Methylation detection reagent and urothelial carcinoma detection kit containing same

Abstract

The invention relates to the technical field of biological detection, in particular to a methylation detection reagent and a urinary tract epithelial cancer detection kit containing the methylation detection reagent. The conversion liquid comprises water, ammonium hydrogen sulfite and sodium hydrogen sulfite, the concentration of the ammonium hydrogen sulfite is 30wt%-90wt%, and the weight ratio of the ammonium hydrogen sulfite to the sodium hydrogen sulfite is (1-50): 1. In the methylation detection process, complete transformation can be realized in a short time by adopting the transformation liquid provided by the invention.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a methylation detection reagent and a urothelial carcinoma detection kit containing the same. Background Art

[0002] Nucleic acid methylation conversion technologies mainly include bisulfite conversion, enzymatic conversion, and methylation-sensitive restriction endonuclease technology. Among them, bisulfite conversion is one of the most commonly used methods for DNA methylation detection. The principle is to treat DNA with bisulfite to convert unmethylated cytosine (C) into uracil (U), while methylated cytosine remains unchanged. Although bisulfite conversion has many advantages over other methods, it still has the disadvantage of being time-consuming for complete conversion.

[0003] In view of this, this application is hereby filed. Summary of the invention

[0004] Based on this, one or more embodiments of the present application provide a methylation detection reagent and a urothelial carcinoma detection kit containing the same, including the following technical solutions:

[0005] One or more embodiments of the present application provide a methylation detection reagent, the reagent comprising a conversion solution;

[0006] The conversion liquid includes water, ammonium bisulfite and sodium bisulfite. The concentration of the ammonium bisulfite is 30wt%-90wt%, and the weight ratio of the ammonium bisulfite to the sodium bisulfite is (1-50):1.

[0007] In some embodiments of the present application, the reagent further comprises a desulfurization liquid;

[0008] The desulfurization liquid comprises a NaOH solution and isopropanol, wherein the volume proportion of the isopropanol in the desulfurization liquid is 50%-90%, and the concentration of NaOH in the NaOH solution is 30mmol / L-600mmol / L.

[0009] In some embodiments of the present application, the reagent further comprises a binding solution; the binding solution comprises water and 10mmol / L-500mmol / L Tris-HCl, 1mol / L-6mol / L guanidine salt and 1mmol / L-100mmol / L EDTA, pH 7.5-8.0;

[0010] Optionally, the guanidine salt includes one or more of guanidine hydrochloride and guanidine isothiocyanate.

[0011] In some embodiments of the present application, the reagent further comprises a rinse solution;

[0012] The rinse solution includes water, pH 7.5-8.0, 10mmol / L-2000mmol / L Tris-HCl, 0.05mol / L-6mol / LNaCl and 20%-82% ethanol by volume, pH 7.5-8.0.

[0013] One or more embodiments of the present application also provide a urothelial carcinoma detection kit, which includes the methylation detection reagent.

[0014] In some embodiments of the present application, the detection kit further comprises:

[0015] Any one of the primer-probe combinations shown in SEQ ID NO.1 to SEQ ID NO.3, the primer-probe combinations shown in SEQ ID NO.4 to SEQ ID NO.6, the primer-probe combinations shown in SEQ ID NO.7 to SEQ ID NO.9, the primer-probe combinations shown in SEQ ID NO.10 to SEQ ID NO.12, and the primer-probe combinations shown in SEQ ID NO.13 to SEQ ID NO.15;

[0016] as well as,

[0017] Any primer-probe combination among the primer-probe combination shown in SEQ ID NO.16 to SEQ ID NO.18, the primer-probe combination shown in SEQ ID NO.19 to SEQ ID NO.21, the primer-probe combination shown in SEQ ID NO.22 to SEQ ID NO.24, the primer-probe combination shown in SEQ ID NO.25 to SEQ ID NO.27, and the primer-probe combination shown in SEQ ID NO.28 to SEQ ID NO.30.

[0018] In some embodiments of the present application, the detection kit further includes a primer-probe combination shown in SEQ ID NO.31 to SEQ ID NO.33.

[0019] One or more embodiments of the present application further provide a method for methylation treatment of urothelial carcinoma samples, the method comprising the step of using the methylation detection reagent to treat the DNA of the sample to be tested.

[0020] In some embodiments of the present application, the sample to be tested includes a urine sample.

[0021] One or more embodiments of the present application further provide a method for detecting a urothelial carcinoma sample, the method comprising: treating the DNA of the sample to be tested by the methylation treatment method, and detecting the treated DNA;

[0022] Optionally, the detection is performed by fluorescent quantitative PCR;

[0023] Further optionally, the fluorescent quantitative PCR method uses the primer-probe combination defined above.

