A sample DNA extraction reagent for InDel molecular markers of Rana spinosa and its preparation method

Abstract

The present invention relates to the field of biotechnology, and particularly to a DNA extraction reagent for a Rana spinosa InDel molecular marker sample and a preparation method thereof. The DNA extraction reagent for a Rana spinosa InDel molecular marker sample provided by the present invention comprises 60-70 parts of a lysate, 8-12 parts of a protease mixture, 10-15 parts of an impurity remover, 6-9 parts of a DNA precipitant, and 10-15 parts of a washing solution. Each component is prepared through specific steps. DNA is extracted using the reagent, and through steps such as sample pretreatment, lysis, impurity removal, precipitation, washing, and dissolution, impurities can be effectively removed, thereby improving DNA purity, concentration, and integrity. PCR amplification has a good effect, and the product band is single and clear, free of primer dimers and non-specific amplification. This greatly improves the accuracy and reliability of Rana spinosa InDel molecular marker detection, and promotes Rana spinosa molecular genetics research and related industries.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a sample DNA extraction reagent for Rana spinosa InDel molecular markers and a preparation method thereof. Background Art

[0002] The spiny-breasted frog (Quasipaa spinosa), a large amphibian endemic to my country, inhabits clear, fast-flowing waters such as mountain streams and rivers. It is widely distributed in southern my country. Not only is it a crucial component of the ecosystem, maintaining a balanced aquatic food chain, but its tender, nutritious meat also makes it valuable for both food and medicinal purposes. In recent years, the scale of its captive breeding has continued to expand.

[0003] Behind the booming development of the spiny-chested frog industry, molecular biotechnology is playing an increasingly prominent role in the management and optimal utilization of its germplasm resources. InDel (Insertion / Deletion) molecular marker technology, with its rich polymorphism, high detection stability, and relatively simple operation, has become a key tool for studying the genetic characteristics of spiny-chested frogs. This technology, based on the insertion or deletion of bases at specific locations in the genome, can accurately reveal subtle yet critical genetic differences between individuals and populations of spiny-chested frogs through PCR amplification and subsequent analysis, providing a powerful basis for species identification, kinship determination, and the exploration of the genetic mechanisms of desirable traits.

[0004] However, the unique characteristics of Rana spinosa samples present significant challenges for DNA extraction, severely restricting the efficient application of InDel molecular labeling technology. First, the skin and muscle tissues of Rana spinosa are rich in adhesive proteins and polysaccharide complexes. These adhesive substances act like a dense network, tightly enveloping cells and nucleic acid components, making it difficult for conventional cell lysis methods to quickly and fully penetrate the cells, greatly hindering the release of DNA. Second, the active biological metabolism in the body produces a wide variety of secondary metabolites, such as polyphenols and terpenoids. Polyphenols are highly susceptible to oxidation, and the resulting quinone products are extremely nucleophilic and can irreversibly bind to DNA molecules, causing DNA damage, resulting in fragmentation of the extracted DNA, and seriously affecting its integrity. Terpenoids, on the other hand, often have unique chemical structures that interfere with the chemical equilibrium in subsequent DNA isolation and purification steps, reducing extraction efficiency and purity.

[0005] Furthermore, the spinous frog's habitat has endowed it with a unique immune defense mechanism. Its body is rich in immunologically active substances such as antimicrobial peptides and lysozymes. These substances can persistently interfere with the normal process of cell lysis during DNA extraction after sample collection, inhibiting the precise regulation of nuclease activity and further complicating the acquisition of high-quality, intact DNA. Conventional DNA extraction reagents and methods are mostly designed for common model organisms or general biological samples, failing to fully account for the complex characteristics of spinous frog samples. Consequently, in practical applications, the extracted DNA is often contaminated with numerous impurities, typically maintaining only low purity levels. The A260 / A280 ratio often deviates from the ideal range (1.8-2.0), and DNA integrity is questionable, with frequent degradation. These DNA cannot meet the stringent requirements of InDel molecular labeling technology for high template purity and integrity. This leads to inefficient subsequent PCR amplification and a significant risk of misinterpretation. This significantly limits the depth and precision of molecular genetic research on spinous frogs and slows the pace of related industries in breeding improved varieties and efficiently managing germplasm resources based on precise molecular information. Therefore, developing an extraction reagent that is specifically adapted to the characteristics of Rana spinosa samples and can stably produce high-quality DNA has become an urgent task to promote molecular research and industrial upgrading of Rana spinosa.

[0006] Therefore, based on the above-mentioned related technologies, it is urgent to develop a sample DNA extraction reagent for InDel molecular markers of Rana spinosa and a preparation method thereof. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to propose a sample DNA extraction reagent for InDel molecular markers of Rana spinosa and its preparation method, aiming to provide a high-quality DNA extraction method for research work such as germplasm identification, genetic diversity analysis and molecular breeding of Rana spinosa.

[0008] Based on the above purpose, the present invention provides a sample DNA extraction reagent for Rana spinosa InDel molecular marker and a preparation method thereof.

[0009] A DNA extraction reagent for a sample of Rana spinosa InDel molecular marker is prepared from the following raw materials in parts by weight: 60-70 parts of a lysate, 8-12 parts of a protease mixture, 10-15 parts of an impurity remover, 6-9 parts of a DNA precipitant, and 10-15 parts of a washing solution;

[0010] The lysate comprises the following raw materials in parts by weight: Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, hexadecyltrimethylammonium bromide and polyvinylpyrrolidone;

[0011] The mass ratio of Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, cetyltrimethylammonium bromide and polyvinylpyrrolidone is 18-20:25-28:8-10:26-32:5-8:14-16:6-10;

[0012] The pH of the Tris-HCl is 8-8.5, and the pH of the EDTA is 7.8-8.3;

[0013] The preparation process of the protease-modified guanidine isothiocyanate is as follows:

[0014] Step A1. Solution preparation: dissolve guanidine thiocyanate in a buffer solution to prepare a 3-5 mol / L guanidine thiocyanate solution, and dilute papain with a buffer solution to 6-10 mg / mL to obtain a papain solution;

[0015] Step A2. Mix the guanidine thiocyanate solution and the papain solution in a reaction vessel, add the buffer, and place the reaction vessel in an environment of 55-65°C and stir at 300-500 rpm for 8-10h;

[0016] Step A3. After the reaction is completed, the reaction system is heated to 80-90° C. and maintained for 20-30 min. The mixture is centrifuged at a speed of 10,000-12,000 rpm for 20-25 min. The supernatant is collected to obtain protease-modified guanidine isothiocyanate.

[0017] The guanidine thiocyanate in step A1 is analytical grade guanidine thiocyanate;

[0018] The enzymatic activity unit of papain in step A1 is 7500-8500 U / g;

[0019] The buffer in step A1 is a Tris-HCl buffer with a pH of 6-7;

[0020] The mass ratio of guanidine thiocyanate to papain in the guanidine thiocyanate solution and the papain solution in step A2 is 40-50:1-3;

[0021] The buffer in step A2 is a Tris-HCl buffer with a pH of 6-7;

[0022] The protease mixture comprises the following raw materials in parts by weight: papain, acid protease, neutral protease, and solvent;

[0023] The mass ratio of papain, acidic protease, neutral protease and solvent is 14-16:10-14:8-12:980-1020;

[0024] The solvent is a Tris-HCl buffer solution containing 9-11 mM CaCl2 at pH = 7-7.5;

[0025] The acidic protease is any one of pepsin and Aspergillus niger acidic protease with a pH of 1.5-2.5;

[0026] The neutral protease is any one of a Bacillus subtilis neutral protease and a thermophilic neutral protease with a pH of 6.5-7.5.

