Deoxyribonucleic acid and pseudovirus preservation solution, preparation method and application thereof

Abstract

This application relates to a deoxyribonucleic acid and pseudovirus preservation solution, its preparation method and application. The deoxyribonucleic acid and pseudovirus preservation solution includes: a buffer solution with a final concentration of 5 mM to 50 mM, a surfactant with a volume percentage of 0.05% to 1.0%, trehalose with a final concentration of 50 mM to 500 mM, a metal ion chelator with a final concentration of 0.5 mM to 5 mM, betaine with a final concentration of 100 mM to 1000 mM, and mannitol with a mass percentage of 1% to 10%. Each specific component in the above-mentioned deoxyribonucleic acid and pseudovirus preservation solution cooperates with each other at a specific concentration, which has the effect of stabilizing the state and structure of nucleic acids. It can achieve the preservation of nucleic acids and pseudoviruses without ultra-low temperature, and can stably preserve nucleic acids and pseudoviruses for up to 12 months at 30°C without affecting subsequent detection tests.

Description

Technical Field

[0001] The present application relates to the technical field of biochemical preparations, and particularly relates to a deoxyribonucleic acid and pseudovirus preservation solution, a preparation method thereof, and an application thereof. Background Art

[0002] Nucleic acids are divided into ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), and are often used in in vitro diagnostic test reagents to replace real clinical samples to prepare calibration products or quality control products. However, as carriers of biological genetic information, nucleic acids have poor stability and are easily affected by physical factors such as external temperature, humidity, and ultraviolet light, chemical factors such as pH value, hydrolysis reaction, and oxidation reaction, and biological factors such as enzymatic hydrolysis and microbial infection.

[0003] In the field of pathogenic microorganism detection, due to the safety risk of biological transmission of un-inactivated viral samples, or the risk of incomplete inactivation even after the viral samples are inactivated, and inactivation may damage viral nucleic acids, affecting the quality or extraction of nucleic acids, resulting in disadvantages such as missed detection in downstream detections, recombinant plasmids containing target genes or pseudoviruses packaged by adenoviruses are often used as quality control products or calibration products for test kits. They are not only simple to prepare, but also have no biological safety problems. Test kits prepared with plasmids or pseudoviruses are usually stored in a frozen manner. However, after long-term frozen storage of test kits prepared with plasmids or pseudoviruses, the state and structure of nucleic acids in the plasmids or pseudoviruses are likely to change, resulting in inaccurate detection results, and frozen storage requires special equipment such as liquid nitrogen tanks, with high costs.

[0004] Therefore, it is of great significance to provide a preservation solution that can be stored at room temperature and can improve the stability of nucleic acids and pseudoviruses. Summary of the Invention

[0005] Based on this, the present application provides a deoxyribonucleic acid and pseudovirus preservation solution that can be stored at room temperature and can improve the stability of nucleic acids and pseudoviruses, a preparation method thereof, and an application thereof.

[0006] The technical solution for the present application to solve the above technical problems is as follows.

[0007] A deoxyribonucleic acid and pseudovirus preservation solution, the deoxyribonucleic acid and pseudovirus preservation solution includes: a buffer solution with a final concentration of 5 mM to 50 mM, a surfactant with a volume percentage of 0.05% to 1.0%, trehalose with a final concentration of 50 mM to 500 mM, a metal ion chelator with a final concentration of 0.5 mM to 5 mM, betaine with a final concentration of 100 mM to 1000 mM, and mannitol with a mass percentage of 1% to 10%; the buffer solution is Tris-HCl buffer solution, and the surfactant is TritonX-100.

[0008] In some of these embodiments, in the deoxyribonucleic acid and pseudovirus preservation solution, the deoxyribonucleic acid and pseudovirus preservation solution comprises: a buffer solution with a final concentration of 5 mM to 30 mM, a surfactant with a volume percentage of 0.05% to 0.5%, trehalose with a final concentration of 50 mM to 300 mM, a metal ion chelator with a final concentration of 0.5 mM to 4 mM, betaine with a final concentration of 200 mM to 800 mM, and mannitol with a mass percentage of 2% to 8%.