[0024] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. DETAILED DESCRIPTION

[0025] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0027] the term

[0028] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0029] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0030] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0031] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0032] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0033] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0034] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0035] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0036] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0037] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0038] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0039] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0040] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0041] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0042] In a first aspect of an embodiment of the present application, a methylation detection reagent is provided, the reagent comprising a conversion solution;

[0043] The conversion liquid includes water, ammonium bisulfite and sodium bisulfite. The concentration of the ammonium bisulfite is 30wt%-90wt%, and the weight ratio of the ammonium bisulfite to the sodium bisulfite is (1-50):1.

[0044] The conversion solution is suitable for an injection volume of 100 pg-1 μg, and complete conversion can be achieved at 95° C. for 5 minutes. Optionally, the concentration of the ammonium bisulfite is 50wt%-90wt%. Optionally, the weight ratio of the ammonium bisulfite to the sodium bisulfite is (10-50):1. Further optionally, the concentration of the ammonium bisulfite is 50wt%-90wt%, and the weight ratio of the ammonium bisulfite to the sodium bisulfite is (10-50):1.

[0045] In some of the examples, the reagent also includes a desulfurization liquid; the desulfurization liquid includes a NaOH solution and isopropanol, the volume proportion of the isopropanol in the desulfurization liquid is 50%-90%, and the concentration of NaOH in the NaOH solution is 30mmol / L-600mmol / L. Optionally, the desulfurization liquid includes a NaOH solution and isopropanol, the volume proportion of the isopropanol in the desulfurization liquid is 60%-90%, and the concentration of NaOH in the NaOH solution is 300mmol / L-600mmol / L. The final methylation conversion rate of the DNA sample treated with the desulfurization liquid is greater than 99.5%, and does not affect the downstream gene amplification detection.

[0046] In some examples, the reagent further comprises a binding solution; the binding solution comprises water, 10 mmol / L-500 mmol / L Tris-HCl, 1 mol / L-6 mol / L guanidine salt and 1 mmol / L-100 mmol / L EDTA, pH 7.5-8.0.

[0047] Optionally, the guanidine salt includes one or more of guanidine hydrochloride and guanidine isothiocyanate.

[0048] Further optionally, the combination solution includes water and 100mmol / L-500mmol / L Tris-HCl, 1mol / L-6mol / L guanidine salt and 1mmol / L-100mmol / L EDTA, pH7.5-8.0. In some examples, the reagent also includes a rinse solution; the rinse solution includes water and pH7.5-8.0, 10mmol / L-2000mmol / L Tris-HCl, 0.05mol / L-6mol / L NaCl and ethanol with a volume ratio of 20%-82%, pH7.5-8.0. Optionally, the rinse solution includes water and pH7.5-8.0, 10mmol / L-100mmol / L Tris-HCl, 0.05mol / L-6mol / L NaCl and ethanol with a volume ratio of 20%-82%, pH7.5-8.0. .

[0049] The water in the embodiments of the present application includes nuclease-free water.

[0050] Urothelial carcinoma (UC) is a common malignant tumor worldwide, including bladder cancer, renal pelvic cancer and ureteral cancer. In China, the incidence of UC ranks first among genitourinary tumors. Early diagnosis is crucial for the prognosis of UC patients, because the five-year survival rate of patients with limited-stage UC can exceed 90%, while once muscle invasion or metastasis occurs, the patient's survival and quality of life will be significantly reduced.

[0051] Currently, the diagnostic methods for urothelial carcinoma include cystoscopy, urine exfoliated cytology, fluorescence in situ hybridization (FISH), etc., but these methods have certain limitations. For example, cystoscopy combined with biopsy, as the "gold standard" for the diagnosis of urothelial carcinoma, although highly accurate, is not suitable for large-scale screening due to its high invasiveness and high price. Therefore, researchers are looking for more convenient, non-invasive, sensitive and specific early diagnosis methods.

[0052] In recent years, urine DNA testing has shown great potential as a non-invasive detection method. By detecting DNA copy number variation and methylation variation in urine, tumors and normal tissues can be accurately predicted. Professor He Zhisong's team from the First Affiliated Hospital of Peking University and Professor Ye Dingwei's team from Shanghai Fudan Cancer Hospital, in collaboration with Golden Oak Medical, published data on the world's first urine early screening and efficacy monitoring product for urothelial cancer, meeting the needs of more accurate, convenient and non-invasive diagnosis and treatment of urothelial cancer.

