Freeze-drying protective agent for DNA polymerase and application of freeze-drying protective agent

Abstract

The invention relates to a freeze-drying protective agent for DNA polymerase and application of the freeze-drying protective agent, and belongs to the technical field of biology. The invention provides a freeze-drying protective agent of DNA polymerase. The freeze-drying protective agent comprises a solvent, potassium chloride, ethylenediamine tetraacetic acid, dithiothreitol, tween-20, trehalose, mannitol, polyethylene glycol and bovine serum albumin. The DNA polymerase freeze-drying preparation obtained by freeze-drying the DNA polymerase by using the freeze-drying protective agent does not need low temperature in the storage and transportation processes, and has the advantage of low cost; the DNA polymerase freeze-drying preparation obtained by freeze-drying the DNA polymerase by using the freeze-drying protective agent is good in formability; the DNA polymerase freeze-drying preparation obtained by freeze-drying the DNA polymerase by using the freeze-drying protective agent is short in redissolution time, and the amplification performance after redissolution is not obviously changed; the DNA polymerase freeze-drying preparation obtained by freeze-drying the DNA polymerase by using the freeze-drying protective agent is good in stability.

Description

Technical Field

[0001] The invention relates to a freeze-dried protective agent for DNA polymerase and application thereof, belonging to the field of biotechnology. Background Art

[0002] Molecular diagnosis refers to the technology of using molecular biology methods to detect changes in the structure or expression level of genetic material in the patient's body to make a diagnosis. As the demand for molecular diagnosis continues to increase, the demand for various molecular detection reagents has also increased accordingly. Molecular detection reagents need to be transported to reach the demand location.

[0003] Traditional molecular detection reagents are generally liquid preparations. Because they contain biologically active enzymes, in order to ensure the stability of liquid molecular detection reagents during transportation and storage, they usually need to be transported through cold chains such as dry ice. The transportation cost is high, and storage in laboratory freezers takes up space and consumes electricity. In addition, changes in ambient temperature during transportation can easily cause repeated freezing and thawing of detection reagents, which will directly affect the performance and shelf life of detection reagents. DNA polymerase is currently the most commonly used and important molecular detection reagent.

[0004] DNA polymerase, also known as DNA-dependent DNA polymerase (DNApol), is a type of enzyme that uses parent DNA as a template to catalyze the polymerization of substrate dNTP molecules to form daughter DNA. There are many types of DNA polymerases, among which Taq polymerase, DNA polymerase I and DNA polymerase II are the more well-researched ones.

[0005] DNA in a room temperature solution will be inactivated by a small temperature change or pH change, and its performance will be greatly reduced. Therefore, in order to protect DNA polymerase and for more convenient and safe storage and transportation, freeze-drying the liquid preparation of DNA polymerase is a good choice. The freeze-dried DNA polymerase can be transported and stored at room temperature and has a longer shelf life. However, how to optimize the formula of the lyoprotectant and the freeze-drying process to maximize the morphology and stability of the DNA polymerase in the freeze-dried state, while ensuring that the amplification performance of the DNA polymerase freeze-dried preparation is not affected after rapid hydration and activation, is still a problem. Summary of the invention

[0006] To solve the above problems, the present invention provides a lyophilized protective agent for DNA polymerase, wherein the components of the lyophilized protective agent include a solvent, potassium chloride (KCL), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), Tween-20 (Tween-20), trehalose, mannitol, polyethylene glycol (PEG) and bovine serum albumin (BSA).

[0007] In one embodiment of the present invention, the concentration of potassium chloride in the solvent is 20-40 mM; the concentration of ethylenediaminetetraacetic acid in the solvent is 0.05-0.1 mM; the concentration of dithiothreitol in the solvent is 0.2-1 mM; in terms of volume percentage, the concentration of Tween-20 in the solvent is 0.02-0.5%; in terms of mass volume percentage (w / v, g / 100 mL), the concentration of trehalose in the solvent is 5-10%; in terms of mass volume percentage (w / v, g / 100 mL), the concentration of mannitol in the solvent is 0.5-2%; in terms of mass volume percentage (w / v, g / 100 mL), the concentration of polyethylene glycol in the solvent is 0.1-1%; in terms of mass volume percentage (w / v, g / 100 mL), the concentration of bovine serum albumin in the solvent is 1-4%.

[0008] In one embodiment of the present invention, the concentration of potassium chloride in the solvent is 40mM; the concentration of ethylenediaminetetraacetic acid in the solvent is 0.1mM; the concentration of dithiothreitol in the solvent is 1mM; in terms of volume percentage, the concentration of Tween-20 in the solvent is 0.5%; in terms of mass volume percentage (w / v, g / 100mL), the concentration of trehalose in the solvent is 10%; in terms of mass volume percentage (w / v, g / 100mL), the concentration of mannitol in the solvent is 2%; in terms of mass volume percentage (w / v, g / 100mL), the concentration of polyethylene glycol in the solvent is 1%; in terms of mass volume percentage (w / v, g / 100mL), the concentration of bovine serum albumin in the solvent is 4%.

[0009] In one embodiment of the present invention, the solvent comprises a buffer; the buffer comprises Tris-HCL buffer, Tris-H2SO4 buffer and / or Tris-acetate buffer.

