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

Abstract

The invention relates to the technical field of biology, in particular to a sample DNA extraction reagent for a giant spiny frog InDel molecular marker and a preparation method thereof.The sample DNA extraction reagent for the giant spiny frog InDel molecular marker is prepared from 60-70 parts of lysate, 8-12 parts of protease mixed liquor, 10-15 parts of impurity removing agent, 6-9 parts of DNA precipitator and 10-15 parts of washing liquid, and all the components are prepared through specific steps. The reagent is used for extracting DNA, through the steps of sample pretreatment, splitting decomposition, impurity removal, precipitation, washing, dissolution and the like, impurities can be effectively removed, the purity, concentration and integrity of the DNA can be improved, the PCR amplification effect is good, a product strip is single and clear, no primer dimer exists, non-specific amplification is achieved, and the accuracy and reliability of detection of the giant spiny frog InDel molecular marker are greatly improved; and molecular genetics research and related industrial development of the giant spiny frogs are promoted.

Description

Technical Field

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

[0002] The spiny-breasted frog (Quasipaa spinosa), as a large amphibian unique to my country, inhabits clear and fast-flowing water environments such as mountain streams and rivers, and is widely distributed in southern my country. It is not only an important part of the ecosystem, maintaining the balance of the aquatic food chain, but also has great value in the fields of food and medicine because of its tender meat and rich nutrition. In recent years, the scale of artificial breeding has continued to expand.

[0003] Behind the booming development of the spiny-chested frog related industry, the role of molecular biotechnology in the management and optimal utilization of its germplasm resources has become increasingly prominent. InDel (Insertion / Deletion) molecular marker technology has gradually become a key tool for studying the genetic characteristics of spiny-chested frogs due to its advantages such as rich polymorphism, high detection stability, and relatively simple operation. This technology is based on base insertion or deletion mutations at specific locations in the genome. Through PCR amplification and subsequent analysis, it can accurately reveal subtle but critical genetic differences between individuals and populations of spiny-chested frogs, providing a strong basis for variety identification, kinship determination, and mining of genetic mechanisms of excellent traits.

[0004] However, the particularity of the spiny-chested frog samples has brought great challenges to DNA extraction, seriously restricting the efficient application of InDel molecular labeling technology. First, the skin and muscle tissues of the spiny-chested frog are rich in a large number of sticky proteins and polysaccharide complexes. These sticky substances are like a dense network, tightly wrapping cells and nucleic acid components, making it difficult for conventional cell lysis methods to quickly and fully penetrate into the cells, greatly hindering the release of DNA. Second, the active biological metabolic activities in its body have spawned a wide variety of secondary metabolites, such as polyphenols and terpenoids. Polyphenols are easily oxidized, and the resulting quinone products have extremely strong nucleophilicity, which will irreversibly bind to DNA molecules, causing DNA damage, resulting in fragmentation of the extracted DNA, and seriously affecting its integrity; terpenoids often have special chemical structures, which interfere with the chemical balance in the subsequent DNA separation and purification steps, and reduce the extraction efficiency and purity.

[0005] Furthermore, the living environment of the spiny-chested frog has endowed it with a unique immune defense mechanism. The body contains a wealth of antimicrobial peptides, lysozymes and other immune active substances. These substances will continue to affect the normal process of cell lysis during the DNA extraction stage after sample collection, inhibiting the accuracy of nuclease activity regulation, further increasing the difficulty of obtaining high-quality and complete DNA. Conventional DNA extraction reagents and methods are mostly designed for common model organisms or general biological samples, without fully considering the complex characteristics of spiny-chested frog samples. Therefore, in actual application, the extracted DNA is often doped with a large amount of impurities, and the purity can usually only be maintained at a low level. The A260 / A280 ratio often deviates from the ideal range (1.8-2.0), and the DNA integrity is worrying, and degradation occurs frequently. It is impossible to meet the strict requirements of InDel molecular labeling technology for high purity and high integrity of template DNA, resulting in low efficiency of subsequent PCR amplification and a sharp increase in the risk of misjudgment of results, which greatly limits the depth and accuracy of molecular genetic research on spiny-chested frogs and delays the pace of related industries in breeding and efficient management of 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 a top priority to promote molecular research and industrial upgrading of Rana spinosa.

[0006] Therefore, according to the above-mentioned related technologies, it is urgent to develop a sample DNA extraction reagent for InDel molecular marker 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 marker of Rana spinosa and its preparation method, aiming to provide high-quality DNA extraction means 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 sample DNA extraction reagent for InDel molecular marker of Rana spinosa is prepared from the following raw materials in parts by weight: 60-70 parts of lysate, 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 isothiocyanate, protease-modified guanidine isothiocyanate, hexadecyltrimethylammonium bromide and polyvinylpyrrolidone; The mass ratio of Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, hexadecyltrimethylammonium bromide and polyvinylpyrrolidone 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 to prepare a 3-5 mol / L guanidine thiocyanate solution, dilute papain with the buffer 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 a buffer solution, and place the reaction vessel in an environment of 55-65°C and stir at 300-500 rpm for 8-10 hours; Step A3. After the reaction is completed, the reaction system is heated to 80-90°C, maintained for 20-30 minutes, centrifuged at a speed of 10000-12000 rpm for 20-25 minutes, and the supernatant is collected to obtain protease-modified guanidine isothiocyanate.

[0010] The guanidine isothiocyanate in step A1 is analytical grade guanidine isothiocyanate; 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; The protease mixture comprises the following raw materials in parts by weight: papain, acid protease, neutral protease, and solvent; The mass ratio of papain, acidic protease, neutral protease and solvent is 14-16:10-14:8-12:980-1020; The solvent is a Tris-HCl buffer 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 neutral protease of Bacillus subtilis and a neutral protease of thermophilic bacteria at pH=6.5-7.5.

