Urine simulation sample and reference substance for urothelial carcinoma as well as preparation method and application of urine simulation sample and reference substance

Abstract

The invention provides a urine simulation sample for urothelial carcinoma, a reference substance and a preparation method and application of the urine simulation sample and the reference substance. A preparation method of a urine simulation sample for urinary tract epithelium cancer comprises the following steps: S1, preparing a urinary tract epithelium cancer cell culture: digesting T24 cells with 0.15%-0.35% v / v trypsin, and culturing to obtain a T24 cell suspension; s2, preparing free DNA of urothelial carcinoma: breaking a human genome DNA standard product NA12878 with a methylation level of 100% into free DNA with a length of 150 bp to 300 bp; s3, mixing the urinary tract epithelium cancer cell culture in the step S1, the free DNA of the urinary tract epithelium cancer in the step S2 and a urine sample of a healthy person to obtain the urine simulation sample of the urinary tract epithelium cancer. The invention provides a urine simulation sample and reference which are clear in background, capable of simulating clinical samples, low in cost, renewable and universal.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a urine simulation sample and reference product for urothelial carcinoma, and a preparation method and application thereof. Background Art

[0002] Urothelial carcinoma (bladder cancer) is one of the most common malignant tumors in the urinary system. The incidence of urothelial carcinoma is related to multiple factors, including genetic susceptibility, environmental factors and lifestyle. Long-term smoking, exposure to certain chemicals (such as aromatic amine compounds) and chronic bladder irritation are all considered to be risk factors for the disease. Urothelial carcinoma not only threatens the physical health of patients, but also imposes a heavy economic burden on society and families. Therefore, how to effectively reduce the disease burden of urothelial carcinoma has become a major public health issue that needs to be addressed urgently.

[0003] Early diagnosis of urothelial carcinoma is crucial to improving the prognosis of patients. At present, urothelial carcinoma is mainly diagnosed clinically using a variety of methods such as cystoscopy, urine cytology and imaging examinations. However, each of these methods has certain defects and requires further attention and improvement. In recent years, researchers have begun to pay attention to the combination of urothelial carcinoma-specific DNA methylation biomarkers. By detecting multiple DNA methylation sites, the problem of low single DNA methylation signals can be effectively overcome, thereby improving the sensitivity and specificity of detection. DNA methylation is an important epigenetic process, and its abnormal changes are closely related to the development of many cancers. Studies have found that there are specific DNA methylation markers in urine, which show significant changes in patients with urothelial carcinoma and therefore become potential diagnostic tools.

[0004] The detection method based on DNA methylation is simple and easy to use, and has objective interpretation. It avoids the subjectivity of human interpretation and improves accuracy. This detection method not only has high sensitivity and specificity, but is also non-invasive, and can effectively avoid complications caused by cystoscopy, thereby improving patient compliance. By detecting DNA methylation markers in urine, researchers can identify patients with urothelial carcinoma at an earlier stage, thereby providing a more timely basis for clinical intervention.

[0005] At present, the detection reference products on the market often have some defects, and the current research and development and application of urine detection reagents face many challenges. First, there are large individual differences in urine samples, which may affect the reliability of the test results. Secondly, in the development of urine detection reagents, the lack of reference products makes performance evaluation uncertain. Many laboratories rely on traditional reference products, and some reference products have unknown sources and lack sufficient background information. These reference products often have unknown sources and perform inconsistently under different experimental conditions. In addition, existing reference products are often costly, difficult to meet the needs of large-scale production, and lack reproducibility, which makes laboratories and corporate R&D face greater economic pressure when using them.

[0006] Therefore, the selection of reference materials is crucial in the development of urine test reagents. It is not only used to verify and calibrate the test methods, but also provides a benchmark for quality control. Suitable reference materials should have the following characteristics: (1) Clear background: The reference material should have a clear source and composition description to ensure its reliability and consistency in the experiment; (2) Simulated clinical samples: The composition of the reference material should be able to effectively simulate the characteristics of real clinical samples to reflect the performance in actual testing; (3) Low cost: The production cost of the reference material should be as low as possible to reduce the economic burden on the laboratory; (4) Renewability: The reference material should have a certain degree of reproducibility to facilitate repeated use in large-scale experiments.

