A sample preservation reagent for an inDel molecular marker of pachyhynobius and a preparation method thereof

Abstract

The application relates to the technical field of biological sample preservation, in particular to a sample preservation reagent for InDel molecular markers of Pachyhynobius and a preparation method thereof. Through multiple action mechanisms such as maintaining pH stability by a buffer system, inhibiting nuclease activity by a nucleic acid protective agent, and removing free radicals by an antioxidant, the DNA degradation in the preservation process can be significantly reduced, the integrity of the DNA is ensured, and a reliable template is provided for the accurate detection of subsequent InDel molecular markers. The use of a cell stabilizer can form a protective film around the cells, prevent ice crystal damage to the cells, and maintain the osmotic pressure balance of the cells, so that the cells can maintain a high activity in the preservation process, and high-quality DNA can be extracted from the cells in the subsequent process; the addition of an antibacterial agent can effectively inhibit the 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 application relates to the technical field of biological sample preservation, and particularly relates to a sample preservation reagent for InDel molecular markers of Quasipaaspinosa and a preparation method thereof. BACKGROUND

[0002] Quasipaaspinosa belongs to Amphibia, Anura, Dicroglossidae and Quasipaa, and has the characteristics of high protein and low fat, and also has the medicinal effects of nourishing yin, tonifying lung, strengthening liver and kidney, etc., so it is known as "King of Frogs" and has high value in the fields of food and medicine.

[0003] However, the survival status of Quasipaaspinosa is not 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 have continuously compressed the natural living space of Quasipaaspinosa. At the same time, the widespread use of herbicides and other chemical pesticides in agricultural production has also caused great pollution to the living environment of Quasipaaspinosa. These chemical substances are continuously accumulated in the body of Quasipaaspinosa through the transmission of food chain, affecting its physiological function and reproductive capacity. In addition, due to the high economic value of Quasipaaspinosa, overexploitation is very serious, and a large number of wild Quasipaaspinosa are illegally captured for market transactions. Based on this, this species has been listed as "vulnerable" species by the International Union for Conservation of Nature (IUCN) and the Chinese Red List, and the task of protecting Quasipaaspinosa is imminent.

[0004] InDel (Insertion / Deletion) molecular marker technology is an important research tool in the study and protection of Pachyhynobius shantungensis. Through the analysis of InDel molecular markers, we can gain a deeper understanding of the genetic structure, population dynamics, and sex determination mechanisms of Pachyhynobius shantungensis. These key information is of great significance for developing scientific and reasonable protection strategies and carrying out effective genetic breeding. For example, in genetic breeding, InDel molecular markers can be used to screen individuals with excellent traits, accelerate the breeding process, and cultivate Pachyhynobius shantungensis varieties that are more suitable for artificial breeding, thereby improving breeding efficiency and reducing dependence on wild resources. In the study of sex determination mechanisms, InDel molecular markers can help accurately identify the sex of Pachyhynobius shantungensis, providing technical support for monosex breeding. Because of the significant differences between males and females in Pachyhynobius shantungensis, males grow faster, are larger, have stronger disease resistance, higher meat yield, and higher commodity recognition, so developing all-male breeding has become an important direction for the development of Pachyhynobius shantungensis breeding industry. However, during the InDel molecular marker-related research, sample preservation is a key step. Currently, there are many problems in the preservation of Pachyhynobius shantungensis samples. Due to the lack of special reagents for preserving InDel molecular marker samples of Pachyhynobius shantungensis, the traditional sample preservation methods are not effective. During the sample preservation process, DNA degradation often occurs. This is because environmental nucleases, temperature changes, oxidation, and other factors can damage the DNA in the sample. For example, when the sample preservation temperature is unstable, the double helix structure of DNA may unwind, leading to DNA fragmentation and degradation. At the same time, cell structures are also easily damaged, and under ordinary preservation conditions, cells may rupture due to osmotic imbalance, ice crystal damage, and other reasons, causing the release of intracellular DNA into the external environment, further increasing the risk of DNA degradation. These problems can seriously affect the subsequent InDel molecular marker detection and analysis results, leading to inaccurate detection data and making it impossible to provide reliable basis for the research and protection of Pachyhynobius shantungensis.

[0005] Therefore, according to the related technology in the above, it is urgent to develop a sample preservation reagent for InDel molecular markers of Pachyhynobius shantungensis and a preparation method thereof. SUMMARY

[0006] Therefore, according to the related technology in the above, it is urgent to develop a sample preservation reagent for InDel molecular markers of Pachyhynobius shantungensis and a preparation method thereof.

[0007] Based on the above purpose, the present application provides a sample preservation reagent for InDel molecular markers of Pachyhynobius shantungensis and a preparation method thereof.

