Genomic RNA protective agent and preparation method thereof

Abstract

The invention relates to a genome RNA protective agent and a preparation method thereof, and belongs to the field of biology, the protective agent comprises 0.1%-0.3% of sucrose, 0.01%-0.02% of sodium chloride, 0.01%-0.05% of ammonium sulfate, 0.01%-0.2% of lactoalbumin hydrolysate, 0.01%-0.05% of disodium ethylene diamine tetraacetate, 0.01%-0.1% of triethanolamine and 0.01%-0.05% of procilin300, and a solvent is nuclease-free DEPC water. The genome RNA protective agent provided by the invention is used for protecting genome RNA, has colorless and transparent characters, has no influence on PCR amplification, and can stably preserve the genome RNA for 18 months or more at-20 DEG C or below.

Description

Technical Field

[0001] This invention belongs to the field of biology, and in particular relates to a genomic RNA protectant and its preparation method. Background Technology

[0002] Genomic RNA mostly exists in single-stranded form, with its base and phosphate backbone exposed, making it susceptible to degradation due to irregular structural pairing. The ubiquitous ribonucleases pose a significant threat to the stability of genomic RNA. Therefore, extracted or purified genomic RNA is extremely unstable and requires protective agents to extend its shelf life. Current techniques generally store genomic RNA at temperatures below -80°C to stabilize it, but even ultra-low temperatures cannot ensure long-term stability. Alternatively, imported genomic RNA preservatives can be purchased, but these are expensive and have poor timeliness. Domestically available preservatives for tissue or blood samples require nucleic acid extraction before downstream PCR experiments; the application of genomic RNA preservatives is rare.

[0003] Chinese patent (CN 116479090A) discloses an RNA preservation solution and its preparation method, which can effectively increase the stability of RNA. The added dithiothreitol is a strong reducing agent, but its inhibitory effect on some RNases is not significant. The added xanthate can covalently modify RNA, which poses a risk of non-specific increase in the concentration of RNA detected by spectrophotometer. No examples have been found of this protectant being directly used in downstream PCR experiments. The stability of RNA samples was only confirmed by gel electrophoresis.

[0004] Chinese patent (CN 11410739B) discloses an RNA protectant and its application, which mainly contains guanidine isothiocyanate or guanidine hydrochloride, tris(2-carboxyethyl)phosphine hydrochloride, PEG, and sulfosalicylic acid. It can effectively maintain the stability of RNA in urine samples, but is not suitable for the protection of genomic RNA samples.

[0005] Chinese patent (CN 113980954A) discloses a viral RNA protectant and its preparation method and application, which mainly contains guanidine isothiocyanate, sodium dodecyl sarcosinate, β-mercaptoethanol, polyethylene glycol 200, Triton X100 and sodium citrate. It is mainly used for the preservation of RNA virus fluid and is not suitable for the protection of genomic RNA samples. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a genomic RNA protectant that can stably preserve RNA for 18 months or more at temperatures below -20°C, has no inhibitory or enhancing effect on PCR reactions, and can be directly used in PCR reactions.

[0007] This invention is achieved through the following technical solution: A genomic RNA protectant comprising 0.1%–0.3% sucrose, 0.01%–0.02% sodium chloride, 0.01%–0.05% ammonium sulfate, 0.01%–0.2% hydrolyzed milk protein, 0.01%–0.05% disodium EDTA, 0.01%–0.1% triethanolamine, and 0.01%–0.05% procilin 300, in a nuclease-free DEPC-free water solvent.

[0008] Furthermore, the RNA mentioned is extracted or purified genomic RNA.

[0009] The present invention also provides a method for preparing the protective agent, as follows: Step 1: Take the solvent DEPC-free water, add sucrose, sodium chloride and ammonium sulfate in sequence to provide a stable solution environment, then add the antioxidant hydrolyzed milk protein, disodium EDTA and triethanolamine to stabilize the pH environment and improve the antioxidant capacity, and finally add the antibacterial agent procilin300, stir to dissolve and mix well to form a mixture; The second step is to take the mixture and filter it through a 0.22μM filter membrane to sterilize it, thus forming a genomic RNA protectant.

