Nucleic acid preserving fluid and preparation method thereof

Abstract

The invention provides a nucleic acid preserving fluid and a preparation method thereof, the nucleic acid preserving fluid comprises a buffer solution, an ionic strength maintaining agent, a chelating agent, a strong denaturing agent and temperature-responsive nanoparticles, and all the components are dissolved in nuclease-free ultrapure water; the temperature responsive nano particles are temperature responsive polymer based nano particles or temperature responsive lipid based nano particles; the particle size of the temperature-responsive nanoparticles is 50-100 nm, and the phase transition temperature of the temperature-responsive nanoparticles is 30-35 DEG C. The temperature-responsive nanoparticles can keep hydrophilic and stably coexist with a nucleic acid sample at a low temperature, when the temperature rises to be higher than the phase change temperature, the temperature-responsive nanoparticles become hydrophobic, a compact physical protection layer is formed on the surfaces of nucleic acid molecules, and the synergistic effect of chemical protection and physical protection is achieved; the nucleic acid preservation stability is remarkably improved, the dependence of nucleic acid preservation on a cold chain is reduced, and the method is particularly suitable for nucleic acid sample protection in long-time storage and transportation environments.

Description

Technical Field

[0001] This invention relates to the field of biological sample preservation technology, and in particular to a nucleic acid preservation solution and its preparation method. Background Technology

[0002] Nucleic acids (including DNA and RNA) serve as carriers of genetic information within organisms and have significant applications in molecular biology research, clinical diagnosis, forensic identification, and vaccine development. However, nucleic acid molecules are complex and extremely unstable, easily degraded by various factors such as temperature, pH, ionic strength, and nucleases, posing a major challenge to the long-term preservation and transportation of nucleic acids.

[0003] Traditional nucleic acid preservation primarily relies on ultra-low temperature freezing technology, typically requiring storage at -20°C or -80°C. While low temperatures effectively inhibit nuclease activity and slow down chemical degradation reactions, this preservation method suffers from high energy consumption, expensive equipment, and reliance on cold chain logistics, making it difficult to scale up in remote areas or resource-scarce environments. To address this issue, researchers have developed room-temperature nucleic acid preservation technology.

[0004] Existing room-temperature nucleic acid preservation solutions typically contain components such as buffer systems, chelating agents, and denaturants to maintain a suitable preservation environment. For example, Chinese patent CN115960990B discloses a preservation solution for deoxyribonucleic acid and pseudoviruses, including buffer solution, surfactant, trehalose, metal ion chelating agent, betaine, and mannitol, which can stably preserve nucleic acids for up to 12 months at 30°C.

[0005] However, existing room-temperature preservation solutions only provide chemical protection and lack physical barriers. When faced with extreme conditions such as temperature fluctuations and exogenous nuclease contamination, nucleic acids are still prone to degradation and cannot meet the requirements for long-term stable preservation. In addition, nucleic acids, especially RNA, are highly susceptible to nuclease degradation, pH fluctuations, and temperature changes, leading to structural damage and sequence loss. Existing preservation technologies are unable to provide comprehensive and effective protection under complex environmental conditions.

[0006] Therefore, there is an urgent need to develop a new nucleic acid preservation technology that can achieve long-term stable preservation at room temperature and provide more comprehensive protection for nucleic acids when facing various adverse environmental factors, so as to meet the needs of modern biotechnology development. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a nucleic acid preservation solution to solve the technical problems of existing room temperature preservation solutions lacking physical barriers, failing to meet the requirements for long-term stable preservation, and nucleic acids being susceptible to degradation by nucleases, pH fluctuations, temperature changes, and other factors that lead to structural damage. At the same time, this invention will also provide a method for preparing the nucleic acid preservation solution.

[0008] To achieve the above and other related objectives, the present invention provides the following technical solutions: In a first aspect, the present invention provides a nucleic acid preservation solution comprising a buffer, an ionic strength maintainer, a chelating agent, a strong denaturant, and temperature-responsive nanoparticles, all of which are dissolved in nuclease-free ultrapure water.

[0009] Furthermore, the temperature-responsive nanoparticles are temperature-responsive polymer-based nanoparticles or temperature-responsive lipid-based nanoparticles, and the particle size of the temperature-responsive nanoparticles is 50~100nm, and the phase transition temperature (LCST) is 30~35℃.