[0008] A sample preservation reagent for an InDel molecular marker of Pelophylax nigromaculatus is prepared from the following raw materials:

[0009] The preservation reagent comprises a buffer system, a nucleic acid protective agent, a cell stabilizer, an antioxidant and an antibacterial agent;

[0010] The nucleic acid protective agent is prepared from nano-silicon dioxide and cationic liposomes;

[0011] The cell stabilizer is a mixture of sucrose, trehalose, poly-N-isopropyl acrylamide and lactic acid-acetic acid copolymer;

[0012] The antioxidant is prepared from idebenone, manganese porphyrin, vitamin C and glutathione;

[0013] The antibacterial agent is nano-silver modified berberine.

[0014] Preferably, the buffer system is EDTA dissolved in Tris-HCl buffer with a concentration of 10-20 mM and a pH value of 7.5-8.5, wherein the concentration of EDTA is 5-10 mM. The Tris-HCl buffer serves to maintain the pH value of the sample solution stable, providing a stable acid-base environment for cells and DNA, preventing DNA degradation and cell structure damage caused by pH value changes. The added ethylenediaminetetraacetic acid can chelate metal ions such as magnesium ions and calcium ions in the sample, which are activators of nucleases. Ethylenediaminetetraacetic acid inhibits the activity of nucleases through chelation, thereby preventing DNA degradation by nucleases. In addition, when cells rupture and release nucleases due to various reasons, EDTA can timely inhibit their activity, preventing the degradation of released nucleic acids. At the same time, the cell protection system maintains the integrity of the cells, reducing the release of nucleases.

[0015] Preferably, the nucleic acid protective agent is prepared as follows:

[0016] Step A1. Disperse nano-silicon dioxide particles in anhydrous ethanol and ultrasonicate to obtain a nano-silicon dioxide ethanol suspension. The nano-silicon dioxide (SiO2) particles have a large specific surface area and good biocompatibility. The nano-SiO2 particles can tightly bind to nucleic acids in the sample through physical adsorption, forming a nano-scale protective barrier that effectively blocks 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 some extent, maintaining the stability of nucleic acids;

[0017] Step A2. Dissolve the dioleoyl trimethyl ammonium chloride in chloroform, evaporate the chloroform on a rotary evaporator to form a lipid film, then add PBS buffer and ultrasonicate to obtain a uniform cationic liposome solution. The cationic liposome can combine with the negatively charged DNA through electrostatic interaction to form a stable liposome-DNA complex. This complex not only protects the DNA from degradation by nucleases, but also enhances the dispersibility and stability of the DNA in solution. In addition, the cationic liposome also has certain membrane fusion properties, which is helpful for the efficient release of DNA from the storage reagent and into the cell or reaction system during subsequent sample processing;

[0018] Step A3. Mix the nano-silicon dioxide ethanol suspension and the cationic liposome solution uniformly to obtain the nucleic acid protection agent. Nano-SiO2 and cationic liposomes mainly protect nucleic acids from the perspective of physical adsorption and complex formation, while EDTA provides protection from the perspective of inhibiting nuclease activity, forming multiple protections.

[0019] Preferably, the ratio of the nano-silicon dioxide particles and anhydrous ethanol in step A1 is 0.4-0.6 g: 45-55 mL.

[0020] The ultrasonic treatment in step A1 is performed at a power of 200-400 W for 30-60 min.

[0021] Preferably, the ratio of the dioleoyl trimethyl ammonium chloride, chloroform and PBS buffer in step A2 is 0.18-0.22 g: 9.5-12 mL: 4-6 mL.

[0022] The temperature of the rotary evaporator in step A2 is set to 30-40℃, and the vacuum degree is 0.05-0.1 MPa.

[0023] The ultrasonic treatment in step A2 is performed at a power of 100-200 W for 15-30 min.

[0024] Preferably, the cell stabilizer is prepared as follows:

[0025] Step B1. Poly-N-isopropyl acrylamide and polylactic acid-glycolic acid copolymer were added into deionized water respectively, heated to 52-58℃, and stirred at a speed of 280-320 rpm until completely dissolved, then the two solutions were mixed evenly to obtain a polymer solution, using temperature-sensitive polymer poly-N-isopropyl acrylamide (PNIPAAm). PNIPAAm is water-soluble at lower temperatures (below its lower critical solution temperature, about 32℃), and can be uniformly dispersed in the preservative. When the sample temperature decreases, PNIPAAm will undergo phase transition to form a gel-like substance, which wraps around the cells and acts 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, reducing the damage caused by changes in osmotic pressure and other factors. Combined with biodegradable polylactic acid-glycolic acid copolymer (PLGA). PLGA has good biocompatibility and biodegradability, and it can form a protective film on the surface of the cells, enhancing the cells' ability to resist damage. Moreover, PLGA can slowly release some cell protective factors (such as amino acids, sugars, etc.), providing nutrients and protection for cells, which helps to maintain the activity and function of cells;

[0026] Step B2. Add sucrose and trehalose to the polymer solution and mix evenly to obtain a cell stabilizer with a sucrose concentration of 100-120 mM and a trehalose concentration of 50-70 mM.

