Urothelial carcinoma markers and applications, primer sets, highly specific kits and detection methods

Abstract

The present invention relates to urothelial carcinoma markers and applications, primer sets, highly specific kits and detection methods; the markers include a combination of SEQ ID NO.1 and SEQ ID NO.2 of a co-methylated region indicated by CG or a combination of their fully complementary sequences. The detection kit of the present invention greatly improves the detection specificity. Among 210 suspected UC individuals, the result sensitivity is still 85%, while the specificity reaches 100%, and no false positive occurs, which is particularly suitable for routine physical examination applications.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a urothelial carcinoma marker and application thereof, a primer set, a highly specific kit and a detection method. Background Art

[0002] Urothelial carcinoma (UC) is one of the most common malignant tumors of the urinary system, including bladder and urethral malignancies in the lower urinary tract and renal pelvis and ureteral malignancies in the upper urinary tract, of which about 90% are bladder UC. According to data from the World Health Organization, there are nearly 800,000 new cases of bladder UC worldwide each year, while there are only nearly 100,000 new cases of bladder UC in my country each year. The number of people with bladder UC in my country has reached 1.3 million, ranking sixth in the world. The incidence rate in men is 4-5 times that in women. Men over 45 years old are the most susceptible group.

[0003] The preoperative qualitative diagnosis of UC mainly relies on ureteroscopy and pathological biopsy. The current non-invasive urine diagnostic techniques such as urine exfoliative cytology and fluorescence in situ hybridization (FISH) have low sensitivities (40-50% and 60-70%, respectively), which are difficult to meet clinical needs. Recently, Guangzhou Benchmark Medical Co., Ltd. (hereinafter referred to as Benchmark Medical) developed a urine DNA methylation qPCR detection kit based on human ONECUT2 and VIM genes (patent number: CN110872631B). It was tested in two groups of suspected bladder UC patients (one group of 192 people and the other group of 98 people) (Clin Epigenet. 2021, 13: 91), with sensitivities of 88.1% and 91.2%, respectively, and specificities of 89.7% and 85.7%, respectively. Next, the kit was tested in two groups of suspected upper urinary tract UC (one group of 153 people and the other group of 75 people) (Chinese Journal of Urology, 2023, 44 (10): 725-730), with sensitivities of 83.0% and 85.2%, and specificities of 92.0% and 85.7%, respectively. In short, the sensitivity and specificity of the kit for detecting UC are both 80-90%, which is much higher than traditional non-invasive techniques (exfoliative cytology and FISH). It is easy to operate and inexpensive, and is expected to be used in clinical practice.

[0004] However, the biggest drawback of this test kit is that its specificity is not high, only 80-90%, which is prone to false positives. The characteristic of UC is that the onset is hidden; even if patients have hematuria, 80% of them do not have UC. Therefore, the low specificity will inevitably lead to a large number of misdiagnoses, especially it cannot be used for routine physical examinations. For example, if there are 1,000 patients with hematuria to be examined clinically, if the specificity is 80%, 200 patients will be misdiagnosed as UC; if the specificity is 90%, 100 patients will be misdiagnosed as UC. As for routine physical examinations, assuming that 100,000 people participate in the test, if the specificity is 90%, 10,000 people will be misdiagnosed as UC. This is absolutely unacceptable in practical applications.

[0005] To this end, we redesigned the detection marker regions of human ONECUT2 and VIM genes and made them into a test kit. The clinical trial results of the obtained test kit showed significantly improved specificity. Summary of the invention

[0006] In view of the above problems, the present invention provides a urothelial carcinoma marker and its application, which can be used for the detection, diagnosis, classification or prediction of urothelial carcinoma risk, treatment monitoring, prognosis or other evaluation. The present invention also provides a primer set suitable for urothelial carcinoma detection, a highly specific kit and a detection method.

[0007] The first aspect of the present invention provides a marker for detecting urothelial carcinoma, wherein the marker is composed of SEQ ID NO. 1 and SEQ ID NO. 2 of the co-methylation region indicated by CG, or is composed of a sequence that is completely complementary to SEQ ID NO. 1 and SEQ ID NO. 2.