[0009] In some of these embodiments, in the deoxyribonucleic acid and pseudovirus preservation solution, the deoxyribonucleic acid and pseudovirus preservation solution consists of the following components: a buffer solution with a final concentration of 5 mM to 50 mM, a surfactant with a volume percentage of 0.05% to 1.0%, trehalose with a final concentration of 50 mM to 500 mM, a metal ion chelator with a final concentration of 0.5 mM to 5 mM, betaine with a final concentration of 100 mM to 1000 mM, mannitol with a mass percentage of 1% to 10%, and the balance being nuclease-free water.

[0010] In some of these embodiments, in the deoxyribonucleic acid and pseudovirus preservation solution, the deoxyribonucleic acid and pseudovirus preservation solution consists of the following components: a buffer solution with a final concentration of 10 mM, a surfactant with a volume percentage of 0.1%, trehalose with a final concentration of 150 mM, a metal ion chelator with a final concentration of 1 mM, betaine with a final concentration of 500 mM, mannitol with a mass percentage of 5%, and the balance being a solvent.

[0011] In some of these embodiments, in the deoxyribonucleic acid and pseudovirus preservation solution, the metal ion chelator is selected from at least one of sodium EDTA and potassium EDTA.

[0012] The present application provides a method for preparing a deoxyribonucleic acid and pseudovirus preservation solution, comprising the following steps:

[0013] Provide raw materials according to the components of the above-mentioned deoxyribonucleic acid and pseudovirus preservation solution;

[0014] After mixing the buffer solution, surfactant, trehalose, chelating agent, betaine, and mannitol, perform sterilization and impurity removal to obtain the deoxyribonucleic acid and pseudovirus preservation solution.

[0015] In some of these embodiments, in the method for preparing the deoxyribonucleic acid and pseudovirus preservation solution, before the mixing step, it further includes adjusting the pH value of the buffer solution to 7.5 to 8.5.

[0016] The present application provides the use of the above-mentioned deoxyribonucleic acid and pseudovirus preservation solution in preserving deoxyribonucleic acid or pseudovirus.

[0017] The present application provides the use of the above-mentioned deoxyribonucleic acid and pseudovirus preservation solution in the preparation of in vitro diagnostic reagents containing deoxyribonucleic acid or pseudovirus.

[0018] Compared with the prior art, the deoxyribonucleic acid and pseudovirus preservation solution of the present application has the following beneficial effects:

[0019] The above-mentioned deoxyribonucleic acid and pseudovirus preservation solution, by combining the above-mentioned specific components in a specific concentration, has the effect of stabilizing the state and structure of nucleic acids, and can achieve the preservation of deoxyribonucleic acid and pseudovirus without ultra-low temperature. Moreover, the deoxyribonucleic acid and pseudovirus can be stably preserved for up to 12 months at 30 °C, without affecting subsequent detection tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is the PCR amplification curve diagram of plasmid DNA stored at 37 °C with the preservation solution prepared in Example 1;

[0022] Figure 2 It is the PCR amplification curve diagram of plasmid DNA stored at 37 °C with the preservation solution prepared in Example 2;

[0023] Figure 3 It is the PCR amplification curve diagram of plasmid DNA stored at 37 °C with the preservation solution prepared in Example 3;

[0024] Figure 4 It is the PCR amplification curve diagram of plasmid DNA stored at 37 °C with the preservation solution prepared in Example 4;

[0025] Figure 5 It is the PCR amplification curve diagram of plasmid DNA stored at 37 °C with the preservation solution prepared in Comparative Example 1;

[0026] Figure 6 It is the PCR amplification curve diagram of plasmid DNA stored at 37 °C with the preservation solution prepared in Comparative Example 2;

[0027] Figure 7 It is the PCR amplification curve diagram of plasmid DNA stored at 37 °C with the preservation solution prepared in Comparative Example 3;

[0028] Figure 8PCR amplification curve of plasmid DNA stored at 37°C using the preservation solution prepared in Comparative Example 4;

[0029] Figure 9 PCR amplification curve of plasmid DNA stored at 37°C using the preservation solution prepared in Comparative Example 5;

[0030] Figure 10 PCR amplification curve of plasmid DNA stored at 37°C using the preservation solution prepared in Comparative Example 6;

[0031] Figure 11 PCR amplification curve of plasmid DNA stored at 37°C using the preservation solution prepared in Comparative Example 7;

[0032] Figure 12 PCR amplification curve of plasmid DNA stored at 37°C using the preservation solution prepared in Comparative Example 8;

[0033] Figure 13 PCR amplification curve of plasmid DNA stored at 37°C using the preservation solution prepared in Comparative Example 9. Detailed implementation manners

[0034] The technical solutions of the present application will be further described in detail below in conjunction with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. It should be understood that the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0036] In the description of this application, it should be understood that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of this application, "a plurality" means two or more unless otherwise specifically defined.

