A cell sample preservation solution and its preparation method and application

Abstract

The present invention discloses a cell sample preservation solution and a preparation method and application thereof. The cell sample preservation solution is an aqueous solution comprising the following substances in percentage by mass: 0.5% to 10% of a cell fixative, 2% to 10% of a DNA enzyme inhibitor, 0.1% to 5% of a preservative, and also comprises a pH buffer for maintaining the pH value of the cell sample preservation solution at 6.5 to 7.5. The preparation method of the cell sample preservation solution is simple, and it can be used to preserve a variety of samples such as urine, cervical exfoliated cells and swabs, and can effectively maintain cell viability and morphology, prevent the sample from deteriorating or being contaminated during the preservation process, and effectively protect the nucleic acid from degradation, so as to avoid affecting the subsequent extraction and detection of the nucleic acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological sample preservation, and in particular relates to a cell sample preservation solution and a preparation method and application thereof. Background Art

[0002] In the field of biology and medicine, the preservation of cell samples is crucial for subsequent experiments and analysis. Human metabolites such as urine sediment cells or cervical exfoliated cells contain many biologically active and nutritious substances, such as cell components, enzymes, and growth factors, as well as microorganisms such as bacteria and fungi. These substances are easily affected by the external environment at room temperature, causing changes in certain chemical components in the sample, thereby causing the nucleic acid in the sample to degrade.

[0003] The existing sample preservation solution has the problems of single sample type preservation, inability to be applied to a variety of biological samples, poor preservation effect, and high cost. A Chinese patent (application number 202010068114.5) discloses a urine preservation solution and its preparation method and application, which is prepared by N-(2,5-dioxo-4-imidazolinyl) urea, 5,5-dimethylhydantoin, Schiff base containing aliphatic azomethine, ethylenediaminetetraacetate, oxalate, glycine-sodium hydroxide-hydrochloric acid buffer, citrate, etc. However, the preservation solution has a complex formula and high cost, and cannot simultaneously meet the preservation of cervical exfoliated cells, and may affect the screening and diagnosis results of cervical cancer. A Chinese patent (application number 202210759176.X) discloses a urine free DNA preservative and a urine preservation tube, which is prepared by dextran, imidazolidinyl urea, diazolidinyl urea, dipotassium ethylenediaminetetraacetate, sodium fluoride, glutathione, etc. However, the preservative and the storage tube using the preservative can only extend the shelf life of 36 hours after being stored at room temperature for a period of time and then encountering a temperature rise, which cannot meet the longer storage time that may be required for transportation to the test. It can be seen that the existing sample preservation solution is often complicated in formula, and the ratio and concentration of various components need to be tested and adjusted many times, which not only increases the complexity and cost of the production process, but also is not conducive to the use and understanding of ordinary users; the preservation time is not ideal: some existing sample preservation solutions are insufficient in protecting the stability and integrity of the samples, and their preservation time is short, which is easily affected by the external environment, resulting in changes in the composition and properties of the samples, thereby affecting the subsequent detection and analysis results; the cost is relatively expensive: high-quality sample preservation solutions usually require special ingredients and manufacturing processes, which leads to high costs, increases the operating costs of the laboratory, and limits its application in some resource-limited areas; inconvenient operation: the operation process of some sample preservation solutions is not simple enough, and users need to go through complex operation steps to complete the preservation of samples, which not only increases the difficulty of use for users, but also may cause loss or contamination of samples due to improper operation.

[0004] Therefore, how to optimize the composition and ratio of the preservation solution to increase the diversity of preserved samples, improve the protection effect, and ensure the stability and reliability of the samples needs to be solved. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a cell sample preservation solution and a preparation method and application thereof, which solve the problems in the above-mentioned background technology.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a cell sample preservation solution is provided, which is an aqueous solution comprising the following substances in percentage by mass: 0.5% to 10% of a cell fixative, 2% to 10% of a DNA enzyme inhibitor, 0.1% to 5% of a preservative, and also comprising a pH buffer for maintaining the pH value of the cell sample preservation solution at 6.5 to 7.5; wherein the cell fixative is at least one of diazolidinyl urea, imidazolidinyl urea, and glucose; and the DNA enzyme inhibitor is at least one of citric acid, sodium phosphate, and disodium ethylenediaminetetraacetic acid.