[0010] Furthermore, the temperature-responsive polymer-based nanoparticles are selected from poly(N-isopropylacrylamide-polyethylene glycol) block copolymer (PNIPAM-PEG) nanoparticles. In the poly(N-isopropylacrylamide-polyethylene glycol) block copolymer, the mass fraction of polyethylene glycol (PEG) is 8-12%, preferably 10%.

[0011] Furthermore, the preparation method of the PNIPAM-PEG nanoparticles includes the following steps: S101. Preparation of macromolecular chain transfer agents based on monomethyl-terminated polyethylene glycol; S102. The macromolecular chain transfer agent, N-isopropylacrylamide monomer (NIPAM) and initiator obtained in step S101 are polymerized in the first solvent to obtain poly(N-isopropylacrylamide-polyethylene glycol block copolymer). S103. The poly(N-isopropylacrylamide-polyethylene glycol) block copolymer obtained in step S102 is dissolved in nuclease-free ultrapure water to prepare a polymer solution. The polymer solution is ultrasonically dispersed under ice bath conditions and filtered through a nuclease-free filter membrane to obtain a temperature-responsive PNIPAM-PEG nanoparticle suspension.

[0012] In step S101, monomethyl-terminated polyethylene glycol and maleic anhydride are added to a reaction vessel at a molar ratio of 1:(1.2~1.5), and dichloromethane is used as a solvent. The mixture is reacted at a constant temperature of 40~50℃ for 4~6h to obtain an addition product. Dithiobenzoic acid is added to the addition product and mixed at a molar ratio of 1:(1.0~1.2). The mixture is reacted at a constant temperature of 60~70℃ for 8~10h under nitrogen protection. After cooling to room temperature, the mixture is dialyzed and freeze-dried to obtain a macromolecular chain transfer agent based on monomethyl-terminated polyethylene glycol.

[0013] Among them, the molecular weight of monomethyl-terminated polyethylene glycol is 2000~5000 Da.

[0014] In step S102, the initiator is selected from azobisisobutyronitrile or benzoyl peroxide, preferably azobisisobutyronitrile.

[0015] In step S102, the mass ratio of macromolecular chain transfer agent, N-isopropylacrylamide monomer, and initiator is 1:(5~8):(0.02~0.05).

[0016] In step S102, the first solvent is selected from tetrahydrofuran, N,N-dimethylformamide (DMF), or toluene; preferably tetrahydrofuran.

[0017] In step S102, the polymerization temperature is 65~75℃ and the reaction time is 12~16h. After the polymerization reaction is completed, the product is poured into excess diethyl ether to precipitate, the precipitate is collected and dialyzed and freeze-dried to obtain poly(N-isopropylacrylamide-polyethylene glycol) block copolymer.

[0018] In step S103, the mass concentration of the polymer in the polymer solution is 1~5 mg / mL.

[0019] Furthermore, the temperature-responsive lipid-based nanoparticles are selected from DPPC / DSPC composite thermosensitive liposomes. In the DPPC / DSPC composite thermosensitive liposomes, the molar ratio of DPPC to DSPC is (8~5):(2~5), preferably 7:3.

[0020] Furthermore, the preparation method of the DPPC / DSPC composite thermosensitive liposomes includes the following steps: S201. Dissolve DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) and DSPC (1,2-distearate-sn-glycerol-3-phosphocholine) in a second solvent to form a homogeneous lipid solution. S202. The lipid solution is subjected to reduced pressure rotary evaporation to remove the solvent, forming a uniform lipid film; S203. Nuclease-free ultrapure water is added to the lipid membrane for full hydration, followed by ultrasonic dispersion in an ice bath and filtration through a nuclease-free filter membrane to obtain the DPPC / DSPC composite thermosensitive liposomes.

[0021] In step S201, the second solvent is selected from chloroform, dichloromethane, or a chloroform / methanol mixture, preferably chloroform.

[0022] In step S201, the molar ratio of DPPC to DSPC is (8~5):(2~5), preferably 7:3.

[0023] In step S202, the rotary evaporation temperature is 37~40℃ and the rotation speed is 50~80 r / min.