[0037] In the description of the embodiments of the present application, the weights of the relevant components mentioned not only can refer to the specific contents of each component, but also can represent the proportional relationship between the weights of each component. Therefore, as long as the contents of the relevant components in the description of the embodiments of the present application are scaled up or down in proportion, they are within the scope disclosed in the description of the embodiments of the present application. Specifically, the weights described in the description of the embodiments of the present application can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

[0038] Nucleic acid is a macromolecular compound polymerized by many nucleotides and is one of the most basic substances of life. It widely exists in animal and plant cells and microorganisms. Nucleic acid in organisms is often combined with proteins to form nucleoproteins. Different nucleic acids have different chemical compositions, nucleotide sequences, etc. According to different chemical compositions, nucleic acids can be divided into ribonucleic acid (abbreviated as RNA) and deoxyribonucleic acid (abbreviated as DNA). DNA consists of a pair of closely related double strands that are intertwined with each other to form a double helix structure. Among them, the deoxyribose-phosphate chain is on the outside of the helix structure, and the bases face inside. The two deoxynucleotide strands are reverse complementary and are connected by base pairing formed by hydrogen bonds between the bases, forming a relatively stable combination. DNA is the main material basis for storing, replicating, and transmitting genetic information. RNA is a long-chain molecule formed by the condensation of ribonucleotides through phosphodiester bonds. It plays an important role in the process of protein synthesis. Among them, transfer ribonucleic acid (abbreviated as tRNA) plays the role of carrying, transferring, and activating amino acids, messenger ribonucleic acid (abbreviated as mRNA) is the template for protein synthesis, and ribosomal ribonucleic acid (abbreviated as rRNA) is the main site for cells to synthesize proteins.

[0039] During the research process, technicians found that traditional deoxyribonucleic acid and pseudovirus preservation solutions need to be stored at low temperatures below zero, which easily changes the nucleic acid state and structure in deoxyribonucleic acid and pseudovirus, resulting in inaccurate detection results, and the preservation and transportation costs are relatively high; and the preservation stability of traditional deoxyribonucleic acid and pseudovirus preservation solutions that can be used at room temperature is poor. After analysis and experiments, the influencing factors are considered to include:

[0040] There are preservatives or guanidinium salts, or strong reducing agents in traditional deoxyribonucleic acid and pseudovirus preservation solutions, and long-term preservation will damage the nucleic acid structure and affect downstream PCR detection; the pH value of traditional deoxyribonucleic acid and pseudovirus preservation solutions < 6.5, and the acidic conditions are likely to damage the nucleic acid structure and cause instability, etc.

[0041] An embodiment of the present application provides a deoxyribonucleic acid and pseudovirus preservation solution, comprising: a buffer solution with a final concentration of 5 mM to 50 mM, a surfactant with a volume percentage of 0.05% to 1.0%, trehalose with a final concentration of 50 mM to 500 mM, a metal ion chelator with a final concentration of 0.5 mM to 5 mM, betaine with a final concentration of 100 mM to 1000 mM, and mannitol with a mass percentage of 1% to 10%; the buffer solution is Tris-HCl buffer solution, and the surfactant is TritonX-100.

[0042] By combining the above specific components in specific concentrations, it has the effect of stabilizing the state and structure of nucleic acids, and can achieve the preservation of nucleic acids and pseudoviruses without ultra-low temperature. Moreover, nucleic acids and pseudoviruses can be stably preserved for up to 12 months at 30 °C without affecting subsequent detection tests.

[0043] Tris-HCl, Chinese alias: Trimethylaminomethane hydrochloride, Tris hydrochloride; English name: TRIS hydrochloride.

[0044] TritonX-100, Chinese alias: Octylphenoxypolyethoxyethanol, Triton X-100; English name: 2-(2-[4-(1,1,3,3-Tetramethylbutyl)phenoxy]ethoxy)ethanol; p-iso-Octyl phenoxypolyethoxyethanol.

[0045] Betaine, chemical name is N,N,N-Trimethylglycine, molecular formula is C5H 11 NO2.