[0011] The impurity remover is obtained by mixing phenol: chloroform: 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; The DNA precipitant is obtained 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 liquid is 74.5%-75.5% ethanol; A method for preparing a sample DNA extraction reagent for InDel molecular marker of Rana spinosa, comprising the following steps: Step S1. Preparation of lysate: accurately weigh Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, hexadecyltrimethylammonium 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 during the process to ensure that all components are fully dissolved and evenly dissolved. After all solid components are completely dissolved and the solution is clear, use a 0.22 μm microporous filter membrane to perform vacuum filtration and sterilization, collect the filtrate into a sterile brown bottle, seal it and store it at room temperature away from light to obtain a lysate; Step S2. Preparation of protease mixture: weigh papain, acid protease and neutral protease powders respectively, place them in pre-cooled centrifuge tubes, add solvent to each centrifuge tube, vortex and oscillate to slowly dissolve the protease to obtain a protease mixture, dispense the prepared protease mixture into sterile EP tubes, freeze and store in a -20°C low-temperature refrigerator, take out as needed when used to avoid repeated freezing and thawing, and obtain a protease mixture; Step S3. Preparation of the impurity remover: In a chemical fume hood with good ventilation and dim light, accurately measure phenol, chloroform and isoamyl alcohol according to the volume ratio, pour them into a clean glass separatory funnel respectively, rotate the separatory funnel slowly, and shake it gently to mix them evenly. Note that the action should be gentle to prevent the liquid from splashing. The mixing process lasts for about 5-10 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and store it in a refrigerator at 4°C to obtain the impurity remover; Step S4. Preparation of precipitant: first accurately prepare 2.4-2.6M potassium acetate solution, carefully adjust the pH to 4.6-5.0 with glacial acetic acid, then slowly add the potassium acetate solution to anhydrous ethanol, stirring while adding to fully mix the two, and operate in an ice bath throughout the process to reduce the impact of solution volatility and temperature on the stability of components. After mixing evenly, transfer to a sterile reagent bottle with a scale, seal and store at 4°C, check whether there is precipitation before use, if there is precipitation, re-prepare to obtain a DNA precipitant; Step S5. Preparation of washing solution: measure anhydrous ethanol with a measuring cylinder, pour it into a clean glass container, and then slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the ethanol concentration after dilution reaches 74.5%-75.5%. After stirring evenly, store at room temperature. Make sure the bottle cap is well sealed before use to prevent ethanol volatilization from causing concentration changes, and obtain washing solution.

[0012] The usage ratio of Tris-HCl to deionized water in step S1 is 18-20 g: 350-360 mL; A method for extracting DNA from a Rana spinosa sample comprises the following steps: Step T1. Sample pretreatment: Take 0.2-0.4g fresh muscle tissue sample, cut it into small particles quickly with sterile scissors, and then transfer it to a 2mL sterile centrifuge tube, making sure that the sample is at the bottom of the tube without any residue on the wall; Step T2. Lysis: Add 700-1000 μL of lysis solution to the centrifuge tube containing the sample, then add 80-120 μL of protease mixture, vortex for 30-60 seconds to fully mix the sample and reagent, then place the centrifuge tube in a 60-65°C constant temperature water bath and incubate for 1.5-2.5 hours. During this period, take out every 30-40 minutes and gently invert and mix 5-8 times to ensure that the sample is evenly heated and fully lysed; Step T3. Impurity removal: After the sample is lysed, cool to room temperature, add an equal volume of impurity remover, invert the centrifuge tube 10-15 times, and then put the centrifuge tube into a high-speed refrigerated centrifuge, centrifuge at 13000-15000rpm for 10-15min, set the temperature to 3-5℃. After the centrifugation, carefully transfer the upper clear water phase to a new sterile centrifuge tube, taking care to avoid aspirating impurities in the middle layer; Step T4. Precipitation: Add 1 / 10 volume of DNA precipitant to a new tube, gently invert and mix 5-8 times, and white flocculent DNA precipitate will gradually precipitate. Place the centrifuge tube in a -20°C refrigerator for 30-45 minutes to further promote complete precipitation, then place it in a centrifuge again, centrifuge at 13000-15000 rpm for 10-15 minutes, operate at 4°C, and discard the supernatant; Step T5. Washing: Add 800-1000 μL of washing solution, invert the centrifuge tube 5-8 times to suspend the precipitate, and then perform the washing operation. Then centrifuge at 12000-14000 rpm for 8-10 min at 4°C, discard the supernatant, and repeat the washing step once to ensure that impurities in the precipitate are washed away; Step T6. Dissolution: Leave the precipitate in the open air at room temperature for 2-3 minutes to allow the residual washing solution to evaporate, add 30-50μLTE buffer (10mM Tris-HCl, 1mM EDTA, pH=8.0) to dissolve the precipitate, and store the resulting DNA solution at -20℃ for a long term to avoid repeated freezing and thawing. Regularly check the DNA concentration and purity for subsequent InDel molecular labeling experiments.

[0013] Beneficial effects of the present invention: The present invention designs a special DNA extraction reagent and method based on the characteristics of Rana spinosa samples. Through the synergistic effect of a specific formula of 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 the existing technology, 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 amount 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 InDel molecular marker detection of the spiny-chested frog, and effectively promotes the molecular genetics research of the spiny-chested frog and the development of related industries. DETAILED DESCRIPTION

