Urine simulation sample, reference product for urothelial carcinoma, and preparation method and application thereof

Abstract

The present invention provides a urine simulation sample, a reference product for urothelial carcinoma, and a preparation method and application thereof. A preparation method of a urine simulation sample for urothelial carcinoma comprises the following steps: S1: preparing a urothelial carcinoma cell culture: digesting T24 cells with trypsin at a concentration of 0.15% - 0.35% v / v, and culturing to obtain a T24 cell suspension; S2: preparing free DNA of urothelial carcinoma: fragmenting the human genomic DNA standard NA12878 with 100% methylation level into free DNA with a length of 150 bp - 300 bp; S3: mixing the urothelial carcinoma cell culture obtained in step S1, the free DNA of urothelial carcinoma obtained in step S2, and a urine sample of a healthy person to obtain a urine simulation sample for urothelial carcinoma. The present invention provides a urine simulation sample and a reference product with a clear background, capable of simulating clinical samples, having low cost, being renewable and having universality.

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] Currently, the detection reference materials available on the market often have some defects, and the research and development and application of current urine detection reagents face many challenges. First, there are significant individual differences in urine samples, which may affect the reliability of test results. Second, during the research and development of urine detection reagents, the lack of reference materials makes performance evaluation uncertain. Many laboratories rely on traditional reference materials, and some of these reference materials have unknown origins and lack sufficient background information. These reference materials often have unknown origins and perform inconsistently under different experimental conditions. In addition, existing reference materials are often costly, difficult to meet the needs of large-scale production, and lack reproducibility, imposing a great economic burden on laboratories and enterprises during research and development.

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

[0007] The lack of detection reference materials not only affects the accuracy and repeatability of experiments but also poses challenges for researchers in developing new detection methods. Therefore, there is an urgent need to develop a urine simulation sample and its detection reference material with a clear background, the ability to simulate clinical samples, low cost, reproducibility, and universality. Summary of the Invention

[0008] Embodiments of the present invention provide a method to at least solve one of the problems existing in the related art. To achieve this purpose, the present invention is realized through the following technical solutions.

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

[0010] S1: Prepare a urothelial carcinoma cell culture: Digest T24 cells with 0.15% - 0.35% v / v trypsin and culture to obtain a T24 cell suspension;

[0011] S2: Prepare free DNA of urothelial carcinoma: Fragment the human genomic DNA standard NA12878 with 100% methylation level into free DNA with a length of 150bp - 300bp;

[0012] S3: Mix the urothelial cancer cell culture in step S1, the cell-free DNA of urothelial cancer in step S2, and the urine sample of a healthy person to obtain a urine mimic sample of urothelial cancer.

[0013] The human bladder transitional cell carcinoma cell line T24 cells, as a cell model of urothelial cancer, can simulate certain biological characteristics of bladder cancer, contribute to the study of 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, facilitating long-term research and experimental repetition. The source of the human genomic DNA standard NA12878 has undergone strict quality control, ensuring the purity and integrity of the 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 various methylation analysis techniques, such as sequencing, fluorescence PCR, etc.

[0014] Preferably, the concentration of trypsin is 0.25% v / v, which can maintain a good cell number and prevent 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 in a 37°C, 5% CO2 incubator for 12 h.

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

[0017] The optimal peak of the cell-free DNA (cfDNA) fragment is 180 bp. The peak of the fragment size of the fragmentation program is approximately 180 bp, and the distribution ratio of the 150 - 300 bp fragments is the highest, 51% (for the human genomic DNA standard NA12878 with 0% methylation level) and 52% (for the human genomic DNA standard NA12878 with 100% methylation level), respectively, which is the optimal fragmentation program.

[0018] In a specific embodiment of the present invention, in step S3, the density of T24 cells in the urine mimic sample is 1 - 2×10 5 cells / mL. The DNA content of cells at this density is more suitable for simulating the urine of urothelial cancer patients.

[0019] The present invention also provides a urine mimic sample of urothelial cancer prepared by the above preparation method.

[0020] The present invention also provides the application of the above urine mimic sample in the preparation of a reference product for detecting urothelial cancer.

