Urine preserving fluid and application thereof

Abstract

The invention relates to a urine preservation solution and application thereof. The urine preserving fluid provided by the invention comprises a pH buffering agent, a DNA enzyme inhibitor, an RNA enzyme inhibitor, a cracking agent, a cell fixing agent, a fixing auxiliary agent, a rinsing agent, an aldehyde group quenching agent and a preservative. The urine preserving fluid is applied to preservation of urine or body fluid, a urine or body fluid sample and the urine preserving fluid are mixed according to the volume ratio of 5: 1, and the urine or body fluid is preserved at room temperature or in a cold storage mode. The urine preservation solution not only can reduce nucleic acid degradation in the urine preservation process, but also can keep the integrity and form of cast-off cells in urine at room temperature so as to support subsequent cytological examination.

Description

Technical Field

[0001] The invention relates to the field of cell biology and medical detection technology, and in particular to a urine preservation solution and application thereof. Background Art

[0002] Urine is a non-invasive sample. Compared with the existing sampling methods of blood, tissues and cells, urine is easy to obtain, can be sampled repeatedly, is simple to operate, causes little damage to the patient, is painless, and is not easily contaminated by other pathogenic microorganisms. It is also non-invasive, truly non-invasive, and highly safe. At the same time, urine also carries genetic material and can detect most cancer information. Changes in urine composition reflect lesions in the urinary system and other tissues and organs, and the accuracy of the test results is also related to the subsequent diagnosis and treatment of the disease.

[0003] Urine exfoliated cell examination is one of the common detection methods for urinary system tumors, especially for tumor diseases related to the urinary system, because it is easier to shed intact cells and cell clusters into the urine. By examining the exfoliated cells in the urine, it can be determined whether there are abnormal cell morphologies, thereby screening and diagnosing urinary system tumors such as bladder cancer and urothelial carcinoma. This method is suitable for comprehensive screening for cancer prevention, finding cancer cells, and early diagnosis of residual pelvic, ureteral and bladder tumors. It is of great significance for the diagnosis and postoperative follow-up of urinary system tumors.

[0004] The accuracy of urine exfoliated cell examination is affected by many factors, such as differences in the examiner's skills, equipment, sample retention method and time, and storage method. Among them, the storage method of urine samples has an important impact on its quality and subsequent test results. Due to the complex composition of urine, there are various metabolic wastes, including various organic and inorganic metabolites. If the exfoliated cells in urine are in a low osmotic pressure, high urea and other toxic substances, and non-physiological pH environment for a long time, it is easy to cause changes in cell morphology and even necrosis. In clinical practice, a certain time interval is required from sample collection to sample testing, and the free nucleic acid content in urine is low and easily degraded, resulting in unsatisfactory accuracy of the test results. In addition, exfoliated cells in urine are easily affected by factors such as changes in urine pH, urine retention in the body, and prolonged placement in vitro, which leads to changes in cell morphology. It is difficult to distinguish its tissue type during cytological examination, thus affecting the accuracy of urine exfoliated cell examination.

[0005] With the continuous advancement of molecular detection technology, the sensitivity and specificity of early diagnosis of urinary system related tumors are also constantly improving. Therefore, in order to achieve the desired effect of molecular detection, the quality requirements for urine samples are getting higher and higher, and the quality requirements for subsequent extracted nucleic acids are also the same, such as DNA point mutation and DNA methylation detection technology. Although the detection specificity and sensitivity of these detection technologies are very high, reaching more than 90%. However, due to the major problems in the processing of urine samples, there are fewer productized detection technologies or kits entering the clinical testing market. This is because urine samples have large individual differences, are extremely easy to degrade, difficult to preserve, and have low success rates for nucleic acid extraction. There are cells in the urine that have fallen off the epithelium but have not begun to degrade. These cells are an important source of nucleic acid extraction, but the current preservatives for the exfoliated cells in urine cannot preserve their complete cell morphology, and cell rupture is prone to occur, and most of the released nucleic acids will be degraded, resulting in low subsequent nucleic acid extraction and poor integrity of nucleic acid samples. Therefore, as the requirements for the quality of urine samples become higher and higher, the preservation of urine also needs to be continuously improved. So the question of how to preserve the exfoliated cells and free nucleic acids in urine has now become a question of how to preserve the exfoliated cells and free nucleic acids in urine more completely and stably.

[0006] Free nucleic acids contained in urine, such as cfDNA and cfRNA, can provide important information about an individual's health status and have potential value for early diagnosis and treatment monitoring of diseases. Preserving free nucleic acids in urine, especially a large number of samples, can provide rich resources for subsequent research, help to further explore the occurrence and development mechanisms of diseases, assess disease risks, and develop new diagnostic and treatment strategies. The preservation of large amounts of free nucleic acids in urine will also provide important support for subsequent clinical diagnosis, disease research, and the construction of biobanks.