[0008] We used the first-generation sequencing technology to analyze the methylation profiles of 23 individuals and found that the detection sites in the prior art CN110872631B just fell into the critical methylation region between non-cancer and cancer, and therefore easily caused false positives. In order to improve the specificity of the detection and to avoid the patent of Benchmark Medical, we redesigned the detection regions of human ONECUT2 and VIM genes, SEQ ID NO. 1 and SEQ ID NO. 2.

[0009] The second aspect of the present invention provides the use of a reagent for detecting the methylation level of the above-mentioned marker in the preparation of a detection system for detecting, diagnosing, classifying or predicting, monitoring treatment, prognosis or other evaluation of urothelial carcinoma, wherein the detection system comprises: a detection reagent, a kit or a detection device.

[0010] The third aspect of the present invention provides a primer set for detecting urothelial carcinoma, the primer set comprising:

[0011] Primer set 1 for SEQ ID NO.1, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.3, a reverse primer with a nucleotide sequence such as SEQ ID NO.4, and a probe with a nucleotide sequence such as SEQ ID NO.5;

[0012] Primer set 2 for SEQ ID NO. 2 consists of a forward primer with a nucleotide sequence such as SEQ ID NO. 6, a reverse primer with a nucleotide sequence such as SEQ ID NO. 7, and a probe with a nucleotide sequence such as SEQ ID NO. 8.

[0013] In one or more embodiments, the primer set also includes: an internal reference ACTB primer set for the internal reference gene ACTB, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.9 and a reverse primer with a nucleotide sequence such as SEQ ID NO.10, and a probe with a nucleotide sequence such as SEQ ID NO.11.

[0014] The fourth aspect of the present invention also provides a highly specific urine methylation detection kit for urothelial carcinoma, comprising the above-mentioned primer set 1 and primer set 2.

[0015] In one or more embodiments, the kit further comprises the above-mentioned internal reference ACTB primer set.

[0016] In one or more embodiments, the kit further comprises at least one of a nucleic acid extraction reagent and a nucleic acid conversion reagent.

[0017] In one or more embodiments, the kit is a qPCR kit.

[0018] The fifth aspect of the present invention provides a highly specific urine methylation qPCR detection method for urothelial carcinoma, comprising the following steps:

[0019] (1) Extract DNA from urine samples;

[0020] (2) Transforming the extracted DNA samples;

[0021] (3) Sample DNA testing: Using the above-mentioned primer set or kit, with ACTB as the internal reference gene, perform triple qPCR testing on the converted DNA sample, and determine the test results based on the test data;

[0022] The judgment criteria are as follows: ① When the Ct value of the reference gene Ct ACTB ≤33, the test result is valid; Ct ACTB>33, the test result is invalid; ② Threshold setting: the difference ∆CT between any CT value of the marker described in the first aspect and the CT value of the internal reference gene ACTB is used as the basis for judgment. When the sample age is less than 70 years old, the threshold ∆CT is 10; for every 10 years increase in age, the threshold ∆CT decreases by 0.5.

[0023] In one or more embodiments, bisulfite is used for conversion in step (2).

[0024] In one or more embodiments, in step (3), the qPCR detection reaction procedure is: 95°C for 30 seconds; 95°C for 15 seconds, 60°C for 15 seconds, collecting fluorescence signals at 60°C, and 45 cycles.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention redesigns the detection regions of human ONECUT2 and VIM genes to achieve high detection specificity. The detection kit of the present invention greatly improves the detection specificity by combining the marker detection regions, the design of primer pairs and probes, and their combined design. The detection was performed on 210 suspected UC individuals (80 UC and 130 non-UC), and the sensitivity was still 85%, while the specificity reached 100%, and no false positives occurred. The detection kit is particularly suitable for routine physical examination applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the design of upstream and downstream primers for the cg12874092 site of the human VIM gene of SEQ ID NO.2 of the present invention;

[0028] Figure 2 The first-generation sequencing results of 4092-1203-4 in 3 UCs and 3 non-UCs; the blue peak represents the cytosine signal;

[0029] Figure 3 The first-generation sequencing results of 4092-1203-2 in 3 UCs and 3 non-UCs; the blue peak represents the cytosine signal;