[0027] The impurity remover is prepared by mixing phenol, chloroform and isoamyl alcohol in a volume ratio of 22-24:23-25:2.5-3.5;

[0028] The phenol is saturated with a Tris-HCl buffer solution having a pH of 7.8-8.0;

[0029] The DNA precipitant is prepared by mixing 2.4-2.6M potassium acetate and anhydrous ethanol in a volume ratio of 0.8-1.1:4.7-5.2, and the pH of the potassium acetate is 4.6-5.0;

[0030] The washing solution is 74.5%-75.5% ethanol;

[0031] A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular markers comprises the following steps:

[0032] Step S1. Preparation of lysis buffer: Accurately weigh Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, cetyltrimethylammonium bromide, and polyvinylpyrrolidone, place in a suitable glass container, add deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8-8.2, and continuously stir to ensure that all components are fully and evenly dissolved. After all solid components are completely dissolved and the solution is clear, vacuum filter and sterilize using a 0.22 μm microporous filter membrane. Collect the filtrate into a sterile brown bottle, seal it, and store it at room temperature in the dark to obtain a lysis buffer;

[0033] Step S2. Preparing a protease mixture: Weigh papain, acidic protease, and neutral protease powders separately and place them in pre-chilled centrifuge tubes. Add solvent to each centrifuge tube and vortex to slowly dissolve the proteases to obtain a protease mixture. Dispense the prepared protease mixture into sterile EP tubes and store frozen in a -20°C freezer. Remove as needed for use to avoid repeated freezing and thawing to obtain a protease mixture.

[0034] Step S3. Preparation of a decontamination agent: In a well-ventilated, dimly lit chemical fume hood, accurately measure phenol, chloroform, and isoamyl alcohol according to their volume ratios and pour them into a clean glass separatory funnel. Slowly rotate the separatory funnel and gently shake to mix thoroughly. Be careful to be gentle to prevent splashing. The mixing process lasts for approximately 5-10 minutes. Transfer the mixed decontamination agent to a brown glass bottle, seal the bottle cap, and refrigerate at 4°C to obtain the decontamination agent.

[0035] Step S4. Preparation of the precipitant: First, accurately prepare a 2.4-2.6 M potassium acetate solution. Carefully adjust the pH to 4.6-5.0 with glacial acetic acid. Slowly add the potassium acetate solution to anhydrous ethanol while stirring to thoroughly mix the two. The entire process is performed in an ice bath to minimize the effects of solution volatility and temperature on component stability. After uniform mixing, transfer the solution to a graduated sterile reagent bottle, seal it, and store at 4°C. Before use, check for precipitation. If precipitation is present, prepare a new solution to obtain the DNA precipitant.

[0036] Step S5. Preparation of washing solution: Measure anhydrous ethanol with a graduated cylinder and pour it into a clean glass container. Slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the diluted ethanol concentration reaches 74.5%-75.5%. After stirring evenly, store at room temperature. Before use, ensure that the bottle cap is well sealed to prevent ethanol volatilization and concentration changes to obtain washing solution.

[0037] The ratio of Tris-HCl to deionized water in step S1 is 18-20 g: 350-360 mL;

[0038] A method for extracting DNA from a Rana spinosa sample comprises the following steps:

[0039] Step T1. Sample pretreatment: Take 0.2-0.4 g of fresh muscle tissue sample and quickly mince it into small particles using sterile scissors. Transfer the sample to a 2 mL sterile centrifuge tube, ensuring that the sample is at the bottom of the tube with no residue on the wall.

[0040] Step T2. Lysis: Add 700-1000 μL of lysis buffer to the centrifuge tube containing the sample, followed by 80-120 μL of protease mix. Vortex for 30-60 seconds to thoroughly mix the sample and reagents. Then, place the centrifuge tube in a constant temperature water bath at 60-65°C and incubate for 1.5-2.5 hours. Remove the tube every 30-40 minutes and gently invert it 5-8 times to ensure even heating and sufficient lysis of the sample.

[0041] Step T3. Impurity Removal: After the sample is lysed and cooled to room temperature, add an equal volume of impurity remover. Invert the centrifuge tube 10-15 times, then place the centrifuge tube in a high-speed refrigerated centrifuge and centrifuge at 13,000-15,000 rpm for 10-15 minutes at 3-5°C. After centrifugation, carefully transfer the upper clear aqueous phase to a new sterile centrifuge tube, taking care to avoid aspirating impurities in the intermediate layer.

[0042] Step T4. Precipitation: Add 1 / 10 volume of DNA precipitant to a fresh tube and gently invert 5-8 times to mix. A white, flocculent DNA precipitate will gradually form. Place the tube in a -20°C refrigerator for 30-45 minutes to further promote complete precipitation. Centrifuge again at 13,000-15,000 rpm for 10-15 minutes at 4°C. Discard the supernatant.

[0043] Step T5. Wash: Add 800-1000 μL of wash buffer and invert the tube 5-8 times to resuspend the pellet and wash. Centrifuge at 12,000-14,000 rpm for 8-10 min at 4°C. Discard the supernatant and repeat the wash step once to ensure that all impurities in the pellet are removed.

[0044] Step T6. Dissolution: Leave the precipitate in the open air at room temperature for 2-3 minutes to allow any residual wash solution to evaporate. Add 30-50 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to dissolve the precipitate. Store the resulting DNA solution at -20°C to avoid repeated freeze-thaw cycles. Regularly monitor the DNA concentration and purity for subsequent InDel labeling experiments.

[0045] Beneficial effects of the present invention:

[0046] The present invention designs a dedicated DNA extraction reagent and method based on the characteristics of Rana spinosa samples. Through the synergistic effect of a specifically formulated lysis solution (containing Tris-HCl, NaCl, EDTA, guanidine isothiocyanate, protease-modified guanidine isothiocyanate, hexadecyltrimethylammonium bromide and polyvinylpyrrolidone, etc.), a protease mixture, a decontaminating agent, a DNA precipitant and a washing solution, the interference of sticky protein and polysaccharide complexes, secondary metabolites and immunoactive substances in Rana spinosa samples on DNA extraction is effectively overcome. Compared with existing technologies, the DNA purity is significantly improved, and the A260 / A280 ratio is stabilized in the ideal range of 1.8-2.0, reducing residual impurities; the DNA concentration is guaranteed to be 280-350ng / μL, meeting the template quantity requirements of subsequent experiments; the DNA integrity is maintained, and the electrophoresis bands are clear without tailing or diffusion; the PCR amplification effect is good, and the product bands are single and clear without primer dimers and non-specific amplification, which greatly improves the accuracy and reliability of the spiny frog InDel molecular marker detection, and effectively promotes the spiny frog molecular genetics research and related industry development. DETAILED DESCRIPTION

[0047] 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 with reference to specific embodiments.

[0048] Example 1: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular markers, comprising the following steps:

[0049] S1. Solution preparation: Dissolve analytical grade guanidine thiocyanate in Tris-HCl buffer at pH 6 to prepare a 3 mol / L guanidine thiocyanate solution. Dilute papain with an activity of 7500 U / g to 6 mg / mL in Tris-HCl buffer at pH 6 to obtain a papain solution.

