A virus stock solution and a method for preparing the same
The virus storage solution, composed of Tris-HCl, guanidine salt lysis agent, composite protectant and chelating agent, solves the problem of easy degradation of viral nucleic acid during storage, realizes rapid inactivation and stable storage of virus, and improves the sensitivity and accuracy of nucleic acid detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HUBEI UNIV OF TECH
- Filing Date
- 2025-03-12
- Publication Date
- 2026-05-22
AI Technical Summary
Existing viral storage solutions are prone to viral nucleic acid degradation during storage and transportation, leading to false negatives and insufficient sensitivity in test results. Furthermore, the production costs are high and the complex processes are not conducive to large-scale production.
A virus storage solution composed of Tris-HCl, guanidine salt lysis agent, composite protectant, chelating agent, and phenol red was used to achieve stable storage of viral nucleic acid by lysing viral cells with guanidine salt, maintaining pH buffer with Tris-HCl, forming nanomicelles to protect viral RNA with the composite protectant, inhibiting RNase activity with the chelating agent, and monitoring pH with phenol red indicator.
The virus is completely inactivated and lysed in a short time, the RNA is prevented from being degraded by nucleases, and the sample is stably stored at room temperature for 48 hours, which improves the sensitivity and accuracy of nucleic acid detection and reduces the risk of false negatives.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of virus storage technology, specifically to a virus storage solution and its preparation method. Background Technology
[0002] With the rapid development of molecular biology techniques, nucleic acid testing plays a crucial role in disease diagnosis, epidemiological investigation, and scientific research. Virus storage solutions, as a key component of nucleic acid testing, are used to protect the integrity of virus samples during collection, transportation, and storage, ensuring the accuracy of subsequent nucleic acid extraction and analysis.
[0003] The primary function of viral storage solutions is to maintain the stability of viral nucleic acids and prevent their degradation. Viral nucleic acids (DNA / RNA) are highly susceptible to degradation by nucleases in the natural environment. Viral storage solutions, by adding specific components (such as lysis salts, buffers, and nuclease inhibitors), can effectively inhibit nuclease activity and protect the integrity of the nucleic acids. Furthermore, viral storage solutions must be adaptable to different testing requirements. For example, in COVID-19 testing, inactivated viral storage solutions can rapidly lyse the virus and inactivate its infectivity, reducing the risk of infection for operators while protecting the nucleic acids from degradation.
[0004] In clinical testing, improper storage of virus samples can cause a variety of problems: the viral capsid is easily broken, releasing nucleic acid; and the samples contain a large number of enzymes that are prone to degradation and contamination, resulting in missed pathogen detection and false negatives due to insufficient sensitivity.
[0005] Viral nucleic acid testing remains the most effective method for detecting viruses. Shortening testing time, reducing testing steps, and improving accuracy and sensitivity remain areas for improvement. Current methods typically require RNA extraction followed by nucleic acid detection using methods such as qRT-PCR. The RNA extraction step increases the operational process and testing time, and also increases the risk of infection for testing personnel. Furthermore, the increased number of steps also increases the risk of false positives or false negatives.
[0006] Chinese patent document CN113736755A discloses a room-temperature-storable inactivated virus preservation solution and its preparation method. The raw materials of the preservation solution include: guanidine isothiocyanate 190-210 g / L; sodium citrate 2-3 g / L; sodium dodecyl sarcosinate 1.8-2.0 g / L; HAc-NaAc buffer 55-65 mL / L; ferrocene-glutathione 10-30 mg / L; and acid-base indicator 30-50 mg / L. This invention adds guanidine isothiocyanate, ferrocene-glutathione, and sodium dodecyl sarcosinate to the preservation solution. Through the combination of these components, rapid inactivation of the virus can be achieved, and the stability of the sample nucleic acid can be maintained. It can be stored at room temperature for a long time without degradation, improving the accuracy of nucleic acid detection. However, the preparation method of ferrocene-glutathione used in this patent is relatively complex, leading to high production costs and process difficulty, which is not conducive to the large-scale production and widespread application of this preservation solution. Summary of the Invention
[0007] The main objective of this invention is to provide a viral storage solution and its preparation method. The preparation process of the viral storage solution of this invention is simple, and the obtained viral storage solution can rapidly inactivate and lyse the collected virus. The RNA released into the solution can be prevented from being degraded by ribonuclease under the action of the storage solution, thereby achieving stable storage of samples containing viral nucleic acid. The viral storage solution of this invention can protect viral RNA samples, reduce the decomposition effect of RNAase on viral RNA, and improve the sensitivity of nucleic acid detection. It is particularly suitable for the collection of highly infectious epidemic virus samples.
