Freeze-drying protectants and their use in the preparation of PCR freeze-dried reagents

Abstract

The present application relates to the field of molecular biology technology, and more particularly to a freeze-drying protective agent and its application in preparing PCR freeze-drying reagents, and the freeze-drying protective agent provided by the present application contains a combination of a sugar and tert-butyl alcohol, which can reduce the water content of the PCR freeze-drying reagent in a common low-humidity environment and improve the moisture resistance of the PCR freeze-drying reagent. The freeze-drying microspheres obtained by the preparation method of the present application have high hardness and are not easy to fall off, which is conducive to manual and automatic dispensing.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a freeze-drying protective agent and its application in preparing PCR freeze-drying reagents. BACKGROUND

[0002] Most of the molecular diagnostic reagents are liquid reagents at present, which have the advantages of high sensitivity and simultaneous detection of multiple targets. However, the enzymes and other substances in the liquid reagents need to be stored at -20℃, and need to be repeatedly frozen and thawed during use, which affects the stability of the reagents. In order to maintain the good performance standard of the reagents and get rid of the cold chain transportation, and reduce the cost, researchers have proposed freeze-drying technology. The freeze-drying technology can now enable the reagents to be stored in solid form at room temperature. Specifically, it is a new and efficient drying technology that the frozen solid ice in the mixture is directly sublimated into gas without being melted into liquid water, and finally the water is removed and the other effective components are retained.

[0003] The freeze-drying technology is widely used in molecular diagnostic reagents. There are in-situ freeze-dried reagents, that is, the PCR reagents added with freeze-drying protective agents are divided into eight rows of tubes, and then put into a freeze-dryer for freeze-drying. The in-situ freeze-dried reagents have the advantages of accurate metering, 4-degree transportation, and easy storage. However, due to the influence of the tube material, the shape is irregular, and only small batches can be produced. It needs to be pre-frozen for 3-4 hours, the freeze-drying process takes a long time, and the experimental operation is difficult to transfer. Another kind is non-in-situ freeze-dried reagents, that is, the PCR reagent is pre-frozen into a ball by liquid nitrogen, and then transferred to a Schlenk bottle, and then transferred to a vacuum drying box for freeze-drying. After the freeze-drying is completed, the freeze-dryer plate layer is lifted to press the cover, and the preliminary sealing is completed. Then, it is transferred to a low-humidity environment for secondary sealing. Because the water content of the freeze-dried product is generally within 3% to 5%, the extremely low water content and the sponge-like structure of the freeze-dried product make it particularly easy to absorb moisture during the unloading process. After absorbing moisture, it has a great influence on the stability and shelf life of the product.

[0004] Common freeze-drying protective agents include combinations of sugars, polymers, BSA, etc. It is very important to select a freeze-drying protective agent that is not easy to make the freeze-dried product absorb moisture for the stability of the product quality in the later stage. Generally, protective agents that can significantly increase the glass transition temperature of the product are used to improve the stability of the freeze-dried product, so that the freeze-dried product is not easy to absorb moisture. However, it is found in actual application that when these freeze-drying agent combinations are used, the freeze-dried product is still easy to absorb moisture and unstable in a conventional RH30% relative humidity environment. At the same time, if the amount of addition is too large, it will cause the freeze-dried product to be poor in strength, easy to crumble during the later dispensing process, and then cause the reagent amount to be reduced and the performance to be unstable. Therefore, at present, it is still necessary to develop a freeze-drying protective agent combination that is not easy to make the freeze-dried product absorb moisture, not easy to crumble, and has high stability. SUMMARY

[0005] Therefore, the present application aims to provide a freeze-drying protective agent and application thereof in preparation of PCR freeze-drying reagents.

[0006] The present application provides a freeze-drying protective agent combination, which comprises lactose, inulin, mannitol, PEG20000, PVP, hydrolyzed gelatin, tert-butyl alcohol, dodecyl polyethylene glycol ether and defoaming agent.