[0014] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0015] Example 1: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular marker, comprising the following steps: 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 enzyme activity of 7500 U / g to 6 mg / mL with Tris-HCl buffer at pH 6 to obtain a papain solution; S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, a Tris-HCl buffer having 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; S3. After the reaction is completed, the reaction system is heated to 80°C, maintained for 20 min, centrifuged at 10000 rpm for 20 min, and the supernatant is collected to obtain protease-modified guanidine isothiocyanate; 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 hexadecyltrimethylammonium bromide and 6 g polyvinylpyrrolidone, place in a suitable glass container, add 350 mL deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8, and continue stirring during the period to ensure that all components are fully dissolved and evenly dissolved. After all solid components are completely dissolved and the solution is clear, use a 0.22 μm microporous filter membrane to vacuum filter and sterilize, collect the filtrate into a sterile brown bottle, seal it and store it at room temperature away from light to obtain a lysate; S5. Weigh 14 g papain, 10 g pepsin at pH 1.5, and 8 g Bacillus subtilis neutral protease at pH 6.5, respectively, and place them in pre-cooled centrifuge tubes. Add Tris-HCl buffer at pH 7 containing 9 mM CaCl2 to each centrifuge tube, vortex and oscillate to slowly dissolve the protease to obtain a protease mixture, wherein the weight of Tris-HCl buffer at pH 7 containing 9 mM CaCl2 is 980 g. Dispense the prepared protease mixture into sterile EP tubes, store them in a -20°C low-temperature refrigerator, and take them out as needed when used to avoid repeated freezing and thawing to obtain a protease mixture; S6. In a well-ventilated and dimly-lit chemical fume hood, accurately measure 22 mL of phenol saturated with Tris-HCl buffer at pH = 7.8, 23 mL of chloroform, and 2.5 mL of isoamyl alcohol according to the volume ratio, and pour them into a clean glass separatory funnel respectively. Slowly rotate the separatory funnel and gently shake it to mix it evenly. Be careful to move gently to prevent the liquid from splashing. The mixing process lasts for about 5 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and store it in a refrigerator at 4°C to obtain the impurity remover; S7. First accurately prepare 2.4M potassium acetate solution, carefully adjust the pH to 4.6 with glacial acetic acid, then slowly add 8mL potassium acetate solution into 47mL anhydrous ethanol, stirring while adding to mix the two thoroughly. The whole process is performed in an ice bath to reduce the effects of solution volatility and temperature on the stability of the components. After mixing evenly, transfer to a sterile reagent bottle with a scale, seal it and store it at 4℃. Check whether there is precipitation before use. If there is precipitation, it needs to be re-prepared to obtain the DNA precipitation agent; S8. Use a measuring cylinder to measure anhydrous ethanol, pour it into a clean glass container, and then 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, and obtain the washing solution.

[0016] Example 2: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular marker, comprising the following steps: 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 having an enzyme activity of 7800 U / g to 7 mg / mL with Tris-HCl buffer at pH 6.5 to obtain a papain solution; S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, a Tris-HCl buffer having a pH of 6.5 was added, and the reaction vessel was placed in an environment of 58° C. and stirred at 350 rpm for 9 h, wherein the mass ratio of guanidine thiocyanate to papain in the guanidine thiocyanate solution and the papain solution was 42:2; S3. After the reaction, the reaction system was heated to 82°C, maintained for 22 min, centrifuged at 10500 rpm for 21 min, and the supernatant was collected to obtain protease-modified guanidine isothiocyanate; S4. Accurately weigh 19g Tris-HCl, 26g NaCl, 9g EDTA, 27g guanidine thiocyanate, 7g protease-modified guanidine thiocyanate, 15g hexadecyltrimethylammonium bromide and 7g polyvinylpyrrolidone, place in a suitable glass container, add 353mL deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8.1, and continue stirring during the period to ensure that all components are fully dissolved and evenly dissolved. After all solid components are completely dissolved and the solution is clear, use a 0.22μm microporous filter membrane to vacuum filter and sterilize, collect the filtrate into a sterile brown bottle, seal it and store it at room temperature away from light to obtain a lysate; S5. Weigh 15g papain, 11g Aspergillus niger acid protease with pH=1.6 and 9g thermophilic neutral protease with pH=6.5 respectively, place them in pre-cooled centrifuge tubes, add Tris-HCl buffer with pH=7.1 containing 10mM CaCl2 to each centrifuge tube, vortex and oscillate to slowly dissolve the protease to obtain a protease mixture, wherein the weight of Tris-HCl buffer with pH=7.1 containing 10mM CaCl2 is 1000g, divide the prepared protease mixture into sterile EP tubes, store them in a -20°C low-temperature refrigerator, take them out as needed when used to avoid repeated freezing and thawing, and obtain a protease mixture; S6. In a well-ventilated and dimly-lit chemical fume hood, accurately measure 23 mL of phenol saturated with Tris-HCl buffer at pH = 7.9, 24 mL of chloroform, and 2.6 mL of isoamyl alcohol according to the volume ratio, and pour them into a clean glass separatory funnel respectively. Slowly rotate the separatory funnel and gently shake it to mix it evenly. Be careful to move gently to prevent the liquid from splashing. The mixing process lasts for about 6 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and store it in a refrigerator at 4°C to obtain the impurity remover; S7. First accurately prepare 2.5M potassium acetate solution, carefully adjust the pH to 4.7 with glacial acetic acid, then slowly add 9mL potassium acetate solution into 48mL anhydrous ethanol, stirring while adding to mix the two thoroughly. The whole process is performed in an ice bath to reduce the effects of solution volatility and temperature on the stability of the components. After mixing evenly, transfer to a sterile reagent bottle with a scale, seal it and store it at 4℃. Check whether there is precipitation before use. If there is precipitation, re-prepare it to obtain the DNA precipitation agent; S8. Use a measuring cylinder to measure anhydrous ethanol, pour it into a clean glass container, and then 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, and obtain the washing solution.