[0007] At present, there are two main methods for preserving urine: refrigeration and adding preservatives: (1) Urine specimens are usually stored at 2-8 degrees Celsius to prevent bacterial growth and changes in urine composition. If stored for a long time, they need to be transferred to an ultra-low temperature refrigerator at -80°C to maximize the original state of free nucleic acids and exfoliated cells in the urine. (2) Adding preservatives is another method for preserving urine, such as concentrated hydrochloric acid, toluene, xylene, glacial acetic acid, thymol, formaldehyde, etc. This is mainly used for urine that needs to be stored for a long time. Although the above preservatives have a certain effect on urine preservation, they are not universal, because different sample preservatives are required for different test items, which is not conducive to clinical processing and selection. Some urine preservatives have been disclosed in the prior art, such as a urine preservative disclosed in CN111869655A in the Chinese patent literature, which includes: pH buffer 6-10mmol / L, DNA enzyme inhibitor 0.2-0.4mol / L, preservative 0.1-0.3mol / L, cell fixative mass fraction 5%-15% and formaldehyde quencher 0.3-1.0mol / L; they still have their respective protective effects between 4℃-40℃, and have no direct effect on free nucleic acids in urine. However, the urine preservative disclosed in the patent only protects free nucleic acids, and has no clear protective effect on exfoliated cells in urine, and exfoliated cells are prone to morphological changes or necrosis in adverse environments, resulting in inaccurate cytological test results. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a urine preservation solution and a preparation method and application thereof.

[0009] The urine preservation solution proposed by the present invention comprises a pH buffer, a DNA enzyme inhibitor, an RNA enzyme inhibitor, a lysing agent, a cell fixative, a fixation auxiliary agent, a rinsing agent, an aldehyde quencher, and a preservative; the pH buffer is one or more of sodium citrate or ammonium sulfate; the DNA enzyme inhibitor is one or more of EDTA-2Na or diethyltriaminepentaacetic acid; the RNA enzyme inhibitor is one or more of diethyl pyrocarbonate or guanidine isothiocyanate; the lysing agent is guanidine hydrochloride; the cell fixative is one or more of formaldehyde, formohydrazide, formamide or isopropanol; the fixation auxiliary agent is sodium acetate NaAc; the rinsing agent is one or more of Tween 20, TritonX-100, and NP-40; the aldehyde quencher is one or more of glycine, arginine, and lysine; the preservative is one or more of imidazolidinyl urea or diazolidinyl urea; the balance is ultrapure water; and the pH value of the urine preservation solution is 7.5-8.0.

[0010] Furthermore, the pH buffer is one or more of sodium citrate or ammonium sulfate with a final concentration of 0.01 mol / L.

[0011] Furthermore, the DNase inhibitor is one or more of EDTA-2Na or diethyltriaminepentaacetic acid with a final concentration of 0.8%.

[0012] Furthermore, the RNase inhibitor is one or more of diethyl pyrocarbonate or guanidine isothiocyanate with a final concentration of 0.02%-0.11%.

[0013] Furthermore, the cell fixative is one or more of formaldehyde, formic hydrazide, formamide, or isopropanol, with a weight of 0.3% of the total weight of the urine preservation solution, or 0.15% of the total weight of the urine preservation solution.

[0014] Furthermore, the rinsing agent is one or more of Tween 20, Triton X-100, and NP-40 with a final concentration of 0.05%.

[0015] Furthermore, the aldehyde quencher is one or more of glycine, arginine, and lysine with a final concentration of 0.02 mol / L.

[0016] Furthermore, the preservative is one or more of imidazolidinyl urea with a final concentration of 0.1% or diazolidinyl urea with a final concentration of 0.09%.

[0017] The present invention also provides a preferred urine preservation solution, comprising a pH buffer, a DNA enzyme inhibitor, an RNA enzyme inhibitor, a lysing agent, a cell fixative, a fixation auxiliary, a rinsing agent, an aldehyde quencher, and a preservative; the pH buffer is sodium citrate with a final concentration of 0.01 mol / L; the DNA enzyme inhibitor is EDTA-2Na with a final concentration of 0.8%; the RNA enzyme inhibitor is diethyl pyrocarbonate with a final concentration of 0.02%; the lysing agent is guanidine hydrochloride with a final concentration of 0.5 mol / L; the cell fixative is formaldehyde with a final concentration of 0.3%; the fixation auxiliary is sodium acetate NaAc with a final concentration of 0.015M; the rinsing agent is Tween 20 with a final concentration of 0.05%; the aldehyde quencher is glycine with a final concentration of 0.02 mol / L; and the preservative is imidazolidinyl urea with a final concentration of 0.1%.

