A conductive nucleic acid bioprotectant

Abstract

This invention relates to a conductive nucleic acid bioprotective agent. Specifically, this invention relates to a conductive nucleic acid bioprotective agent comprising [Mg2(thd)4(dmeda)(edta)]. The conductive nucleic acid bioprotective agent of this invention can effectively protect nucleic acids and has excellent conductivity.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection, and more specifically, to a conductive nucleic acid bioprotectant. Background Technology

[0002] Viruses are composed of a nucleic acid molecule and proteins, or only proteins. Nucleic acid detection kits are in vitro diagnostic reagents used for the rapid qualitative detection of specific fragments of the novel coronavirus, such as the RdRp gene, N gene, and E gene. SARS-CoV-2 is a single-stranded, positive-sense RNA-enveloped beta-coronavirus whose genome encodes non-structural proteins, structural proteins, and accessory proteins. RNA detection is the most crucial step in detecting the novel coronavirus; however, RNA itself is highly susceptible to degradation, with endogenous RNases and exogenous RNases being the most significant causes. Therefore, regardless of whether the sample is tissue, cell, or liquid, it should be properly preserved as soon as possible after leaving its optimal cellular state to prevent RNA or DNA contamination and damage. However, current technology lacks an effective reagent for protecting nucleic acids.

[0003] Therefore, there is a need in this field to develop a reagent that can effectively protect nucleic acids. Summary of the Invention

[0004] The purpose of this invention is to provide a conductive nucleic acid bioprotectant that effectively protects nucleic acids.

[0005] The first aspect of the present invention provides a conductive nucleic acid bioprotective agent, wherein the conductive nucleic acid bioprotective agent comprises [Mg2(thd)4(dmeda)(edta)].

[0006] Preferably, the [Mg2(thd)4(dmeda)(edta)] is a coordination compound.

[0007] Preferably, the [Mg2(thd)4(dmeda)(edta)] is a crystal.

[0008] Preferably, the conductive nucleic acid bioprotectant also includes Trizol.

[0009] Preferably, the nucleic acid includes RNA and / or DNA.

[0010] A second aspect of the present invention provides a method for preparing [Mg2(thd)4(dmeda)(edta)], the method comprising the steps of:

[0011] (1) Dissolve dimethylethylenediamine in n-hexane to obtain a dimethylethylenediamine solution;

[0012] (2) Add [Mg2(thd)4] to the dimethyl ethylenediamine solution obtained in step (1), stir and mix, evaporate the solution under nitrogen protection, wash the resulting crystals with water and n-hexane in sequence, and then sublimate and purify to collect the standard crystals of [Mg2(thd)4(dmeda)].

[0013] (3) Weigh the standard crystals of [Mg2(thd)4(dmeda)] and ethylenediaminetetraacetic acid (EDTA), mix them evenly, and then disperse them in a mixture of Trizol and water. After the reaction, a transparent solution is obtained.

[0014] (4) After slowly cooling the transparent solution obtained in step (3) to 10-30℃, filter to remove impurities, and crystallize at 1-6℃ under argon protection to separate [Mg2(thd)4(dmeda)(edta)].

[0015] Preferably, in step (3), the water includes deionized water.

[0016] Preferably, in step (3), the volume ratio of Trizol to water is (0.5-1.5):(0.5-1.5), more preferably (0.8-1.2):(0.8-1.2), and even more preferably 1:1.

[0017] Preferably, in step (4), after crystallization, the obtained crystals are collected, washed, refined, and dried in a vacuum to obtain [Mg2(thd)4(dmeda)(edta)].

[0018] Preferably, in step (4), the crystallization temperature is 1-3℃, more preferably 1.5-2.5℃.

[0019] Preferably, the method includes:

[0020] (1) Dissolve 1.0-1.4 mol of dimethylethylenediamine in 190-210 ml of n-hexane to obtain a dimethylethylenediamine solution;

[0021] (2) Add 1.3-1.7 mol [Mg2(thd)4] to the dimethyl ethylenediamine solution obtained in step (1), stir at 18-22℃ for 13-17 min, evaporate the solution under nitrogen protection, wash the resulting crystals with water and n-hexane in sequence, and then sublimate to purify and collect the standard crystals of [Mg2(thd)4(dmeda)].

[0022] (3) Weigh 0.6-1.0 mol of [Mg2(thd)4(dmeda)] standard crystals and 0.8-1.2 mol of ethylenediaminetetraacetic acid (EDTA), mix them evenly, and then disperse them in 130-150 ml of a mixed solution of Trizol and water with a volume ratio of (0.8-1.2):(0.8-1.2). React at 55-65℃ for 18-22 min to obtain a transparent solution.