[0007] In a preferred embodiment of the present invention, the composition is composed of the following components by mass percentage: 0.5% to 10% of a cell fixative, 2% to 10% of a DNA enzyme inhibitor, 0.1% to 5% of a preservative and 10% to 50% of a pH buffer, and the remainder is water.

[0008] In a preferred embodiment of the present invention, the preservative is one or more of sodium chloride, Proclin 300, K-100, and citric acid.

[0009] In a preferred embodiment of the present invention, the pH buffer is Tris-HCl solution or potassium dihydrogen phosphate.

[0010] The present invention also provides a method for preparing the above-mentioned cell sample preservation solution, comprising the following steps:

[0011] 1) Weigh the cell fixative and DNase inhibitor according to the formula, add distilled water to dissolve, and obtain a mixed solution;

[0012] 2) adding a prescribed amount of preservative and pH buffer to the mixed solution, and stirring to mix;

[0013] 3) The pH is adjusted to 6.5-7.5, and distilled water is added to make up to the final volume to obtain a cell sample preservation solution.

[0014] In a preferred embodiment of the present invention, in step 1), heating to 60° C. is performed to accelerate dissolution.

[0015] In a preferred embodiment of the present invention, in step 2), the volume ratio of the preservative to the pH buffer is 1:25.

[0016] The present invention also provides an application of the above-mentioned cell sample preservation solution, which is used to preserve urine, cervical exfoliated cells and swabs; wherein, when used to preserve urine, the cell sample preservation solution and urine are evenly mixed in a volume ratio of 1:9.

[0017] Compared with the background technology, this technical solution has the following advantages:

[0018] 1. Simplify the formula: Through scientific proportioning and optimized ingredients, the formula of cell sample preservation fluid is simplified to make it simpler and easier to produce and use.

[0019] 2. Extend the shelf life: By adopting stable ingredients and ratios, the shelf life of the sample can be extended to ensure the stability and integrity of the sample.

[0020] 3. Reduce costs: By optimizing the production and manufacturing processes, the cost of sample preservation fluid can be reduced, making it more economical and suitable for a wider range of application scenarios.

[0021] 4. Convenient operation: Simplify the operation process, allowing users to collect, process and preserve samples more conveniently. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the gDNACt value trend chart when the urine sample is stored for 7 days without adding preservation solution;

[0023] Figure 2 This is a trend chart of gDNA Ct values ​​when the storage time is 20 days in Example 1;

[0024] Figure 3 This is a gDNA Ct value trend chart of clinical urine samples selected in Example 1 when stored for 20 days;

[0025] Figure 4 This is a trend chart of gDNA Ct values ​​when the storage time in Example 2 is 20 days. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Unless defined otherwise, all technical and scientific terms used herein are relevant to the technical field of the present invention.

[0028] Comparative Example No Preservative Solution Added

[0029] The group without preservative solution added refers to a group that does not use the sample preservative solution of the present invention as a preservation solution during the experiment. In the study, in order to compare and verify the effects of other preservative solutions, one or more control groups are usually set up, of which the group without preservative solution added is one of them.

[0030] Such a setup helps researchers understand the state and changes of samples in the absence of specific preservation solutions, thereby better evaluating and comparing the effects of other treatment methods.

[0031] Sample collection: Collect urine samples from volunteers, and each collection tube is filled with 4.5mL of urine. Store at room temperature (15-25℃) for 0 days, 3 days, 5 days, and 7 days. Take the corresponding samples at each time point for DNA extraction.

[0032] Experimental method: Sample pretreatment conditions: 10000g, centrifugation for 5min, discard the supernatant, collect urine precipitate cells into a centrifuge tube, use Tiangen (DP304) blood / cell / tissue genomic DNA extraction kit to extract gDNA in urine, and use qPCR instrument to perform conserved gene β-actin fluorescence quantitative analysis.

[0033] Detection method: Fluorescence quantitative analysis was used to compare and analyze the Ct values ​​of gDNA in urine samples stored at different time periods.

[0034] Table 1 Test results of the group without added preservation solution

[0035]

[0036] The samples without added preservation solution showed obvious turbidity after being placed at room temperature for 3 days. As the storage time prolonged, the Ct value of β-actin amplification increased significantly. The ΔCt value of the samples after 3 days was greater than 3 compared with the Ct value of 0 day, and the ΔCt value of the samples after 5 and 7 days was greater than 4-5 compared with the Ct value of 0 day, indicating that the samples underwent significant degradation during the room temperature storage process without the addition of preservation solution.