[0024] In step S202, the obtained lipid film is vacuum dried for 2-4 hours to completely remove residual solvent.

[0025] In step S203, the final lipid concentration during hydration is 1~5 mg / mL.

[0026] In step S203, the lipid film is fully hydrated by shaking in a water bath at 37~40℃ for 30~60 minutes.

[0027] Furthermore, the final concentration of the temperature-responsive nanoparticles in the nucleic acid preservation solution is 0.1~0.5 mg / mL, preferably 0.2 mg / mL.

[0028] Furthermore, the buffer solution is selected from Tris-HCl buffer or PBS buffer. This buffer solution is used to maintain the pH of the preservation solution within the range of 7.5-8.5 to prevent nucleic acid hydrolysis under excessively acidic or alkaline conditions. The buffer solution is preferably Tris-HCl buffer with a final concentration of 10-50 mM, preferably 10 mM.

[0029] Furthermore, the ionic strength maintaining agent includes sodium acetate and / or NaCl, used to provide ionic strength, reduce the aggregation between nucleic acid molecules, and inhibit some nuclease activity. The final concentration of sodium acetate in the nucleic acid preservation solution is 20-100 mM, preferably 50 mM. The final concentration of NaCl in the nucleic acid preservation solution is 50-150 mM, preferably 50 mM.

[0030] Furthermore, the chelating agent is selected from ethylenediaminetetraacetic acid (EDTA) or its sodium salt. This chelating agent can bind to metal ions in the sample, and since metal ions are essential cofactors for nucleases, it inhibits the degradation by nucleases. Preferably, the chelating agent is disodium EDTA, with a final concentration of 0.5–5 mM in the nucleic acid preservation solution, preferably 1.0 mM.

[0031] Furthermore, the strong denaturant is selected from guanidine isothiocyanate, which can rapidly inactivate nucleases in the sample, especially RNases, and prevent them from degrading RNA. The final concentration of guanidine isothiocyanate in the nucleic acid preservation solution is 1-5M, preferably 2M.

[0032] Furthermore, betaine is added to the nucleic acid preservation solution. Betaine helps maintain the stable structure of nucleic acids and enhances the efficiency of subsequent nucleic acid extraction or amplification. The final concentration of betaine in the nucleic acid preservation solution is 0.5~1M, preferably 0.5M.

[0033] A second aspect of the present invention provides a method for preparing a nucleic acid preservation solution, comprising the following steps: (1) Weigh each component according to the proportion, and first mix and dissolve the buffer, ionic strength maintainer, chelating agent, strong denaturant and nuclease-free ultrapure water; (2) After adjusting the pH value, filter out insoluble impurities; (3) Finally, add the temperature-responsive nanoparticle suspension and mix well to obtain the nucleic acid preservation solution.

[0034] Furthermore, in step (1), betaine is added, and the final concentration of betaine in the nucleic acid preservation solution is 0.5~1M, preferably 0.5M.

[0035] As described above, the nucleic acid preservation solution and its preparation method of the present invention have the following beneficial effects: 1. The temperature-responsive nanoparticles provided by this invention can automatically adjust their hydrophilicity and hydrophobicity when the temperature changes. At low temperatures, the nanoparticles remain hydrophilic and coexist stably with nucleic acid samples in the aqueous phase. When the temperature rises above the phase transition temperature, the nanoparticles become hydrophobic, spontaneously aggregate, and form a dense physical protective layer on the surface of nucleic acid molecules. This prevents exogenous nucleases and impurities from contacting the nucleic acid, while simultaneously reducing the influence of ions in the solution on the nucleic acid structure, achieving a synergistic effect of chemical and physical protection. Polymer-based nanoparticles improve dispersibility by introducing hydrophilic segments (PEG), while lipid-based nanoparticles, with their excellent biocompatibility, are suitable for a wider range of applications.

[0036] 2. The temperature-responsive nanoparticles of this invention have good compatibility with the components of the nucleic acid preservation solution. The hydrophilic segments of the polymer-based nanoparticles can prevent them from agglomerating and precipitating in high-salt, high-concentration strong denaturant (guanidine isothiocyanate) environments. The lipid-based nanoparticles improve their compatibility with the preservation solution by optimizing the lipid ratio. Compatibility tests have verified that all types of nanoparticles can remain clear and transparent in the preservation solution for 72 hours, with a stable pH of 7.5-8.5 and no abnormal aggregation.