[0027] Preferably, the ratio of poly-N-isopropyl acrylamide to deionized water in step B1 is 1.8-2.2 g:48-52 mL; and the ratio of polylactic acid-glycolic acid copolymer to deionized water is 0.8-1.3 g:48-52 mL.

[0028] Preferably, the antioxidant is prepared as follows:

[0029] Idarubicin and manganese porphyrin were dissolved in DMSO respectively, then vitamin C and glutathione were added, and finally PBS buffer was added for dilution, so that the concentration of vitamin C was 50-60 μM and the concentration of glutathione was 20-30 μM, to obtain an antioxidant. Idarubicin has strong antioxidant capacity and can effectively scavenge free radicals in the sample, reducing oxidative stress damage to DNA and cells. Compared with traditional antioxidants, idarubicin has a smaller molecular structure and can penetrate cell membranes more easily, thus more efficiently exerting antioxidant effects. Manganese porphyrin can simulate the activity of superoxide dismutase, catalyzing the dismutation of superoxide anion radicals to convert them into oxygen and hydrogen peroxide, further reducing oxidative damage in the sample;

[0030] The ratio of idarubicin to DMSO is 0.008-0.012 g:0.9-1.3 mL.

[0031] The use amount ratio of the manganese porphyrin and DMSO is 0.0025-0.0035g:0.9-1.3mL.

[0032] Preferably, the preparation process of the antibacterial agent is as follows:

[0033] The berberine and the nano-silver particles are respectively added into the deionized water, and are ultrasonically treated in an ultrasonic cleaner at a power of 140-150W for 18-22min to make them uniformly dispersed, and then are uniformly mixed to obtain an antibacterial agent mixed solution.

[0034] The use amount ratio of the berberine, the nano-silver particles and the deionized water is 0.075-0.085g:0.0045-0.0055g:46-53mL.

[0035] A preparation method of a sample preservation reagent for an InDel molecular marker of Pachigrogylus, comprising the following steps:

[0036] Step S1. 14-16mL of a nucleic acid protection agent, 18-22mL of a cell stabilizer, 8-13mL of an antioxidant and 8-12mL of an antibacterial agent are added into a 500mL volumetric flask;

[0037] Step S2. The PBS buffer is used to make up to 500mL, and a magnetic stirrer is used to uniformly stir at a rotating speed of 380-410rpm;

[0038] Step S3. Filtration sterilization is performed through a 0.22μm filter membrane, so as to obtain the sample preservation reagent for the InDel molecular marker of Pachigrogylus.

[0039] The buffer system is matched with the nucleic acid protection system of the nanomaterials, and the pH value stabilizing effect of the buffer is helpful to maintain the combined state of the nanomaterials and the nucleic acids in the process of stabilizing the nucleic acids by the nanometer SiO2 and the cationic liposome.

[0040] Sucrose and trehalose can form a protective film around the cells, the principle is to form a layer of glass-like material on the cell surface by forming hydrogen bonds with water molecules. During freezing and thawing, this protective film can prevent the formation of ice crystals, which can cause cell rupture. At the same time, they can also maintain the osmotic pressure balance of the cells and keep the cells active. PNIPAAm and PLGA mainly protect the 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 cooperation with the nucleic acid protection component, the stable cell structure can reduce the release of intracellular nucleases, thereby indirectly protecting the nucleic acids. At the same time, the stability of nucleic acids is also conducive to the better function of cells after preservation.

[0041] Vitamin C is a common antioxidant that can provide electrons to neutralize free radicals in the sample, such as superoxide anion radicals (O2⁻) and hydroxyl radicals (・OH). Glutathione can remove free radicals through its own redox reaction and participate in the intracellular antioxidant defense system, and works synergistically with idebenone and MnP. Multiple antioxidants can remove free radicals from different mechanisms. For example, idebenone mainly plays an antioxidant role in cell membranes and other parts, vitamin C and glutathione can remove free radicals in aqueous environments inside and outside the cells, and MnP focuses on dismutation of superoxide anion radicals. They work together to more comprehensively reduce the impact of oxidative damage on DNA and cells. At the same time, the role of antioxidants also helps to maintain the stability of other components such as nanomaterials and intelligent polymers.

[0042] Penicillin mainly acts on the synthesis process of bacterial cell wall to inhibit the growth and reproduction of bacteria; streptomycin exerts antibacterial effect by interfering with the synthesis of bacterial proteins, and works synergistically with berberine and nano-silver particles. Berberine mainly destroys microbial cell membranes, nano-silver 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 the sample from being contaminated by microorganisms during preservation, and also helps to maintain the stability of other components (such as nucleic acid protection components, cell protection components, etc.), thereby prolonging the preservation time of the sample.