[0017] Example 3: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular marker, comprising the following steps: 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 enzyme activity of 8000 U / g to 8 mg / mL with Tris-HCl buffer at pH 7 to obtain a papain solution; S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, a Tris-HCl buffer having 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; S3. After the reaction is completed, the reaction system is heated to 85°C, maintained for 25 min, centrifuged at 11000 rpm for 22 min, and the supernatant is collected to obtain protease-modified guanidine isothiocyanate; S4. Accurately weigh 20g Tris-HCl, 27g NaCl, 10g EDTA, 28g guanidine thiocyanate, 6g protease-modified guanidine thiocyanate, 16g hexadecyltrimethylammonium bromide and 8g polyvinylpyrrolidone, place in a suitable glass container, add 356mL deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8.2, and continue stirring during the period to ensure that all components are fully dissolved and evenly dissolved. After all solid components are completely dissolved and the solution is clear, use a 0.22μm microporous filter membrane to vacuum filter and sterilize, collect the filtrate into a sterile brown bottle, seal it and store it at room temperature away from light to obtain a lysate; S5. Weigh 16 g papain, 12 g pepsin at pH = 1.8, and 10 g Bacillus subtilis neutral protease at pH = 6.7, respectively, and place them in pre-cooled centrifuge tubes. Add Tris-HCl buffer containing 11 mM CaCl2 at pH = 7.2 to each centrifuge tube, vortex and oscillate to slowly dissolve the protease to obtain a protease mixture, wherein the weight of Tris-HCl buffer containing 11 mM CaCl2 at pH = 7.2 is 1010 g. Dispense the prepared protease mixture into sterile EP tubes, store them in a -20°C low-temperature refrigerator, and take them out as needed when used to avoid repeated freezing and thawing to obtain a protease mixture; S6. In a well-ventilated and 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 according to the volume ratio, and pour them into a clean glass separatory funnel respectively. Slowly rotate the separatory funnel and gently shake it to mix it evenly. Be careful to move gently to prevent the liquid from splashing. The mixing process lasts for about 7 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and store it in a refrigerator at 4°C to obtain the impurity remover; S7. First accurately prepare 2.6M potassium acetate solution, carefully adjust the pH to 4.8 with glacial acetic acid, then slowly add 10mL potassium acetate solution into 49mL anhydrous ethanol, stirring while adding to mix the two thoroughly. The whole process is performed in an ice bath to reduce the effects of solution volatility and temperature on the stability of the components. After mixing evenly, transfer to a sterile reagent bottle with a scale, seal it and store it at 4℃. Check whether there is precipitation before use. If there is precipitation, re-prepare it to obtain the DNA precipitation agent; S8. Use a measuring cylinder to measure anhydrous ethanol, pour it into a clean glass container, and then slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the ethanol concentration after dilution 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, and obtain the washing solution.

[0018] Example 4: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular marker, comprising the following steps: 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 having an enzyme activity of 8200 U / g to 9 mg / mL with Tris-HCl buffer at pH 6 to obtain a papain solution; S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, a Tris-HCl buffer having 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; S3. After the reaction is completed, the reaction system is heated to 86°C, maintained for 26 min, centrifuged at 11500 rpm for 23 min, and the supernatant is collected to obtain protease-modified guanidine isothiocyanate; 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 hexadecyltrimethylammonium bromide and 9 g polyvinylpyrrolidone, place in a suitable glass container, add 358 mL deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8, and continue stirring during the period to ensure that all components are fully dissolved and evenly dissolved. After all solid components are completely dissolved and the solution is clear, use a 0.22 μm microporous filter membrane to vacuum filter and sterilize, collect the filtrate into a sterile brown bottle, seal it and store it at room temperature away from light to obtain a lysate; S5. Weigh 14g papain, 13g Aspergillus niger acid protease with pH=2.0 and 11g thermophilic neutral protease with pH=6.8 respectively, place them in pre-cooled centrifuge tubes, add Tris-HCl buffer with pH=7.3 containing 9mM CaCl2 to each centrifuge tube, vortex and oscillate to slowly dissolve the protease to obtain a protease mixture, wherein the weight of Tris-HCl buffer with pH=7.3 containing 9mM CaCl2 is 1008g, and the prepared protease mixture is dispensed into sterile EP tubes, stored in a -20°C low-temperature refrigerator, and taken out as needed when used to avoid repeated freezing and thawing to obtain a protease mixture; S6. In a well-ventilated and 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 according to the volume ratio, and pour them into a clean glass separatory funnel respectively. Slowly rotate the separatory funnel and gently shake it to mix it evenly. Be careful to move gently to prevent the liquid from splashing. The mixing process lasts for about 8 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and store it in a refrigerator at 4°C to obtain the impurity remover; S7. First accurately prepare 2.4M potassium acetate solution, carefully adjust the pH to 4.9 with glacial acetic acid, then slowly add 11mL potassium acetate solution into 50mL anhydrous ethanol, stirring while adding to mix the two thoroughly. The whole process is performed in an ice bath to reduce the effects of solution volatility and temperature on the stability of the components. After mixing evenly, transfer to a sterile reagent bottle with a scale, seal it and store it at 4℃. Check whether there is precipitation before use. If there is precipitation, re-prepare it to obtain the DNA precipitation agent; S8. Use a measuring cylinder to measure anhydrous ethanol, pour it into a clean glass container, and then slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the concentration of the diluted ethanol 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, and obtain the washing solution.

[0019] Example 5: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular marker, comprising the following steps: 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 having an enzyme activity of 8300 U / g to 7 mg / mL with Tris-HCl buffer at pH 6.5 to obtain a papain solution; S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, a Tris-HCl buffer having a pH of 6.5 was added, and the reaction vessel was placed in an environment of 63° C. 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; S3. After the reaction is completed, the reaction system is heated to 88°C, maintained for 28 min, centrifuged at 11800 rpm for 24 min, and the supernatant is collected to obtain protease-modified guanidine isothiocyanate; S4. Accurately weigh 19g Tris-HCl, 27g NaCl, 9g EDTA, 30g guanidine thiocyanate, 7g protease-modified guanidine thiocyanate, 15g hexadecyltrimethylammonium bromide and 8g polyvinylpyrrolidone, place in a suitable glass container, add 355mL deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8.1, and continue stirring during the period to ensure that all components are fully dissolved and evenly dissolved. After all solid components are completely dissolved and the solution is clear, use a 0.22μm microporous filter membrane to perform vacuum filtration and sterilization, collect the filtrate into a sterile brown bottle, seal it and store it at room temperature away from light to obtain a lysate; S5. Weigh 15g papain, 12g pepsin at pH=2.2, and 10g Bacillus subtilis neutral protease at pH=7.2 respectively, place them in pre-cooled centrifuge tubes, add Tris-HCl buffer containing 10mM CaCl2 at pH=7.4 to each centrifuge tube, vortex and oscillate to slowly dissolve the protease to obtain a protease mixture, wherein the weight of Tris-HCl buffer containing 10mM CaCl2 at pH=7.4 is 988g, and the prepared protease mixture is dispensed into sterile EP tubes, stored in a -20°C low-temperature refrigerator, and taken out as needed when used to avoid repeated freezing and thawing to obtain a protease mixture; S6. In a well-ventilated and dimly-lit chemical fume hood, accurately measure 23 mL of phenol saturated with Tris-HCl buffer at pH = 7.9, 24 mL of chloroform, and 3.3 mL of isoamyl alcohol according to the volume ratio, and pour them into a clean glass separatory funnel respectively. Slowly rotate the separatory funnel and gently shake it to mix it evenly. Be careful to move gently to prevent the liquid from splashing. The mixing process lasts for about 9 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and store it in a refrigerator at 4°C to obtain the impurity remover; S7. First accurately prepare 2.5M potassium acetate solution, carefully adjust the pH to 4.9 with glacial acetic acid, then slowly add 9mL potassium acetate solution into 51mL anhydrous ethanol, stirring while adding to mix the two thoroughly. The whole process is performed in an ice bath to reduce the effects of solution volatility and temperature on the stability of the components. After mixing evenly, transfer to a sterile reagent bottle with a scale, seal it and store it at 4℃. Check whether there is precipitation before use. If there is precipitation, re-prepare it to obtain the DNA precipitation agent; S8. Use a measuring cylinder to measure anhydrous ethanol, pour it into a clean glass container, and then slowly add deionized water while stirring. Use an alcohol meter to monitor the ethanol concentration in real time to ensure that the ethanol concentration after dilution 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, and obtain the washing solution.