[0037] 3. The temperature-responsive nanoparticles of the present invention are suitable for use in nucleic acid preservation solutions. They can not only prevent nucleic acid degradation, but also provide intelligent protection. The stable preservation time of RNA can be extended from 7-14 days to 21-28 days, and the stable preservation time of DNA can be extended from 1 month to 2 months. They can also withstand short-term temperature fluctuations of 25-35°C, which significantly reduces the dependence of nucleic acid preservation on the cold chain. They are particularly suitable for the protection of nucleic acid samples in long-term storage and transportation environments.

[0038] 4. This invention designs specific preparation methods for different types of nanoparticles. Polymer-based nanoparticles are prepared using reversible addition-fragmentation chain transfer polymerization (RAFT) technology, while lipid-based nanoparticles are prepared using rotary evaporation-hydration method. Both methods achieve precise control of nanoparticle size (50-100 nm) and uniform particle size distribution (PDI < 0.3) by precisely controlling reaction conditions. The preparation process does not require the use of highly toxic reagents, and the products are highly pure after purification by dialysis, filtration, etc., and are free from nuclease contamination, and can be directly used for nucleic acid preservation. Detailed Implementation

[0039] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0040] A nucleic acid preservation solution is provided, comprising a buffer, an ionic strength maintainer, a chelating agent, a strong denaturant, betaine, and temperature-responsive nanoparticles, all of which are dissolved in nuclease-free ultrapure water.

[0041] The buffer solution is selected from Tris-HCl buffer or PBS buffer, with a final concentration of 10-50 mM. This buffer is used to maintain the pH of the preservation solution within the range of 7.5-8.5, preventing hydrolysis of nucleic acids under excessively acidic or alkaline conditions. Ionic strength maintainers include sodium acetate and / or NaCl, used to provide ionic strength, reduce aggregation between nucleic acid molecules, and inhibit the activity of some nucleases. The final concentration of sodium acetate in the nucleic acid preservation solution is 20-100 mM, and the final concentration of NaCl is 50-150 mM. The chelating agent is selected from ethylenediaminetetraacetic acid (EDTA) or its sodium salt, with a final concentration of 0.5-5 mM. The chelating agent binds to metal ions in the sample, which are essential cofactors for nucleases, thereby inhibiting nuclease degradation. The strong denaturing agent is selected from guanidine isothiocyanate, with a final concentration of 1-5 M, which rapidly inactivates nucleases in the sample, especially RNase, preventing their degradation of RNA. The final concentration of betaine in nucleic acid preservation solution is 0.5~1M. Betaine can help maintain the stable structure of nucleic acids and enhance the efficiency of subsequent nucleic acid extraction or amplification.

[0042] The temperature-responsive nanoparticles are temperature-responsive polymer-based nanoparticles or temperature-responsive lipid-based nanoparticles with a particle size of 50~100nm, a phase transition temperature of 30~35℃, and a final concentration of 0.1~0.5mg / mL in the nucleic acid preservation solution.

[0043] Preferably, the temperature-responsive polymer-based nanoparticles are selected from poly(N-isopropylacrylamide-polyethylene glycol) block copolymer nanoparticles, and their preparation method includes the following steps: S101. Monomethyl-terminated polyethylene glycol and maleic anhydride are added to a reaction vessel at a molar ratio of 1:(1.2~1.5), and dichloromethane is used as a solvent. The mixture is reacted at a constant temperature of 40~50℃ for 4~6h to obtain an addition product. Dithiobenzoic acid is added to the addition product and mixed at a molar ratio of 1:(1.0~1.2). The mixture is reacted at a constant temperature of 60~70℃ for 8~10h under nitrogen protection. After cooling to room temperature, the mixture is dialyzed and freeze-dried to obtain a macromolecular chain transfer agent based on monomethyl-terminated polyethylene glycol. S102. The macromolecular chain transfer agent obtained in step S101, N-isopropylacrylamide monomer (NIPAM), and initiator are polymerized in a first solvent at a mass ratio of 1:(5~8):(0.02~0.05). The polymerization temperature is 65~75℃ and the reaction time is 12~16h. After the polymerization reaction is completed, the product is poured into excess diethyl ether to precipitate. The precipitate is collected, dialyzed, and freeze-dried to obtain poly(N-isopropylacrylamide-polyethylene glycol) block copolymer. The initiator is selected from azobisisobutyronitrile or benzoyl peroxide, and the first solvent is selected from tetrahydrofuran, N,N-dimethylformamide (DMF), or toluene. S103. The poly(N-isopropylacrylamide-polyethylene glycol) block copolymer obtained in step S102 is dissolved in nuclease-free ultrapure water to prepare a polymer solution. The polymer solution is ultrasonically dispersed under ice bath conditions and filtered through a nuclease-free filter membrane to obtain a temperature-responsive PNIPAM-PEG nanoparticle suspension.