[0043] The beneficial effects of the present application are as follows:

[0044] The present application provides a sample preservation reagent for an InDel molecular marker of an Odorrana tormota and a preparation method thereof, which has the following significant advantages:

[0045] Effectively protect DNA integrity: through the buffering system to maintain the pH value stability, nucleic acid protective agent to inhibit the nuclease activity and antioxidant to remove free radicals and other mechanisms, can significantly reduce the degradation of DNA in the preservation process, ensure the integrity of DNA, provide a reliable template for the subsequent InDel molecular marker accurate detection;

[0046] Maintain cell activity: the use of cell stabilizers can form a protective film around the cells, prevent ice crystals from damaging cells, and maintain the osmotic pressure balance of cells, so that the cells remain high activity during the preservation process, which is beneficial to the subsequent extraction of high-quality DNA from cells;

[0047] Prevent microbial contamination: the addition of antibacterial agents can effectively inhibit the possible microbial contamination of the sample during the preservation process, prolong the preservation time of the sample, and ensure the quality and safety of the sample;

[0048] Simple operation: the preparation method of the preservation reagent of the application is simple, easy to operate, low in cost, and convenient to use. Only by mixing the collected Pachymedusa samples with the preservation reagent in a certain proportion can the samples be preserved under conventional low-temperature conditions, which is suitable for sample preservation in different laboratories and on-site collection. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below with specific examples.

[0050] The sources and properties of some raw materials used in the present application are as follows:

[0051] Example 1: A preparation method of a sample preservation reagent for InDel molecular markers of Pachymedusa, comprising the following steps:

[0052] S1. Dissolve EDTA in Tris-HCl buffer with a concentration of 10 mM and a pH value of 7.5 to obtain a buffer system, wherein the concentration of EDTA is 5-7.5-10 mM;

[0053] S2. Disperse 0.4 g of nano-silicon dioxide particles in 45 mL of anhydrous ethanol, and treat with ultrasonic waves at 200 W for 30 min to obtain a nano-silicon dioxide ethanol suspension;

[0054] S3. Dissolve 0.18 g of dioleoyl trimethyl ammonium chloride in 9.5 mL of chloroform, evaporate the chloroform on a rotary evaporator, set the temperature of the rotary evaporator to 30 DEG C and the vacuum degree to 0.05 MPa, form a lipid film, then add 4 mL of PBS buffer, and treat with ultrasonic waves at 100 W for 15 min to obtain a uniform cationic liposome solution;

[0055] S4. Mix the nano-silica ethanol suspension and the cationic liposome solution evenly to obtain a nucleic acid protection agent;

[0056] S5. Add 1.8 g of poly-N-isopropyl acrylamide and 0.8 g of poly-lactic acid-glycolic acid copolymer into 48 mL of deionized water respectively, heat to 52℃, and stir at a speed of 280 rpm until completely dissolved, then mix the two solutions evenly to obtain a polymer solution;

[0057] S6. Add sucrose and trehalose into the polymer solution and mix evenly so that the concentration of sucrose is 100 mM and the concentration of trehalose is 50 mM to obtain a cell stabilizer;

[0058] S7. Dissolve 0.008 g of idebenone and 0.0025 g of manganese porphyrin in 0.9 mL of DMSO respectively, then add vitamin C and glutathione, and finally add PBS buffer for dilution so that the concentration of vitamin C is 50 μM and the concentration of glutathione is 20 μM to obtain an antioxidant;

[0059] S8. Add 0.075 g of berberine and 0.0045 g of nano-silver particles into 46 mL of deionized water respectively, and ultrasonically treat in an ultrasonic cleaner at a power of 140 W for 18 min to make them evenly dispersed, then mix evenly to obtain an antibacterial agent;

[0060] S9. Add 14 mL of the nucleic acid protection agent, 18 mL of the cell stabilizer, 8 mL of the antioxidant, and 8 mL of the antibacterial agent into a 500 mL volumetric flask;

[0061] S10. Dilute to 500 mL with the buffer system, and stir evenly with a magnetic stirrer at a speed of 380 rpm;

[0062] S11. Filter sterilization through a 0.22 μm filter membrane to obtain a sample preservation reagent for InDel molecular markers of Paa.

[0063] Embodiment 2: A preparation method of a sample preservation reagent for InDel molecular markers of Paa, comprising the following steps:

[0064] S1. Dissolve EDTA in a Tris-HCl buffer with a concentration of 15 mM and a pH value of 8 to obtain a buffer system, wherein the concentration of EDTA is 7.5 mM;

[0065] S2. Disperse 0.5 g of nano-silica particles in 50 mL of absolute ethanol, and ultrasonically treat at 300 W for 45 min to obtain a nano-silica ethanol suspension;

[0066] S3. Dissolve 0.2 g of dioleoyl trimethyl ammonium chloride in 10.5 mL of chloroform, evaporate the chloroform on a rotary evaporator, set the temperature of the rotary evaporator to 35 DEG C and the vacuum degree to 0.07 MPa, form a lipid film, then add 5 mL of PBS buffer, and treat with ultrasonic waves at 150 W for 20 min to obtain a uniform cationic liposome solution;