[0007] The mass ratio of each component is (0.03-0.25):(0.02-0.07):(0.01-0.15):(0.005-0.05):(0.002-0.05):(0.001-0.01):(0.001-0.02):(0.001-0.01):(0.001-0.005).

[0008] Specifically, in some embodiments, the mass ratio of lactose, inulin, mannitol, PEG20000, PVP, hydrolyzed gelatin, tert-butyl alcohol, dodecyl polyethylene glycol ether and defoaming agent is 0.03:0.03:0.02:0.01:0.005:0.005:0.005:0.005:0.002.

[0009] In some specific embodiments, the defoaming agent is SE-15.

[0010] The tert-butyl alcohol in the freeze-drying protective agent combination of the present application can be arbitrarily mixed with water, and forms needle-like structure in freezing, and loose skeleton structure and tubular channel after sublimation, which can make the freeze-dried microspheres maintain good appearance and not easy to fall off, and reduce the water content. In specific embodiments of the present application, the freeze-dried microspheres added with the freeze-drying protective agent combination of the present application have less water content, no change in size and performance, and no falling off phenomenon after dispensing after exposure in ordinary low-humidity environment (relative humidity 30%) for different time.

[0011] The present application provides application of the freeze-drying protective agent in preparation of PCR freeze-drying reagents.

[0012] The present application provides a PCR freeze-drying reagent, which comprises PCR amplification reagent and the freeze-drying protective agent of the present application.

[0013] Further, the concentration of each component in the PCR amplification reagent is as follows:

[0014] 20mmol / L~200mmol / L buffer system, 10mmol / L~100mmol / L cation, 30μmol / L~400μmol / L deoxyribonucleotide, 0.5U / μL~10U / μL DNA polymerase or isothermal amplification enzyme, 0.5U / μL~5U / μL reverse transcriptase, 0.3μmol / L~1.0μmol / L upstream and downstream primers and 0.15μmol / L~0.5μmol / L probe;

[0015] Preferably, the concentration of the buffer system is 100mmol / L, the concentration of the cation is 50mmol / L, the concentration of the deoxyribonucleotide is 200μmol / L, the concentration of the DNA polymerase or isothermal amplification enzyme is 1U / μL, the concentration of the reverse transcriptase is 1U / μL, the concentration of the upstream and downstream primers is 0.8μmol / L, and the concentration of the probe is 0.4μmol / L.

[0016] Further, the mass fraction of the freeze-drying protective agent in the PCR freeze-dried reagent is 10wt%~30wt%;

[0017] Preferably, the mass fraction of the freeze-drying protective agent is 11.2wt%.

[0018] The PCR freeze-dried reagent provided by the application comprises at least one of Tris-HCl, HEPES and Tricine as the buffer system.

[0019] The cation comprises at least one of K + , Mg 2+ and Mn 2+ .

[0020] The DNA polymerase or isothermal amplification enzyme comprises at least one of Taq polymerase, Pfu polymerase, KOD polymerase, Bst polymerase and Phi29 polymerase.

[0021] The reverse transcriptase comprises at least one of MMLV reverse transcriptase and T7 reverse transcriptase.

[0022] Specifically, in the embodiments of the application, the components in the PCR freeze-dried reagent comprise:

[0023] Tricine 100mmol / L, Mg 2+ 50mmol / L, dNTP 200μmol / L, Taq polymerase 1U / μL, MMLV reverse transcriptase 1U / μL, upstream primer 0.4μmol / L, downstream primer 0.4μmol / L, probe 0.2μmol / L and freeze-drying protective agent 11.2wt%.

[0024] The application further provides a preparation method of the PCR freeze-dried reagent, comprising pre-freezing the prepared PCR reagent by liquid nitrogen, and then performing vacuum freeze-drying to obtain the PCR freeze-dried reagent.