[0010] The present invention also provides a method for protecting genomic RNA using the RNA protectant, wherein the method involves adding the RNA protectant to extracted or purified RNA.

[0011] As a preferred technical solution, 10-100 times the volume of the RNA protectant is added to the extracted or purified genomic RNA solution.

[0012] The beneficial effects of this invention compared to the prior art are as follows: The genomic RNA protectant of this invention uses reagents with lower concentrations, resulting in higher safety. It has no inhibitory or promoting effect on PCR reactions and can maintain the most accurate concentration of genomic RNA. The key technical feature is that it can be stored at -20°C or below for up to 18 months for the protection of genomic RNA. Attached Figure Description

[0013] Figure 1 This is a gel electrophoresis image; Figure 2A This is a fluorescent RT-PCR amplification image of the diluted solution of "sterile purified water" in Experiment 2 of Example 4; Figure 2B This is a fluorescent RT-PCR amplification image of experimental group 2 in Example 4, using "DEPC-free water without nuclease"; Figure 2C This is a fluorescent RT-PCR amplification image of "genomic RNA protectant" in Experimental Group 2 of Example 4; Figure 3 This is a diagram showing the results of digital fluorescent RT-PCR amplification. Figure 4A This is a monitoring graph for "Long-term stability of genomic RNA diluted 10-fold" in Example 5; Figure 4B This is a stability monitoring graph of the genomic RNA protectant in Example 5, "Long-term stability of genomic RNA diluted 100 times". Detailed Implementation

[0014] To facilitate understanding of the present invention, a more comprehensive description will be provided below. The present invention can be implemented in many different reagent formulations and is not limited to the embodiments described herein. Rather, these embodiments are provided to make the disclosure of the present invention more thorough and complete.

[0015] Example 1: Screening test of key components of a genomic RNA protectant To screen for the most critical components, DEPC water, sodium chloride, ammonium sulfate, disodium EDTA, and procilin 300 were first identified as fixed components. Then, the optimal components for carbohydrates and inert proteins, as well as the most critical components, were screened, with solutions prepared according to Tables 1 and 3. Genomic RNA was extracted from foot-and-mouth disease virus nucleic acid sensitivity control samples using a viral RNA extraction kit. The extracted genomic RNA was diluted 10-fold with a genomic RNA protectant and stored at 2–8°C. Fluorescent RT-PCR amplification was performed according to primers and probes in GB / T 18935-2018 Foot-and-Mouth Disease Diagnostic Techniques. Results showed that sucrose was the optimal carbohydrate component, hydrolyzed milk protein was the optimal inert protein component, and triethanolamine was the most critical component. The optimal concentration of triethanolamine was found to be 0.01%–0.1%.

[0016] Table 1. Screening of optimal components from carbohydrate compounds and inert proteins. ; Table 2. Fluorescent RT-PCR validation results (average Ct values) of the optimal components of carbohydrate compounds and inert proteins. ; Table 3 Screening of the most critical components ; Table 4. Results of fluorescent RT-PCR validation of key components (average Ct value) .

[0017] Example 2 A genomic RNA protectant comprising 0.1%–0.3% sucrose, 0.01%–0.02% sodium chloride, 0.01%–0.05% ammonium sulfate, 0.01%–0.2% hydrolyzed milk protein (Sinopharm Reagent), 0.01%–0.05% disodium EDTA, 0.01%–0.1% triethanolamine (Sinopharm Reagent), and 0.01%–0.05% procilin 300, in a nuclease-free DEPC-free water solvent.

[0018] One specific embodiment is a genomic RNA protectant comprising 0.1% sucrose, 0.01% sodium chloride, 0.01% ammonium sulfate, 0.01% hydrolyzed milk protein, 0.01% disodium EDTA, 0.03% triethanolamine, and 0.05% procilin 300, in a nuclease-free DEPC-free water solution (substrate composition of Example 2).