[0044] Preferably, the temperature-responsive lipid-based nanoparticles are selected from DPPC / DSPC composite thermosensitive liposomes, and their preparation method includes the following steps: S201. Dissolve DPPC and DSPC in a molar ratio of (8~5):(2~5) in a second solvent, the second solvent being selected from chloroform, dichloromethane or a chloroform / methanol mixture. Stir on a magnetic stirrer for 30 minutes to completely dissolve the lipid raw materials and form a uniform lipid solution. S202. The lipid solution is subjected to reduced pressure rotary evaporation at 37~40℃ to remove the solvent, forming a uniform lipid film. The lipid film is then vacuum dried for 2~4 hours to completely remove residual solvent. S203. Add nuclease-free ultrapure water to the lipid membrane and oscillate in a water bath at 37-40℃ for 30-60 minutes to fully hydrate it. Then place it in an ultrasonic cell disruptor and sonicate it in an ice bath for 10-15 minutes (ultrasonic power 200-300W, sonication time 3s, interval time 5s). Filter it through a 0.22μm nuclease-free filter membrane to obtain the DPPC / DSPC composite thermosensitive liposome.

[0045] Example 1 Preparation of temperature-responsive PNIPAM-PEG nanoparticles: S101. Add 5.0 g of monomethyl-terminated polyethylene glycol and 2.1 g of maleic anhydride to a 250 mL three-necked flask, add 100 mL of dichloromethane to dissolve, and react at 45 °C for 5 h. After the reaction is complete, add 2.3 g of dithiobenzoic acid to the flask, purge the air with nitrogen three times, and react at 65 °C for 9 h. After cooling to room temperature, place the reaction solution in a dialysis bag (molecular weight cutoff 3500 Da), dialyze with dichloromethane for 3 days, changing the dialysate twice a day. After dialysis, freeze-dry to obtain a macromolecular chain transfer agent.

[0046] S102. Add 2.0g of macromolecular chain transfer agent, 12.0g of N-isopropylacrylamide monomer, and 0.06g of azobisisobutyronitrile to a 250mL three-necked flask, add 150mL of tetrahydrofuran to dissolve, purge the air with nitrogen three times, and polymerize at 70℃ for 14h. After the reaction is complete, slowly pour the reaction solution into 500mL of excess diethyl ether, let it stand to precipitate for 2h, filter and collect the precipitate. Place the precipitate in a dialysis bag (molecular weight cutoff 5000Da), dialyze with tetrahydrofuran for 3 days, changing the dialysate twice a day. After dialysis, freeze-dry to obtain PNIPAM-PEG block copolymer.

[0047] S103. Dissolve 1.0 g of PNIPAM-PEG block copolymer in 300 mL of nuclease-free ultrapure water to prepare a polymer solution with a mass concentration of 3.3 mg / mL. Place the polymer solution in an ice bath and disperse it by ultrasonic cell disruption for 12 min (ultrasonic power 250 W, ultrasonic time 3 s, interval time 5 s). After ultrasonication, filter it through a 0.22 μm nuclease-free filter membrane to obtain a temperature-responsive nanoparticle suspension with a particle size of 75 nm and a phase transition temperature of 32 °C determined by dynamic light scattering (DLS).

