A DNA protective agent, its preparation method and application

Abstract

The application discloses a DNA protection agent and a preparation method and application thereof. The DNA protection agent is composed of EDTA-disodium salt, Tris, anhydrous ethanol, sodium dodecyl sulfate and an antifoaming agent, and has a pH of 8.5-9.5. The formula uses high-concentration ethanol to quickly fix samples, irreversibly inactivates nucleases, EDTA completely chelates metal ions, SDS assists protein denaturation, Tris stabilizes an alkaline environment, and an antifoaming agent is introduced to solve the mixed foam problem. The DNA protection agent can maintain the integrity of the high-molecular-weight main band of genomic DNA when storing a fecal sample for 30 days under a high-temperature condition of 50 DEG C, and the effect is obviously superior to that of existing commercial and general formulas, and the DNA protection agent is particularly suitable for sample collection and transportation under hot weather.

Description

Technical Field

[0001] This invention belongs to the field of microbial sample preservation technology, specifically relating to a DNA protectant, its preparation method, and its application. Background Technology

[0002] DNA, as the carrier of genetic information, has important applications in life science research, medical diagnosis, genetic engineering, forensic identification, and other fields. However, DNA molecules are extremely susceptible to physical, chemical, and biological factors in the in vitro environment, such as enzymatic degradation, oxidative damage, and ultraviolet radiation.

[0003] Currently, commonly used DNA protection methods include cryopreservation, adding protective agents, or regulating the solution environment, but they have the following drawbacks: (1) Cryopreservation is costly and not suitable for large-scale transportation and long-term storage; (2) The types of chemical protective agents are limited, the effects are not ideal, and some even interfere with subsequent experiments; (3) Environmental regulation has limited effect on pH and ionic strength, and cannot completely avoid degradation.

[0004] Therefore, there is an urgent need for a highly efficient, stable, and safe DNA protectant to protect DNA molecules from external damage in various application scenarios. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient, safe, and stable DNA protectant, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: providing a DNA protectant, comprising the following components: Enzyme inhibitor: EDTA disodium salt, wherein the molar concentration of EDTA disodium salt in the DNA protectant is 200-400 mM; Buffer: Tris, wherein the molar concentration of Tris in the DNA protectant is 40-60 mM; Antibacterial agent: anhydrous ethanol, wherein the volume percentage of anhydrous ethanol in the DNA protectant is 15%-30%; Surfactant: Sodium dodecyl sulfate, wherein the sodium dodecyl sulfate constitutes 0.1%-1.0% by mass / volume in the DNA protectant; Defoamer: The volume percentage of the defoamer in the DNA protectant is 0.05%-0.2%; The solvent is water. Another technical solution of the present invention is: providing a method for preparing the above-mentioned DNA protectant, comprising the following steps: (1) Weigh out EDTA-disodium salt, dissolve it in sodium hydroxide solution, and stir until completely dissolved; (2) Add buffer, surfactant and defoamer to the solution obtained in step (1) in sequence, and continue stirring until completely dissolved; (3) Add the antibacterial agent to the solution obtained in step (2) and mix well; (4) Adjust the pH of the solution obtained in step (3) to 8.5-9.5 using acid or base; (5) Add water to make up the volume, filter to remove bacteria, and obtain DNA protectant.

[0007] Another technical solution of the present invention is to provide the application of the above-mentioned DNA protectant in the preservation of fecal sample DNA.

[0008] Beneficial effects of this invention: This invention is the first to propose the combined use of high-concentration ethanol (15-30%) with EDTA-disodium salt, Tris, and SDS to form a composite protection system. Compared with existing DNA protectant formulations, the DNA protectant of this invention uses high-concentration ethanol as the core antibacterial and fixative component. High-concentration ethanol can rapidly denature and inactivate microorganisms and nucleases in the sample. Combined with EDTA chelating metal ions, SDS assisting denaturation, and Tris maintaining an alkaline environment, it can achieve immediate fixation and long-term protection of DNA at both room temperature and 50°C. Attached Figure Description

[0009] Figure 1 This is a comparative experimental result diagram of the protective effect of DNA protectant on DNA integrity of fecal samples under different temperature conditions in Example 2 of the specific implementation of the present invention; Figure 2 This is a comparative experimental result diagram of the protective effect of different DNA protectants on the DNA integrity of fecal samples at 50°C for 3 days in Example 3 of the specific implementation of the present invention. Detailed Implementation

[0010] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0011] This invention provides a highly efficient, safe, and stable DNA protectant that can effectively protect DNA in fecal samples from degradation under normal or even high temperature conditions.