[0067] S4. Mix the nano-silicon dioxide ethanol suspension and the cationic liposome solution to obtain a nucleic acid protection agent;

[0068] S5. Add 2 g of poly-N-isopropyl acrylamide and 1 g of polylactic acid-glycolic acid copolymer into 50 mL of deionized water respectively, heat to 55 DEG C, and stir at a speed of 300 rpm until completely dissolved, then mix the two solutions to obtain a polymer solution;

[0069] S6. Add sucrose and trehalose to the polymer solution, mix well to obtain a cell stabilizer, so that the concentration of sucrose is 110 mM and the concentration of trehalose is 60 mM;

[0070] S7. Dissolve 0.01 g of idebenone and 0.003 g of manganese porphyrin in 1.1 mL of DMSO respectively, then add vitamin C and glutathione, and finally add PBS buffer to dilute, so that the concentration of vitamin C is 55 μM and the concentration of glutathione is 25 μM, to obtain an antioxidant;

[0071] S8. Add 0.08 g of berberine and 0.005 g of nano-silver particles into 50 mL of deionized water respectively, treat with ultrasonic waves in an ultrasonic cleaner at a power of 145 W for 20 min to disperse uniformly, then mix well to obtain an antibacterial agent;

[0072] S9. Add 15 mL of nucleic acid protection agent, 20 mL of cell stabilizer, 10 mL of antioxidant and 10 mL of antibacterial agent into a 500 mL volumetric flask;

[0073] S10. Dilute to 500 mL with the buffer system, and stir uniformly with a magnetic stirrer at a speed of 400 rpm;

[0074] S11. Filter sterilization through a 0.22 μm filter membrane to obtain a sample preservation reagent for InDel molecular markers of Paa.

[0075] Example 3: A preparation method of a sample preservation reagent for InDel molecular markers of Paa, comprising the following steps:

[0076] S1. Dissolve EDTA in Tris-HCl buffer with a concentration of 20 mM and a pH value of 8.5 to obtain a buffer system, wherein the concentration of EDTA is 10 mM;

[0077] S2. 0.6 g of nano-silica particles were dispersed in 55 mL of anhydrous ethanol, and ultrasonic treatment was performed at 400 W for 60 min to obtain a nano-silica ethanol suspension;

[0078] S3. 0.22 g of dioleoyl trimethyl ammonium chloride was dissolved in 12 mL of chloroform, and the chloroform was removed by evaporation on a rotary evaporator, the temperature of the rotary evaporator was set to 40 DEG C, and the vacuum degree was 0.1 MPa, a lipid film was formed, 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;

[0079] S4. The nano-silica ethanol suspension and the cationic liposome solution were mixed uniformly to obtain a nucleic acid protection agent;

[0080] S5. 2.2 g of poly-N-isopropyl acrylamide and 1.3 g of poly-lactic acid-glycolic acid copolymer were added to 52 mL of deionized water respectively, heated to 58 DEG C, and stirred at a speed of 320 rpm until completely dissolved, then the two solutions were mixed uniformly to obtain a polymer solution;

[0081] S6. Sucrose and trehalose were added to the polymer solution and mixed uniformly to obtain a cell stabilizer, so that the concentration of sucrose was 120 mM and the concentration of trehalose was 70 mM;

[0082] S7. 0.012 g of idebenone and 0.0035 g of manganese porphyrin were dissolved in 1.3 mL of DMSO respectively, then vitamin C and glutathione were added, and finally PBS buffer was added for dilution, so that the concentration of vitamin C was 60 μM and the concentration of glutathione was 30 μM, to obtain an antioxidant;

[0083] S8. 0.085 g of berberine and 0.0055 g of nano-silver particles were added to 53 mL of deionized water respectively, and ultrasonic treatment was performed in an ultrasonic cleaner at a power of 150 W for 22 min to uniformly disperse them, and then mixed uniformly to obtain an antibacterial agent;

[0084] S9. 16 mL of the nucleic acid protection agent, 22 mL of the cell stabilizer, 13 mL of the antioxidant, and 12 mL of the antibacterial agent were added to a 500 mL volumetric flask;

[0085] S10. The buffer system was used to make up to 500 mL, and a magnetic stirrer was used to stir uniformly at a speed of 410 rpm;

[0086] S11. Filtration sterilization was performed through a 0.22 μm filter membrane to obtain a sample preservation reagent for Ileodromus InDel molecular markers.

[0087] Comparative Example 1:

[0088] The comparative example is compared with example 1, and no nano-silicon dioxide is added in the preparation process of the sample preservation reagent for the InDel molecular marker of the Pachyhynobius, and the remaining steps and parameters are the same. The comparative example will not be repeated, and the sample preservation reagent for the InDel molecular marker of the Pachyhynobius is finally obtained.

