An in-situ vitrification-semi-solid nucleic acid room temperature stable preservation solution, its preservation method, and reagent kit

Abstract

This invention discloses an in-situ vitrification-semi-solid nucleic acid room-temperature stable preservation solution, its preservation method, and a reagent kit, belonging to the field of biological sample preservation technology. The preservation solution comprises a double buffer system, a mild chelation system, a vitrification framework, a thermoresponsive gelling system, an oxidative protection system, a low-toxicity antibacterial system, and water. The thermoresponsive gelling system forms a weakly reversible gel network within the range of 18-37°C after the sample is mixed with the preservation solution, placing the sample nucleic acid in a confined microenvironment with low diffusion and low water activity. This invention achieves long-term stable preservation of nucleic acid samples at room temperature through the synergistic effect of a triple mechanism of "low water activity - thermoresponsive gelling - multi-target passivation." This invention eliminates the need for cold chain transportation, is simple to operate, and has high safety, making it suitable for the room-temperature collection, transportation, and preservation of various biological samples such as blood, saliva, oral swabs, sputum, and tissue homogenates.

Description

Technical Field

[0001] This invention belongs to the field of biological sample preservation technology, and particularly relates to an in-situ vitrification-semi-solid nucleic acid room temperature stable preservation solution, its preservation method, and a kit. Background Technology

[0002] Nucleic acid samples are highly susceptible to degradation due to various internal and external factors during collection, transportation, and preservation. Specifically, endogenous nucleases present in the sample, exogenous nucleases introduced by microbial contamination, oxidative stress, pH fluctuations, mechanical shear forces, and repeated freeze-thaw cycles can all cause irreversible damage to the integrity of nucleic acids. Traditionally, -20°C or -80°C cryopreservation is used to ensure the long-term stability of nucleic acids. While this method is stable, it is highly dependent on cold chain transportation and specialized freezing equipment, resulting in high costs and complex operations, making it difficult to meet the practical needs of large-scale screening, primary healthcare units, field operations, and home collection. Therefore, developing a technical solution that can stably preserve nucleic acid samples at room temperature and is easy to operate has significant application value and market demand.

[0003] To address the aforementioned issues, various room-temperature nucleic acid preservation methods have been developed in existing technologies, which can be mainly categorized into three technical routes. The first category is liquid preservation systems based on high salt or denaturing agents. These systems use high concentrations of liquid salts or strong denaturing agents (such as guanidine salts, SDS, etc.) to denature and inactivate nucleases, thereby inhibiting degradation reactions. However, these methods often suffer from residual denaturing agents that strongly inhibit downstream molecular detection processes such as polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), and library construction, requiring cumbersome purification steps before use, thus limiting their applicability in rapid detection fields. The second category is dry preservation systems on paper-based, membrane-based, or porous substrates. These systems adsorb nucleic acids onto solid carriers and then dry them to achieve room-temperature preservation at low water activity. However, these methods typically rely on specialized carrier materials and suffer from problems such as unstable nucleic acid elution efficiency, low sample recovery rates, and poor adaptability to different types of samples (such as blood, saliva, and tissue homogenates). The third category is direct stabilization storage systems for bodily fluid samples such as saliva and blood. These systems are mostly aqueous buffer formulations. Although they offer some convenience for both preservation and detection, they are still mostly homogeneous liquid phases. After the sample is added, nucleic acid molecules still have a high degree of diffusion freedom in the liquid phase, and the probability of collision with residual nucleases is relatively high. In addition, the water activity is high, making it difficult to achieve both high stability and low detection interference during long-term preservation.

[0004] From the perspective of existing technologies, there are already relatively abundant solutions in the field of nucleic acid room temperature preservation, mainly including the aforementioned liquid preservation, dry preservation, and body fluid-specific preservation. However, existing technologies generally share a common deficiency: their technical concepts are mostly limited to a simple functional combination of "buffer + chelating agent + sugar stabilizer + surfactant," failing to make fundamental innovations in the microenvironment structure of sample preservation. These solutions often rely solely on the stacking of chemical components, lacking the ability to regulate the physical microenvironment of nucleic acid molecules, and are unable to form a synergistic protective effect across multiple dimensions such as inhibiting nuclease diffusion, reducing water activity, passivating metal ion catalysis, and scavenging free radicals.