[0008] To achieve the above objectives, the present invention proposes a virus storage solution comprising the following components: Tris-HCl 0.5-1 g / L, guanidine salt lysis agent 80-150 g / L, composite protectant 10-40 g / L, chelating agent 0.1-1 g / L, phenol red 0.005-0.1 g / L, and water as the balance.
[0009] More preferably, the virus storage solution comprises the following components: Tris-HCl 0.6-0.8 g / L, guanidine salt lysis agent 100-120 g / L, composite protectant 20-30 g / L, chelating agent 0.3-0.5 g / L, phenol red 0.03-0.05 g / L, and water as the balance.
[0010] Preferably, the guanidine salt cleavage agent is guanidine isothiocyanate and / or guanidine chloride; the guanidine salt cleavage agent has the functions of dissolving proteins and promoting cell disruption, and can also denature RNase, thereby protecting RNA molecules.
[0011] Preferably, the composite protective agent includes tea tree oil, hydrogenated lecithin, gallic acid, dithioerythritol, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
[0012] More preferably, the composite protective agent is prepared by mixing tea tree oil, hydrogenated lecithin, gallic acid, dithioerythritol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and glycerol until homogeneous to obtain the composite protective agent.
[0013] Preferably, the mass ratio of tea tree oil, hydrogenated lecithin, gallic acid, dithioerythritol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and glycerol is 1-3:0.3-0.8:0.5-1.5:1.2-2:0.1-0.5:20.
[0014] Preferably, the chelating agent is at least one selected from ethylenediaminetetraacetic acid, citric acid, ethylene glycol diethyl ether diaminetetraacetic acid, and trisodium hypotriacetate monohydrate;
[0015] More preferably, the chelating agent is ethylenediaminetetraacetic acid (EDTA); the chelating agent can inhibit the reactivity of RNAase, prolong the storage time of RNA, and improve the sensitivity of detection.
[0016] Preferably, the method for preparing the virus storage solution of the present invention is as follows:
[0017] Tris-HCl, guanidine salt lysis agent, composite protectant, chelating agent, phenol red, and water are mixed and stirred evenly, then dispensed and stored to obtain the virus storage solution.
[0018] The viral capsid is composed of proteins or lipoproteins. Under the action of guanidine salt lysis agents, cells lyse, proteins denature, and viral nucleic acid is released. Tris-HCl maintains a pH buffer environment, keeping the released nucleic acid stable. A composite protectant effectively protects viral RNA nucleic acid from degradation, achieving stable storage of samples containing viral nucleic acid. Among the composite protectants, tea tree oil and hydrogenated lecithin can form a nano-micelle protective layer on the nucleic acid surface, inhibiting the proliferation of living and contaminating microorganisms and reducing the risk of false negatives. Gallic acid can bind to nucleic acid molecules through π-π stacking, enhancing their stability and clearing environmental contaminants. The oxidizing factor, erythritol, can reduce the disulfide bonds in RNAase, further disrupting the RNAase structure and extending sample storage time. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide can prevent excessive cross-linking of nucleic acids. Its addition has a good shaping effect on the higher-order structure of nucleic acids, making the storage capacity of the virus storage solution stronger. Gallic acid, erythritol, tea tree oil, hydrogenated lecithin, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide form a synergistic defense, effectively protecting the integrity of viral nucleic acids and ensuring the stability and safety of samples during storage and transportation.
[0019] The present invention also provides the application of the virus storage solution, the applicable samples of which include respiratory extracts, in vitro samples of human tissues, and in vitro samples of animal and plant tissues.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0021] 1) The virus storage solution disclosed in this invention is an inactivating type, which can completely inactivate and lyse the virus in a short time. The RNA released into the solution can be prevented from being degraded by ribonuclease under the action of the storage solution, thereby achieving stable storage of samples containing viral nucleic acid. The virus storage solution of this invention can protect viral RNA samples, reduce the decomposition effect of RNAase on viral RNA, and improve the sensitivity of nucleic acid detection.
[0022] 2) This invention adds a composite protectant, which can effectively protect viral RNA nucleic acid from degradation, and achieve stable storage of samples containing viral nucleic acid. Even under the interference of human skin cells, it can still achieve relatively accurate determination within 24 hours, with virtually no false negatives.
[0023] 3) The virus preservation solution disclosed in this invention allows the preserved samples to be stored at room temperature without the need for storage at -20°C or lower, and the nucleic acids therein can remain stable within 48 hours. Detailed Implementation
[0024] To avoid unnecessary details, unless otherwise specified, all items used in the following examples are commercially available products, and all methods used are conventional methods unless otherwise specified.
[0025] Example 1
[0026] A virus storage solution, the preparation method of which is as follows:
[0027] Mix 0.7g Tris-HCl, 110g guanidine isothiocyanate, 25g composite protectant, 0.4g ethylenediaminetetraacetic acid, 0.04g phenol red, and 600mL purified water until homogeneous, then bring the volume to 1L. The mixture is then dispensed and stored to obtain the virus storage solution.