[0053] In addition, the Urothelial Carcinoma Expert Committee of the Chinese Society of Clinical Oncology (CSCO) released the "Guidelines for the Diagnosis and Treatment of Urothelial Carcinoma (2020 Edition)", which provides important reference for the diagnosis and treatment of urothelial carcinoma in China, including guidance for early diagnosis.

[0054] In general, the research background of early diagnosis of urothelial carcinoma focuses on improving diagnostic accuracy, reducing invasiveness, and increasing patient compliance, while emphasizing the combination of domestic and foreign research results and China's actual clinical situation in order to improve patients' prognosis and quality of life.

[0055] The methylation detection reagent provided in the embodiments of the present application can be used for the detection of urothelial carcinoma, etc., and provides a new solution for the clinical detection of urothelial carcinoma.

[0056] A second aspect of an embodiment of the present application provides a urothelial carcinoma detection kit, wherein the detection kit comprises the methylation detection reagent.

[0057] In some examples, the detection kit further includes: any one of the primer-probe combinations shown in SEQ ID NO.1 to SEQ ID NO.3, the primer-probe combinations shown in SEQ ID NO.4 to SEQ ID NO.6, the primer-probe combinations shown in SEQ ID NO.7 to SEQ ID NO.9, the primer-probe combinations shown in SEQ ID NO.10 to SEQ ID NO.12, and the primer-probe combinations shown in SEQ ID NO.13 to SEQ ID NO.15; and,

[0058] Any primer-probe combination among the primer-probe combination shown in SEQ ID NO.16 to SEQ ID NO.18, the primer-probe combination shown in SEQ ID NO.19 to SEQ ID NO.21, the primer-probe combination shown in SEQ ID NO.22 to SEQ ID NO.24, the primer-probe combination shown in SEQ ID NO.25 to SEQ ID NO.27, and the primer-probe combination shown in SEQ ID NO.28 to SEQ ID NO.30.

[0059] In some examples, the detection kit also includes a primer-probe combination shown in SEQ ID NO.31 to SEQ ID NO.33.

[0060] According to a third aspect of the embodiments of the present application, a method for methylation treatment of a urothelial carcinoma sample is provided, wherein the method comprises the step of using the methylation detection reagent to treat the DNA of the sample to be tested.

[0061] In some examples, the sample to be tested includes a urine sample.

[0062] In a fourth aspect of the embodiments of the present application, a method for detecting a urothelial carcinoma sample is provided, the method comprising: treating the DNA of the sample to be tested by the methylation treatment method, and detecting the treated DNA;

[0063] Optionally, the detection is performed by fluorescent quantitative PCR;

[0064] Further optionally, the fluorescent quantitative PCR method uses the primer-probe combination defined above.

[0065] Table 1. Primer pairs and probes for detecting TWIST1 gene

[0066]

[0067]

[0068] Table 2. Primer pairs and probes for detecting PENK gene

[0069]

[0070] Table 3. Primer pairs and probes for detecting GAPDH gene

[0071]

[0072] In Tables 1 to 3, all primer sequences are from 5' end to 3' end.

[0073] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0074] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0075] Example 1

[0076] 1. Take 100 μL of conversion solution and add it to a 1.5 mL centrifuge tube containing 50 μL of DNA sample, mix well, and centrifuge briefly to ensure that there is no residual liquid on the tube cap and tube wall.

[0077] If the volume of the DNA sample is less than 50 μL, it needs to be made up to 50 μL with water. The volume of the conversion solution added to the DNA sample must also be reduced by the same volume as the amount of DNA sample is increased, and the total reaction volume remains 150 μL.

[0078] 2. Place the above 1.5 mL centrifuge tube on a PCR instrument or metal bath instrument and run the following program: 95°C / 5min.

[0079] 3. Add 500 μL of binding solution to the centrifuge tube, shake to mix and centrifuge briefly.

[0080] 4. Add all the reaction solution in the tube directly to the purification column, centrifuge at ≥10,000×g for 1 minute, and discard the filtrate.

[0081] 5. Add 400 μL of the prepared rinse solution to the purification column and centrifuge at ≥10,000 × g for 1 minute.

[0082] 6. Add 200 μL of desulfurization solution to the purification column, place at room temperature (20-30°C) for 15-20 minutes, and centrifuge at ≥10,000×g for 1 minute.

[0083] 7. Add 400 μL of the prepared rinse buffer to the purification column and centrifuge at ≥10,000 × g for 1 minute.

[0084] 8. Repeat step 7.