[0005] In response to the aforementioned technological limitations, this invention departs from traditional aqueous or dry preservation approaches and proposes a novel structural design. This design employs a technical approach of "liquid-fillable, in-situ gelation upon sample contact, and microenvironment vitrification." The aim is to construct a semi-solid protective microenvironment with low diffusivity, low water activity, and multi-target passivation capabilities, thereby achieving long-term stable preservation of nucleic acid samples at room temperature and ensuring direct compatibility with downstream molecular detection processes. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes an in-situ vitrification-semi-solid nucleic acid room-temperature stable preservation solution, its preservation method, and a reagent kit. The purpose of this invention is to provide a room-temperature stable nucleic acid preservation solution that eliminates the need for cryogenic transportation, forms a stable semi-solid protective microenvironment at room temperature, synergistically inhibits nucleases and oxidative damage, and is directly compatible with routine molecular detection procedures. Another objective of this invention is to provide methods for applying this preservation solution to samples such as blood, saliva, oral swabs, sputum, and tissue homogenates, as well as a nucleic acid sample preservation kit containing this preservation solution.

[0007] To achieve the above objectives, the present invention provides an in-situ vitrified-semi-solid nucleic acid room temperature stable preservation solution, comprising: a double buffer system, a mild chelation system, a vitrification framework, a thermally responsive gelling system, an oxidative protection system, a low-toxicity antibacterial system, and water; The thermally responsive gelling system forms a weakly reversible gel network within the range of 18-37°C after the sample is mixed with the preservation solution, thereby reducing the diffusion and degradation of nucleic acids in the sample.

[0008] Furthermore, the dual buffer system comprises 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and 1,3-bis(tris(hydroxymethyl)amino)propane (BIS-TRIS propane), and the pH of the preservation solution is 7.4-8.0.

[0009] Furthermore, the mild chelation system is ethylenediamine disuccinic acid (EDDS), with a concentration of 0.5-2.5 mM in the preservation solution.

[0010] Furthermore, the vitrified framework comprises trehalose and sorbitol, wherein the mass-volume percentage of trehalose is 2-8% and the mass-volume percentage of sorbitol is 1-5%.

[0011] Furthermore, the thermoresponsive gelling system includes poloxamer 407 and poloxamer 188, wherein the mass-volume percentage of poloxamer 407 is 8-16% and the mass-volume percentage of poloxamer 188 is 1-4%. Preferably, the thermoresponsive gelling system further includes 0.05-0.5% by weight / volume of hydroxypropyl methylcellulose (HPMC).

[0012] Furthermore, the oxidative protection system is ergothioneine or its salt, and its mass-volume percentage in the preservation solution is 0.01-0.05%.

[0013] Furthermore, the low-toxicity antibacterial system is phenoxyethanol, p-hydroxyacetophenone, or a combination thereof, and its mass-volume percentage in the preservation solution is 0.1-0.6%.

[0014] Furthermore, the preservation solution does not contain guanidine salts, sodium azide, or high-concentration dissociation agents.

[0015] The present invention also provides a method for preserving nucleic acid samples at room temperature, comprising the following steps: adding biological samples to the above-mentioned preservation solution at a volume ratio of 1:1 to 1:2, mixing well, and letting stand for 1-5 minutes to form a semi-solid protective microenvironment, and then transporting or preserving them at 18-37°C.

[0016] The present invention also provides a nucleic acid sample preservation kit, comprising the above-mentioned preservation solution and a sampling container, wherein the sampling container is pre-filled with the preservation solution; Preferably, the sampling container is selected from blood sampling tubes, saliva sampling tubes, oral swab tubes, sputum sampling tubes, or tissue homogenate sampling tubes.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: This invention proposes a novel approach to nucleic acid preservation: in-situ vitrification-semi-solid. A thermally responsive gelling system spontaneously forms a semi-solid gel network at room temperature upon sample addition, transforming the preservation system from a liquid state to a confined microenvironment with low diffusion and low water activity. This phase transition has significant technical implications: the semi-solid gel network significantly reduces the diffusion coefficient of nucleic acid molecules within the system, minimizing collisions between nucleic acids and residual nucleases, thus physically inhibiting degradation reactions. Experimental data show that after 180 days of preservation at 25°C, the DNA retention rate in the embodiments of this invention remains above 90%, with ΔCt less than 0.8, while the retention rate in the control group (with the gelling system removed) drops to 64.8%, with ΔCt reaching 1.98. This demonstrates that the physical barrier effect of the gel network plays a crucial role in long-term stable preservation.

