Sample preservation reagent for giant spiny frog InDel molecular marker and preparation method thereof

Abstract

The invention relates to the technical field of biological sample preservation, in particular to a sample preservation reagent for a giant spiny frog InDel molecular marker and a preparation method of the sample preservation reagent, degradation of DNA in the preservation process can be remarkably reduced through multiple action mechanisms of maintaining the pH value stable through a buffer solution system, inhibiting nuclease activity through a nucleic acid protective agent, scavenging free radicals through an antioxidant and the like, and the preservation effect is good. The method has the advantages that the cell stabilizer is added, DNA completeness is guaranteed, a reliable template is provided for follow-up accurate detection of InDel molecular markers, protective films can be formed around cells by the aid of the cell stabilizer, the cells are prevented from being damaged by ice crystals, osmotic pressure balance of the cells is maintained, high activity of the cells can be kept in the storage process, and follow-up extraction of high-quality DNA from the cells is benefited; the addition of the antibacterial agent can effectively inhibit possible microbial contamination of the sample in the preservation process, prolong the preservation time of the sample, and ensure the quality and safety of the sample.

Description

Technical Field

[0001] The present invention relates to the technical field of biological sample preservation, and in particular to a sample preservation reagent for Rana spinosa InDel molecule marker and a preparation method thereof. Background Art

[0002] The spiny-chested frog (Quasipaaspinosa) belongs to the class Amphibia, order Anura, family Dicroglossidae, and genus Quasipaa. It has delicious meat, rich in nutrients, high in protein and low in fat. It also has medicinal effects such as nourishing yin and lungs, strengthening the liver and kidneys. Therefore, it is known as the "king of frogs" and has high value in both food and medicinal fields.

[0003] However, the survival status of the spiny-chested frog has not been optimistic in recent years. With the continuous expansion of human activities, its habitat has been seriously disturbed by human activities. For example, large-scale development and construction in mountainous areas has continuously compressed the natural living space of the spiny-chested frog. At the same time, the widespread use of chemical pesticides such as herbicides in agricultural production has also caused great pollution to the living environment of the spiny-chested frog. These chemicals are continuously accumulated in the spiny-chested frog through the transmission of the food chain, affecting its physiological functions and reproductive capacity. In addition, due to the high economic value of the spiny-chested frog, the phenomenon of over-catching is very serious, and a large number of wild spiny-chested frogs are illegally caught for market transactions. Based on this, this species has been listed as a "vulnerable" species by the International Union for Conservation of Nature (IUCN) and the China Red List, and the task of protecting the spiny-chested frog is imminent.

[0004] In the research and protection of spiny-chested frogs, InDel (Insertion / Deletion) molecular marker technology is an important research method. Through the analysis of InDel molecular markers, we can deeply understand the key information such as the genetic structure, population dynamics and sex determination mechanism of spiny-chested frogs. This information has important guiding significance for formulating scientific and reasonable protection strategies and carrying out effective genetic breeding. For example, in genetic breeding, the use of InDel molecular markers can screen individuals with excellent traits, accelerate the breeding process, and cultivate spiny-chested frog varieties that are more suitable for artificial breeding, thereby improving breeding efficiency and reducing dependence on wild resources. In the study of sex determination mechanism, InDel molecular markers can help accurately identify the sex of spiny-chested frogs and provide technical support for the realization of monosex breeding. Because it is found in breeding practice that there are significant differences between the sexes of spiny-chested frogs, males grow fast, are large, have strong disease resistance, high meat killing rate, and high commodity recognition, all-male breeding has become an important direction for the development of spiny-chested frog breeding industry. However, when conducting research related to InDel molecular markers, sample preservation is a key link. At present, there are many problems in the preservation of spiny-chested frog samples. Due to the lack of preservation reagents specifically for spiny-chested frog InDel molecular marker samples, traditional sample preservation methods are not effective. During the sample preservation process, DNA degradation often occurs. This is because factors such as nucleases, temperature changes, and oxidation in the environment can damage the DNA in the sample. For example, when the sample storage temperature is unstable, the double helix structure of DNA may unwind, leading to DNA breakage and degradation. At the same time, the cell structure is also easily damaged. Under ordinary storage conditions, cells may rupture due to osmotic pressure imbalance, ice crystal damage, etc., causing the DNA in the cells to be released into the external environment, further increasing the risk of DNA degradation. These problems will seriously affect the subsequent InDel molecular marker detection and analysis results, resulting in inaccurate detection data and unable to provide a reliable basis for the research and protection of spiny-chested frogs.

[0005] Therefore, according to the above-mentioned related technologies, it is urgent to develop a sample preservation reagent for InDel molecular markers of Rana spinosa and a preparation method thereof. Summary of the invention

[0006] In view of this, the purpose of the present invention is to propose a sample preservation reagent for InDel molecular markers of Rana spinosa and a preparation method thereof, so as to solve the problems existing in the sample preservation process of Rana spinosa in the prior art, ensure the DNA integrity and cell activity of the sample, thereby improving the accuracy and reliability of InDel molecular marker detection.