[0025] Specifically, in the embodiment of the application, the specific steps of the liquid nitrogen pre-freezing are as follows: the prepared PCR reagent is filled into a point bead machine, and then is dripped into liquid nitrogen after adjusting the dispensing volume and dispensing rate; the pre-frozen microspheres are quickly transferred to a pre-frozen Schlenk flask, and then are transferred to a pre-frozen vacuum freeze-drying machine after half-capping; during the transferring process, the freeze-dried microspheres are ensured to be always in the liquid nitrogen; the liquid nitrogen pre-freezing is mainly to prevent the freeze-dried microspheres from melting into liquid at normal temperature.

[0026] The freeze-drying includes two steps of sublimation drying and resolution drying, and specifically is as follows:

[0027] (1) Sublimation drying: the first stage is set to a pre-freezing temperature of-60±2℃, and the duration is 120-360 min; the second stage is set to a temperature rising of 0.5±0.1℃ / min, and the temperature is raised to-40±2℃; after reaching the temperature, the third stage is automatically entered; the third stage is set to a temperature of-40±2℃, and the duration is 720-1000 min.

[0028] (2) Resolution drying: the first stage is set to a temperature rising of 0.5±0.1℃ / min, and the temperature is raised to 40±2℃; after reaching the temperature, the second stage is automatically entered; the second stage is set to a temperature of 40±2℃, and the duration is 120-600 min.

[0029] The PCR freeze-dried reagent obtained by the preparation method has the advantages of not needing to be re-prepared during use, simple operation, and being capable of being used for multiple detection technology platforms and having good compatibility.

[0030] The application has the following beneficial effects:

[0031] 1. The freeze-dried microspheres prepared by adding the freeze-dried protective agent combination to the PCR reagent have a significantly reduced water absorption in the environment, and have good moisture resistance; in a normal low-humidity environment (relative humidity of 30%), the water content of the freeze-dried microspheres almost does not change within 1 hour, and the performance detection is qualified.

[0032] 2. The freeze-dried microspheres prepared by adding the freeze-dried protective agent combination can be subjected to warehouse-out, sub-packaging and other operations in a low-humidity environment, and the water content and activity of the freeze-dried microspheres are not affected, so it is not necessary to strictly control the extremely low humidity in the workshop, and the energy consumption is greatly reduced.

[0033] 3. After adding the freeze-dried protective agent combination, the freeze-dried microspheres have high hardness and are not easy to crumble, and are more suitable for manual or automatic dispensing. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Figure 1 shows the moisture resistance detection diagram of freeze-dried microspheres, 1 is freeze-dried microspheres added with a combination of two sugar freeze-drying protectants, 2, 3 and 4 are freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%, 1% and 2%);

[0035] Figure 2 Figure 1 shows the moisture resistance detection diagram of freeze-dried microspheres, 1 is freeze-dried microspheres added with a combination of two sugar freeze-drying protectants, 2, 3 and 4 are freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%, 1% and 2%);

[0036] Figure 3 Figure 1 shows the moisture resistance detection diagram of freeze-dried microspheres, 1 is freeze-dried microspheres added with a combination of two sugar freeze-drying protectants, 2, 3 and 4 are freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%, 1% and 2%);

[0037] Figure 4 Figure 1 shows the moisture resistance detection diagram of freeze-dried microspheres, 1 is freeze-dried microspheres added with a combination of two sugar freeze-drying protectants, 2, 3 and 4 are freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%, 1% and 2%);

[0038] Figure 5 Figure 1 shows the moisture resistance detection diagram of freeze-dried microspheres, 1 is freeze-dried microspheres added with a combination of two sugar freeze-drying protectants, 2, 3 and 4 are freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%, 1% and 2%);

[0039] Figure 6 Figure 1 shows the moisture resistance detection diagram of freeze-dried microspheres, 1 is freeze-dried microspheres added with a combination of two sugar freeze-drying protectants, 2, 3 and 4 are freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%, 1% and 2%);

