setting: Manually set the baseline. The start cycle of the baseline is usually set at 3, and the end cycle is usually set at 15. ② Threshold setting: Manually set the threshold, which is usually set at 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 get the Ct value of each sample.

[0284] ΔCt value of methylated fragment = Ct value of methylated fragment - Ct value of reference gene.

[0285] 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 highly methylated, otherwise the target gene is hypomethylated.

[0286] II. Reading the results of the PCR reaction mixture of point mutation probes and primers: ① Baseline setting: Manually set the baseline. The start point (Start Cycle) of the ACTB-M baseline (Baseline) is generally set at 3, and the end point (End Cycle) is generally set at 15. ② Fluorescence threshold setting: Manually set the threshold (Threshold), which is generally set at 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 get the Ct value of each sample.

[0287] ΔCt value of point mutation = Ct value of point mutation - Ct value of reference gene.

[0288] 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.

[0289] Example 2 Detection results of two gene markers in early diagnosis of urothelial carcinoma

[0290] 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. The 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 (ΔCt = Ct of the detection gene - Ct of the internal reference gene ACTB) was calculated.

[0291] Judgment criteria: The gene marker combination includes gene mutation and methylation. If either or both of the gene mutation and methylation are positive, the patient is judged to have a positive test result. For a gene marker combination that includes more than one gene site mutation, if one or two site mutations are judged to be positive, the gene mutation of the gene marker combination is judged to be positive.

[0292] Compared with the gold standard cystoscopic pathology test results, the cystoscopic pathology test results were positive in 131 cases and negative in 121 cases. The combination of gene markers used is shown in Table 5, and the test results are shown in Tables 6-1 to 6-5 and Table 7. The results showed that compared with the control group, the test sensitivity and specificity of the experimental group were higher, at more than 80%.

[0293] Table 5 Two-gene marker combination

[0294]

[0295] Table 6-1 Test results of experimental group 1

[0296]

[0297] Table 6-2 Test results of experimental group 2

[0298]

[0299]

[0300] Table 6-3 Test results of experimental group 3

[0301]

[0302] Table 6-4 Test results of control group 1

[0303]

[0304] Table 6-5 Test results of control group 2

[0305]

[0306] Table 7 Summary of the test results of two-gene combination for early diagnosis

[0307]

[0308] Example 3 Detection results of two gene markers in monitoring recurrence of urothelial carcinoma

[0309] 47 patients with suspected recurrence of urothelial carcinoma were collected and tested by the method of Example 2, and compared with the gold standard cystoscopic pathological test results. The gene marker combination used is shown in Table 5, and the test results are shown in Table 8-1, Table 8-2, Table 8-3, Table 8-4 and Table 9. The results show that compared with the control group, the test sensitivity and specificity of the experimental group are higher, at more than 80%.

[0310] Table 8-1 Test results of experimental group 1

[0311]

[0312] Table 8-2 Test results of experimental group 2

[0313]

[0314] Table 8-3 Test results of control group 1

[0315]

[0316] Table 8-4 Test results of control group 2

[0317]

[0318] Table 9 Summary of the results of the two-gene combination test for recurrence monitoring

[0319] Group Gene marker combination Sensitivity Specificity Experimental Group 1 VIM methylation site 1+TERT C228T 91.6％ 82.6％ Experimental Group 2 VIM methylation site 2+TERT C228T 87.5％ 82.6％ Control group 1 EOMES methylation + TERT C228T 58.3％ 65.2％ Control group 2 VIM methylation site 2+TP53: chr17 7577085_7577085_C>T 45.5％ 78.3％

[0320] Example 4 Detection results of three gene markers in early diagnosis of urothelial carcinoma

[0321] 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 examined by endoscopy, abdominal ultrasound, CT scan or abdominal and pelvic MRI, and pathologically confirmed. 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. The 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 (ΔCt = Ct of the detection gene-Ct of the internal reference gene ACTB) was calculated.

[0322] Judgment criteria: The gene marker combination includes gene mutation and methylation. If either or both of the gene mutation and methylation are positive, the patient is judged to have a positive test result. For a gene marker combination that includes more than one gene site mutation, if one or two site mutations are judged to be positive, the gene mutation of the gene marker combination is judged to be positive.

[0323] Compared with the gold standard cystoscopic pathology test results, the cystoscopic pathology test results were positive in 131 cases and negative in 121 cases. The gene marker combination used is shown in Table 10, and the test results are shown in Table 11-1, Table 11-2 and Table 12. The results showed that the detection sensitivity and specificity of experimental group 1 and experimental group 2 were both high, above 70%, and experimental group 1 was above 85%.

[0324] Table 10 Three-gene marker combination

[0325]

[0326]

[0327] Table 11-1 Test results of experimental group 1

[0328]

[0329] Table 11-2 Test results of experimental group 2

[0330]

[0331] Table 12 Summary of the test results of the three-gene combination for early diagnosis

[0332] Group Gene marker combination Sensitivity Specificity Experimental Group 1 VIM methylation site 1+TERT C228T+FGFR3 R248C 87.7％ 93.3％ Experimental Group 2 ONECUT2 methylation + TERT C228T + TERT C250T 74.8％ 81.8％

[0333] Example 5 Detection results of six gene markers in early diagnosis of urothelial carcinoma

[0334] 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 examined by endoscopy, abdominal ultrasound, CT scan or abdominal and pelvic MRI, and pathologically confirmed. 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. The 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 (ΔCt = Ct of the detection gene-Ct of the internal reference gene ACTB) was calculated.

