Freeze-drying protective agent, PCR amplification reagent freeze-drying cake and preparation method of PCR amplification reagent freeze-drying cake

Abstract

The invention discloses a freeze-drying protective agent, a PCR (Polymerase Chain Reaction) amplification reagent freeze-dried cake and a preparation method of the PCR amplification reagent freeze-dried cake. The freeze-drying protective agent comprises beta-cyclodextrin, sorbitol, glutamic acid, BSA (Bovine Serum Albumin) and TritonX-100. The freeze-drying protective agent disclosed by the invention can improve the stability of the PCR amplification reagent, is beneficial to improving the liquid dispensing success rate, stabilizes the form of a freeze-dried cake of the PCR amplification reagent, and ensures the forming rate and yield; the freeze-drying protective agent provided by the invention is a PCR amplification reagent system and has no inhibition effect on amplification reaction. The freeze-drying protective agent provided by the invention can be used for preparing a freeze-dried cake suitable for the shape of a molecular POC card box amplification reaction chamber, can be used in card boxes or channels of various narrow reaction areas, and can also realize multi-channel detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology diagnosis and detection, and more specifically, the present invention relates to a freeze-dried protective agent, a PCR amplification reagent freeze-dried cake and a preparation method thereof. Background Art

[0002] The DNA polymerase, reverse transcriptase, nucleic acid, etc. contained in PCR amplification reagents are mostly very sensitive to temperature and easily inactivated. Their biological activity is relatively active and they need to be stored at low temperatures to prevent their activity from decreasing. Therefore, the preservation of PCR amplification reagents is a major problem in the in vitro diagnostic industry. They need to be stored at low temperatures, otherwise their activity will quickly decrease and the reagents will become ineffective.

[0003] With the development of biotechnology, many PCR amplification reagents are dehydrated by freeze-drying technology in order to be stored at room temperature. Freeze-drying technology is to freeze the PCR amplification reagents in liquid form under low temperature conditions and then sublimate and dehydrate them, turning unstable DNA polymerase, reverse transcriptase, and nucleic acid substances into a stable solid form. Due to the low water content of the solid form, the activity of the PCR amplification reagent can be maintained to the maximum extent, achieving the effect of long-term stable storage at room temperature. However, some components of the freeze-dried protective agent may inhibit the amplification reaction of the PCR amplification reagent, resulting in target missed detection.

[0004] In addition, for the freeze-drying of PCR amplification reagents (lyophilized cakes) in molecular POC cartridges with small amplification reaction chambers and irregular flat and shaped chambers, it is not easy to form a complete liquid surface tension on the lyophilized cake mold, and point-liquid molding is difficult. In addition, the lyophilized cakes are very thin and brittle and are easily broken during demolding. Therefore, the yield of the lyophilized cakes is also low. Summary of the invention

[0005] Based on this, the object of the present invention is to provide a lyophilized protective agent, a PCR amplification reagent lyophilized cake and a preparation method thereof. The lyophilized protective agent of the present invention can be used to prepare a PCR amplification reagent lyophilized cake for the reaction chamber of the aptamer POC cartridge, with a high molding yield and good lyophilized cake amplification performance.

[0006] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] In a first aspect of the present invention, a lyoprotectant is provided, which comprises 5wt% to 15wt% of β-cyclodextrin, 2.0wt% to 6.0wt% of sorbitol, 1.0wt% to 3.0wt% of glutamic acid, 0.05wt% to 0.15wt% of BSA and 0.5wt% to 1.5wt% of Triton X-100.

[0008] The second aspect of the present invention provides the use of the above-mentioned lyoprotectant in preparing a lyophilized cake of a PCR amplification reagent.

[0009] The third aspect of the present invention provides a PCR amplification reagent freeze-dried cake, which is obtained by mixing a PCR amplification reagent premix with the above-mentioned freeze-dried protective agent at a volume ratio of 1:1.8-2.2 and then freeze-drying.

[0010] A fourth aspect of the present invention provides a method for preparing a PCR amplification reagent freeze-dried cake, comprising the following steps:

[0011] (1) Mixing the PCR amplification reagent premix with the above-mentioned lyophilized protective agent at a volume ratio of 1:1.8 to 2.2 to obtain a mixed solution;

[0012] (2) applying the mixed solution to a freeze-dried cake mold;

[0013] (3) Pre-freeze the freeze-dried cake mold, freeze-dry it in a freeze dryer, and tap the freeze-dried cake mold to obtain it.