[0037] Example 1

[0038] The present embodiment provides a cell sample preservation solution, the components of which are as follows: by mass percentage: 1 g sodium chloride, 1 g diazolidinyl urea, 5 g disodium ethylenediaminetetraacetic acid, 3 g citric acid; by volume percentage: 50 uL 1% K-100 solution, 50 uL K-100, 2.5 mL 1M Tris-Hcl, and 80 mL distilled water.

[0039] The preparation method of this embodiment comprises the following steps:

[0040] 1) According to the formula, weigh 1g sodium chloride, 1g diazolidinyl urea, 5g disodium ethylenediaminetetraacetic acid, and 3g citric acid into a glass, add distilled water within the formula, stir evenly, and heat to 60°C to accelerate dissolution;

[0041] 2) Add 2.5 mL of 1 M Tris-HCl and 50 uL of K-100 to the mixed solution and stir to mix;

[0042] 3) About 2.32 mL of 10 M sodium hydroxide solution was added to the resulting mixed solution, the pH was adjusted to 6.5, and the final volume was made up to 100 mL with distilled water.

[0043] The cell sample preservation solution of this embodiment is applied to urine preservation. During preservation, the cell preservation solution and urine are mixed evenly in a volume ratio of 1:9, and the preservation effect is tested:

[0044] 1. Add the cell preservation solution (0.5 mL) of this example into the collection tube, collect the volunteer urine sample (4.5 mL) and put it into the collection tube corresponding to Example 1. Store at room temperature (15-25°C) for 0 days, 3 days, 5 days, 7 days, 10 days, and 20 days respectively. Take the corresponding sample at each time point and use the Tiangen (DP304) blood / cell / tissue genomic DNA extraction kit to extract the gDNA in the urine, and use the qPCR instrument to perform the conserved gene β-actin fluorescence quantitative analysis.

[0045] Detection method: Fluorescence quantitative analysis was used to compare and analyze the Ct values ​​of gDNA in urine samples stored at different time periods:

[0046] Table 2 Test results of Example 1

[0047]

[0048] The preservation solution of Example 1 can be preserved at room temperature for up to 20 days. Compared with the Ct value at day 0, ΔCt<1. Compared with the group without preservation solution, it is fully proved that the preservation solution of the present invention has excellent effect in preserving samples.

[0049] 2. 12 clinical urine samples were selected, and the preservation solution in Example 1 was added respectively, and stored at room temperature (15-25°C) for 0 days, 7 days, and 20 days. The corresponding samples were taken at each time point, and the gDNA in the urine was extracted using the Tiangen (DP304) blood / cell / tissue genomic DNA extraction kit, and the conserved gene β-actin fluorescence quantitative analysis was performed using a qPCR instrument.

[0050] Detection method: Fluorescence quantitative analysis was used to compare and analyze the Ct values ​​of gDNA in urine samples stored at different time periods:

[0051] Table 3 Test results of clinical sample preservation effect of Example 1

[0052]

[0053] The 12 clinical samples added with the preservation solution of Example 1 can be preserved at room temperature for up to 20 days. Compared with the Ct value at day 0, ΔCt<1. Compared with the group without the preservation solution, it is fully proved that the preservation solution of the present invention has excellent effect in preserving samples.

[0054] Example 2

[0055] The present embodiment provides a cell sample preservation solution, the components of which are: 2 g sodium phosphate, 10 g diazolidinyl urea, 8 g disodium ethylenediaminetetraacetic acid, 1 g citric acid; and by volume percentage: 600 uL Proclin 300, 0.5 mL 1 M potassium dihydrogen phosphate solution, 2 mL 1 M Tris-Hcl, and 80 mL distilled water.

[0056] The preparation method is as in Example 1.

[0057] The cell sample preservation solution of this embodiment is applied to urine preservation. During preservation, the cell preservation solution and urine are mixed evenly at a volume ratio of 1:9, and the preservation effect is measured:

[0058] The cell sample preservation solution (0.5 mL) of this example was added to the collection tube, and the volunteer urine sample (4.5 mL) was collected and placed in the collection tube corresponding to Example 2. The samples were stored at room temperature (15-25°C) for 0 days, 3 days, 5 days, 7 days, 10 days, and 20 days, respectively. The corresponding samples were taken at each time point and the gDNA in the urine was extracted using the Tiangen (DP304) blood / cell / tissue genomic DNA extraction kit, and the conserved gene β-actin fluorescence quantitative analysis was performed using a qPCR instrument.

