RNA (Ribonucleic Acid) protective agent as well as preparation method and application thereof

Abstract

The invention relates to an RNA (Ribonucleic Acid) protective agent as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention provides an RNA (Ribonucleic Acid) protective agent. The RNA protective agent is prepared from beta-mercaptoethanol, guanidine hydrochloride, graphene oxide, carboxymethyl chitosan, glycine, ethylenediamine tetraacetic acid, sodium citrate, glycerol and water. According to the RNA protective agent disclosed by the invention, all components have synergy, and the graphene oxide and the carboxymethyl chitosan react to form a compound, so that the adsorption and protection capabilities of the graphene oxide on nucleic acid are further enhanced; then beta-mercaptoethanol, guanidine hydrochloride, glycine, ethylenediaminetetraacetic acid, sodium citrate and glycerol are matched, so that the protection effect of the protective agent on nucleic acid can be remarkably improved, and a new thought is provided for nucleic acid protection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an RNA protectant, its preparation method, and its application. Background Technology

[0002] RNA viruses are a class of viruses that use ribonucleic acid (RNA) as their genetic material. They are widely distributed in the biological world, with hosts including humans, animals, plants, and microorganisms. Their biological characteristics differ significantly from those of DNA viruses, with core features concentrated in their genome structure, replication mechanism, and adaptive evolution.

[0003] RNA viruses are major pathogens of serious infectious diseases in humans, animals, and plants. Common human RNA viruses include influenza virus, SARS-CoV-2, measles virus, poliovirus, and hepatitis C virus. These viruses are mostly transmitted through the respiratory tract, digestive tract, or bodily fluids. Their pathogenic mechanisms are often related to the massive replication of the virus within host cells, leading to cell damage and triggering an immune inflammatory response. Among animal RNA viruses, classical swine fever virus, porcine epidemic diarrhea virus, and porcine rotavirus pose serious threats to the livestock industry. Plant RNA viruses (such as tobacco mosaic virus and rice stripe virus) can cause crop yield reduction or even crop failure. Meanwhile, RNA viruses have wide applications in scientific research, medicine, agriculture, and industry. Their unique genomic characteristics and replication mechanisms provide crucial support for biotechnology innovation and disease control. In the scientific research field, as ideal models with small genomes, rapid replication, and high mutation rates, RNA viruses (such as tobacco mosaic virus and influenza virus) facilitate viral entry... Research on the mechanisms of host-pathogen interaction and gene function has provided important tools for elucidating cross-species transmission patterns and adaptive evolutionary pathways. In the medical field, key antigens (such as spike proteins and capsid proteins) have become core targets for vaccine development, driving the successful launch of multiple vaccines, including live attenuated influenza vaccines, COVID-19 mRNA vaccines, inactivated / attenuated polio vaccines, and live attenuated measles vaccines. In agriculture, plant RNA viruses can be modified into transient expression vectors for the rapid production of medicinal proteins and vaccines. Furthermore, based on RNA interference (RNAi) technology targeting viral genes, transgenic crops resistant to rice stripe virus and potato virus Y have been successfully cultivated, effectively reducing agricultural disease losses. In industry, genetically engineered RNA virus particles, with their unique nanoscale structure and biological characteristics, have developed into a highly modular and powerful nanocarrier platform. However, in the application of RNA, due to its fragile single-stranded structure and susceptibility to degradation by RNases, RNA viruses generally exhibit weak stability in the environment. While conventional disinfection methods (such as alcohol, chlorine-containing disinfectants, and heating to 56°C) can rapidly destroy their nucleic acid structure and achieve inactivation, this characteristic also presents challenges for sample detection—nucleic acid degradation must be prevented through methods such as cryopreservation and the addition of RNase inhibitors, but current methods have many limitations. Therefore, providing RNA preservatives that can effectively prevent RNA degradation without affecting subsequent detection results is crucial for the molecular biological detection of RNA and represents a pressing technical problem that needs to be solved in RNA preservation. Summary of the Invention

[0004] The purpose of this invention is to provide an RNA protectant, its preparation method, and its application, so as to solve the many limitations of existing RNA protection methods and products.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an RNA protectant, which comprises the following components in parts by weight: 10-20 parts β-mercaptoethanol, 11-21 parts guanidine hydrochloride, 4-12 parts graphene oxide, 1-10 parts carboxymethyl chitosan, 5-15 parts glycine, 5-15 parts ethylenediaminetetraacetic acid, 4-12 parts sodium citrate, 1-10 parts glycerol and 50-150 parts water.

[0006] Preferably, the RNA protectant comprises the following components in parts by weight: 13-17 parts β-mercaptoethanol, 14-18 parts guanidine hydrochloride, 6-10 parts graphene oxide, 3-7 parts carboxymethyl chitosan, 8-12 parts glycine, 8-12 parts ethylenediaminetetraacetic acid, 6-10 parts sodium citrate, 3-7 parts glycerol and 80-120 parts water.