[0046] It can be understood that the above concentrations are the final concentrations in the DNA and pseudovirus preservation solution; further, it can be understood that the final concentration of the buffer includes, but is not limited to, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM; the volume percentage of the surfactant includes, but is not limited to, 0.05%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, 0.3%, 0.5%, 0.8%, 1.0%; the final concentration of trehalose includes, but is not limited to, 50 mM, 60 mM, 80 mM, 100 mM, 120 mM, 130 mM, 150 mM, 160 mM, 180 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM; the final concentration of the metal ion chelator includes, but is not limited to, 0.5 mM, 0.8 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM; the final concentration of betaine includes, but is not limited to, 100 mM, 120 mM, 130 mM, 150 mM, 160 mM, 180 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM; the mass percentage of mannitol includes, but is not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 10%.

[0047] Optionally, in some of these examples, in the DNA and pseudovirus preservation solution, the DNA and pseudovirus preservation solution includes: a buffer with a final concentration of 5 mM to 30 mM, a surfactant with a volume percentage of 0.05% to 0.5%, trehalose with a final concentration of 50 mM to 300 mM, a metal ion chelator with a final concentration of 0.5 mM to 4 mM, betaine with a final concentration of 200 mM to 800 mM, and mannitol with a mass percentage of 2% to 8%.

[0048] In the DNA and pseudovirus preservation solution, there is no need to add preservatives, guanidinium salts or strong reducing agents, effectively avoiding the problem of preservatives, guanidinium salts or strong reducing agents damaging the nucleic acid structure, thereby effectively improving the stability of nucleic acids in the DNA and pseudovirus preservation solution.

[0049] In some of these examples, in the DNA and pseudovirus preservation solution, the DNA and pseudovirus preservation solution consists of the following components: a buffer with a final concentration of 5 mM to 50 mM, a surfactant with a volume percentage of 0.05% to 1.0%, trehalose with a final concentration of 50 mM to 500 mM, a metal ion chelator with a final concentration of 0.5 mM to 5 mM, betaine with a final concentration of 100 mM to 1000 mM, mannitol with a mass percentage of 1% to 10%, and the balance being nuclease-free water.

[0050] In some of these examples, in the DNA and pseudovirus preservation solution, the DNA and pseudovirus preservation solution consists of the following components: a buffer with a final concentration of 10 mM, a surfactant with a volume percentage of 0.1%, trehalose with a final concentration of 150 mM, a metal ion chelator with a final concentration of 1 mM, betaine with a final concentration of 500 mM, mannitol with a mass percentage of 5%, and the balance being a solvent.

[0051] In some of these examples, in the DNA and pseudovirus preservation solution, the metal ion chelator is selected from at least one of sodium EDTA and potassium EDTA.

[0052] EDTA, ethylenediaminetetraacetic acid, has the chemical formula C 10 H 16 N2O8.

[0053] It can be understood that sodium EDTA includes but is not limited to disodium EDTA and tetrasodium EDTA; potassium EDTA includes but is not limited to dipotassium EDTA and tripotassium EDTA.

[0054] Correspondingly, an embodiment of the present application provides a method for preparing a DNA and pseudovirus preservation solution, including step S10:

[0055] Provide raw materials according to the components of the above DNA and pseudovirus preservation solution;

[0056] After mixing the buffer, surfactant, trehalose, chelator, betaine, and mannitol, perform sterilization and impurity removal to obtain the DNA and pseudovirus preservation solution.

[0057] In some of these examples, before the mixing step in step S10, there is also step S00:

[0058] Adjust the pH value of the buffer to 7.5 to 8.5.

[0059] It is understandable that the pH value of the buffer solution is first adjusted to 7.5 - 8.5, and then the buffer solution, surfactant, trehalose, chelating agent, betaine and mannitol are mixed; it can be further understood that the pH value of the buffer solution includes but is not limited to 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5.

[0060] In some of these examples, in step S10, sterilization and impurity removal are carried out by filtration.

[0061] Furthermore, the pore size of the filter membrane used for filtration is ≤0.22 μM.

[0062] In some specific examples, in step S10, solid-liquid separation includes the following steps:

[0063] The mixed solution obtained from the mixing step is subjected to suction filtration using a filter membrane with a pore size ≤0.22 μM.

