Protective agent for nucleic acid substances and application of protective agent

Abstract

The invention discloses a protective agent for nucleic acid substances and application of the protective agent. The protective agent comprises nanoparticles, trehalose, mannitol and bovine serum albumin, wherein the nanoparticles are provided with cavities capable of wrapping nucleic acid substances. The protective agent can maintain the structure of nucleic acid substances in the high-temperature drying process and reduce the water content at the same time, and normal-temperature storage and transportation of the nucleic acid substances are achieved; the stability of nucleic acid substances at lower concentration can be improved, and the sensitivity or the detection rate of the PCR detection kit is improved.

Description

Technical Field

[0001] The present application belongs to the field of molecular biology detection, and specifically relates to a protective agent for nucleic acid substances and its application, and further relates to a protective agent for nucleic acid substances, a high-temperature drying protection method for nucleic acid substances, a nucleic acid detection system and a nucleic acid detection kit. Background Art

[0002] Nucleic acid sample processing usually involves high temperatures. From the perspective of drying technology, high-temperature drying uses simple equipment, short drying time, low drying cost, and simple operation. However, high temperatures may cause instability in the nucleic acid structure. Therefore, it is necessary to develop a protective agent for nucleic acid substances. Summary of the invention

[0003] Based on this, it is necessary to provide a protective agent for nucleic acid substances and its application, and further provide a protective agent for nucleic acid substances, a high-temperature drying protection method for nucleic acid substances, a nucleic acid detection system and a nucleic acid detection kit.

[0004] The first aspect of the present application provides a protective agent for nucleic acid substances, the protective agent comprising nanoparticles, trehalose, mannitol and bovine serum albumin; wherein the nanoparticles have a cavity capable of encapsulating nucleic acid substances.

[0005] In some embodiments, the protective agent further includes an aqueous solvent, the volume percentage concentration of the nanoparticles in the protective agent is 0.1%~2%, the mass volume fraction of the trehalose in the protective agent is 20%~30%, the mass volume fraction of the mannitol in the protective agent is 5%~10%, and the mass volume fraction of bovine serum albumin in the protective agent is 0.1%~2%.

[0006] In some embodiments, the nanoparticle is a lipid nanoparticle.

[0007] In some embodiments, the nanoparticles satisfy one or more of the following characteristics:

[0008] The particle size of the lipid nanoparticles is 18nm~22nm;

[0009] The lipid nanoparticles include ionizable lipids, phospholipids and cholesterol. Optionally, in the nanoparticles, the weight ratio of the ionizable lipids, phospholipids and cholesterol is (0.9~1.1): (2.9~3.1): (0.9~1.1).

[0010] The second aspect of the present application provides a high-temperature drying protection method for nucleic acid substances, the method comprising: mixing the nucleic acid substances with the protective agent described in the first aspect of the present application to obtain a mixture, and high-temperature drying the mixture, wherein the temperature of the high-temperature drying is 70°C to 95°C.

[0011] In some embodiments, the high temperature drying protection method satisfies one or more of the following characteristics:

[0012] The volume of the nucleic acid substance is 10 μL to 20 μL;

[0013] The nucleic acid substance includes one or more of plasmid, bacterial solution, DNA, RNA and pseudovirus, and the concentration of the nucleic acid substance in the mixture is 1×10 3 IU / mL or more; the high temperature drying time is 15min~120min.

[0014] The third aspect of the present application provides a nucleic acid detection system, which includes a nucleic acid substance and a protective agent for the nucleic acid substance described in the first aspect of the present application.

[0015] In some embodiments, the nucleic acid detection system meets one or more of the following characteristics:

[0016] Nucleic acid substances include one or more of DNA, RNA, plasmid, bacterial solution and pseudovirus;

[0017] The concentration of nucleic acid substances in the nucleic acid detection system is greater than or equal to 1×10 3 IU / mL.