[0010] In one embodiment of the present invention, the solvent is a Tirs-HCL buffer solution; the concentration of the Tirs-HCL buffer solution is 20-30 mM and the pH is 7.8-8.5.

[0011] In one embodiment of the present invention, the concentration of the Tirs-HCL buffer is 20 mM and the pH is 8.0.

[0012] In one embodiment of the present invention, the components of the lyoprotectant are composed of a solvent, potassium chloride (KCL), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), Tween-20 (Tween-20), trehalose, mannitol, polyethylene glycol (PEG) and bovine serum albumin (BSA).

[0013] In one embodiment of the present invention, the polyethylene glycol comprises PEG8000.

[0014] In one embodiment of the present invention, the DNA polymerase comprises Taq DNA polymerase, DNA polymerase I and / or DNA polymerase II.

[0015] The present invention also provides a freeze-dried preparation of DNA polymerase, the components of which include the freeze-dried protective agent and DNA polymerase.

[0016] In one embodiment of the present invention, the preparation method of the lyophilized DNA polymerase preparation comprises: adding the DNA polymerase to a lyophilization protectant to obtain a mixture; and freeze-drying the mixture to obtain the lyophilized DNA polymerase preparation.

[0017] In one embodiment of the present invention, in the mixture, the volume ratio of DNA polymerase to lyophilization protectant is 1:9-20.

[0018] In one embodiment of the present invention, the DNA polymerase comprises Taq DNA polymerase, DNA polymerase I and / or DNA polymerase II.

[0019] In one embodiment of the present invention, the freeze-drying sequentially comprises a pre-freezing stage, a quick freezing stage, a primary sublimation drying (vacuuming) stage, a secondary sublimation drying stage, a primary desorption drying stage, a secondary desorption drying stage and a storage stage.

[0020] In one embodiment of the present invention, the temperature of the pre-freezing stage is -20 to -40°C, the slope temperature is 60 to 90 minutes, and the constant temperature time is 60 to 90 minutes;

[0021] The temperature of the quick freezing stage is -40 to -50°C, the slope temperature is 60 to 90 minutes, and the constant temperature time is 60 to 90 minutes;

[0022] The temperature of the primary sublimation drying stage is -20 to -30°C, the slope temperature is 60 to 90 minutes, and the constant temperature time is 180 to 360 minutes;

[0023] The temperature of the secondary sublimation drying stage is -15 to -25°C, the slope temperature is 60 to 90 minutes, and the constant temperature time is 360 to 560 minutes;

[0024] The temperature of the primary desorption drying stage is 20-25°C, the slope temperature is 120-150min, and the constant temperature time is 90-120min;

[0025] The temperature of the secondary desorption drying stage is 25-35°C, the slope temperature is 30-60min, and the constant temperature time is 90-120min;

[0026] The temperature of the storage stage is 0-8°C, the slope temperature is 60-190 minutes, and the constant temperature time is 30-150 minutes. The slope temperature refers to the speed and amplitude of the temperature drop during the freeze-drying process. After setting the time on the freeze-drying instrument, the freeze-drying instrument will drop to the specified temperature at the specified time.

[0027] In one embodiment of the present invention, the temperature of the pre-freezing stage is -30°C, the slope temperature is 60min, and the constant temperature time is 60min;

[0028] The temperature of the quick freezing stage is -45°C, the slope temperature is 30min, and the constant temperature time is 30min;

[0029] The temperature of the first sublimation drying stage is -25°C, the slope temperature is 60min, and the constant temperature time is 180min;

[0030] The temperature of the secondary sublimation drying stage is -20°C, the slope temperature is 60min, and the constant temperature time is 480min;

[0031] The temperature of the first desorption drying stage is 20°C, the slope temperature is 120min, and the constant temperature time is 90min;

[0032] The temperature of the secondary desorption drying stage is 30°C, the slope temperature is 30min, and the constant temperature time is 90min;

[0033] The temperature of the storage stage is 4° C., the slope temperature is 60 min, and the constant temperature time is 30 min.

[0034] The present invention also provides a method for preparing the above-mentioned DNA polymerase freeze-dried preparation, the method comprising: adding DNA polymerase to the above-mentioned freeze-dried protective agent to obtain a mixture; freeze-drying the mixture to obtain the above-mentioned DNA polymerase freeze-dried preparation.

[0035] In one embodiment of the present invention, in the mixture, the volume ratio of DNA polymerase to lyophilization protectant is 1:9-20.

[0036] In one embodiment of the present invention, the DNA polymerase comprises Taq DNA polymerase, DNA polymerase I and / or DNA polymerase II.

[0037] In one embodiment of the present invention, the freeze-drying sequentially comprises a pre-freezing stage, a quick freezing stage, a primary sublimation drying (vacuuming) stage, a secondary sublimation drying stage, a primary desorption drying stage, a secondary desorption drying stage and a storage stage.