[0018] This invention ensures preservation effectiveness while fully considering compatibility with downstream molecular detection. Unlike traditional preservation solutions that rely on high-concentration guanidine salts and strong denaturants such as SDS, this invention uses the mild chelating agent EDDS instead of EDTA, selects low-toxicity antibacterial agents such as phenoxyethanol and p-hydroxyacetophenone instead of sodium azide, and avoids the use of high-concentration dissociation salts. EDDS at room temperature has a good effect on Mg... 2+ The chelating ability of this product is relatively mild, and its impact on the PCR reaction after dilution is significantly lower than that of EDTA. Experiments have shown that the preservation solution of this invention can be directly used for qPCR detection in its undiluted state, with no significant difference in Ct value compared to the purified template. In contrast, the Ct value of the EDTA control group was delayed by 2-3 cycles in its undiluted state, and the SDS control group was delayed by as much as 8 cycles. This advantage allows for direct detection of samples after preservation without complex purification steps, significantly simplifying the operation process.

[0019] This invention demonstrates excellent preservation effects on various sample types, including blood, saliva, oral swabs, sputum, and tissue homogenates, reflecting its broad sample versatility. By adjusting the ratio of Poloxamer 407 to Poloxamer 188 and the amount of HPMC, gel strength and gelation speed can be precisely controlled, thereby optimizing its adaptability to samples with different viscosities and protein contents.

[0020] More importantly, this invention is not a simple functional stacking of "buffer + chelating agent + sugar + surfactant" as in existing technologies. Instead, it achieves a multi-target, multi-dimensional synergistic protective amplification effect through a chain-like synergistic mechanism of "gelation limiting diffusion - sugar alcohol reducing water activity - EDDS inhibiting metal catalysis - antioxidant eliminating free radicals - buffer system stabilizing pH". DNase I challenge experiments show that the embodiments of this invention maintain a DNA retention rate of over 86% under nuclease stress, with a protection factor of over 4.3 times, significantly superior to Comparative Example 1 (degelatinized) (3.2 times), Comparative Example 2 (de-sugar alcoholized) (2.9 times), and Comparative Example 4 (de-antioxidantized) (3.7 times). Further analysis revealed that the average DNA retention rate of Comparative Examples 1, 2, and 4 at 180 days was only 65.3%, far lower than the 92.3% of Example 1. This difference fully demonstrates that the synergistic effect of each component is far beyond what can be achieved by simply adding up individual functions. This synergistic effect allows this invention to achieve a comprehensive preservation effect superior to existing technologies without relying on strong inhibitory components.

[0021] Furthermore, this invention also boasts significant advantages in terms of safety. The selected antibacterial agents, such as phenoxyethanol and p-hydroxyacetophenone, are widely used mild preservatives with low toxicity and low irritation; ergothioneine is a natural antioxidant; and EDDS is a biodegradable chelating agent. The entire formulation system is free of highly toxic substances such as sodium azide and thimerosal, and contains no guanidine salts or high-concentration dissociation agents, making it friendly to operators and the environment. It aligns with the development principles of green chemistry and facilitates its widespread application in primary healthcare and large-scale screening scenarios. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0024] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0025] In this invention, the in-situ vitrified-semi-solid nucleic acid room-temperature stable preservation solution comprises the following components: a dual buffer system consisting of HEPES (4-hydroxyethylpiperazine ethanesulfonic acid, an amphoteric biological buffer with excellent biocompatibility) and BIS-TRISpropane (1,3-bis(tris(hydroxymethyl)amino)propane, with a wide buffering range and low metal ion chelating ability), which, when combined within a pH range of 7.4-8.0, form a stable environment with high buffering capacity; and a mild chelation system using EDDS (ethylenediamine disuccinic acid, a biodegradable amino polycarboxylic acid chelating agent), which chelates Ca through moderate strength. 2+ Mg 2+ Metal ions inhibit nuclease activity and simultaneously affect the Mg content in the PCR reaction. 2+ The impact is significantly lower than that of traditional EDTA; the glass transition framework is composed of trehalose (a natural non-reducing disaccharide with excellent glass transition properties) and sorbitol (a natural hexaglycol with good hydration and glass-forming ability), which construct a low-water-activity glass transition microenvironment through a complementary hydrogen bond network; the thermoresponsive gelling system includes Poloxamer 407 (a triblock copolymer of polyethylene oxide, polypropylene oxide, and polyethylene oxide with a low critical gelation temperature) and Poloxamer Poloxamer 188 (a similar copolymer with a relatively high critical gelation temperature) and its combination can precisely control the gelation temperature within the range of 18-37℃. HPMC (hydroxypropyl methylcellulose, a semi-synthetic cellulose derivative, a water-soluble nonionic thickener) can be further added to enhance the mechanical strength of the gel. The oxidative protection system uses ergothioneine (a natural sulfur-containing amino acid derivative, mainly found in mushrooms and other microorganisms, with extremely strong free radical scavenging ability), which effectively removes reactive oxygen species such as hydroxyl radicals and inhibits nucleic acid oxidative damage. The low-toxicity antibacterial system uses phenoxyethanol (an aromatic alcohol compound, a mild preservative widely used in the cosmetics industry) and hydroxyacetophenone (a phenolic compound naturally found in herbs, a safe and mild preservative synergist). The combination of these two inhibits microbial proliferation and is compatible with molecular detection. In addition, for highly viscous samples, xanthan gum (an extracellular polysaccharide produced by microbial fermentation, a natural polymeric thickener) can be selectively added as an auxiliary thickener. Through a chain-like synergistic mechanism of "gelation limiting diffusion - sugar alcohol reducing water activity - EDDS inhibiting metal catalysis - antioxidants eliminating free radicals - buffer system stabilizing pH - antibacterial agents preventing microbial contamination", the above components achieve long-term stable preservation of nucleic acid samples at room temperature and good compatibility with downstream detection without the need for strong denaturants, highly toxic preservatives and high concentrations of liquid salt.