[0028] The composite protective agent is prepared by mixing and stirring 20g of tea tree oil, 5g of hydrogenated lecithin, 10g of gallic acid, 15g of dithioerythritol, 3g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 200g of glycerol until homogeneous.
[0029] Example 2
[0030] A virus storage solution, the preparation method of which is as follows:
[0031] Mix 0.6g Tris-HCl, 100g guanidine isothiocyanate, 20g composite protective agent, 0.3g ethylenediaminetetraacetic acid, 0.03g phenol red, and 600mL purified water until homogeneous, then bring the volume to 1L. The mixture is then dispensed and stored to obtain the virus storage solution.
[0032] The composite protective agent is prepared by mixing and stirring 10g of tea tree oil, 3g of hydrogenated lecithin, 5g of gallic acid, 12g of dithioerythritol, 1g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 200g of glycerol until homogeneous.
[0033] Example 3
[0034] A virus storage solution, the preparation method of which is as follows:
[0035] Mix 0.8g Tris-HCl, 120g guanidine isothiocyanate, 30g composite protectant, 0.3g ethylenediaminetetraacetic acid, 0.05g phenol red, and 600mL purified water until homogeneous, then bring the volume to 1L. The mixture is then dispensed and stored to obtain the virus storage solution.
[0036] The composite protective agent is a mixture of tea tree oil, hydrogenated lecithin, gallic acid, dithioerythritol, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a mass ratio of 3:0.8:1.5:2:0.5.
[0037] The composite protective agent is prepared by mixing and stirring 30g of tea tree oil, 8g of hydrogenated lecithin, 15g of gallic acid, 20g of dithioerythritol, 5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 200g of glycerol until homogeneous.
[0038] Comparative Example 1
[0039] A virus storage solution, prepared in a method similar to that of Example 1, differs in that gallic acid is not added to the composite protectant, as detailed below:
[0040] Mix 0.7g Tris-HCl, 110g guanidine isothiocyanate, 25g composite protectant, 0.4g ethylenediaminetetraacetic acid, 0.04g phenol red, and 600mL purified water until homogeneous, then bring the volume to 1L. The mixture is then dispensed and stored to obtain the virus storage solution.
[0041] The composite protective agent is prepared by mixing and stirring 20g of tea tree oil, 5g of hydrogenated lecithin, 15g of dithioerythritol, 3g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 200g of glycerol until homogeneous.
[0042] Comparative Example 2
[0043] A virus storage solution, prepared in a method similar to that of Example 1, differs in that dithioerythritol is not added to the composite protectant, as detailed below:
[0044] Mix 0.7g Tris-HCl, 110g guanidine isothiocyanate, 25g composite protectant, 0.4g ethylenediaminetetraacetic acid, 0.04g phenol red, and 600mL purified water until homogeneous, then bring the volume to 1L. The mixture is then dispensed and stored to obtain the virus storage solution.
[0045] The composite protective agent is prepared by mixing and stirring 20g of tea tree oil, 5g of hydrogenated lecithin, 10g of gallic acid, 3g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 200g of glycerol until homogeneous.
[0046] Comparative Example 3
[0047] A virus storage solution, prepared in a method similar to that of Example 1, differs in that tea tree oil is not added to the composite protectant, as detailed below:
[0048] Mix 0.7g Tris-HCl, 110g guanidine isothiocyanate, 25g composite protectant, 0.4g ethylenediaminetetraacetic acid, 0.04g phenol red, and 600mL purified water until homogeneous, then bring the volume to 1L. The mixture is then dispensed and stored to obtain the virus storage solution.
[0049] The composite protective agent is prepared by mixing and stirring 25g hydrogenated lecithin, 10g gallic acid, 15g dithioerythritol, 3g 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 200g glycerol until homogeneous.
[0050] Comparative Example 4
[0051] A virus storage solution, prepared in a method similar to that of Example 1, differs in that hydrogenated lecithin is not added to the composite protectant, as detailed below:
[0052] Mix 0.7g Tris-HCl, 110g guanidine isothiocyanate, 25g composite protectant, 0.4g ethylenediaminetetraacetic acid, 0.04g phenol red, and 600mL purified water until homogeneous, then bring the volume to 1L. The mixture is then dispensed and stored to obtain the virus storage solution.
[0053] The composite protective agent is prepared by mixing and stirring 25g of tea tree oil, 10g of gallic acid, 15g of dithioerythritol, 3g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 200g of glycerol until homogeneous.