[0064] It is understandable that the filtrate obtained from solid-liquid separation is the deoxyribonucleic acid and pseudovirus preservation solution.

[0065] The above-mentioned deoxyribonucleic acid and pseudovirus preservation solution is convenient and simple to store, without the need for additional low-temperature storage equipment; the components are simple and the content is low; the preparation method is simple, without the need for high-temperature treatment, and it is easy to mass-produce; the cost is low.

[0066] One embodiment of the present application provides the application of the above-mentioned deoxyribonucleic acid and pseudovirus preservation solution in preserving deoxyribonucleic acid or pseudovirus.

[0067] In some of these examples, in the application of the deoxyribonucleic acid and pseudovirus preservation solution, the deoxyribonucleic acid is DNA extracted from a biological sample, and the pseudovirus is a pseudovirus with DNA nucleic acid genetic material.

[0068] The above-mentioned deoxyribonucleic acid and pseudovirus preservation solution can store DNA or pseudovirus samples for a long time under normal temperature conditions, and can maintain the integrity of the DNA structure for at least 12 months at 30°C, ensuring the stability of DNA.

[0069] One embodiment of the present application provides the application of the above-mentioned deoxyribonucleic acid and pseudovirus preservation solution in the preparation of in vitro diagnostic reagents containing deoxyribonucleic acid or pseudovirus. Another embodiment of the present application provides an in vitro diagnostic reagent, which contains deoxyribonucleic acid or pseudovirus, and the above-mentioned deoxyribonucleic acid and pseudovirus preservation solution.

[0070] The above-mentioned deoxyribonucleic acid and pseudovirus preservation solution is used for preparing in vitro diagnostic reagents, which can stably preserve the calibrators and quality control products in the in vitro diagnostic reagents at room temperature, without the need for freezing and thawing during use, facilitating convenient use, and avoiding the instability of calibrators and quality control products caused by freezing and thawing. In addition, when stored at room temperature, the in vitro diagnostic reagents are free from the limitations of freezing conditions during use, transportation, and storage, greatly reducing the economic cost. Specific embodiments

[0072] The following are examples of the deoxyribonucleic acid and pseudovirus preservation solution of the present application, its preparation method, and application. It can be understood that the deoxyribonucleic acid and pseudovirus preservation solution of the present application, its preparation method, and application are not limited to the following embodiments.

[0073] The pH value of the Tris-HCl buffer solution used in the following examples and comparative examples is 8.3.

[0074] Example 1

[0075] By final concentration, the components are: 10 mM Tris-HCl buffer, 150 mM trehalose, 1 mM EDTA, 5 wt% mannitol, 500 mM betaine, 0.1 v / v% Triton X-100;

[0076] Raw materials are provided according to the components of the above specific concentrations. The buffer solution, surfactant, trehalose, chelating agent, betaine, and mannitol are mixed, and then subjected to solid-liquid separation and sterilization and impurity removal in sequence to obtain the preservation solution.

[0077] Example 2

[0078] It is basically the same as Example 1, except that the components are as follows:

[0079] 30 mM Tris-HCl buffer, 300 mM trehalose, 4 mM EDTA, 8 wt% mannitol, 800 mM betaine, 0.50 v / v% Triton X-100.

[0080] Example 3

[0081] It is basically the same as Example 1, except that the components are as follows:

[0082] 5 mM Tris-HCl buffer, 500 mM trehalose, 0.5 mM EDTA, 1 wt% mannitol, 100 mM betaine, 1 v / v% Triton X-100.

[0083] Example 4

[0084] It is basically the same as Example 1, except that the components are as follows:

[0085] 50 mM Tris-HCl buffer, 50 mM trehalose, 5 mM EDTA, 10 wt% mannitol, 1000 mM betaine, 0.05 v / v% Triton X-100.

[0086] Comparative Example 1

[0087] Same as Example 1, except that the components are as follows:

[0088] 50 mM Tris-HCl buffer, 3 wt% bovine serum albumin (BSA), 100 mM trehalose, 3 mM EDTA.

[0089] Comparative Example 2

[0090] Same as Example 1, except that the components are as follows:

[0091] 10 mM Tris-HCl buffer, 3 wt% bovine serum albumin (BSA), 100 mM trehalose, 1 mM EDTA.

[0092] Comparative Example 3

[0093] Same as Example 1, except that the components are as follows:

[0094] 10 mM Tris-HCl buffer, 3 wt% bovine serum albumin (BSA), 100 mM trehalose, 1 mM EDTA, 10 v / v% glycerol.