[0026] The preparation method of the preservation solution of the present invention is as follows: The following formulations are based on the preparation of 100 mL of preservation solution. For preparation, first place 40-60 mL of water for injection (resistivity ≥18.2 MΩ·cm) in a suitable container. Under magnetic stirring, add the buffer salt, chelating agent, and sugar alcohol components sequentially, stirring until completely dissolved to form a clear solution. Then, transfer the container to a low-temperature environment of 4-8°C. While continuously stirring, slowly add the thermoresponsive gelling system components (including Poloxamer 188, Poloxamer 407, and optional HPMC), maintaining low-temperature stirring until the system is uniformly clear or uniformly translucent. This low-temperature operation step aims to ensure that the thermoresponsive polymer remains in a sol state during preparation, avoiding premature gel formation. After the above components are completely dissolved, add the oxidative protection system and low-toxicity antibacterial system components sequentially. Monitor and adjust the pH to the target value (typically 7.6±0.1) using a pre-calibrated pH meter. Finally, add water to bring the volume to 100 mL. After the prepared preservation solution is filtered through a 0.22 μm sterile filter membrane for sterilization, it is aseptically dispensed into preservation containers, sealed, and refrigerated at 2-8℃ for later use.

[0027] When using, add the collected nucleic acid sample to the preservation solution at a volume ratio of 1:1 or 1:2, gently invert and mix 5-10 times, then let it stand for 1-5 minutes. During the standing process, the mixture of preservation solution and sample spontaneously forms a stable semi-solid gel at room temperature. If direct PCR or qPCR detection is required, the gel system can be diluted in a suitable buffer or water at a volume ratio of 1:5 to 1:20, vortexed, and then a suitable amount can be used for routine reactions. For highly viscous samples (such as sputum or tissue homogenate), a brief vortexing can be performed first to promote sample dispersion, followed by standing to form a gel network.

[0028] Example 1 This embodiment provides a preservation solution suitable for room temperature preservation of DNA from blood and oral swab samples, with the following composition: HEPES 20 mM; BIS-TRIS propane 10 mM; EDDS 1.0 mM; trehalose 5 wt%; sorbitol 2 wt%; Poloxamer 407 12 wt%; Poloxamer 188 2 wt%; HPMC 0.15 wt%; ergothioneine 0.02 wt%; phenoxyethanol 0.25 wt%; the balance being water for injection.

[0029] This formulation forms a semi-solid gel within approximately 1-2 minutes after being mixed with the sample at 25°C, exhibiting excellent gelation speed and embedding effect.

[0030] Example 2 This embodiment provides a preservation solution suitable for preserving DNA from saliva and sputum samples at room temperature, with the following composition: HEPES 25 mM; BIS-TRIS propane 8 mM; EDDS 1.5 mM; trehalose 4 wt%; sorbitol 3 wt%; Poloxamer 407 10 wt%; Poloxamer 188 3 wt%; HPMC 0.10 wt%; ergothionein 0.03 wt%; p-hydroxyacetophenone 0.20 wt%; the balance is water for injection.