[0023] (4) After slowly cooling the transparent solution obtained in step (3) to 18-22℃, filter to remove impurities, and crystallize at 1.5-2.5℃ under argon protection to separate [Mg2(thd)4(dmeda)(edta)].

[0024] Preferably, the method includes:

[0025] (1) Dissolve 1.2 mol of dimethylethylenediamine in 200 ml of n-hexane to obtain a dimethylethylenediamine solution;

[0026] (2) Add 1.5 mol [Mg2(thd)4] to the dimethyl ethylenediamine solution obtained in step (1), stir at 20°C for 15 min, evaporate the solution under nitrogen protection, wash the obtained crystals with water and n-hexane in sequence, and then sublimate to purify and collect the standard crystals of [Mg2(thd)4(dmeda)].

[0027] (3) Weigh 0.8 mol of [Mg2(thd)4(dmeda)] standard crystals and 1.0 mol of ethylenediaminetetraacetic acid (EDTA), mix them evenly and disperse them in 140 ml of Trizol and water mixed solution with a volume ratio of 1:1, react at 60 °C for 20 min to obtain a transparent solution;

[0028] (4) After slowly cooling the transparent solution obtained in step (3) to 20°C, filter to remove impurities, and crystallize at 2°C under argon protection to obtain [Mg2(thd)4(dmeda)(edta)].

[0029] A third aspect of the present invention provides a composition comprising a conductive nucleic acid bioprotectant as described in the first aspect of the present invention.

[0030] Preferably, the composition is a reagent composition.

[0031] Preferably, the composition further includes a reagent-acceptable carrier.

[0032] Preferably, the dosage form of the composition is a liquid formulation.

[0033] A fourth aspect of the present invention provides the use of the conductive nucleic acid bioprotectant as described in the first aspect of the present invention for preparing reagents for protecting nucleic acids.

[0034] Preferably, the nucleic acid includes RNA and / or DNA.

[0035] Preferably, the nucleic acid includes viral nucleic acid.

[0036] Preferably, the nucleic acid includes Sars-cov-2 RNA.

[0037] The fifth aspect of the present invention provides a nucleic acid detection kit, wherein the nucleic acid detection kit is a conductive nucleic acid biological protectant as described in the first aspect of the present invention.

[0038] Preferably, the nucleic acid detection kit further includes nucleic acid detection reagents.

[0039] The sixth aspect of the present invention provides a method for protecting nucleic acids by contacting the nucleic acids with a conductive nucleic acid bioprotectant as described in the first aspect of the present invention, thereby protecting the nucleic acids.

[0040] Preferably, the contact is in vivo or in vitro.

[0041] Preferably, the method includes in vitro or in vivo methods.

[0042] Preferably, the method is a non-diagnostic and non-therapeutic method.

[0043] Preferably, the nucleic acid includes RNA and / or DNA.

[0044] Within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below can be combined with each other to form new or preferred technical solutions. Attached Figure Description

[0045] Figure 1 To investigate the protective effect of [Mg2(thd)4(dmeda)(edta)] prepared in Example 1 on nucleic acids.

[0046] Figure 2 To investigate the electrical conductivity of [Mg2(thd)4(dmeda)(edta)] prepared in Example 1. Detailed Implementation

[0047] This invention develops a conductive nucleic acid bioprotective agent, comprising [Mg2(thd)4(dmeda)(edta)]. The conductive nucleic acid bioprotective agent of this invention can effectively protect nucleic acids and exhibits excellent conductivity.

[0048] the term

[0049] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only open-ended definitions but also semi-closed and closed definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0050] As used herein, the terms “TRIZOL,” “Trizol,” “TRIZOL reagent,” and “TRIZOL” are used interchangeably. @ "Regent" is an interchangeable term and is a total RNA extraction reagent that can directly extract total RNA from cells or tissues. Trizol contains substances such as guanidine isothiocyanate, which can rapidly lyse cells and inhibit the activity of nucleases released by cells. In homogenizing or dissolving samples, Trizol reagent can maintain the integrity of RNA while destroying cells and dissolving cell components.

[0051] As used in this article, the term "DMEDA" refers to dimethylethylenediamine, abbreviated as dmeda, and its English name is N,N'-Dimethylethylenediamine.

[0052] As used in this article, the term "EDTA" refers to ethylenediaminetetraacetic acid, abbreviated as edta.

[0053] As used in this article, the term "thd" refers to 2,2,6,6-tetramethyl-3,5-heptadecane.

[0054] [Mg2(thd)4(dmeda)(edta)] and its preparation method

[0055] This invention provides a [Mg2(thd)4(dmeda)(edta)], which can effectively protect nucleic acids and has excellent conductivity.