[0089] Comparative example 2:

[0090] The comparative example is compared with example 1, and no cationic liposome is added in the preparation process of the sample preservation reagent for the InDel molecular marker of the Pachyhynobius, and the remaining steps and parameters are the same. The comparative example will not be repeated, and the sample preservation reagent for the InDel molecular marker of the Pachyhynobius is finally obtained.

[0091] Comparative example 3:

[0092] The comparative example is compared with example 1, and no poly-N-isopropyl acrylamide is added in the preparation process of the sample preservation reagent for the InDel molecular marker of the Pachyhynobius, and the remaining steps and parameters are the same. The comparative example will not be repeated, and the sample preservation reagent for the InDel molecular marker of the Pachyhynobius is finally obtained.

[0093] Comparative example 4:

[0094] The comparative example is compared with example 1, and no glutathione is added in the preparation process of the sample preservation reagent for the InDel molecular marker of the Pachyhynobius, and the remaining steps and parameters are the same. The comparative example will not be repeated, and the sample preservation reagent for the InDel molecular marker of the Pachyhynobius is finally obtained.

[0095] Comparative example 5:

[0096] The comparative example is compared with example 1, and only “14 mL of nucleic acid protection agent, 18 mL of cell stabilizer, 8 mL of antioxidant and 8 mL of antibacterial agent” are adjusted to “16 mL of nucleic acid protection 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. The comparative example will not be repeated, and the sample preservation reagent for the InDel molecular marker of the Pachyhynobius is finally obtained.

[0097] Comparative example 6:

[0098] The comparative example is compared with example 1, and no EDTA is added in the preparation process of the sample preservation reagent for the InDel molecular marker of the Pachyhynobius, and the remaining steps and parameters are the same. The comparative example will not be repeated, and the sample preservation reagent for the InDel molecular marker of the Pachyhynobius is finally obtained.

[0099] Performance test:

[0100] DNA integrity test:

[0101] Test method: Agarose gel electrophoresis method. Extract DNA from the preserved Rhacophorus ornatus samples after different time (1 week, 2 weeks, 4 weeks, 8 weeks), mix the DNA sample with loading buffer, and add it to the 1%-1.5% agarose gel loading well, and perform electrophoresis in 1xTAE buffer, voltage 100-120V, electrophoresis time 30-60min. After electrophoresis, observe and take pictures under the gel imaging system to record the integrity of the DNA band. The clear DNA band without obvious tailing is the standard of good DNA integrity. Quantitative analysis of band brightness and width is performed by image analysis software (such as ImageJ), and the degradation degree of DNA is calculated.

[0102] Test standard: DNA degradation degree = (initial DNA band brightness - preserved DNA band brightness) / initial DNA band brightness x 100%. The lower the degradation degree, the better the DNA integrity.

[0103] Cell activity test:

[0104] Test method: Trypan blue staining method. The preserved Rhacophorus ornatus cell samples are digested into single cell suspension with trypsin, and an appropriate amount of cell suspension is mixed with 0.4% trypan blue solution at a ratio of 1:1, incubated at room temperature for 2-3min, and then counted under a microscope with a hemocytometer. Live cells are not stained and are colorless; dead cells are stained blue. Calculate cell activity = (number of live cells / total number of cells) x 100%.

[0105] Test standard: The higher the cell activity, the better the protective effect of the sample preservation reagent on cells.

[0106] Microbial contamination test:

[0107] Test method: Plate count method. The preserved sample is gradient diluted with sterile normal saline, and an appropriate amount of diluent is spread on nutrient agar medium plates, which are incubated at 37℃ for 24-48h, and then the number of colonies on the plates is counted. At the same time, set up a blank control plate (only spread sterile normal saline).

[0108] Test standard: The fewer the colonies, the lower the degree of microbial contamination of the sample. If colonies appear on the blank control plate, the experiment is invalid and needs to be repeated.

[0109]

[0110]

[0111]

[0112] Data analysis:

[0113] As can be seen from Tables 1-3, the degree of DNA degradation of Examples 1-3 gradually increases with the extension of the storage time, but the degree of degradation is lower than that of the Comparative Examples under the same storage time, which shows that the complete reagent formula in the Examples, including nano-silica, cationic liposome, EDTA and other components, can effectively protect the integrity of DNA through synergistic effect. The degree of DNA degradation of Comparative Example 1 (without adding nano-silica) and Comparative Example 2 (without adding cationic liposome) is obviously higher than that of the Examples, which shows that nano-silica and cationic liposome play a key role in nucleic acid protection. Nano-silica forms a protective barrier through physical adsorption, and cationic liposome forms a complex with DNA, both of which are indispensable. The degree of DNA degradation of Comparative Example 6 (without adding EDTA) is also relatively high, which shows that the effect of EDTA chelating metal ions to inhibit the activity of nucleases is crucial to maintain the integrity of DNA.