[0085] 9. Transfer the purification column to a clean 1.5 mL centrifuge tube, draw ≥ 10 μL of rinse solution and add it directly to the column matrix. Place at room temperature for 3-5 minutes, then centrifuge at full speed for 1 minute to elute the DNA.

[0086] The DNA can be analyzed immediately or stored at -20°C or lower (-80°C) for later use. Use 1-5 μL of eluted DNA per PCR, up to 10 μL can be used. The elution volume can be ≥10 μL, depending on the experimental requirements, but smaller elution volumes will produce higher DNA concentrations.

[0087] In this embodiment:

[0088] Conversion solution: Taking 50mL conversion solution as an example, take 48mL of ammonium bisulfite solution, of which the mass of ammonium bisulfite is 33.6g, and add 2mL of 1g / mL sodium bisulfite solution, of which the weight of sodium bisulfite is 2g. The weight ratio of ammonium bisulfite to sodium bisulfite in the conversion solution is 16.8:1.

[0089] Binding solution: Taking the preparation of 50 mL of binding solution as an example, weigh 11.82 g of guanidine thiocyanate (2 mol / L), measure 5 mL of 1 M pH 7.5 Tris-HCl (100 mmol / L pH 7.5 Tris-HCl), 500 μL of 0.5 M EDTA (5 mmol / L EDTA) and 44.5 mL of nuclease-free water, and mix them.

[0090] Desulfurization liquid: 300mmol / L NaOH solution and isopropanol, the volume proportion of isopropanol in the desulfurization liquid is 90%.

[0091] Rinsing solution: Take the preparation of 50 mL of rinsing solution as an example: mix 500 μL of 1M pH 7.5 Tris-HCl (10 mmol / L pH7.5 Tris-HCl), 1.67 mL of 3M NaCl, 7.83 mL of nuclease-free water and 40 mL of 80% ethanol. The resulting rinsing solution contains 10 mM Tris-HCl, about 0.1 M NaCl and about 80.8% ethanol by volume.

[0092] Results: The reagent formula in this embodiment is the best when compared with other cross-experimental conditions. The specific process is as follows: In order to obtain the DNA conversion efficiency of the converted and purified DNA samples, the unconverted DNA samples are used as the blank control group to calculate their sulfite conversion efficiency. Specifically, in the above-mentioned converted and purified DNA samples, only the step of supplementing the reaction volume to 100 μL with nuclease-free water is used to replace the conversion solution, and the other steps are the same as the above-mentioned conversion and purification of DNA samples to obtain an unconverted DNA solution. Take 5 μL of the above-mentioned converted and purified DNA samples and unconverted DNA samples, add primer pairs and probes for detecting the TWIST1 gene, respectively, and perform fluorescent PCR quantitative analysis.

[0093] The standard mutation rate of 10% in the table is a mixed sample of TWIST1 gene methylation 10% solution and PENK gene methylation 10% solution; the standard mutation rate of 5% in the table is a mixed sample of TWIST1 gene methylation 5% solution and PENK gene methylation 5% solution. The results are as follows:

[0094] Table 4

[0095] Standard (mutation frequency) Ct value of DNA samples after transformation and purification Ct value of unconverted DNA sample 10％ 30.78 38.86 5％ 32.15 39.19

[0096] For the DNA samples with 10% mutation frequency and 5% mutation frequency, the difference between the Ct value of the converted and purified DNA samples and the Ct value of the unconverted DNA samples was greater than 7, thus judging that the conversion rate was greater than 99%.

[0097] Example 2

[0098] 1. Methylation treatment

[0099] 1. Take 100 μL of conversion solution and add it to a 1.5 mL centrifuge tube containing 50 μL of DNA sample, mix well, and centrifuge briefly to ensure that there is no residual liquid on the tube cap and tube wall.

[0100] If the volume of the DNA sample is less than 50 μL, it needs to be made up to 50 μL with water. The volume of the conversion solution added to the sample must also be reduced by the same volume as the sample, and the total reaction volume remains 150 μL.

[0101] 2. Place the 1.5 mL centrifuge tube on a PCR instrument or metal bath instrument and run the following program: 95°C / 5 min.

[0102] 3. Reagent plate adding method (32-bit nucleic acid extraction instrument)

[0103] Table 5

[0104]

[0105] In this embodiment:

[0106] Conversion solution: Take 50mL conversion solution as an example: take 48mL of ammonium bisulfite solution, of which the mass of ammonium bisulfite is 33.6g, and add 2mL of 1g / mL sodium bisulfite solution, of which the weight of sodium bisulfite is 2g. The weight ratio of ammonium bisulfite to sodium bisulfite is 16.8:1.