[0007] Preferably, the RNA protectant comprises the following components in parts by weight: 15 parts β-mercaptoethanol, 16 parts guanidine hydrochloride, 8 parts graphene oxide, 5 parts carboxymethyl chitosan, 10 parts glycine, 10 parts ethylenediaminetetraacetic acid, 8 parts sodium citrate, 5 parts glycerol and 100 parts water.

[0008] Preferably, the pH value of the RNA protectant is 6.6 to 8.6.

[0009] This invention provides a method for preparing the aforementioned RNA protectant, comprising the following steps: Graphene oxide and carboxymethyl chitosan were dissolved in water, sonicated for 25-35 minutes, stirred for 1-3 hours, and then β-mercaptoethanol, guanidine hydrochloride, glycine, ethylenediaminetetraacetic acid, sodium citrate and glycerol were added to adjust the pH value to obtain an RNA protectant.

[0010] Preferably, the temperature of the ultrasound is 25~35℃ and the power of the ultrasound is 100~200W.

[0011] Preferably, the stirring speed is 100~200 rpm.

[0012] This invention provides the application of the RNA protectant described herein or the RNA protectant prepared by the method described herein in maintaining the stability of RNA in a sample.

[0013] This invention provides a method for protecting viral samples, which involves mixing and preserving the viral sample with an RNA preservative. The storage temperature is -80~37℃; The RNA protectant is the RNA protectant described above or the RNA protectant prepared by the preparation method described above.

[0014] Preferably, the volume ratio of the virus sample to the RNA protectant is 2-4:1-3.

[0015] The present invention has the following technical effects and advantages: This application provides an RNA protectant in which both graphene oxide and carboxymethyl chitosan can physically adsorb nucleic acids to protect them. This application allows graphene oxide and carboxymethyl chitosan to react under certain conditions to obtain a complex, which further enhances the adsorption and protection of nucleic acids. Then, when combined with β-mercaptoethanol, guanidine hydrochloride, glycine, ethylenediaminetetraacetic acid, sodium citrate, and glycerol, the protective effect of the protectant on nucleic acids can be significantly improved, providing a new approach to nucleic acid protection methods. Detailed Implementation

[0016] This invention provides an RNA protectant, which comprises the following components in parts by weight: 10-20 parts β-mercaptoethanol, 11-21 parts guanidine hydrochloride, 4-12 parts graphene oxide, 1-10 parts carboxymethyl chitosan, 5-15 parts glycine, 5-15 parts ethylenediaminetetraacetic acid, 4-12 parts sodium citrate, 1-10 parts glycerol and 50-150 parts water; The β-mercaptoethanol is preferably 13 to 17 parts by mass, and more preferably 15 parts by mass. The mass fraction of guanidine hydrochloride is preferably 14 to 18 parts, more preferably 16 parts; The preferred mass fraction of the graphene oxide is 6 to 10 parts, more preferably 8 parts; The preferred mass fraction of the carboxymethyl chitosan is 3 to 7 parts, more preferably 5 parts; The preferred mass fraction of the glycine is 8 to 12 parts, more preferably 10 parts; The preferred mass fraction of the ethylenediaminetetraacetic acid is 8 to 12 parts, more preferably 10 parts; The sodium citrate is preferably 6 to 10 parts by mass, and more preferably 8 parts by mass. The glycerol is preferably 3 to 7 parts by weight, more preferably 5 parts by weight; The water is preferably in the form of 80 to 120 parts by mass, and more preferably 100 parts by mass.

[0017] In this invention, the pH value of the RNA protectant is 6.6 to 8.6, preferably 7.6.

[0018] This invention provides a method for preparing the aforementioned RNA protectant, comprising the following steps: Graphene oxide and carboxymethyl chitosan were dissolved in water, sonicated for 25-35 minutes, stirred for 1-3 hours, and then β-mercaptoethanol, guanidine hydrochloride, glycine, ethylenediaminetetraacetic acid, sodium citrate and glycerol were added to adjust the pH value to obtain an RNA protectant. The preferred time for ultrasound is 30 minutes; the preferred time for stirring is 2 hours.

[0019] In this invention, the temperature of the ultrasound is 25~35℃, preferably 30℃; the power of the ultrasound is 100~200W, preferably 150W.

[0020] In this invention, the stirring speed is 100~200 rpm, preferably 150 rpm.

[0021] This invention provides the application of the RNA protectant described herein or the RNA protectant prepared by the method described herein in maintaining the stability of RNA in a sample.