[0007] The lack of detection references not only affects the accuracy and repeatability of the experiment, but also makes it challenging for researchers to develop new detection methods. Therefore, it is urgent to develop a urine simulation sample and its detection reference with a clear background, the ability to simulate clinical samples, low cost, reproducibility and universality. Summary of the invention

[0008] The embodiments of the present invention provide a method to at least solve one of the problems existing in the related art. To achieve this object, the present invention is implemented by the following technical solutions.

[0009] The present invention provides a method for preparing a simulated urine sample of urothelial carcinoma, comprising the following steps:

[0010] S1: preparing urothelial cancer cell culture: digesting T24 cells with 0.15% to 0.35% v / v trypsin, culturing, and obtaining a T24 cell suspension;

[0011] S2: Preparation of free DNA of urothelial carcinoma: The human genomic DNA standard NA12878 with 100% methylation level was broken into free DNA with a length of 150 bp to 300 bp;

[0012] S3: The urothelial cancer cell culture of step S1, the free DNA of urothelial cancer of step S2 and the urine sample of a healthy person are mixed to obtain a simulated urine sample of urothelial cancer.

[0013] As a cell model of urothelial carcinoma, human bladder transitional cell carcinoma T24 cells can simulate certain biological characteristics of bladder cancer, which is helpful for studying the pathogenesis and treatment strategies of bladder cancer. It is also easy to grow and reproduce under laboratory conditions, suitable for various cell biology studies, and has good genetic stability, which is convenient for long-term research and experimental repetition. The source of human genome DNA standard NA12878 has undergone strict quality control to ensure the purity and integrity of DNA. It is a reference material recognized by the International Organization for Standardization, ensuring the comparability of results between different laboratories and studies, and can be applied to a variety of methylation analysis techniques, such as sequencing, fluorescent PCR, etc.

[0014] Preferably, the trypsin concentration is 0.25% v / v, which can maintain a good cell number without cell clumping.

[0015] In a specific embodiment of the present invention, T24 cells are cultured in DMEM high-glucose medium containing 10% (v / v) fetal bovine serum at 60 r / min, 37° C., 5% CO 2 incubator for 12 h.

[0016] In a specific embodiment of the present invention, in step S2, the peak power of the interruption program of the interruption instrument is set to 50w, the working coefficient is 30%, the number of cycles of each interruption is 200, the processing time is 300s, the temperature is 20°C, and the sample volume is 50μL.

[0017] The optimal peak value of free DNA (cfDNA) fragments is 180bp. The peak fragment size of the shearing program is about 180bp, and the distribution of 150-300bp fragments accounts for the highest proportion, which are 51% (0% methylation level of human genome DNA standard NA12878) and 52% (100% methylation level of human genome DNA standard NA12878), respectively, which is the optimal shearing program.

[0018] In a specific embodiment of the present invention, in step S3, the density of T24 cells in the urine simulation sample is 1 to 2×10 5 The content of cellular DNA at this density is more suitable for simulating urine of patients with urothelial carcinoma.

[0019] The present invention also provides a simulated urine sample of urothelial carcinoma prepared by the preparation method.

[0020] The present invention also provides the use of the simulated urine sample in preparing a reference material for detecting urothelial carcinoma.

[0021] The present invention provides a reference substance for detecting urothelial carcinoma, wherein the reference substance contains DNA of a urine simulation sample of urothelial carcinoma and / or free DNA of a human genomic DNA standard substance NA12878 with a 0% methylation level.

[0022] Furthermore, the DNA methylation level of the reference substance = the DNA mass of the urine simulation sample of urothelial carcinoma / (the DNA mass of the urine simulation sample of urothelial carcinoma + the free DNA mass of the human genomic DNA standard substance NA12878 with a 0% methylation level)×100%.

[0023] In a specific embodiment of the present invention, the reference includes a negative reference and / or a positive reference, the DNA methylation level of the negative reference is 0%, which is the free DNA of the human genomic DNA standard NA12878 with a methylation level of 0%; the DNA methylation level of the positive reference is 30%, and the mass ratio of the DNA of the urine simulation sample of urothelial carcinoma and the free DNA of the human genomic DNA standard NA12878 with a methylation level of 0% is 3:7.