[0007] Based on the above purpose, the present invention provides a sample preservation reagent for Rana spinosa InDel molecule marker and a preparation method thereof.

[0008] A sample preservation reagent for InDel molecular marker of Rana spinosa is prepared from the following raw materials: The preservation reagent includes a buffer system, a nucleic acid protectant, a cell stabilizer, an antioxidant and an antibacterial agent; The nucleic acid protective agent is prepared from nano-silicon dioxide and cationic liposomes; The cell stabilizer is obtained by mixing sucrose, trehalose, poly (N-isopropylacrylamide) and lactic acid and acetic acid copolymer; The antioxidant is prepared from idebenone, manganese porphyrin, vitamin C and glutathione; The antibacterial agent is nano-silver modified berberine.

[0009] Preferably, the buffer system is obtained by dissolving EDTA in a Tris-HCl buffer with a concentration of 10-20 mM and a pH value between 7.5 and 8.5, wherein the concentration of EDTA is 5-10 mM. The function of the Tris-HCl buffer is to maintain the pH value of the sample solution stable, provide a stable acid-base environment for cells and DNA, and prevent DNA degradation and cell structure damage caused by pH changes. The added ethylenediaminetetraacetic acid can chelate metal ions in the sample, such as magnesium ions and calcium ions. These metal ions are activators of nucleases. Ethylenediaminetetraacetic acid inhibits the activity of nucleases through chelation, thereby preventing DNA from being degraded by nucleases. In addition, when cells rupture for various reasons and release nucleases, EDTA can promptly inhibit their activity to prevent the released nucleic acids from being degraded. At the same time, the cell protection system maintains the integrity of the cells and reduces the release of nucleases.

[0010] Preferably, the preparation process of the nucleic acid protective agent is as follows: Step A1. Disperse nano-silica particles in anhydrous ethanol and perform ultrasonic treatment to obtain a nano-silica ethanol suspension. Nano-silica (SiO2) particles are used, which have a large specific surface area and good biocompatibility. Nano-SiO2 particles can be tightly combined with nucleic acids in the sample through physical adsorption to form a nano-scale protective barrier, effectively blocking the contact between nucleases and nucleic acids, thereby inhibiting the degradation of DNA by nucleases. At the same time, nano-SiO2 can also buffer the effects of external environmental factors (such as temperature and pH changes) on nucleic acids to a certain extent, maintaining the stability of nucleic acids; Step A2. Dissolve dioleoyltrimethylammonium chloride in chloroform, evaporate the chloroform on a rotary evaporator to form a lipid film, then add PBS buffer and perform ultrasonic treatment to obtain a uniform cationic liposome solution. The cationic liposome can bind to the negatively charged DNA through electrostatic interaction to form a stable liposome-DNA complex. This complex can not only protect DNA from nuclease degradation, but also enhance the dispersibility and stability of DNA in the solution. In addition, cationic liposomes also have certain membrane fusion properties, which can help DNA to be efficiently released from the preservation reagent and enter the cell or reaction system during subsequent sample processing; Step A3. Evenly mix the nano-silica ethanol suspension and the cationic liposome solution to obtain a nucleic acid protective agent. Nano-SiO2 and cationic liposomes protect nucleic acids mainly from the perspective of physical adsorption and complex formation, while EDTA provides protection from the perspective of inhibiting nuclease activity, forming multiple protections.

[0011] Preferably, the ratio of the nano-silicon dioxide particles to anhydrous ethanol in step A1 is 0.4-0.6 g: 45-55 mL; The power of the ultrasonic treatment in step A1 is 200-400 W, and the time is 30-60 min.

[0012] Preferably, the usage ratio of dioleoyltrimethylammonium chloride, chloroform and PBS buffer in step A2 is 0.18-0.22 g: 9.5-12 mL: 4-6 mL.

[0013] The temperature of the rotary evaporator in step A2 is set to 30-40° C. and the vacuum degree is 0.05-0.1 MPa; The power of the ultrasonic treatment in step A2 is 100-200 W, and the time is 15-30 min.

[0014] Preferably, the preparation process of the cell stabilizer is as follows: Step B1. Add poly (N-isopropylacrylamide) and poly (lactic acid-co-glycolic acid) to deionized water, heat to 52-58°C, and stir at a speed of 280-320rpm until completely dissolved, then mix the two solutions evenly to obtain a polymer solution, and use the thermosensitive polymer poly (N-isopropylacrylamide) (PNIPAAm). PNIPAAm is water-soluble at a lower temperature (below its lower critical solution temperature, about 32°C) and can be evenly dispersed in the preservation reagent. When the sample temperature is lowered, PNIPAAm undergoes a phase transition to form a gel-like substance that wraps around the cells, acting as a physical barrier to prevent the cells from being damaged by ice crystal formation at low temperatures. At the same time, this gel-like substance can also maintain the stability of the microenvironment around the cells and reduce cell damage caused by factors such as changes in osmotic pressure. Combined with biodegradable poly (lactic acid-co-glycolic acid) (PLGA). PLGA has good biocompatibility and degradability. It can form a protective film on the cell surface to enhance the cell's resistance to damage. Moreover, PLGA can slowly release some cell protective factors (such as amino acids, sugars, etc.), provide nutrition and protection for cells, and help maintain cell activity and function; Step B2. Add sucrose and trehalose to the polymer solution and mix them evenly so that the concentration of sucrose is 100-120 mM and the concentration of trehalose is 50-70 mM to obtain a cell stabilizer.