[0020] Example 6: A method for preparing a sample DNA extraction reagent for Rana spinosa InDel molecular marker, comprising the following steps: 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 having an enzyme activity of 8500 U / g to 10 mg / mL with Tris-HCl buffer at pH 7 to obtain a papain solution; S2. The guanidine thiocyanate solution and the papain solution were mixed in a reaction vessel, a Tris-HCl buffer having a pH of 7 was added, and the reaction vessel was placed in an environment of 65° C. 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; S3. After the reaction is completed, the reaction system is heated to 90°C, maintained for 30 min, centrifuged at 12000 rpm for 25 min, and the supernatant is collected to obtain protease-modified guanidine isothiocyanate; S4. Accurately weigh 20g Tris-HCl, 28g NaCl, 10g EDTA, 32g guanidine thiocyanate, 8g protease-modified guanidine thiocyanate, 16g hexadecyltrimethylammonium bromide and 10g polyvinylpyrrolidone, place in a suitable glass container, add 360mL deionized water to dissolve, slowly add sodium hydroxide solution dropwise using a pH meter, carefully adjust the pH to 8.2, and continue stirring during the period to ensure that all components are fully dissolved and evenly dissolved. After all solid components are completely dissolved and the solution is clear, use a 0.22μm microporous filter membrane to vacuum filter and sterilize, collect the filtrate into a sterile brown bottle, seal it and store it at room temperature away from light to obtain a lysate; S5. Weigh 16 g papain, 14 g Aspergillus niger acid protease at pH = 2.5, and 12 g thermophilic neutral protease at pH = 7.5, respectively, and place them in pre-cooled centrifuge tubes. Add Tris-HCl buffer containing 11 mM CaCl2 at pH = 7.5 to each centrifuge tube, vortex and oscillate to slowly dissolve the protease to obtain a protease mixture, wherein the weight of Tris-HCl buffer containing 11 mM CaCl2 at pH = 7.5 is 1020 g. Dispense the prepared protease mixture into sterile EP tubes, store them in a -20°C low-temperature refrigerator, and take them out as needed when used to avoid repeated freezing and thawing to obtain a protease mixture; S6. In a well-ventilated and 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 according to the volume ratio, and pour them into a clean glass separatory funnel respectively. Slowly rotate the separatory funnel and gently shake it to mix it evenly. Be careful to move gently to prevent the liquid from splashing. The mixing process lasts for about 10 minutes. Transfer the mixed impurity remover to a brown glass bottle, seal the bottle cap, and store it in a refrigerator at 4°C to obtain the impurity remover; S7. First accurately prepare 2.6M potassium acetate solution, carefully adjust the pH to 5.0 with glacial acetic acid, then slowly add 10mL potassium acetate solution into 52mL anhydrous ethanol, stirring while adding to mix the two thoroughly. The whole process is performed in an ice bath to reduce the effects of solution volatility and temperature on the stability of the components. After mixing evenly, transfer to a sterile reagent bottle with a scale, seal it and store it at 4℃. Check whether there is precipitation before use. If there is precipitation, re-prepare it to obtain the DNA precipitation agent; S8. Use a measuring cylinder to measure anhydrous ethanol, pour it into a clean glass container, and then 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, and obtain the washing solution.

[0021] Example 7: A method for extracting DNA from a Rana spinosa sample, comprising the following steps: S1. Sample pretreatment: Take 0.2g of fresh muscle tissue sample, cut it into small particles with sterile scissors, and transfer it to a 2mL sterile centrifuge tube, making sure that the sample is at the bottom of the tube without any residue on the wall; S2. Lysis: Add 700 μL of the lysis solution in Example 1 to the centrifuge tube containing the sample, and then add 80 μL of the protease mixture in Example 1, vortex and oscillate for 30 seconds to fully mix the sample and the reagent, then place the centrifuge tube in a 60°C constant temperature water bath and incubate for 1.5 hours. During this period, take out every 30 minutes and gently invert and mix 5 times to ensure that the sample is evenly heated and fully lysed; S3. Impurity removal: After the sample is lysed, cool to room temperature, add an equal volume of the impurity remover in Example 1, invert the centrifuge tube 10 times, and then put the centrifuge tube into a high-speed refrigerated centrifuge, centrifuge at 13000 rpm for 10 min, and set the temperature to 3°C. After the centrifugation is completed, carefully transfer the upper clear water phase to a new sterile centrifuge tube, taking care to avoid sucking impurities in the middle layer; S4. Precipitation: Add 1 / 10 volume of the DNA precipitant in Example 1 to a new tube, gently invert and mix 5 times, and white flocculent DNA precipitate gradually precipitates. Place the centrifuge tube in a -20°C refrigerator for 30 minutes to further promote complete precipitation, then place it in a centrifuge again, centrifuge at 13000 rpm for 10 minutes, operate at 4°C, and discard the supernatant; S5. Washing: Add 800 μL of the washing solution in Example 1, invert the centrifuge tube 5 times to suspend the precipitate, 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; S6. Dissolution: Leave the precipitate in the open air at room temperature for 2 minutes to allow the residual washing solution to evaporate, add 30μLTE buffer (10mM Tris-HCl, 1mM EDTA, pH=8.0) to dissolve the precipitate, and store the resulting DNA solution at -20℃ for a long term to avoid repeated freezing and thawing. Regularly check the DNA concentration and purity for subsequent InDel molecular labeling experiments.

