Virus storage liquid and preparation method thereof

Abstract

The invention discloses a virus storage solution and a preparation method thereof, and belongs to the technical field of virus storage. The virus storage solution prepared by the invention can be used for quickly inactivating and cracking collected viruses, and RNA (Ribonucleic Acid) dissociating into the solution can be prevented from being degraded by ribonuclease under the action of the storage solution, so that a sample containing viral nucleic acid is stably stored; the virus storage liquid can protect a virus RNA sample, reduce the decomposition effect of RNAase on the virus RNA and improve the sensitivity of nucleic acid detection, and is particularly suitable for collecting an epidemic virus sample with strong infectivity.

Description

Technical Field

[0001] The present invention relates to the technical field of virus storage, and particularly relates to a virus storage solution and a preparation method thereof. Background Art

[0002] With the rapid development of molecular biology technology, nucleic acid detection plays a crucial role in disease diagnosis, epidemiological investigation, and scientific research. As one of the key links in nucleic acid detection, virus storage solution is 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 main function of virus storage solution is to maintain the stability of virus nucleic acid and prevent its degradation. Virus nucleic acid (DNA / RNA) is extremely easy to be degraded by nucleases in the natural environment. By adding specific components (such as lysis salts, buffers, nuclease inhibitors, etc.), virus storage solution can effectively inhibit the activity of nucleases and protect the integrity of nucleic acid. In addition, virus storage solution also needs to adapt to different detection requirements. For example, in the detection of novel coronavirus, inactivated virus storage solution can quickly lyse the virus and inactivate its infectivity, reducing the infection risk of operators while protecting nucleic acid from degradation.

[0004] In clinical testing work, various problems are caused by improper storage of virus samples: the virus outer shell is easy to rupture, and the released nucleic acid is easily degraded and contaminated due to the large amount of enzyme substances in the sample, resulting in problems such as missed detection of pathogens and insufficient sensitivity leading to false negatives.

[0005] Virus nucleic acid detection remains the most effective detection method for viruses at present. Shortening the detection time, reducing the detection steps, and improving the detection accuracy and sensitivity are still the directions that need to be worked on currently. The current common detection methods usually require the extraction of RNA first, and then nucleic acid detection based on methods such as qRT-PCR. The existence of the RNA extraction step will increase the operation process and detection time, and also increase the risk of virus infection for the testers. At the same time, the increase in operation steps will also increase the risk of false positives or false negatives in the detection results.

[0006] Chinese Patent Document CN113736755A discloses an inactivated virus preservation solution that can be stored at room temperature 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 lauroyl sarcosinate 1.8 - 2.0 g / L; HAc-NaAc buffer solution 55 - 65 mL / L; ferrocene-glutathione 10 - 30 mg / L; acid-base indicator 30 - 50 mg / L. In the present invention, guanidine isothiocyanate, ferrocene-glutathione and sodium lauroyl sarcosinate are added to the preservation solution. Through the compounding of each component, 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. The preparation method of ferrocene-glutathione used in this patent is relatively complex, resulting in high production costs and great process difficulty, which is not conducive to the large-scale production and popularization of this preservation solution. Summary of the Invention

[0007] The main object of the present invention is to propose a virus storage solution and its preparation method. The preparation process of the virus storage solution of the present invention is simple, and the prepared virus storage solution can rapidly inactivate and lyse the collected virus. The RNA free in the solution can be prevented from being degraded by ribonuclease under the action of the storage solution, thereby realizing the stable storage of the sample containing virus nucleic acid. The virus storage solution of the present invention can protect the virus RNA sample, reduce the decomposition of RNAase on the virus RNA, improve the sensitivity of nucleic acid detection, and is particularly suitable for the collection of highly infectious epidemic virus samples.

[0008] To achieve the above object, the present invention proposes a virus storage solution, which includes the following components: Tris-HCl 0.5 - 1 g / L, guanidine salt lysing agent 80 - 150 g / L, composite protecting agent 10 - 40 g / L, chelating agent 0.1 - 1 g / L, phenol red 0.005 - 0.1 g / L, and the balance is water.

[0009] Further preferably, the virus storage solution includes the following components: Tris-HCl 0.6 - 0.8 g / L, guanidine salt lysing agent 100 - 120 g / L, composite protecting agent 20 - 30 g / L, chelating agent 0.3 - 0.5 g / L, phenol red 0.03 - 0.05 g / L, and the balance is water.

[0010] Preferably, the guanidine salt lysing agent is guanidine isothiocyanate and / or guanidine chloride; the guanidine salt lysing agent has the functions of dissolving proteins, promoting cell breakage, and also can denature RNAase, thereby playing a role in protecting RNA molecules.