[0015] Preferably, in step B1, the ratio of the poly (N-isopropylacrylamide) to deionized water is 1.8-2.2 g: 48-52 mL; the ratio of the poly (lactic acid-glycolic acid copolymer) to deionized water is 0.8-1.3 g: 48-52 mL.

[0016] Preferably, the preparation process of the antioxidant is as follows: Dissolve idebenone and manganese porphyrin in DMSO, add vitamin C and glutathione, and finally dilute with PBS buffer to make the concentration of vitamin C 50-60μM and the concentration of glutathione 20-30μM to obtain antioxidants. Idebenone has strong antioxidant capacity and can effectively remove free radicals in samples and reduce the damage of oxidative stress to DNA and cells. Compared with traditional antioxidants, idebenone has a smaller molecular structure and is easier to penetrate cell membranes, thereby exerting an antioxidant effect more efficiently. Manganese porphyrin can simulate the activity of superoxide dismutase, catalyze the dismutation reaction of superoxide anion free radicals, convert them into oxygen and hydrogen peroxide, and further reduce oxidative damage in samples. The dosage ratio of idebenone to DMSO is 0.008-0.012 g: 0.9-1.3 mL; The dosage ratio of the manganese porphyrin to DMSO is 0.0025-0.0035 g: 0.9-1.3 mL.

[0017] Preferably, the preparation process of the antibacterial agent is as follows: Berberine and nano-silver particles are added to deionized water respectively, and ultrasonically treated in an ultrasonic cleaner at a power of 140-150W for 18-22 minutes to make them evenly dispersed, and then mixed evenly to obtain an antibacterial agent mixed solution. Berberine has a broad-spectrum antibacterial activity and has inhibitory effects on a variety of bacteria, fungi and other microorganisms. It can achieve the purpose of antibacterial by combining with the phospholipids on the cell membrane of microorganisms and destroying the integrity of the cell membrane. At the same time, berberine also has certain anti-inflammatory and antioxidant properties, which helps to maintain the stability of the sample. Nano-silver particles have unique antibacterial properties. Their large specific surface area enables them to fully contact with microorganisms. The released silver ions can combine with biomacromolecules such as proteins and nucleic acids in microorganisms to interfere with the metabolism and growth of microorganisms, thereby achieving high-efficiency antibacterial properties. The dosage ratio of the berberine, the nano-silver particles and the deionized water is 0.075-0.085 g: 0.0045-0.0055 g: 46-53 mL.

[0018] A method for preparing a sample preservation reagent for InDel molecular marker of Rana spinosa, comprising the following steps: Step S1. Add 14-16 mL of nucleic acid protectant, 18-22 mL of cell stabilizer, 8-13 mL of antioxidant and 8-12 mL of antibacterial agent into a 500 mL volumetric flask; Step S2. The volume was adjusted to 500 mL with PBS buffer and stirred evenly with a magnetic stirrer at a speed of 380-410 rpm; Step S3: Filter and sterilize the sample through a 0.22 μm filter membrane to obtain a sample preservation reagent for Rana spinosa InDel molecule labeling.

[0019] The buffer system cooperates with the nucleic acid protection system of nanomaterials. In the process of stabilizing nucleic acids, the pH stabilization of the buffer helps maintain the binding state of these nanomaterials and nucleic acids. At the same time, when the smart polymer cell protection system works, the appropriate pH value is also conducive to the formation of a stable structure of PNIPAAm and PLGA, which can better wrap and protect cells.

[0020] Sucrose and trehalose can form a protective film around cells. The principle is to form a layer of glass-like substance on the cell surface by forming hydrogen bonds with water molecules. During the freezing and thawing process, this protective film can prevent the formation of ice crystals, which will cause cells to rupture. At the same time, they can also maintain the osmotic pressure balance of cells and keep cells active. PNIPAAm and PLGA mainly protect cells from the perspective of temperature response and nutrient supply, while sucrose and trehalose focus on preventing ice crystal damage and osmotic pressure balance. In conjunction with nucleic acid protection components, the stable cell structure can reduce the release of intracellular nucleases, thereby indirectly protecting nucleic acids. At the same time, the stability of nucleic acids is also conducive to cells to better perform their functions after preservation.

