Preserving fluid for stabilizing viruses at normal temperature and high temperature as well as preparation method and application of preserving fluid

Abstract

The invention relates to a preserving fluid for stabilizing viruses at normal temperature and high temperature as well as a preparation method and application of the preserving fluid. The preserving fluid is an aqueous solution containing the following components in concentration: 2mM-15mM of ethylenediamine tetraacetic acid tetrasodium salt, 5mM-20mM of sodium citrate, 0.1 wt%-10wt% of tween-20, 10wt%-30wt% of a structure stabilizer, 0.5 wt%-10wt% of an amide solution, 0.5 wt%-10wt% of a hemolysis inhibitor, 0.05 wt%-10wt% of a reducing agent and 0.1 mM-2mM of 3-morpholine propanesulfonic acid. According to the preservation solution, the formula is optimized, and particularly, the amide solution and the reducing agent component are matched with MOPS for use, so that the activity of viruses in a sample under normal-temperature and high-temperature conditions can be greatly maintained, and the stability of nucleic acid is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to virus preservation, specifically to a preservation solution that stabilizes viruses at room temperature and high temperature, its preparation method, and its application. Background Technology

[0002] In the field of molecular diagnostics for infectious diseases, DNA / RNA viral nucleic acids in biological samples (saliva, serum, plasma, urine, etc.) are key biomarkers. On the other hand, DNA / RNA viral particles, genetically engineered viruses, or naked nucleic acids preserved in biological samples are essential quality control materials, calibrators, and standard substances for the development and application of molecular diagnostic products. Therefore, the preservation quality of DNA / RNA viruses in biological samples, especially the integrity of nucleic acids, is crucial for the clinical application, development evaluation, quality control effectiveness, and system maintenance and calibration of molecular diagnostic products for infectious diseases, and may potentially affect individual health and public health prevention and control.

[0003] The preservation of DNA / RNA viruses in biological samples, especially the stable preservation of nucleic acids, faces numerous challenges, primarily stemming from two aspects: chemical instability and interference from the biological sample. Various factors can induce the degradation of DNA / RNA virus particles, genetically engineered viruses, or naked nucleic acids preserved in samples, and increased temperature exacerbates the degradation process. Compared to DNA viruses, RNA viruses are more unstable, facing more stringent chemical challenges (the 2' hydroxyl group of the sugar ring easily attacks the phosphodiester bond of the 3' hydroxyl group, readily leading to RNA breakage) and interference from the biological sample (such as the widespread presence of RNases). In summary, current methods for preserving DNA / RNA viruses in biological samples mainly involve 2-8℃ (short-term refrigeration), -15℃ (frozen storage and cold chain transportation), and -70℃ (long-term ultra-low temperature storage). Taking Abbott Laboratories' Hepatitis C Virus (HCV, positive-sense RNA virus) assay kit (National Medical Device Registration Certificate No. 20193402038) as an example, the main components of the kit's quality control and calibrators are non-infectious RNA pseudoviruses embedded with HCV sequences dissolved in negative plasma. These pseudoviruses require storage at -10°C or lower, and can only be stored for 24 hours at 2-8°C. The applicable serum or plasma samples for this kit require storage at -10°C or lower, and can only be stored for 3 days at 2-8°C. This results in stringent quality control, calibration, and applicable sample storage conditions for such products, leading to high costs and cumbersome usage procedures. This makes them unsuitable for transportation, storage, application, and quality control evaluation in resource-constrained remote areas.

[0004] Preserving DNA / RNA viral nucleic acids in biological samples at room temperature or even high temperature is of great significance. It can ensure continuous operation from sample collection, sample transportation, to detection and quality control calibration, and reduce dependence on expensive low-temperature equipment, thereby improving the efficiency of global prevention and control of infectious diseases. However, there are also high technical barriers, especially in achieving stable room temperature preservation of RNA viral nucleic acids in biological samples. Existing room temperature preservation technologies mainly include the following: (1) Guanidine salt-dependent inactivation protection technology, such as CN111363729B which describes a technology using guanidine isothiocyanate or ammonium salt, combined with reducing agents, metal ion chelating agents, surfactants Tween and lithium chloride. Taking the novel coronavirus (RNA virus) as an example, this scheme can achieve preservation at 25°C for 3 weeks or at 37°C for 1 week. The feature of this technology is that it lyses and inactivates RNA viruses in human samples and inhibits RNase activity, thereby preserving the integrity of RNA and can be well connected with downstream molecular diagnostics, but it will affect the isolation and culture of the virus and the preservation effect is generally poor. (2) Stabilizer combination protection technology that does not rely on guanidine salts, such as the technical solution described in US9376709B2, which uses metal ion chelating agents, N-acetylcysteine, polymers, and ethanol. Taking RNA in whole blood samples as an example, this solution can achieve stable preservation and transportation at room temperature for 2 weeks. The feature of this technical solution is that, without introducing guanidine salts or similar substances to inactivate the virus, it inhibits nuclease activity and maintains osmotic pressure through the combination of protective agents, thus better protecting the integrity of DNA / RNA. However, since more than 20% of low alcohols are used as fixatives, it will affect the isolation and culture of the virus and downstream molecular diagnostics. (3) Physical protection technology based on freeze-drying, such as the technical solution described in CN108070583B, which uses hydroxypropyl-β-cyclodextrin and DEPC as freeze-drying protectants and combines them with freeze-drying process to change the storage form. Taking duck Tembusu virus (RNA virus) as an example, this solution can achieve stable preservation and transportation at room temperature for 6 months. The feature of this technical solution is that it preserves nucleic acid at room temperature in the form of dry solid, thus isolating it from adverse factors such as nucleases and oxidative stress. However, the introduction of freeze-drying increases preparation costs and complicates operations. Furthermore, in practical applications, this technology can only address the room-temperature storage and transportation of quality control materials, calibrators, and standard substances; it cannot be applied to the preservation of DNA / RNA viral nucleic acids in biological samples, thus lacking universal applicability. Currently, DNA / RNA viruses can only be stored and transported at low temperatures, or even ultra-low temperatures.