[0011] Preferably, the composite protecting agent includes tea tree oil, hydrogenated lecithin, gallic acid, dithiothreitol and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0012] Further preferably, the preparation method of the composite protective agent is as follows: Mix tea tree oil, hydrogenated lecithin, gallic acid, dithiothreitol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and glycerol evenly by stirring to obtain the composite protective agent.

[0013] Preferably, the mass ratio of the tea tree oil, hydrogenated lecithin, gallic acid, dithiothreitol, 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 of ethylenediaminetetraacetic acid, citric acid, ethylene glycol diethyl ether diamine tetraacetic acid, and nitrilotriacetic acid trisodium salt monohydrate;

[0015] Further preferably, the chelating agent is ethylenediaminetetraacetic acid; The chelating agent can inhibit the reaction activity of RNAase, extend the preservation time of RNA, and improve the detection sensitivity at the same time.

[0016] Preferably, the preparation method of the virus storage solution of the present invention is as follows:

[0017] Mix Tris-HCl, guanidine salt lysing agent, composite protective agent, chelating agent, phenol red, and water evenly by stirring, and store them in aliquots to obtain the virus storage solution.

[0018] The outer shell of the virus is composed of protein or lipoprotein. Under the action of the guanidine salt lysing agent, the cells are lysed, the protein is denatured, and the viral nucleic acid is released; Tris-HCl maintains the pH buffer environment and keeps the released nucleic acid stable; The composite protective agent can effectively protect the viral RNA nucleic acid from degradation, realize the stable storage of the sample containing viral nucleic acid. Among them, tea tree oil and hydrogenated lecithin in the composite protective agent can form a nano micelle protective layer on the surface of the nucleic acid, inhibit the proliferation of living microorganisms and contaminated microorganisms, reduce the risk of false negatives. Gallic acid can bind to nucleic acid molecules through π-π stacking, enhance its stability and remove oxidation factors in the environment. Dithiothreitol can reduce the disulfide bond in RNAase, further destroy the structure of RNAase, and extend the sample preservation time. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide can avoid excessive cross-linking of nucleic acid, and has a good shaping effect on the higher-order structure of nucleic acid after addition, making the storage capacity of the virus storage solution stronger. Gallic acid, dithiothreitol, tea tree oil, hydrogenated lecithin, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide form a synergistic defense to effectively protect the integrity of viral nucleic acid and ensure the stability and safety of the sample during storage and transportation.

[0019] The present invention also provides an application of the virus storage solution. The applicable samples of the virus storage solution include respiratory extracts, ex vivo samples of human tissues, and ex vivo 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 by the present invention belongs to the inactivated type. The virus can be completely inactivated and lysed in a short time, and the RNA released into the solution can be prevented from being degraded by ribonuclease under the action of the storage solution, so as to realize the stable storage of samples containing virus nucleic acid. The virus storage solution of the present invention can protect the virus RNA sample, reduce the decomposition effect of RNAase on the virus RNA, and improve the sensitivity of nucleic acid detection.

[0022] 2) By adding a composite protective agent, the composite protective agent can effectively protect the virus RNA nucleic acid from degradation, realize the stable storage of samples containing virus nucleic acid, and still be able to achieve a relatively accurate determination within 24 hours under the interference of human skin cells, with basically no false negative phenomenon.

[0023] 3) The virus preservation solution disclosed by the present invention can store the preserved samples at room temperature without the need to store them at -20°C or lower, and the nucleic acid therein can remain stable within 48 hours. Detailed implementation manners

[0024] To avoid unnecessary repetition, unless otherwise specified, the items used in the following examples are all commercially available products, and the methods used are all conventional methods unless otherwise specified.

[0025] Example 1

[0026] A virus storage solution, and its preparation method is as follows:

[0027] Mix 0.7 g of Tris-HCl, 110 g of guanidine isothiocyanate, 25 g of composite protective agent, 0.4 g of ethylenediaminetetraacetic acid, 0.04 g of phenol red, and 600 mL of pure water evenly, then make up the volume to 1 L, and then store them in aliquots to obtain the virus storage solution.

[0028] The preparation method of the composite protective agent is: mix 20 g of tea tree oil, 5 g of hydrogenated lecithin, 10 g of gallic acid, 15 g of dithiothreitol, 3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 200 g of glycerol evenly to obtain the composite protective agent.