[0030] Figure 4 This is a schematic diagram of the design of upstream and downstream primers for the cg10835584 site of human ONECUT2 gene of SEQ ID NO.1 of the present invention;

[0031] Figure 5 The first-generation sequencing results of 5584-1105-2 in 3 UCs and 3 non-UCs; the blue peak represents the cytosine signal;

[0032] Figure 6The first-generation sequencing results of 5584-1126-1 in 3 UCs and 3 non-UCs; the blue peak represents the cytosine signal;

[0033] Figure 7 The first-generation sequencing results of 5584-1105-1 in 3 UCs and 3 non-UCs; the blue peak represents the cytosine signal;

[0034] Figure 8 The figure is a schematic diagram comparing the performance of the kit of the present invention and the kit in CN110872631B. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0036] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0037] In the description of the present invention, the description with reference to the terms "some embodiments", "examples", etc. means that the specific methods and materials described in conjunction with the embodiments or examples are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific methods and materials described can be combined in any one or more embodiments or examples in a suitable manner.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0039] the term:

[0040] As used in the present invention, "marker" refers to a biological molecule or a biological molecule fragment, whose change and / or detection can be associated with a specific physical condition or state. The terms "marker", "molecular marker" or "biomarker" are used interchangeably throughout the disclosure of the invention. In the present invention, unless otherwise stated, the "marker" refers to a "cancer (tumor) marker". In the present invention, unless otherwise stated, the cancer is urothelial carcinoma.

[0041] As used in the present invention, the "detection" includes "assessment", "determination", "analysis", "prediction" and "evaluation"; the "evaluation" or "assessment" also includes "scoring".

[0042] As used in the present invention, the "patient", "subject" or "individual" may refer to an organism, and in some aspects, the subject may be a human. The subject providing the sample may include a population at potential risk of the disease or a population diagnosed with the disease. The disease in the present invention refers to urothelial carcinoma.

[0043] As used in the present invention, the "sample" and "specimen" are used interchangeably, which include substances obtained from individual or separated tissues, cells (such as epithelial cells) or body fluids (such as urine, serum, plasma) that are suitable for tumor marker detection.

[0044] As used in the present invention, the "urothelial carcinoma" refers to a cancer that occurs in the epithelial cells of the urinary tract, including the bladder, renal pelvis, ureter, and urethra. Urothelial carcinoma is multiple in nature or synchrony, so the "urothelial carcinoma" of the present invention includes primary cancer and cancer that has metastasized in the tissue. In some specific embodiments, the urothelial carcinoma is classified according to the site of tumor occurrence, and includes but is not limited to: renal pelvic cancer, ureteral cancer, bladder cancer, and urethral cancer. It should be understood that the urothelial carcinoma includes high-grade urothelial carcinoma and low-grade urothelial carcinoma.

[0045] As used herein, "complementary" and "complementarity" refer to a nucleotide (e.g., 1 nucleotide) or polynucleotide (e.g., a sequence of nucleotides) associated with the base pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Complementarity can be "partial", in which only some of the nucleic acid bases are matched according to the base pairing rules. Alternatively, there may be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid chains affects the efficiency and intensity of hybridization between nucleic acid chains. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

[0046] As used herein, "methylation" refers to methylation of cytosine at cytosine position C5 or N4, N6 site of adenine or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated, because in vitro DNA amplification methods generally do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" may also refer to amplified DNA that is unmethylated or methylated from the original template, respectively.

[0047] The methylation state can optionally be represented or indicated by a "methylation value" (e.g., representing a methylation frequency, fraction, ratio, percentage, etc.). The methylation value can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification spectra after a bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. Therefore, a value such as a methylation value represents the methylation state and can therefore be used as a quantitative indicator of the methylation state in multiple copies of a locus. The degree of co-methylation is represented or indicated by the methylation state of more than one methylation site, and co-methylation is defined as when the methylation state of more than one methylation site is methylated within a methylated region.

[0048] Co-methylation detection methods include: methylation-specific PCR (MSP), DNA methylation chip, targeted DNA methylation sequencing, digital PCR quantification and fluorescence quantitative PCR.