[0019] One specific embodiment of this invention is a genomic RNA protectant comprising 0.3% sucrose, 0.02% sodium chloride, 0.05% ammonium sulfate, 0.2% hydrolyzed milk protein, 0.05% disodium EDTA, 0.05% triethanolamine, and 0.05% procilin 300, in a solvent of nuclease-free DEPC water.

[0020] One specific embodiment of this invention is a genomic RNA protectant comprising 0.2% sucrose, 0.02% sodium chloride, 0.05% ammonium sulfate, 0.2% hydrolyzed milk protein, 0.03% disodium EDTA, 0.1% triethanolamine, and 0.01% procilin 300, in a nuclease-free DEPC-free water solution.

[0021] The present invention also provides a method for preparing the composition, wherein the amount of each component is any value of the mass percentage of each component in the composition, wherein any ratio can achieve the technical effect of the composition of the present invention, namely, the solution is colorless and transparent, has no effect on PCR reaction, and has good stability.

[0022] The specific preparation method is as follows: Step 1: Take the solvent DEPC-free water, add sucrose, sodium chloride and ammonium sulfate in sequence to provide a stable solution environment, then add the antioxidant hydrolyzed milk protein, disodium EDTA and triethanolamine to stabilize the pH environment and improve the antioxidant capacity, and finally add the antibacterial agent procilin300, stir to dissolve and mix well to form a mixture; Step 2: Take the mixture and filter it through a 0.22 μM filter membrane for sterilization to form a genomic RNA protectant. (Example 2: Genomic RNA Protectant).

[0023] Example 3: Phenotypic testing of a genomic RNA protectant A genomic RNA protectant comprising 0.1%–0.3% sucrose, 0.01%–0.02% sodium chloride, 0.01%–0.05% ammonium sulfate, 0.01%–0.2% hydrolyzed milk protein, 0.01%–0.05% disodium EDTA, 0.01%–0.1% triethanolamine, and 0.01%–0.05% procilin 300, in a nuclease-free DEPC-free water solvent.

[0024] The specific preparation method is as follows: Step 1: Take the solvent DEPC-free water, add sucrose, sodium chloride and ammonium sulfate in sequence to provide a stable solution environment, then add the antioxidant hydrolyzed milk protein, disodium EDTA and triethanolamine to stabilize the pH environment and improve the antioxidant capacity, and finally add the antibacterial agent procilin300, stir to dissolve and mix well to form a mixture; Step 2: Take the mixture and filter it through a 0.22 μM filter membrane for sterilization to form a genomic RNA protectant. (Example 3: Genomic RNA Protectant).

[0025] The phenotypic testing method was as follows: 100 ml of the prepared genomic RNA protectant of Example 2 and Example 3 were placed in transparent beakers for observation. Both the genomic RNA protectant of Example 2 and Example 3 were colorless and transparent liquids.

[0026] Example 4: Applicability testing of a genomic RNA protectant The required components include 0.1% sucrose, 0.01% sodium chloride, 0.01% ammonium sulfate, 0.01% hydrolyzed milk protein, 0.01% disodium EDTA, 0.03% triethanolamine, and 0.05% procilin 300, with DEPC-free water as the solvent. This example demonstrates application testing through the following three experimental groups: Experimental Group 1: The Effect of Genomic RNA Protectants on Conventional PCR Genomic RNA was extracted from inactivated classical swine fever virus (China / 2018LX strain) standard material using a viral RNA extraction kit. The extracted genomic RNA was diluted 2-fold with genomic RNA protectant, nuclease-free DEPC water, and sterile purified water to serve as the detection template. Specific primers were designed based on the complete genome sequence of classical swine fever virus (accession number NC_002657.1) for RT-PCR amplification, and the results were analyzed by gel electrophoresis imaging. Figure 1 The protective agent has no effect on the amplification of ordinary RT-PCR.