[0024] In a specific embodiment of the present invention, the reference material includes a detection limit reference material, the DNA methylation level of the detection limit reference material is 5%, and the mass ratio of the DNA of the urine simulation sample of urothelial carcinoma and the free DNA of the human genome DNA standard NA12878 with a methylation level of 0% is 1:19.

[0025] Furthermore, the reference material includes a precision reference material, and the DNA methylation level of the precision reference material is 0% to 20%.

[0026] In a specific embodiment of the present invention, the DNA methylation level of the precision reference can be at least one of 0%, 10% and 20%. The DNA methylation level of the precision reference is 0%, that is, the free DNA of the human genome DNA standard NA12878 with a methylation level of 0%; the DNA methylation level of the precision reference is 10%, and the mass ratio of the DNA of the urine simulation sample of urothelial carcinoma to the free DNA of the human genome DNA standard NA12878 with a methylation level of 0% is 1:9; the DNA methylation level of the precision reference is 20%, and the mass ratio of the DNA of the urine simulation sample of urothelial carcinoma to the free DNA of the human genome DNA standard NA12878 with a methylation level of 0% is 1:4.

[0027] The present invention also provides the use of the reference material in preparing a product for detecting urothelial carcinoma.

[0028] The embodiments of the present invention have the following beneficial effects:

[0029] The present invention provides a urine simulation sample and a reference material for detecting urothelial carcinoma that has a clear background, can simulate clinical samples, is low-cost, reproducible and universal, and provides a new idea and solution for the research and development of urine detection reagents.

[0030] It has the following advantages:

[0031] (1) Standardization: The simulated samples have standardized components. The substances they contain are consistent, which can ensure the repeatability and reliability of the test results. Ensuring the consistency and repeatability of the test results effectively reduces the misjudgment rate and the occurrence of false positive and false negative results, thereby providing accuracy that matches the clinical results.

[0032] (2) Controllability: Since the composition of the simulated sample is known and controllable, researchers can accurately simulate a specific state or concentration level, flexibly simulate the characteristics of different clinical samples, facilitate diagnostic test verification, and meet different testing needs. This preparation method can be processed and produced in large quantities in a sustainable and stable manner, and has good inter-batch and intra-batch stability.

[0033] (3) No ethical issues: The use of simulated samples avoids the privacy and ethical issues that may be involved in collecting and using real patient urine.

[0034] (4) Safety: The substances in the simulated samples are known and do not contain pathogens that may be present in real biological samples, so they do not pose a health risk to laboratory personnel during processing and analysis.

[0035] (5) Economic benefits: The simulated urine sample of the present invention uses renewable raw materials and meets environmental protection standards. This not only reduces resource waste, but also helps promote the concept of sustainable development. The preparation cost of simulated samples is usually lower than the cost of collecting and processing real urine samples, which is particularly suitable for situations where a large number of samples are required for testing or research. It exhibits high stability during storage and transportation, avoiding the degradation problem that may occur in traditional urine samples under temperature and time changes. This feature provides greater convenience for laboratories.

[0036] (6) Facilitate teaching and training: The simulated sample can be used for education and training to help students and medical personnel learn and practice urine analysis and testing techniques without risk. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 The results of the 0% methylation level human genomic DNA standard NA12878 shearing and 2100 quality inspection;

[0039] Figure 2 The results of the 100% methylation level human genomic DNA standard NA12878 shearing and 2100 quality inspection. DETAILED DESCRIPTION

[0040] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. However, it will be appreciated by those skilled in the art that in the embodiments of the present invention, many technical details are proposed in order to enable the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in the present application can be implemented. The division of the following embodiments is for the convenience of description, and should not constitute any limitation on the specific implementation of the present invention. The various embodiments can be combined and referenced with each other without contradiction.