[0031] This formulation is optimized for low-viscosity samples (saliva). By appropriately reducing the concentration of Poloxamer 407 and increasing the proportion of Poloxamer 188, the mixing uniformity of the sample is improved while ensuring gelation performance.

[0032] Example 3 This embodiment provides a DNA preservation solution suitable for high-temperature transportation, with the following composition: HEPES 15 mM; BIS-TRIS propane 15 mM; EDDS 0.8 mM; trehalose 6 wt%; sorbitol 1.5 wt%; Poloxamer 407 14 wt%; Poloxamer 188 1.5 wt%; HPMC 0.20 wt%; ergothioneine 0.01 wt%; phenoxyethanol 0.30 wt%; the balance being water for injection.

[0033] This formulation enhances gel network strength by increasing the concentration of Poloxamer 407 and strengthens vitrification protection by increasing the proportion of trehalose to cope with high temperature stress environments of 37°C and above.

[0034] Example 4 This embodiment provides a preservation solution suitable for room temperature preservation of DNA from tissue homogenate samples, with the following composition: HEPES 20 mM; BIS-TRIS propane 10 mM; EDDS 1.0 mM; trehalose 3 wt%; sorbitol 4 wt%; Poloxamer 407 11 wt%; Poloxamer 188 2 wt%; HPMC 0.25 wt%; ergothioneine 0.02 wt%; phenoxyethanol 0.25 wt%; the balance being water for injection.

[0035] This formulation enhances the mechanical strength of the gel network by increasing the sorbitol ratio and HPMC concentration to accommodate the higher protein and cell debris content in tissue homogenate samples.

[0036] Example 5 This embodiment provides a room-temperature DNA preservation solution suitable for highly viscous body fluid samples (such as highly viscous sputum and joint effusion), with the following composition: HEPES 20 mM; BIS-TRIS propane 10 mM; EDDS 1.2 mM; trehalose 5 wt%; sorbitol 2 wt%; Poloxamer 407 12 wt%; Poloxamer 188 2 wt%; HPMC 0.15 wt%; ergothioneine 0.02 wt%; phenoxyethanol 0.25 wt%; xanthan gum 0.03 wt%; the balance being water for injection.

[0037] This formulation adds a trace amount of xanthan gum as a thickener to the original formulation, further enhancing the gel's retention and its ability to accommodate highly viscous samples.

[0038] In Examples 1 to 5 above, the preservation solution can form a semi-solid or high-viscosity state within 1-5 minutes after being mixed with the corresponding sample. Among them, Examples 1 and 3 have a faster gelation speed (about 1-2 minutes), which is suitable for routine rapid sampling scenarios; Examples 4 and 5 have higher gel network strength and stronger embedding ability, which are particularly suitable for sample types with high protein content or high viscosity.

[0039] Example 6 This embodiment provides a preservation solution with an adjusted proportion of a thermally responsive gelling system, the composition of which is as follows: HEPES 20 mM; BIS-TRIS propane 10 mM; EDDS 1.0 mM; trehalose 5 wt%; sorbitol 2 wt%; Poloxamer 4079 wt%; Poloxamer 188 4 wt%; HPMC 0.10 wt%; ergothioneine 0.02 wt%; phenoxyethanol 0.25 wt%; the balance being water for injection.

[0040] Compared to Example 1, this example reduces the concentration of Poloxamer 407 and increases the proportion of Poloxamer 188, resulting in a relatively weak gel network.

[0041] Example 7 This embodiment provides a preservation solution that replaces trehalose with sucrose, with the following composition: HEPES 20 mM; BIS-TRIS propane 10 mM; EDDS 1.0 mM; sucrose 5 wt%; sorbitol 2 wt%; Poloxamer 407 12 wt%; Poloxamer 188 2 wt%; HPMC 0.15 wt%; ergothioneine 0.02 wt%; phenoxyethanol 0.25 wt%; the balance being water for injection.

[0042] Example 8 This embodiment provides a preservation solution formulation that replaces ergothioneine with vitamin C, and its composition is as follows: HEPES 20 mM; BIS-TRIS propane 10 mM; EDDS 1.0 mM; trehalose 5 wt%; sorbitol 2 wt%; Poloxamer 4071 2 wt%; Poloxamer 188 2 wt%; HPMC 0.15 wt%; vitamin C 0.02 wt%; phenoxyethanol 0.25 wt%; the balance is water for injection.