[0022] Example 8: A method for extracting DNA from a Rana spinosa sample, comprising the following steps: S1. Sample pretreatment: Take 0.3 g of fresh muscle tissue sample, cut it into small particles with sterile scissors, and transfer it to a 2 mL sterile centrifuge tube, making sure that the sample is at the bottom of the tube without any residue on the wall; S2. Lysis: Add 825 μL of the lysis solution in Example 2 to the centrifuge tube containing the sample, and then add 100 μL of the protease mixture in Example 2, vortex and oscillate for 45 seconds to fully mix the sample and the reagent, then place the centrifuge tube in a 62.5°C constant temperature water bath and incubate for 2 hours. During this period, take out every 35 minutes and gently invert and mix 7 times to ensure that the sample is evenly heated and fully lysed; S3. Impurity removal: After the sample is lysed, cool to room temperature, add an equal volume of the impurity remover in Example 2, invert the centrifuge tube 12 times, and then put the centrifuge tube into a high-speed refrigerated centrifuge, centrifuge at 14000 rpm for 12.5 min, and set the temperature to 4°C. After the centrifugation is completed, carefully transfer the upper clear water phase to a new sterile centrifuge tube, taking care to avoid aspirating impurities in the middle layer; S4. Precipitation: Add 1 / 10 volume of the DNA precipitant in Example 2 to a new tube, gently invert and mix 6 times, and white flocculent DNA precipitate gradually precipitates. Place the centrifuge tube in a -20°C refrigerator for 40 min to further promote complete precipitation, then place it in a centrifuge again, centrifuge at 14000 rpm for 12 min, operate at 4°C, and discard the supernatant; S5. Washing: Add 900 μL of washing solution, invert the centrifuge tube 7 times to suspend the precipitate, and then perform the washing operation. Then centrifuge at 13000 rpm for 9 min at 4°C, discard the supernatant, and repeat the washing step once to ensure that impurities in the precipitate are washed away; S6. Dissolution: Leave the precipitate in the open air at room temperature for 2.5 minutes to allow the residual washing solution to evaporate. Add 40μLTE buffer (10mM Tris-HCl, 1mM EDTA, pH=8.0) to dissolve the precipitate. Store the resulting DNA solution at -20℃ for a long term to avoid repeated freezing and thawing. Regularly check the DNA concentration and purity for subsequent InDel molecular labeling experiments.

[0023] Example 9: A method for extracting DNA from a Rana spinosa sample, comprising the following steps: S1. Sample pretreatment: Take 0.4 g of fresh muscle tissue sample, cut it into small particles quickly with sterile scissors, and then transfer it to a 2 mL sterile centrifuge tube, making sure that the sample is at the bottom of the tube without any residue on the wall; S2. Lysis: Add 1000 μL of the lysis solution in Example 3 to the centrifuge tube containing the sample, and then add 120 μL of the protease mixture in Example 3, vortex and oscillate for 60 seconds to fully mix the sample and the reagent, then place the centrifuge tube in a 65°C constant temperature water bath and incubate for 2.5 hours. During this period, take out every 40 minutes and gently invert and mix 8 times to ensure that the sample is evenly heated and fully lysed; S3. Impurity removal: After the sample is lysed, cool to room temperature, add an equal volume of the impurity remover in Example 3, invert the centrifuge tube 15 times, and then put the centrifuge tube into a high-speed refrigerated centrifuge, centrifuge at 15000 rpm for 15 minutes, and set the temperature to 5°C. After the centrifugation is completed, carefully transfer the upper clear water phase to a new sterile centrifuge tube, and be careful to avoid sucking impurities in the middle layer; S4. Precipitation: Add 1 / 10 volume of the DNA precipitant in Example 3 to a new tube, gently invert and mix 8 times, and white flocculent DNA precipitate gradually precipitates. Place the centrifuge tube in a -20°C refrigerator for 45 minutes to further promote complete precipitation, then place it in a centrifuge again, centrifuge at 15000 rpm for 15 minutes, operate at 4°C, and discard the supernatant; S5. Washing: Add 1000 μL of the washing solution in Example 3, invert the centrifuge tube 8 times to suspend the precipitate, 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; S6. Dissolution: Leave the precipitate open at room temperature for 3 minutes to allow the residual washing solution to evaporate, add 50μLTE buffer (10mM Tris-HCl, 1mM EDTA, pH=8.0) to dissolve the precipitate, and store the resulting DNA solution at -20℃ for a long term to avoid repeated freezing and thawing. Regularly check the DNA concentration and purity for subsequent InDel molecular labeling experiments.

[0024] Comparative Example 1: Compared with Example 1, this comparative example only replaces the "protease mixture" with "papain", and the remaining steps and parameters are the same, which will not be repeated in this comparative example. Finally, a sample DNA extraction reagent for InDel molecular marker of Rana spinosa was obtained.

[0025] Comparative Example 2: Compared with Example 1, this comparative example did not add "guanidine isothiocyanate" during the preparation of the sample DNA extraction reagent for the InDel molecular marker of Rana spinosa. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, the sample DNA extraction reagent for the InDel molecular marker of Rana spinosa was obtained.

[0026] Comparative Example 3: 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 InDel molecular marker of Rana spinosa. The remaining steps and parameters are the same and will not be repeated in this comparative example. Finally, the sample DNA extraction reagent for the InDel molecular marker of Rana spinosa was obtained.

[0027] Comparative Example 4: 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, which will not be repeated in this comparative example. Finally, a sample DNA extraction reagent for InDel molecular marker of Rana spinosa was obtained.

[0028] Comparative Example 5: Compared with Example 1, this comparative example only adjusts the dosage of "protease-modified guanidine isothiocyanate" from "5g" to "26g", and adjusts the dosage 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.