[0041] Example 1 Preparation of reference material for detecting urothelial carcinoma

[0042] 1. Preparation of Urothelial Cancer Cell Cultures

[0043] The frozen human bladder transitional cell carcinoma T24 cells were taken out, and thawed quickly in a 37°C water bath, and gently shaken to accelerate thawing to obtain thawed T24 cells, and the thawed T24 cells were transferred to a preheated DMEM high-glucose medium containing 10% (v / v) fetal bovine serum, and the cells were resuspended by centrifugation to obtain a T24 cell suspension, and the T24 cell suspension was transferred to a culture bottle and placed in an incubator for culture. When the cells grow to a density of about 80% in the culture flask, add a 0.25% (v / v) trypsin solution of the same volume as the cell culture medium, gently shake the culture flask to allow the trypsin solution to completely immerse the cells, put the culture flask in an incubator and incubate for 2 minutes to digest the cells, wait until most of the cells are observed to be rounded and not attached to the wall under a microscope, and when a large number of cells are detached by gently shaking and tapping the sides of the culture flask, add 2 times the volume of trypsin in DMEM high-glucose medium containing 10% (v / v) fetal bovine serum to terminate the digestion, and gently blow the cells several times to completely detach all the cells from the wall, thereby terminating the digestion of the cells; centrifuge at room temperature at 1100 rpm to terminate the digestion of the cells for 4 minutes, discard the supernatant, add DMEM high-glucose medium containing 10% (v / v) fetal bovine serum to resuspend the cells, set the stirring speed to 60 r / min, and culture in a 37°C, 5% CO2 incubator for 12 hours to obtain a urothelial cancer cell culture.

[0044] 2. Preparation of free DNA of urothelial carcinoma

[0045] The human genomic DNA standard NA12878 with 0% methylation level and the human genomic DNA standard NA12878 with 100% methylation level were sheared to prepare free DNA (cfDNA).

[0046] The method for preparing cell-free DNA (cfDNA) is as follows:

[0047] (1) Turn on the computer, turn on the power of the interrupter (Covaris M-Series), open the SonoLab7.2 software, and select the corresponding program.

[0048] (2) Place a test tube plate labeled microTUBE 130μL on the Tube Holder and add AFA-grade water to the WaterSense Aperture until the water level reaches the bottom of the microTUBE 130μL test tube plate.

[0049] (3) Take out the frozen sample (0% methylation level human genomic DNA standard NA12878 or 100% methylation level human genomic DNA standard NA12878), add 500 ng of sample to the test tube to make the total sample system volume 50 μL. If it is less than 50 μL, use ethidium bromide (EB) to make up to 50 μL.

[0050] (4) The sample system in step (3) was mixed by oscillation, and then briefly centrifuged. The test tube was placed on the microTUBE 130 μL test tube plate treated in step (2). The test tube was pressed down with a sliding weight, and the Safety Cover was closed.

[0051] (5) Set the interruption program and check whether the program is correct: set the peak power to 50W, the working coefficient to 30%, the number of cycles for each interruption to 200, the processing time to 300s, the temperature to 20℃, and the sample volume to 50μL; after all three statuses in the Instrument Status in the software interface are lit with "√", you can click "RUN" in the software interface to start running the program and obtain the interrupted sample, which is the free DNA of urothelial carcinoma.

[0052] 3. Preparation of reference materials

[0053] (1) DNA from urine simulation samples: Urothelial cancer cell culture, free DNA from human genomic DNA standard NA12878 with 100% methylation level, and urine samples from healthy people were mixed to obtain urine simulation samples. The volume ratio of urothelial cancer cell culture to urine samples from healthy people was 1:4-9, so that the density of T24 cells in the urine simulation sample was 1-2×10 5 The concentration of free DNA after shearing with the 100% methylated human genomic DNA standard NA12878 was 50ng / μL. TM The free DNA isolation kit was used to extract DNA from urine simulation samples to obtain DNA from urine simulation samples. After digital PCR verification, the methylation level of the DNA was 100%.

[0054] (2) Positive reference: The DNA of the urine simulation sample and the free DNA of the human genomic DNA standard NA12878 with a 0% methylation level were mixed at a mass ratio of 3:7 to obtain a positive reference with a DNA concentration of 50 ng / μL and a methylation level of 30%.

[0055] (3) Negative reference: Take free DNA of the human genomic DNA standard NA12878 with a methylation level of 0%, dilute it with TE (1×, Tris-EDTA buffer) to obtain a negative reference with a concentration of 50 ng / μL and a methylation level of 0%.