[0005] Existing technologies cannot effectively address the degradation of DNA / RNA viral nucleic acids in biological samples at room temperature, especially RNA viruses, which exhibit extremely poor preservation. This hinders the widespread adoption and application of molecular diagnostic technologies globally. Therefore, in the field of molecular diagnostics for infectious diseases, there is an urgent need for a technological solution that can truly achieve stable storage and transportation of DNA / RNA viruses in biological samples at room temperature. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a preservation solution for stabilizing viruses at room temperature and high temperature, as well as its preparation method and application. The preservation solution can effectively maintain the activity and nucleic acid integrity of viruses (including DNA viruses and RNA viruses) in biological samples under both room temperature (10℃-30℃) and high temperature (45±1℃) conditions.

[0007] The first aspect of the present invention is to provide a virus preservation solution, said preservation solution being an aqueous solution comprising the following components at concentrations: 2mM-15mM tetrasodium ethylenediaminetetraacetate, 5mM-20mM sodium citrate, 0.1wt%-10wt% Tween-20, 10wt%-30wt% structural stabilizer, 0.5wt%-10wt% amide solution, 0.5wt%-10wt% hemolysis inhibitor, 0.05wt%-10wt% reducing agent, and 0.1mM-2mM 3-morpholinopropanesulfonic acid (MOPS).

[0008] In some embodiments, the preservation solution is an aqueous solution containing the following components at concentrations: 4 mM-10 mM tetrasodium ethylenediaminetetraacetate, 8 mM-16 mM sodium citrate, 0.2 wt%-5 wt% Tween-20, 15 wt%-25 wt% structural stabilizer, 2 wt%-5 wt% amide solution, 1 wt%-5 wt% hemolysis inhibitor, 0.05 wt%-1 wt% reducing agent, and 0.5 mM-1 mM 3-morpholinopropanesulfonic acid.

[0009] In some embodiments, the preservation solution is an aqueous solution containing the following components at concentrations: 4 mM-8 mM tetrasodium ethylenediaminetetraacetate, 10 mM-16 mM sodium citrate, 0.2 wt%-2 wt% Tween-20, 15 wt%-20 wt% structural stabilizer, 2 wt%-5 wt% amide solution, 2 wt%-5 wt% hemolysis inhibitor, 0.05 wt%-0.5 wt% reducing agent, and 0.5 mM-1 mM 3-morpholine propanesulfonic acid.

[0010] In some embodiments, the amide solution is selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylpropionamide, and N-methylpyrrolidone.

[0011] In some embodiments, the reducing agent is selected from at least one of N-acetylcysteine, tris(2-carbonylethyl)phosphohydrochloride, and water-soluble vitamins.

[0012] In some embodiments, the hemolytic inhibitor is selected from at least one of polyvinyl alcohol, polyvinylpyrrolidone, and dextran.

[0013] In some embodiments, the structural stabilizer is selected from at least one of sucrose, trehalose, galactose, lactose, and polyethylene glycol.

[0014] In some embodiments, the preservation solution is an aqueous solution containing the following components at concentrations: 4 mM-8 mM tetrasodium ethylenediaminetetraacetate, 10 mM-16 mM sodium citrate, 0.2 wt%-2 wt% Tween-20, 15 wt%-20 wt% trehalose, 2 wt%-5 wt% N,N-dimethylformamide, 2 wt%-5 wt% polyvinylpyrrolidone, 0.05 wt%-0.5 wt% N-acetylcysteine, and 0.5 mM-1 mM 3-morpholinopropanesulfonic acid.

[0015] In some embodiments, the relative molecular weight of the polyvinyl alcohol is 2,000 to 100,000, preferably 2,000 to 50,000.

[0016] In some embodiments, the relative molecular weight of the polyvinyl alcohol is 8,000 to 100,000, preferably 18,000 to 58,000.

[0017] In some embodiments, the relative molecular weight of the dextran is 3,000 to 40,000, preferably 3,000 to 30,000.

[0018] In some embodiments, the preservation solution also contains a preservative.

[0019] In some embodiments, the preservative is selected from ProClin. ® 300 and / or ProClin ® 950.

[0020] In some embodiments, the concentration of the preservative in the preservation solution is 0.05wt%-5wt%, preferably 0.05wt%-2wt%.

[0021] In some embodiments, the pH of the preservation solution is 5.5-7.2.

[0022] In some embodiments, the pH of the preservation solution is 5.5-6.5.

[0023] A second aspect of the present invention is to provide a method for preparing the preservation solution as described above, comprising the following steps: weighing each component according to the required amount, adding them sequentially to water, dissolving them completely, and mixing them evenly; adjusting the pH value; and filtering to obtain the solution.

[0024] A third aspect of the invention is to provide the application of the preservation solution described above in the transportation and preservation of virus samples.

[0025] A fourth aspect of the present invention is to provide a method for preserving a virus sample, comprising the following steps: adding the preservation solution as described above to the virus sample and mixing them evenly.

[0026] In some embodiments, the volume ratio or mass-volume ratio of the virus sample to the preservation solution is 1:(1~4).

[0027] In some embodiments, the virus sample includes any human tissue or material containing the virus and a virus quality control sample preserved using any human tissue or material as a matrix. The human tissue or material includes throat swabs, nasal swabs, bronchoalveolar lavage fluid, serum, plasma, whole blood, peripheral blood, sputum, urine, feces, lymph nodes, and other bodily fluids or materials.

[0028] In some embodiments, the virus comprises any DNA or RNA virus derived from humans, animals, or other organisms, including adenovirus (ADV), herpesvirus (HSV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), other positive / negative strand, single / double strand viruses, viral genetic engineering vectors, or materials containing target nucleic acids.

[0029] This invention has developed a preservation solution that is highly suitable for preserving the activity and nucleic acid integrity of viruses (including DNA viruses and RNA viruses) under both ambient temperature (10℃-30℃) and high temperature (45±1℃) conditions. Through optimization of the formulation, especially by combining specific types of amide solutions and reducing agent components with MOPS, the preservation solution can greatly maintain the activity of viruses under both ambient temperature and high temperature conditions, as well as improve the stability of viral nucleic acids.

[0030] The preservation solution of this invention can stably preserve quality control RNA virus nucleic acid in serum matrix for at least 60 days at 45°C; it can stably preserve quality control RNA virus nucleic acid in serum matrix for 18 months at 30°C, RNA virus nucleic acid in clinical serum samples for 12 months, and DNA / RNA virus nucleic acid in clinical urine samples for 18 months. The preservation time at room temperature and high temperature is significantly longer than that of the preservation solutions of existing technologies.