[0095] Comparative Example 4

[0096] Same as Example 1, except that the components are as follows:

[0097] 10 mM Tris-HCl buffer, 3 wt% bovine serum albumin (BSA), 100 mM trehalose, 1 mM EDTA, 10 v / v% glycerol, 5 wt% mannitol.

[0098] Comparative Example 5

[0099] Same as Example 1, except that the components are as follows:

[0100] 10 mM Tris-HCl buffer, 3 wt% bovine serum albumin (BSA), 75 mM trehalose, 1 mM EDTA, 100 mM KCl.

[0101] Comparative Example 6

[0102] Same as Example 1, except that the components are as follows:

[0103] 10 mM Tris-HCl buffer, 3 wt% bovine serum albumin (BSA), 120 mM trehalose, 1 mM EDTA, 100 mM KCl.

[0104] Comparative Example 7

[0105] Same as Example 1, except that the components are as follows:

[0106] 10 mM Tris-HCl buffer, 3 wt% bovine serum albumin (BSA), 150 mM trehalose, 1 mM EDTA, 100 mM KCl.

[0107] Comparative Example 8

[0108] Same as Example 1, except that the components are as follows:

[0109] 10 mM Tris-HCl buffer, 150 mM trehalose, 1 mM EDTA, 5 v / v% glycerol, 500 mM betaine.

[0110] Comparative Example 9

[0111] Same as Example 1, except that the components are as follows:

[0112] 10 mM Tris-HCl buffer, 60 mM trehalose, 1 mM EDTA, 2 wt% mannitol, 100 mM KCl, 2 wt% glycine.

[0113] Experiment 1: Test the accelerated stability of the preservation solutions prepared in each example and comparative example on plasmid DNA

[0114] 1. Instruments, reagents, samples

[0115] 1) Main instruments: BioRad CFX Opus 96 Real-Time PCR Systems, palm centrifuge, biological safety cabinet, laminar flow hood, constant temperature incubator;

[0116] 2) Main reagents: Primers for detecting hepatitis B virus PreS2 gene, primers for detecting hepatitis B virus P gene, primers for internal reference gene RNase P gene, qPCR reaction detection reagents.

[0117] 2. Experimental protocol:

[0118] 1) The mixed plasmid containing the hepatitis B virus gene and the internal reference gene RNaseP fragment was prepared with the preservation solution prepared in each embodiment and comparative example, and stored at ≤-20°C and 37°C for 5 days, respectively. After reaching the treatment time point, it was taken out and detected by PCR fluorescent probe method. Each sample was tested 3 times, with the treatment at ≤-20°C as the control group and the treatment at 37°C as the test group. The absolute value of the deviation of the mean Ct value of the test was calculated.

[0119] 丨△Ct丨≤1.5Ct is the acceptance standard for evaluating the quality of the preservation solution formula (the stability of plasmids and pseudoviruses is judged according to the provisions of 5.4.2 of the nucleic acid amplification detection reagents (kits) in the "Pharmaceutical Industry Standards of the People's Republic of China". The absolute deviation of accuracy does not exceed ±0.5 logarithmic orders of magnitude; in PCR amplification, the template concentration is 2 n Increase, after about 3.3 cycles (2 3.3 ≈10), the template concentration increases 10 times, that is, the template concentration increases by one logarithmic order of magnitude; in the real-time fluorescence quantitative PCR reaction, the Ct value increases by 1 Ct value for each additional cycle number. Therefore, according to the above accuracy requirements, the absolute deviation of the accuracy shall not exceed ±1.5Ct).

[0120] 2) The qPCR reaction system was prepared as follows:

[0121]

[0122] The amplification reaction program is shown in Table 1:

[0123] Table 1

[0124]