[0056] (4) Detection limit reference material: The DNA of the urine simulation sample and the negative human genomic DNA were mixed at a mass ratio of 1:19 to obtain a detection limit reference material with a concentration of 50 ng / μL and a methylation level of 5%.

[0057] (5) Precision reference 1: negative reference, with a methylation level of 0%.

[0058] (6) Precision reference material 2: The DNA of the urine simulation sample and the negative human genomic DNA were mixed at a mass ratio of 1:9 to obtain a reference material with a concentration of 50 ng / μL and a detection limit of 10% methylation level.

[0059] (7) Precision reference material 3: The DNA of the urine simulation sample and the negative human genomic DNA were mixed at a mass ratio of 1:4 to obtain a detection limit reference material with a concentration of 50 ng / μL and a methylation level of 20%.

[0060] Example 2 Effects of different concentrations of trypsin on the number and size of T24 cells exfoliated from urine

[0061] 1. Experimental Methods

[0062] The concentrations of trypsin solution were set to 0.15% (v / v), 0.25% (v / v) and 0.35% (v / v), and 3 portions of 1×10 6 T24 cells were digested and cultured according to the method of Example 1 to obtain urothelial cancer cell cultures.

[0063] The cells were counted and the particle size distribution of cell agglomerates was detected. When the cell agglomerates were loose, trypan blue staining was used to count the cells using a hemocytometer. When the cell agglomerates were severe, crystal violet staining was used to count the cells. The particle size distribution of cell agglomerates was measured using a MALVERN laser particle counter.

[0064] 2. Experimental Results

[0065] As shown in Table 1, 0.25% (v / v) trypsin has the best effect on digesting T24 cells. When the trypsin concentration exceeds 0.25% (v / v), the cell agglomeration particle size exceeds the cell diameter, indicating that cell agglomeration occurs. When the trypsin concentration is lower than 0.25% (v / v), the number of cells after digestion decreases.

[0066] Table 1

[0067]

[0068] Example 3 Determining the amount of urine exfoliated cells added

[0069] Different numbers of T24 cells were selected, and DNA was extracted and DNA concentration was detected using the Ezup column adherent culture cell genomic DNA extraction kit (manufacturer: Sangon Biotechnology, catalog number: B518251).

[0070] Table 2

[0071]

[0072] 200 mL of urine from a tumor patient corresponds to 500 to 1000 ng of total DNA. According to the data provided in Table 2, 1 to 2 × 10 5 T24 cells.

[0073] Example 4 Shearing conditions for preparing free DNA

[0074] 1. Experimental Methods

[0075] According to the method of Example 1, the human genomic DNA standard NA12878 with a methylation level of 0% and the human genomic DNA standard NA12878 with a methylation level of 100% were sheared to prepare free DNA (cfDNA), and 2100 quality inspection was performed to check the proportion of the corresponding fragments after shearing.

[0076] The sample information of the human genomic DNA standard NA12878 with a 0% methylation level is shown in Table 3, and the sample information of the human genomic DNA standard NA12878 with a 100% methylation level is shown in Table 4. The interruption program set is shown in Table 5.

[0077] Table 3

[0078]

[0079] Table 4

[0080]

[0081] Table 5

[0082]

[0083] 2. Experimental Results

[0084] The interruption procedure and results of the 0% methylation level human genomic DNA standard sample NA12878 are shown in Table 6 and Figure 1 Among the 11 interruption programs shown in Table 6, the peak values ​​of the fragment sizes of No. 5, 6, 10 and 11 are around 180 bp, among which the fragment size of No. 10 is the highest in the range of 150 bp to 300 bp.

[0085] The sample information of 100% methylated human genomic DNA standard NA12878 is shown in Table 6 and Figure 2 Among the 11 fragmentation programs shown in Table 6, the peak values ​​of the fragment sizes of No. 5, 6, 10 and 11 are around 180 bp, and the fragment size of No. 10 has the highest proportion of 150 bp to 300 bp.