[0018] The urine preservation solution of the present invention is used in the preservation of urine or body fluids. The preservation method is to mix the urine or body fluid sample with the urine preservation solution in a volume ratio of 5:1 and store it at room temperature or in a refrigerator.

[0019] The embodiments of the present invention have the following beneficial effects:

[0020] 1. The urine preservation solution of the present invention can not only reduce nucleic acid degradation during urine preservation, but also maintain the integrity and morphology of exfoliated cells in urine at room temperature to support subsequent cytological examinations, such as cell morphology examination and fluorescence in situ hybridization (FISH).

[0021] 2. The urine preservation solution of the present invention can maintain urine without deterioration at room temperature or in a refrigerated environment, such as 4°C-40°C, thereby ensuring the relative content of cfDNA and maintaining the integrity of cells.

[0022] 3. In the urine preservation solution of the present invention, the pH buffer is used to maintain the pH value and salt ion concentration of the solution to avoid cell rupture or cfDNA degradation in urine; the DNA enzyme inhibitor is mainly a metal ion chelator, which is used to bind to metal ions such as Mg2+, Ca2+, Fe3+ in urine to inhibit the activity of nucleases in urine, maintain the stability of cells in urine, and also avoid the degradation of cfDNA by DNA enzymes in urine; the RNA inhibitor is mainly diethyl pyrocarbonate, which is a strong but not thorough RNA enzyme inhibitor, which denatures proteins by binding to the imidazole ring of the active genomic amino acid of RNA enzyme, thereby inhibiting the activity of the enzyme; the preservative is mainly used to inhibit the activity of microorganisms originally present in urine to prevent further reproduction of microorganisms and cause urine deterioration; the cell fixative is mainly used to maintain the morphology of cells in urine to avoid cell rupture and release of genomic DNA into urine, affecting the actual concentration of cfDNA; the rinse agent is used as a non-ionic surfactant to dissociate the mucus secreted by the bladder and increase the collection rate of cells; the aldehyde quencher is mainly used to eliminate the free aldehydes released from the preservative to avoid affecting the subsequent PCR process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 This is a comparison chart of the nucleic acid concentration measurement results of the urine mixed sample in Example 9;

[0025] Figure 2 This is a comparison chart of CT values ​​for DNA methylation detection of urine mixed sample cells in Example 9;

[0026] Figure 3 This is a diagram showing the gene detection results of different samples stored at room temperature for 0 days in Example 10;

[0027] Figure 4 This is a graph showing the gene detection results of different samples stored at room temperature for 7 days in Example 10. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1: The basic urine preservation solution of the present invention is prepared according to a commercially available urine preservation reagent:

[0030] A urine preservation reagent purchased from the market was analyzed to be composed of nucleic acid buffer, metal ion chelator, preservative, cell fixative and formaldehyde quencher, wherein the nucleic acid buffer was Tris-HCl with a final concentration of 2 mmol / L and a working pH of 8; the metal ion chelator was EDTA with a final concentration of 10 mmol / L; the preservative was composed of citric acid with a final concentration of 1.25 mmol / L and sodium citrate with a final concentration of 3.75 mmol / L; the cell fixative was composed of equal amounts of paraformaldehyde, diazolidinyl urea and imidazolidinyl urea, and the amount of the cell fixative was 0.1% of the total weight of the urine preservation reagent; the formaldehyde quencher was composed of equal amounts of glycine, lysine and ethylenediamine, and the final concentration of the mixed total formaldehyde quencher was 40 mmol / L.

[0031] The preparation method of the basic urine preservation solution of the present invention is as follows:

[0032] Dissolve Tris in water, adjust the pH to 7-8 with hydrochloric acid, and prepare 80mmol / L Tris-HCl solution; dissolve EDTA solid in the above-mentioned Tris-HCl buffer to prepare 0.4mol / L EDTA solution 1; dissolve paraformaldehyde, diazolidinyl urea and imidazolidinyl urea in water respectively to prepare cell fixative solution 2 with a concentration of 10% of each component; dissolve glycine, lysine and ethylenediamine in water to prepare formaldehyde quencher solution 3 with a concentration of 4mol / L of each component; dissolve citric acid and sodium citrate in water to prepare preservative solution 4 with concentrations of citric acid and sodium citrate of 50mmol / L and 150mmol / L respectively; mix solution 1, solution 2, solution 3, solution 4 and solution 5 according to the above-mentioned dosage ratio of a urine preservation reagent, filter, and adjust the pH to 8 with hydrochloric acid or sodium hydroxide to obtain the basic urine preservation solution (urine preservation solution 1) of the present invention.