[0040] Figure 7 Figure 1 shows the moisture resistance detection diagram of freeze-dried microspheres, 1 is freeze-dried microspheres added with a combination of two sugar freeze-drying protectants, 2, 3 and 4 are freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%, 1% and 2%);

[0041] Figure 8 Figure 1 shows the moisture resistance detection diagram of freeze-dried microspheres, 1 is freeze-dried microspheres added with a combination of two sugar freeze-drying protectants, 2, 3 and 4 are freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%, 1% and 2%);

[0042] Figure 9 Figure 1 shows the moisture resistance detection diagram of freeze-dried microspheres, 1 is freeze-dried microspheres added with a combination of two sugar freeze-drying protectants, 2, 3 and 4 are freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%, 1% and 2%); DETAILED DESCRIPTION

[0043] The present application provides a freeze-drying protectant and its application in the preparation of PCR freeze-dried reagents. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to achieve. It is particularly important to note that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0044] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market.

[0045] The application is further described below in connection with examples:

[0046] Example 1

[0047] 1. Preparation of PCR reagent

[0048] The fluorescent quantitative PCR reagent is KAPA3G HotStart DNA Polymerase (without glycerol) and NxtScript 2G RT enzyme (without glycerol) of Roche Company, 10x PCR buffer, primers and probes are synthesized by Thermo Fisher. Other materials, reagents, etc. used, if not specially stated, can be obtained from commercial channels.

[0049] Table 1 is a PCR freeze-dried reagent containing two kinds of sugar freeze-dried protectant combinations.

[0050] Table 1

[0051]

[0052]

[0053] Table 2 is a PCR freeze-dried reagent containing the freeze-dried protectant combination of the application, and tert-butyl alcohol is 0.5%.

[0054] Table 2

[0055] Component Final Concentration 20 μL System (μL) 10 x PCR buffer 1× 2 Syncytial virus upstream primer 0.4 μM 0.08 Syncytial virus downstream primer 0.4 μM 0.08 Syncytial virus probe 0.2 μM 0.04 dNTP 200 μM 0.2 KAPA 3G HotStart DNA Polymerase (glycerol free) 1 U / μL 0.4 NxtScript 2G RT enzyme (glycerol free) 1 U / μL 0.4 Lactose 3％ 1.5 Inulin 3％ 1.5 Mannitol 2％ 1.3 PEG 1％ 0.5 PVP 0.5％ 0.25 Hydrolyzed gelatin 0.5％ 0.25 Tert-butanol 0.5％ 1 Brij-35 0.5％ 1 SE-15 0.2％ 0.4 ddH2O 9.1 Total 20

[0056] Table 3 is a PCR freeze-dried reagent containing the freeze-dried protectant combination of the application, and tert-butyl alcohol is 1%.

[0057] Table 3

[0058]

[0059]

[0060] Table 4 is a PCR freeze-dried reagent containing the freeze-dried protectant combination of the application, and tert-butyl alcohol is 2%.

[0061] Table 4

[0062]

[0063]

[0064] 2. Preparation of freeze-dried microspheres

[0065] The PCR reagent was prepared according to the above formula, filled into a beading machine, and dropped into liquid nitrogen at 20 μL / drop. The pre-frozen microspheres were quickly transferred into a pre-frozen vial, and then transferred into a pre-frozen vacuum freeze dryer after half-lid closing. Vacuum freeze drying was carried out according to Table 5.

[0066] Table 5

[0067]

[0068] After freeze-drying, the freeze-dried microspheres were exposed to an environment with a relative humidity of 20%, 30% and 50%, and the water content was measured.