[0107] Binding solution: Taking the preparation of 50 mL of binding solution as an example, weigh 11.82 g of guanidine thiocyanate, measure 5 mL of 1 M pH 7.5 Tris-HCl (100 mmol / L pH 7.5 Tris-HCl), 500 μL of 0.5 M EDTA (5 mmol / L EDTA) and 44.5 mL of nuclease-free water, and mix them.

[0108] Desulfurization liquid: 300mmol / L NaOH solution and isopropanol, the volume proportion of isopropanol in the desulfurization liquid is 90%.

[0109] Rinsing solution: Take the preparation of 50mL of rinsing solution as an example: mix 500μL 1M pH 7.5 Tris-HCl (10mmol / L pH7.5 Tris-HCl), 1.67mL 3M NaCl, 7.83mL nuclease-free water and 40mL 80% ethanol. The Tris-HCl in the resulting rinsing solution is 10mM, NaCl is about 0.1M, and the volume of ethanol accounts for about 80.8%. 4. Place the 96-well plate loaded with reagents flat on the table.

[0110] 5. Add 150 μL of the mixed solution and 20 μL of magnetic beads in the centrifuge tube in step 2 to well 1 / 7.

[0111] 6. Place the 96-well plate on the base of the nucleic acid extractor, insert the 8-position magnetic sleeve rod into the card slot of the nucleic acid extractor (do not forget to put in the magnetic sleeve), and run the corresponding automated purification and extraction program.

[0112] 7. After the program is completed, transfer the eluted product in well 6 / 12 to a new 1.5mL centrifuge tube and store it at -20℃.

[0113] Table 6. Parameters of purification program of 32-bit nucleic acid extractor

[0114] step Hole Position name Waiting time Mixing time Magnetic suction time Volume Mixing speed temperature 1 1 / 7 Cleavage + Binding 0s 120s 30s 800μL 3 OFF 2 2 / 8 rinsing 0s 20s 30s 600μL 3 OFF 3 3 / 9 Desulfurization 0s 300s 30s 400μL 3 OFF 4 4 / 10 rinsing 0s 20s 30s 600μL 3 OFF 5 5 / 11 rinsing 0s 60s 30s 600μL 3 OFF 6 6 / 12 Elution 300s 180s 60s 100μL 2 60℃ 7 4 / 10 Discard magnetic beads 0s 15s 0s 600μL 3 OFF

[0115] 2. Urothelial Carcinoma Detection

[0116] 1. Sample selection

[0117] 370 urine samples were selected, including 104 urine samples from patients with urothelial carcinoma, 114 urine samples from patients with interfering diseases, and 152 urine samples from healthy people. Sample inclusion criteria:

[0118] The following conditions were selected for inclusion in the urothelial carcinoma group: patients with clinical or case-diagnosed urothelial carcinoma;

[0119] Interference sample inclusion group: subjects clinically diagnosed with benign urinary system diseases;

[0120] The healthy group was selected under three conditions: (1) according to the questionnaire, subjects with common chronic diseases (hypertension, diabetes, coronary heart disease), tumor treatment history and major surgery history were excluded; (2) self-reported no obvious clinical symptoms; (3) physical examination results were within the normal range and no obvious abnormalities were found; and the examination results were beyond the normal range or abnormal but judged by the doctor to have no clinical significance. (Physical examination items include: LDCt, abdominal color Doppler ultrasound, 4 tumor markers, blood pressure, etc.).

[0121] Exclusion criteria: under 18 years old; pregnant; duplicate samples, undiagnosed, or unqualified quality inspection.

[0122] The steps for collecting urine sediment and urine supernatant samples are as follows: transfer 10-200 mL of urine to a suitable centrifuge tube (50 mL centrifuge tube) and centrifuge at 3000×g for 15 min; transfer the supernatant to a new centrifuge tube for storage, and keep 100-200 μL of urine cell sediment, blow it evenly, and transfer it to a 2 mL centrifuge tube.

[0123] 2. DNA Extraction

[0124] Cellular DNA / cell-free DNA can be easily purified from up to 40 mL of urine using the Quick-DNATM Urine Kit Catalog No. D3061 (ZYMO RESEARCH). DNA Yield - The DNA binding capacity of the column is 5 μg.

[0125] Please note that DNA yields may vary depending on the urine itself. Female urine generally yields more DNA than male urine. The average range of urine DNA amounts for healthy female individuals is 6ng / mL-1000ng / mL. The average range for healthy male individuals is 2-20ng / mL.