[0031] The preservation solution of this invention does not contain denaturing agents (such as guanidine salts and SDS), cross-linking fixatives (such as formaldehyde and lower alcohols), or hazardous preservatives (such as sodium azide). It can better maintain the viral activity and nucleic acid integrity in biological samples under ambient and high temperature conditions, without affecting downstream virus isolation and culture, nucleic acid extraction and purification, and molecular diagnostics. It can also inhibit the growth and contamination of bacteria and other microorganisms. It is particularly suitable for the room temperature preservation and transportation of DNA / RNA viruses and molecular diagnostic quality control products, calibrators, and standard substances with similar biological properties in complex clinical samples. It is beneficial for the preservation, transportation, and molecular diagnostics of samples worldwide, especially in resource-limited areas. Attached Figure Description

[0032] Figure 1 To investigate the preservation effects of different preservation solutions on serum and urine samples.

[0033] Figure 2 The Ct values ​​for nucleic acid detection of HCV strong positive control samples in different preservation solutions under storage conditions of 45℃.

[0034] Figure 3 The Ct values ​​for nucleic acid detection of HCV critical positive quality control samples in different preservation solutions under storage conditions of 45℃.

[0035] Figure 4 The Ct values ​​for nucleic acid detection of HCV strong positive control samples in different preservation solutions under storage conditions of 30℃.

[0036] Figure 5 The Ct values ​​for nucleic acid detection of HCV critical positive control samples in different preservation solutions under storage conditions of 30℃.

[0037] Figure 6 The Ct values ​​for nucleic acid detection in HCV clinical serum sample A in different preservation solutions under storage conditions of 30℃.

[0038] Figure 7 The Ct values ​​for nucleic acid detection in HCV clinical serum sample B in different preservation solutions under storage conditions of 30℃.

[0039] Figure 8 The Ct values ​​for nucleic acid detection in HCV clinical serum sample C in different preservation solutions under storage conditions of 30℃.

[0040] Figure 9 The Ct values ​​for nucleic acid detection in clinical urine samples of HSV-2, UU, and ZIKA in different preservation solutions under storage conditions of 30℃.

[0041] Figure 10 The infection of HEK293T cells with lentivirus in different preservation solutions under storage conditions of 16℃. Detailed Implementation

[0042] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0043] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0045] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0046] Some embodiments of the present invention relate to a virus preservation solution, which is an aqueous solution containing the following components at concentrations: 2mM-15mM tetrasodium ethylenediaminetetraacetate, 5mM-20mM sodium citrate, 0.1wt%-10wt% Tween-20, 10wt%-30wt% structural stabilizer, 0.5wt%-10wt% amide solution, 0.5wt%-10wt% hemolysis inhibitor, 0.05wt%-10wt% reducing agent, and 0.1mM-2mM 3-morpholinopropanesulfonic acid.

[0047] This invention, through research and optimization, yielded a preservation solution highly suitable for preserving the activity and nucleic acid integrity of viruses (including DNA and RNA viruses) under ambient and high-temperature conditions. The preservation solution comprises appropriate concentrations of tetrasodium ethylenediaminetetraacetate (EDTA), sodium citrate, Tween-20, a structural stabilizer, an amide solution, a hemolysis inhibitor, a reducing agent, and 3-morpholinopropanesulfonic acid buffer. Through formulation optimization, particularly the combination of specific types of amide solutions and reducing agents with MOPS, the generation of ROS in samples can be better inhibited, RNase activity reduced, and the preservation solution exhibits superior thermal stability, thereby significantly maintaining viral activity under ambient and high-temperature conditions and improving viral nucleic acid stability. The scientific combination and concentration optimization of raw materials form an organic and synergistic whole. The components work together synergistically to inhibit viral nucleic acid degradation through multiple pathways under ambient and high-temperature conditions, maintaining viral activity without affecting downstream viral isolation and culture, nucleic acid extraction and purification, and molecular diagnostics.

[0048] The combination of raw materials for the preservation solution of this invention is not a simple addition of components, but an optimized combination based on sample characteristics, virus properties and nucleic acid molecule properties, which significantly improves the overall room temperature and high temperature preservation performance of the preservation solution.

[0049] The present invention will be further described in detail below with reference to specific embodiments.

[0050] Example 1

[0051] This embodiment has conducted extensive research and optimization based on sample characteristics, viral properties, nucleic acid molecular properties, and multi-pathway anti-degradation mechanisms, and finally obtained a preservation solution that has a good protective effect against DNA / RNA viruses in complex clinical samples under room temperature and high temperature conditions. The preservation solution formulation in Table 1 is used as an example for comparison and illustration.

[0052] In Table 1, formulations 1 and 5 are formulations of the present invention, and formulations 2-4 are control formulations. Compared with formulation 1, N-acetylcysteine ​​is replaced with mannitol in formulation 2; N,N-dimethylformamide is replaced with DMSO in formulation 3; and 3-morpholinopropanesulfonic acid buffer system is replaced with D-HANKS buffer in formulation 4.

[0053] Table 1

[0054]

[0055] The preparation methods for the preservation solutions in Table 1 are as follows: 1. According to the formula, EDTA tetrasodium salt, sodium citrate, Tween-20, trehalose, N,N-dimethylformamide (or DMSO), PVP K30, N-acetylcysteine ​​(or mannitol), preservative, and 3-morpholinopropanesulfonic acid (or D-HANKS buffer) are mixed in the following order, with the remainder being pure water. The mixture is placed in a container equipped with a stirrer, dissolved, stirred, and mixed evenly to obtain a homogeneous solution; 2. HCl is added to the above mixture, stirred evenly, the pH value is adjusted, and pure water is added to make up to the final volume; 3. The pH-adjusted solution is filtered through a 0.22 μm aqueous filter membrane for sterilization to obtain the preservation solution. The obtained preservation solution is stored at room temperature (10-30℃).

[0056] Figure 1 This demonstrates the use of the above-mentioned preservation solution to preserve serum and urine samples at a 1:1 v / v ratio. The control preservation solution used was DNA / RNA Shield from ZYMO RESEARCH, USA. TM Product. (By) Figure 1 It is known that the preservation solutions of this invention (Formula 1 and Formula 5) are compatible with multiple sample types and maintain good compatibility in different sample matrices, which is beneficial for maintaining the original state of the sample. The preservation solution of Formula 2 is more turbid when preserving serum, which is quite different from the original state of the sample; the preservation solution of Formula 3 causes protein precipitation in the serum sample; the preservation solution of Formula 4 is slightly viscous when preserving serum.