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

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

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

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

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

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

[0027] The present invention also provides an application of the reference product 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 product for detecting urothelial carcinoma with a clear background, capable of simulating clinical samples, having low cost, being renewable and universal, providing new ideas and solutions for the research and development of urine detection reagents.

[0030] It has the following advantages:

[0031] (1) Standardization: The simulation sample has standardized components, and the substances they contain are consistent, which can ensure the repeatability and reliability of test results. Ensure the consistency and repeatability of detection results, effectively reduce the misjudgment rate, reduce the occurrence of false positive and false negative results, and thus provide accuracy matching clinical results.

[0032] (2) Controllability: Since the components of the simulation sample are known and controllable, researchers can precisely simulate specific states or concentration levels, flexibly simulate the characteristics of different clinical samples, facilitate the verification of diagnostic tests, and meet different detection requirements. The preparation method can be industrialized, continuously and stably mass-produced, and has good stability between batches and within batches.

[0033] (3) No ethical issues: Using the simulation sample avoids privacy and ethical issues that may be involved in collecting and using real patients' urine.

[0034] (4) Safety: The substances in the simulation sample are known, and there are no pathogens that may be contained in real biological samples. Therefore, it does not pose a health risk to laboratory personnel during processing and analysis.

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

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

[0037] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

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

[0039] Figure 2 Fragmentation and 2100 quality inspection results of the human genomic DNA standard NA12878 with 100% methylation level. Detailed implementation manners

[0040] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following will elaborate on each implementation manner of the present invention with reference to the accompanying drawings. However, those of ordinary skill in the art can understand that in each implementation manner of the present invention, many technical details are proposed to help readers better understand the present application. However, even without these technical details and various changes and modifications based on the following implementation manners, the technical solutions claimed in the present application can still be implemented. The division of the following embodiments is for convenience of description and should not constitute any limitation to the specific implementation manners of the present invention. The various embodiments can be combined and cross-referenced with each other on the premise of no contradiction.

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

[0042] 1. Preparation of urothelial carcinoma cell culture

[0043] Take out the cryopreserved human bladder transitional cell carcinoma T24 cells, quickly place them in a 37°C water bath to thaw, gently shake to accelerate thawing, obtain the thawed T24 cells, transfer the thawed T24 cells to a preheated DMEM high-glucose medium containing 10% (v / v) fetal bovine serum, centrifuge and resuspend the cells to obtain a T24 cell suspension, and transfer the T24 cell suspension to a culture flask and place it in an incubator for culture. When the cells grow to about 80% density in the culture flask, add a 0.25% (v / v) trypsin solution with the same volume as the cell culture medium, gently shake the culture flask to make the trypsin solution completely cover the cells, place the culture flask in the incubator for 2 minutes to digest the cells. When most cells become round and non-adherent under the microscope and a large number of cells detach after gently shaking and tapping both sides of the culture flask, add a DMEM high-glucose medium containing 10% (v / v) fetal bovine serum with twice the volume of trypsin to terminate digestion, and gently pipette the cells several times to completely detach all cells to obtain the cells with digestion terminated; centrifuge the cells with digestion terminated at 1100 rpm at room temperature for 4 minutes, discard the supernatant, resuspend the cells with a DMEM high-glucose medium containing 10% (v / v) fetal bovine serum, set the stirring speed to 60 r / min, and culture in a 37°C, 5% CO2 incubator for 12 hours to obtain a urothelial carcinoma cell culture.

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

[0045] Fragment the human genomic DNA standard NA12878 with 0% methylation level and the human genomic DNA standard NA12878 with 100% methylation level respectively to prepare cell-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 fragmenter (Covaris M-Series), turn on the SonoLab 7.2 software, and select the corresponding program.

[0048] (2) Place the test tube plate marked as microTUBE 130μL on the Tube Holder, 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 cryopreserved sample (human genomic DNA standard NA12878 with 0% methylation level or human genomic DNA standard NA12878 with 100% methylation level), add 500 ng of the sample to the test tube to make the total sample system volume 50 μL. If it is less than 50 μL, make it up to 50 μL with ethidium bromide (EB).