[0056] This invention also provides a method for preparing [Mg2(thd)4(dmeda)(edta)], the method comprising the steps of:

[0057] (1) Dissolve dimethylethylenediamine in n-hexane to obtain a dimethylethylenediamine solution;

[0058] (2) Add [Mg2(thd)4] to the dimethyl ethylenediamine solution obtained in step (1), stir and mix, evaporate the solution under nitrogen protection, wash the resulting crystals with water and n-hexane in sequence, and then sublimate and purify to collect the standard crystals of [Mg2(thd)4(dmeda)].

[0059] (3) Weigh the standard crystals of [Mg2(thd)4(dmeda)] and ethylenediaminetetraacetic acid (EDTA), mix them evenly, and then disperse them in a mixture of Trizol and water. After the reaction, a transparent solution is obtained.

[0060] (4) After slowly cooling the transparent solution obtained in step (3) to 10-30℃, filter to remove impurities, and crystallize at 1-6℃ under argon protection to separate [Mg2(thd)4(dmeda)(edta)].

[0061] Specifically, the method is as described in the first aspect of the present invention above.

[0062] Conductive nucleic acid bioprotectants

[0063] This invention provides a conductive nucleic acid bioprotectant, wherein the conductive nucleic acid bioprotectant comprises [Mg2(thd)4(dmeda)(edta)].

[0064] The conductive nucleic acid bioprotectant described in this invention may also include Trizol.

[0065] use

[0066] The present invention also provides the use of the conductive nucleic acid bioprotectant described herein in the preparation of reagents for protecting nucleic acids.

[0067] The nucleic acids described in this invention may include RNA and / or DNA.

[0068] Preferably, the nucleic acid includes viral nucleic acid.

[0069] Preferably, the nucleic acid includes Sars-cov-2 RNA.

[0070] The main superior technical effects of this invention include:

[0071] (1) This invention provides a conductive nucleic acid bioprotectant, comprising [Mg2(thd)4(dmeda)(edta)]. The conductive nucleic acid bioprotectant of this invention can protect nucleic acids from degradation by degrading enzymes, improve the conductivity of the solution, and can be applied in various fields. It is suitable for preparing various nucleic acid activity preservation agents requiring a certain level of conductivity and is suitable for the transport and preservation of nucleic acid detection samples. The conductive nucleic acid bioprotectant of this invention can maintain the protective properties of nucleic acids, providing long-term protection for viral nucleic acids at room temperature, preventing viral nucleic acid degradation and contamination, thereby enabling operation with microcurrent reagent kits even at room temperature.

[0072] (2) This invention provides a method for preparing [Mg2(thd)4(dmeda)(edta)]. The method has a simple synthesis process, high purity of chelates, and excellent comprehensive performance of products. The reaction conditions are mild, the reaction process is controllable and reproducible, and the production cost is low.

[0073] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the following specific embodiments are based on the present technical solution and provide detailed implementation methods and specific operation processes, but the scope of protection of the present invention is not limited to these embodiments.

[0074] Example 1

[0075] DMEDA stands for dimethylethylenediamine, also abbreviated as dmeda, and its English name is N,N'-Dimethylethylenediamine.

[0076] EDTA stands for ethylenediaminetetraacetic acid, also abbreviated as edta.

[0077] THD is 2,2,6,6-tetramethyl-3,5-heptadecane.

[0078] 1. Preparation of [Mg2(thd)4(dmeda)(edta)] crystals

[0079] (1.1) Dissolve 1.2 mol of dimethyl ethylenediamine (DMEDA) in 200 ml of n-hexane to obtain a DMEDA solution.

[0080] (1.2) 1.5 mol of [Mg2(thd)4] was added to the DMEDA solution obtained in step (1.1), and stirred at 20 °C for 15 min. After the solution was evaporated under nitrogen protection, the resulting crystals were washed with water and n-hexane in sequence, and then purified by sublimation in a glove box to remove small molecule impurities. The standard crystals of [Mg2(thd)4(dmeda)] were collected.

[0081] (1.3) Weigh 0.8 mol of [Mg2(thd)4(dmeda)] standard crystals and 1 mol of ethylenediaminetetraacetic acid (EDTA), mix them evenly, and disperse them in 140 ml of a mixed solution of Trizol and deionized water in a volume ratio of 1:1. React at 60 °C for 20 min to obtain a transparent solution.

[0082] (1.4) After slowly cooling the transparent solution obtained in step (1.3) to 20°C, filter to remove impurities, crystallize at 2°C under argon protection, collect the obtained crystals, wash and refine them, and dry them in vacuum to obtain the target crystal [Mg2(thd)4(dmeda)(edta)], with a final yield of 78.4%.

[0083] During use, the reagent is prepared according to the required concentration ratio and diluted to obtain a transparent and colorless biological protection reagent containing Trizol and the coordination compound [Mg2(thd)4(dmeda)(edta)].