[0018] The fourth aspect of the present application provides the use of the protective agent described in the first aspect of the present application in the preparation of a nucleic acid detection kit.

[0019] The fifth aspect of the present application provides a nucleic acid detection kit, which includes the protective agent and nucleic acid detection reagent described in the first aspect of the present application.

[0020] In some embodiments, the nucleic acid detection reagent is used for PCR detection.

[0021] Among the aforementioned protective agents, the synergistic effect of the combined use of nanoparticles, trehalose, mannitol and bovine serum albumin (BSA) has a good protective effect on nucleic acid substances such as DNA, RNA, plasmids, bacterial liquid, pseudoviruses, etc. commonly used in the field of molecular biology detection after high-temperature drying, as well as nucleic acids after extraction from clinical samples, thereby achieving room temperature storage and transportation of nucleic acid substances.

[0022] Furthermore, through the synergistic effect of the combined use of nanoparticles, trehalose, mannitol and BSA, the stability of nucleic acid substances such as plasmids, bacterial liquids, pseudoviruses, DNA and RNA at lower concentrations can be improved, and the sensitivity or detection rate of PCR detection kits can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0024] Figure 1 This is a real-time stability monitoring diagram of different nucleic acid substances at room temperature after drying in one embodiment of the present application;

[0025] Figure 2 This is a comparative test diagram of three different systems after drying in one embodiment of the present application;

[0026] Figure 3 This is an accelerated stability monitoring diagram of three different systems after drying in one embodiment of the present application;

[0027] Figure 4 This is a real-time stability monitoring diagram of three different systems at room temperature after drying in one embodiment of the present application;

[0028] Figure 5 A in FIG. 1 is a diagram of nucleic acid extraction detection using an eluent containing a protective agent in one embodiment of the present application. Figure 5 B in the figure is a nucleic acid extraction detection diagram using an eluent without a protective agent in one embodiment of the present application. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application 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.

[0031] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0032] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0033] The terms "having", "containing", "including" and "comprising" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0034] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0035] In this application, if the unit of the data range is followed only by the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 10-20 μL means that the units of the left endpoint "10" and the right endpoint "20" are both μL, which have the same meaning as 10 μL-20 μL. In addition, similar descriptions involving other parameters also apply to the above understanding.

[0036] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0037] In this application, "greater than or equal to", "greater than or equal to" and "≥" have the same meaning and can be used interchangeably; "less than or equal to", "less than or equal to" and "≤" have the same meaning and can be used interchangeably; "greater than" can be equivalently expressed as ">" and "less than" can be equivalently expressed as "<". In this application, unless otherwise specified, "greater than or equal to" and "≥" can be regarded as providing two solutions of "greater than" and "equal to". In this application, unless otherwise specified, "less than or equal to" and "≤" can be regarded as providing two solutions of "less than" and "equal to".

[0038] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form a new technical solution.

[0039] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", "the fifth aspect", etc., the terms "first", "second", "third", "fourth", "fifth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", "fifth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0040] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0041] In this application, the term "room temperature" or "normal temperature" generally refers to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 10°C to 30°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0042] In this application, unless otherwise specified, the parameter unit "IU / mL" means international unit per milliliter, and is used to indicate the concentration of a bioactive substance. In some embodiments of this application, 1 IU / mL = 5.8 copies / μL.

[0043] At present, the comparison of technologies used for drying nucleic acid substances is shown in Table 1:

[0044] Table 1

[0045]

[0046] In terms of drying process, high temperature drying has the advantages of simpler equipment, shorter drying time, lower drying cost, and simpler operation than freeze drying.

[0047] The embodiments of the present application at least provide a protective agent for nucleic acid substances and its application. The protective agent includes nanoparticles, trehalose, mannitol and bovine serum albumin; wherein the nanoparticles have a cavity capable of encapsulating nucleic acid substances. The protective agent can maintain the structure of nucleic acid substances and reduce the water content during high-temperature drying, thereby achieving room-temperature storage and transportation of nucleic acid substances; it can also improve the stability of nucleic acid substances at lower concentrations, and improve the sensitivity or detection rate of PCR detection kits.