[0038] In one embodiment of the present invention, the temperature of the pre-freezing stage is -20 to -40°C, the slope temperature is 60 to 90 minutes, and the constant temperature time is 60 to 90 minutes;

[0039] The temperature of the quick freezing stage is -40 to -50°C, the slope temperature is 60 to 90 minutes, and the constant temperature time is 60 to 90 minutes;

[0040] The temperature of the primary sublimation drying stage is -20 to -30°C, the slope temperature is 60 to 90 minutes, and the constant temperature time is 180 to 360 minutes;

[0041] The temperature of the secondary sublimation drying stage is -15 to -25°C, the slope temperature is 60 to 90 minutes, and the constant temperature time is 360 to 560 minutes;

[0042] The temperature of the primary desorption drying stage is 20-25°C, the slope temperature is 120-150min, and the constant temperature time is 90-120min;

[0043] The temperature of the secondary desorption drying stage is 25-35°C, the slope temperature is 30-60min, and the constant temperature time is 90-120min;

[0044] The temperature of the storage stage is 0-8°C, the slope temperature is 60-190 minutes, and the constant temperature time is 30-150 minutes.

[0045] In one embodiment of the present invention, the temperature of the pre-freezing stage is -30°C, the slope temperature is 60min, and the constant temperature time is 60min;

[0046] The temperature of the quick freezing stage is -45°C, the slope temperature is 30min, and the constant temperature time is 30min;

[0047] The temperature of the first sublimation drying stage is -25°C, the slope temperature is 60min, and the constant temperature time is 180min;

[0048] The temperature of the secondary sublimation drying stage is -20°C, the slope temperature is 60min, and the constant temperature time is 480min;

[0049] The temperature of the first desorption drying stage is 20°C, the slope temperature is 120min, and the constant temperature time is 90min;

[0050] The temperature of the secondary desorption drying stage is 30°C, the slope temperature is 30min, and the constant temperature time is 90min;

[0051] The temperature of the storage stage is 4° C., the slope temperature is 60 min, and the constant temperature time is 30 min.

[0052] The present invention also provides a kit, the components of which include the above-mentioned DNA polymerase freeze-dried preparation.

[0053] In one embodiment of the present invention, the kit is a nucleic acid amplification reaction kit or a molecular detection kit.

[0054] The present invention also provides a nucleic acid amplification method, which is not for the purpose of disease diagnosis and treatment, and uses the above-mentioned DNA polymerase freeze-dried preparation or the above-mentioned kit to amplify the target nucleic acid.

[0055] The present invention also provides a molecular detection method, which is not for the purpose of disease diagnosis and treatment, and uses the above-mentioned DNA polymerase freeze-dried preparation or the above-mentioned kit to perform molecular detection on the target nucleic acid.

[0056] The present invention also provides the use of the above lyophilized protective agent or the above lyophilized preparation of DNA polymerase or the above kit in nucleic acid amplification or molecular detection, and the application is not for the purpose of diagnosis and treatment of diseases.

[0057] The technical solution of the present invention has the following advantages:

[0058] 1. The present invention provides a lyophilization protective agent for DNA polymerase, the components of which include solvent, potassium chloride (KCL), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), Tween-20 (Tween-20), trehalose, mannitol, polyethylene glycol (PEG) and bovine serum albumin (BSA). Among them, EDTA mainly plays an acid-base regulating role, and can adjust the pH of the biological product to the most stable region of the active substance during the freeze-drying process and storage process; Tween-20 and trehalose mainly play a lyophilization protective role, and can prevent the active components from denaturing during the freezing and drying process; mannitol, polyethylene glycol and BSA mainly play a filling role, which can prevent the effective components from sublimating and escaping with water vapor, and make the effective components take shape. Studies have shown that the lyophilization protective agent has the following advantages:

[0059] First, the freeze-dried DNA polymerase preparation obtained by freeze-drying the DNA polymerase using the freeze-dried protective agent does not require low temperature during storage and transportation, and has the advantage of low cost;

[0060] Second, the freeze-dried DNA polymerase preparation obtained by freeze-drying the DNA polymerase using the freeze-drying protective agent has good formability and presents a white, uniform, compact and smooth cake shape;

[0061] Third, the DNA polymerase freeze-dried preparation obtained by freeze-drying the DNA polymerase using the freeze-dried protective agent has a short reconstitution time, and the amplification performance after reconstitution does not change significantly;

[0062] Fourth, the lyophilized preparation of DNA polymerase obtained by lyophilizing the DNA polymerase using the lyophilization protective agent has good stability, and the amplification performance does not change significantly after being stored at 37°C for 2 months or at 4°C for one year.

[0063] Therefore, the lyoprotectant has great application prospects in the preparation of freeze-dried preparations of DNA polymerase, and can ensure the morphology and stability of Taq polymerase in the freeze-dried state to the greatest extent, while ensuring that the amplification performance of the freeze-dried preparation of Taq polymerase is not affected after rapid hydration and activation.