[0043] Comparative Example 1 This comparative example differs from Example 1 by removing Poloxamer 407, Poloxamer 188, and HPMC, while keeping the remaining components unchanged.

[0044] Comparative Example 2 This comparative example is based on Example 1, with trehalose and sorbitol removed, while the remaining components remain unchanged.

[0045] Comparative Example 3 In this comparative example, the EDDS in Example 1 was replaced with equimolar concentrations of EDTA (approximately 1.0 mM), while the other components remained unchanged.

[0046] Comparative Example 4 This comparative example is based on Example 1, with ergothioneine removed, while the remaining components remain unchanged.

[0047] Comparative Example 5 This comparative example uses a traditional liquid preservation formula: 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Tween-20, and 10% glycerol.

[0048] Comparative Example 6 This comparative example uses a preservation formula containing a strong denaturant: 0.5% SDS, 20 mM Tris-HCl (pH 8.0).

[0049] Comparative Example 7 In this comparative example, the dual buffer system (HEPES+BIS-TRIS propane) in Example 1 was replaced with a single HEPES buffer (30 mM) of equal total concentration, while the other components remained unchanged.

[0050] Comparative Example 8 In this comparative example, the Poloxamer 407 / 188 combination in Example 1 was replaced with a single Poloxamer 407 (14 wt%) at the same total concentration, and Poloxamer 188 was not added. The remaining components remained unchanged.

[0051] Test case 1 Experimental Methods (1) Sample preparation Peripheral blood (EDTA anticoagulated), saliva, and oral swab samples were collected from healthy volunteers. Blood samples were gently inverted and mixed after collection, then aliquoted into 200 μL portions. Saliva samples were collected after the volunteers fasted for 30 minutes prior to collection, were gently mixed by pipetting, and aliquoted into 200 μL portions. Oral swab samples were collected by wiping both buccal mucosa with a sterile swab, cutting off the swab tip and placing it in a centrifuge tube containing 200 μL of physiological saline. After vortexing and elution, the swab tip was removed, and the eluent was used as the sample to be preserved. Each type of sample was aliquoted after mixing, and an equal volume (200 μL) of preservation solution (for the example group, supplementary example group, and comparative example group) was added to each portion. The samples were gently inverted and mixed 5-10 times, then allowed to stand for 5 minutes. The gelation process was recorded, and the samples were placed under the appropriate preservation conditions. Three replicates were set up for each group.

[0052] (2) Storage conditions Two storage conditions were set up: room temperature (25℃±2℃, relative humidity 60%±10%) and accelerated aging (37℃±1℃). Samples were taken at 0 days, 7 days, 30 days, 90 days, 180 days and 360 days for testing.

[0053] (3) DNA extraction and quantification After sampling, for Example Groups and Comparative Examples 1-4 and 7-8, 200 μL of the mixed system was taken, and DNA was extracted using a commercial magnetic bead DNA extraction kit (e.g., Company A) according to the manufacturer's instructions. For Comparative Example 5 (conventional liquid preservation) and Comparative Example 6 (strong denaturant control), the same volume was directly extracted. The concentration, A260 / 280, and A260 / 230 ratios of the extracted DNA were determined using a micro-volume UV spectrophotometer. DNA retention rate was expressed as a percentage of the DNA concentration extracted at each time point to the DNA concentration extracted on day 0.

[0054] (4) Real-time quantitative PCR detection Primers and probes with amplification fragment length of 100-150 bp were designed targeting human single-copy genes (such as RNase P or β-actin). Commercial qPCR premix (e.g., from Company B) was used for detection. The reaction volume was 20 μL, containing 2 μL of template DNA. The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing and extension for 30 s, for 40 cycles. Each sample was set up in triplicate, and the amplification result was expressed as Ct value. ΔCt represents the difference between the Ct value at each time point and the Ct value on day 0. The PCR success rate was defined as the percentage of samples with a Ct value <35 out of the total number of samples in the group.

[0055] (5) DNA integrity assessment 200 ng of DNA samples were extracted at each time point and subjected to electrophoresis on a 1.5% agarose gel (5 V / cm, 45 min). After photographing with a gel imaging system, the integrity of the bands was semi-quantitatively scored based on the grayscale distribution. Scoring criteria: 5 points, clear and complete bands without tailing; 4 points, clear bands with slight tailing; 3 points, blurry bands with obvious tailing; 2 points, diffuse bands, main band indistinguishable; 1 point, no visible bands.