[0029] The InDel molecular marker samples of Rana spinosa obtained in Comparative Examples 1 to 5 were respectively subjected to DNA extraction operations using DNA extraction reagents.

[0030] Performance test: The extraction results in Examples 7-9 and Comparative Examples 1-5 were tested as follows: DNA purity test: Use an ultraviolet 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, pH8.0) to ensure that the absorbance value is within the accurate measurement range of the instrument (generally, the absorbance value is more accurate between 0.1-1.0), and 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 1.8-2.0. A ratio close to 1.8 indicates that the content of impurities such as protein is low; if the ratio is greater than 2.0, RNA contamination may exist; if the ratio is less than 1.8, it means that there are more residual proteins or other impurities, which will affect subsequent molecular biology experiments; DNA concentration determination: Also use a UV spectrophotometer to calculate the DNA concentration based on the absorbance value at 260nm. It is known that at 260nm, when the absorbance is 1, the concentration of double-stranded DNA is about 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, such as between 100-500ng / μL, which can ensure that there is enough template for accurate detection in subsequent PCR amplification experiments; DNA integrity test: agarose gel electrophoresis. Prepare 0.8%-1.2% agarose gel, mix the extracted DNA sample with an appropriate amount of loading buffer (such as 6× loading buffer, containing bromophenol blue, sucrose, etc.), then add the mixed sample to the loading well of the gel, and add a DNA molecular weight standard of known molecular weight (such as λ-HindIIIdigest, etc.) as a control. In 1×TAE or 1×TBE electrophoresis buffer, electrophoresis is performed at a voltage of 80-120V for 30-60min. After the electrophoresis, the gel is stained in a solution containing ethidium bromide (EB) or other safe nucleic acid dyes (such as SYBR Green, etc.) for 15-30min, and then the integrity of the DNA band is observed on a UV transilluminator. The intact DNA should show clear and bright bands without obvious tailing or diffusion. The position of the band should be consistent with the position of the DNA fragment of the corresponding size in the molecular weight standard, indicating that the DNA has not been severely 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 the detection of InDel molecular markers; PCR amplification effect detection: Design PCR primer pairs for the specific InDel site of Rana spinosa, and use the extracted DNA as a template for PCR amplification reaction. The reaction system generally includes: a certain concentration of template DNA (such as 50-200ng), upstream and downstream primers (0.2-0.5μM each), dNTPs (0.2-0.4mM), TaqDNA polymerase (1-2.5U) and corresponding PCR buffer (such as 1×PCR buffer, containing Mg²⁺, etc.), with a total volume of 20-50μL. PCR reaction conditions can be optimized according to the Tm value of the primers and the length of the amplified fragment, generally including pre-denaturation (94-95℃, 3-5min), followed by 30-35 cycles of denaturation (94-95℃, 30-45s), annealing (according to the Tm value of the primers, 50-65℃, 30-45s), extension (72℃, the extension time is determined according to the length of the amplified fragment, generally 1-1.5min per 1kb), and finally 72℃ extension for 5-10min. The PCR amplification products were detected by 2%-3% agarose gel electrophoresis to observe the specificity, brightness and clarity of the amplified bands. The ideal PCR amplification results should show a single, clear and bright specific band, without obvious non-specific amplification bands or primer dimers. The position of the band should be consistent with the expected amplified fragment size, indicating that the extracted DNA can be effectively used as a template for PCR amplification, and the primer specificity is good. The reaction system and conditions are suitable, which can meet the requirements of PCR amplification for the detection of InDel molecular markers of Rana spinosa. If multiple nonspecific bands or a large number of primer dimers appear, or the target band is not amplified, it may be due to insufficient DNA purity, the presence of inhibitors, or unreasonable primer design.

[0031] The results are shown in Table 1 below: , Data Analysis: From Table 1 we can see that: The A260 / A280 ratios of Examples 7-9 are between 1.88-1.95, all within the ideal range of 1.8-2.0. This shows that in these examples, the extraction reagents can effectively remove impurities such as proteins and polysaccharides, so that the extracted DNA purity is relatively high, and the strict requirements of the InDel molecular marker detection of Rana spinosa for DNA purity can be met. For example, the ratio of Example 9 is 1.95, indicating that the DNA extraction process of this example performs well in removing impurities and provides a high-purity DNA template for subsequent experiments, while the A260 / A280 ratio of Comparative Example 1 is 1.65, which is significantly lower than the ideal range. This means that the extraction process does not remove proteins or other impurities well, and these impurities may interfere with subsequent experiments, such as inhibiting enzyme activity in PCR reactions; the ratio of comparative example 2 is 2.10, which is greater than 2.0, indicating that RNA contamination may exist. 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 are close to or within an acceptable range, they are still not as ideal as those of the embodiments, that is, embodiments 7-9 are significantly better than comparative examples 1-5 in terms of DNA purity, indicating that the reagent formula 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; The DNA concentrations of Examples 7-9 are between 280-350 ng / μL, which are all within the range suitable for subsequent InDel molecular marker detection. Sufficient DNA concentration can ensure sufficient template amount in experiments such as PCR amplification, thereby improving the accuracy and sensitivity of detection. For example, the DNA concentration of Example 7 is 320 ng / μL, which provides sufficient DNA template for subsequent experimental operations, while the DNA concentration of Comparative Example 1 is 150 ng / μL, which is relatively low and may affect the accuracy of experimental results due to insufficient template amount in subsequent experiments; the concentrations of Comparative Examples 2-5 are between 200-400 ng / μL. Although the concentrations of some comparative examples can meet the requirements, there are still some problems considering other factors such as purity, that is, the DNA concentrations of Examples 7-9 can stably reach a level suitable for experiments, while Comparative Examples 1-5 have certain volatility and deficiencies in DNA concentration, which further reflects the advantages of the present invention in ensuring effective DNA extraction amount; The agarose gel electrophoresis results of Examples 7-9 show that the DNA bands are clear and bright, without obvious tailing or diffusion. This shows that in these examples, the DNA was not significantly mechanically sheared or chemically degraded during the extraction process, and the integrity was good, which could provide complete gene sequence information for InDel molecular marker detection, indicating that the extraction methods of Examples 7-9 caused less damage to the DNA, while the DNA bands of Comparative Example 1 had a more serious tailing phenomenon, indicating that the DNA was degraded to a certain extent; Comparative Examples 3-4 also had tailing or diffusion phenomena to varying degrees, indicating that the extraction methods of these comparative examples had defects in maintaining DNA integrity; Comparative Example 2 may be an abnormal phenomenon caused by RNA residues, which will also cause certain interference to the judgment 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 problems to varying degrees, highlighting the superiority of the extraction method of the present invention in protecting DNA integrity; The PCR amplification products of Examples 7-9 showed a single, clear specific band after electrophoresis, without primer dimers and non-specific amplification bands, which indicates that the extracted DNA can be well used as a template for PCR amplification, and the primer specificity is good, the reaction system and conditions are suitable, and can meet the requirements of PCR amplification for the detection of InDel molecular markers of Rana spinosa. For example, the electrophoresis bands of the PCR amplification product of Example 9 are highly specific and bright, indicating that the quality of the DNA extracted in this example is very high and is an ideal template for InDel molecular marker detection. However, the PCR amplification of Comparative Example 1 showed multiple non-specific bands, and the target band was blurred. This is due to the low purity of the DNA, poor integrity, and the possible presence of inhibitors. This situation will seriously affect the detection and analysis of the InDel site. Although Comparative Examples 2-5 can partially obtain the target bands, there are also non-specific bands to varying degrees, indicating that there are certain problems with the quality of the DNA extracted in these comparative examples, which will affect the resolution and accuracy of the InDel site detection. That is, Examples 7-9 are significantly better than Comparative Examples 1-5 in terms of PCR amplification effect, proving 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, and the DNA quality problems of the comparative examples will have an adverse effect on PCR amplification and subsequent detection.