[0012] Specifically, the DNA protectant provided by this invention comprises the following components: Enzyme inhibitor: EDTA disodium salt, wherein the molar concentration of EDTA disodium salt in the DNA protectant is 200-400 mM; Buffer: Tris, wherein the molar concentration of Tris in the DNA protectant is 40-60 mM; Antibacterial agent: anhydrous ethanol, wherein the volume percentage of anhydrous ethanol in the DNA protectant is 15%-30%; Surfactant: Sodium dodecyl sulfate, wherein the sodium dodecyl sulfate constitutes 0.1%-1.0% by mass / volume in the DNA protectant; Defoamer: The volume percentage of the defoamer in the DNA protectant is 0.05%-0.2%; The solvent is water.

[0013] As described above, this invention is the first to propose the combined use of high-concentration ethanol (15-30%) with EDTA-disodium salt, Tris, and SDS to form a composite protection system. Compared with existing DNA protectant formulations, this invention uses high-concentration ethanol as the core antibacterial and fixative component. High-concentration ethanol can rapidly denature and inactivate microorganisms and nucleases in samples. Combined with EDTA chelating metal ions, SDS assisting denaturation, and Tris maintaining an alkaline environment, it can achieve immediate fixation and long-term protection of DNA at both room temperature and 50°C.

[0014] Furthermore, in the above-mentioned DNA protectant, the molar concentration of the EDTA-disodium salt in the DNA protectant is 300 mM.

[0015] As described above, this concentration of EDTA-disodium salt is relatively high, and it can more thoroughly chelate divalent metal ions (such as Mg²⁺). + Ca² + This concentration inhibits the activity of DNA enzymes. The preferred concentration ensures the inhibitory effect while avoiding potential interference from excessively high salt concentrations in subsequent experiments such as PCR.

[0016] Furthermore, in the above-mentioned DNA protectant, the molar concentration of Tris in the DNA protectant is 50 mM.

[0017] As described above, this concentration of Tris can provide sufficient buffer capacity to stably maintain the pH of the protectant at around 9.0 under accelerated degradation conditions at 50°C, effectively resisting pH drops caused by microbial metabolism or acidic substances in the sample itself, thereby protecting DNA from acid hydrolysis.

[0018] Furthermore, in the above-mentioned DNA protectant, the volume percentage of anhydrous ethanol in the DNA protectant is 23.5%.

[0019] As described above, this concentration is the optimal value for balancing the protective effect and the compatibility of subsequent applications. 23.5% ethanol can effectively inhibit the growth of bacteria and fungi, and can denature and coagulate proteins such as nucleases. At the same time, this concentration will not cause excessive hardening of fecal samples or inhibit the enzyme system of PCR amplification after large dilution, thus taking into account both the sample fixation effect and the smoothness of downstream experiments.

[0020] Furthermore, in the above-mentioned DNA protectant, the sodium dodecyl sulfate accounts for 0.5% of the DNA protectant by mass volume.

[0021] As described above, this concentration of SDS, as a protein denaturant, works synergistically with ethanol to accelerate and enhance the inactivation of microorganisms and nucleases.

[0022] Furthermore, in the above-mentioned DNA protectant, the pH value range of the DNA protectant is 8.5-9.5.

[0023] As described above, DNA conformation is more stable at this alkaline pH and acidic hydrolysis is inhibited.