[0126] 3. Methylation detection

[0127] The extracted and purified DNA was subjected to methylation conversion according to the first part above. The PCR reaction system of TWIST1, PENK and internal reference gene GAPDH was prepared according to Table 6, and the primer pairs and probes used were as shown in Table 1. Nucleic acid combination 5 (TWIST1-F5: SEQ ID NO.13; TWIST1-R5: SEQ ID NO.14; TWIST1-P5: SEQ ID NO.15), Table 2. Nucleic acid combination 10 (PENK-F4: SEQ ID NO.28; PENK-R4: SEQ ID NO.29; PENK-P4: SEQ ID NO.30), and Table 3. Nucleic acid combination 11.

[0128] Table 7. Reaction system

[0129] Element Dosage 2×qPCR Mix (including taq enzyme, reaction buffer, dNTP, etc.) 12.5μL Forward primer (10 μM) 0.5μL Reaction primer (10 μM) 0.5μL Probe (10 μM) 0.5μL Nuclease-free water 6μL Bis-DNA (amplification template, concentration 0.2ng / μL) 5.0μL Total volume 25μL

[0130] Table 8. Reaction conditions

[0131]

[0132] Standard reference: The positive control is bladder cancer tissue DNA; the negative control is leukocyte DNA. The reaction system and reaction conditions of the corresponding controls are carried out according to Table 7 and Table 8.

[0133] 4. Judgment basis

[0134] The Ct values ​​of the internal reference gene, TWIST1 gene, and PENK gene were expressed as Ct0, Ct1, and Ct2, respectively, ΔCt1 = |Ct1-Ct0|, ΔCt2 = |Ct2-Ct0|, and a Ct0 value greater than 35 was considered unqualified. The result was determined under the condition that the Ct0 value was ≤35.

[0135] Table 9. Judgment basis for combined detection of TWIST1 gene and PENK gene

[0136]

[0137] Under the condition of single PENK gene testing, the following judgment criteria are used: Ct2≤35 and △Ct2<8, it is judged as positive; other situations are judged as negative.

[0138] Table 10. Urine sediment test results

[0139]

[0140] Table 11. Urine supernatant test results

[0141]

[0142] The DNA extracted from all samples was sent to a third party for testing, and the methylation mutation frequencies of TWIST1 and PENK in the samples were evaluated by first-generation sequencing. The results were consistent with the test results of this example.

[0143] Embodiment 3, Embodiment 4

[0144] Example 3 and Example 4 are respectively variations of Example 1, and the only difference from Example 1 is the formulation of the detection reagent. For details, please refer to the following table:

[0145] Table 12

[0146]

[0147] The standards were processed and tested respectively, and the conversion time was 5 minutes. Among them, the standard mutation rate 10% in the table is a mixed sample of TWIST1 gene methylation 10% solution and PENK gene methylation 10% solution; the standard mutation rate 5% in the table is a mixed sample of TWIST1 gene methylation 5% solution and PENK gene methylation 5% solution.

[0148] Table 13

[0149] Standard (mutation frequency) Example 1 / Ct Example 3 / Ct Example 4 / Ct 10％ 30.23 30.47 29.86 5％ 32.05 31.97 32.41

[0150] Using standards with different mutation frequencies as test templates, in the gene amplification after treatment, the difference between the Ct value of Example 1 and the Ct values ​​in Examples 3 and 4 is within 0.5, so Examples 1, 3 and 4 can all achieve complete transformation in a short time.

[0151] Example 5

[0152] This embodiment is a variation of embodiment 1, and the main difference from embodiment 1 is that isopropanol in the desulfurization solution is replaced by ethanol. The standard mutation rate 10% in the table is a mixed sample of a 10% solution of TWIST1 gene methylation and a 10% solution of PENK gene methylation; the standard mutation rate 5% in the table is a mixed sample of a 5% solution of TWIST1 gene methylation and a 5% solution of PENK gene methylation.

[0153] Table 14

[0154] Standard (mutation frequency) Example 1 Example 5 10％ 30.23 34.67 5％ 32.63 37.01

[0155] Using standards with different mutation frequencies as test templates, the Ct value of Example 1 in the gene amplification after treatment is significantly better than that of Example 5. Therefore, the desulfurization liquid of Example 5 is not within the reference range of subsequent experiments.

[0156] Comparative Example 1

[0157] This comparative example is a comparative example of Example 1, and the main difference from Example 1 is that the ammonium bisulfite in the conversion solution is replaced by ammonium sulfite. The standard mutation rate 10% in the table is a mixed sample of a 10% solution of TWIST1 gene methylation and a 10% solution of PENK gene methylation; the standard mutation rate 5% in the table is a mixed sample of a 5% solution of TWIST1 gene methylation and a 5% solution of PENK gene methylation.