[0049] In some embodiments, DNA (eg, genomic DNA, such as extracted genomic DNA or processed genomic DNA) is isolated by any standard means in the art, including use of commercially available kits.

[0050] As used herein, "methylation assay" refers to any assay used to determine the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0051] Unless otherwise specified, the reagents, materials, and equipment used in the examples of the present invention are all commercially available; the test methods, unless otherwise specified, are all conventional test methods in the art.

[0052] Materials and Methods

[0053] 1. Sample collection

[0054] 210 urine samples (80 UC and 130 non-UC) were collected from Zhujiang Hospital of Southern Medical University and tested at Guangzhou Zhongxin Gene Medical Technology Co., Ltd. Each sample was 50ml-100ml of urine, all of which were suspected UC cases and untreated. The procedures followed in this study were in accordance with the requirements of the World Medical Association's Declaration of Helsinki, revised in 2013.

[0055] 2. DNA extraction from urine exfoliated cells

[0056] In the embodiment, HiPure Tissue DNA MicroKits micro-DNA extraction kit (column method) produced by Guangzhou Meiji Biotechnology Co., Ltd. was used to extract bladder exfoliated cell DNA. The recovered DNA fragments were tested for concentration using qubit. Other commercially available reagents can also be used for DNA extraction.

[0057] 3. DNA transformation

[0058] Generally, DNA converted with bisulfite is used for whole genome amplification and then MSP sequencing. In the present embodiment, the bisulfite conversion kit of Lightning Conversion Reagent of zymo research is used to convert DNA for downstream methylation qPCR MSP detection (detection of methylated fragments). Bisulfite conversion is performed according to the instructions.

[0059] 4. First-generation sequencing analysis.

[0060] BSP primers were designed using olig7 or methyl express software. The amplified products were sent to Guangzhou Aiki Biotechnology Co., Ltd. for first-generation sequencing. The sequencing results were analyzed using SnapGene 4.3.6 software.

[0061] 5. Statistical analysis:

[0062] Data were processed using R language 4.3.1 statistical software. The efficacy of the two examination methods was expressed as a rate, and the inter-group comparison was performed using the χ2 test. Statistical bar graph data were expressed as mean and 95% CI, and the 95% CI was calculated using the Wald method. P < 0.05 was considered statistically significant.

[0063] Example 1 Sequencing analysis of existing technology benchmark medical patent sites

[0064] In order to clarify the methylation status of the detection sites in the benchmark medical patent CN110872631B in UC and non-UC individuals, we first used first-generation sequencing to detect the methylation status around the benchmark medical sites in 6 individuals (three UC and three non-UC). The sites detected in the human ONECUT2 and VIM genes in the benchmark medical patent CN110872631B are: cg10835584 (SEQ ID NO.1 in CN110872631B) and cg12874092 (SEQ ID NO.2 in CN110872631B). BSP primers were designed upstream and downstream to amplify the converted urine DNA and perform first-generation sequencing.

[0065] The BSP primers designed for SEQ ID NO.1 and SEQ ID NO.2 in CN110872631B are shown in Table 1.

[0066] Table 1 BSP sequencing primers

[0067]

[0068] Sequencing results:

[0069] 1. The first-generation sequencing results showed that the reason for the false positive of the benchmark medical kit (CN110872631B) was that one of the detection sites was located at the edge of the CpG island and had low specificity.

[0070] (1) The detection site cg12874092 is located in the middle of the CpG island and has high specificity.

[0071] Two pairs of primers were designed upstream and downstream of cg12874092: 4092-1203-4 and 4092-1203-2 (Table 1 and Figure 1 ).like Figure 1 As shown: 1) cg12874092 is located in the middle of the CpG island; 2) the upstream and downstream amplification products are all within the CpG island. First-generation sequencing results: The upstream and downstream CpG sites of the cg12874092 site only show cytosine signals in the three UCs, while the three non-UCs have no signals, so they are all cancer-specific methylation ( Figure 2 ,3).

[0072] (2) The detection site cg10835584 is located at the edge of the CpG island and has low specificity.