[0048] In some embodiments, the protective agent for nucleic acid substances provided in the present application has a good protective effect on the nucleic acids of DNA, RNA, plasmids, bacterial liquids, pseudoviruses commonly used in the field of molecular biology detection after high-temperature drying and after extraction from clinical samples.

[0049] In some embodiments, the protective agent for nucleic acid substances provided in the present application is mainly used for high-temperature drying of DNA, RNA, plasmids, bacterial liquids and pseudoviruses. After plasmids, bacterial liquids and pseudoviruses are dried at high temperature using the protective agent of the present application, they can be stored and transported at room temperature, freeing these heat-sensitive nucleic acid substances from temperature restrictions. If frozen in liquid form, the shelf life of plasmids, bacterial liquids and pseudoviruses can also be extended, reducing storage conditions.

[0050] In some embodiments, after the protective agent of nucleic acid substances provided in the present application is diluted to a certain proportion, it can be used as a plasmid, bacterial solution and pseudovirus dilution solution to replace TE Buffer or DEPC water, which can improve the detection rate of PCR detection kits when using these low-concentration templates and improve the sensitivity of the detection kit.

[0051] The first aspect of the present application provides a protective agent for nucleic acid substances.

[0052] In some embodiments, the protective agent includes nanoparticles, sugars, sugar alcohols and bovine serum albumin (BSA); wherein the nanoparticles have a cavity capable of encapsulating nucleic acid substances.

[0053] In some embodiments, the sugar may be, but is not limited to, one or more of trehalose, sucrose, and maltose.

[0054] In some embodiments, the sugar alcohol may be, but is not limited to, one or more of mannitol, xylitol, and maltitol.

[0055] In some embodiments, a protective agent for nucleic acid substances is provided, the protective agent comprising nanoparticles, trehalose, mannitol and bovine serum albumin (BSA); wherein the nanoparticles have a cavity capable of encapsulating the nucleic acid substances.

[0056] In some embodiments, the nucleic acid material includes one or more of DNA, RNA, plasmid, bacterial solution and pseudovirus.

[0057] In the present application, unless otherwise specified, "trehalose" can form a protective film on the surface of nucleic acid molecules to prevent the structure of the nucleic acid from being destroyed when it is dried under high temperature conditions and maintain its structural integrity.

[0058] In the present application, unless otherwise specified, "mannitol" can inhibit the activity of nucleases and can also form a protective film on the surface of nucleic acid molecules, which not only protects the integrity of the nucleic acid structure but also stabilizes the nucleic acid.

[0059] In the present application, unless otherwise specified, "bovine serum albumin" has a blocking effect on nucleases, reduces the activity of nucleases, and can protect nucleic acid molecules.

[0060] In some embodiments, the protectant is a drying protectant.

[0061] In this application, unless otherwise specified, "drying protective agent" refers to a agent mainly used to protect nucleic acids or nucleic acid analogs during the drying process. In some embodiments, the drying temperature can be 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or any temperature or range between the two temperatures.

[0062] In some embodiments, the protective agent further comprises an aqueous solvent. If not otherwise specified, the "aqueous solvent" at least comprises sterile purified water, and non-limitingly, the aqueous solvent may also comprise one or more of DEPC water, a buffer, etc. A non-limiting example of a buffer is 1×TE buffer_.

[0063] In some embodiments, the volume percentage concentration of the nanoparticles in the protective agent is 0.1% to 2%. Further, the volume percentage concentration of the nanoparticles in the protective agent can be 0.1%, 0.5%, 1%, 1.5%, 2% or a value or range between any two of the above values.