[0158] Example 6

[0159] This embodiment is a variation of embodiment 1. The main difference from embodiment 1 is the length of the transformation incubation time. The transformation time of embodiment 1 is 5 minutes, and the transformation time of this embodiment is 2 hours. The standard mutation rate 10% in the table is a mixed sample of TWIST1 gene methylation 10% solution and PENK gene methylation 10% solution; the standard mutation rate 5% in the table is a mixed sample of TWIST1 gene methylation 5% solution and PENK gene methylation 5% solution.

[0160] Table 15

[0161] Standard (mutation frequency) Example 1 / Ct Comparative Example 1 / Ct Example 6 / Ct 10％ 30.23 33.19 32.56 5％ 32.05 35.38 36.72

[0162] Using standards with different mutation frequencies as test templates, the Ct value of Example 1 in the gene amplification after treatment is significantly better than that of Comparative Example 1 and Example 6. Therefore, the transformation conditions of Comparative Example 1 and Example 6 are not within the reference range of subsequent experiments.

[0163] Comparative Example 2

[0164] This comparative example is a comparative example of Example 1, and the main difference from Example 1 is that the sodium bisulfite in the conversion solution is replaced by sodium metabisulfite. The standard mutation rate 10% in the table is a mixed sample of a 10% solution of TWIST1 gene methylation and a 10% solution of PENK gene methylation; the standard mutation rate 5% in the table is a mixed sample of a 5% solution of TWIST1 gene methylation and a 5% solution of PENK gene methylation.

[0165] Example 7

[0166] This embodiment is a variation of embodiment 1. The main difference from embodiment 1 is the length of the transformation incubation time. The transformation time of embodiment 1 is 5 minutes, and the transformation time of this embodiment is 1 hour. The standard mutation rate 10% in the table is a mixed sample of TWIST1 gene methylation 10% solution and PENK gene methylation 10% solution; the standard mutation rate 5% in the table is a mixed sample of TWIST1 gene methylation 5% solution and PENK gene methylation 5% solution.

[0167] Table 16

[0168]

[0169]

[0170] Using standards with different mutation frequencies as test templates, the Ct value of Example 1 in the gene amplification after treatment is significantly better than that of Comparative Example 2 and Example 7. Therefore, the transformation conditions of Comparative Example 2 and Example 7 are not within the reference range of subsequent experiments.

[0171] Comparative Example 3

[0172] This comparative example is a comparative example of Example 1, and the main difference from Example 1 is that the weight ratio of ammonium bisulfite to sodium bisulfite in the conversion solution is 0.5:1. The standard mutation rate 10% in the table is a mixed sample of a 10% solution of TWIST1 gene methylation and a 10% solution of PENK gene methylation; the standard mutation rate 5% in the table is a mixed sample of a 5% solution of TWIST1 gene methylation and a 5% solution of PENK gene methylation.

[0173] Example 8

[0174] This example is an example of Example 1, and the main difference from Example 1 is the length of the transformation incubation time. The transformation time of Example 1 is 5 minutes, and the transformation time of this example is 30 minutes. The standard mutation rate 10% in the table is a mixed sample of TWIST1 gene methylation 10% solution and PENK gene methylation 10% solution; the standard mutation rate 5% in the table is a mixed sample of TWIST1 gene methylation 5% solution and PENK gene methylation 5% solution.

[0175] Table 17

[0176] Standard (mutation frequency) Example 1 / Ct Comparative Example 3 / Ct Example 8 / Ct 10％ 30.23 35.00 31.85 5％ 32.05 36.20 34.26

[0177] Using standards with different mutation frequencies as test templates, the Ct value of Example 1 in the gene amplification after treatment is significantly better than that of Comparative Example 3 and Example 8. Therefore, the transformation conditions of Comparative Example 3 and Example 8 are not within the reference range of subsequent experiments.

[0178] Comparative Example 4

[0179] This comparative example is a comparative example of Example 1, and the main difference from Example 1 is that the weight ratio of ammonium bisulfite to sodium bisulfite in the conversion solution is 55:1. The standard mutation rate 10% in the table is a mixed sample of a 10% solution of TWIST1 gene methylation and a 10% solution of PENK gene methylation; the standard mutation rate 5% in the table is a mixed sample of a 5% solution of TWIST1 gene methylation and a 5% solution of PENK gene methylation.