[0021] Vitamin C is a common antioxidant that can provide electrons to neutralize free radicals in samples, such as superoxide anion radicals (O2⁻), hydroxyl radicals (・OH), etc. Glutathione can scavenge free radicals through its own redox reaction, and participate in the antioxidant defense system in cells, synergizing with idebenone and MnP. Various antioxidants can scavenge free radicals through different mechanisms. For example, idebenone mainly exerts its antioxidant effect in parts such as cell membranes, vitamin C and glutathione can scavenge free radicals in aqueous solution environments inside and outside cells, and MnP focuses on the dismutation reaction of superoxide anion radicals. Together, they can more comprehensively reduce the effects of oxidative damage on DNA and cells. At the same time, the action of antioxidants also helps maintain the stability of other components such as nanomaterials and smart polymers.

[0022] Penicillin mainly acts on the synthesis process of bacterial cell walls, inhibiting the growth and reproduction of bacteria; streptomycin exerts its antibacterial effect by interfering with the synthesis of bacterial proteins. It works in synergy with berberine and nanosilver particles. Berberine mainly destroys microbial cell membranes, nanosilver particles interfere with microbial metabolism, and penicillin and streptomycin inhibit microbial growth from the perspective of cell wall and protein synthesis, forming a multi-target antibacterial system. This powerful antibacterial system can effectively prevent samples from being contaminated by microorganisms during storage, and also helps maintain the stability of other components (such as nucleic acid protection components, cell protection components, etc.), extending the storage time of samples.

[0023] Beneficial effects of the present invention: The present invention provides a sample preservation reagent for InDel molecular marker of Rana spinosa and a preparation method thereof, which has the following significant advantages: Effectively protect DNA integrity: Through multiple mechanisms such as maintaining pH stability through the buffer system, inhibiting nuclease activity through nucleic acid protectants, and scavenging free radicals through antioxidants, it can significantly reduce DNA degradation during storage, ensure DNA integrity, and provide a reliable template for the accurate detection of subsequent InDel molecular markers; Maintain cell activity: The use of cell stabilizers can form a protective film around the cells to prevent ice crystals from damaging the cells and maintain the osmotic pressure balance of the cells, so that the cells maintain high activity during the storage process, which is conducive to the subsequent extraction of high-quality DNA from the cells; Prevent microbial contamination: The addition of antimicrobial agents can effectively inhibit the microbial contamination that may occur to samples during storage, extend the storage time of samples, and ensure the quality and safety of samples; Easy operation: The preparation method of the preservation reagent of the present invention is simple, easy to operate, low in cost, and easy to use. The preservation reagent only needs to be mixed with the collected spiny-chested frog sample in a certain proportion, and then it can be preserved under conventional low-temperature conditions. It is suitable for the preservation of samples collected in different laboratories and on-site. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0025] The sources and properties of some of the raw materials used in the present invention are as follows: Example 1: A method for preparing a sample preservation reagent for Rana spinosa InDel molecular marker, comprising the following steps: S1. Dissolve EDTA in a Tris-HCl buffer having a concentration of 10 mM and a pH value between 7.5 to obtain a buffer system in which the concentration of EDTA is 5-7.5-10 mM; S2. 0.4 g of nano-silica particles were dispersed in 45 mL of anhydrous ethanol and ultrasonically treated at 200 W for 30 min to obtain a nano-silica ethanol suspension; S3. 0.18 g of dioleoyltrimethylammonium chloride was dissolved in 9.5 mL of chloroform, and the chloroform was evaporated on a rotary evaporator. The temperature of the rotary evaporator was set to 30°C and the vacuum degree was 0.05 MPa to form a lipid film, and then 4 mL of PBS buffer was added. Ultrasonic treatment was performed at 100 W for 15 min to obtain a uniform cationic liposome solution; S4. The nano-silica ethanol suspension and the cationic liposome solution are mixed evenly to obtain a nucleic acid protective agent; S5. 1.8 g of poly (N-isopropylacrylamide) and 0.8 g of poly (lactic acid-co-glycolic acid) were added to 48 mL of deionized water, respectively, heated to 52° C., and stirred at 280 rpm until completely dissolved, and then the two solutions were mixed to obtain a polymer solution; S6. Add sucrose and trehalose to the polymer solution and mix well so that the concentration of sucrose is 100 mM and the concentration of trehalose is 50 mM to obtain a cell stabilizer; S7. 0.008 g of idebenone and 0.0025 g of manganese porphyrin were dissolved in 0.9 mL of DMSO, vitamin C and glutathione were added, and finally PBS buffer was added to dilute the mixture so that the concentration of vitamin C was 50 μM and the concentration of glutathione was 20 μM, thereby obtaining an antioxidant; S8. 0.075 g of berberine and 0.0045 g of nanosilver particles were added to 46 mL of deionized water, respectively, and ultrasonically treated for 18 min at a power of 140 W in an ultrasonic cleaner to uniformly disperse them, and then mixed to obtain an antibacterial agent; S9. Add 14 mL of nucleic acid protectant, 18 mL of cell stabilizer, 8 mL of antioxidant and 8 mL of antibacterial agent into a 500 mL volumetric flask; S10. The volume was adjusted to 500 mL with a buffer system and stirred evenly at 380 rpm using a magnetic stirrer; S11. Sterilize by filtering through a 0.22 μm filter membrane to obtain a sample preservation reagent for Rana spinosa InDel molecule labeling.