[0064] In some embodiments, the mass volume fraction of trehalose in the protective agent is 20% to 30%. Further, the mass volume fraction of trehalose in the protective agent can be 20%, 22%, 24%, 26%, 28%, 30% or a value or range between any two of the above values.

[0065] In some embodiments, the mass volume fraction of mannitol in the protective agent is 5% to 10%. Further, the mass volume fraction of mannitol in the protective agent can be 5%, 6%, 7%, 8%, 9%, 10% or a value or range between any two of the above values.

[0066] In some embodiments, the mass volume fraction of BSA in the protective agent is 0.1% to 2%. Further, the mass volume fraction of BSA in the protective agent can be 0.1%, 0.5%, 1%, 1.5%, 2% or a value or range between any two of the above values.

[0067] In some embodiments, the nanoparticles are lipid nanoparticles.

[0068] In some embodiments, the particle size of the lipid nanoparticles can be 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, or a value or range between any two of the above values.

[0069] In some embodiments, the lipid nanoparticles include ionizable lipids, phospholipids and cholesterol. In a non-limiting manner, the ionizable lipids are selected from at least one of 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester and ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate); the phospholipids are selected from at least one of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine.

[0070] In some embodiments, the weight ratio of ionizable lipids, phospholipids and cholesterol is (0.9~1.1):(2.9~3.1):(0.9~1.1), and further, the weight ratio of ionizable lipids, phospholipids and cholesterol is 1:3:1.

[0071] In some embodiments, the preparation of lipid nanoparticles comprises the following steps: mixing and dissolving ionizable lipids, phospholipids and cholesterol in an organic solvent, ethanol, according to a weight ratio.

[0072] Among the aforementioned protective agents, the synergistic effect of the combined use of nanoparticles, trehalose, mannitol and BSA has a good protective effect on the DNA, RNA, plasmids, bacterial liquids, pseudoviruses commonly used in the field of molecular biology detection after high-temperature drying and nucleic acids extracted from clinical samples.

[0073] Furthermore, through the synergistic effect of the combined use of nanoparticles, trehalose, mannitol and BSA, the stability of nucleic acid substances such as plasmids, bacterial liquids, pseudoviruses, DNA and RNA at lower concentrations can be improved, and the sensitivity or detection rate of PCR detection kits can be improved.

[0074] In one embodiment of the present application, a method for preparing a protective agent for nucleic acid substances is provided, comprising the following steps: mixing nanoparticles, trehalose, mannitol and BSA according to a weight ratio.

[0075] The second aspect of the present application provides a high-temperature drying protection method for nucleic acid substances, comprising: mixing the nucleic acid substances with the protective agent of the first aspect of the present application to obtain a mixture, and drying the mixture at a high temperature of 70°C to 95°C.

[0076] In some embodiments, the nucleic acid material includes one or more of DNA, RNA, plasmid, bacterial solution and pseudovirus.

[0077] In some embodiments, the temperature of high temperature drying can be 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., or any temperature or range between any two of the above temperatures.

[0078] In some embodiments, during high temperature drying, the volume of the nucleic acid substance is 10 μL to 20 μL. Furthermore, the volume of the nucleic acid substance can be 10 μL, 12 μL, 14 μL, 16 μL, 18 μL, 20 μL or a value or range between any two of the above values.

[0079] In some embodiments, during high temperature drying, the concentration of nucleic acid substances is 1×10 3 IU / mL and above. In this application, unless otherwise specified, when “above” is used to describe a value, it includes the value itself. For example, 1×10 3 IU / mL or more means greater than or equal to 1×10 3 IU / mL; without limitation, the concentration of nucleic acid substances can be 1×10 3 IU / mL, 1×10 4 IU / mL, 1×10 5 IU / mL, 1×10 6 IU / mL, 2×10 6IU / mL or a value between or above any two of the above values. In some embodiments, the time of high temperature drying is 15min to 120min, and further, the time of high temperature drying can be 15min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min or any time or range between any two of the above times.