[0057] Studies have found that both Formula 1 and Formula 5 preservation solutions exhibit good virus preservation effects under both room temperature and high temperature conditions, with Formula 1 showing better results. Compared to Formula 1, Formula 2 virus preservation solution replaces N-acetylcysteine ​​with mannitol, leading to a decrease in the preservation effect of the solution on the virus in the sample. This invention has found that in the preservation solution system of this invention, the combined use of N,N-dimethylformamide and N-acetylcysteine ​​can form a sustained reducing microenvironment, better inhibiting ROS production and reducing RNase activity. Mannitol, as a commonly used ROS scavenging component in preservation solutions, cannot combine with N,N-dimethylformamide to construct a sustained reducing microenvironment, thus failing to cope with viral nucleic acid degradation caused by unstable reactive oxygen species in biological samples, resulting in a significantly reduced nucleic acid protection effect. Formula 3, the virus preservation solution, replaces N,N-dimethylformamide with DMSO. While DMSO is also a highly efficient cell membrane permeabilizer and a polar aprotic solvent, capable of entering cells and influencing metabolism, it cannot effectively inhibit RNase activity in combination with N-acetylcysteine. Furthermore, it does not work well with tetrasodium EDTA to reduce permeability regulation, lower metabolic levels, or inhibit enzyme activity, thus weakening its protective effect on viral nucleic acids in the sample. Moreover, it exhibits poor compatibility in some real biological samples (e.g., ...). Figure 1This resulted in protein precipitation in serum samples; Formula 4, which replaced the 3-morpholinopropanesulfonic acid buffer system with the D-HANKS buffer system, led to a decrease in the overall thermal stability of the preservation solution and a weakening of the viral nucleic acid protection effect. The preservation effects of each formulation on the virus in the samples will be documented in subsequent studies.

[0058] Example 2

[0059] This embodiment tests the stability of different preservation solutions for RNA pseudovirus quality control samples at 45°C.

[0060] 2.1 Main experimental materials, instruments and reagents

[0061] Experimental materials: Non-infectious RNA pseudovirus particles embedded with HCV-specific gene sequences (Fobo Biopharmaceutical Technology Co., Ltd., catalog number FNRV7471), after being dissolved in negative serum at different final concentrations, can be used as strong positive control and borderline positive control materials, and can be used in conjunction with the "Hepatitis C Virus (HCV) Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method)" (Guangzhou Wondfo Biotech Co., Ltd., catalog number WZA7).

[0062] Preservative solution: Preservative solutions from formulations 1-5 in Example 1; control preservation solution: DNA / RNA Shield. TM (ZYMORESEARCH, USA, part number R1100-250).

[0063] Nucleic acid extraction and purification: Nucleic acid extraction or purification kit (Guangzhou Wondfo Biotech Co., Ltd., catalog number WE03).

[0064] Real-time quantitative PCR instrument: Fully automated medical PCR analysis system (Hongshi SLAN-96S).

[0065] 2.2 Preparation of experimental samples

[0066] Preparation of positive control: The RNA pseudovirus particles embedded with the HCV-specific gene sequence were valued using the "Hepatitis C Virus (HCV) Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method)". Then, they were dissolved in negative serum at different final concentrations to prepare strong positive control (2 × 10⁻⁶). 4 IU / mL) and critical positive control (1×10⁻⁶) 2 (IU / mL) to evaluate the preservation effect of the preservation solution on different viral loads.

[0067] Sample processing: Strong positive control and borderline positive control were divided into 6 equal parts. Experimental groups 1-5 were prepared by mixing the control with preservation solution of formulations 1-5 at a volume ratio (v / v) of 1:1. The commercial control group was prepared by mixing the control with DNA / RNAShield™ preservation solution at a volume ratio (v / v) of 1:1. The mixtures were then aliquoted into nuclease-free preservation tubes. The concentration of the strong positive control after mixing was 1×10⁻⁶. 4 IU / mL, critical positive control concentration is 1×10⁻⁶ 2 IU / mL.

[0068] 2.3 Constant temperature storage

[0069] The above-mentioned aliquoted samples were placed in a constant temperature incubator and stored at 45℃±1℃ to simulate the harsh transportation and storage environment. The corresponding experimental samples were taken out on day 0 (starting point), day 15, day 30, day 45 and day 60 of storage, and nucleic acid extraction and detection were performed immediately afterwards.

[0070] 2.4 Detection of Experimental Samples

[0071] Following the testing procedure outlined in the "Hepatitis C Virus (HCV) Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method)," 300 μL of experimental samples taken at each time point were mixed with 20 μL of internal standard solution from the kit. When the batch internal standard solution concentration was 8000 IU / mL, total nucleic acid was extracted from each time point using a nucleic acid extraction or purification kit, followed by real-time quantitative qRT-PCR detection. The target detection Ct value was recorded, and the experimental sample concentration was calculated using the SLAN-96S internal standard quantification plugin. Four technical replicates were set up for the experimental samples to ensure data reliability.

[0072] 2.5 Experimental Results

[0073] (1) Preservation effect of strong positive control products

[0074] The preservation results of each group of preservation solutions for strong positive control samples are shown in Table 2 and... Figure 2 As shown.

[0075] Table 2

[0076]

[0077] As shown in Table 2, the detection rates and target Ct values ​​of the preservation solutions in Table 1 after 45 days and 60 days of storage at 45℃ were superior to those of the commercial control group. Furthermore, the preservation effects of formulations 1 and 5 were superior to those of formulations 2-4. Among these, formulation 1 showed the best preservation effect. The detection rate of formulation 1 was 100% on days 0, 15, 30, 45, and 60, with a target Ct precision range of 0.8%–1.25%. The mean target Ct value on day 45 was 27.73, a deviation of 3.32 from the starting point; the mean logarithm (log10 C) of the measured concentration on day 45 was 3.105, a deviation of 0.99 from the starting point.

[0078] The nucleic acid assay results of the preservation solution of Formula 1 and the commercial control preservation solution are shown in Table 3.

[0079] Table 3

[0080]

[0081] (2) Preservation effect of critical positive control samples

[0082] The preservation results of each group of preservation solutions for critical positive quality control samples are shown in Table 4 and... Figure 3 As shown.