[0014] The inventors of the present invention conducted extensive research on freeze-dried cakes of PCR amplification reagents and found that a freeze-dried cake of PCR amplification reagents prepared by organically combining five ingredients, namely, β-cyclodextrin, sorbitol, glutamic acid, BSA and TritonX-100, at a certain concentration as a freeze-dried cake protectant has a high molding rate and yield, and the PCR amplification reagent has high stability and the amplification performance is not affected.

[0015] The present invention provides a way to match the special-shaped chambers of different molecular POC cartridges currently on the market with PCR freeze-dried reagents. By preparing a hole mold that is adapted to the special-shaped chamber of the POC cartridge and then freeze-drying the PCR amplification reagent, the yield rate in the production process of the molecular POC cartridge is improved.

[0016] The freeze-dried cake preparation process of the present invention is simple, the freeze-drying efficiency is effectively improved, and it is conducive to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the structure of the freeze-dried cake mold (circular) in the present invention.

[0018] Figure 2 for Figure 1 A partial enlarged view of the freeze-dried cake mold.

[0019] Figure 3 It is a schematic diagram of the structure of the freeze-dried cake mold (square) in the present invention.

[0020] Figure 4 for Figure 3 A partial enlarged view of the freeze-dried cake mold.

[0021] Figure 5 This is the amplification curve of each PCR amplification reagent freeze-dried cake in Experimental Example 2 of the present invention.

[0022] Figure 6 This is the amplification curve of the in situ freeze-dried powder and freeze-dried cake for influenza A virus nucleic acid samples in Experimental Example 3 of the present invention.

[0023] Figure 7 This is the amplification curve of the in situ freeze-dried powder and freeze-dried cake for the syncytial virus nucleic acid sample in Experimental Example 3 of the present invention.

[0024] Figure 8 These are the amplification curves of the lyophilized cakes in Experimental Example 3 of the present invention at the 0th day and the 3rd month of acceleration. DETAILED DESCRIPTION

[0025] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0026] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0027] Unless otherwise specified, the examples are all based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions. The raw materials and reagents used in the present invention are all commercially available, and any biological germplasm material can be provided to the outside for scientific research.

[0028] In some embodiments of the present invention, a lyoprotectant for a lyophilized cake of a PCR amplification reagent is disclosed, which includes 5wt% to 15wt% of β-cyclodextrin, 2.0wt% to 6.0wt% of sorbitol, 1.0wt% to 3.0wt% of glutamic acid, 0.05wt% to 0.15wt% of BSA and 0.5wt% to 1.5wt% of Triton X-100.

[0029] When preparing the freeze-dried cake of PCR amplification reagent, a freeze-dried protective agent needs to be added for protection. However, on the one hand, due to the large variety of freeze-dried protective agents and the large total specific gravity, it is not easy to form a complete liquid surface tension on the freeze-dried cake mold, that is, it is difficult to form the freeze-dried cake mold with liquid, and when the freeze-dried cake is prepared, it is easy to break when demolding because the freeze-dried cake is brittle; on the other hand, some components of the freeze-dried protective agent may inhibit the amplification reaction of the PCR amplification reagent, thereby causing the target to be missed. The inventors of the present invention have found that when β-cyclodextrin, sorbitol, glutamic acid, BSA and Triton X-100 are mixed at a certain concentration and used as a freeze-dried protective agent to prepare the freeze-dried cake of PCR amplification reagent, not only the yield of the freeze-dried cake of the PCR amplification reagent can be guaranteed, but also the amplification performance of the PCR amplification reagent will not be affected.

[0030] By using the freeze-dried protective agent of the present invention, after freeze-drying the PCR amplification reagent premix, a freeze-dried cake suitable for the shape of the molecular POC cartridge amplification reaction chamber can be prepared, which has a stable structure, is easy to clamp, and is small in size and convenient to store; since there is no interference from other freeze-dried carrier materials, the cartridge amplification reaction chamber can have the same background fluorescence background and PCR reaction efficiency as the fluorescent PCR instrument, so it can be well matched with the rapid detection cartridge to give full play to the performance of the PCR amplification reagent in the cartridge, so that it can be used in cartridges or channels in various narrow reaction areas, and multi-channel detection can also be achieved. The freeze-dried cake prepared by the present invention has a size slightly smaller than the PCR chamber, and will not shake excessively in the PCR chamber. Since the thickness of the freeze-dried cake is lower than that of the PCR chamber, it can be placed in the PCR chamber without affecting the assembly of the PP film (polypropylene, used to encapsulate the amplification reaction chamber). When the sample to be tested enters the amplification reaction chamber, the sample to be tested can fill the amplification reaction chamber well, and the freeze-dried cake can be quickly redissolved from all sides; when the PCR amplification reaction is performed, the sample to be tested in the amplification reaction chamber and the redissolved PCR amplification reagent can be fully mixed by thermal drive, so as to achieve the purpose of simplifying the process.