[0080] The third aspect of the present application provides a nucleic acid detection system, which includes a nucleic acid substance and a protective agent for the nucleic acid substance of the first aspect of the present application.

[0081] In some embodiments, the nucleic acid material includes one or more of DNA, RNA, plasmid, bacterial solution and pseudovirus.

[0082] In some embodiments, the concentration of nucleic acid substances in the nucleic acid detection system is greater than or equal to 1×10 3 IU / mL. Furthermore, the concentration of nucleic acid substances can be 1×10 3 IU / mL, 1×10 4 IU / mL, 1×10 5 IU / mL, 1×10 6 IU / mL, 2×10 6 IU / mL or a value between or above any two of the above values.

[0083] The fourth aspect of the present application provides the use of the protective agent of the first aspect of the present application in the preparation of a nucleic acid detection kit.

[0084] The fifth aspect of the present application provides a nucleic acid detection kit, comprising the protective agent and the nucleic acid detection reagent of the first aspect of the present application.

[0085] In some embodiments, the nucleic acid detection reagent can be used for PCR detection. Further, the nucleic acid detection reagent can include enzyme reagents, primers, probes, and nuclease-free water.

[0086] Some examples are provided below.

[0087] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0088] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0089] Trehalose in the following examples was purchased from Aladdin, mannitol was purchased from Sangon Biotech (Shanghai) Co., Ltd., and BSA was purchased from Roche.

[0090] The HBV sample DNA and HCV sample RNA in the following examples were provided by Hubei Provincial People's Hospital, the HBV plasmid and group B Streptococcus (GBS) culture were synthesized by Shanghai Shenggong Biotechnology, and the HBV pseudovirus was synthesized by Shandong Weizhen Biotechnology Co., Ltd.

[0091] Example 1 Preparation of protective agent

[0092] Ionizable lipids, phospholipids, and cholesterol are selected and mixed in a certain weight ratio (1:3:1), dissolved in an organic solvent miscible with water to form an oil phase, and then the oil phase and the aqueous solution are mixed and emulsified through mechanical shearing and other methods to form lipid nanoparticles with an average particle size of 20nm. Finally, impurities are removed by centrifugation and ultrafiltration.

[0093] The lipid nanoparticles, trehalose, mannitol and BSA were mixed to obtain a protective agent, wherein the final concentration of the lipid nanoparticles was 2% (v / v), the final concentration of trehalose was 30% (w / v), the final concentration of mannitol was 10% (w / v), and the final concentration of BSA was 2% (w / v).

[0094] Example 2 Real-time stability study of nucleic acid substances containing protective agents

[0095] The diluent containing the protective agent prepared in Example 1 was used to dilute the high-concentration HBV sample DNA, HCV sample RNA, artificially synthesized HBV plasmid, group B streptococcus (GBS) bacterial solution, and artificially synthesized HBV pseudovirus at least 100 times to obtain a concentration of 1×10 6 IU / mL of HBV DNA, 1×10 7 IU / mL HCV RNA, 2×10 5 IU / mL of HBV plasmid, 2×10 5 IU / mL of HBV pseudovirus, 5×10 4 CFU / mL of GBS bacterial solution, and then batch drying treatment was carried out. The specific experimental steps of drying were as follows: the solutions of the above-mentioned nucleic acid substances were placed in a constant temperature metal bath, the volume of the solution was 10 μL, and high-temperature drying was carried out at 85°C for 45 minutes.

[0096] Real-time stability monitoring was performed on each nucleic acid substance after drying. The reaction procedures of each nucleic acid substance are shown in Tables 2 to 4. The test was performed once every 3 months. Three tubes of each nucleic acid substance were tested in parallel each time. The Ct values ​​of the three test results were counted and the average was taken for plotting. The results are shown in Figure 1 The results show that using the protective agent prepared in Example 1 as the diluent can make the dried nucleic acid material present good room temperature stability.