[0026] Example 2: A method for preparing a sample preservation reagent for Rana spinosa InDel molecular marker, comprising the following steps: S1. Dissolve EDTA in a Tris-HCl buffer having a concentration of 15 mM and a pH value between 8 to obtain a buffer system in which the concentration of EDTA is 7.5 mM; S2. 0.5 g of nano-silica particles were dispersed in 50 mL of anhydrous ethanol and ultrasonically treated at 300 W for 45 min to obtain a nano-silica ethanol suspension; S3. 0.2 g of dioleoyltrimethylammonium chloride was dissolved in 10.5 mL of chloroform, and the chloroform was evaporated on a rotary evaporator. The temperature of the rotary evaporator was set to 35°C and the vacuum degree was 0.07 MPa to form a lipid film, and then 5 mL of PBS buffer was added. The mixture was ultrasonically treated at 150 W for 20 min to obtain a uniform cationic liposome solution; S4. The nano-silica ethanol suspension and the cationic liposome solution are mixed evenly to obtain a nucleic acid protective agent; S5. 2 g of poly (N-isopropylacrylamide) and 1 g of poly (lactic acid-co-glycolic acid) were added to 50 mL of deionized water, respectively, heated to 55° C., and stirred at 300 rpm until completely dissolved, and then the two solutions were mixed to obtain a polymer solution; S6. Add sucrose and trehalose to the polymer solution and mix well so that the concentration of sucrose is 110 mM and the concentration of trehalose is 60 mM to obtain a cell stabilizer; S7. 0.01 g of idebenone and 0.003 g of manganese porphyrin were dissolved in 1.1 mL of DMSO, vitamin C and glutathione were added, and finally PBS buffer was added to dilute the mixture so that the concentration of vitamin C was 55 μM and the concentration of glutathione was 25 μM, thereby obtaining an antioxidant; S8. 0.08 g of berberine and 0.005 g of nanosilver particles were added to 50 mL of deionized water, respectively, and ultrasonically treated at a power of 145 W for 20 min in an ultrasonic cleaner to uniformly disperse them, and then mixed to obtain an antibacterial agent; S9. Add 15 mL of nucleic acid protectant, 20 mL of cell stabilizer, 10 mL of antioxidant and 10 mL of antibacterial agent into a 500 mL volumetric flask; S10. The volume was adjusted to 500 mL with a buffer system and stirred evenly at a speed of 400 rpm using a magnetic stirrer; S11. Sterilize by filtering through a 0.22 μm filter membrane to obtain a sample preservation reagent for Rana spinosa InDel molecule labeling.

[0027] Example 3: A method for preparing a sample preservation reagent for Rana spinosa InDel molecule marker, comprising the following steps: S1. Dissolve EDTA in a Tris-HCl buffer having a concentration of 20 mM and a pH value between 8.5 to obtain a buffer system in which the concentration of EDTA is 10 mM; S2. 0.6 g of nano-silica particles were dispersed in 55 mL of anhydrous ethanol and ultrasonically treated at 400 W for 60 min to obtain a nano-silica ethanol suspension; S3. 0.22 g of dioleoyltrimethylammonium chloride was dissolved in 12 mL of chloroform, and the chloroform was evaporated on a rotary evaporator. The temperature of the rotary evaporator was set to 40°C and the vacuum degree was 0.1 MPa to form a lipid film. Then 6 mL of PBS buffer was added and ultrasonic treatment was performed at 200 W for 30 min to obtain a uniform cationic liposome solution; S4. The nano-silica ethanol suspension and the cationic liposome solution are mixed evenly to obtain a nucleic acid protective agent; S5. 2.2 g of poly (N-isopropylacrylamide) and 1.3 g of poly (lactic acid-co-glycolic acid) were added to 52 mL of deionized water, respectively, heated to 58° C., and stirred at a speed of 320 rpm until completely dissolved, and then the two solutions were mixed to obtain a polymer solution; S6. Add sucrose and trehalose to the polymer solution and mix well so that the concentration of sucrose is 120 mM and the concentration of trehalose is 70 mM to obtain a cell stabilizer; S7. 0.012 g of idebenone and 0.0035 g of manganese porphyrin were dissolved in 1.3 mL of DMSO, vitamin C and glutathione were added, and PBS buffer was added to dilute the mixture so that the concentration of vitamin C was 60 μM and the concentration of glutathione was 30 μM, thereby obtaining an antioxidant; S8. 0.085 g of berberine and 0.0055 g of nanosilver particles were added to 53 mL of deionized water, respectively, and ultrasonically treated in an ultrasonic cleaner at a power of 150 W for 22 min to uniformly disperse them, and then mixed to obtain an antibacterial agent; S9. Add 16 mL of nucleic acid protectant, 22 mL of cell stabilizer, 13 mL of antioxidant and 12 mL of antibacterial agent into a 500 mL volumetric flask; S10. The volume was adjusted to 500 mL with a buffer system and stirred evenly with a magnetic stirrer at a speed of 410 rpm; S11. Sterilize by filtering through a 0.22 μm filter membrane to obtain a sample preservation reagent for Rana spinosa InDel molecule labeling.