[0050] S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, Tris-HCl buffer with a pH of 6 was added, and the reaction vessel was placed in a 55°C environment and stirred at 300 rpm for 8 h, wherein the mass ratio of guanidine thiocyanate to papain in the guanidine thiocyanate solution and the papain solution was 40:1;

[0051] S3. After the reaction, the reaction system was heated to 80°C for 20 min, centrifuged at 10,000 rpm for 20 min, and the supernatant was collected to obtain protease-modified guanidine isothiocyanate;

[0052] S4. Accurately weigh 18 g Tris-HCl, 25 g NaCl, 8 g EDTA, 26 g guanidine thiocyanate, 5 g protease-modified guanidine thiocyanate, 14 g cetyltrimethylammonium bromide, and 6 g polyvinylpyrrolidone, place in a suitable glass container, add 350 mL of deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8, and continuously stir to ensure that all ingredients are fully dissolved. After all solid components are completely dissolved and the solution is clear, vacuum filter and sterilize using a 0.22 μm microporous membrane. Collect the filtrate into a sterile brown bottle, seal it, and store it at room temperature in the dark to obtain the lysate;

[0053] S5. Weigh 14 g of papain, 10 g of pepsin (pH 1.5), and 8 g of Bacillus subtilisin (pH 6.5) into pre-chilled centrifuge tubes. Add Tris-HCl buffer (pH 7) containing 9 mM CaCl2 to each tube and vortex to slowly dissolve the proteases to obtain a protease mixture. The weight of the Tris-HCl buffer (pH 7) containing 9 mM CaCl2 is 980 g. Aliquot the prepared protease mixture into sterile EP tubes and store in a -20°C freezer. Remove as needed for use to avoid repeated freeze-thaw cycles. This provides a protease mixture.

[0054] S6. In a well-ventilated, dimly lit chemical fume hood, accurately measure 22 mL of phenol saturated with pH 7.8 Tris-HCl buffer, 23 mL of chloroform, and 2.5 mL of isoamyl alcohol, based on the volume ratio. Pour each into a clean glass separatory funnel. Slowly rotate the separatory funnel and gently shake to mix thoroughly. Be careful to avoid splashing the liquid. Mix for approximately 5 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and refrigerate at 4°C to obtain the impurity remover.

[0055] S7. First, accurately prepare a 2.4 M potassium acetate solution. Carefully adjust the pH to 4.6 with glacial acetic acid. Slowly add 8 mL of potassium acetate solution to 47 mL of anhydrous ethanol, stirring to thoroughly mix the two. This should be done in an ice bath throughout the process to minimize the effects of evaporation and temperature on the stability of the components. After mixing, transfer the solution to a graduated sterile reagent bottle, seal it, and store at 4°C. Before use, check for precipitation. If precipitation is present, prepare a new solution to obtain the DNA precipitation agent.

[0056] S8. Use a graduated cylinder to measure anhydrous ethanol and pour it into a clean glass container. Slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the diluted ethanol concentration reaches 74.5%. After stirring evenly, store at room temperature. Before use, ensure that the bottle cap is well sealed to prevent ethanol volatilization and concentration changes. Obtain the washing solution.

[0057] Example 2: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular markers, comprising the following steps:

[0058] S1. Solution preparation: Dissolve analytical grade guanidine thiocyanate in Tris-HCl buffer at pH 6.5 to prepare a 4 mol / L guanidine thiocyanate solution. Dilute papain with an activity of 7800 U / g to 7 mg / mL in Tris-HCl buffer at pH 6.5 to obtain a papain solution.

[0059] S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, Tris-HCl buffer with a pH of 6.5 was added, and the reaction vessel was placed in a 58°C environment and stirred at 350 rpm for 9 hours, wherein the mass ratio of guanidine thiocyanate to papain in the guanidine thiocyanate solution and the papain solution was 42:2;

[0060] S3. After the reaction, the reaction system was heated to 82°C for 22 min, centrifuged at 10,500 rpm for 21 min, and the supernatant was collected to obtain protease-modified guanidine isothiocyanate;

[0061] S4. Accurately weigh 19 g Tris-HCl, 26 g NaCl, 9 g EDTA, 27 g guanidine thiocyanate, 7 g protease-modified guanidine thiocyanate, 15 g cetyltrimethylammonium bromide, and 7 g polyvinylpyrrolidone, place in a suitable glass container, add 353 mL of deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8.1, and continuously stir to ensure that all ingredients are fully dissolved. After all solid components are completely dissolved and the solution is clear, vacuum filter and sterilize using a 0.22 μm microporous membrane. Collect the filtrate into a sterile brown bottle, seal it, and store it at room temperature in the dark to obtain the lysate;

[0062] S5. Weigh 15 g of papain, 11 g of Aspergillus niger acid protease (pH 1.6), and 9 g of Thermophilic neutral protease (pH 6.5) respectively, place them in pre-chilled centrifuge tubes, add Tris-HCl buffer (pH 7.1) containing 10 mM CaCl2 to each centrifuge tube, and vortex to slowly dissolve the proteases to obtain a protease mixture, wherein the weight of Tris-HCl buffer (pH 7.1) containing 10 mM CaCl2 is 1000 g. Aliquot the prepared protease mixture into sterile EP tubes and store in a -20°C freezer. Remove as needed for use to avoid repeated freeze-thaw cycles, to obtain a protease mixture.

[0063] S6. In a well-ventilated, dimly lit chemical fume hood, accurately measure 23 mL of phenol saturated with pH 7.9 Tris-HCl buffer, 24 mL of chloroform, and 2.6 mL of isoamyl alcohol, based on the volume ratio. Pour each into a clean glass separatory funnel. Slowly rotate the separatory funnel and gently shake to mix thoroughly. Be careful to avoid splashing the liquid. Mix for approximately 6 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and refrigerate at 4°C to obtain the impurity remover.

[0064] S7. First, accurately prepare a 2.5 M potassium acetate solution. Carefully adjust the pH to 4.7 with glacial acetic acid. Slowly add 9 mL of potassium acetate solution to 48 mL of anhydrous ethanol, stirring while adding to thoroughly mix the two. This should be done in an ice bath throughout the process to minimize the effects of evaporation and temperature on the stability of the components. After mixing, transfer the solution to a graduated sterile reagent bottle, seal it, and store at 4°C. Before use, check for precipitation. If precipitation is present, prepare a new solution to obtain the DNA precipitation agent.

[0065] S8. Use a graduated cylinder to measure anhydrous ethanol and pour it into a clean glass container. Slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the diluted ethanol concentration reaches 74.6%. After stirring evenly, store at room temperature. Before use, ensure that the bottle cap is well sealed to prevent ethanol volatilization and concentration changes. Obtain the washing solution.

[0066] Example 3: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular markers, comprising the following steps:

[0067] S1. Solution preparation: Dissolve analytical grade guanidine thiocyanate in Tris-HCl buffer at pH 7 to prepare a 5 mol / L guanidine thiocyanate solution. Dilute papain with an activity of 8000 U / g to 8 mg / mL in Tris-HCl buffer at pH 7 to obtain a papain solution.