[0024] Another technical solution of the present invention is: providing a method for preparing the above-mentioned DNA protectant, comprising the following steps: (1) Weigh out EDTA-disodium salt, dissolve it in sodium hydroxide solution, and stir until completely dissolved; (2) Add buffer, surfactant and defoamer to the solution obtained in step (1) in sequence, and continue stirring until completely dissolved; (3) Add the antibacterial agent to the solution obtained in step (2) and mix well; (4) Adjust the pH of the solution obtained in step (3) to 8.5-9.5 using acid or base; (5) Add water to make up the volume, filter to remove bacteria, and obtain DNA protectant.

[0025] Another technical solution of the present invention is to provide the application of the above-mentioned DNA protectant in the preservation of fecal sample DNA. The fecal sample DNA is preserved at a temperature of 50°C.

[0026] Example 1 A DNA protectant, based on the preparation of 1L, accurately weigh the following components: Disodium ethylenediaminetetraacetate (EDTA-disodium salt): 112g; Tris(hydroxymethyl)aminomethane (Tris): 6g; Anhydrous ethanol: 235 mL; Sodium dodecyl sulfate (SDS): 5g; Antifoam A defoamer: 1 mL; Deionized water: Add to 1L.

[0027] The method for preparing the DNA protectant includes the following steps: (1) Add sodium hydroxide to 500 mL of deionized water and stir to dissolve to prepare a solution of about 1 M; add 112 g of EDTA-disodium salt to the alkaline solution in portions and stir vigorously until completely dissolved.

[0028] (2) Add 6g Tris, 5g SDS and 1mL Antifoam A to the above solution in sequence and stir until completely dissolved.

[0029] (3) Take 235 mL of anhydrous ethanol, slowly add it to the solution, and stir until well mixed.

[0030] (4) Adjust the pH of the solution to 9.0 with 10M NaOH.

[0031] (5) Transfer the solution into a 1L volumetric flask, dilute to the mark with deionized water, mix well, filter to sterilize, and then dispense and store.

[0032] The resulting DNA protectant comprises the following components: (1) Enzyme inhibitor: 300mM (11.2%, w / v) EDTA disodium salt, used to inhibit the degradation of nucleases.

[0033] (2) Buffer: 50mM (0.6%, w / v) Tris, maintain pH 9.0.

[0034] (3) Antibacterial agent: 23.5% (v / v) ethanol, used to inhibit the growth of microorganisms.

[0035] (4) Surfactant: 0.5% (w / v) sodium dodecyl sulfate (SDS), used to denature proteins and inhibit microbial growth.

[0036] (5) Defoamer: 0.1% (v / v) Antifoam A, used to reduce foam generated during sample mixing.

[0037] The DNA protectant obtained above can be used as follows: mix it with the DNA protectant at a ratio of 1:5 based on the sample volume. This mixture can preserve DNA samples for a long time at room temperature and is also suitable for high-temperature transportation environments.

[0038] Experiments show that this DNA protectant can effectively protect DNA under both room temperature and high temperature (50℃) conditions, with minimal concentration drop and high DNA integrity, which is superior to commercial preservation solutions A and B.

[0039] Example 2 A comparative experiment of the DNA protectant obtained in Example 1 with other existing protectants; 1. According to the test conditions, aliquot 200 μL of fecal sample into centrifuge tubes containing 1 mL of sample DNA preservation solution; aliquot another 200 μL of fecal sample into an empty centrifuge tube as a control. (2) Use a vortex mixer at its maximum speed for 30 seconds to thoroughly mix the sample; (3) Store the test samples in incubators at room temperature and 50°C for 1 day / 3 days / 1 month respectively; store the control samples and the tested samples in a -80°C refrigerator. (4) Extract DNA from the sample and detect the concentration (Qubit fluorescence quantification) and integrity of the DNA (agarose gel electrophoresis analysis).

[0040] 2. Interpretation of Qubit fluorescence quantitative quantification results Table 1 shows the comparative experimental results of DNA protectants on the concentration of DNA in fecal samples under different temperature conditions.

[0041] Table 1 RT: room temperature; D1: day 1; D3: day 3; M1: one month; -80℃ (a or b): two independent experiments.