[0086] Table 6

[0087]

[0088] Table 7

[0089]

[0090] As shown in Table 7, the results of the interruption program No. 10 are shown. The median concentration of cfDNA in urine is 16.70 ng / μL, and cfDNA fragments less than 10 kb are mainly distributed around 180 bp. The peak value of cfDNA fragments is 180 bp. In the experiment, the peak value of the fragment size of the interruption program No. 10 is about 180 bp, and the distribution of 150-300 bp fragments accounts for the highest proportion, 51% and 52% respectively, which is the most suitable interruption program.

[0091] Example 5 Verification of reference materials for detecting urothelial carcinoma

[0092] 1. Experimental Methods

[0093] The concentration of the reference product prepared in Example 1 was verified: 3 batches of reference products were prepared continuously, 3 reference products in each batch, and each reference product was tested for concentration using a Qubit fluorescence quantifier. The methylation level was detected using the Human Twist1 Gene Methylation Detection Kit (Fluorescence PCR Method) of Anhui Dajian Medical Technology Co., Ltd., and the test was repeated 5 times for each batch.

[0094] 2. Experimental Results

[0095] As shown in Table 8, the results of re-determination of the concentration of the reference product were all within the error range of 10%, and the CV values ​​were all less than 5%, indicating that the inter-batch and intra-batch stability was good.

[0096] As shown in Table 9, the positive reference samples prepared in three batches were all positive, with a compliance rate of 100%; the negative reference samples were all negative, with a compliance rate of 100%; the detection limit reference samples were all detectable, with a detection rate of 100%; the precision reference sample 1 was repeated 15 times, and the results were all negative; the precision reference sample 2 was repeated 15 times, and the results were all positive; the precision reference sample 3 was repeated 15 times, and the results were all positive. This shows that the reference samples prepared by the present invention are effective through detection and confirmation, and can be used as a unified standard for experiments or production processes, which is helpful to establish comparability between different times, places and operators, and can ensure the traceability and reproducibility of test results.

[0097] Table 8 Concentration determination results of enterprise reference products (concentration unit: ng / μL)

[0098]

[0099] Table 9 Test results of the test kit of the enterprise reference product

[0100]

[0101] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a simulated urine sample for urothelial carcinoma, characterized in that: The following steps are involved: S1: preparing urothelial cancer cell culture: digesting T24 cells with 0.15% to 0.35% v / v trypsin, culturing, and obtaining a T24 cell suspension; S2: Preparation of free DNA of urothelial carcinoma: The human genomic DNA standard NA12878 with 100% methylation level was broken into free DNA with a length of 150 bp to 300 bp; S3: The urothelial cancer cell culture of step S1, the free DNA of urothelial cancer of step S2 and the urine sample of a healthy person are mixed to obtain a simulated urine sample of urothelial cancer.

2. The preparation method according to claim 1, characterized in that: In step S1, the density of T24 cells in the T24 cell suspension is 1-2×10 5 Pieces / mL.

3. A simulated urine sample of urothelial carcinoma prepared by the preparation method according to claim 1 or 2.

4. Use of the simulated urine sample according to claim 1 or 2 in preparing a reference material for detecting urothelial carcinoma.

5. A reference substance for detecting urothelial carcinoma, characterized in that: The reference material contains DNA of the urine simulation sample of urothelial carcinoma according to claim 3 and / or free DNA of the human genomic DNA standard NA12878 with a 0% methylation level.

6. The reference product according to claim 5, characterized in that: The DNA methylation level of the reference product = the DNA mass of the urine simulation sample of urothelial carcinoma / (DNA mass of the urine simulation sample of urothelial carcinoma + the free DNA mass of the human genomic DNA standard product NA12878 with a 0% methylation level)×100%.

7. The reference product according to claim 6, characterized in that: The reference includes a negative reference and / or a positive reference, the DNA methylation level of the negative reference is 0%, and the DNA methylation level of the positive reference is 30%.

8. The reference product according to claim 6, characterized in that: The reference material includes a detection limit reference material, and the DNA methylation level of the detection limit reference material is 5%.

9. The reference product according to claim 6, characterized in that: The reference material includes a precision reference material, and the DNA methylation level of the precision reference material is 0% to 20%.

10. Use of the reference substance according to any one of claims 5 to 9 in the preparation of a product for detecting urothelial carcinoma.

Images