[0180] Example 9

[0181] This embodiment is a variation of embodiment 1. The main difference from embodiment 1 is the length of the transformation incubation time. The transformation time of embodiment 1 is 5 minutes, and the transformation time of this embodiment is 20 minutes. The standard mutation rate of 10% in the table is a mixed sample of TWIST1 gene methylation 10% solution and PENK gene methylation 10% solution; the standard mutation rate of 5% in the table is a mixed sample of TWIST1 gene methylation 5% solution and PENK gene methylation 5% solution.

[0182] Table 18

[0183] Standard (mutation frequency) Example 1 / Ct Comparative Example 4 / Ct Example 9 / Ct 10％ 30.23 33.6 31.03 5％ 32.05 34.55 32.97

[0184] Using standards with different mutation frequencies as test templates, the Ct value of Example 1 in the gene amplification after treatment is significantly better than that of Comparative Example 4 and Example 9. Therefore, the transformation conditions of Comparative Example 4 and Example 9 are not within the reference range of subsequent experiments.

[0185] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples 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.

[0186] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A methylation detection reagent, characterized in that: The reagents include conversion solution; The conversion liquid comprises water, ammonium bisulfite and sodium bisulfite, the concentration of the ammonium bisulfite is 30wt%-90wt%, and the weight ratio of the ammonium bisulfite to the sodium bisulfite is (1-50):1; Optionally, the concentration of the ammonium bisulfite is 50wt%-90wt%; Optionally, the weight ratio of the ammonium bisulfite to the sodium bisulfite is (10-50):

1.

2. The methylation detection reagent according to claim 1, characterized in that The reagent also includes desulfurization liquid; The desulfurization liquid comprises a NaOH solution and isopropanol, wherein the volume proportion of the isopropanol in the desulfurization liquid is 50%-90%, and the concentration of NaOH in the NaOH solution is 30mmol / L-600mmol / L.

3. The methylation detection reagent according to any one of claims 1 to 2, characterized in that The reagents also include a binding solution; The binding solution includes water, 10mmol / L-500mmol / L Tris-HCl, 1mol / L-6mol / L guanidine salt and 1mmol / L-100mmol / L EDTA, pH 7.5-8.0; Optionally, the guanidine salt includes one or more of guanidine hydrochloride and guanidine isothiocyanate.

4. The methylation detection reagent according to any one of claims 1 to 2, characterized in that The reagent also includes a rinse solution; The rinse solution includes water, pH 7.5-8.0, 10mmol / L-2000mmol / L Tris-HCl, 0.05mol / L-6mol / LNaCl and 20%-82% ethanol by volume, pH 7.5-8.

0.

5. A kit for detecting urothelial carcinoma, characterized in that: The detection kit comprises the methylation detection reagent according to any one of claims 1 to 4.

6. The urothelial carcinoma detection kit according to claim 5, characterized in that: The detection kit also includes: Any one of the primer-probe combinations shown in SEQ ID NO.1 to SEQ ID NO.3, the primer-probe combinations shown in SEQ ID NO.4 to SEQ ID NO.6, the primer-probe combinations shown in SEQ ID NO.7 to SEQ ID NO.9, the primer-probe combinations shown in SEQ ID NO.10 to SEQ ID NO.12, and the primer-probe combinations shown in SEQ ID NO.13 to SEQ ID NO.15; as well as, Any primer-probe combination among the primer-probe combination shown in SEQ ID NO.16 to SEQ ID NO.18, the primer-probe combination shown in SEQ ID NO.19 to SEQ ID NO.21, the primer-probe combination shown in SEQ ID NO.22 to SEQ ID NO.24, the primer-probe combination shown in SEQ ID NO.25 to SEQ ID NO.27, and the primer-probe combination shown in SEQ ID NO.28 to SEQ ID NO.

30.

7. The urothelial carcinoma detection kit according to claim 6, characterized in that: The detection kit also includes a primer-probe combination shown in SEQ ID NO.31 to SEQ ID NO.

33.

8. A method for methylation treatment of urothelial carcinoma samples, characterized in that: The methylation treatment method comprises the step of using the methylation detection reagent according to any one of claims 1 to 4 to treat the DNA of the sample to be tested.

9. The method for methylation treatment of urothelial carcinoma samples according to claim 8, characterized in that: The sample to be tested includes a urine sample.

10. A method for detecting urothelial carcinoma samples, characterized in that: The detection method comprises: Treating the DNA of the sample to be tested by the methylation treatment method according to any one of claims 8 to 9, and detecting the treated DNA; Optionally, the detection is performed by fluorescent quantitative PCR; Further optionally, the fluorescent quantitative PCR method adopts the primer-probe combination defined in any one of claims 6 to 7.

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