[0033] Example 2: Comparison of different cell fixatives:

[0034] Urine preservation solutions 2-5 were prepared in the same manner as in Example 1, except that the fixative components used and their final concentrations in the urine preservation solutions were as follows:

[0035] Urine preservation solution 2: formaldehyde in an amount of 0.3% by weight of the total weight of the urine preservation solution;

[0036] Urine preservation solution 3: formohydrazide, equal to formaldehyde;

[0037] Urine preservation solution 4: formamide, equal to formaldehyde;

[0038] Urine preservation solution 5: isopropanol in an amount of 1.5% by weight of the total weight of the urine preservation solution;

[0039] The same urine sample was mixed with the urine preservative solution 1 of Example 1 and the urine preservative solution 2-5 of this example, respectively, in a mixing ratio of urine sample: urine preservative solution volume ratio of 5:1, and the samples were divided into 2 mL centrifuge tubes and stored at room temperature to obtain test groups 2-1, 2-2, 2-3, 2-4, and 2-5, respectively; another portion of the same urine sample was taken without adding any urine preservative solution, and was directly divided into 2 mL centrifuge tubes and stored at room temperature to obtain test group 2-6.

[0040] MagMAX by ThermoFisher TM The free DNA isolation kit was used to collect samples of urine mixed samples from the above six test groups on the first, fourth and seventh days of storage at room temperature, centrifuged at 16000rpm for 10min, and the supernatant was taken for nucleic acid extraction. Three parallel extractions were performed for each test, and the concentration of the extracted nucleic acid was measured. The nucleic acid concentration was measured using Q-bit, and the results are shown in Table 1.

[0041] Table 1 Results of nucleic acid concentrations extracted from mixed urine samples (concentration unit: ng / μL)

[0042]

[0043]

[0044] As can be seen from Table 1, as the storage time increases, the concentration of nucleic acids extracted from the urine mixed samples decreases; in test groups 2-6 where no urine protective solution was added, the nucleic acid degradation was serious, and the nucleic acid concentration was already below the detection limit on the 7th day.

[0045] Comparing the urine preservation solutions with different cell fixatives added, after 7 days of preservation, the cfDNA concentration of test group 2-2 corresponding to urine preservation solution 2 with 0.3% formaldehyde as the cell fixative was the highest, reaching 0.117 ng / μL in this embodiment, which was significantly better than the other three commonly used cell fixatives, indicating that the urine preservation solution with 0.3% formaldehyde added can more effectively preserve free DNA in urine and avoid its degradation compared with other urine preservation solutions.

[0046] Example 3, comparison of different DNase inhibitors:

[0047] Urine preservation solutions 6 and 7 were prepared in the same manner as urine preservation solution 2, except that the DNase inhibitor components used and their final concentrations were as follows:

[0048] Urine preservation solution 6: EDTA-2Na, 0.8%;

[0049] Urine preservation solution 7: diethyltriaminepentaacetic acid (DTPA), 0.8%;

[0050] The same urine sample was mixed with the urine preservative solution 2 of Example 2 and the urine preservative solution 6-7 of this example, respectively, in a mixing ratio of urine sample: urine preservative solution volume ratio of 5:1, and the mixture was divided into 2 mL centrifuge tubes and stored at room temperature to obtain test groups 3-1, 3-2, and 3-3, respectively; another portion of the same urine sample was taken without adding any urine preservative solution, and was directly divided into 2 mL centrifuge tubes and stored at room temperature to obtain test group 3-4.

[0051] The nucleic acid was extracted and the concentration was measured using the method of Example 2. The results are shown in Table 2.

[0052] Table 2 Results of nucleic acid concentrations extracted from urine mixed samples (concentration unit: ng / μL)

[0053]

[0054] As can be seen from Table 2, 3-4, which did not add any urine protection solution, had severe nucleic acid degradation, and the nucleic acid extracted was already below the detection limit on the 7th day. The urine preservation solution 6 with the added DNA enzyme inhibitor EDTA-2Na was better than 3-1 without the addition of DNA enzyme inhibitor. After 7 days of preservation, the free DNA (cfDNA) concentration of the test group 3-2 was the highest, reaching 0.133ng / μL in this embodiment, while the effect of diethyltriaminepentaacetic acid (DTPA), another DNA enzyme inhibitor commonly used in the industry, was not obvious, indicating that the urine preservation solution with the addition of the DNA enzyme inhibitor EDTA-2Na at a final concentration of 0.8% was more effective than other urine preservation solutions in preserving free DNA in urine and preventing its degradation.