[0056] (6) DNase I challenge experiment Take 100 μL of each preservation solution and sample mixture (day 0 after preservation), add DNase I to a final concentration of 0.1 U / μL, and incubate at 25°C for 1 hour. After incubation, immediately perform DNA extraction, using parallel samples without DNase I as blank controls. Calculate the DNA residue rate (%) = (DNA concentration extracted from the enzyme-added group / DNA concentration extracted from the blank control group) × 100%. The protection fold = DNA residue rate of the blank control group / DNA residue rate of the enzyme-added group, used to evaluate the tolerance of each preservation system to nuclease attack.

[0057] (7) Evaluation of PCR inhibitors Take the mixture of each preservation solution and sample (day 0 after preservation) and perform serial dilutions (stock solution, 1:5 dilution, 1:10 dilution, 1:20 dilution). Take 2 μL of each solution and add it to the qPCR reaction system. Use the purified DNA template without preservation solution as a positive control and the reaction system without template as a negative control. Calculate the changes in Ct values ​​at each dilution to evaluate the degree of inhibition of PCR reaction by the preservation solution.

[0058] (8) Statistical methods All experimental data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed using SPSS software. Tukey's test was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0059] 2 Experimental Data Table 1. DNA retention rate (%) at different time points under storage conditions at 25℃ and ΔCt after 180 days

[0060] As shown in Table 1, the long-term storage data at 25°C indicates that Examples 1 to 6 maintained a DNA retention rate of over 85% after 180 days of storage. Examples 1 to 5 showed retention rates exceeding 90%, with ΔCt values ​​less than 0.8, indicating that the amplifiable nucleic acids in the samples did not undergo significant degradation after six months of storage at room temperature. In contrast, the retention rates of the comparative groups at 180 days were significantly lower than those of the example groups (P < 0.01), especially Comparative Example 2 (removal of trehalose / sorbitol) and Comparative Example 5 (traditional liquid control), where retention rates decreased to 58.3% and 49.6%, respectively, with ΔCt values ​​of 2.34 and 2.76. While the retention rates of Supplementary Examples 7 and 8 (replacing the vitrification framework and antioxidant, respectively) (83.4% and 85.7%) were better than the comparative examples, they were still significantly lower than that of Example 1 (92.3%), indicating that the combination of trehalose and ergothioneine had the optimal protective effect.

[0061] Table 2. DNA integrity and PCR compatibility after 30 days of storage under accelerated conditions at 37℃.

[0062] As shown in Table 2, the accelerated storage data at 37°C indicates that the example groups maintained a high DNA recovery rate (89.7%-92.4%), a good integrity score (4.5-4.8), and a 100% PCR success rate after 30 days of accelerated aging. This fully demonstrates that the system not only effectively protects the total amount of nucleic acid but also maintains the amplifiability of the template molecules, and has no inhibitory interference with downstream detection. In contrast, the comparative groups showed varying degrees of performance decline. The PCR success rate of Comparative Example 2 (de-sugarol) dropped to 66.7%, and the recovery rate of Comparative Example 5 (traditional liquid) was less than 50%, further highlighting the superiority of the multi-component synergistic protection of this invention.

[0063] Table 3 DNA Residual Rate (%) and Relative Protection Factor after DNase I Challenge Experiment

[0064] Table 3 shows the DNase I challenge experiment results, further revealing the synergistic protective mechanism of each component. The Example group maintained over 86% DNA residue under nuclease stress, with a protection factor exceeding 4.3 times, indicating that the semi-solid gel network and the chemical passivation system together constitute a highly efficient nuclease barrier. The protection factors of Comparative Example 1 (removal of the gelling system) and Comparative Example 2 (removal of sugar alcohols) decreased to 3.2 times and 2.9 times, respectively, indicating that both the physical barrier (gel network) and the chemical barrier (reduction of water activity) are indispensable. Although the protection factor of Comparative Example 4 (removal of antioxidants) (3.7 times) was better than Comparative Examples 1 and 2, it was still significantly lower than that of the Example group, suggesting that oxidative damage is also an important pathway for nucleic acid degradation, and the addition of the antioxidant module further improved the overall protective efficacy.