[0031] In some embodiments, the lyoprotectant includes 8wt% to 10wt% of β-cyclodextrin, 2.5wt% to 3.5wt% of sorbitol, 1.8wt% to 2.2wt% of glutamic acid, 0.08wt% to 0.12wt% of BSA and 0.7wt% to 0.9wt% of Triton X-100.

[0032] In other embodiments of the present invention, the use of the above-mentioned lyoprotectant in preparing lyophilized cakes of PCR amplification reagents is disclosed.

[0033] In other embodiments of the present invention, a PCR amplification reagent freeze-dried cake is disclosed, which is obtained by mixing a PCR amplification reagent premix with the above-mentioned freeze-dried protective agent in a volume ratio of 1:1.8-2.2 and then freeze-drying.

[0034] In some embodiments, the PCR amplification reagent premix includes RT enzyme, Taq enzyme, RNase inhibitor, dNTP, buffer and primer probe; and / or, in the PCR amplification reagent premix, the concentration of RT enzyme is 1U / μL~5U / μL, the concentration of Taq enzyme is 0.1U / μL~1U / μL, the concentration of RNase inhibitor is 1U / μL~5U / μL, the concentration of dNTP is 100nm~300nm, and the concentration of primer probe is 100nm~900nm respectively.

[0035] In other embodiments of the present invention, a method for preparing a PCR amplification reagent freeze-dried cake is disclosed, comprising the following steps:

[0036] (1) Mixing the PCR amplification reagent premix with the lyophilized protective agent in a volume ratio of 1:1.8 to 2.2 to obtain a mixed solution;

[0037] (2) applying the mixed solution to a freeze-dried cake mold;

[0038] (3) Pre-freeze the freeze-dried cake mold, freeze-dry it in a freeze dryer, and tap the freeze-dried cake mold to obtain it.

[0039] In some embodiments, the pre-freezing in step (3) includes: maintaining at -20°C to -30°C for 1.5h to 2.5h, and then heating to -40°C to -50°C and maintaining for 0.5h to 1.5h.

[0040] In some embodiments, the freeze-drying procedure of the freeze dryer in step (3) includes: vacuuming at -40°C to -50°C and 0.133 mbar±0.01 mbar for 8 min to 12 min; main drying at -30°C to -40°C and 0.133 mbar±0.01 mbar for 7.5 h to 8.5 h; final drying at 20°C to 30°C and 0.133 mbar±0.01 mbar for 7.5 h to 8.5 h.

[0041] In some embodiments, the freeze-dried cake mold in step (2) is pre-ultrasonic cleaned and dried; and / or the frequency of the ultrasound is 350W to 450W, and the ultrasound time is 25min to 35min; the drying temperature is 45°C to 55°C, and the drying time is 1.5h to 2.5h.

[0042] In some embodiments, the freeze-dried cake mold in step (2) is round or square.

[0043] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] Example 1 Lyophilized Protectant, PCR Amplification Reagent Lyophilized Cake and Preparation Method Thereof

[0045] The freeze-dried cake of the PCR amplification reagent of this embodiment comprises: 9wt% of β-cyclodextrin, 3wt% of sorbitol, 2wt% of glutamic acid, 0.1wt% of BSA, and 0.8wt% of TritonX-100. β-cyclodextrin, sorbitol, glutamic acid, BSA and TritonX-100 are dissolved in water to obtain the freeze-dried cake.

[0046] Using the lyophilized protective agent of this embodiment, preparing a PCR amplification reagent lyophilized cake comprises the following steps:

[0047] 1. Clean the freeze-dried cake mold (such as Figure 1 to Figure 4 Then, the mold was dried at 50°C for 2 hours;

[0048] 2. Mix the PCR amplification reagent premix with the lyophilized protective agent in a volume ratio of 1:2 to obtain a mixed solution;

[0049] 3. Use a pipette or electric dispenser to apply the above mixed solution on the freeze-dried cake mold (e.g., the diameter of the circular hole mold is 4 mm, the thickness is 1.4 mm, and the solution is 15 μL). The mixed solution maintains a fixed shape through the tension between liquid molecules;

[0050] 4. Pre-freeze using liquid nitrogen or an ultra-low temperature refrigerator, and transfer to a freeze dryer as soon as possible after pre-freezing (the freeze dryer plate layer must be pre-frozen to below -30°C in advance) and run the freeze-drying program to obtain a freeze-dried cake. The pre-freezing and freeze-drying procedures are shown in Table 1.