[0042] As shown in Table 1 above, compared to fecal sample 1 directly frozen at -80℃, the concentrations of the protective agent of this invention and commercial preservation solution B were higher at room temperature, regardless of whether the samples were stored for 1 day or 3 days. This suggests that these two protective agents may improve the degree of bacterial lysis in fecal samples, thereby increasing DNA extraction efficiency. The concentration of commercial preservation solution A decreased significantly, especially on the 3rd day, with a 12.9% decrease in DNA concentration. This indicates that at room temperature, both the protective agent of this invention and commercial preservation solution B provide good protection for fecal sample DNA.

[0043] It is noteworthy that the concentration of fecal samples stored at room temperature for one month in the protective agent of the present invention was still higher than that of direct freezing, indicating that the protective agent of the present invention has good DNA protection effect at room temperature and can be used for long-term room temperature DNA storage, reducing dependence on -80°C refrigerators.

[0044] Under high-temperature conditions, the concentration decrease of the protective agent of this invention is relatively small, remaining at 16.50 ng / μL (-3.0%) after 3 days of storage. Commercial preservation solutions A and B exhibit more significant concentration decreases at high temperatures (commercial preservation solution A: -11.2%, commercial preservation solution B: -14.4%).

[0045] The results for fecal sample 2 were consistent with those for fecal sample 1. This indicates that the protective agent solution of this invention provides the best DNA protection under high temperature conditions.

[0046] 3. Interpretation of agarose gel electrophoresis results Please refer to Figure 1 The figure shows the results of a comparative experiment on the effect of DNA protectants on the DNA integrity of fecal samples under different temperature conditions. Figure 1 In this context, RT represents room temperature; D1 represents day 1; D3 represents day 3; M1 represents one month; and -80℃ (a or b) represents two independent experiments.

[0047] from Figure 1 It can be known that: Gel brightness and band intensity: The brightness of each sample band reflects the integrity and concentration of DNA. Bright and clear bands (without diffusion) indicate good DNA stability, while blurred or dimmed bands indicate DNA degradation or decreased concentration.

[0048] Fecal samples 1 (a and b) frozen at -80℃: The band intensity is the brightest, indicating high DNA stability.

[0049] At room temperature and 50°C, the band brightness gradually decreased over time (D1 to D3), especially under commercial preservation solutions A and B, indicating that DNA may be degraded at high temperatures.

[0050] The D3 band intensity of commercial preservation solutions A and B was significantly reduced at 50°C.

[0051] The results for fecal sample 2 were similar to those for fecal sample 1. Similarly, the sample stored at -80°C showed the strongest band brightness. The degradation of commercial preservation solutions A and B was more significant, especially at time point D3 at 50°C, where the band brightness was significantly weaker than that of the protective agent of this invention.

[0052] 4. Conclusion: Both DNA concentration quantification analysis and gel electrophoresis indicate that the protective agent of this invention provides better protection for DNA stability under different conditions than commercial preservation solutions A and B. Furthermore, it can be concluded from the above that the DNA protective agent of this invention is particularly suitable for transporting samples during hot summer months.

[0053] Example 3 1. All other conditions are the same as in Example 2, except that the protective agent used for comparison is different; The protective agents of this invention are shown in Table 2, and the pH is controlled at 9.0; Table 2 Comparative Example 1: Low concentration of EDTA protective agent as shown in Table 2, pH controlled at 9.0; Table 3 The concentrations of NaCl protective agents, such as those used in Comparative Example 2 and ethanol, are shown in Table 3, with pH controlled at 9.0. Table 4 Comparative Example 3, pH=8.0, protectant as shown in Table 4; Table 5 Comparative Example 4: An existing protective agent is shown in Table 5, with pH controlled at 8.0; Table 6 2. Interpretation of Qubit fluorescence quantitative quantification results Table 7 shows the comparative experimental results of the protective effects of different DNA protectants on the DNA concentration of fecal samples at 50℃ for 3 days. Table 7 Interpretation of Results: (1) Degradation background under high temperature stress (the control group used phosphate-buffered saline (PBS) as a negative control). -80℃ vs 50℃ PBS: DNA concentration in sample 3 dropped sharply from 17.30 ng / μL to 3.70 ng / μL, and in sample 4 it dropped from 23.40 ng / μL to 2.24 ng / μL.