[0114] Cell activity: The cell activity of Examples 1-3 gradually decreases during the storage process, but always remains at a relatively high level, which shows that the reagent with complete formula can better maintain the cell activity. The cell activity of Comparative Example 3 (without adding poly-N-isopropyl acrylamide) decreases relatively greatly, which shows that poly-N-isopropyl acrylamide plays an important role in forming a gel-like substance to wrap the cells and maintain the stability of the cell microenvironment at low temperature. The cell activity of Comparative Example 4 (without adding glutathione) also decreases to a certain extent, which shows that glutathione participates in the intracellular antioxidant defense system and has a positive impact on maintaining cell activity.

[0115] Microbial contamination: The number of colonies of Examples 1-3 is relatively small, which shows that the antibacterial system (nano-silver modified berberine, etc.) can effectively inhibit the growth of microorganisms and prevent sample contamination. The number of colonies of Comparative Examples 1-6 is relatively large, which shows that the lack of some key components will affect the antibacterial effect. For example, the absence of nano-materials in Comparative Examples 1-2 may affect the dispersion and effect of the antibacterial agent; the absence of EDTA in Comparative Example 6 may indirectly affect the stability of the overall reagent, thereby causing microorganisms to grow more easily.

[0116] The Tris-HCl buffer in it plays a role in maintaining the pH value of the sample solution stable, providing a stable acid-base environment for cells and DNA, preventing DNA degradation and cell structure damage caused by pH value change, and adding ethylenediaminetetraacetic acid can chelate metal ions such as magnesium ions and calcium ions in the sample. These metal ions are activators of nucleases, and ethylenediaminetetraacetic acid inhibits the activity of nucleases through chelation, thereby preventing DNA from being degraded by nucleases. In addition, when cells rupture and release nucleases for various reasons, EDTA can inhibit their activity in time to prevent the released nucleic acids from being degraded, and the cell protection system maintains the integrity of the cells and reduces the release of nucleases;

[0117] The adopted nano-silicon dioxide (SiO2) particles 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, forming a nanoscale protective barrier that effectively blocks 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 influence of external environmental factors (such as temperature and pH changes) on nucleic acids to some extent, maintaining the stability of nucleic acids;

[0118] Cationic liposomes can combine with negatively charged DNA through electrostatic interaction to form stable liposome-DNA complexes. Such complexes 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 help DNA to be efficiently released from the preservation reagent and enter cells or reaction systems during subsequent sample processing;

[0119] Thermosensitive polymer poly-N-isopropyl acrylamide (PNIPAAm) is used. PNIPAAm is water-soluble at lower temperatures (below its lower critical solution temperature, about 32°C), and can be uniformly dispersed in the preservation reagent. When the sample temperature decreases, PNIPAAm undergoes 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, reducing damage caused by changes in osmotic pressure and other factors. Combined with biodegradable polylactic acid-glycolic acid copolymer (PLGA). PLGA has good biocompatibility and biodegradability, and it can form a protective film on the surface of the cells, enhancing the cells' resistance to damage. Moreover, PLGA can slowly release some cell-protecting factors (such as amino acids and sugars), providing nutrients and protection for the cells, which helps to maintain the activity and function of the cells;

[0120] Idarubicin has strong antioxidant capacity and can effectively scavenge free radicals in the sample, reducing oxidative stress damage to DNA and cells. Compared with traditional antioxidants, idarubicin has a smaller molecular structure and can more easily penetrate cell membranes, thereby more efficiently exerting antioxidant effects. Manganese porphyrin can mimic the activity of superoxide dismutase, catalyzing the dismutation of superoxide anion radicals, converting them into oxygen and hydrogen peroxide, further reducing oxidative damage in the sample;

[0121] Berberine has broad-spectrum antibacterial activity and can inhibit a variety of bacteria, fungi and other microorganisms. It can combine with phospholipids on the cell membrane of microorganisms, destroy the integrity of the cell membrane, and achieve the purpose of antibiosis. At the same time, berberine also has certain anti-inflammatory and antioxidant properties, which helps to maintain the stability of the sample. Silver nanoparticles have unique antibacterial properties. Their large specific surface area enables them to come into full contact with microorganisms, and the released silver ions can combine with biological macromolecules such as proteins and nucleic acids in the microorganisms, interfere with the metabolism and growth of the microorganisms, and thus achieve high-efficiency antibiosis.

[0122] It should be understood by those of ordinary skill in the art that the above discussion of any of the embodiments is merely exemplary and is not intended to suggest that the scope of the present application is limited to these examples; under the idea of the present application, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other changes of different aspects of the present application as described above. In order to be brief, they are not provided in details.

[0123] The present application is intended to cover all such alternatives, modifications and variations as fall within the broad scope of the appended claims. Accordingly, any and all such modifications, variations, omissions, and equivalents are intended to be encompassed by the present application.