[0051] Table 1

[0052] step Set temperature(℃) Set vacuum degree (mbar) Setting time (min) Duration (h) Pre-freeze -25 / 30 2 Pre-freeze -45 / 20 1 Vacuum -45 0.133 10 0 dry -35 0.133 10 6 dry 25 0.133 60 7

[0053] 5. Gently tap the freeze-drying mold and the freeze-dried cake will fall off the mold.

[0054] The freeze-dried cake prepared in this embodiment has a stable structure, is easy to clamp, and is small in size and convenient to store; freeze-dried cakes of different shapes can be assembled by matching the cartridge chamber.

[0055] Example 2 Lyophilized Protectant, PCR Amplification Reagent Lyophilized Cake and Preparation Method Thereof

[0056] The lyophilization protectant of the PCR amplification reagent lyophilized cake of this embodiment includes: 8 wt % of β-cyclodextrin, 3.5 wt % of sorbitol, 1.8 wt % of glutamic acid, 0.12 wt % of BSA, and 0.7 wt % of Triton X-100.

[0057] The freeze-dried cake of PCR amplification reagent was prepared using the freeze-dried protective agent of this example, using the same method as in Example 1.

[0058] Example 3 Lyophilized Protectant, PCR Amplification Reagent Lyophilized Cake and Preparation Method Thereof

[0059] The lyophilization protective agent of the PCR amplification reagent freeze-dried cake of this embodiment includes: 10wt% of β-cyclodextrin, 2.5wt% of sorbitol, 2.2wt% of glutamic acid, 0.08wt% of BSA, and 0.9wt% of TritonX-100.

[0060] The freeze-dried cake of PCR amplification reagent was prepared using the freeze-dried protective agent of this example, using the same method as in Example 1.

[0061] Comparative Example 1

[0062] The lyophilized protective agent of the PCR amplification reagent in this comparative example includes: 9wt% of β-cyclodextrin, 2wt% of glutamic acid, and 0.1wt% of BSA.

[0063] The freeze-dried cake of PCR amplification reagent was prepared by using the freeze-dried protective agent of this comparative example, using the same method as in Example 1.

[0064] Comparative Example 2

[0065] The lyophilized protective agent of the PCR amplification reagent in this comparative example includes: 9wt% of β-cyclodextrin, 2wt% of glutamic acid, 0.1wt% of BSA, and 3wt% of sorbitol.

[0066] The freeze-dried cake of PCR amplification reagent was prepared by using the freeze-dried protective agent of this comparative example, using the same method as in Example 1.

[0067] Comparative Example 3

[0068] The lyophilized protective agent of the PCR amplification reagent of this comparative example comprises: 9wt% of β-cyclodextrin, 2wt% of glutamic acid, 0.1wt% of BSA, 6wt% of sucrose, and 1.2wt% of TritonX-100.

[0069] The freeze-dried cake of PCR amplification reagent was prepared by using the freeze-dried protective agent of this comparative example, using the same method as in Example 1.

[0070] Comparative Example 4

[0071] The lyophilized protective agent of the PCR amplification reagent of this comparative example comprises: 9wt% of β-cyclodextrin, 2wt% of glutamic acid, 0.1wt% of BSA, 6wt% of cellobiose, and 1.2wt% of Triton X-100.

[0072] The freeze-dried cake of PCR amplification reagent was prepared by using the freeze-dried protective agent of this comparative example, using the same method as in Example 1.

[0073] Comparative Example 5

[0074] The lyophilized protective agent of the PCR amplification reagent of this comparative example comprises: 9 wt % of cellobiose, 2 wt % of glutamic acid, 0.1 wt % of BSA, 3 wt % of sorbitol, and 0.8 wt % of glycerol.

[0075] The freeze-dried cake of PCR amplification reagent was prepared by using the freeze-dried protective agent of this comparative example, using the same method as in Example 1.

[0076] Comparative Example 6

[0077] The lyophilized protective agent of the PCR amplification reagent of this comparative example comprises: 9wt% of sucrose, 2wt% of glutamic acid, 0.1wt% of BSA, 3wt% of sorbitol, and 0.8wt% of polyvinyl alcohol.