[0068] S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, Tris-HCl buffer with a pH of 7 was added, and the reaction vessel was placed in a 60°C environment and stirred at 400 rpm for 10 h, wherein the mass ratio of guanidine thiocyanate to papain in the guanidine thiocyanate solution and the papain solution was 45:3;

[0069] S3. After the reaction, the reaction system was heated to 85°C for 25 min, centrifuged at 11,000 rpm for 22 min, and the supernatant was collected to obtain protease-modified guanidine isothiocyanate;

[0070] S4. Accurately weigh 20 g Tris-HCl, 27 g NaCl, 10 g EDTA, 28 g guanidine thiocyanate, 6 g protease-modified guanidine thiocyanate, 16 g cetyltrimethylammonium bromide, and 8 g polyvinylpyrrolidone, place in a suitable glass container, add 356 mL of deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8.2, and continuously stir to ensure that all ingredients are fully dissolved. After all solid components are completely dissolved and the solution is clear, vacuum filter and sterilize using a 0.22 μm microporous membrane. Collect the filtrate into a sterile brown bottle, seal it, and store it at room temperature in the dark to obtain the lysate;

[0071] S5. Weigh 16 g of papain, 12 g of pepsin (pH 1.8), and 10 g of Bacillus subtilisin (pH 6.7) into pre-chilled centrifuge tubes. Add Tris-HCl buffer (pH 7.2) containing 11 mM CaCl2 to each tube and vortex to slowly dissolve the proteases to obtain a protease mixture, wherein the weight of Tris-HCl buffer (pH 7.2) containing 11 mM CaCl2 is 1010 g. Aliquot the prepared protease mixture into sterile EP tubes and store in a -20°C freezer. Remove as needed for use to avoid repeated freeze-thaw cycles. This provides a protease mixture.

[0072] S6. In a well-ventilated, dimly lit chemical fume hood, accurately measure 24 mL of phenol saturated with pH 8.0 Tris-HCl buffer, 25 mL of chloroform, and 2.8 mL of isoamyl alcohol, based on the volume ratio. Pour each into a clean glass separatory funnel. Slowly rotate the separatory funnel and gently shake to mix thoroughly. Be careful to avoid splashing the liquid. The mixing process continues for approximately 7 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and refrigerate at 4°C to obtain the impurity remover.

[0073] S7. First, accurately prepare a 2.6 M potassium acetate solution. Carefully adjust the pH to 4.8 with glacial acetic acid. Slowly add 10 mL of potassium acetate solution to 49 mL of anhydrous ethanol, stirring to thoroughly mix the two. This should be done in an ice bath throughout the process to minimize the effects of evaporation and temperature on the stability of the components. After mixing, transfer the solution to a graduated sterile reagent bottle, seal it, and store at 4°C. Before use, check for precipitation. If precipitation is present, prepare a new solution to obtain the DNA precipitation agent.

[0074] S8. Use a graduated cylinder to measure anhydrous ethanol and pour it into a clean glass container. Slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the diluted ethanol concentration reaches 74.8%. After stirring evenly, store at room temperature. Before use, ensure that the bottle cap is well sealed to prevent ethanol volatilization and concentration changes. Obtain the washing solution.

[0075] Example 4: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular markers, comprising the following steps:

[0076] S1. Solution preparation: Dissolve analytical grade guanidine thiocyanate in Tris-HCl buffer at pH 6 to prepare a 3 mol / L guanidine thiocyanate solution. Dilute papain with an activity of 8200 U / g to 9 mg / mL in Tris-HCl buffer at pH 6 to obtain a papain solution.

[0077] S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, Tris-HCl buffer with a pH of 6 was added, and the reaction vessel was placed in a 62°C environment and stirred at 450 rpm for 8 h, wherein the mass ratio of guanidine thiocyanate to papain in the guanidine thiocyanate solution and the papain solution was 46:1;

[0078] S3. After the reaction, the reaction system was heated to 86°C for 26 min, centrifuged at 11500 rpm for 23 min, and the supernatant was collected to obtain protease-modified guanidine isothiocyanate;

[0079] S4. Accurately weigh 18 g Tris-HCl, 28 g NaCl, 8 g EDTA, 29 g guanidine thiocyanate, 5 g protease-modified guanidine thiocyanate, 14 g cetyltrimethylammonium bromide, and 9 g polyvinylpyrrolidone, place in a suitable glass container, add 358 mL of deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8, and continuously stir to ensure that all ingredients are fully dissolved. After all solid components are completely dissolved and the solution is clear, vacuum filter and sterilize using a 0.22 μm microporous membrane. Collect the filtrate into a sterile brown bottle, seal it, and store it at room temperature in the dark to obtain the lysate;

[0080] S5. Weigh 14 g of papain, 13 g of Aspergillus niger acid protease (pH 2.0), and 11 g of Thermophilic neutral protease (pH 6.8) separately and place them in pre-chilled centrifuge tubes. Add Tris-HCl buffer (pH 7.3) containing 9 mM CaCl2 to each centrifuge tube and vortex to slowly dissolve the proteases to obtain a protease mixture, wherein the weight of Tris-HCl buffer (pH 7.3) containing 9 mM CaCl2 is 1008 g. Aliquot the prepared protease mixture into sterile EP tubes and store in a -20°C freezer. Remove as needed for use to avoid repeated freeze-thaw cycles to obtain a protease mixture.

[0081] S6. In a well-ventilated, dimly lit chemical fume hood, accurately measure 22 mL of phenol saturated with pH 7.8 Tris-HCl buffer, 23 mL of chloroform, and 3.0 mL of isoamyl alcohol, based on the volume ratio. Pour the mixture into a clean glass separatory funnel. Slowly rotate the separatory funnel and gently shake it to mix thoroughly. Be careful to avoid splashing the liquid. The mixing process continues for approximately 8 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and refrigerate at 4°C to obtain the impurity remover.

[0082] S7. First, accurately prepare a 2.4 M potassium acetate solution. Carefully adjust the pH to 4.9 with glacial acetic acid. Slowly add 11 mL of potassium acetate solution to 50 mL of anhydrous ethanol, stirring to thoroughly mix the two. This should be done in an ice bath throughout the process to minimize the effects of evaporation and temperature on the stability of the components. After mixing, transfer the solution to a graduated sterile reagent bottle, seal it, and store at 4°C. Before use, check for precipitation. If precipitation is present, prepare a new solution to obtain the DNA precipitation agent.

[0083] S8. Use a graduated cylinder to measure anhydrous ethanol and pour it into a clean glass container. Slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the diluted ethanol concentration reaches 75%. After stirring evenly, store at room temperature. Before use, ensure that the bottle cap is well sealed to prevent ethanol volatilization and concentration changes. Obtain the washing solution.

[0084] Example 5: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular markers, comprising the following steps:

[0085] S1. Solution preparation: Dissolve analytical grade guanidine thiocyanate in Tris-HCl buffer at pH 6.5 to prepare a 4 mol / L guanidine thiocyanate solution. Dilute papain with an activity of 8300 U / g to 7 mg / mL in Tris-HCl buffer at pH 6.5 to obtain a papain solution.