[0125] The PCR amplification results of Examples 1 to 4 and Comparative Examples 1 to 9 are as follows: Figures 1 to 13 As shown. Among them, Figures 1 to 4 A in the figure represents the amplification curve of the FAM fluorescence channel, B in the figure represents the amplification curve of the VIC fluorescence channel, and C in the figure represents the amplification curve of the CY5 fluorescence channel; Figures 5 to 13 (a) in the figure represents the amplification curve of the FAM fluorescence channel, (b) in the figure represents the amplification curve of the CY5 fluorescence channel, and (c) in the figure represents the amplification curve of the VIC fluorescence channel; "Before treatment" represents the amplification curve stored at ≤-20°C and not accelerated at 37°C, and "After treatment" represents the amplification curve accelerated at 37°C; the vertical axis RFU in the figure is the fluorescence intensity. Figures 1 to 4 The results showed that the Ct value after treatment was slightly lower than that before treatment, which showed a good effect on the preservation effect of the preservation solution on the DNA sample; Figures 5 to 13The results show that the Ct values after treatment are significantly increased compared with those before treatment, and are significantly greater than 1.5 Ct, indicating that the preservation solutions of Comparative Examples 1-9 have poor preservation effects on plasmids, and there is a large degree of degradation after treatment at 37 °C for 5 days.

[0126] The amplified Ct values are shown in Table 2.

[0127] Table 2

[0128]

[0129]

[0130]

[0131] From Figures 1 to 13 and Table 2, it can be seen that after 5 days of accelerated treatment at 37 °C, the absolute deviation of Ct before and after sample treatment in Examples 1-4, |ΔCt| < 1.5, indicating that the preservation solutions of Examples 1-4 have the effect of stabilizing the structural integrity of plasmid DNA. While for the samples of Comparative Examples 1-9, the absolute deviation of Ct before and after sample treatment, |ΔCt| > 1.5, all do not meet the requirements.

[0132] Experiment 2: Test the effect of the preservation solution prepared in Example 1 on the stability of plasmid DNA

[0133] 1. The instruments, reagents, and samples are the same as in Experiment 1.

[0134] 2. Experimental protocol:

[0135] 1) Prepare three groups of mixed plasmids containing hepatitis B virus gene and internal reference gene RNase P fragment with high concentration (10 -3 ng / μL), medium concentration (10 -5 ng / μL), and low concentration (10 - 7 ng / μL) of the preservation solution of Example 1, and store them at ≤ -20 °C and 30 °C respectively for 1 month, 3 months, 6 months, 9 months, and 12 months. After reaching the treatment time point, take them out for detection. The fluorescence probe method is used for detection, and each sample is tested 3 times. The sample treated at ≤ -20 °C for 0 months is used as the control group, and the sample treated at 30 °C is used as the experimental group. Calculate the absolute value of the deviation of the average Ct value of the test, |ΔCt|; |ΔCt| ≤ 1.5 Ct is used as the acceptance criterion to evaluate the stability effect of the preservation solution on plasmid DNA.

[0136] 2) The preparation of the qPCR reaction system and the amplification reaction program are the same as in Experiment 1;

[0137] The stability results of the preservation solution of Example 1 for plasmid DNA stored at 30 °C are shown in Table 3.

[0138] Table 3

[0139]

[0140]

[0141] As can be seen from Table 3, the preservation solution of Example 1 was stored at 30°C for 12 months. The detected Ct values of plasmid DNA at high, medium, and low concentrations were compared with those stored at -20°C for 0 months, and |ΔCt| < 1.5, indicating that the preservation solution of Example 1 can stabilize plasmid DNA for at least 12 months.

[0142] Experiment 3: Testing the effect of the preservation solution prepared in Example 1 on the stability of pseudovirus samples

[0143] 1. Instruments, reagents, samples

[0144] 1) Main instruments: BioRad CFX Opus 96 Real-Time PCR Systems, hand-held centrifuge, biological safety cabinet, ultra-clean workbench, constant temperature incubator;

[0145] 2) Main reagents: Primers for detecting hepatitis B virus PreS2 gene, primers for detecting hepatitis B virus P gene, primers for internal reference gene RNase P gene, qPCR reaction detection reagent, TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 (product number 9766) nucleic acid extraction reagent.

[0146] 2. Experimental protocol:

[0147] 1) Dilute hepatitis B virus pseudovirus into high (10 5 copies / mL), medium (10 3 copies / mL), and low (500 copies / mL) concentration pseudovirus samples using the preservation solution of Example 1, and store them at ≤ -20°C and 30°C for 1 month, 3 months, 6 months, 9 months, and 12 months respectively. After reaching the treatment time point, take them out, equilibrate to room temperature, and then use the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 nucleic acid extraction reagent of Takara company to extract hepatitis B virus DNA for detection according to the instructions. Fluorescent probe method is used for detection, and each sample is tested 3 times. Samples treated at ≤ -20°C for 0 months are used as the control group, and samples treated at 30°C are used as the experimental group, and |ΔCt| is calculated. |ΔCt| ≤ 1.5 Ct is used as the acceptance criterion to evaluate the stability effect of the preservation solution on pseudovirus.