[0054] Comparative Example 5
[0055] A virus storage solution, prepared in a method similar to that of Example 1, differs in that 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is not added to the composite protective agent, as detailed below:
[0056] Mix 0.7g Tris-HCl, 110g guanidine isothiocyanate, 25g composite protectant, 0.4g ethylenediaminetetraacetic acid, 0.04g phenol red, and 600mL purified water until homogeneous, then bring the volume to 1L. The mixture is then dispensed and stored to obtain the virus storage solution.
[0057] The composite protective agent is prepared by mixing and stirring 20g of tea tree oil, 5g of hydrogenated lecithin, 10g of gallic acid, 15g of dithioerythritol and 200g of glycerol until homogeneous.
[0058] Performance testing
[0059] For the virus storage solutions prepared in Examples 1-3 and Comparative Examples 1-5, the COVID-2019 pseudovirus purchased from novoprotein was diluted to a final concentration of 10⁵ copies / mL using the above virus storage solutions. After being stored for 24 h and 48 h according to the experimental method described below, RT-PCR experiments were performed, using primers ORF1ab and primer N, and the Ct value was measured.
[0060] In the RT-PCR experiment, the samples were amplified using the reaction system and reaction procedure shown in Table 1.
[0061] Table 1. Reaction system and reaction procedure for RT-PCR experiments.
[0062]
[0063]
[0064] In the above reaction, the FAM and VIC channels were selected for detection, and the detection data were retained to obtain the Ct values of the sample for the ORF1ab and N genes. If the CT value of each channel is ≤34 and the amplification curve is a typical "S" shape, the corresponding target gene detection result is positive. If there is no obvious amplification curve in the FAM and VIC channels, or the Ct value is >40, the corresponding target gene detection result is negative; if other situations occur, retesting is required.
[0065] If a sample that was originally positive receives a negative result during testing, the sample is recorded as a "false negative" sample.
[0066] The storage effect of the above-mentioned sample storage solution was determined by the following comparative experiments.
[0067] Experiment 1, RNase tolerance test: Human epithelial cells (purchased from Mingzhou Biotechnology, catalog number MZ-M0498) were added to the above-mentioned sample storage solution containing COVID-19 pseudovirus. Twenty samples were used in each sample storage solution. The samples were stored at room temperature and parallel experiments were conducted. The number of negative samples was recorded after 24 h and 48 h. The test results are shown in Table 2.
[0068] Table 2 Performance test results of the storage fluid
[0069]
[0070]
[0071] As can be seen from the data in Table 2, the viral RNA stored in the viral storage solution prepared in this application can still achieve relatively accurate detection within 24 hours even under the interference of human skin cells, with virtually no false negatives.
[0072] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.
Claims
1. A virus storage solution, characterized in that, It consists of the following components: Tris-HCl 0.5-1 g / L, guanidine salt cleavage agent 80-150 g / L, composite protective agent 10-40 g / L, chelating agent 0.1-1 g / L, phenol red 0.005-0.1 g / L, and water balance; The composite protective agent is composed of the following components: tea tree oil, hydrogenated lecithin, gallic acid, dithioerythritol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and glycerol; The guanidine salt cleavage agent is guanidine isothiocyanate; The mass ratio of tea tree oil, hydrogenated lecithin, gallic acid, dithioerythritol, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1-3:0.3-0.8:0.5-1.5:1.2-2:0.1-0.5; The chelating agent is ethylenediaminetetraacetic acid; The virus in question is COVID-19.
2. The virus storage solution according to claim 1, characterized in that, It consists of the following components: Tris-HCl 0.6-0.8 g / L, guanidine salt cleavage agent 100-120 g / L, composite protective agent 20-30 g / L, chelating agent 0.3-0.5 g / L, phenol red 0.03-0.05 g / L, and water balance.
3. The virus storage solution according to claim 1, characterized in that, The composite protective agent is prepared as follows: tea tree oil, hydrogenated lecithin, gallic acid, dithioerythritol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and glycerol are mixed and stirred evenly to obtain the composite protective agent.
4. A method for preparing the virus storage solution according to any one of claims 1-3, characterized in that, The process includes the following steps: mixing Tris-HCl, guanidine salt lysis agent, composite protectant, chelating agent, phenol red, and water until homogeneous, then dispensing and storing the mixture to obtain the virus storage solution.
5. The use of the virus storage solution according to any one of claims 1-3 in the storage of samples containing viral nucleic acid, characterized in that: The virus is COVID-19, and the applicable samples for the virus storage fluid include ex vivo samples of animal tissue.
6. The application of the virus storage solution according to any one of claims 1-3 in the storage of samples containing viral nucleic acid, characterized in that: The virus is COVID-19, and the applicable samples for the virus storage fluid include ex vivo samples of human tissue.
7. The application of the virus storage solution according to any one of claims 1-3 in the storage of samples containing viral nucleic acid, characterized in that: The virus is COVID-19, and the applicable samples for the virus storage fluid include respiratory extracts.