[0083] Table 4

[0084]

[0085]

[0086] Table 4 shows that the detection rates and target Ct values ​​of Formula 1 and Formula 5 preservation solutions after 45 days and 60 days of storage at 45℃ were superior to those of the commercial control group and Formulas 2-4. Among them, Formula 1 preservation solution showed the best preservation effect. The detection rate of Formula 1 preservation solution group was 100% on days 0, 15, 30, 45, and 60, and the precision range of target Ct value was 0.64%~4.77%. The mean target Ct value on day 45 was 35.34, which deviated from the starting point by 3.71. The mean logarithm of the measured concentration (log10 C) on day 45 was 1.14, which deviated from the starting point by 1.

[0087] The nucleic acid assay results of the preservation solution of Formula 1 and the commercial control preservation solution are shown in Table 5.

[0088] Table 5

[0089]

[0090] 2.6 Experimental Conclusions

[0091] Under harsh conditions of 45°C, the preservation solutions of formulations 1 and 5 exhibit good protective effects on strong positive and borderline positive quality controls containing serum matrix. They meet the quantitative efficacy requirements for strong positive quality controls on day 45 and the positive detection requirements for positive quality controls on day 60, demonstrating superior performance compared to commercially available comparative products. This indicates that the preservation solutions of this invention can achieve stable storage and transportation of quality control products under high-temperature conditions.

[0092] Example 3

[0093] This embodiment studies the stability preservation effect of different preservation solutions on RNA pseudovirus quality control samples at 30℃.

[0094] 3.1 Main experimental materials, instruments and reagents

[0095] Same as Example 2.

[0096] 3.2 Preparation of experimental samples

[0097] Same as Example 2.

[0098] 3.3 Constant temperature storage

[0099] The above-mentioned aliquoted samples were placed in a constant temperature incubator and stored at 30℃±1℃ to simulate the harsh transportation and storage environment. The corresponding experimental samples were taken out on day 0 (starting point), month 1, month 3, month 6, month 12 and month 18 of storage, and nucleic acid extraction and detection were performed immediately afterwards.

[0100] 3.4 Detection of Experimental Samples

[0101] Following the testing procedure outlined in the "Hepatitis C Virus (HCV) Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method)," 300 μL of experimental samples taken at each time point were mixed with 20 μL of internal standard solution from the kit. When the batch internal standard solution concentration was 12000 IU / mL, total nucleic acid was extracted from each time point using a nucleic acid extraction or purification kit, followed by real-time quantitative qRT-PCR detection. The target detection Ct value was recorded, and the experimental sample concentration was calculated using the SLAN-96S internal standard quantification plugin. Four technical replicates were set up for the experimental samples to ensure data reliability.

[0102] 3.5 Experimental Results

[0103] (1) Preservation effect of strong positive control products

[0104] The preservation results of each group of preservation solutions for strong positive control samples are shown in Table 6 and... Figure 4 As shown.

[0105] Table 6

[0106]

[0107] As shown in Table 6, the detection rate and target Ct value of the experimental group after 12 and 18 months of storage at 30℃ were superior to those of the commercial control group, and the preservation effects of formulas 1 and 5 were better than those of formulas 2-4. Among them, the preservation effect of formula 1 was the best. The detection rate of formula 1 was 100% on day 0, month 1, month 3, month 6, month 12, and month 18, and the precision range of the target Ct value was 0.1%~0.37%. The mean target Ct value at month 12 was 26.66, which deviated from the starting point by 2.00. The mean logarithm of the measured concentration (log10 C) at month 18 was 3.67, which deviated from the starting point by 0.81.

[0108] The nucleic acid assay results of the preservation solution of Formula 1 and the commercial control preservation solution are shown in Table 7.

[0109] Table 7

[0110]

[0111] (2) Preservation effect of critical positive control samples

[0112] The preservation results of each group of preservation solutions for critical positive quality control samples are shown in Table 8 and... Figure 5 As shown.

[0113] Table 8

[0114]

[0115] As shown in Table 8, the detection rates and target Ct values ​​of Formula 1 and Formula 5 preservation solutions after 12 and 18 months of storage at 30℃ were superior to those of the commercial control group and Formulas 2-4. Among them, Formula 1 preservation solution showed the best preservation effect. The detection rate of Formula 1 preservation solution group was 100% on day 0, month 1, month 3, month 6, month 12, and month 18, and the precision range of target Ct value was 1.02%~4.43%. The mean target Ct value at month 12 was 33.89, which was 2.65 away from the starting point. The mean logarithm of the measured concentration (log10 C) at month 18 was 1.52, which was 0.99 away from the starting point.

[0116] The nucleic acid assay results of the preservation solution of Formula 1 and the commercial control preservation solution are shown in Table 9.

[0117] Table 9

[0118]

[0119] 3.6 Experimental Conclusions

[0120] Under long-term storage conditions at 30°C, the preservation solutions of formulations 1 and 5 of this invention exhibit excellent protective effects on both strongly positive and borderline positive quality controls containing serum matrix. Even after 18 months, they still meet the quantitative efficacy and positive detection requirements for strongly positive quality controls, demonstrating significantly superior performance compared to commercially available comparative products. This indicates that the preservation solutions of this invention can achieve stable storage and transportation of quality controls at room temperature for up to 18 months.

[0121] Example 4

[0122] This embodiment examines the stability of different preservation solutions for real clinical HCV serum samples at 30°C.

[0123] 4.1 Main Experimental Materials, Instruments, and Reagents

[0124] Experimental materials: Three anonymous clinical serum samples known to be HCV positive were used, numbered A, B, and C. The dilution matrix was a mixture of serum from HCV-negative healthy individuals.

[0125] Preservative solutions: Preservative solutions 1 and 5, which performed better in the aforementioned examples, were used for verification. The control solution was DNA / RNA Shield. TM (ZYMO RESEARCH, USA, part number R1100-250).

[0126] Nucleic acid extraction and purification, nucleic acid detection kits, and real-time fluorescence quantitative PCR instruments are the same as in Example 2.

[0127] 4.2 Preparation of Experimental Samples

[0128] To simulate a real clinical scenario as closely as possible, the positive sera from the three HCV patients (A, B, and C) were appropriately diluted using a mixed serum sample from HCV-negative healthy individuals. Each diluted positive sample was then divided into four aliquots and processed as follows:

[0129] Experimental group 1: Mixed with the preservation solution of formula 1 of the present invention at a volume ratio of 1:1 (sample: preservation solution).