[0029] Example 2

[0030] A virus storage solution, and its preparation method is as follows:

[0031] Mix 0.6 g of Tris-HCl, 100 g of guanidine isothiocyanate, 20 g of compound protective agent, 0.3 g of ethylenediaminetetraacetic acid, 0.03 g of phenol red, and 600 mL of pure water, stir evenly, make up the volume to 1 L, and then store in aliquots to obtain the virus storage solution.

[0032] The preparation method of the compound protective agent is as follows: Mix 10 g of tea tree oil, 3 g of hydrogenated lecithin, 5 g of gallic acid, 12 g of dithiothreitol, 1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 200 g of glycerol evenly to obtain the compound protective agent.

[0033] Example 3

[0034] A virus storage solution, and its preparation method is as follows:

[0035] Mix 0.8 g of Tris-HCl, 120 g of guanidine isothiocyanate, 30 g of compound protective agent, 0.3 g of ethylenediaminetetraacetic acid, 0.05 g of phenol red, and 600 mL of pure water, stir evenly, make up the volume to 1 L, and then store in aliquots to obtain the virus storage solution.

[0036] The compound protective agent is a mixture of tea tree oil, hydrogenated lecithin, gallic acid, dithiothreitol, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide with a mass ratio of 3:0.8:1.5:2:0.5.

[0037] The preparation method of the compound protective agent is as follows: Mix 30 g of tea tree oil, 8 g of hydrogenated lecithin, 15 g of gallic acid, 20 g of dithiothreitol, 5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 200 g of glycerol evenly to obtain the compound protective agent.

[0038] Comparative Example 1

[0039] A virus storage solution, the preparation method of which is similar to that of Example 1, the difference is that gallic acid is not added to the compound protective agent, and the specific method is as follows:

[0040] Mix 0.7 g of Tris-HCl, 110 g of guanidine isothiocyanate, 25 g of compound protective agent, 0.4 g of ethylenediaminetetraacetic acid, 0.04 g of phenol red, and 600 mL of pure water, stir evenly, make up the volume to 1 L, and then store in aliquots to obtain the virus storage solution.

[0041] The preparation method of the compound protective agent is as follows: Mix 20 g of tea tree oil, 5 g of hydrogenated lecithin, 15 g of dithiothreitol, 3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 200 g of glycerol evenly to obtain the compound protective agent.

[0042] Comparative Example 2

[0043] A virus storage solution, the preparation method of which is similar to that of Example 1, except that dithiothreitol is not added to the composite protective agent, specifically as follows:

[0044] Mix 0.7 g of Tris-HCl, 110 g of guanidine isothiocyanate, 25 g of composite protective agent, 0.4 g of ethylenediaminetetraacetic acid, 0.04 g of phenol red, and 600 mL of pure water, stir evenly, make up the volume to 1 L, and then store in aliquots to obtain the virus storage solution.

[0045] The preparation method of the composite protective agent is: mix 20 g of tea tree oil, 5 g of hydrogenated lecithin, 10 g of gallic acid, 3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 200 g of glycerol evenly to obtain the composite protective agent.

[0046] Comparative Example 3

[0047] A virus storage solution, the preparation method of which is similar to that of Example 1, except that tea tree oil is not added to the composite protective agent, specifically as follows:

[0048] Mix 0.7 g of Tris-HCl, 110 g of guanidine isothiocyanate, 25 g of composite protective agent, 0.4 g of ethylenediaminetetraacetic acid, 0.04 g of phenol red, and 600 mL of pure water, stir evenly, make up the volume to 1 L, and then store in aliquots to obtain the virus storage solution.

[0049] The preparation method of the composite protective agent is: mix 25 g of hydrogenated lecithin, 10 g of gallic acid, 15 g of dithiothreitol, 3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 200 g of glycerol evenly to obtain the composite protective agent.

[0050] Comparative Example 4

[0051] A virus storage solution, the preparation method of which is similar to that of Example 1, except that hydrogenated lecithin is not added to the composite protective agent, specifically as follows:

[0052] Mix 0.7 g of Tris-HCl, 110 g of guanidine isothiocyanate, 25 g of composite protective agent, 0.4 g of ethylenediaminetetraacetic acid, 0.04 g of phenol red, and 600 mL of pure water, stir evenly, make up the volume to 1 L, and then store in aliquots to obtain the virus storage solution.

[0053] The preparation method of the composite protective agent is: mix 25 g of tea tree oil, 10 g of gallic acid, 15 g of dithiothreitol, 3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 200 g of glycerol evenly to obtain the composite protective agent.