[0032] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0033] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A sample DNA extraction reagent for Rana spinosa InDel molecular marker, characterized in that: The method is prepared from the following raw materials in parts by weight: 60-70 parts of lysate, 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 isothiocyanate, protease-modified guanidine isothiocyanate, hexadecyltrimethylammonium bromide and polyvinylpyrrolidone.

2. The sample DNA extraction reagent for InDel molecular marker of Rana spinosa according to claim 1, characterized in that: The mass ratio of Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, hexadecyltrimethylammonium 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.

3. The sample DNA extraction reagent for InDel molecular marker of Rana spinosa according to claim 1, characterized in that: The preparation process of the protease-modified guanidine isothiocyanate is as follows: Step A1. Solution preparation: dissolve guanidine thiocyanate in a buffer to prepare a 3-5 mol / L guanidine thiocyanate solution, dilute papain with the buffer 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 a buffer solution, and place the reaction vessel in an environment of 55-65°C and stir at 300-500 rpm for 8-10 hours; Step A3. After the reaction is completed, the reaction system is heated to 80-90°C, maintained for 20-30 minutes, centrifuged at a speed of 10000-12000 rpm for 20-25 minutes, and the supernatant is collected to obtain protease-modified guanidine isothiocyanate.

4. The sample DNA extraction reagent for InDel molecular marker of Rana spinosa according to claim 3, characterized in that: The guanidine isothiocyanate in step A1 is analytical grade guanidine isothiocyanate; 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.

5. The sample DNA extraction reagent for InDel molecular marker of Rana spinosa according to claim 3, characterized in that: 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.

6. The sample DNA extraction reagent for InDel molecular marker of Rana spinosa 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.

7. The sample DNA extraction reagent for InDel molecular marker of Rana spinosa according to claim 6, characterized in that: The solvent is a Tris-HCl buffer 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 neutral protease of Bacillus subtilis and a neutral protease of thermophilic bacteria at pH=6.5-7.

5.

8. The sample DNA extraction reagent for InDel molecular marker of Rana spinosa according to claim 1, characterized in that: The impurity remover is obtained by mixing phenol: chloroform: 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.

9. The sample DNA extraction reagent for InDel molecular marker of Rana spinosa according to claim 1, characterized in that: The DNA precipitant is obtained 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 liquid is 74.5%-75.5% ethanol.

10. The method for preparing a sample DNA extraction reagent for InDel molecular marker of Rana spinosa according to any one of claims 1 to 9, characterized in that: The following steps are involved: Step S1. Preparation of lysate: weigh Tris-HCl, NaCl, EDTA, guanidine thiocyanate, protease-modified guanidine thiocyanate, hexadecyltrimethylammonium bromide and polyvinylpyrrolidone, place in a suitable glass container, add deionized water to dissolve, dropwise add sodium hydroxide solution, adjust the pH to 8-8.2, and then perform vacuum filtration with a 0.22 μm microporous filter membrane. Collect the filtrate into a sterile brown bottle, seal it and store it at room temperature away from light to obtain a lysate; Step S2. Preparation of protease mixture: Weigh papain, acid protease and neutral protease powders respectively, place them in pre-cooled centrifuge tubes, add solvent to each centrifuge tube, vortex and oscillate to dissolve the protease to obtain a protease mixture, dispense the prepared protease mixture into sterile EP tubes, and freeze and store them in a -20°C low-temperature refrigerator to obtain a protease mixture; Step S3. Preparation of a de-impurity agent: Phenol, chloroform and isoamyl alcohol are measured and poured into clean glass separatory funnels respectively, mixed evenly, and then transferred to a brown glass bottle, sealed with a bottle cap, and stored in a refrigerator at 4° C. to obtain a de-impurity agent; Step S4. Preparation of precipitant: first prepare 2.5M potassium acetate solution, adjust the pH to 4.6-5.0 with glacial acetic acid, then add the potassium acetate solution to anhydrous ethanol, stirring while adding, and operate in an ice bath throughout the process. After mixing evenly, transfer to a sterile reagent bottle with a scale, seal and store at 4°C to obtain a DNA precipitant; Step S5. Preparation of washing liquid: measure anhydrous ethanol, pour it into a glass container, add deionized water, stir while adding, make the ethanol concentration reach 74.5%-75.5%, stir evenly, and store at room temperature to obtain washing liquid; The usage ratio of Tris-HCl to deionized water in step S1 is 18-20 g: 350-360 mL.