[0055] Example 4, comparison of different pH buffers:

[0056] Urine preservation solutions 8 and 9 were prepared in the same manner as urine preservation solution 6, except that the pH buffer components used and their final concentrations were as follows:

[0057] Urine preservation solution 8: sodium citrate, 0.01 mol / l;

[0058] Urine preservation solution 9: ammonium sulfate, 0.01 mol / l;

[0059] The same urine sample was mixed with the urine preservative solution 6 of Example 3 and the urine preservative solution 8-9 of this example, respectively, in a mixing ratio of urine sample: urine preservative solution volume ratio of 5:1, and the mixture was divided into 2 mL centrifuge tubes and stored at room temperature to obtain test groups 4-1, 4-2, and 4-3, respectively; another portion of the same urine sample was taken without adding any urine preservative solution, and was directly divided into 2 mL centrifuge tubes and stored at room temperature to obtain test group 4-4.

[0060] The nucleic acid was extracted and the concentration was measured using the method of Example 2. The results are shown in Table 3.

[0061] Table 3 Results of nucleic acid concentrations extracted from urine mixed samples (concentration unit: ng / μL)

[0062]

[0063] As can be seen from Table 3, urine preservation solution 8 in which the pH buffer added was sodium citrate with a final concentration of 0.01 mol / l was the best. After 7 days of preservation, the free DNA (cfDNA) concentration of test group 4-2 was the highest, reaching 0.150 ng / μL in this embodiment, which was better than the pH buffer Tris-HCl used in commercial products and ammonium sulfate, another pH buffer commonly used in the industry. This shows that urine preservation solution in which the pH buffer added was sodium citrate with a final concentration of 0.01 mol / l was more able to effectively preserve free DNA in urine and avoid its degradation than other urine preservation solutions.

[0064] Example 5, comparison of different rinse agents:

[0065] Urine preservation solutions 10-12 were prepared in the same manner as urine preservation solution 8, except that the rinse agent components used and their final concentrations were as follows:

[0066] Urine preservation solution 10: Tween 20, 0.05% (v / v);

[0067] Urine preservation solution 11: TritonX-100, 0.05% (v / v);

[0068] Urine preservation solution 12: NP-40, 0.05% (v / v);

[0069] The same urine sample was mixed with the urine preservative solution 8 of Example 4 and the urine preservative solution 10-12 of this example, respectively, in a mixing ratio of urine sample: urine preservative solution volume ratio of 5:1, and the mixture was divided into 2 mL centrifuge tubes and stored at room temperature to obtain test groups 5-1, 5-2, 5-3, and 5-4, respectively; another portion of the same urine sample was taken without adding any urine preservative solution, and it was directly divided into 2 mL centrifuge tubes and stored at room temperature to obtain test group 5-5.

[0070] The nucleic acid was extracted and the concentration was measured using the method of Example 2. The results are shown in Table 4.

[0071] Table 4 Results of nucleic acid concentration determination extracted from urine mixed samples (concentration unit: ng / μL)

[0072]

[0073] As can be seen from Table 4, the urine preservation solution 8 with Tween 20 added as a rinsing agent with a final concentration of 0.05% has the best effect. After 7 days of storage, the free DNA (cfDNA) concentration of the test group 4-2 is the highest, reaching 0.165 ng / μL in this embodiment, which is better than the other two commonly used rinsing agents TritonX-100 and NP-40 in the industry. This shows that the urine preservation solution with Tween 20 added at a final concentration of 0.05% can more effectively preserve the free DNA in the urine and avoid its degradation compared with other urine preservation solutions.

[0074] Example 6, comparison of different aldehyde quenchers:

[0075] Urine preservation solutions 13-15 were prepared in the same manner as urine preservation solution 10, except that the aldehyde quencher components used and their final concentrations were as follows:

[0076] Urine preservation solution 13: glycine, 0.02 mol / L;

[0077] Urine preservation solution 14: arginine, 0.02 mol / L;

[0078] Urine preservation solution 15: lysine, 0.02 mol / L;

[0079] The same urine sample was mixed with the urine preservative solution 10 of Example 5 and the urine preservative solutions 13-15 of this example, respectively, in a mixing ratio of urine sample: urine preservative solution volume ratio of 5:1, and the samples were divided into 2 mL centrifuge tubes and stored at room temperature to obtain test groups 6-1, 6-2, 6-3, and 6-4, respectively; another portion of the same urine sample was taken without adding any urine preservative solution, and was directly divided into 2 mL centrifuge tubes and stored at room temperature to obtain test group 6-5.

[0080] The nucleic acid was extracted and the concentration was measured using the method of Example 2. The results are shown in Table 5.