[0064] 2. The present invention provides a lyophilized preparation of DNA polymerase, the ingredients of which include a lyophilized protective agent and DNA polymerase, and the ingredients of the lyophilized protective agent include a solvent, potassium chloride (KCL), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), Tween-20 (Tween-20), trehalose, mannitol, polyethylene glycol (PEG) and bovine serum albumin (BSA). Among the lyophilized protective agents, EDTA mainly plays an acid-base regulating role, and can adjust the pH of the biological product to the most stable region of the active substance during the freeze-drying process and storage process; Tween-20 and trehalose mainly play a lyophilization protective role, and can prevent the active components from denaturing during the freezing and drying process; mannitol, polyethylene glycol and BSA mainly play a filling role, which can prevent the effective components from sublimating and escaping with water vapor, and make the effective components take shape. Studies have shown that the lyophilized preparation of DNA polymerase has the following advantages:

[0065] First, the lyophilized DNA polymerase preparation does not require low temperature during storage and transportation, and has the advantage of low cost;

[0066] Second, the freeze-dried DNA polymerase preparation has good formability and presents a white, uniform, compact and smooth cake shape;

[0067] Third, the reconstitution time of the lyophilized DNA polymerase preparation is short, and the amplification performance after reconstitution does not change significantly;

[0068] Fourth, the lyophilized preparation of DNA polymerase has good stability, and the amplification performance does not change significantly after being stored at 37°C for 2 months or at 4°C for one year.

[0069] Therefore, the lyophilized preparation of DNA polymerase has great application prospects in the fields of nucleic acid amplification or molecular detection.

[0070] Furthermore, the preparation method of the lyophilized DNA polymerase preparation comprises: adding the DNA polymerase to a lyophilization protective agent to obtain a mixture; freeze-drying the mixture to obtain the lyophilized DNA polymerase preparation;

[0071] The freeze drying sequentially comprises a pre-freezing stage, a quick freezing stage, a primary sublimation drying (vacuuming) stage, a secondary sublimation drying stage, a primary desorption drying stage, a secondary desorption drying stage and a storage stage.

[0072] This freeze-drying process can further ensure the morphology and stability of the DNA polymerase in the freeze-dried state, and further ensure that the amplification performance of the DNA polymerase freeze-dried preparation is not affected after rapid hydration and activation, thereby reducing the storage and transportation costs of the DNA polymerase and the kit composed of various DNA polymerases. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 :Effects of different potassium ion concentrations on freeze-dried morphology.

[0074] Figure 2 :Effects of different potassium ion concentrations on reconstitution after freeze-drying.

[0075] Figure 3 :The influence of different freeze-drying formulas on freeze-dried morphology (lyoprotectants 1-5).

[0076] Figure 4 :The influence of different freeze-drying formulas on freeze-dried morphology (lyoprotectants 6-10).

[0077] Figure 5 :The influence of different freeze-drying formulas on freeze-dried morphology (lyoprotectants 11-15).

[0078] Figure 6 :The influence of different freeze-drying processes on freeze-dried morphology. DETAILED DESCRIPTION

[0079] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0080] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0081] The qPCR primers used in the following examples are shown in Table 1.

[0082] Table 1 qPCR primers

[0083]

[0084]

[0085] Example 1 to Example 10: A freeze-dried protective agent for DNA polymerase

[0086] The present embodiment provides a lyophilized protective agent for DNA polymerase, wherein the lyophilized protective agent consists of Tirs-HCL buffer, potassium chloride (KCL), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), Tween-20 (Tween-20), trehalose, mannitol, PEG8000 and bovine serum albumin (BSA), and its specific formula is shown in Tables 2 and 3.

[0087] Table 2 Formula of freeze-drying protective agent

[0088]

[0089] Table 3 Formula of lyophilization protective agent

[0090]

[0091] Comparative Examples 1 to 5: A freeze-dried protective agent for DNA polymerase

[0092] This comparative example provides a lyophilized protective agent for DNA polymerase. The specific formula of the lyophilized protective agent is shown in Table 4.

[0093] Table 4 Formula of lyophilized protective agent

[0094]

[0095] Experimental Example 1: Effect of Lyoprotectant on the Performance of Taq Polymerase

[0096] The experimental process is as follows:

[0097] Experiment 1: Effect of mannitol concentration on Taq polymerase amplification performance

[0098] Experimental group 1: Taq polymerase and the lyophilized protective agent 5 (mannitol concentration 2%) of Example 5 were mixed at a volume ratio of 1:9 (Taq polymerase: lyophilized protective agent = 1:9) to obtain a mixture; the mixture was freeze-dried to obtain a lyophilized Taq polymerase preparation; the lyophilized Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, and 293 cells, pigs and Arabidopsis cDNA were used as samples (cDNA was obtained by reverse transcription after RNA was extracted from cell samples, pork samples and Arabidopsis leaf samples), and the amplification performance of Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1), and the detection results are shown in Table 5;

[0099] Experimental groups 2-5: Based on experimental group 1, the concentration of mannitol in the lyophilized protective agent was replaced with 0%, 0.5%, 1%, and 4%, respectively, and the rest remained unchanged. The test results are shown in Table 5;

[0100] The freeze drying procedure is as follows:

[0101] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0102] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0103] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0104] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0105] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0106] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0107] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0108] As shown in Table 5, after adding 0.5-2% mannitol, the CT value obtained by qPCR was significantly reduced. It can be seen that 0.5-2% mannitol can improve the amplification performance of Taq polymerase.