[0028] Comparative Example 1: Compared with Example 1, nano-silica was not added during the preparation of the sample preservation reagent for InDel molecular marker of Rana spinosa. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, the sample preservation reagent for InDel molecular marker of Rana spinosa was obtained.

[0029] Comparative Example 2: Compared with Example 1, cationic liposomes were not added in the preparation process of the sample preservation reagent for InDel molecular marker of Rana spinosa in this comparative example. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, the sample preservation reagent for InDel molecular marker of Rana spinosa was obtained.

[0030] Comparative Example 3: Compared with Example 1, poly (N-isopropylacrylamide) was not added during the preparation of the sample preservation reagent for InDel molecule marker of Rana spinosa. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, the sample preservation reagent for InDel molecule marker of Rana spinosa was obtained.

[0031] Comparative Example 4: Compared with Example 1, glutathione was not added during the preparation of the sample preservation reagent for InDel molecule marker of Rana spinosa in this comparative example. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, the sample preservation reagent for InDel molecule marker of Rana spinosa was obtained.

[0032] Comparative Example 5: Compared with Example 1, this comparative example only adjusts "14 mL of nucleic acid protective agent, 18 mL of cell stabilizer, 8 mL of antioxidant and 8 mL of antibacterial agent" to "16 mL of nucleic acid protective agent, 25 mL of cell stabilizer, 8 mL of antioxidant and 8 mL of antibacterial agent", and the remaining steps and parameters are the same. This comparative example will not be repeated, and finally a sample preservation reagent for InDel molecular marking of Rana spinosa is obtained.

[0033] Comparative Example 6: Compared with Example 1, EDTA was not added during the preparation of the sample preservation reagent for InDel molecular marker of Rana spinosa in this comparative example, and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, the sample preservation reagent for InDel molecular marker of Rana spinosa was obtained.

[0034] Performance Testing: DNA Integrity Test: Test method: Agarose gel electrophoresis was used. DNA was extracted from Rana spinosa samples after different storage times (1 week, 2 weeks, 4 weeks, and 8 weeks). An appropriate amount of DNA sample was mixed with the loading buffer and added to the 1%-1.5% agarose gel loading well. Electrophoresis was performed in 1×TAE buffer at a voltage of 100-120V for 30-60 minutes. After the electrophoresis, the integrity of the DNA bands was observed and photographed under a gel imaging system. Clear DNA bands without obvious tailing were considered good standards for DNA integrity. Image analysis software (such as ImageJ) was used to quantify the brightness and width of the bands and calculate the degree of DNA degradation.

[0035] Test standard: DNA degradation degree = (initial DNA band brightness - DNA band brightness after storage) / initial DNA band brightness × 100%. The lower the degree of degradation, the better the DNA integrity.

[0036] Cell viability test: Test method: Trypan blue staining method is used. The preserved R. spinosa cell samples are digested with trypsin to form a single cell suspension. An appropriate amount of the cell suspension is mixed with 0.4% trypan blue solution at a ratio of 1:1, incubated at room temperature for 2-3 minutes, and then counted under a microscope using a hemocytometer. Living cells are colorless due to staining, while dead cells are stained blue. Calculate cell activity = (number of living cells / total number of cells) × 100%.

[0037] Test standard: The higher the cell activity, the better the protection effect of the sample preservation reagent on the cells.

[0038] Microbiological contamination test: Test method: Plate count method is used. The stored sample is diluted with sterile saline solution in a gradient manner, and an appropriate amount of the dilution is spread on a nutrient agar medium plate, cultured at 37°C for 24-48 hours, and then the number of colonies on the plate is counted. At the same time, a blank control plate (only sterile saline solution) is set up.

[0039] Test standard: The fewer the number of colonies, the less contamination the sample has with microorganisms. If colonies appear on the blank control plate, the experiment is invalid and needs to be repeated.

[0040]

[0041]

[0042]

[0043] Data Analysis: As can be seen from Tables 1-3, the degree of DNA degradation in Examples 1-3 gradually increases with the extension of storage time, but at the same storage time, the degree of degradation is lower than that in the comparative example, which shows that the complete reagent formula in the embodiment, including the synergistic effect of nano-silica, cationic liposomes, EDTA and other components, can effectively protect the integrity of DNA. The degree of DNA degradation in Comparative Example 1 (without the addition of nano-silica) and Comparative Example 2 (without the addition of cationic liposomes) is significantly higher than that in the embodiment, indicating that nano-silica and cationic liposomes play a key role in nucleic acid protection. Nano-silica forms a protective barrier through physical adsorption, and cationic liposomes form a complex with DNA, and both are indispensable. The degree of DNA degradation in Comparative Example 6 (without the addition of EDTA) is also high, indicating that the role of EDTA in chelating metal ions to inhibit nuclease activity is crucial to maintaining DNA integrity.