[0065] It is worth noting that simply adding the effects of each component together is insufficient to achieve the comprehensive level demonstrated in Examples 1 to 5. Taking Comparative Examples 1, 2, and 4 as examples, these three lack the gelation, vitrification, and antioxidant modules, respectively, and their 180-day retention rates are 64.8%, 58.3%, and 72.8%, all significantly lower than the 92.3% of Example 1. Furthermore, the simple average of the three (65.3%) differs significantly from the measured value of Example 1. This comparison fully demonstrates that the present invention does not simply pile up traditional components, but rather achieves a multi-target, multi-dimensional synergistic protective amplification effect through a chain-like synergistic mechanism of "gelation limiting diffusion - sugar alcohol reducing water activity - EDDS inhibiting metal catalysis - antioxidant eliminating free radicals - buffer system stabilizing pH."

[0066] Table 4 Evaluation results of PCR inhibitors (Ct values)

[0067] Table 4 shows that the example group exhibited Ct values ​​close to those of the positive control in its original state, indicating that the preservation solution of the present invention did not significantly inhibit the PCR reaction; while Comparative Example 3 (EDTA replacement), Comparative Example 5 (traditional liquid) and Comparative Example 6 (SDS denaturation) all showed significant Ct value delays in their original state, requiring dilution to eliminate the inhibition, further verifying the superiority of the mild chelation system and low-inhibition formulation of the present invention.

[0068] Table 4 shows the evaluation results of PCR inhibitors. The example group could be directly used for qPCR detection in its stock solution state, with no significant difference in Ct values ​​compared to the positive control (P>0.05). However, formulations containing EDTA or traditional liquid preservation showed significant inhibitory effects (Ct values ​​delayed by 1-3 cycles), and the strongly denaturing formulation containing SDS showed severe inhibition (Ct values ​​delayed by up to 8 cycles). These results further validate the technical advantages of this invention, which uses a mild chelating agent (EDDS) and avoids the use of strong denaturing agents, ensuring high compatibility between the preservation solution and downstream molecular detection processes.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cryopreservation solution for in situ vitrification of semi-solid nucleic acids at room temperature, characterized by comprising: It comprises: a double buffering system, a mild chelating system, a vitrification skeleton, a thermoresponsive gelation system, an oxidation protection system, a low-toxicity bacteriostatic system, and water; The thermoresponsive gelation system forms a weakly reversible gel network in the range of 18-37℃ after the sample is mixed with the preservative solution, so as to reduce the diffusion and degradation of nucleic acids in the sample.

2. The preservative solution according to claim 1, wherein The double buffering system comprises 4-hydroxyethylpiperazine ethanesulfonic acid and 1,3-bis(trihydroxymethylmethylamino)propane, and the pH of the preservative solution is 7.4-8.

0.

3. The preservative solution of claim 1, wherein The mild chelating system is ethylenediamine disuccinic acid, and the concentration thereof in the preservative solution is 0.5-2.5 mM.

4. The preservative solution of claim 1, wherein The vitrification skeleton comprises trehalose and sorbitol, wherein the mass-volume percentage of trehalose is 2-8%, and the mass-volume percentage of sorbitol is 1-5%.

5. The preservative solution of claim 1, wherein The thermoresponsive gelation system comprises poloxamer 407 and poloxamer 188, wherein the mass-volume percentage of poloxamer 407 is 8-16%, and the mass-volume percentage of poloxamer 188 is 1-4%. Preferably, the thermoresponsive gelation system further comprises hydroxypropyl methyl cellulose with a mass-volume percentage of 0.05-0.5%.

6. The preservative solution of claim 1, wherein The oxidation protection system is ergothioneine or a salt thereof, and the mass-volume percentage thereof in the preservative solution is 0.01-0.05%.

7. The preservative solution of claim 1, wherein The low-toxicity bacteriostatic system is phenoxyethanol, p-hydroxyacetophenone, or a combination thereof, and the mass-volume percentage thereof in the preservative solution is 0.1-0.6%.

8. The preservative solution of claim 1, wherein The preservative solution does not contain guanidium salt, sodium azide, and high-concentration chaotropic agent.

9. A method for the room temperature preservation of a nucleic acid sample, characterized by, It comprises the following steps: adding a biological sample into the preservative solution of any one of claims 1-8 at a volume ratio of 1:1 to 1:2, mixing and standing for 1-5 minutes to form a semi-solid protective microenvironment, and then transporting or storing at 18-37℃.

10. A nucleic acid sample preservation kit, characterized by, It comprises the preservative solution of any one of claims 1-8 and a sampling container, wherein the sampling container is pre-filled with the preservative solution. Preferably, the sampling container is selected from a blood sampling tube, a saliva sampling tube, a buccal swab tube, a sputum sampling tube, or a tissue homogenate sampling tube.

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