[0109] Table 5 Effect of mannitol concentration on Taq polymerase amplification performance

[0110]

[0111] 2. Effect of trehalose concentration on Taq polymerase amplification performance

[0112] Experimental group 1: Taq polymerase and the lyophilized protective agent 5 (trehalose concentration 10%) of Example 5 were mixed at a volume ratio of 1:9 (Taq polymerase: lyophilized protective agent = 1:9) to obtain a mixture; the mixture was freeze-dried to obtain a lyophilized Taq polymerase preparation; the lyophilized Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, and then mouse and tomato cDNA were used as samples (mice were purchased from Rat Leybold, and RNA was extracted from mouse liver samples and tomato leaf samples and then reverse transcribed to obtain cDNA), and the amplification performance of Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1), and the detection results are shown in Table 6;

[0113] Experimental groups 2-5: Based on experimental group 1, the concentration of trehalose in the lyophilized protective agent was replaced with 0%, 1%, 5%, and 20%, respectively, and the rest remained unchanged. The test results are shown in Table 6;

[0114] The freeze drying procedure is as follows:

[0115] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0116] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0117] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0118] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0119] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0120] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0121] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0122] As shown in Table 6, after adding 5-10% trehalose, the CT value obtained by qPCR was significantly reduced. It can be seen that 5-10% trehalose can improve the amplification performance of Taq polymerase.

[0123] Table 6 Effect of trehalose concentration on Taq polymerase amplification performance

[0124]

[0125] 3. Effect of PEG8000 concentration on Taq polymerase amplification performance

[0126] Experimental group 1: Taq polymerase and the lyophilized protective agent 5 (PEG8000 concentration 1%) of Example 5 were mixed at a volume ratio of 1:9 (Taq polymerase: lyophilized protective agent = 1:9) to obtain a mixture; the mixture was freeze-dried to obtain a lyophilized Taq polymerase preparation; the lyophilized Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, and wheat and rice cDNA were used as samples (wheat leaf samples and rice leaf samples were extracted RNA and then reverse transcribed to obtain cDNA) as samples, and the amplification performance of Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1), and the detection results are shown in Table 7;

[0127] Experimental groups 2-5: Based on experimental group 1, the concentration of PEG8000 in the lyophilized protective agent was replaced with 0%, 0.1%, 0.5%, and 2%, respectively, and the rest remained unchanged. The test results are shown in Table 7;

[0128] The freeze drying procedure is as follows:

[0129] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0130] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0131] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0132] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0133] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0134] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0135] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0136] As shown in Table 7, after adding 0.1-1% PEG8000, the CT value obtained by qPCR was significantly reduced. It can be seen that 0.1-1% PEG8000 can improve the amplification performance of Taq polymerase.

[0137] Table 7 Effect of PEG8000 concentration on Taq polymerase amplification performance

[0138]

[0139] 4. Effect of bovine serum albumin concentration on Taq polymerase amplification performance

[0140] Experimental group 1: Taq polymerase and the lyophilized protective agent 5 (bovine serum albumin concentration 4%) of Example 5 were mixed at a volume ratio of 1:9 (Taq polymerase: lyophilized protective agent = 1:9) to obtain a mixture; the mixture was freeze-dried to obtain a lyophilized Taq polymerase preparation; the lyophilized Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, and the amplification performance of the Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1) using pig and Arabidopsis cDNA as samples, respectively. The test results are shown in Table 8;

[0141] Experimental groups 2-5: Based on experimental group 1, the concentration of bovine serum albumin in the lyophilized protective agent was replaced with 0%, 1%, 2%, and 5%, respectively, and the rest remained unchanged. The test results are shown in Table 8;

[0142] The freeze drying procedure is as follows:

[0143] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0144] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0145] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0146] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0147] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0148] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0149] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0150] As shown in Table 8, after adding 1-4% BSA, the CT value obtained by qPCR was significantly reduced. It can be seen that 1-4% BSA can improve the amplification performance of Taq polymerase.

[0151] Table 8 Effect of bovine serum albumin concentration on Taq polymerase amplification performance

[0152]

[0153] 5. Effect of EDTA concentration on Taq polymerase amplification performance

[0154] Experimental group 1: Taq polymerase and the lyophilized protective agent 5 (EDTA concentration 0.1 mM) of Example 5 were mixed at a volume ratio of 1:9 (Taq polymerase: lyophilized protective agent = 1:9) to obtain a mixture; the mixture was freeze-dried to obtain a lyophilized Taq polymerase preparation; the lyophilized Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, and the amplification performance of Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1) using wheat and rice cDNA as samples, respectively. The detection results are shown in Table 9;

[0155] Experimental groups 2-4: Based on experimental group 1, the concentration of EDTA in the lyophilized protective agent was replaced with 0mM, 0.05mM, and 0.2mM respectively, and the rest remained unchanged. The test results are shown in Table 9;

[0156] The freeze drying procedure is as follows:

[0157] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0158] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0159] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0160] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0161] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0162] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0163] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0164] As shown in Table 9, after adding 0.05-0.2 mM EDTA, the CT value obtained by qPCR was significantly reduced. It can be seen that 0.05-0.2 mM EDTA can improve the amplification performance of Taq polymerase.