[0044] Cell activity: The cell activity of Examples 1-3 gradually decreased during the storage process, but always remained at a relatively high level, indicating that the reagent with a complete formula can maintain cell activity well. The cell activity of Comparative Example 3 (without the addition of poly N-isopropylacrylamide) decreased relatively significantly, indicating that poly N-isopropylacrylamide played an important role in forming a gel-like substance to wrap cells at low temperatures and maintaining the stability of the cell microenvironment. The cell activity of Comparative Example 4 (without the addition of glutathione) also decreased to a certain extent, indicating that glutathione participates in the intracellular antioxidant defense system and has a positive effect on maintaining cell activity.

[0045] Microbial contamination: The colony counts of Examples 1-3 are relatively small, indicating that their antibacterial systems (nanosilver-modified berberine, etc.) can effectively inhibit microbial growth and prevent sample contamination. The colony counts of Comparative Examples 1-6 are relatively large, indicating that the lack of certain key components will affect the antibacterial effect. For example, the lack of nanomaterials in Comparative Examples 1-2 may affect the dispersion and effect of the antibacterial agent; the lack of EDTA in Comparative Example 6 may indirectly affect the stability of the overall reagent, making it easier for microorganisms to grow.

[0046] The function of the Tris-HCl buffer is to maintain the pH value of the sample solution stable, provide a stable acid-base environment for cells and DNA, and prevent DNA degradation and cell structure damage caused by pH changes. The added ethylenediaminetetraacetic acid can chelate metal ions in the sample, such as magnesium ions and calcium ions. These metal ions are activators of nucleases. EDTA inhibits the activity of nucleases through chelation, thereby preventing DNA from being degraded by nucleases. In addition, when cells rupture for various reasons and release nucleases, EDTA can inhibit their activity in time to prevent the released nucleic acids from being degraded. At the same time, the cell protection system maintains the integrity of the cells and reduces the release of nucleases. The nano-silicon dioxide (SiO2) particles used have a large specific surface area and good biocompatibility. Nano-SiO2 particles can be closely combined with nucleic acids in the sample through physical adsorption, forming a nano-level protective barrier, effectively blocking the contact between nucleases and nucleic acids, thereby inhibiting the degradation of DNA by nucleases. At the same time, nano-SiO2 can also buffer the effects of external environmental factors (such as temperature and pH changes) on nucleic acids to a certain extent, maintaining the stability of nucleic acids; Cationic liposomes can bind to negatively charged DNA through electrostatic interaction to form a stable liposome-DNA complex. This complex can not only protect DNA from degradation by nucleases, but also enhance the dispersibility and stability of DNA in solution. In addition, cationic liposomes also have certain membrane fusion properties, which can help DNA to be efficiently released from the preservation reagent and enter the cell or reaction system during subsequent sample processing; Use the thermosensitive polymer poly N-isopropylacrylamide (PNIPAAm). PNIPAAm is water-soluble at lower temperatures (below its lower critical solution temperature, about 32°C) and can be evenly dispersed in the preservation reagent. When the sample temperature drops, PNIPAAm undergoes a phase transition to form a gel-like substance that wraps around the cells, acting as a physical barrier to prevent cells from being damaged by ice crystal formation at low temperatures. At the same time, this gel-like substance can also maintain the stability of the microenvironment around the cells and reduce damage to the cells caused by factors such as changes in osmotic pressure. Combined with biodegradable polylactic acid-glycolic acid copolymer (PLGA). PLGA has good biocompatibility and degradability. It can form a protective film on the cell surface to enhance the cell's resistance to damage. Moreover, PLGA can slowly release some cell protective factors (such as amino acids, sugars, etc.) to provide nutrition and protection for cells, helping to maintain cell activity and function; Idebenone has strong antioxidant capacity and can effectively remove free radicals in samples, reducing the damage of oxidative stress to DNA and cells. Compared with traditional antioxidants, the molecular structure of idebenone is smaller and easier to penetrate the cell membrane, thus playing an antioxidant role more efficiently. Manganese porphyrin can simulate the activity of superoxide dismutase, catalyze the dismutation reaction of superoxide anion free radicals, convert them into oxygen and hydrogen peroxide, and further reduce the oxidative damage in samples. Berberine has a broad-spectrum antibacterial activity and has inhibitory effects on a variety of bacteria, fungi and other microorganisms. It can achieve the purpose of antibacterial by combining with the phospholipids on the cell membrane of microorganisms and destroying the integrity of the cell membrane. At the same time, berberine also has certain anti-inflammatory and antioxidant properties, which helps to maintain the stability of the sample. Nanosilver particles have unique antibacterial properties. Their large specific surface area enables them to fully contact with microorganisms. The released silver ions can combine with biomacromolecules such as proteins and nucleic acids in microorganisms, interfering with the metabolism and growth of microorganisms, thereby achieving high antibacterial efficiency.