[0050] (4) Vortex and mix the sample system in step (3), centrifuge briefly, place the test tube on the microTUBE 130μL test tube plate processed in step (2), press the test tube with the Sliding weight, and close the Safety Cover.

[0051] (5) Set the fragmentation program and check whether the program is correct: set the peak power to 50 w, the duty cycle to 30%, the number of cycles for each fragmentation to 200, the processing time to 300 s, the temperature to 20 °C, and the sample volume to 50 μL; after the 3 statuses in Instrument Status on the software interface all show "√", click "RUN" on the software interface to start running the program and obtain the fragmented sample, which is the cell-free DNA of urothelial carcinoma.

[0052] 3. Prepare the reference

[0053] (1) DNA of urine simulation sample: The urine simulation sample was obtained by mixing urothelial carcinoma cell culture, free DNA after fragmentation of the human genomic DNA standard NA12878 with 100% methylation level, and urine samples from healthy individuals. The volume ratio of urothelial carcinoma cell culture to urine samples from healthy individuals was 1:4 - 9, so that the density of T24 cells in the urine simulation sample was 1 - 2×10 5 cells / ml, and the concentration of free DNA after fragmentation of the human genomic DNA standard NA12878 with 100% methylation level was 50 ng / μL. The DNA of the urine simulation sample was extracted using the MagMAX TM Free DNA Isolation Kit from ThermoFisher, and the DNA of the urine simulation sample was obtained. After verification by digital PCR, its methylation level was 100%.

[0054] (2) Positive reference: The DNA of the urine simulation sample and free DNA of the human genomic DNA standard NA12878 with 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: Free DNA of the human genomic DNA standard NA12878 with 0% methylation level was diluted 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: The DNA of the urine simulation sample and negative human genomic DNA were mixed at a mass ratio of 1:19 to obtain a detection limit reference with a concentration of 50 ng / μL and a methylation level of 5%.

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

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

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

[0060] Example 2 Effects of Trypsin at Different Concentrations on the Number and Aggregate Particle Size of Shed Urinary Cells T24

[0061] I. Experimental Method

[0062] The concentrations of the trypsin solution were set to 0.15% (v / v), 0.25% (v / v), and 0.35% (v / v) respectively. Three portions of 1×10 6 T24 cells were selected and urothelial carcinoma cell cultures were obtained by digestion and culture according to the method of Example 1.

[0063] The cells were counted and the particle size distribution of cell aggregates was detected. When the cell aggregates were loose, they were stained with trypan blue and counted using a hemocytometer. When the cell aggregates were severe, they were counted by the crystal violet staining method. The particle size distribution of cell aggregates was measured using a MALVERN laser particle size analyzer.

[0064] II. Experimental Results

[0065] As shown in Table 1, the effect of trypsin at 0.25% (v / v) on digesting T24 cells was the best. When the concentration of trypsin exceeded 0.25% (v / v), the particle size of cell aggregates exceeded the cell diameter, indicating the occurrence of cell aggregation. When the concentration of trypsin was lower than 0.25% (v / v), the number of digested cells decreased.

[0066] Table 1

[0067]

[0068] Example 3 Determination of the addition amount of urine exfoliated cells

[0069] Different numbers of T24 cells were selected respectively, and DNA extraction and DNA concentration detection were carried out using an Ezup column adherent cell genomic DNA extraction kit (manufacturer: Sangon Biotech, product number: B518251).

[0070] Table 2

[0071]

[0072] For tumor patients, 200 mL of urine corresponds to a total DNA amount of 500 - 1000 ng. According to the data provided in Table 2, 1 - 2×10 5 T24 cells can be added.

[0073] Example 4 Preparation of fragmentation conditions for free DNA

[0074] I. Experimental Method

[0075] According to the method of Example 1, the human genomic DNA standard NA12878 with 0% methylation level and the human genomic DNA standard NA12878 with 100% methylation level were fragmented respectively to prepare cell-free DNA (cfDNA), and 2100 quality inspection was carried out to check the proportion of the corresponding fragments after fragmentation.