[0165] Table 9 Effect of EDTA concentration on Taq polymerase amplification performance

[0166]

[0167] 6. Effect of Tween-20 concentration on Taq polymerase amplification performance

[0168] Experimental group 1: Taq polymerase and the lyophilized protective agent 5 (Tween-20 concentration 0.5%) of Example 5 were mixed at a volume ratio of 1:9 (Taq polymerase: lyophilized protective agent = 1:9) to obtain a mixture; the mixture was freeze-dried to obtain a lyophilized Taq polymerase preparation; the lyophilized Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, and the amplification performance of the Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1) using 293 cells and porcine cDNA as samples, respectively. The test results are shown in Table 10;

[0169] Experimental groups 2-5: Based on experimental group 1, the concentration of Tween-20 in the lyophilized protective agent was replaced with 0%, 0.05%, 0.1%, and 1%, respectively, and the rest remained unchanged. The test results are shown in Table 10;

[0170] The freeze drying procedure is as follows:

[0171] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0172] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0173] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0174] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0175] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0176] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0177] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0178] As shown in Table 10, after adding 0.05% to 0.5% Tween-20, the CT value obtained by qPCR was significantly reduced. It can be seen that 0.05% to 0.5% Tween-20 can improve the amplification performance of Taq polymerase.

[0179] Table 10 Effect of Tween-20 concentration on Taq polymerase amplification performance

[0180]

[0181] 7. Effect of pH on Taq polymerase amplification performance

[0182] Experimental group 1: Taq polymerase and the lyophilized protective agent 5 (pH 8.0) of Example 5 were mixed at a volume ratio of 1:9 (Taq polymerase: lyophilized protective agent = 1:9) to obtain a mixture; the mixture was freeze-dried to obtain a lyophilized Taq polymerase preparation; the lyophilized Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, and the amplification performance of Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1) using tomato and rice cDNA as samples, respectively. The detection results are shown in Table 11;

[0183] Experimental groups 2-5: Based on experimental group 1, the pH of the buffer solution in the lyophilization protective agent was replaced with 7.8, 8.3, 8.5, and 8.8, respectively, and the rest remained unchanged. The test results are shown in Table 11;

[0184] The freeze drying procedure is as follows:

[0185] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0186] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0187] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0188] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0189] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0190] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0191] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0192] As shown in Table 11, within the pH range of 7.8 to 8.5, the CT value obtained by qPCR is significantly reduced. It can be seen that the pH of 7.8 to 8.5 can improve the amplification performance of Taq polymerase.

[0193] Table 11 Effect of pH on Taq polymerase amplification performance

[0194]

[0195] 8. Effect of potassium ion concentration on freeze-dried morphology and amplification performance of Taq polymerase

[0196] Experimental group 1: Taq polymerase and lyophilized protective agent 5 (potassium ion concentration 40 mM) of Example 5 were mixed at a volume ratio of 1:9 (Taq polymerase: lyophilized protective agent = 1:9) to obtain a mixture; the mixture was freeze-dried to obtain a lyophilized Taq polymerase preparation; the phenotype of the lyophilized Taq polymerase preparation in the lyophilized state and the reconstituted state was observed, and the observation results are shown in Figure 1-2 After the freeze-dried Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, the amplification performance of Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1) using Arabidopsis and rice cDNA as samples, respectively. The test results are shown in Table 12;

[0197] Experimental groups 2-5: Based on experimental group 1, the concentration of potassium chloride in the lyophilized protective agent was replaced with 20mM, 30mM, 50mM, and 60mM respectively, and the rest remained unchanged. The test results are shown in Table 12;

[0198] The freeze drying procedure is as follows:

[0199] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0200] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0201] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0202] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0203] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0204] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0205] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0206] Depend on Figure 1-2 As shown in Table 12, the freeze-dried morphology of 20 mM to 40 mM potassium ions is better and the CT value is reduced. It can be seen that 20 mM to 40 mM potassium ions can improve the freeze-dried morphology of Taq polymerase without affecting the amplification performance of Taq polymerase.

[0207] Table 12 Effect of potassium ion concentration on Taq polymerase amplification performance

[0208]

[0209] 9. Effect of dithiothreitol on the stability of lyophilized Taq polymerase preparations

[0210] Experimental group 1: Taq polymerase and the lyophilized protective agent 5 (dithiothreitol concentration 1 mM) of Example 5 were mixed at a volume ratio of 1:9 (Taq polymerase: lyophilized protective agent = 1:9) to obtain a mixture; the mixture was freeze-dried to obtain a Taq polymerase lyophilized preparation; the Taq polymerase lyophilized preparation was stored at 37° C., and the Taq polymerase lyophilized preparation was sampled on the 0th day, the 7th day, the 10th day and the 14th day of storage; after sampling, the Taq polymerase lyophilized preparation was reconstituted to the original volume with enzyme-free water, and mouse and Arabidopsis cDNA were used as samples, respectively, and the amplification performance of Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1), and the detection results are shown in Tables 13 and 14;

[0211] Experimental groups 2-5: Based on experimental group 1, the concentration of dithiothreitol in the lyophilized protective agent was replaced with 0mM, 0.1mM, 0.2mM, and 2mM respectively, and the rest remained unchanged. The test results are shown in Tables 13-14;

[0212] The freeze drying procedure is as follows:

[0213] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0214] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0215] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0216] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0217] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0218] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0219] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0220] It can be seen from Tables 13 and 14 that the CT value of the freeze-dried reagent Taq enzyme that was reconstituted 14 days after adding 0.2-1 mM dithiothreitol did not increase significantly. It can be seen that 0.2-1 mM dithiothreitol can improve the stability of the freeze-dried Taq polymerase.