[0022] This invention provides a method for protecting viral samples, which involves mixing and preserving the viral sample with an RNA preservative. The storage temperature is -80~37℃; The RNA protectant is the RNA protectant described above or the RNA protectant prepared by the preparation method described above.

[0023] In this invention, the volume ratio of the virus sample to the RNA protectant is 2-4:1-3, preferably 3:2.

[0024] In this invention, the virus sample includes one or more of porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), and classical swine fever virus (CSFV).

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1

[0027] An RNA protectant comprising 15 mg of β-mercaptoethanol, 16 mg of guanidine hydrochloride, 8 mg of graphene oxide, 5 mg of carboxymethyl chitosan, 10 mg of glycine, 10 mg of ethylenediaminetetraacetic acid, 8 mg of sodium citrate, 5 mg of glycerol and 100 mg of water.

[0028] Preparation method of RNA protectant: Graphene oxide and carboxymethyl chitosan were dissolved in water and sonicated at 30°C (150W) for 30 minutes. Then, the mixture was stirred at 150 rpm for 2 hours. β-mercaptoethanol, guanidine hydrochloride, glycine, ethylenediaminetetraacetic acid, sodium citrate, and glycerol were added and mixed to adjust the pH to 7.6 to obtain the RNA protectant.

[0029] Example 2

[0030] An RNA protectant comprising 12 mg of β-mercaptoethanol, 19 mg of guanidine hydrochloride, 10 mg of graphene oxide, 3 mg of carboxymethyl chitosan, 7 mg of glycine, 13 mg of ethylenediaminetetraacetic acid, 10 mg of sodium citrate, 3 mg of glycerol and 100 mg of water.

[0031] Preparation method of RNA protectant: Graphene oxide and carboxymethyl chitosan were dissolved in water and sonicated at 27°C (power 200W) for 32 min. Then, the mixture was stirred at 200 rpm for 1.5 h. β-mercaptoethanol, guanidine hydrochloride, glycine, ethylenediaminetetraacetic acid, sodium citrate and glycerol were added and mixed to adjust the pH to 7.6 to obtain the RNA protectant.

[0032] Example 3

[0033] A method for protecting viral RNA samples involves mixing the viral RNA sample with the RNA protectant prepared in Example 1 at a volume ratio of 3:2 and storing it at a temperature of -80 to 37°C.

[0034] Comparative Example 1

[0035] The difference from Example 1 is that graphene oxide is not added to the protective agent, and the preparation method does not involve ultrasonication and stirring. Instead, the components are directly added to water, mixed, and the pH value is adjusted.

[0036] Comparative Example 2

[0037] The difference from Example 1 is that trehalose is used instead of glycerol; otherwise, they are exactly the same.

[0038] Comparative Example 3

[0039] The difference from Example 1 is that guanidine isothiocyanate is used instead of guanidine hydrochloride, otherwise they are exactly the same.

[0040] Comparative Example 4

[0041] The difference from Example 1 lies in the preparation method. The preparation method does not involve ultrasonication and stirring; instead, the components are directly added to water, mixed, and the pH value is adjusted.

[0042] Experiment 1: The protective effect of different protective agents on RNA in samples

[0043] Test Sample: While oral fluid contains oral epithelial cells and leukocytes from salivary glands, making it possible to extract RNA samples, its complex composition, including large amounts of RNase, proteins, and other organic matter, easily leads to RNA degradation. Therefore, oral fluid is used as the test sample. Specifically, a piece of cotton rope is suspended in the pigpen, allowing a pig to chew freely for 30 minutes. The rope is collected, squeezed into a plastic bag, and a corner of the bag is cut off. The oral fluid is then placed in test tubes and diluted with sterile water to obtain 18 diluted oral fluid samples. To further ensure the presence of virus in each oral fluid sample, live porcine reproductive and respiratory syndrome virus (PRRSV) (isolated from saliva samples of pigs infected with PRRSV) is mixed with the diluted oral fluid at a volume ratio of 1:2 as the test sample.

[0044] Experimental Groups: The experiment was divided into 6 groups, with each group being repeated three times. These were the control group and treatment groups 1-5. The control group consisted of 3 mL of the test sample plus 2 mL of the RNA protectant from Example 1 in CN112760360A. Treatment group 1 consisted of 3 mL of the test sample plus 2 mL of the RNA protectant from Example 1. Treatment group 2 consisted of 3 mL of the test sample plus 2 mL of the RNA protectant from Comparative Example 1. Treatment group 3 consisted of 3 mL of the test sample plus 2 mL of the RNA protectant from Comparative Example 2. Treatment group 4 consisted of 3 mL of the test sample plus 2 mL of the RNA protectant from Comparative Example 3. Treatment group 5 consisted of 3 mL of the test sample plus 2 mL of the RNA protectant from Comparative Example 4.