[0130] Experimental group 2: Mixed with the preservation solution of formula 5 of the present invention at a volume ratio of 1:1 (sample: preservation solution).

[0131] Control group 1: No preservation solution was added.

[0132] Control group 2: Compared with commercial DNA / RNA Shield TM The preservation solution was mixed at a volume ratio of 1:1.

[0133] All processed samples were aliquoted into sterile centrifuge tubes.

[0134] 4.3 Constant temperature storage

[0135] The control group 1 (frozen control) samples were immediately stored in an ultra-low temperature freezer at -70℃±5℃ as a baseline control. All samples from experimental group 1, experimental group 2, and control group 2 (commercial control) were placed in a constant temperature incubator and stored at 30℃±1℃ to simulate a high-temperature transportation or storage environment. Samples were retrieved on day 0 (immediately), month 1, month 6, and month 12 of storage, and nucleic acid extraction and detection were performed immediately.

[0136] 4.4 Detection of Experimental Samples

[0137] Following the testing procedure outlined in the "Hepatitis C Virus (HCV) Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method)," 300 μL of experimental samples taken at each time point were mixed with 20 μL of internal standard solution from the kit. When the batch internal standard solution concentration was 7500 IU / mL, total nucleic acid was extracted from each time point using a nucleic acid extraction or purification kit, followed by real-time quantitative qRT-PCR detection. The target detection Ct value was recorded, and the experimental sample concentration was calculated using the SLAN-96S internal standard quantification plugin. Four technical replicates were set up for the experimental samples to ensure data reliability.

[0138] 4.5 Experimental Results

[0139] The results of preserving real clinical samples are shown in Table 10. Figure 6 , Figure 7 and Figure 8 As shown.

[0140] Table 10

[0141]

[0142]

[0143]

[0144]

[0145] It is evident that the variation in HCV target Ct values ​​in the three clinical samples preserved with Formula 1 was significantly smaller than that in the commercial control group and similar to that in the frozen control group 1. The mean deviation of the logarithm (log10 C) of the concentration measured in Formula 1 at month 12 was 0.82 for sample A, 1.32 for sample B, and 0.93 for sample C. In contrast, at month 12, the mean deviation of the logarithm (log10 C) of the concentration measured in the three samples preserved in the commercial control group was greater than 2.

[0146] 4.6 Experimental Conclusions

[0147] Under long-term storage conditions at 30°C, the preservation solutions of formulations 1 and 5 of this invention have good protective effects on three real clinical HCV serum samples, and can still be accurately detected at 12 months. Their performance is significantly better than that of the commercial control group and close to that of the frozen control group.

[0148] Combined with Examples 2 and 3, it is confirmed that the preservation solutions of Formulas 1 and 5 of the present invention overcome the defect of poor preservation effect of RNA viruses in biological samples at room temperature. They can be used as quality control products for serum-containing RNA viruses and for room temperature storage and transportation of clinical RNA virus serum samples, ensuring continuous operation from sample collection to detection to quality control calibration, reducing dependence on expensive low-temperature equipment, and improving the efficiency of prevention and control of infectious diseases worldwide.

[0149] Example 5

[0150] This study investigated the stability of different preservation solutions for real clinical DNA / RNA pathogen urine samples at 30°C.

[0151] 5.1 Main materials and reagents:

[0152] Experimental materials: Three clinical urine samples containing Zika virus (ZIKA), ureaplasma urealyticum (UU), and herpes simplex virus type 2 (HSV-2) were selected as test samples. They were diluted to different final concentrations using negative urine matrix to simulate clinical urine samples with low viral load.

[0153] Preservative solutions: In this embodiment, preservation solutions 1 and 5, which performed better in the previous embodiments, were selected for verification. Specific preparation methods are detailed in Example 1. The control preservation solution was DNA / RNA Shield. TM (ZYMO RESEARCH, USA, part number R1100-250).

[0154] Nucleic acid extraction and purification, real-time fluorescence quantitative PCR instrument, and nucleic acid detection kit are the same as in Example 2.

[0155] 5.2 Preparation of Experimental Samples

[0156] To simulate low-load samples that may be encountered in real-world testing and to ensure sufficient volume for long-term studies, midstream morning urine from healthy volunteers was collected. After initial centrifugation to remove sediment, this was used as the basal urine matrix. Using this treated basal urine matrix, the three clinical urine samples containing Zika virus (ZIKA), Ureaplasma urealyticum (UU), and herpes simplex virus type 2 (HSV-2), respectively, were appropriately diluted several times and thoroughly mixed. The diluted samples were then divided into four equal portions and processed as follows:

[0157] Experimental group 1: Mixed with the preservation solution of formula 1 of the present invention at a volume ratio of 1:1 (sample: preservation solution).

[0158] Experimental group 2: Mixed with the preservation solution of formula 5 of the present invention at a volume ratio of 1:1 (sample: preservation solution).

[0159] Control group 1 (no added control): No preservation solution was added.

[0160] Control group 2 (commercial control): compared with commercial DNA / RNA Shield TM The preservation solution was mixed at a volume ratio of 1:1 (sample: preservation solution).

[0161] All processed samples were aliquoted into nuclease-free centrifuge tubes.

[0162] 5.3 Preservation and Sampling

[0163] Samples from experimental groups 1, 2, and 2 (control group 2) were placed in a constant temperature incubator and stored at 30℃±1℃ to simulate room temperature sample retention, mailing, or transportation scenarios. Samples from control group 1 were placed in an ultra-low temperature freezer at -70℃±5℃ to simulate long-term sample storage conditions.

[0164] To comprehensively evaluate the protective effect against multiple nucleic acids, samples were retrieved on day 0 (immediately), 12 months, and 18 months of storage, and nucleic acid extraction and testing were performed immediately.

[0165] 5.4 Detection Methods

[0166] Before sample extraction, the samples must be thoroughly mixed. Use a nucleic acid extraction and purification instrument and a magnetic bead nucleic acid extraction or purification kit to extract total nucleic acid from samples at each time point. Use a pipette to aspirate 200 μL of urine samples diluted with the above four sets of preservation solutions into a deep well plate. For detailed operating steps and extraction procedures, please refer to the extraction kit instructions. The extracted nucleic acid samples should be used immediately.