[0086] S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, Tris-HCl buffer with a pH of 6.5 was added, and the reaction vessel was placed in a 63°C environment and stirred at 460 rpm for 9 h, wherein the mass ratio of guanidine thiocyanate to papain in the guanidine thiocyanate solution and the papain solution was 48:2;

[0087] S3. After the reaction, the reaction system was heated to 88°C for 28 min, centrifuged at 11800 rpm for 24 min, and the supernatant was collected to obtain protease-modified guanidine isothiocyanate;

[0088] S4. Accurately weigh 19 g Tris-HCl, 27 g NaCl, 9 g EDTA, 30 g guanidine thiocyanate, 7 g protease-modified guanidine thiocyanate, 15 g cetyltrimethylammonium bromide, and 8 g polyvinylpyrrolidone, place in a suitable glass container, add 355 mL of deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8.1, and continuously stir to ensure that all ingredients are fully dissolved. After all solid components are completely dissolved and the solution is clear, vacuum filter and sterilize using a 0.22 μm microporous membrane. Collect the filtrate into a sterile brown bottle, seal it, and store it at room temperature in the dark to obtain the lysate;

[0089] S5. Weigh 15 g of papain, 12 g of pepsin (pH 2.2), and 10 g of Bacillus subtilisin (pH 7.2) into pre-chilled centrifuge tubes. Add Tris-HCl buffer (pH 7.4) containing 10 mM CaCl2 to each tube and vortex to slowly dissolve the proteases to obtain a protease mixture, wherein the weight of Tris-HCl buffer (pH 7.4) containing 10 mM CaCl2 is 988 g. Aliquot the prepared protease mixture into sterile EP tubes and store in a -20°C freezer. Remove as needed for use to avoid repeated freeze-thaw cycles. This provides a protease mixture.

[0090] S6. In a well-ventilated, dimly lit chemical fume hood, accurately measure 23 mL of phenol saturated with pH 7.9 Tris-HCl buffer, 24 mL of chloroform, and 3.3 mL of isoamyl alcohol, based on the volume ratio. Pour each into a clean glass separatory funnel. Slowly rotate the separatory funnel and gently shake to mix thoroughly. Be careful to avoid splashing the liquid. The mixing process lasts for approximately 9 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and refrigerate at 4°C to obtain the impurity remover.

[0091] S7. First, accurately prepare a 2.5 M potassium acetate solution. Carefully adjust the pH to 4.9 with glacial acetic acid. Slowly add 9 mL of potassium acetate solution to 51 mL of anhydrous ethanol, stirring to thoroughly mix the two. This should be done in an ice bath throughout the process to minimize the effects of evaporation and temperature on the stability of the components. After mixing, transfer the solution to a graduated sterile reagent bottle, seal it, and store at 4°C. Before use, check for precipitation. If precipitation is present, prepare a new solution to obtain the DNA precipitation agent.

[0092] S8. Use a graduated cylinder to measure anhydrous ethanol and pour it into a clean glass container. Slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the diluted ethanol concentration reaches 75.2%. After stirring evenly, store at room temperature. Before use, ensure that the bottle cap is well sealed to prevent ethanol volatilization and concentration changes. Obtain the washing solution.

[0093] Example 6: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular markers, comprising the following steps:

[0094] S1. Solution preparation: Dissolve analytical grade guanidine thiocyanate in Tris-HCl buffer at pH 7 to prepare a 5 mol / L guanidine thiocyanate solution. Dilute papain with an activity of 8500 U / g to 10 mg / mL in Tris-HCl buffer at pH 7 to obtain a papain solution.

[0095] S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, Tris-HCl buffer with a pH of 7 was added, and the reaction vessel was placed in a 65°C environment and stirred at 500 rpm for 10 h, wherein the mass ratio of guanidine thiocyanate to papain in the guanidine thiocyanate solution and the papain solution was 50:3;

[0096] S3. After the reaction, the reaction system was heated to 90°C for 30 min, centrifuged at 12,000 rpm for 25 min, and the supernatant was collected to obtain protease-modified guanidine isothiocyanate;

[0097] S4. Accurately weigh 20 g Tris-HCl, 28 g NaCl, 10 g EDTA, 32 g guanidine thiocyanate, 8 g protease-modified guanidine thiocyanate, 16 g cetyltrimethylammonium bromide, and 10 g polyvinylpyrrolidone, place in a suitable glass container, add 360 mL of deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8.2, and continuously stir to ensure that all ingredients are fully dissolved. After all solid components are completely dissolved and the solution is clear, vacuum filter and sterilize using a 0.22 μm microporous membrane. Collect the filtrate into a sterile brown bottle, seal it, and store it at room temperature in the dark to obtain the lysate;

[0098] S5. Weigh 16 g of papain, 14 g of Aspergillus niger acid protease (pH 2.5), and 12 g of Thermophilic neutral protease (pH 7.5) respectively, place them in pre-chilled centrifuge tubes, add Tris-HCl buffer (pH 7.5) containing 11 mM CaCl2 to each centrifuge tube, and vortex to slowly dissolve the proteases to obtain a protease mixture, wherein the weight of Tris-HCl buffer (pH 7.5) containing 11 mM CaCl2 is 1020 g. Aliquot the prepared protease mixture into sterile EP tubes and store in a -20°C freezer. Remove as needed for use to avoid repeated freeze-thaw cycles, to obtain a protease mixture.

[0099] S6. In a well-ventilated, dimly lit chemical fume hood, accurately measure 24 mL of phenol saturated with pH 8.0 Tris-HCl buffer, 25 mL of chloroform, and 3.5 mL of isoamyl alcohol, based on the volume ratio. Pour each into a clean glass separatory funnel. Slowly rotate the separatory funnel and gently shake to mix thoroughly. Be careful to avoid splashing the liquid. Mix for approximately 10 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and refrigerate at 4°C to obtain the impurity remover.

[0100] S7. First, accurately prepare a 2.6 M potassium acetate solution. Carefully adjust the pH to 5.0 with glacial acetic acid. Slowly add 10 mL of potassium acetate solution to 52 mL of anhydrous ethanol, stirring while adding to thoroughly mix the two. This should be done in an ice bath throughout the process to minimize the effects of evaporation and temperature on the stability of the components. After mixing, transfer the solution to a graduated sterile reagent bottle, seal it, and store at 4°C. Before use, check for precipitation. If precipitation is present, prepare a new solution to obtain the DNA precipitation agent.

[0101] S8. Use a graduated cylinder to measure anhydrous ethanol and pour it into a clean glass container. Slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the diluted ethanol concentration reaches 75.5%. After stirring evenly, store at room temperature. Before use, ensure that the bottle cap is well sealed to prevent ethanol volatilization and concentration changes. Obtain the washing solution.

[0102] Example 7: A method for extracting DNA from a Rana spinosa sample, comprising the following steps:

[0103] S1. Sample pretreatment: Take a 0.2 g fresh muscle tissue sample and quickly mince it into small particles using sterile scissors. Transfer the sample to a 2 mL sterile centrifuge tube, ensuring that the sample is at the bottom of the tube with no residue on the wall.

[0104] S2. Lysis: Add 700 μL of the lysis buffer described in Example 1 to the centrifuge tube containing the sample, followed by 80 μL of the protease mixture described in Example 1. Vortex for 30 seconds to thoroughly mix the sample and reagents. Incubate the tube in a 60°C water bath for 1.5 hours, removing the tube from the water bath every 30 minutes and gently inverting it five times to ensure uniform heating and adequate lysis.

[0105] S3. Impurity Removal: After the sample is lysed and cooled to room temperature, an equal volume of the impurity remover described in Example 1 is added. The centrifuge tube is inverted 10 times, and then the centrifuge tube is placed in a high-speed refrigerated centrifuge and centrifuged at 13,000 rpm for 10 minutes at 3°C. After centrifugation, the upper clear aqueous phase is carefully aspirated and transferred to a new sterile centrifuge tube, taking care to avoid aspirating impurities in the intermediate layer.