[0078] The freeze-dried cake of PCR amplification reagent was prepared using the freeze-dried protective agent of this comparative example, and the method was the same as that of Example 1. Test Example 1 Yield of freeze-dried cake of PCR amplification reagent prepared using different freeze-dried protective agents

[0079] The yields of the PCR amplification reagent freeze-dried cakes prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were statistically analyzed, and the results are shown in Table 2.

[0080] Table 2

[0081] No. Yield Example 1 100％(50 / 50) Example 2 100％(50 / 50) Example 3 100％(50 / 50) Comparative Example 1 72％(36 / 50) Comparative Example 2 86％(43 / 50) Comparative Example 3 90％(45 / 50) Comparative Example 4 76％(38 / 50) Comparative Example 5 80％(40 / 50) Comparative Example 6 70％(35 / 50)

[0082] From the results in Table 2, it can be seen that using β-cyclodextrin, sorbitol, glutamic acid, BSA, and TritonX-100 as raw materials as freeze-dried protective agents for PCR amplification reagent freeze-dried cakes can form a complete liquid surface tension, so that the freeze-dried cakes can be formed by spotting liquid on the freeze-dried cake mold, so that the freeze-dried cakes have a complete and stable shape, and the freeze-dried cakes are not easy to break when they fall off the freeze-dried mold. The yield of the prepared freeze-dried cakes can reach 100% (Examples 1 to 3). When β-cyclodextrin, glutamic acid and BSA are used as freeze-drying protective agents (Comparative Example 1); β-cyclodextrin, glutamic acid, BSA and Triton X-100 are used as freeze-drying protective agents (Comparative Example 2); β-cyclodextrin, sucrose / cellobiose, glutamic acid, BSA and Triton X-100 are used as freeze-drying protective agents (Comparative Examples 3 and 4); sucrose / cellobiose, sorbitol, glutamic acid, BSA and glycerol / polyvinyl alcohol are used as freeze-drying protective agents (Comparative Examples 5 and 6), the yield of the PCR amplification reagent freeze-dried cake is greatly reduced (Comparative Examples 1 to 6). Experimental Example 2 Amplification performance of PCR amplification reagent freeze-dried cakes prepared using different freeze-drying protective agents

[0083] Using the PCR amplification reagent freeze-dried cakes prepared in Examples 1 to 3 and Comparative Examples 1 to 6 (the same batch of PCR amplification reagent premixes), 600 copies / mL of influenza A virus, influenza B virus and syncytial virus nucleic acid throat swab samples were subjected to PCR amplification, and the amplification curves were detected. The amplification procedures are shown in Table 3. The results are shown in Table 3. Figure 5 shown.

[0084] Table 3

[0085]

[0086] from Figure 5 It can be seen from each amplification curve that when the lyophilized cakes of PCR amplification reagents prepared by the lyophilized protective agents of Examples 1 to 3 are used for amplification, they all have good amplification performance, and when the lyophilized cakes of PCR amplification reagents prepared by the lyophilized protective agents of Comparative Example 4 are used for amplification, they also have good amplification performance; while when the lyophilized cakes of PCR amplification reagents prepared by the lyophilized protective agents of Comparative Examples 1 to 3 and Comparative Examples 5 to 6 are used for amplification, there is a phenomenon of target missed detection, and the amplification performance is reduced. Therefore, using β-cyclodextrin, sorbitol, glutamic acid, BSA, and Triton X-100 as raw materials as lyophilized protective agents for PCR amplification reagent lyophilized cakes will not affect the amplification performance of the PCR amplification reagent lyophilized cakes while ensuring the molding yield.

[0087] Experimental Example 3 Study on the transfer performance of PCR amplification reagent freeze-dried cakes prepared using different freeze-dried protective agents

[0088] This example compares the conversion performance of in-situ freeze-dried powder (i.e., eight-tube freeze-dried, PCR amplification reagent premix and freeze-dried protective agent are the same as in Example 1, and the freeze-drying procedure is also the same as in Example 1) and PCR amplification reagent freeze-dried cake (Example 1). The method is as follows:

[0089] 1. Amplification performance

[0090] The lyophilized cake was divided into eight tubes (the same batch as the in situ lyophilized powder), and nucleic acid samples (1000 copies / mL influenza A virus, 1000 copies / mL syncytial virus) were added for PCR amplification. At the same time, nucleic acid samples (1000 copies / mL influenza A virus, 1000 copies / mL syncytial virus) were added to the in situ lyophilized powder for PCR amplification. The amplification procedure is shown in Table 2, and the amplification results are shown in Tables 4 and Figure 6-7 .