[0027] Experimental Group 2: Effects of Genomic RNA Protectants on Fluorescent RT-PCR Genomic RNA was extracted from inactivated classical swine fever virus (China / 2018LX strain) standard material using a viral RNA extraction kit. The RNA was then extracted with sterile purified water (…). Figure 2A DEPC-free water () Figure 2B ), Genomic RNA protectants ( Figure 2C The extracted genomic RNA was diluted 10-fold, 100-fold...1,000,000-fold to serve as templates for detection. Fluorescent RT-PCR amplification was performed according to GB / T 27540-2011, "Real-time Fluorescent RT-PCR Detection Method for Classical Swine Fever Virus". Analysis of the fluorescent RT-PCR amplification results showed that the protective agent had no effect on the amplification.

[0028] Table 5 Results of Fluorescent RT-PCR Detection .

[0029] Experimental Group 3: The Effect of Genomic RNA Protectants on Digital PCR Genomic RNA was extracted from inactivated classical swine fever virus (China / 2018LX strain) using a viral RNA extraction kit. The extracted genomic RNA was diluted 10-fold with a genomic RNA protectant to serve as the detection template. Digital RT-PCR amplification was performed according to the primers and probes specified in GB / T 27540-2011, "Real-time Fluorescent RT-PCR Detection Method for Classical Swine Fever Virus". The results of the digital RT-PCR amplification were analyzed. Figure 3 The protectant has no effect on digital RT-PCR amplification.

[0030] Table 6. Digital RT-PCR detection results (copies / μl) .

[0031] Example 5: Stability test of a genomic RNA protectant The preparation included 0.1% sucrose, 0.01% sodium chloride, 0.01% ammonium sulfate, 0.01% hydrolyzed milk protein, 0.01% disodium EDTA, 0.03% triethanolamine, and 0.05% procilin 300, with DEPC-free water as the solvent. Stability was tested using the following experimental groups in this embodiment: Experimental Group 1: Storage stability test below -20℃ Genomic RNA was extracted from foot-and-mouth disease virus nucleic acid sensitivity control samples using a viral RNA extraction kit. The extracted genomic RNA was then diluted 10-fold with a genomic RNA protectant. Figure 4A ), 100 times ( Figure 4BThe samples were stored at -20°C or below. Long-term stability monitoring results were obtained from fluorescent RT-PCR amplification analysis using primers and probes according to GB / T 18935-2018 "Foot-and-Mouth Disease Diagnostic Techniques". The results showed that the genomic RNA was stable for 18 months when stored at -20°C or below.

[0032] The genomic RNA protectant of the present invention includes sucrose, sodium chloride, ammonium sulfate, hydrolyzed milk protein, disodium EDTA, triethanolamine, procilin 300, and nuclease-free DEPC water, all of which are commercially available, safe and non-toxic reagents, and are existing technologies. Those skilled in the art only need to follow the accompanying instructions for use to perform the experiments, without requiring any creative effort from those skilled in the art.

[0033] 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 technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A genomic RNA protectant, characterized in that, The protective agent comprises 0.1%–0.3% sucrose, 0.01%–0.02% sodium chloride, 0.01%–0.05% ammonium sulfate, 0.01%–0.2% hydrolyzed milk protein, 0.01%–0.05% disodium EDTA, 0.01%–0.1% triethanolamine, and 0.01%–0.05% procilin 300, and the solvent is nuclease-free DEPC water. The percentages are by mass.

2. The method for preparing the protective agent according to claim 1, characterized in that, The method is as follows: Step 1: Take DEPC water (a solvent without nuclease), add sucrose, sodium chloride, and ammonium sulfate in sequence to provide a stable solution environment, then add the antioxidants hydrolyzed milk protein, disodium EDTA, and triethanolamine to stabilize the pH environment and improve antioxidant capacity, and finally add the antibacterial agent procilin 300, stir to dissolve and mix well to form a mixture. The second step is to take the mixture and filter it through a 0.22μM filter membrane to sterilize it, thus forming a genomic RNA protectant.

3. A method for protecting RNA using the genomic RNA protectant of claim 1, characterized in that, The RNA protectant described above is added to the extracted or purified genomic RNA solution.

4. The method according to claim 3, characterized in that, Add 10-100 times the volume of the RNA protectant to the extracted or purified genomic RNA solution.

Images