[0167] Nucleic acid samples must be thoroughly mixed before use. Four groups of ZIKA samples were tested using the "7-Item Nucleic Acid Detection Kit for Mosquito-borne Pathogens (Fluorescent PCR Method)" (Guangzhou Wondfo Biotech Co., Ltd., Catalog No.: WR41), and four groups of UU and HSV-2 samples were tested using the "Multiple Nucleic Acid Detection Kit for Reproductive Tract Pathogens (Fluorescent PCR Method)" (Guangzhou Wondfo Biotech Co., Ltd., Catalog No.: WRA4). For detailed operating procedures, please refer to the fluorescent PCR kit instructions. Each sample was tested in four replicate wells.

[0168] The ABI 7500 was used for testing and data analysis, and the Ct values ​​detected in the samples at each time point were recorded.

[0169] 5.5 Experimental Results

[0170] The results of clinical urine sample preservation are shown in Table 11 and Figure 9 As shown.

[0171] Table 11

[0172]

[0173] It is evident that both formulations 1 and 5 of the preservation solutions ensured stable detection of all three representative pathogens after 18 months of storage at 30°C. For the three pathogens stored at 30°C for 18 months using formulation 1, the Ct value shift (ΔCt) was controllable, all less than 0.5. In contrast, the control group samples without preservation solution showed relatively large Ct value shifts for all three pathogens after 18 months of storage at -70°C. In comparison, the commercial control group samples showed stable detection of HSV-2 after 18 months of storage at 30°C, with relatively controllable Ct value shifts. However, ZIKA and UU showed larger Ct value shifts, with some ZIKA samples showing Ct values ​​close to 40, indicating a risk of missed detection.

[0174] 5.6 Experimental Conclusions

[0175] Under long-term storage conditions at 30°C, the preservation solutions of formulations 1 and 5 of this invention have good protective effects on urine samples of three real clinical DNA / RNA pathogens, and can still be accurately detected after 18 months. Their performance is significantly better than that of the commercial control group.

[0176] In this embodiment, viruses with single-stranded positive-sense RNA as their genetic material, mycoplasma with double-stranded circular DNA, and pathogens with double-stranded linear DNA were selected to achieve broad-spectrum, highly efficient, and stable protection for up to 18 months. This demonstrates that its long-term preservation performance is superior to commercial preservation technologies, solves the problem of DNA / RNA viral nucleic acid degradation in biological samples at room temperature, and can truly achieve stable storage and transportation of DNA / RNA viral nucleic acid in biological samples at room temperature. This is conducive to the popularization and application of molecular diagnostic technology worldwide.

[0177] Example 6

[0178] This embodiment studies the protective effects of different preservation solutions on viral infectivity.

[0179] 6.1 Main Materials and Reagents

[0180] Experimental materials: Lentiviral particles LV-EGFP-Puro-CON expressing green fluorescent protein (GFP) (FLV109, catalog number FLV109, Fubai Biopharmaceutical Technology Co., Ltd.), HEK293T cells.

[0181] Preservative solutions: In this embodiment, the preservative solutions 1 and 5, which performed better in the previous embodiments, were selected for verification. For specific preparation methods, please refer to Example 1. The control preservative solution was DNA / RNA Shield. TM (ZYMO RESEARCH, USA, part number R1100-250).

[0182] Cell culture reagents: HEK293T cells are cultured using high-glucose DMEM (Gibcol) containing fetal bovine serum (Gibcol) and penicillin (sigma).

[0183] Biological incubator: Carbon dioxide incubator (Thermo Fisher Scientific, Forma 3111).

[0184] Fluorescence microscope: Fluorescence microscope and imaging system (Nikon Eclipse Ni DS-Ri2).

[0185] 6.2 Preparation of Experimental Samples

[0186] Lentiviral sample preparation: Take samples with a concentration of 9×10 8 0.05 mL of LV-EGFP-Puro-CON (Fobo Biopharmaceutical Technology Co., Ltd., catalog number FLV109) at a concentration of TU / mL was added to 4.45 mL of PBS to dilute the lentivirus stock solution to 1×10⁻⁶. 7 TU / mL to be used.

[0187] Sample processing: Four 1mL aliquots were taken from the lentivirus sample and dispensed into nuclease-free preservation tubes. Experimental groups 1-2 consisted of lentivirus mixed with preservation solutions from formulations 1 and 5 of this invention at a volume ratio (v / v) of 1:4. The concentration of this experimental sample was 2 × 10⁻⁶. 6 TU / mL, 4mL per group; the commercial control group consisted of lentivirus and DNA / RNA Shield. ™ The preservation solution was mixed and preserved at a volume ratio (v / v) of 1:4. The concentration of the sample in this experiment was 2 × 10⁻⁶. 6 TU / mL, 4 mL per group; the control group without additives consisted of lentivirus and PBS mixed at a volume ratio (v / v) of 1:4 and stored. The sample concentration for this experiment was 2 × 10⁻⁶. 6 TU / mL, 4mL per group.

[0188] 6.3 Preservation and Sampling

[0189] Experimental groups 1 and 2, along with the commercial control group, were placed in a constant temperature incubator and stored at 16±0.5℃. The additive-free control group was stored at -70℃. Samples diluted with different preservation solutions from all four groups were retrieved on day 7 for cell infection experiments.

[0190] 6.4 Detection Methods

[0191] Resuscitated cryopreserved HEK293T cells were passaged in DMEM cell culture medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to the logarithmic growth phase. After trypsin digestion, the cells were resuspended in cell culture medium and the cell concentration was adjusted to 1×10⁶ cells / year. 5 cells / mL, and the cell suspension was prepared at 2×10 5 Cells were seeded per well into 6-well cell culture plates (9.6 cm² / well), for a total of 15 wells. The seeded plates were incubated at 37°C for 4 hours. After cell adhesion, the culture medium was discarded, and the cells were washed twice with PBS. The medium was then replaced with serum-free and antibiotic-free DMEM, and incubated at 37°C for 4 hours. The culture medium was discarded, and 1 mL of serum-free and antibiotic-free DMEM containing the infection-enhancing agent LV-EnhanceI was added to each well. The 15 wells were divided into 5 groups of 3 wells each. Group 1 received 1 mL of DMEM as a negative control for lentivirus infection; Group 2 received 1 mL of lentivirus preserved in PBS stored at -70°C as a positive control; Group 3 received 1 mL of lentivirus preserved in the preservation solution of Formula 1 of this invention as experimental group 1; Group 4 received 1 mL of lentivirus preserved in the preservation solution of Formula 5 of this invention as experimental group 2; and Group 5 received 1 mL of DNA / RNAShield. ™ Lentiviral cells preserved in the preservation solution served as a commercial control group for lentivirus infection. After incubating the well plates at 37°C for 24 hours, the culture medium was discarded, the plates were washed twice with PBS, and the culture medium was replaced with DMEM (containing 10% fetal bovine serum and 1% penicillin and streptomycin). The plates were then cultured for another 72 hours, and bright-field and GFP fluorescence were observed under a fluorescence microscope.