[0148] 2) The preparation of the qPCR reaction system and the amplification reaction program were the same as those in Experiment 1;

[0149] The stability results of the storage solution of Example 1 for the pseudovirus stored at 30 °C are shown in Table 4.

[0150] Table 4

[0151]

[0152]

[0153] As can be seen from Table 4, when the storage solution of Example 1 was stored at 30 °C for 12 months, the absolute deviation of the detected Ct values of the pseudoviruses at high, medium, and low concentrations from those stored at -20 °C for 0 months was less than 1.5, i.e., |ΔCt| < 1.5, indicating that the storage solution of Example 1 can stabilize the pseudovirus for at least 12 months.

[0154] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0155] The above-described embodiments only represent several implementation manners of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the protection scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A deoxyribonucleic acid and pseudovirus preservation solution, characterized in that, The DNA and pseudovirus preservation solution consists of the following components: a buffer with a final concentration of 5 mM to 50 mM, a surfactant with a volume percentage of 0.05% to 1.0%, trehalose with a final concentration of 50 mM to 500 mM, a metal ion chelator with a final concentration of 0.5 mM to 5 mM, betaine with a final concentration of 100 mM to 1000 mM, mannitol with a mass percentage of 1% to 10%, and the balance being nuclease-free water; the buffer is Tris-HCl buffer, the surfactant is TritonX-100, and the metal ion chelator is selected from at least one of sodium EDTA and potassium EDTA.

2. The DNA and pseudovirus preservation solution according to claim 1, characterized in that, The DNA and pseudovirus preservation solution consists of the following components: a buffer with a final concentration of 5 mM to 30 mM, a surfactant with a volume percentage of 0.05% to 0.5%, trehalose with a final concentration of 50 mM to 300 mM, a metal ion chelator with a final concentration of 0.5 mM to 4 mM, betaine with a final concentration of 200 mM to 800 mM, mannitol with a mass percentage of 2% to 8%, and the balance being nuclease-free water.

3. The deoxyribonucleic acid and pseudovirus preservation solution according to claim 1, characterized in that, The DNA and pseudovirus preservation solution consists of the following components: a buffer with a final concentration of 5 mM to 15 mM, a surfactant with a volume percentage of 0.05% to 0.3%, trehalose with a final concentration of 50 mM to 200 mM, a metal ion chelator with a final concentration of 0.5 mM to 2 mM, betaine with a final concentration of 300 mM to 600 mM, mannitol with a mass percentage of 3% to 6%, and the balance being nuclease-free water.

4. The deoxyribonucleic acid and pseudovirus preservation solution according to claim 3, wherein, The DNA and pseudovirus preservation solution consists of the following components: a buffer with a final concentration of 10 mM, a surfactant with a volume percentage of 0.1%, trehalose with a final concentration of 150 mM, a metal ion chelator with a final concentration of 1 mM, betaine with a final concentration of 500 mM, mannitol with a mass percentage of 5%, and the balance being nuclease-free water.

5. A method for preparing a deoxyribonucleic acid and pseudovirus preservation solution, characterized in that, It includes the following steps: Providing raw materials according to the components of the DNA and pseudovirus preservation solution described in any one of claims 1 to 4; After mixing the buffer, surfactant, trehalose, chelator, betaine and mannitol, performing sterilization and impurity removal to obtain the DNA and pseudovirus preservation solution.

6. The preparation method of the deoxyribonucleic acid and pseudovirus preservation solution according to claim 5, characterized in that, Before the mixing step, it further includes adjusting the pH value of the buffer to 7.5 to 8.

5.

7. The preparation method of the deoxyribonucleic acid and pseudovirus preservation solution according to claim 5, characterized in that, The sterilization and impurity removal are performed by filtration.

8. Use of the DNA and pseudovirus preservation solution according to any one of claims 1 to 4 for preserving DNA or pseudovirus.

9. Use of the DNA and pseudovirus preservation solution according to any one of claims 1 to 4 in the preparation of in vitro diagnostic reagents containing DNA or pseudovirus.

Images