[0084] The corresponding RNA was extracted from pre-packaged Sars-cov-2 pseudoviruses, and its concentration was determined. The samples were divided into six groups to determine the protective ability of [Mg2(thd)4(dmeda)(edta)] against nucleic acids. The results are as follows: Figure 1 As shown. From Figure 1 As can be seen, when RNA was added to [Mg2(thd)4(dmeda)(edta)] solution, the RNA degradation rate was significantly lower than that of the H2O group. Moreover, the combination of RNA + [Mg2(thd)4(dmeda)(edta)] maintained a high concentration of RNA for a period of time, and the degradation rate was significantly better than other combinations, similar to the EDTA group. Therefore, [Mg2(thd)4(dmeda)(edta)] has an excellent protective effect on nucleic acids.

[0085] A 500mA current was applied to an equal volume (2ml) of experimental sample (EDTA solution and [Mg2(thd)4(dmeda)(edta)] solution) using a DC regulated power supply. The probes of both a digital oscilloscope and a conventional oscilloscope were inserted into the experimental specimen. The sample impedance information was obtained by observing the digital oscilloscope reading and the voltage waveform received by the oscilloscope. The results are as follows: Figure 2 As shown. From Figure 2 As can be seen, the resistivity of the [Mg2(thd)4(dmeda)(edta)] solution is significantly lower than that of the EDTA solution. Furthermore, the resistivity of the [Mg2(thd)4(dmeda)(edta)] solution is approximately one-third that of the EDTA solution, indicating that [Mg2(thd)4(dmeda)(edta)] possesses excellent electrical conductivity.

Claims

1. A conductive nucleic acid biological protective agent, characterized in that, The conductive nucleic acid bioprotectant mentioned above includes [Mg2(thd)4(dmeda)(edta)]; In this context, "thd" refers to 2,2,6,6-tetramethyl-3,5-heptadecane; "dmeda" refers to dimethylethylenediamine; and "edta" refers to ethylenediaminetetraacetic acid. The [Mg2(thd)4(dmeda)(edta)] is prepared by the following method, which includes the following steps: (1) Dissolve dimethylethylenediamine in n-hexane to obtain a dimethylethylenediamine solution; (2) Add [Mg2(thd)4] to the dimethyl ethylenediamine solution obtained in step (1), stir and mix, evaporate the solution under nitrogen protection, wash the resulting crystals with water and n-hexane in sequence, and then sublimate and purify to collect the standard crystals of [Mg2(thd)4(dmeda)]. (3) Weigh the standard crystals of [Mg2(thd)4(dmeda)] and ethylenediaminetetraacetic acid (EDTA), mix them evenly, and then disperse them in a mixture of Trizol and water. After the reaction, a transparent solution is obtained. (4) After slowly cooling the transparent solution obtained in step (3) to 10-30℃, filter to remove impurities, and crystallize at 1-6℃ under argon protection to separate [Mg2(thd)4(dmeda)(edta)].

2. The conductive nucleic acid bioprotectant as described in claim 1, characterized in that, The method includes: (1) Dissolve 1.0-1.4 mol of dimethylethylenediamine in 190-210 ml of n-hexane to obtain a dimethylethylenediamine solution; (2) Add 1.3-1.7 mol [Mg2(thd)4] to the dimethyl ethylenediamine solution obtained in step (1), stir at 18-22℃ for 13-17 min, evaporate the solution under nitrogen protection, wash the resulting crystals with water and n-hexane in sequence, and then sublimate to purify and collect the standard crystals of [Mg2(thd)4(dmeda)]. (3) Weigh 0.6-1.0 mol of [Mg2(thd)4(dmeda)] standard crystals and 0.8-1.2 mol of ethylenediaminetetraacetic acid (EDTA), mix them evenly, and then disperse them in 130-150 ml of a mixed solution of Trizol and water with a volume ratio of (0.8-1.2):(0.8-1.2). React at 55-65℃ for 18-22 min to obtain a transparent solution. (4) After slowly cooling the transparent solution obtained in step (3) to 18-22℃, filter to remove impurities, and crystallize at 1.5-2.5℃ under argon protection to separate [Mg2(thd)4(dmeda)(edta)].

3. A composition, characterized in that, The composition includes the conductive nucleic acid bioprotectant as described in claim 1.

4. The composition according to claim 3, characterized in that, The composition described is a reagent composition.

5. The use of the conductive nucleic acid bioprotectant as described in claim 1, characterized in that, Reagents used to prepare protective nucleic acids.

6. The use as described in claim 5, characterized in that, The nucleic acids mentioned include RNA and / or DNA.

7. A method for protecting nucleic acids, characterized in that, The conductive nucleic acid bioprotectant as described in claim 1 is brought into contact with the nucleic acid to protect the nucleic acid.

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