Claims

1. A sample preservation reagent for an InDel molecular marker of Pelophylax lessonae, characterized by comprising, Prepared from the following raw materials: The preservation reagent includes a buffer system, a nucleic acid protective agent, a cell stabilizer, an antioxidant and an antibacterial agent; The amounts of the nucleic acid protective agent, the cell stabilizer, the antioxidant and the antibacterial agent are 14-16 mL, 18-22 mL, 8-13 mL and 8-12 mL, respectively; The buffer system is EDTA dissolved in Tris-HCl buffer with a concentration of 10-20 mM and a pH value of 7.5-8.5, wherein the concentration of EDTA is 5-10 mM; The nucleic acid protective agent is prepared from nano-silicon dioxide and cationic liposomes; The cell stabilizer is a mixture of sucrose, trehalose, poly-N-isopropyl acrylamide and lactic acid-acetic acid copolymer; The antioxidant is prepared from idebenone, manganese porphyrin, vitamin C and glutathione; The antibacterial agent is nano-silver modified berberine; The preparation process of the nucleic acid protective agent is as follows: Step A1. Disperse nano-silicon dioxide particles in anhydrous ethanol, and ultrasonically treat to obtain a nano-silicon dioxide ethanol suspension; Step A2. Dissolve dioleoyl trimethyl ammonium chloride in chloroform, evaporate the chloroform on a rotary evaporator to form a lipid film, then add PBS buffer and ultrasonically treat to obtain a uniform cationic liposome solution; Step A3. Mix the nano-silicon dioxide ethanol suspension and the cationic liposome solution uniformly to obtain the nucleic acid protective agent; The preparation process of the cell stabilizer is as follows: Step B1. Add poly-N-isopropyl acrylamide and poly-lactic acid-glycolic acid copolymer to deionized water respectively, heat to 52-58°C, and stir at a speed of 280-320 rpm until completely dissolved, then mix the two solutions uniformly to obtain a polymer solution; Step B2. Add sucrose and trehalose to the polymer solution, mix uniformly, so that the concentration of sucrose is 100-120 mM and the concentration of trehalose is 50-70 mM, to obtain the cell stabilizer; The amount ratio of the nano-silicon dioxide particles to anhydrous ethanol in step A1 is 0.4-0.6 g:45-55 mL; The amount ratio of dioleoyl trimethyl ammonium chloride, chloroform and PBS buffer in step A2 is 0.18-0.22 g:9.5-12 mL:4-6 mL; The amount ratio of poly-N-isopropyl acrylamide to deionized water in step B1 is 1.8-2.2 g:48-52 mL; the amount ratio of poly-lactic acid-glycolic acid copolymer to deionized water is 0.8-1.3 g:48-52 mL; The preparation process of the antioxidant is as follows: Dissolve idebenone and manganese porphyrin in DMSO respectively, then add vitamin C and glutathione, and finally add PBS buffer for dilution, so that the concentration of vitamin C is 50-60 μM and the concentration of glutathione is 20-30 μM, to obtain the antioxidant; The amount ratio of idebenone to DMSO is 0.008-0.012 g:0.9-1.3 mL; The amount ratio of manganese porphyrin to DMSO is 0.0025-0.0035 g:0.9-1.3 mL; The preparation process of the antibacterial agent is as follows: The berberine and nano-silver particles are respectively added into deionized water, and ultrasonic treatment is carried out in an ultrasonic cleaner to make them uniformly dispersed, and then they are uniformly mixed to obtain an antibacterial agent mixed solution. The dosage ratio of the berberine, nano-silver particles and deionized water is 0.075-0.085 g: 0.0045-0.0055 g: 46-53 mL.

2. The sample preservation reagent for Pachina floresa InDel molecular marker according to claim 1, characterized in that, The power of the ultrasonic treatment in step A1 is 200-400 W, and the time is 30-60 min.

3. The sample preservation reagent for Pachina floresa InDel molecular marker according to claim 1, characterized in that, In step A2, the temperature of the rotary evaporator is set to 30-40 DEG 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.

4. The sample preservation reagent for Pachina floresa InDel molecular marker according to claim 1, characterized in that, The power of the ultrasonic cleaner in the preparation process of the antibacterial agent is 140-150 W, and the ultrasonic treatment time is 18-22 min.

5. The method for preparing a sample preservation reagent for an InDel molecular marker of P. spinimacula according to any one of claims 1-4, characterized in that, The preparation process comprises the following steps: Step S1. 14-16 mL of the nucleic acid protection agent, 18-22 mL of the cell stabilizer, 8-13 mL of the antioxidant and 8-12 mL of the antibacterial agent are added into a 500 mL volumetric flask; Step S2. The buffer solution is used to make up to 500 mL, and a magnetic stirrer is used to stir uniformly at a speed of 380-410 rpm; Step S3. Filtration sterilization is carried out through a 0.22 μm filter membrane, and a sample preservation reagent for an InDel molecular marker of Paa spinosa is obtained.

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