[0221] Table 13 Effect of dithiothreitol concentration on the stability of Taq polymerase after lyophilization - mouse

[0222] Days of 37℃ pressurization 0 7 10 14 Comparison 0 2.5054 2.0657 2.9933 DTT-0.1mM 0 1.7131 1.6737 2.5269 DTT-0.2mM 0 0.2954 0.3948 0.3885 DTT-1mM 0 0.1914 0.3891 0.3928 DTT-2mM 0 2.1914 1.5891 1.5928

[0223] Table 14 Effect of dithiothreitol concentration on the stability of Taq polymerase after lyophilization - Arabidopsis

[0224] Days of 37℃ pressurization 0 7 10 14 Comparison 0 2.2925 2.5240 2.7955 DTT-0.1mM 0 1.766 1.1690 2.558 DTT-0.2mM 0 0.697 0.6708 0.7545 DTT-1mM 0 0.4131 0.47378 0.5269 DTT-2mM 0 2.3525 2.0227 2.871

[0225] 10. Effect of lyoprotectant formulation on the freeze-dried morphology and amplification performance of Taq polymerase

[0226] The Taq polymerase was mixed with the lyophilized protective agents 1 to 10 of Examples 1 to 10 and the lyophilized protective agents 11 to 15 of Comparative Examples 1 to 5 at a volume ratio of 1:9 (Taq polymerase:lyophilized protective agent=1:9) to obtain a mixture; the mixture was freeze-dried to obtain a lyophilized Taq polymerase preparation; the phenotype of the lyophilized Taq polymerase preparation in the lyophilized state was observed, and the observation results are shown in Figure 3 to Figure 5 After the freeze-dried Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, the amplification performance of Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1) using 293 cells and tomato cDNA as samples, respectively. The test results are shown in Tables 15 to 18;

[0227] The freeze drying procedure is as follows:

[0228] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0229] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0230] The primary sublimation drying stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0231] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0232] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0233] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0234] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0235] Depend on Figure 3 to Figure 5 As shown in Tables 15 to 18, compared with lyophilized protectants 11 to 15, lyophilized protectants 1 to 10 have better lyophilized forms, and the Taq enzyme performance is not significantly different from that of the unlyophilized ones. It can be seen that the lyophilized forms of the Taq polymerase lyophilized preparations prepared by the lyophilized protectants 1 to 10 of Examples 1 to 10 are the best without affecting the amplification performance of the Taq polymerase.

[0236] Table 15 Effects of lyophilized protective agents 1 to 5 on Taq polymerase amplification performance

[0237]

[0238] Table 16 Effect of lyophilized protective agents 6 to 10 on Taq polymerase amplification performance

[0239]

[0240] Table 17 Effects of lyophilized protective agents 11 to 13 on Taq polymerase amplification performance

[0241]

[0242] Table 18 Effect of lyophilized protective agents 14-15 on Taq polymerase amplification performance

[0243]

[0244] Example 11: A lyophilized Taq polymerase preparation and its preparation method

[0245] This embodiment provides a lyophilized preparation of Taq polymerase, wherein the ingredients of the lyophilized preparation of Taq polymerase consist of the lyoprotectant 2 of Example 2 and Taq polymerase.

[0246] The preparation method of the lyophilized Taq polymerase preparation is as follows:

[0247] Mixing Taq polymerase and the lyophilized protective agent 5 of Example 5 at a volume ratio of 1:9 (Taq polymerase:lyophilized protective agent=1:9) to obtain a mixture; freeze-drying the mixture to obtain a lyophilized Taq polymerase preparation;

[0248] The freeze drying procedure is as follows:

[0249] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0250] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0251] The first sublimation drying (vacuuming) stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0252] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0253] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0254] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0255] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0256] Example 12: A lyophilized Taq polymerase preparation and its preparation method

[0257] This example provides a lyophilized Taq polymerase preparation, wherein the lyophilized Taq polymerase preparation is based on Example 6, except that the freeze-drying procedure is replaced by:

[0258] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0259] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0260] The primary sublimation drying (vacuuming) stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 140min;

[0261] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 360min;

[0262] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0263] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0264] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 150min.

[0265] Example 13: A lyophilized Taq polymerase preparation and its preparation method

[0266] This example provides a lyophilized Taq polymerase preparation, wherein the lyophilized Taq polymerase preparation is based on Example 6, except that the freeze-drying procedure is replaced by:

[0267] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0268] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0269] The first sublimation drying (vacuuming) stage: temperature is -30°C, slope temperature is 60min, constant temperature time is 240min;

[0270] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 360min;

[0271] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0272] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0273] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0274] Example 14: A lyophilized Taq polymerase preparation and its preparation method

[0275] This example provides a lyophilized Taq polymerase preparation, wherein the lyophilized Taq polymerase preparation is based on Example 6, except that the freeze-drying procedure is replaced by:

[0276] Pre-freezing stage: temperature is -25℃, slope temperature is 60min, constant temperature time is 60min;

[0277] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 30min;

[0278] The first sublimation drying (vacuuming) stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0279] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0280] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0281] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0282] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0283] Example 15: A lyophilized Taq polymerase preparation and its preparation method