[0106] S4. Precipitation: Add 1 / 10 the volume of the DNA precipitant from Example 1 to a new tube and gently invert five times to mix. A white, flocculent DNA precipitate will gradually form. Place the tube in a -20°C refrigerator for 30 minutes to further promote complete precipitation. Then, centrifuge again at 13,000 rpm for 10 minutes at 4°C. Discard the supernatant.

[0107] S5. Washing: Add 800 μL of the washing solution in Example 1, invert the centrifuge tube five times to resuspend the precipitate and perform the washing operation, then centrifuge at 12000 rpm for 8 min at 4°C, discard the supernatant, and repeat the washing step once to ensure that impurities in the precipitate are washed away;

[0108] S6. Dissolution: Leave the precipitate in the open air at room temperature for 2 minutes to allow the residual wash solution to evaporate. Add 30 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0) to dissolve the precipitate. Store the resulting DNA solution at -20°C to avoid repeated freeze-thaw cycles. Regularly check the DNA concentration and purity for subsequent InDel molecular labeling experiments.

[0109] Example 8: A method for extracting DNA from a Rana spinosa sample, comprising the following steps:

[0110] S1. Sample Preparation: Take a 0.3 g fresh muscle tissue sample and quickly mince it into small particles using sterile scissors. Transfer the sample to a 2 mL sterile centrifuge tube, ensuring that the sample is at the bottom of the tube with no residue on the wall.

[0111] S2. Lysis: Add 825 μL of the lysis buffer described in Example 2 to the centrifuge tube containing the sample, followed by 100 μL of the protease mixture described in Example 2. Vortex for 45 seconds to thoroughly mix the sample and reagents. Incubate the tube in a 62.5°C water bath for 2 hours, removing the tube from the water bath every 35 minutes and gently inverting it seven times to ensure uniform heating and adequate lysis.

[0112] S3. Impurity Removal: After the sample is lysed and cooled to room temperature, an equal volume of the impurity remover described in Example 2 is added. The centrifuge tube is inverted 12 times. The centrifuge tube is then placed in a high-speed refrigerated centrifuge and centrifuged at 14,000 rpm for 12.5 minutes at 4°C. After centrifugation, the upper clear aqueous phase is carefully aspirated and transferred to a new sterile centrifuge tube, taking care to avoid aspirating impurities in the intermediate layer.

[0113] S4. Precipitation: Add 1 / 10 the volume of the DNA precipitant from Example 2 to a new tube and gently invert six times to mix. A white, flocculent DNA precipitate will gradually form. Place the tube in a -20°C refrigerator for 40 minutes to further promote complete precipitation. Then, centrifuge again at 14,000 rpm for 12 minutes at 4°C. Discard the supernatant.

[0114] S5. Wash: Add 900 μL of wash buffer and invert the tube seven times to resuspend the pellet. Wash the pellet, then centrifuge at 13,000 rpm for 9 minutes at 4°C. Discard the supernatant and repeat the wash step once to ensure that all impurities in the pellet are removed.

[0115] S6. Dissolution: Leave the precipitate in the open air at room temperature for 2.5 min to allow the residual wash solution to evaporate. Add 40 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0) to dissolve the precipitate. Store the resulting DNA solution at -20°C to avoid repeated freeze-thaw cycles. Regularly check the DNA concentration and purity for subsequent InDel molecular labeling experiments.

[0116] Example 9: A method for extracting DNA from a Rana spinosa sample, comprising the following steps:

[0117] S1. Sample Preparation: Take a 0.4 g fresh muscle tissue sample and quickly mince it into small particles using sterile scissors. Transfer the sample to a 2 mL sterile centrifuge tube, ensuring that the sample is at the bottom of the tube with no residue on the wall.

[0118] S2. Lysis: To the centrifuge tube containing the sample, add 1000 μL of the lysis buffer described in Example 3, followed by 120 μL of the protease mixture described in Example 3. Vortex for 60 seconds to thoroughly mix the sample and reagents. The centrifuge tube was then placed in a 65°C water bath and incubated for 2.5 hours. The tube was removed from the water bath every 40 minutes and gently inverted eight times to ensure uniform heating and adequate lysis of the sample.

[0119] S3. Impurity Removal: After the sample is lysed and cooled to room temperature, an equal volume of the impurity remover described in Example 3 is added. The centrifuge tube is inverted 15 times, and then the centrifuge tube is placed in a high-speed refrigerated centrifuge and centrifuged at 15,000 rpm for 15 minutes at 5°C. After centrifugation, the upper clear aqueous phase is carefully aspirated and transferred to a new sterile centrifuge tube, taking care to avoid aspirating impurities in the intermediate layer.

[0120] S4. Precipitation: Add 1 / 10 the volume of the DNA precipitant from Example 3 to a new tube and gently invert to mix eight times. A white, flocculent DNA precipitate will gradually form. Place the tube in a -20°C refrigerator for 45 minutes to further promote complete precipitation. Then, centrifuge again at 15,000 rpm for 15 minutes at 4°C. Discard the supernatant.

[0121] S5. Washing: Add 1000 μL of the washing solution in Example 3, invert the centrifuge tube 8 times to resuspend the precipitate and perform the washing operation, then centrifuge at 14000 rpm for 10 min at 4°C, discard the supernatant, and repeat the washing step once to ensure that impurities in the precipitate are washed away;

[0122] S6. Dissolution: Leave the precipitate in the open air at room temperature for 3 minutes to allow the residual wash solution to evaporate. Add 50 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0) to dissolve the precipitate. Store the resulting DNA solution at -20°C to avoid repeated freeze-thaw cycles. Regularly check the DNA concentration and purity for subsequent InDel molecular labeling experiments.

[0123] Comparative Example 1:

[0124] Compared with Example 1, this comparative example only replaced the "protease mixture" with "papain", and the remaining steps and parameters were the same, which will not be repeated in this comparative example. Finally, a sample DNA extraction reagent for Rana spinosa InDel molecular marker was obtained.

[0125] Comparative Example 2:

[0126] Compared with Example 1, this comparative example did not add "guanidine isothiocyanate" during the preparation of the sample DNA extraction reagent for the spinous chest frog InDel molecular marker. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, the sample DNA extraction reagent for the spinous chest frog InDel molecular marker was obtained.

[0127] Comparative Example 3:

[0128] Compared with Example 1, this comparative example did not add "protease-modified guanidine isothiocyanate" during the preparation of the sample DNA extraction reagent for the spinous chest frog InDel molecular marker. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, the sample DNA extraction reagent for the spinous chest frog InDel molecular marker was obtained.

[0129] Comparative Example 4:

[0130] Compared with Example 1, this comparative example only replaces "protease-modified guanidine isothiocyanate" with "Bacillus subtilis neutral protease-modified guanidine isothiocyanate", and the remaining steps and parameters are the same. This comparative example will not be repeated, and finally a sample DNA extraction reagent for InDel molecular marker of Rana spinosa is obtained.

[0131] Comparative Example 5:

[0132] Compared with Example 1, this comparative example only adjusted the amount of "protease-modified guanidine isothiocyanate" from "5g" to "26g", and the amount of "guanidine isothiocyanate" from "26g" to "5g". The remaining steps and parameters are the same, and this comparative example will not be repeated. Finally, a sample DNA extraction reagent for InDel molecular marker of Rana spinosa was obtained.