[0081] Table 5 Results of nucleic acid concentration determination extracted from urine mixed samples (concentration unit: ng / μL)

[0082]

[0083] As can be seen from Table 5, the urine preservation solution 13 with glycine added as an aldehyde quencher at a final concentration of 0.02% has the best effect. After 7 days of preservation, the free DNA (cfDNA) concentration of the test group 6-2 is the highest, reaching 0.220 ng / μL in this embodiment, which is better than the other two aldehyde quenchers arginine and lysine commonly used in the industry. The protective effect of the urine preservation solution with arginine or lysine on the free DNA in the urine is not even as good as the urine preservation solution 10 without the addition of the aldehyde quencher, indicating that the urine preservation solution with glycine added at a final concentration of 0.02% can more effectively preserve the free DNA in the urine and avoid its degradation compared with other urine preservation solutions.

[0084] Example 7, comparison of different preservatives:

[0085] Urine preservation solutions 16 and 17 were prepared in the same manner as the urine preservation solution 13 in Example 6, except that the preservative components and final concentrations used were as follows:

[0086] Urine preservation solution 16: imidazolidinyl urea, 0.1%;

[0087] Urine preservation solution 17: diazolidinyl urea, 0.09%;

[0088] The same urine sample was mixed with the urine preservative solution 13 of Example 6 and the urine preservative solution 16-17 of this example, respectively, in a mixing ratio of urine sample: urine preservative solution volume ratio of 5:1, and the mixture was divided into 2 mL centrifuge tubes and stored at room temperature to obtain test groups 7-1, 7-2, and 7-3, respectively; another portion of the same urine sample was taken without adding any urine preservative solution, and was directly divided into 2 mL centrifuge tubes and stored at room temperature to obtain test group 7-4.

[0089] The nucleic acid was extracted and the concentration was measured using the method of Example 2. The results are shown in Table 6.

[0090] Table 6 Results of nucleic acid concentration determination extracted from urine mixed samples (concentration unit: ng / μL)

[0091]

[0092] As can be seen from Table 6, the urine preservation solution 16 with an added preservative of 0.1% imidazolidinyl urea had the best effect. After 7 days of preservation, the free DNA (cfDNA) concentration of the test group 7-2 was the highest, reaching 0.238 ng / μL in this embodiment, while the urine preservation solution with a final concentration of 0.09% diazolidinyl urea was not even as protective of free DNA in urine as the urine preservation solution 13 without adding a preservative, indicating that the urine preservation solution with a final concentration of 0.1% imidazolidinyl urea can more effectively preserve free DNA in urine and avoid its degradation compared with other urine preservation solutions.

[0093] Example 8, Improvement of urine storage solution:

[0094] Urine preservation solution 18 was prepared in the same manner as urine preservation solution 16 in Example 7, except that sodium acetate (NaAc) with a final concentration of 0.015 M as a cell fixation auxiliary, guanidine hydrochloride with a final concentration of 0.5 mol / L as a lysis agent (for lysing bacteria and viruses in cell samples), and diethyl pyrocarbonate (DEPC) with a final concentration of 0.02% as an RNase inhibitor were additionally added.

[0095] The same urine sample was mixed with the urine preservative solution 16 of Example 7 and the urine preservative solution 18 of this example, respectively, in a mixing ratio of urine sample: urine preservative solution volume ratio of 5:1, and the mixture was divided into 2 mL centrifuge tubes and stored at room temperature to obtain test groups 8-1 and 8-2, respectively; another portion of the same urine sample was taken without adding any urine preservative solution, and was directly divided into 2 mL centrifuge tubes and stored at room temperature to obtain test group 8-3.

[0096] The nucleic acid was extracted and the concentration was measured using the method of Example 2. The results are shown in Table 7.

[0097] Table 7 Results of nucleic acid concentration determination extracted from urine mixed samples (concentration unit: ng / μL)

[0098]

[0099] As can be seen from Table 7, urine preservation solution 18 with additional additions of cell fixation auxiliary agent with a final concentration of 0.015M sodium acetate (NaAc), lysing agent (for lysing bacteria and viruses in cell samples) with a final concentration of 0.5mol / L guanidine hydrochloride, and RNase inhibitor with a final concentration of 0.02% diethyl pyrocarbonate (DEPC) has better effect. After 7 days of preservation, the free DNA (cfDNA) concentration of the test group 8-2 is the highest, reaching 0.250ng / μL in this embodiment, indicating that urine preservation solution 18 is more capable of effectively preserving free DNA in urine and avoiding its degradation than other urine preservation solutions.