[0048] Example 2 Preparation of nucleic acid preservation solution with added temperature-responsive PNIPAM-PEG nanoparticles: (1) Weigh 1.21 g of Tris-HCl, 4.10 g of sodium acetate, 0.37 g of EDTA (disodium salt), 2.92 g of NaCl, ~190 g of guanidine isothiocyanate, and ~58 g of betaine using an electronic balance. Add all the above reagents to a 1L nuclease-free beaker in sequence, add about 800 mL of nuclease-free ultrapure water, stir slowly until the reagents are completely dissolved, and make up to 940 mL with nuclease-free ultrapure water. (2) After adjusting the pH to 7.5-8.5, filter out insoluble impurities to obtain a mixed solution; (3) Take 9.4 mL of the above mixed solution and add it to a 15 mL nuclease-free centrifuge tube. Add 0.6 mL of the PNIPAM-PEG nanoparticle suspension prepared in Example 1 and gently invert and mix 5 times to obtain a nucleic acid preservation solution with a final nanoparticle concentration of 0.2 mg / mL, which is referred to as preservation solution 1.

[0049] Example 3 Preparation of DPPC / DSPC composite thermosensitive liposomes: S201. Weigh 2.0g DPPC and 1.3g DSPC, add them to a 250mL round-bottom flask, add 80mL chloroform, and stir on a magnetic stirrer for 30min to completely dissolve the lipid raw materials and form a homogeneous lipid solution.

[0050] S202. Fix the round-bottom flask on the rotary evaporator, set the rotation speed to 60 r / min and the water bath temperature to 38℃, and rotary evaporate under reduced pressure for 30 min to remove the chloroform solvent, so that the lipid forms a uniform and smooth film on the inner wall of the flask; then transfer the round-bottom flask to the vacuum drying oven and vacuum dry at 38℃ for 3 h to completely remove the residual chloroform solvent.

[0051] S203. Add 1000 mL of nuclease-free ultrapure water to a round-bottom flask and place it in a 38°C water bath. Gently shake for 45 min to allow the lipid film to fully hydrate and detach, forming a milky white suspension. Transfer the suspension to an ultrasonic cell disruptor and sonicate for 12 min under ice bath conditions (ultrasonic power 250 W, sonication time 3 s, interval 5 s). After sonication, filter the suspension through a 0.22 μm nuclease-free filter membrane to remove undispersed large lipid particles, obtaining a temperature-responsive lipid-based nanoparticle suspension with a nanoparticle mass concentration of 3.3 mg / mL. The nanoparticle size is 82 nm, and the phase transition temperature (LCST) measured by dynamic light scattering (DLS) is 33°C.

[0052] Example 4 Preparation of nucleic acid preservation solution with added temperature-responsive lipid-based nanoparticles: (1) Weigh 1.21 g of Tris-HCl, 4.10 g of sodium acetate, 0.37 g of EDTA (disodium salt), 2.92 g of NaCl, ~190 g of guanidine isothiocyanate, and ~58 g of betaine using an electronic balance. Add all the above reagents to a 1L nuclease-free beaker in sequence, add about 800 mL of nuclease-free ultrapure water, stir slowly until the reagents are completely dissolved, and make up to 940 mL with nuclease-free ultrapure water. (2) After adjusting the pH to 7.5-8.5, filter out insoluble impurities to obtain a mixed solution; (3) Take 9.4 mL of the above mixed solution and add it to a 15 mL nuclease-free centrifuge tube. Add 0.6 mL of the DPPC / DSPC composite thermosensitive liposome suspension prepared in Example 3. Gently invert and mix 5 times to obtain a nucleic acid preservation solution with a final nanoparticle concentration of 0.2 mg / mL, which is referred to as preservation solution 2.

[0053] Example 5 Performance testing: 1. Stability testing: Preservative solution 1 was left to stand at room temperature (25℃), and its appearance, pH value, and particle size distribution were observed at 0h, 24h, and 72h. The results showed that the mixed system was clear and transparent within 72h, with no precipitation or stratification, and the pH was maintained between 7.7 and 7.9. The PDI value of the nanoparticles was 0.22-0.28, and there was no abnormal aggregation.

[0054] The preservation solution 2 was left to stand at room temperature (25℃), and its appearance, pH value, and particle size distribution were observed at 0h, 24h, and 72h. The results showed that the mixed system was clear and transparent within 72h, with no precipitation or stratification, and the pH was maintained between 7.7 and 7.9. The PDI value of the nanoparticles was 0.23-0.29, and there was no abnormal aggregation.