[0133] The Rana spinosa InDel molecular markers obtained in Comparative Examples 1 to 5 were respectively subjected to DNA extraction using a sample DNA extraction reagent.

[0134] Performance test: The following tests were performed on the extraction results of Examples 7-9 and Comparative Examples 1-5:

[0135] DNA purity testing: Use a UV spectrophotometer to measure the absorbance of the extracted DNA solution at wavelengths of 260nm and 280nm. First, dilute the extracted DNA solution with an appropriate amount of TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0) to ensure that the absorbance value is within the accurate measurement range of the instrument (generally, an absorbance value between 0.1-1.0 is more accurate). Then, record the absorbance readings at 260nm and 280nm, respectively, and calculate the A260 / A280 ratio. The A260 / A280 ratio of a high-purity DNA solution should be between 1.8 and 2.0. A ratio close to 1.8 indicates a low content of impurities such as protein. If the ratio is greater than 2.0, RNA contamination may be present. If the ratio is less than 1.8, it indicates that a large amount of protein or other impurities remain, which will affect subsequent molecular biology experiments.

[0136] DNA concentration determination: Also use a UV spectrophotometer to calculate the DNA concentration based on the absorbance at 260nm. It is known that at 260nm, when the absorbance is 1, the concentration of double-stranded DNA is approximately 50μg / mL. By measuring the absorbance value A260 of the sample at 260nm, the concentration of the original DNA solution is calculated according to the formula: DNA concentration (μg / mL) = A260 × dilution factor × 50. For the detection of InDel molecular markers in Rana spinosa, the DNA concentration is generally required to reach a certain level, for example, between 100-500ng / μL is more appropriate. This can ensure that there is sufficient template amount for accurate detection in subsequent PCR amplification experiments;

[0137] DNA integrity testing: Agarose gel electrophoresis is used. Prepare a 0.8%-1.2% agarose gel and mix the extracted DNA sample with an appropriate amount of loading buffer (e.g., 6x loading buffer containing bromophenol blue and sucrose). The mixed sample is then added to the wells of the gel, along with a DNA molecular weight standard of known molecular weight (e.g., λ-HindIII digest) as a control. Electrophoresis is performed in 1x TAE or 1x TBE running buffer at 80-120 V for 30-60 minutes. After electrophoresis, the gel is stained with ethidium bromide (EB) or another safe nucleic acid dye (e.g., SYBR Green) for 15-30 minutes. DNA band integrity is then observed on a UV transilluminator. Intact DNA should appear as clear, bright bands without noticeable smearing or smearing. The band positions should correspond to DNA fragments of corresponding sizes in the molecular weight standard, indicating that the DNA has not been significantly degraded. If a large amount of smear or tailing occurs, it indicates that the DNA has been degraded to varying degrees, which may affect its accuracy as a template in InDel molecular marker detection;

[0138] PCR amplification assay: Design PCR primer pairs targeting specific InDel sites in Rana spinosa and perform PCR amplification using the extracted DNA as a template. The reaction system generally includes: a certain concentration of template DNA (e.g., 50-200 ng), upstream and downstream primers (0.2-0.5 μM each), dNTPs (0.2-0.4 mM), Taq DNA polymerase (1-2.5 U), and the appropriate PCR buffer (e.g., 1× PCR buffer containing Mg²⁺), in a total volume of 20-50 μL. PCR reaction conditions can be optimized based on the primer Tm values ​​and amplicon length. These conditions generally include initial denaturation (94-95°C, 3-5 minutes), followed by 30-35 cycles of denaturation (94-95°C, 30-45 seconds), annealing (50-65°C, 30-45 seconds, depending on the primer Tm values), and extension (72°C; the extension time is determined by the amplicon length, generally 1-1.5 minutes per kb), followed by a final extension at 72°C for 5-10 minutes. PCR products are analyzed by electrophoresis on 2%-3% agarose gels to assess the specificity, brightness, and clarity of the amplified bands. Ideal PCR results should show a single, clear, bright, specific band, with no obvious nonspecific amplified bands or primer dimers. Band positions should be consistent with the expected amplicon size, indicating that the extracted DNA can effectively serve as a template for PCR amplification, that the primers are specific, and that the reaction system and conditions are appropriate, meeting the PCR amplification requirements for detecting InDel markers in Rana spinosa. If multiple nonspecific bands or primer dimers are present, or the target band is not amplified, it may be due to insufficient DNA purity, the presence of inhibitors, or unreasonable primer design.

[0139] The results are shown in Table 1 below:

[0140] ,

[0141] Data Analysis:

[0142] From Table 1 we can see that:

[0143] The A260 / A280 ratios for Examples 7-9 ranged from 1.88 to 1.95, all falling within the ideal range of 1.8 to 2.0. This demonstrates that the extraction reagents in these examples were able to effectively remove impurities such as proteins and polysaccharides, resulting in high-purity DNA extraction that met the stringent DNA purity requirements for Rana spinosa InDel molecular marker detection. For example, the ratio for Example 9 was 1.95, demonstrating that the DNA extraction process in this example performed well in removing impurities and provided a high-purity DNA template for subsequent experiments. However, the A260 / A280 ratio for Comparative Example 1 was 1.65, significantly below the ideal range. This means that the extraction process did not effectively remove proteins or other impurities, which may interfere with subsequent experiments, such as inhibiting enzyme activity in PCR reactions. The ratio of Comparative Example 2 was 2.10, which was greater than 2.0, suggesting the possibility of RNA contamination. The presence of RNA may compete with DNA for reaction reagents in subsequent experiments, affecting the accuracy of experimental results. Although the ratios of Comparative Examples 3-5 were close or within an acceptable range, they were still not as ideal as those of the Examples. That is, Examples 7-9 were significantly superior to Comparative Examples 1-5 in terms of DNA purity, indicating that the reagent formulation and extraction method of the present invention have significant advantages in ensuring DNA purity and can effectively reduce the impact of impurities on DNA quality.

[0144] The DNA concentrations of Examples 7-9 ranged from 280 to 350 ng / μL, all within a range suitable for subsequent InDel molecular marker detection. A sufficient DNA concentration ensures sufficient template quantity in experiments such as PCR amplification, thereby improving detection accuracy and sensitivity. For example, the DNA concentration of Example 7 was 320 ng / μL, providing sufficient DNA template for subsequent experimental operations, while the DNA concentration of Comparative Example 1 was relatively low at 150 ng / μL, which may result in insufficient template quantity in subsequent experiments and affect the accuracy of experimental results. The concentrations of Comparative Examples 2-5 ranged from 200 to 400 ng / μL. Although the concentrations of some comparative examples met the requirements, some issues remained when considering other factors such as purity. Specifically, the DNA concentrations of Examples 7-9 were able to stably reach a level suitable for experiments, while the DNA concentrations of Comparative Examples 1-5 showed certain volatility and deficiencies, further demonstrating the advantages of the present invention in ensuring effective DNA extraction yields.