[0073] Three pairs of primers were designed upstream and downstream of cg10835584: 5584-1105-2, 5584-1126-1, and 5584-1105-1 (Table 4 and Figure 4 ).like Figure 4 As shown: 1) cg10835584 is located at the edge of the CpG island; 2) 5584-1105-2 is downstream of the cg10835584 site and outside the CpG island; 3) 5584-1126-1 contains the cg10835584 site; 4) 5584-1105-1 is located upstream of the cg10835584 site and within the CpG island.

[0074] Result analysis:

[0075] 1) The CpG site of 5584-1105-2 showed cytosine signals in both UC and non-UC, so this region is universally methylated ( Figure 5); 2) The CpG site of 5584-1126-1 showed cytosine signals in 3 UCs and 1 non-UC, so this region was only partially cancer-specifically methylated ( Figure 6 ); 3) The CpG site of 5584-1105-1 showed cytosine signals only in 3 UCs, so this region is cancer-specifically methylated ( Figure 7 ).

[0076] In the first-generation sequencing of 17 expanded samples, it was further confirmed that cg10835584 was nonspecifically amplified, while its upstream 5584-1105-1 region was 100% cancer-specifically methylated.

[0077] To verify the above findings, we further performed first-generation sequencing using primers 5584-1126-1 and 5584-1105-1 in 4 healthy subjects, 7 non-UC patients, and 6 UC patients. Results: Both primers were negative in 4 healthy subjects; 2 of the 7 non-UC patients were positive for 5584-1126-1 (28.6%) and all of the 5584-1105-1 were negative; 5 of the 6 UC patients were positive for both primers (83.3%), and the remaining 1 was negative for both primers.

[0078] In conclusion, cg10835584 is located at the edge of the CpG island, and some non-cancer individuals also have methylation in this area, which may be the reason why the detection specificity in the benchmark medical patent is not high. Designing primer probes in the middle of the CpG island may improve specificity.

[0079] Example 2 Determining new gene detection regions and establishing new markers

[0080] On the basis of Example 1, in order to improve the specificity of detection and to avoid the patent of Benchmark Medical, the present application redesigned the detection region of human ONECUT2 and VIM genes and proposed new markers for urothelial carcinoma detection: 4092-0204 (SEQ ID NO. 2) and 5584-1224-4 (SEQ ID NO. 1) ( Figure 1 and 4 , and as shown in Table 2).

[0081] Table 2 New marker gene detection regions

[0082]

[0083] Among them, the 4092-0204 region (128: 1748-1875) overlaps with the detection region of SEQ ID NO. 2 in the benchmark medical patent CN110872631B (87: 1822-1908) by 54 bp (62%, which is lower than the patent requirement of benchmark medical> 70%) ( Figure 1 ); The 5584-1224-4 region has no intersection with the detection region of SEQ ID NO.1 in the benchmark medical patent CN110872631B ( Figure 4 ).

[0084] Example 3 Primer probe screening

[0085] Multiple pairs of MSP primer probes were designed on the CpG islands of the two genes of the new markers, and a qPCR system was established. Then the designed probe primer combinations were used to detect clinical samples, screen and filter based on sensitivity and specificity. After screening the probe primer combination, the two target genes of the newly designed markers and the primer probes of the internal reference ACTB were combined to perform blank control experiments, negative control experiments and positive clinical sample tests. The reagent used was Kangwei Century's FastStarProbe Kit to screen out the best triple probe primer combination. Table 3 shows the best primer and probe combinations screened out.

[0086] Table 3 Primer-probe combinations for triplex qPCR

[0087]

[0088] Example 4 qPCR test

[0089] A kit comprises the triple primer-probe combination screened in Example 3, and matching reagents.

[0090] The kit is then used to perform urothelial carcinoma qPCR detection, which includes the following steps:

[0091] (1) Extract DNA from urine samples;

[0092] (2) performing bisulfite conversion on the extracted DNA samples;

[0093] (3) Sample DNA testing: Using the assembled test kit and ACTB as the internal reference gene, triple qPCR tests were performed on the converted DNA samples, and the test results were determined based on the test data;

[0094] The qPCR reaction volume was 25 μl. The primer / probe concentration of the primer set 5584-1224-4 was 0.2 μM / 0.1 μM, and the primer / probe concentration of the internal reference ACTB and 4092-0204 primer set was twice that of the 5584-1224-4 primer set (0.4 μM / 0.2 μM).