[0055] 2. Temperature response testing: 1 mL of the preservation solution was placed in environments of 25℃ (below LCST) and 37℃ (above LCST) for 30 min, respectively, and the particle size change was detected using dynamic light scattering (DLS). The results showed that the particle size was 75 nm at 25℃ and 118 nm at 37℃, an increase of 60%, indicating that PNIPAM-PEG nanoparticles can achieve precise temperature-responsive transitions.

[0056] Take 21 mL of the preservation solution and equilibrate it for 30 min at 25℃ (below LCST) and 40℃ (above LCST), respectively. The particle size change was detected by dynamic light scattering (DLS). The results showed that the particle size was 82 nm at 25℃ and 125 nm at 40℃, an increase of 52%, indicating that lipid-based nanoparticles can achieve precise temperature-responsive transitions.

[0057] 3. Nucleic acid preservation integrity test: Standard RNA samples (β-actin mRNA, final concentration 100 ng / μL) were added to preservation solution 1 of Example 2, preservation solution 2 of Example 4, and negative control (9.4 mL mixed solution + 0.6 mL nuclease-free ultrapure water), respectively. The samples were stored at 4℃, 25℃, and 37℃ for 28 days. Samples were taken on days 0, 7, 14, and 28, and three parallel groups were set up. The Ct values ​​of the samples were recorded by qPCR. The test results are shown in Table 1.

[0058] Table 1. Integrity test results of RNA samples at different temperatures

[0059] Note: D0 refers to the Ct value on the day the sample was saved, D7 refers to the Ct value on the 7th day, and so on.

[0060] The results showed that the negative control (without temperature-responsive nanoparticles) showed a significant increase in Ct value (nucleic acid degradation) after 14 days at 25℃ and 7 days at 37℃, and no effective signal after 28 days. In contrast, the preservation solution 1 (with added PNIPAM-PEG nanoparticles) and preservation solution 2 (with added DPPC / DSPC liposomes) of the present invention showed only a slight increase in Ct value after 28 days at 25℃, and still had a stable effective signal (Ct value < 27) after 28 days at 37℃. This proves that the stable preservation time of RNA is extended from 7-14 days in the prior art to 21-28 days, which fully meets the requirements for long-term room temperature preservation.

[0061] Standard DNA samples (λ-DNA, final concentration 200 ng / μL) were added to preservation solution 1 of Example 2, preservation solution 2 of Example 4, and negative control (9.4 mL mixed solution + 0.6 mL nuclease-free ultrapure water), respectively. The samples were stored at 4℃, 25℃, and 37℃ for 28 days. Samples were taken on days 0, 7, 14, 30, and 60, and three parallel groups were set up. The Ct values ​​of the samples were recorded by qPCR. The test results are shown in Table 2.

[0062] Table 2. Integrity test results of DNA samples at different temperatures

[0063] The results showed that the negative control had a Ct value of 30.16 after 30 days at 25°C and no effective signal after 60 days; while the Ct values ​​of preservation solution 1 and preservation solution 2 were 27.72 and 27.65 after 60 days at 25°C, respectively, which were only 1.47~1.55 higher than the initial value (D0), proving that the stable preservation time of DNA was extended from 1 month in the existing technology to 2 months, and the integrity of nucleic acids was not significantly damaged.

[0064] At 37°C, the negative control showed no effective signal after 30 days, while the Ct values ​​of preservation solution 1 and preservation solution 2 were 27.85 and 28.00 respectively after 60 days, which are still within the detectable and stable range. This indicates that the preservation solution of the present invention can withstand short-term temperature fluctuations (25~35°C), greatly reducing the dependence on cold chain logistics and making it suitable for application scenarios in remote areas or resource-scarce environments.

[0065] In summary, this invention utilizes temperature-responsive nanoparticles that maintain hydrophilicity and stably coexist with nucleic acid samples at low temperatures. When the temperature rises above the phase transition temperature, they become hydrophobic, forming a dense physical protective layer on the surface of nucleic acid molecules, achieving a synergistic effect of chemical and physical protection. This significantly improves the stability of nucleic acid preservation, reduces the dependence on cold chains, and is particularly suitable for the protection of nucleic acid samples under long-term storage and transportation conditions. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and has high industrial application value.