[0335] Judgment criteria: The gene marker combination includes gene mutation and methylation. If either or both of the gene mutation and methylation are positive, the patient is judged to have a positive test result. For a gene marker combination that includes more than one gene site mutation, if one or two site mutations are judged to be positive, the gene mutation of the gene marker combination is judged to be positive.

[0336] Compared with the gold standard cystoscopic pathology test results, the cystoscopic pathology test results were positive in 131 cases and negative in 121 cases. The gene marker combination used is shown in Table 13, and the test results are shown in Table 14-1, Table 14-2 and Table 15. The results showed that compared with the control group, the test sensitivity and specificity of experimental group 1 were higher, at more than 90%, while the sensitivity and specificity of the control group were both below 70%.

[0337] Table 13 Six gene marker combinations

[0338]

[0339] Table 14-1 Test results of experimental group 1

[0340]

[0341] Table 14-2 Test results of control group 1

[0342]

[0343] Table 15 Summary of the test results of the six-gene combination for early diagnosis

[0344]

[0345] Example 6 Detection results of six gene markers in monitoring recurrence of urothelial carcinoma

[0346] 47 patients with suspected recurrence of urothelial carcinoma were collected and tested by the method of Example 5, and compared with the gold standard cystoscopic pathological test results. The gene marker combination used is shown in Table 13, and the test results are shown in Table 16-1, Table 16-2 and Table 17. The results show that compared with the control group, the test sensitivity and specificity of the experimental group are higher, at more than 90%, while the control group is less than 60%.

[0347] Table 16-1 Test results of experimental group 1

[0348]

[0349] Table 16-2 Test results of control group 1

[0350]

[0351] Table 17 Summary of the test results of the six-gene combination for early diagnosis

[0352]

[0353]

[0354] Example 7 Detection results of different primer / probe sequences in six gene combinations

[0355] According to the multiple methylation regions of VIM or ONECUT2, different primer probe combinations were designed, and urine samples of 252 suspected patients with urothelial carcinoma and 47 recurrent patients were tested according to the method of Example 5 or Example 6. The primer probe combination of the six-gene marker combination is as in Example 5, except that the primer probe of VIM or ONECUT2 methylation is replaced with the primer probe in Table 18, and the test results are shown in Table 19. The results show that for different methylation fragments, the detection sensitivity and specificity of the six genes are high, both above 85%.

[0356] Table 18

[0357]

[0358] Table 19

[0359]

[0360] Example 8 Detection results of six gene markers in early diagnosis of urothelial carcinoma

[0361] 529 suspected patients with urothelial carcinoma (296 positive by pathological diagnosis and 233 negative by pathological diagnosis) were collected and tested using the method of Example 5. The gene marker combination used is shown in Table 13, and the test results are shown in Table 20. The results showed that the detection sensitivity and specificity of the six-gene detection experimental group were both high, at more than 85%.

[0362] Table 20 Test results of experimental group

[0363]

[0364] The sensitivity and specificity of the detection results of the present application are respectively: 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 a gene mutation of TERT. The kit is used to confirm the presence of urothelial carcinoma, assess the risk of urothelial carcinoma formation and / or assess the progression of urothelial carcinoma, wherein the sample to be tested is urine, 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 a nucleic acid sequence selected from any one of the following groups: (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, (5) Primers: SEQ ID NO. 25-26, probe: SEQ ID NO. 27, and And the TERT gene mutation is C228T.

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 claim 1, wherein the reagent combination comprises primers and / or probes that can specifically recognize and / or bind to the TERT gene mutation C228T.

5. The use according to claim 4, 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. The use according to claim 1 , wherein the reagent combination comprises a reagent capable of detecting a gene mutation of FGFR3.

7. The use according to claim 6, wherein the gene mutation of FGFR3 comprises one or more selected from R248C and S249C.

8. The use according to claim 7, wherein the reagent combination comprises primers and / or probes that can specifically recognize and / or bind to the FGFR3 gene mutation R248C.

9. The use according to claim 8, 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 NOs. 32, 34 and 35.

10. The use according to claim 1, wherein the reagent combination comprises primers and / or probes that can specifically recognize and / or bind to the FGFR3 gene mutation S249C.

11. The use according to claim 10, 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.

12. The use according to claim 1, wherein the reagent combination further comprises a reagent for detecting the methylation level of the DNA region or fragment thereof where the ONECUT2 gene is located in the sample to be tested.

13. The use according to claim 12, 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.

14. The use according to claim 12, 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 primers and / or probes capable of specifically recognizing and / or binding to the DNA region or fragment thereof where the ONECUT2 gene is located.

15. The use according to claim 14, 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.

16. The use according to claim 14, 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) 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; and (4) Primers: SEQ ID NOs. 10-11, probe: SEQ ID NO.

12.

17. The use according to claim 1, 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 17 , wherein the reference gene is the ACTB gene.

19. The use according to claim 17, 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.

20. The method according to claim 1, further comprising a deamination agent and / or a methylation-sensitive restriction endonuclease.

21. The use according to claim 20, wherein the deamination agent comprises bisulfite.

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