[0100] Example 9: Nucleic acid quality detection experiment in urine storage solution:

[0101] The same urine sample was mixed with the urine preservative solution 1 of Example 1 and the urine preservative solution 18 of Example 8, respectively, in a urine sample: urine preservative solution volume ratio of 5:1, and the mixture was divided into 2 mL centrifuge tubes and stored at room temperature to obtain test groups 9-1 and 9-2, respectively; another portion of the same urine sample was taken without adding any urine preservative solution, and was directly divided into 2 mL centrifuge tubes and stored at room temperature to obtain test group 9-3.

[0102] The nucleic acid was extracted and the concentration was measured using the method of Example 2. The results are shown in Table 8.

[0103] Table 8 Results of nucleic acid concentration determination extracted from urine mixed samples (concentration unit: ng / μL)

[0104]

[0105]

[0106] As can be seen from Table 8, urine preservation solution 18 has the best effect. After 7 days of preservation, the free DNA (cfDNA) concentration of test group 9-2 is the highest, reaching 0.250 ng / μL in this embodiment. However, urine preservation solution 1 prepared according to a commercially available urine preservation solution formula, after 7 days of preservation, the free DNA (cfDNA) concentration of test group 9-1 is only 0.074 ng / μL. The nucleic acid of test group 9-3 without urine preservation solution is severely degraded, and the extracted nucleic acid is already below the detection limit on the 7th day. This indicates that urine preservation solution 18 can effectively preserve free DNA in urine and prevent its degradation, and the preservation effect is 3.38 times better than that of existing urine preservation solution products. The average values ​​of repeat 1, repeat 2 and repeat 3 of each test group are plotted, as shown in the figure. Figure 1 As shown, the nucleic acid in the test group 9-3 was significantly degraded, and the commercially available urine preservation solution had a certain preservation effect, but the effect was not significant. However, the urine preservation solution 18 was able to effectively preserve the free DNA in the urine, prevent its degradation, and maintain the integrity of the cells.

[0107] While detecting nucleic acid, human ONECUT2 / VIM gene methylation detection kit (fluorescence PCR method) was used to perform qPCR on the test groups 9-1, 9-2, and 9-3 to detect the nucleic acid quality. The DNA samples were quantified using the same Qubit fluorometer, and the total amount was not less than 50 ng. The subsequent detection and data analysis were strictly carried out in accordance with the instructions. The results are shown in Table 9. The CT value indicates the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold. The CT value can reflect the concentration of the test sample. The lower the CT value, the higher the nucleic acid concentration of the test sample, and the higher the CT value, the lower the nucleic acid concentration in the test sample; the difference of 1 CT value in the nucleic acid concentration of the sample is two times, and the difference of 2 CT values ​​is four times, and so on.

[0108] Table 9 Comparison of CT values ​​of cell DNA methylation detection in different storage solutions

[0109]

[0110] The average value of replicate 1, replicate 2 and replicate 3 of each test group was taken as Figure 2 , from Table 9 and Figure 2 It can be seen that the CT value of the test group 9-1 was abnormal when it was stored for 7 days, which was about 1 higher than the theoretical value. It is speculated that the growth of microorganisms in the sample was not effectively inhibited. The CT values ​​of DNA extracted from other preservation solutions were consistent with the concentration ratio in Table 8. This result shows that the effect of urine samples preserved with the urine preservative 18 of the present invention is more stable than that with the existing urine preservation solution 1.

[0111] Example 10, Cell preservation experiment:

[0112] The human cell line suspension was added to the urine preservation solution 18 of Example 8 as sample 1, and added to the urine preservation solution 1 of Example 1 as sample 2, and stored at room temperature for 7 days. Equal amounts of cell samples were taken on days 0 and 7 and DNA was extracted using the same method. After polymerase chain reaction (PCR) amplification, agarose gel electrophoresis was performed to detect human B globulin gene (250 bp). The test results can be seen in Figure 3 and Figure 4 .

[0113] from Figure 3 It can be seen that the cell states in sample 1 to which urine preservation solution was initially added and sample 2 to which urine preservation solution was not added were basically the same; Figure 4 The results show that after being stored at room temperature for 7 days, mixed bands appeared in the electrophoresis results of sample 2, indicating that the cells in urine preservation solution 1 were damaged; while the state of the cells in urine preservation solution 18 after 7 days of storage was basically the same as the initial state, indicating that the urine preservative 18 of the present invention can maintain the integrity and morphology of the exfoliated cells in the urine at room temperature.

[0114] In summary, after multiple rounds of screening and comparison experiments of different components, the urine preservation solution 18 in Example 8 was selected as the optimal option, and its formula is shown in Table 10. The urine preservation solution has a unique function of avoiding mutual interference between the components. Under refrigeration and room temperature conditions, such as 4°C-40°C, it still has its corresponding protective effect, and can effectively preserve the free DNA in the urine to avoid its degradation while maintaining the integrity of the cells.