[0192] 6.5 Experimental Results

[0193] Infection test results after 7 days of storage are as follows Figure 10 As shown.

[0194] Fluorescence microscopy revealed that, in bright-field observation, the 293T cells in the negative control group (without lentivirus), the positive control group (lentivirus diluted with PBS), and the experimental group 2 (lentivirus diluted with preservation solution of formulation 5 of this invention) showed good growth, intact cell morphology, and good refractive properties. A small number of blank areas of cells could be observed in experimental group 1 (lentivirus diluted with preservation solution of formulation 1 of this invention). The commercial control group (lentivirus diluted with DNA / RNA Shield™ preservation solution) showed almost complete cell apoptosis, with only cell debris and impurities remaining.

[0195] Fluorescence microscopy revealed that, in the green fluorescent field, the positive control group with lentivirus diluted in PBS had the highest infection efficiency, while experimental group 1 with lentivirus diluted in the preservation solution of formulation 1 of this invention had a relatively high infection efficiency. Experimental group 2 with lentivirus diluted in the preservation solution of formulation 5 of this invention had a relatively low infection efficiency, but infection was still successful. No fluorescence signal was observed in the commercial control group with lentivirus diluted in DNA / RNA Shield™ preservation solution.

[0196] 6.6 Experimental Conclusions

[0197] This embodiment demonstrates that the preservation solutions provided by this invention, specifically Formula 1 and Formula 5 (especially Formula 1), exhibit significantly superior protective effects compared to commercially available preservation solutions using other technologies at 16°C. Formula 1 of this invention exhibits lower cytotoxicity and is more conducive to preserving the activity of viral samples at 16°C. The preservation solution of this invention not only stabilizes nucleic acids, but its mild components also provide a protective microenvironment for intact cells over a longer period. This embodiment confirms the technical superiority of this invention, which is superior to solutions using denaturing agents such as guanidine salts and SDS, cross-linking and immobilizing agents such as formaldehyde and lower alcohols, and hazardous preservatives such as sodium azide. It utilizes only specific concentrations of metal ion chelating agents, amide solutions, hemolytic inhibitors, and other sub-components in conjunction with MOPS buffer to maintain pH stability, ensuring the integrity and stability of DNA / RNA viral nucleic acids in biological samples without affecting downstream virus isolation and culture, nucleic acid extraction and purification, and molecular diagnostics.

[0198] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A virus preservation solution, characterized in that, The preservation solution is an aqueous solution containing the following components at the following concentrations: ethylenediaminetetraacetic acid tetrasodium salt 2mM-15mM, sodium citrate 5mM-20mM, Tween-20 0.1wt%-10wt%, structure stabilizer 10wt%-30wt%, amide solution 0.5wt%-10wt%, hemolysis inhibitor 0.5wt%-10wt%, reducing agent 0.05wt%-10wt%, and 3-morpholinopropanesulfonic acid 0.1mM-2mM.

2. The preservation solution as described in claim 1, characterized in that, The preservation solution is an aqueous solution containing the following components at the following concentrations: 4mM-10mM tetrasodium ethylenediaminetetraacetate, 8mM-16mM sodium citrate, 0.2wt%-5wt% Tween-20, 15wt%-25wt% structural stabilizer, 2wt%-5wt% amide solution, 1wt%-5wt% hemolysis inhibitor, 0.05wt%-1wt% reducing agent, and 0.5mM-1mM 3-morpholinopropanesulfonic acid.

3. The preservation solution as described in claim 1 or 2, characterized in that, The amide solution is selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylpropionamide, and N-methylpyrrolidone; and / or, The reducing agent is selected from at least one of N-acetylcysteine, tris(2-carbonylethyl)phosphohydrochloride, and water-soluble vitamins; and / or, The hemolytic inhibitor is selected from at least one of polyvinyl alcohol, polyvinylpyrrolidone, and dextran; and / or, The structural stabilizer is selected from at least one of sucrose, trehalose, galactose, lactose, and polyethylene glycol.

4. The preservation solution as described in claim 3, characterized in that, The preservation solution is an aqueous solution containing the following components at the following concentrations: 4 mM-8 mM tetrasodium ethylenediaminetetraacetate, 10 mM-16 mM sodium citrate, 0.2 wt%-2 wt% Tween-20, 15 wt%-20 wt% trehalose, 2 wt%-5 wt% N,N-dimethylformamide, 2 wt%-5 wt% polyvinylpyrrolidone, 0.05 wt%-0.5 wt% N-acetylcysteine, and 0.5 mM-1 mM 3-morpholinopropanesulfonic acid.

5. The preservation solution as described in claim 1, characterized in that, The preservation solution also contains preservatives; Preferably, the preservative is selected from ProClin. ® 300 and / or ProClin ® 950; and / or, The concentration of the preservative in the preservation solution is 0.05wt%-5wt%.

6. The preservation solution as described in claim 1, characterized in that, The pH value of the preservation solution is 5.5-7.2, preferably 5.5-6.

5.

7. The method for preparing the preservation solution according to any one of claims 1 to 6, characterized in that, Includes the following steps: Weigh each component according to the dosage, add them to water in sequence, dissolve them completely, mix well, adjust the pH value, and filter to obtain the final product.

8. The use of the preservation solution as described in any one of claims 1 to 6 in the transportation and / or preservation of virus samples.

9. A method for preserving a virus sample, characterized in that, The method includes the following steps: adding the preservation solution as described in any one of claims 1 to 6 to the virus sample and mixing them thoroughly.

10. The preservation method as described in claim 9, characterized in that, The volume ratio or mass-volume ratio of the virus sample to the preservation solution is 1:(1~4).

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