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

[0077] Table 3

[0078]

[0079] Table 4

[0080]

[0081] Table 5

[0082]

[0083] II. Experimental Results

[0084] The fragmentation procedure and results of the human genomic DNA standard NA12878 sample with 0% methylation level are shown in Table 6 and Figure 1 as shown. Among the 11 fragmentation procedures shown in Table 6, the peak fragment sizes of numbers 5, 6, 10, and 11 are around 180 bp. Among them, the proportion of 150 bp - 300 bp in the fragmentation procedure numbered 10 is the highest.

[0085] The sample information of the human genomic DNA standard NA12878 sample with 100% methylation level is shown in Table 6 and Figure 2 as shown. Among the 11 fragmentation procedures shown in Table 6, the peak fragment sizes of numbers 5, 6, 10, and 11 are around 180 bp. Among them, the proportion of 150 bp - 300 bp in the fragmentation procedure numbered 10 is the highest.

[0086] Table 6

[0087]

[0088] Table 7

[0089]

[0090] As shown in Table 7, it is the result of the fragmentation procedure numbered 10. The median concentration of cfDNA in urine is 16.70 ng / μL. The cfDNA fragments less than 10 kb are mainly distributed around 180 bp. The best peak of the cfDNA fragment is 180 bp. In the experiment, the peak fragment size of the fragmentation procedure numbered 10 is about 180 bp, and the distribution proportion of 150 - 300 bp fragments is the highest, 51% and 52% respectively, which is the most suitable fragmentation procedure.

[0091] Example 5 Verifying the Reference Product for Detecting Urothelial Carcinoma

[0092] I. Experimental Methods

[0093] Verify the concentration of the reference product prepared in Example 1: Take 3 batches of reference products prepared continuously, with 3 reference products in each batch. The concentration of each reference product is detected using a Qubit fluorescence quantitative analyzer. The methylation level is detected using the Human Twist1 Gene Methylation Detection Kit (fluorescent PCR method) of Anhui Dajian Medical Technology Co., Ltd., and the experiment is repeated 5 times for each batch.

[0094] II. Experimental Results

[0095] As shown in Table 8, the results of the re-determined concentration of the reference products are all within the error range of 10%, and the CV values are all less than 5%, indicating good stability between batches and within batches.

[0096] As shown in Table 9, all the positive reference products prepared in 3 consecutive batches are positive, with a compliance rate of 100%; all the negative reference products are negative, with a compliance rate of 100%; all the detection limit reference products can be detected, with a detection rate of 100%; for the precision reference product 1, the experiment is repeated 15 times, and all the results are negative; for the precision reference product 2, the experiment is repeated 15 times, and all the results are positive; for the precision reference product 3, the experiment is repeated 15 times, and all the results are positive. It shows that the reference products prepared by the present invention are detected and confirmed to be effective, can provide a unified standard for the experimental or production process, help to establish comparability among different times, places and operators, and can ensure the traceability and reproducibility of test results.

[0097] Table 8 Results of Concentration Determination of Enterprise Reference Products (Concentration Unit: ng / μL)

[0098]

[0099] Table 9 Results of Kit Detection of Enterprise Reference Products

[0100]

[0101] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made therein without departing from the principles and spirit of the present invention, and 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: Preparation of urothelial cancer cell culture: T24 cells were digested with 0.25% v / v trypsin and cultured to obtain a T24 cell suspension; S2: Preparation of free DNA of urothelial carcinoma: The 100% methylated human genomic DNA standard NA12878 was broken into free DNA with a length of 150 bp to 300 bp; S3: mixing 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, so as to obtain a simulated urine sample of urothelial cancer; the volume ratio of the urothelial cancer cell culture to the urine sample of a healthy person is 1:4-9; In step S1, the density of T24 cells in the T24 cell suspension is 1-2×10 5 Pieces / mL; The concentration of free DNA after shearing from the 100% methylated human genomic DNA standard NA12878 is 50ng / μL.

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

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

4. 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 2 and free DNA of the human genomic DNA standard NA12878 with a 0% methylation level.

5. The reference product according to claim 4, characterized in that: 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 NA12878 with a 0% methylation level) × 100%.

6. Use of the reference substance according to any one of claims 4 to 5 in the preparation of a product for detecting urothelial carcinoma.

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