[0115] Table 10 Main components of urine preservation solution 18

[0116] Reagents content Paraformaldehyde 0.3％ Sodium acetate NaAc 0.015M EDTA-2Na 0.8％ Sodium Citrate 0.01mol / l Twain 20 0.05%(v / v) Glycine 0.02mol / L Imidazolidinyl urea 0.1％ DEPC 0.02％-0.11％ Guanidine hydrochloride 0.5mol / L

[0117] In some embodiments, the method for preserving urine includes the following steps: morning urine and the first urine after getting up in the morning need to be taken. Women are required to be tested during the non-menstrual period. If the menstruation has just ended, the test needs to be performed 3 days after the menstrual period ends. Use a sampling cup to collect the morning urine, pour the urine into a urine bottle with urine preservation solution, the volume ratio of urine to urine preservation solution is 5:1, and after fully mixing, the mixed sample is stored at room temperature or the recommended temperature for refrigeration. As needed, subsequent cytological analysis, molecular biology experiments, etc. are performed within the prescribed storage time.

[0118] 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 urine preservation solution, characterized in that: The invention comprises a pH buffer, a DNA enzyme inhibitor, an RNA enzyme inhibitor, a lysing agent, a cell fixative, a fixation auxiliary agent, a rinsing agent, an aldehyde quencher, and a preservative; the pH buffer is one or more of sodium citrate or ammonium sulfate; the DNA enzyme inhibitor is one or more of EDTA-2Na or diethyltriaminepentaacetic acid; the RNA enzyme inhibitor is one or more of diethyl pyrocarbonate or guanidine isothiocyanate; the lysing agent is guanidine hydrochloride; the cell fixative is one or more of formaldehyde, formohydrazide, formamide or isopropanol; the fixation auxiliary agent is sodium acetate NaAc; the rinsing agent is one or more of Tween 20, TritonX-100, and NP-40; the aldehyde quencher is one or more of glycine, arginine, and lysine; the preservative is one or more of imidazolidinyl urea or diazolidinyl urea; the balance is ultrapure water; and the pH value of the urine preservation solution is 7.5-8.

0.

2. The urine preservation solution according to claim 1, characterized in that: The pH buffer is one or more of sodium citrate or ammonium sulfate with a final concentration of 0.01 mol / L.

3. The urine preservation solution according to claim 1, characterized in that: The DNase inhibitor is one or more of EDTA-2Na or diethyltriaminepentaacetic acid with a final concentration of 0.8%.

4. The urine preservation solution according to claim 1, characterized in that: The RNase inhibitor is one or more of diethyl pyrocarbonate or guanidine isothiocyanate with a final concentration of 0.02%-0.11%.

5. The urine preservation solution according to claim 1, characterized in that: The cell fixative is one or more of formaldehyde, formic hydrazide, formamide, or isopropanol, with a weight of 0.3% of the total weight of the urine preservation solution, or 0.15% of the total weight of the urine preservation solution.

6. The urine preservation solution according to claim 1, characterized in that: The rinsing agent is one or more of Tween 20, Triton X-100, and NP-40 with a final concentration of 0.05%.

7. The urine preservation solution according to claim 1, characterized in that: The aldehyde quencher is one or more of glycine, arginine and lysine with a final concentration of 0.02 mol / L.

8. The urine preservation solution according to claim 1, characterized in that: The preservative is one or more of imidazolidinyl urea with a final concentration of 0.1% or diazolidinyl urea with a final concentration of 0.09%.

9. The urine preservation solution according to claim 1, characterized in that: The pH buffer is sodium citrate with a final concentration of 0.01 mol / L; the DNA enzyme inhibitor is EDTA-2Na with a final concentration of 0.8%; the RNA enzyme inhibitor is diethyl pyrocarbonate with a final concentration of 0.02%; the lysing agent is guanidine hydrochloride with a final concentration of 0.5 mol / L; the cell fixative is formaldehyde with a final concentration of 0.3%; the fixation auxiliary is sodium acetate NaAc with a final concentration of 0.015M; the rinsing agent is Tween 20 with a final concentration of 0.05%; the aldehyde quencher is glycine with a final concentration of 0.02 mol / L; and the preservative is imidazolidinyl urea with a final concentration of 0.1%.

10. Use of the urine preservation solution according to any one of claims 1 to 9 in the preservation of urine or body fluids, characterized in that: The urine or body fluid sample is mixed with the urine preservation solution according to any one of claims 1 to 9 at a volume ratio of 5:1 and stored at room temperature or in a refrigerator.

Images