[0045] Experimental Protocol: Different storage conditions were set, and different treatment groups were stored under different conditions. Specific parameters are shown in Table 1. After storage, RNA was extracted using a kit, and then quantitative real-time RT-PCR detection was performed using a universal real-time fluorescent RT-PCR detection kit for Porcine reproductive and respiratory syndrome virus (PRRSV) (purchased from Hangzhou Bori Technology Co., Ltd., catalog number BSL33S1). The Ct values ​​were recorded, and the Ct value change rate was calculated. A higher Ct value change rate indicates more severe RNA degradation. The calculation method for the Ct value change rate is as follows: ; In the formula, Ct1 represents the Ct value after the processing group has been stored for a certain period of time; Ct2 represents the Ct value after the processing group has been saved for 0 days.

[0046] Table 1 Different storage conditions

[0047] The results of the rate of change of Ct values ​​for different treatment groups under different storage conditions are shown in Table 2.

[0048] Table 2. Results of Ct value change rate for different treatment groups under different storage conditions.

[0049] As shown in Table 2, the RNA preservative in treatment group 1 has a preservation effect on the test samples comparable to that of the preservative in Example 1 of patent CN112760360A. The RNA preservatives in treatment groups 2-5 show different preservation effects on the test samples, but are significantly lower than those in treatment group 1. This indicates that the components of the RNA preservative prepared according to the present application have a synergistic effect, and the interaction between the components results in a significant protective effect on RNA.

[0050] As can be seen from the above embodiments, the present application provides an RNA protectant in which both graphene oxide and carboxymethyl chitosan can physically adsorb nucleic acids to protect them. The present application enables graphene oxide and carboxymethyl chitosan to react under certain conditions to obtain a complex, which further enhances the adsorption and protection of nucleic acids. Then, when combined with β-mercaptoethanol, guanidine hydrochloride, glycine, ethylenediaminetetraacetic acid, sodium citrate and glycerol, the protective effect of the protectant on nucleic acids can be significantly improved, providing a new approach to nucleic acid protection methods.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An RNA protectant, characterized in that, The RNA protectant comprises the following components in parts by weight: 10-20 parts β-mercaptoethanol, 11-21 parts guanidine hydrochloride, 4-12 parts graphene oxide, 1-10 parts carboxymethyl chitosan, 5-15 parts glycine, 5-15 parts ethylenediaminetetraacetic acid, 4-12 parts sodium citrate, 1-10 parts glycerol and 50-150 parts water.

2. The RNA protectant according to claim 1, characterized in that, The RNA protectant comprises the following components in parts by weight: 13-17 parts β-mercaptoethanol, 14-18 parts guanidine hydrochloride, 6-10 parts graphene oxide, 3-7 parts carboxymethyl chitosan, 8-12 parts glycine, 8-12 parts ethylenediaminetetraacetic acid, 6-10 parts sodium citrate, 3-7 parts glycerol and 80-120 parts water.

3. The RNA protectant according to claim 2, characterized in that, The RNA protectant comprises the following components in parts by weight: 15 parts β-mercaptoethanol, 16 parts guanidine hydrochloride, 8 parts graphene oxide, 5 parts carboxymethyl chitosan, 10 parts glycine, 10 parts ethylenediaminetetraacetic acid, 8 parts sodium citrate, 5 parts glycerol and 100 parts water.

4. The RNA protectant according to any one of claims 1 to 3, characterized in that, The pH value of the RNA protectant is 6.6~8.

6.

5. A method for preparing the RNA protectant according to any one of claims 1 to 4, characterized in that, Includes the following steps: Graphene oxide and carboxymethyl chitosan were dissolved in water, sonicated for 25-35 minutes, stirred for 1-3 hours, and then β-mercaptoethanol, guanidine hydrochloride, glycine, ethylenediaminetetraacetic acid, sodium citrate and glycerol were added to adjust the pH value to obtain an RNA protectant.

6. The preparation method according to claim 5, characterized in that, The temperature of the ultrasound is 25~35℃, and the power of the ultrasound is 100~200W.

7. The preparation method according to claim 5, characterized in that, The stirring speed is 100~200 rpm.

8. The use of the RNA protectant according to any one of claims 1 to 4 or the RNA protectant prepared by the preparation method according to any one of claims 5 to 7 in maintaining the stability of RNA in a sample.

9. A method for protecting a virus sample, characterized in that, Simply mix the virus sample with an RNA preservative and store it. The storage temperature is -80~37℃; The RNA protectant is the RNA protectant according to any one of claims 1 to 4 or the RNA protectant prepared by the preparation method according to any one of claims 5 to 7.

10. The method according to claim 9, characterized in that, The volume ratio of the virus sample to the RNA protectant was 2-4:1-3.

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