[0054] Comparative Example 5

[0055] A virus storage solution was prepared in a similar manner to Example 1, except that 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was not added to the composite protective agent, and the specific preparation method was as follows:

[0056] 0.7 g of Tris-HCl, 110 g of guanidine isothiocyanate, 25 g of composite protective agent, 0.4 g of ethylenediaminetetraacetic acid, 0.04 g of phenol red, and 600 mL of pure water were mixed and stirred evenly, then made up to 1 L, and then aliquoted and stored to obtain the virus storage solution.

[0057] The preparation method of the composite protective agent was: 20 g of tea tree oil, 5 g of hydrogenated lecithin, 10 g of gallic acid, 15 g of dithiothreitol, and 200 g of glycerol were mixed and stirred evenly to obtain the composite protective agent.

[0058] Performance test

[0059] For the virus storage solutions prepared in Examples 1-3 and Comparative Examples 1-5, the above virus storage solution was used to dilute the COVID-2019 pseudovirus purchased from novoprotein to a final concentration of 105 copies / mL. After storing for 24 h and 48 h according to the following experimental method, RT-PCR experiments were carried out. Primers ORF1ab and primer N were selected, and their Ct values were measured.

[0060] In the RT-PCR experiment, the samples were amplified through the reaction system and reaction program shown in Table 1.

[0061] Table 1 Reaction system and reaction program in RT-PCR experiment

[0062]

[0063]

[0064] In the above reaction, the FAM channel and the VIC channel were selected for detection, and the detection data were retained to obtain the Ct values of the samples for the ORF1ab and N genes. If the CT value of each channel ≤ 34 and the amplification curve is a typical "S" shape, the detection result of the corresponding target gene is positive. If there is no obvious amplification curve in the FAM and VIC channels, or the Ct value > 40, the detection result of the corresponding target gene is negative; if other situations occur, re-detection is required.

[0065] If a sample that was originally positive gives a negative result during detection, the sample is recorded as a "false negative" sample.

[0066] For the above sample storage solution, its storage effect was determined through the following experimental comparison.

[0067] Experiment 1, RNAase tolerance experiment: Human epithelial cells (purchased from Mingzhou Bio, product number MZ-M0498) were added to the above sample storage solution containing COVID-19 pseudovirus. There were twenty samples in each group of sample storage solutions, which were stored at room temperature for parallel experiments. The number of negatives measured after the samples were placed for 24 h and 48 h was recorded. The test results are shown in Table 2:

[0068] Table 2 Performance test results of the storage solution

[0069]

[0070]

[0071] It can be seen from the data in Table 2 that the virus storage solution prepared by the present application can store viral RNA and still achieve relatively accurate measurement within 24 h under the interference of human skin cells, with basically no false negative phenomenon.

[0072] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A virus storage solution, characterized in that: The invention comprises the following components: Tris-HCl 0.5-1g / L, guanidine salt lysing agent 80-150g / L, composite protective agent 10-40g / L, chelating agent 0.1-1g / L, phenol red 0.005-0.1g / L and the balance of water.

2. The virus storage solution according to claim 1, characterized in that The invention comprises the following components: 0.6-0.8 g / L of Tris-HCl, 100-120 g / L of a guanidine salt lysing agent, 20-30 g / L of a composite protective agent, 0.3-0.5 g / L of a chelating agent, 0.03-0.05 g / L of phenol red and the balance of water.

3. The virus storage solution according to claim 1, characterized in that: The guanidine salt lysing agent is guanidine isothiocyanate and / or guanidine chloride.

4. The virus storage solution according to claim 1, characterized in that: The composite protective agent comprises tea tree oil, hydrogenated lecithin, gallic acid, dithioerythritol and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

5. The virus storage solution according to claim 4, characterized in that: The mass ratio of the 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.

6. The virus storage solution according to claim 4, characterized in that The preparation method of the composite protective agent is 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.

7. The virus storage solution according to claim 1, characterized in that: The chelating agent is at least one of ethylenediaminetetraacetic acid, citric acid, ethylene glycol diethyl ether diaminetetraacetic acid, and nitrilotriacetic acid trisodium salt monohydrate.

8. The virus storage solution according to claim 7, characterized in that: The chelating agent is ethylenediaminetetraacetic acid.

9. A method for preparing the virus storage solution according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: mixing Tris-HCl, a guanidine salt lysing agent, a composite protective agent, a chelating agent, phenol red and water, stirring the mixture evenly, and packaging the mixture for storage to obtain the virus storage solution.

10. A use of the virus storage solution according to any one of claims 1 to 8, characterized in that: Applicable samples of the virus storage solution include respiratory tract extracts, ex vivo samples of human tissues, and ex vivo samples of animal and plant tissues.

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