[0047] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0048] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A sample preservation reagent for Rana spinosa InDel molecular marker, characterized in that: Prepared from the following raw materials: The preservation reagent includes a buffer system, a nucleic acid protectant, a cell stabilizer, an antioxidant and an antibacterial agent; The nucleic acid protective agent is prepared from nano-silicon dioxide and cationic liposomes; The cell stabilizer is obtained by mixing sucrose, trehalose, poly (N-isopropylacrylamide) and lactic acid and acetic acid copolymer; The antioxidant is prepared from idebenone, manganese porphyrin, vitamin C and glutathione; The antibacterial agent is nano-silver modified berberine.

2. The sample preservation reagent for InDel molecular marker of Rana spinosa according to claim 1, characterized in that: The buffer system is obtained by dissolving EDTA in a Tris-HCl buffer with a concentration of 10-20 mM and a pH value between 7.5 and 8.5, wherein the concentration of EDTA is 5-10 mM.

3. The sample preservation reagent for InDel molecular marker of Rana spinosa according to claim 1, characterized in that: The preparation process of the nucleic acid protective agent is as follows: Step A1. dispersing nano-silicon dioxide particles in anhydrous ethanol and ultrasonically treating to obtain a nano-silicon dioxide ethanol suspension; Step A2. dissolving dioleoyltrimethylammonium chloride in chloroform, evaporating the chloroform on a rotary evaporator to form a lipid film, then adding PBS buffer and ultrasonically treating to obtain a uniform cationic liposome solution; Step A3. Evenly mix the nano-silica ethanol suspension and the cationic liposome solution to obtain a nucleic acid protective agent.

4. The sample preservation reagent for InDel molecular marker of Rana spinosa according to claim 3, characterized in that: The ratio of the nano-silicon dioxide particles to anhydrous ethanol in step A1 is 0.4-0.6 g: 45-55 mL; The power of the ultrasonic treatment in step A1 is 200-400 W, and the time is 30-60 min.

5. The sample preservation reagent for InDel molecular marker of Rana spinosa according to claim 3, characterized in that: The usage ratio of dioleoyltrimethylammonium chloride, chloroform and PBS buffer in step A2 is 0.18-0.22 g: 9.5-12 mL: 4-6 mL; The temperature of the rotary evaporator in step A2 is set to 30-40° C. and the vacuum degree is 0.05-0.1 MPa; The power of the ultrasonic treatment in step A2 is 100-200 W, and the time is 15-30 min.

6. The sample preservation reagent for InDel molecular marker of Rana spinosa according to claim 1, characterized in that: The preparation process of the cell stabilizer is as follows: Step B1. Add poly (N-isopropylacrylamide) and poly (lactic acid-co-glycolic acid) into deionized water, respectively, heat to 52-58° C., and stir at a speed of 280-320 rpm until completely dissolved, and then mix the two solutions to obtain a polymer solution; Step B2. Add sucrose and trehalose to the polymer solution and mix them evenly so that the concentration of sucrose is 100-120 mM and the concentration of trehalose is 50-70 mM to obtain a cell stabilizer.

7. The sample preservation reagent for InDel molecular marker of Rana spinosa according to claim 6, characterized in that: In step B1, the dosage ratio of the poly (N-isopropylacrylamide) to deionized water is 1.8-2.2 g: 48-52 mL; the dosage ratio of the poly (lactic acid-glycolic acid copolymer) to deionized water is 0.8-1.3 g: 48-52 mL.

8. The sample preservation reagent for InDel molecular marker of Rana spinosa according to claim 1, characterized in that: The preparation process of the antioxidant is as follows: Dissolve idebenone and manganese porphyrin in DMSO, add vitamin C and glutathione, and finally add PBS buffer to dilute, so that the concentration of vitamin C is 50-60 μM and the concentration of glutathione is 20-30 μM, to obtain an antioxidant; The dosage ratio of idebenone to DMSO is 0.008-0.012 g: 0.9-1.3 mL; The dosage ratio of the manganese porphyrin to DMSO is 0.0025-0.0035 g: 0.9-1.3 mL.

9. The sample preservation reagent for InDel molecular marker of Rana spinosa according to claim 1, characterized in that: The preparation process of the antibacterial agent is as follows: The berberine and the nano-silver particles are added to deionized water respectively, and ultrasonically treated for 18-22 minutes at a power of 140-150 W in an ultrasonic cleaner to make them uniformly dispersed, and then mixed to obtain an antibacterial agent mixed solution; The dosage ratio of the berberine, the nano-silver particles and the deionized water is 0.075-0.085 g: 0.0045-0.0055 g: 46-53 mL.

10. The method for preparing a sample preservation reagent for Rana spinosa InDel molecular marker according to any one of claims 1 to 9, characterized in that: The following steps are involved: Step S1. Add 14-16 mL of nucleic acid protectant, 18-22 mL of cell stabilizer, 8-13 mL of antioxidant and 8-12 mL of antibacterial agent into a 500 mL volumetric flask; Step S2. The volume was adjusted to 500 mL with PBS buffer and stirred evenly with a magnetic stirrer at a speed of 380-410 rpm; Step S3: Filter and sterilize the sample through a 0.22 μm filter membrane to obtain a sample preservation reagent for Rana spinosa InDel molecule labeling.

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