[0145] The agarose gel electrophoresis results of Examples 7-9 showed clear, bright DNA bands with no obvious tailing or smearing. This indicates that in these examples, the DNA was not significantly mechanically sheared or chemically degraded during the extraction process, and its integrity was good, capable of providing complete gene sequence information for InDel molecular marker detection. This indicates that the extraction methods of Examples 7-9 caused less damage to the DNA, while the DNA bands of Comparative Example 1 showed more severe tailing, indicating a certain degree of DNA degradation. Comparative Examples 3-4 also showed varying degrees of tailing or smearing, indicating that the extraction methods of these comparative examples had defects in maintaining DNA integrity. Comparative Example 2 may be an abnormality caused by residual RNA, which would also interfere with the determination of DNA integrity. That is, Examples 7-9 performed well in terms of DNA integrity, while the DNA integrity of Comparative Examples 1-5 had varying degrees of problems, highlighting the superiority of the extraction method of the present invention in protecting DNA integrity.

[0146] After electrophoresis, the PCR amplification products of Examples 7-9 showed a single, clear specific band, without primer dimers and non-specific amplification bands. This indicates that the extracted DNA can be well used as a template for PCR amplification, the primers have good specificity, and the reaction system and conditions are suitable, which can meet the PCR amplification requirements for the detection of InDel molecular markers in Rana spinosa. For example, the electrophoretic bands of the PCR amplification product of Example 9 were highly specific and bright, indicating that the DNA extracted in this example was of very high quality and was an ideal template for InDel molecular marker detection. However, the PCR amplification of Comparative Example 1 showed multiple nonspecific bands, and the target band was relatively blurred. This was due to low DNA purity, poor integrity, and the possible presence of inhibitors, which seriously affected the detection and analysis of InDel sites. Although Comparative Examples 2-5 partially obtained the target bands, they also had varying degrees of nonspecific bands, indicating that there were certain problems with the quality of the DNA extracted in these comparative examples, which would affect the resolution and accuracy of InDel site detection. That is, Examples 7-9 were significantly superior to Comparative Examples 1-5 in terms of PCR amplification results, demonstrating that the reagents and extraction methods of the present invention can provide high-quality DNA templates for PCR amplification, which is conducive to accurate InDel molecular marker detection. However, the DNA quality problems of the comparative examples would have an adverse effect on PCR amplification and subsequent detection.

[0147] 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. Within the scope 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.

[0148] 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 within the scope of protection of the present invention.

Claims

1. A DNA extraction reagent for sample of Rana spinosa InDel molecular marker, characterized in that: Prepared from the following raw materials in parts by weight: 60-70 parts of lysis solution, 8-12 parts of protease mixture, 10-15 parts of impurity remover, 6-9 parts of DNA precipitant, and 10-15 parts of washing solution; The lysate comprises the following raw materials in parts by weight: Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, hexadecyltrimethylammonium bromide and polyvinylpyrrolidone; The mass ratio of Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, cetyltrimethylammonium bromide and polyvinylpyrrolidone in the lysate is 18-20:25-28:8-10:26-32:5-8:14-16:6-10; The pH of the Tris-HCl is 8-8.5, and the pH of the EDTA is 7.8-8.3; The preparation process of the protease-modified guanidine isothiocyanate is as follows: Step A1. Solution preparation: dissolve guanidine thiocyanate in a buffer solution to prepare a 3-5 mol / L guanidine thiocyanate solution, and dilute papain with a buffer solution to 6-10 mg / mL to obtain a papain solution; Step A2. Mix the guanidine thiocyanate solution and the papain solution in a reaction vessel, add the buffer, and place the reaction vessel in an environment of 55-65°C and stir at 300-500 rpm for 8-10h; Step A3. After the reaction is completed, the reaction system is heated to 80-90 ° C, maintained for 20-30 min, centrifuged at 10000-12000 rpm for 20-25 min, and the supernatant is collected to obtain protease-modified guanidine isothiocyanate; The guanidine thiocyanate in step A1 is analytical grade guanidine thiocyanate; The enzymatic activity unit of papain in step A1 is 7500-8500 U / g; The buffer in step A1 is a Tris-HCl buffer with a pH of 6-7; The mass ratio of guanidine thiocyanate to papain in the guanidine thiocyanate solution and the papain solution in step A2 is 40-50:1-3; The buffer in step A2 is a Tris-HCl buffer with a pH of 6-7.

2. The sample DNA extraction reagent for Rana spinosa InDel molecular marker according to claim 1, characterized in that The protease mixture comprises the following raw materials in parts by weight: papain, acid protease, neutral protease, and solvent; The mass ratio of the papain, the acidic protease, the neutral protease and the solvent is 14-16:10-14:8-12:980-1020.

3. The sample DNA extraction reagent for Rana spinosa InDel molecular marker according to claim 2, characterized in that The solvent is a Tris-HCl buffer solution containing 9-11 mM CaCl2 at pH = 7-7.5; The acidic protease is any one of pepsin and Aspergillus niger acidic protease with a pH of 1.5-2.5; The neutral protease is any one of a Bacillus subtilis neutral protease and a thermophilic neutral protease with a pH of 6.5-7.

5.

4. The sample DNA extraction reagent for Rana spinosa InDel molecular marker according to claim 1, characterized in that The impurity remover is prepared by mixing phenol, chloroform and isoamyl alcohol in a volume ratio of 22-24:23-25:2.5-3.5; The phenol is saturated with a Tris-HCl buffer solution with a pH of 7.8-8.

0.

5. The sample DNA extraction reagent for Rana spinosa InDel molecular marker according to claim 1, characterized in that The DNA precipitant is prepared by mixing 2.4-2.6M potassium acetate and anhydrous ethanol in a volume ratio of 0.8-1.1:4.7-5.2, and the pH of the potassium acetate is 4.6-5.0; The washing solution is 74.5%-75.5% ethanol.

6. The method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular marker according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step S1. Preparation of lysate: Weigh Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, cetyltrimethylammonium bromide, and polyvinylpyrrolidone, place in a suitable glass container, add deionized water to dissolve, add sodium hydroxide solution dropwise, adjust the pH to 8-8.2, and then vacuum filter through a 0.22 μm microporous filter membrane. Collect the filtrate into a sterile brown bottle, seal it, and store it at room temperature in the dark to obtain a lysate; Step S2. Preparing a protease mixture: Weigh papain, acidic protease, and neutral protease powders separately and place them in pre-chilled centrifuge tubes. Add solvent to each centrifuge tube and vortex to dissolve the proteases to obtain a protease mixture. Dispense the prepared protease mixture into sterile EP tubes and store them in a -20°C freezer to obtain a protease mixture. Step S3. Preparation of a decontamination agent: Phenol, chloroform, and isoamyl alcohol were measured and poured into a clean glass separatory funnel, mixed evenly, and then transferred to a brown glass bottle, sealed with a cap, and refrigerated at 4°C to obtain a decontamination agent; Step S4. Preparation of a precipitant: Prepare a 2.5 M potassium acetate solution, adjust the pH to 4.6-5.0 with glacial acetic acid, then add the potassium acetate solution to anhydrous ethanol while stirring in an ice bath. After mixing thoroughly, transfer the solution to a graduated sterile reagent bottle, seal, and store at 4°C to obtain a DNA precipitant. Step S5. Preparation of washing solution: Anhydrous ethanol was measured and poured into a glass container. Deionized water was then added while stirring until the ethanol concentration reached 74.5%-75.5%. After stirring evenly, the mixture was stored at room temperature to obtain a washing solution. The usage ratio of Tris-HCl and deionized water in step S1 is 18-20 g:350-360 mL.