[0059] Detection method: Fluorescence quantitative analysis was used to compare and analyze the Ct values ​​of gDNA in urine samples stored at different time periods:

[0060] Table 4 Example 2 test results

[0061]

[0062] The preservation solution of Example 2 can be preserved at room temperature for up to 20 days. Compared with the Ct value at day 0, ΔCt<1. Compared with the group without preservation solution, it is fully proved that the preservation solution of the present invention has excellent effect in preserving samples.

[0063] The cell sample preservation solution can preserve urine at room temperature (15-25°C) for 20 days. It is also applicable to cervical exfoliated cells or swab samples, and can be preserved at room temperature (15-25°C) for 20 days. Among them, diimidazole lignoceramide as a cell fixative has good stability and biocompatibility, and can interact with components such as proteins or sugars on the cell membrane, thereby fixing the cells in a certain position or shape; disodium ethylenediaminetetraacetic acid as a DNA enzyme inhibitor effectively prevents DNA enzymes from degrading DNA; the preservative ProClin 300 has an antibacterial effect to ensure that the sample does not mildew during storage. The pH buffer Tris-HCl is a weak base that can bind to H+ ions to adjust and stabilize the pH value of the solution. Adding an appropriate amount of Tris-HCl to the preservation solution can effectively maintain the pH value of the solution within an appropriate range, preventing damage and degradation of biological samples, cells or tissues caused by pH fluctuations. And it does not affect the interaction between Tris-HCl and other components to form a stable complex, further enhancing the protective effect of the preservation solution.

[0064] In summary, the present invention adopts unique ingredients and ratios. Through scientific ratios, the cell fixative, DNA enzyme inhibitor, preservative, pH buffer and other ingredients are optimized and combined to solve the problem that alcohols contained in the preservation solution on the market are harmful to the human body. Excessive ingredients may cause false positives or false negatives in the experimental results, affecting the judgment and interpretation of the experimental results. The raw material components used in the cell sample preservation solution of the present invention are simple, low-cost, easy to prepare, and achieve high efficiency and stability of the sample preservation solution.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell sample preservation solution, characterized in that: The invention relates to an aqueous solution comprising the following substances in percentage by mass: 0.5% to 10% of a cell fixative, 2% to 10% of a DNA enzyme inhibitor, and 0.1% to 5% of a preservative, and also comprising a pH buffer for maintaining the pH value of the cell sample preservation solution at 6.5 to 7.5; wherein the cell fixative is at least one of diazolidinyl urea, imidazolidinyl urea, and glucose; and the DNA enzyme inhibitor is at least one of citric acid, sodium phosphate, and disodium ethylenediaminetetraacetic acid.

2. A cell sample preservation solution according to claim 1, characterized in that: The invention is composed of the following components in percentage by mass: 0.5% to 10% of a cell fixative, 2% to 10% of a DNA enzyme inhibitor, 0.1% to 5% of a preservative and 10% to 50% of a pH buffer, and the balance is water.

3. The cell sample preservation solution according to claim 1, characterized in that: The preservative is one or more of sodium chloride, Proclin 300, K-100, and citric acid.

4. The cell sample preservation solution according to claim 1, characterized in that: The pH buffer is Tris-HCl solution or potassium dihydrogen phosphate.

5. The method for preparing a cell sample preservation solution according to any one of claims 1 to 4, characterized in that: The steps include: 1) Weigh the cell fixative and DNase inhibitor according to the formula, add distilled water to dissolve, and obtain a mixed solution; 2) adding a prescribed amount of preservative and pH buffer to the mixed solution, and stirring to mix; 3) The pH is adjusted to 6.5-7.5, and distilled water is added to make up to the final volume to obtain a cell sample preservation solution.

6. The method for preparing a cell sample preservation solution according to claim 5, characterized in that: In step 1), heating to 60° C. accelerates dissolution.

7. The method for preparing a cell sample preservation solution according to claim 5, characterized in that: In step 2), the volume ratio of the preservative to the pH buffer is 1:

25.

8. The use of a cell sample preservation solution according to any one of claims 1 to 4, characterized in that: Used to preserve urine, cervical exfoliated cells and swabs; wherein, the cell sample preservation fluid and urine are evenly mixed in a volume ratio of 1:9.

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