[0095] The PCR reaction conditions are as follows:

[0096]

[0097] The judgment criteria are as follows: the CT difference between the two target genes of the marker and the internal reference gene ACTB (target gene CT value - internal reference CT value) is used as the judgment basis. According to the combined analysis of the test results of clinical samples and clinical pathological conditions, the ACTB CT value less than 33 is the standard for effective detection, and the threshold is set as follows: the difference between the CT value of any target gene and the internal reference ACTB CT value ∆CT is the threshold, and the ∆CT is 10 for samples under 70 years old (<70); according to the test results and sample information and the literature, methylation is also present in older people. Through analysis, the threshold ∆CT is set to decrease by 0.5 for every 10 years of age, such as 70-79 years old, the threshold ∆CT is 9.5, 80-89 years old, the threshold ∆CT is 9.

[0098] A total of 210 suspected UC cases (80 UC cases and 130 non-UC cases) were tested. Figure 8 shown.

[0099] Comparative Example 1

[0100] The kit was reproduced according to the second and third sets of primers and probes in Example 2 of the patent specification CN110872631B of Benchmark Medical and the experimental conditions of Example 6. The primers and probes are SEQ ID NO.155 and SEQ ID NO.177 for SEQ ID NO.1 in CN110872631B; SEQ ID NO.90 and SEQ ID NO.112 for SEQ ID NO.2, and SEQ ID NO.134; and the internal reference primers and probes are SEQ ID NO.221 and SEQ ID NO.222, and SEQ ID NO.223.

[0101] The same 210 suspected UCs (80 UCs and 130 non-UCs) were tested and compared with the results of Example 4. The results are as follows: Figure 8 As shown, the sensitivity of the benchmark medical treatment is 87.5% and the specificity is 90%, which is consistent with the results reported previously; while the sensitivity of the kit of the embodiment of the present invention is 85% (equivalent to the benchmark medical treatment) and the specificity is as high as 100%. Therefore, it is shown that after using the primer-probe combination of the present invention, the specificity of detecting new markers is significantly improved, and the kit of the embodiment of the present invention will not have false positives, and is particularly suitable for routine physical examinations.

[0102] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A marker for detecting urothelial carcinoma, characterized in that: The marker consists of SEQ ID NO. 1 and SEQ ID NO. 2 of the co-methylated region indicated by CG, or consists of a sequence that is completely complementary to SEQ ID NO. 1 and SEQ ID NO.

2.

2. Use of a reagent for detecting the methylation level of a marker according to claim 1 in the preparation of a detection system for detecting, diagnosing, classifying or predicting, monitoring treatment, prognosing or otherwise evaluating urothelial carcinoma, the detection system comprising: A detection reagent, a kit or a detection device.

3. A primer set for detecting urothelial carcinoma, characterized in that: The primer set comprises: Primer set 1 for SEQ ID NO.1, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.3, a reverse primer with a nucleotide sequence such as SEQ ID NO.4, and a probe with a nucleotide sequence such as SEQ ID NO.5; Primer set 2 for SEQ ID NO. 2 consists of a forward primer with a nucleotide sequence such as SEQ ID NO. 6, a reverse primer with a nucleotide sequence such as SEQ ID NO. 7, and a probe with a nucleotide sequence such as SEQ ID NO.

8.

4. The primer set for detecting urothelial carcinoma according to claim 3, characterized in that: The primer set also includes: an internal reference ACTB primer set for the internal reference gene ACTB, which consists of a forward primer with a nucleotide sequence such as SEQ ID NO.9 and a reverse primer with a nucleotide sequence such as SEQ ID NO.10, and a probe with a nucleotide sequence such as SEQ ID NO.

11.

5. A highly specific urine methylation kit for detecting urothelial carcinoma, characterized in that: Comprising the primer set as described in any one of claims 3-4.

6. The kit according to claim 5, characterized in that The kit further comprises at least one of a nucleic acid extraction reagent and a nucleic acid conversion reagent.

7. The kit according to claim 5, characterized in that The kit is a qPCR kit.

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