[0097] Table 2 HBV amplification procedure

[0098]

[0099] Table 3 HCV amplification procedure

[0100]

[0101] Table 4 GBS amplification procedure

[0102]

[0103] Comparative Example 1: Performance comparison of a protective agent containing nanoparticles (Example 1), a protective agent without nanoparticles, and nucleic acid without a protective agent after high-temperature drying

[0104] The protective agent was prepared by the method of Example 1, except that lipid nanoparticles were not added, thereby obtaining a protective agent without nanoparticles.

[0105] High-concentration HBV DNA was diluted with a protective agent containing nanoparticles, a protective agent without nanoparticles, and a normal diluent without a protective agent at the same time, and then dried simultaneously. After drying, they were tested together on the machine. The test results are as follows: Figure 2 As shown. Figure 2 It can be seen that after the nucleic acid substances without protective agents are dried, the Ct value of the nucleic acid substances without protective agents is the largest at the same concentration of the starting template, and its performance is significantly deteriorated. The results show that the diluent containing protective agents has a good protective effect on the drying of nucleic acid substances, and the protective agent with lipid nanoparticles has a better protective effect after drying than the protective agent without lipid nanoparticles.

[0106] Comparative Example 2: Accelerated stability study at 50°C after drying of protective agent containing nanoparticles (Example 1), protective agent without nanoparticles, and nucleic acid-based substances without protective agent

[0107] The protective agent containing nanoparticles (Example 1), the protective agent without nanoparticles, and the diluent without protective agent were diluted to 2×10 6IU / mL, synchronous batch drying treatment was performed, the drying steps were consistent with Example 2, and after drying, accelerated stability monitoring was performed at 50°C, once a week, and 3 tubes of each dilution were tested in parallel each time. The Ct values ​​of the 3 tube test results were counted and the average was taken for plotting. The results are shown in Figure 3 The results show that the nucleic acid containing the protective agent of nanoparticles has better accelerated stability at 50°C after drying than the nucleic acid without the protective agent and the nucleic acid without the protective agent of nanoparticles.

[0108] Comparative Example 3: Study on the room temperature stability of protective agents containing nanoparticles (Example 1), protective agents without nanoparticles, and nucleic acid substances without protective agents after drying

[0109] The protective agent containing nanoparticles (Example 1), the protective agent without nanoparticles, and the diluent without protective agent were diluted to 2×10 6 IU / mL, and then batch drying was performed synchronously. After drying, real-time stability monitoring was performed. The test was performed once every 3 months. Each time, 3 tubes of each dilution were tested in parallel. The Ct values ​​of the 3 tube test results were counted and the average was taken for plotting. The results are shown in the figure. Figure 4 The results show that the room temperature stability of the nucleic acid containing the protective agent of nanoparticles after drying is better than that of the nucleic acid without the protective agent and the nucleic acid without the protective agent of nanoparticles after drying.

[0110] Comparative Example 4: Comparative detection of nucleic acids containing the protective agent prepared in Example 1 using different drying methods

[0111] The HBV DNA containing the protective agent prepared in Example 1 was freeze-dried, naturally air-dried, and dried at high temperature. The freeze-drying method used pre-freezing at -40°C for 3 hours, sublimation at -38°C 20Pa for 8 hours, and analytical drying at 35°C for 3 hours; the natural air-drying method used room temperature for 48 hours; the experimental steps and parameters of high-temperature drying were the same as those in Example 2. The nucleic acid was dried by these three drying methods and then compared and detected through the FAM channel. The reaction procedure was the same as that in Example 2, and the detection results were shown in Table 5 below.

[0112] Table 5

[0113]

[0114] The room temperature stability of the nucleic acids dried by the above three drying methods was studied, and the test results are shown in Table 6. The results show that although there is no significant difference in the initial test performance results of the nucleic acids dried by the three drying methods, the long-term stability of the nucleic acids dried at high temperature is better than that of freeze-dried and naturally air-dried nucleic acids at room temperature.