[0054] Microbiological explanation: Feces are rich in DNase I (deoxyribonuclease I) and microbial endonucleases. 50°C is close to the optimal reaction temperature for many mesophilic bacteria and residual pancreatic DNase in feces. Without a protective agent, DNA will be enzymatically cleaved into short oligonucleotide fragments that cannot be detected by quantitative fluorescence and electrophoresis within 3 days.

[0055] (2) Comparison of the protective effects of each formulation is shown in Table 8 (sample 3 is the main one, and sample 4 shows the same trend). Table 8 3. Agarose gel electrophoresis results Figure 2 A figure showing the comparative experimental results of different DNA protectants on the protection of DNA integrity in fecal samples at 50°C for 3 days; Figure 2 middle: 1: Store at -80℃ without preservative solution; 2: Store at 50℃ in PBS; 3: Store at 50℃ using the preservation solution of this invention; 4: Store at 50℃ in Comparative Example 1 (low concentration EDTA) preservation solution; 5: Store at 50℃ in Comparative Example 2 (NaCl with ionic concentrations similar to ethanol); 6: Store at 50℃ in the preservation solution of Comparative Example 3 (pH=8.0); 7: Store at 50℃ using the preservation solution from Comparative Example 4; Interpretation of Results: (1) Lane 1 (-80℃): Displayed as a clear high molecular weight band, which is the baseline for the sample.

[0056] (2) Lane 2 (PBS): The bands almost completely disappeared, confirming complete degradation.

[0057] (3) Lane 3 (in this invention): The band is closest to the baseline, indicating that the DNA integrity is well maintained.

[0058] (4) Lane 7 (Comparative Example 4): The bands were clearly smearing, indicating significant fragmentation.

[0059] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A DNA protectant, characterized in that, Includes the following components: Enzyme inhibitor: EDTA disodium salt, wherein the molar concentration of EDTA disodium salt in the DNA protectant is 200-400 mM; Buffer: Tris, wherein the molar concentration of Tris in the DNA protectant is 40-60 mM; Antibacterial agent: anhydrous ethanol, wherein the volume percentage of anhydrous ethanol in the DNA protectant is 15%-30%; Surfactant: Sodium dodecyl sulfate, wherein the sodium dodecyl sulfate constitutes 0.1%-1.0% by mass / volume in the DNA protectant; Defoamer: The volume percentage of the defoamer in the DNA protectant is 0.05%-0.2%; The solvent is water.

2. The DNA protectant according to claim 1, characterized in that, The molar concentration of the EDTA-disodium salt in the DNA protectant is 300 mM.

3. The DNA protectant according to claim 1, characterized in that, The molar concentration of Tris in the DNA protectant is 50 mM.

4. The DNA protectant according to claim 1, characterized in that, The volume percentage of anhydrous ethanol in the DNA protectant is 23.5%.

5. The DNA protectant according to claim 1, characterized in that, The sodium dodecyl sulfate contains 0.5% by mass and volume in the DNA protectant.

6. The DNA protectant according to claim 1, characterized in that, The pH range of the DNA protectant is 8.5-9.

5.

7. A method for preparing the DNA protectant according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Weigh out EDTA-disodium salt, dissolve it in sodium hydroxide solution, and stir until completely dissolved; (2) Add buffer, surfactant and defoamer to the solution obtained in step (1) in sequence, and continue stirring until completely dissolved; (3) Add the antibacterial agent to the solution obtained in step (2) and mix well; (4) Adjust the pH of the solution obtained in step (3) to 8.5-9.5 using acid or base; (5) Add water to make up the volume, filter to remove bacteria, and obtain DNA protectant.

8. The use of the DNA protectant according to any one of claims 1-6 in the preservation of DNA from fecal samples.

9. The application according to claim 8, characterized in that, The fecal sample DNA was preserved at a temperature of 50°C.

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