[0091] Table 4

[0092]

[0093]

[0094] from Figure 6-7 As shown in the results of Table 4, there is no significant difference in the performance between the freeze-dried cake and the in-situ freeze-dried powder, indicating that the process for preparing the freeze-dried cake of the present invention meets the requirements of stable conversion performance.

[0095] 2. 50℃ Accelerated Aging Performance

[0096] The lyophilized cake was divided into eight tubes (the same batch of in situ lyophilized powder), and nucleic acid samples (600 copies / mL influenza A virus, 600 copies / mL influenza B virus, 600 copies / mL syncytial virus, and 600 copies / mL internal reference RNaseP nucleic acid sample) were added. The PCR amplification performance was compared at 50°C on the 0th day and the 3rd month of acceleration. The amplification program is shown in Table 2, and the amplification results are shown in Tables 5 and Figure 8 .

[0097] Table 5

[0098]

[0099] Figure 8 The results in Table 5 show that the performance of the freeze-dried cake did not change significantly after being accelerated at 50°C for 3 months, indicating that the process for preparing the freeze-dried cake of the present invention meets the requirements of stable conversion performance.

[0100] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0101] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A freeze-drying protective agent, characterized in that The invention comprises 5wt% to 15wt% of beta-cyclodextrin, 2.0wt% to 6.0wt% of sorbitol, 1.0wt% to 3.0wt% of glutamic acid, 0.05wt% to 0.15wt% of BSA and 0.5wt% to 1.5wt% of Triton X-100.

2. The lyoprotectant according to claim 1, characterized in that The invention comprises 8wt% to 10wt% of beta-cyclodextrin, 2.5wt% to 3.5wt% of sorbitol, 1.8wt% to 2.2wt% of glutamic acid, 0.08wt% to 0.12wt% of BSA and 0.7wt% to 0.9wt% of Triton X-100.

3. Use of the lyoprotectant according to claim 1 or 2 in preparing a lyophilized cake of a PCR amplification reagent.

4. A PCR amplification reagent freeze-dried cake, characterized in that: The PCR amplification reagent premix is ​​mixed with the freeze-drying protective agent according to claim 1 or 2 in a volume ratio of 1:1.8-2.2, and then freeze-dried.

5. The PCR amplification reagent freeze-dried cake according to claim 1, characterized in that: The PCR amplification reagent premix includes RT enzyme, Taq enzyme, RNase inhibitor, dNTP, buffer and primer probe.

6. A method for preparing a PCR amplification reagent freeze-dried cake, characterized in that: The following steps are involved: (1) Mixing a PCR amplification reagent premix with the lyophilized protective agent according to claim 1 or 2 at a volume ratio of 1:1.8 to 2.2 to obtain a mixed solution; (2) applying the mixed solution to a freeze-dried cake mold; (3) Pre-freeze the freeze-dried cake mold, freeze-dry it in a freeze dryer, and tap the freeze-dried cake mold to obtain it.

7. The method for preparing the PCR amplification reagent freeze-dried cake according to claim 6, characterized in that: The pre-freezing in step (3) includes: maintaining at -20°C to -30°C for 1.5h to 2.5h, and then heating to -40°C to -50°C and maintaining for 0.5h to 1.5h.

8. The method for preparing the PCR amplification reagent freeze-dried cake according to claim 6, characterized in that: The freeze-drying procedure of the freeze dryer in step (3) includes: vacuuming at -40°C to -50°C and 0.133 mbar±0.01 mbar for 8min to 12min; main drying at -30°C to -40°C and 0.133 mbar±0.01 mbar for 7.5h to 8.5h; final drying at 20°C to 30°C and 0.133 mbar±0.01 mbar for 7.5h to 8.5h.

9. The method for preparing the PCR amplification reagent freeze-dried cake according to claim 8, characterized in that: The freeze-dried cake mold in step (2) is pre-ultrasonic cleaned and dried; the frequency of the ultrasound is 350W to 450W, and the time of the ultrasound is 25min to 35min; the temperature of the drying is 45°C to 55°C, and the time of the drying is 1.5h to 2.5h.

10. The method for preparing the PCR amplification reagent freeze-dried cake according to claim 8, characterized in that: The freeze-dried cake mold in step (2) is round or square.

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