[0284] This example provides a lyophilized Taq polymerase preparation, wherein the lyophilized Taq polymerase preparation is based on Example 6, except that the freeze-drying procedure is replaced by:

[0285] Pre-freezing stage: temperature is -30℃, slope temperature is 60min, constant temperature time is 60min;

[0286] Quick freezing stage: temperature is -45°C, slope temperature is 30min, constant temperature time is 120min;

[0287] The first sublimation drying (vacuuming) stage: temperature is -25°C, slope temperature is 60min, constant temperature time is 180min;

[0288] Secondary sublimation drying stage: temperature is -20°C, slope temperature is 60min, constant temperature time is 480min;

[0289] Primary desorption and drying stage: temperature is 20°C, slope temperature is 120min, constant temperature time is 90min;

[0290] Secondary desorption and drying stage: temperature is 30°C, slope temperature is 30min, constant temperature time is 90min;

[0291] Storage stage: temperature is 4°C, slope temperature is 60min, and constant temperature time is 30min.

[0292] Experimental Example 2: Effect of freeze-drying process on the performance of Taq polymerase

[0293] The experimental process is as follows:

[0294] The phenotypes of the freeze-dried Taq polymerase preparations prepared in Examples 11 to 15 were observed. Figure 6 After the lyophilized Taq polymerase preparation was reconstituted to the original volume with enzyme-free water, rapeseed and Arabidopsis cDNA were used as samples (cDNA was obtained by reverse transcription after RNA was extracted from rapeseed leaf samples and Arabidopsis leaf samples), and the amplification performance of Taq polymerase was detected by qPCR (qPCR primers are shown in Table 1). The test results are shown in Table 19.

[0295] Depend on Figure 6 As shown in Table 19, the freeze-dried form of the Taq polymerase freeze-dried preparation prepared by the freeze-drying process of Example 11 is the best without affecting the amplification performance of the Taq polymerase.

[0296] Table 19 Effect of freeze-drying process on Taq polymerase amplification performance

[0297]

[0298] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A freeze-dried protective agent for DNA polymerase, characterized in that: The freeze-drying protective agent comprises solvent, potassium chloride, ethylenediaminetetraacetic acid, dithiothreitol, Tween-20, trehalose, mannitol, polyethylene glycol and bovine serum albumin.

2. The lyoprotectant according to claim 1, characterized in that The concentration of potassium chloride in the solvent is 20-40 mM; the concentration of ethylenediaminetetraacetic acid in the solvent is 0.05-0.1 mM; the concentration of dithiothreitol in the solvent is 0.2-1 mM; in terms of volume percentage, the concentration of Tween-20 in the solvent is 0.02-0.5%; in terms of mass volume percentage, the concentration of trehalose in the solvent is 5-10%; in terms of mass volume percentage, the concentration of mannitol in the solvent is 0.5-2%; in terms of mass volume percentage, the concentration of polyethylene glycol in the solvent is 0.1-1%; and in terms of mass volume percentage, the concentration of bovine serum albumin in the solvent is 1-4%.

3. The lyoprotectant according to claim 1 or 2, characterized in that The concentration of potassium chloride in the solvent is 40mM; the concentration of ethylenediaminetetraacetic acid in the solvent is 0.1mM; the concentration of dithiothreitol in the solvent is 1mM; in terms of volume percentage, the concentration of Tween-20 in the solvent is 0.5%; in terms of mass volume percentage, the concentration of trehalose in the solvent is 10%; in terms of mass volume percentage, the concentration of mannitol in the solvent is 2%; in terms of mass volume percentage, the concentration of polyethylene glycol in the solvent is 1%; and in terms of mass volume percentage, the concentration of bovine serum albumin in the solvent is 4%.

4. The lyoprotectant according to any one of claims 1 to 3, characterized in that The solvent comprises a buffer; the buffer comprises Tris-HCL buffer, Tris-H2SO4 buffer and / or Tris-acetate buffer.

5. A lyophilized preparation of DNA polymerase, characterized in that: The components of the lyophilized preparation of DNA polymerase include the lyoprotectant according to any one of claims 1 to 4 and DNA polymerase.

6. A method for preparing the lyophilized preparation of DNA polymerase according to claim 5, characterized in that: The method comprises: adding DNA polymerase to the lyophilized protective agent according to any one of claims 1 to 4 to obtain a mixture; and freeze-drying the mixture to obtain the lyophilized preparation of DNA polymerase according to claim 5.

7. A kit, characterized in that: The components of the kit include the lyophilized preparation of DNA polymerase according to claim 5.

8. A method for nucleic acid amplification, characterized in that: The method is not for the purpose of disease diagnosis and treatment, and the method uses the lyophilized preparation of DNA polymerase according to claim 5 or the kit according to claim 7 to amplify the target nucleic acid.

9. A molecular detection method, characterized in that The method is not for the purpose of disease diagnosis and treatment, and uses the lyophilized preparation of DNA polymerase according to claim 5 or the kit according to claim 7 to perform molecular detection of the target nucleic acid.

10. Use of the lyoprotectant according to any one of claims 1 to 4, the lyophilized preparation of DNA polymerase according to claim 5, or the kit according to claim 7 in nucleic acid amplification or molecular detection, characterized in that: The application is not for the purpose of diagnosis and treatment of diseases.

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