[0066] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A nucleic acid preservation solution, characterized in that, The product contains buffer solution, ionic strength maintainer, chelating agent, strong denaturant, and temperature-responsive nanoparticles, all of which are dissolved in nuclease-free ultrapure water. The temperature-responsive nanoparticles are temperature-responsive polymer-based nanoparticles or temperature-responsive lipid-based nanoparticles.

2. The nucleic acid preservation solution according to claim 1, characterized in that, The temperature-responsive nanoparticles have a particle size of 50~100nm and a phase transition temperature of 30~35℃.

3. The nucleic acid preservation solution according to claim 1, characterized in that, The temperature-responsive polymer-based nanoparticles are selected from poly(N-isopropylacrylamide-polyethylene glycol) block copolymer nanoparticles, and the preparation method of the poly(N-isopropylacrylamide-polyethylene glycol) block copolymer nanoparticles includes the following steps: S101. Preparation of macromolecular chain transfer agents based on monomethyl-terminated polyethylene glycol; S102. The macromolecular chain transfer agent, N-isopropylacrylamide monomer and initiator obtained in step S101 are subjected to a polymerization reaction in the first solvent to obtain poly(N-isopropylacrylamide-polyethylene glycol block copolymer). S103. Dissolve the poly(N-isopropylacrylamide-polyethylene glycol) block copolymer obtained in step S102 in nuclease-free ultrapure water to prepare a polymer solution. Disperse the polymer solution ultrasonically under ice bath conditions and filter it through a nuclease-free filter membrane to obtain a temperature-responsive poly(N-isopropylacrylamide-polyethylene glycol) block copolymer nanoparticle suspension.

4. The nucleic acid preservation solution according to claim 3, characterized in that, In step S102, the initiator is selected from azobisisobutyronitrile or benzoyl peroxide, and the mass ratio of macromolecular chain transfer agent, N-isopropylacrylamide monomer and initiator is 1:(5~8):(0.02~0.05).

5. The nucleic acid preservation solution according to claim 3, characterized in that, In step S102, the first solvent is selected from tetrahydrofuran, N,N-dimethylformamide (DMF) or toluene; the polymerization temperature is 65~75℃ and the reaction time is 12~16h.

6. The nucleic acid preservation solution according to claim 1, characterized in that, The temperature-responsive lipid-based nanoparticles are selected from DPPC / DSPC composite thermosensitive liposomes, and the preparation method of the DPPC / DSPC composite thermosensitive liposomes includes the following steps: S201. Dissolve DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) and DSPC (1,2-distearate-sn-glycerol-3-phosphocholine) in a second solvent to form a homogeneous lipid solution. S202. The lipid solution is subjected to reduced pressure rotary evaporation to remove the solvent, forming a uniform lipid film; S203. Nuclease-free ultrapure water is added to the lipid membrane for full hydration, followed by ultrasonic dispersion in an ice bath and filtration through a nuclease-free filter membrane to obtain the DPPC / DSPC composite thermosensitive liposomes.

7. The nucleic acid preservation solution according to claim 1, characterized in that, The final concentration of the temperature-responsive nanoparticles in the nucleic acid preservation solution is 0.1~0.5 mg / mL.

8. The nucleic acid preservation solution according to claim 1, characterized in that, The ionic strength maintaining agent includes sodium acetate and / or NaCl; the final concentration of sodium acetate in the nucleic acid preservation solution is 20-100 mM; the final concentration of NaCl in the nucleic acid preservation solution is 50-150 mM.

9. The nucleic acid preservation solution according to claim 1, characterized in that, The nucleic acid preservation solution also contains betaine, and the final concentration of betaine in the nucleic acid preservation solution is 0.5~1M.

10. A method for preparing a nucleic acid preservation solution, characterized in that, Includes the following steps: (1) Weigh each component according to the proportion, and first mix and dissolve the buffer, ionic strength maintainer, chelating agent, strong denaturant and nuclease-free ultrapure water; (2) After adjusting the pH value, filter out insoluble impurities; (3) Finally, add the temperature-responsive nanoparticle suspension and mix well to obtain the nucleic acid preservation solution.

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