[0115] Table 6

[0116]

[0117] Comparative Example 5: Adding the protective agent prepared in Example 1 to the extraction reagent eluent

[0118] The same whole blood sample (clinical sample 1 and clinical sample 2) was extracted using a nucleic acid extraction reagent eluent containing the protective agent prepared in Example 1 and an eluent without the protective agent. Clinical sample 1 and clinical sample 2 were samples from different patients from Guangzhou Women and Children's Medical Center (Central Laboratory). The extracted nucleic acid was simultaneously tested for apolipoprotein E (Apolipoprotein E, APOE) genotyping using a human APOE genotyping detection kit (produced by Guangzhou Baochuang Biotechnology Co., Ltd.). The test results are as follows: Figure 5 As shown in A and B in the figure. The results show that when the protective agent prepared in Example 1 is added to the eluent ( Figure 5 A in the figure can not only greatly improve the quality of extracted nucleic acid, but also improve the anti-interference ability of the amplification system.

[0119] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0120] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the scope of the claims.

Claims

1. A protective agent for nucleic acid substances, characterized in that: The protective agent comprises nanoparticles, trehalose, mannitol and bovine serum albumin; wherein the nanoparticles have cavities capable of encapsulating nucleic acid substances.

2. The protective agent according to claim 1, characterized in that The protective agent also includes an aqueous solvent, the volume percentage concentration of the nanoparticles in the protective agent is 0.1%-2%, the mass volume fraction of the trehalose in the protective agent is 20%-30%, the mass volume fraction of the mannitol in the protective agent is 5%-10%, and the mass volume fraction of bovine serum albumin in the protective agent is 0.1%-2%.

3. The protective agent according to claim 1 or 2, characterized in that The nanoparticles are lipid nanoparticles.

4. The protective agent according to claim 3, characterized in that The nanoparticles satisfy one or more of the following characteristics: The particle size of the lipid nanoparticles is 18nm~22nm; The lipid nanoparticles include ionizable lipids, phospholipids and cholesterol. Optionally, in the nanoparticles, the weight ratio of the ionizable lipids, phospholipids and cholesterol is (0.9~1.1): (2.9~3.1): (0.9~1.1).

5. A high temperature drying protection method for nucleic acid substances, characterized in that: The high-temperature drying protection method comprises: mixing a nucleic acid substance with the protective agent according to any one of claims 1 to 4 to obtain a mixture, and drying the mixture at a high temperature of 70°C to 95°C.

6. The high temperature drying protection method according to claim 5, characterized in that: The high temperature drying protection method meets one or more of the following characteristics: The volume of the nucleic acid substance is 10 μL to 20 μL; The nucleic acid substance includes one or more of plasmid, bacterial solution, DNA, RNA and pseudovirus, and the concentration of the nucleic acid substance in the mixture is 1×10 3 IU / mL or more; The high temperature drying time is 15min to 120min.

7. A nucleic acid detection system, characterized in that: The nucleic acid detection system comprises a nucleic acid substance and a protective agent for the nucleic acid substance according to any one of claims 1 to 4.

8. The nucleic acid detection system according to claim 7, characterized in that: Meet one or more of the following characteristics: The nucleic acid substance includes one or more of DNA, RNA, plasmid, bacterial solution and pseudovirus; The concentration of the nucleic acid substance in the nucleic acid detection system is greater than or equal to 1×10 3 IU / mL.

9. Use of the protective agent according to any one of claims 1 to 4 in the preparation of a nucleic acid detection kit.

10. A nucleic acid detection kit, characterized in that: The nucleic acid detection kit comprises the protective agent and the nucleic acid detection reagent according to any one of claims 1 to 4; Optionally, the nucleic acid detection reagent is used for PCR detection.

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