[0069] 3. Experimental results

[0070] 3.1. Measurement of water content

[0071] (1) Measurement of moisture absorption water content of freeze-dried microspheres containing a combination of two sugar freeze-drying protectants (shown in Table 1) in a normal environment, and the results are shown in Table 6 below:

[0072] Table 6

[0073]

[0074]

[0075] (2) Measurement of moisture absorption water content of freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 0.5%) (shown in Table 2) in a normal environment, and the results are shown in Table 7 below:

[0076] Table 7

[0077]

[0078] (3) Measurement of moisture absorption water content of freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 1%) (shown in Table 3) in a normal environment, and the results are shown in Table 8 below:

[0079] Table 8

[0080]

[0081] (4) Measurement of moisture absorption water content of freeze-dried microspheres added with the freeze-drying protectant combination of the present application (t-butyl alcohol 2%) (shown in Table 4) in a normal environment, and the results are shown in Table 9 below:

[0082] Table 9

[0083]

[0084] As described above, comparing the change in water content of the four kinds of lyophilized microspheres exposed to an environment with a relative humidity of 30%, it can be found that the water content of the lyophilized microspheres added with the lyophilization protective agent combination (t-butyl alcohol 0.5%, 1%, 2%) of the present application is significantly reduced, and the moisture resistance is good.

[0085] 3.2, appearance detection

[0086] The four kinds of lyophilized microspheres were exposed to an environment with a relative humidity of 30% for 3 hours, and the changes of the lyophilized microspheres were observed, and the results are shown in Figure 1 .

[0087] The results show that the lyophilized microspheres added with two kinds of sugar lyophilization protective agent combinations become smaller, while the lyophilized microspheres added with the lyophilization protective agent combination of the present application (t-butyl alcohol 0.5%, 1%, 2%) have no change in appearance, indicating good moisture resistance.

[0088] 3.3, performance detection

[0089] The four kinds of lyophilized microspheres were exposed to an environment with a relative humidity of 30% for 3 hours and compared with the lyophilized microspheres without exposure, and the results are shown in Figure 2 .

[0090] The results show that after the lyophilized microspheres added with two kinds of sugar lyophilization protective agent combinations are exposed to an environment with a relative humidity of 30% for 3 hours, the CT value is delayed and the performance is decreased, while the performance of the lyophilized microspheres added with the lyophilization protective agent combination of the present application (t-butyl alcohol 0.5%, 1%, 2%) exposed to an environment with a relative humidity of 30% for 3 hours is consistent with that of the lyophilized microspheres without exposure, indicating good moisture resistance and no effect on the activity of the reagent.

[0091] 3.4, verification of chipping phenomenon

[0092] The lyophilized microspheres added with two kinds of sugar lyophilization protective agent combinations and the lyophilized microspheres added with the lyophilization protective agent combination (t-butyl alcohol 0.5%, 1%, 2%) of the present application after dispensing in a general low-humidity environment are shown in Figure 3 .

[0093] The results show that the lyophilized microspheres added with the lyophilization protective agent combination of the present application (t-butyl alcohol 0.5%, 1%, 2%) do not have chipping phenomenon when artificially dispensed, the surface is smooth without wrinkles, and the performance is stable.

[0094] Example 2

[0095] 1. Preparation of PCR reagent

[0096] The reagents for the quantitative PCR were KAPA3G HotStart DNA Polymerase (without glycerol), NxtScript 2G RT enzyme (without glycerol), 10x PCR buffer, primers and probes synthesized by Thermo Fisher. Other materials and reagents used, if not otherwise specified, were obtained commercially.

[0097] Table 10 is a PCR lyophilized reagent containing no lyophilization protectant of the present application, only one protectant (raffinose):

[0098] Table 10

[0099] Component Final Concentration 20 μL System (μL) 10 x PCR buffer 1× 2 Syncytial virus upstream primer 0.4 μM 0.08 Syncytial virus downstream primer 0.4 μM 0.08 Syncytial virus probe 0.2 μM 0.04 dNTP 200 μM 0.2 KAPA 3G HotStart DNA Polymerase (glycerol free) 1 U / μL 0.4 NxtScript 2G RT enzyme (glycerol free) 1 U / μL 0.4 Raffinose 3％ 2 ddH2O 14.8 Total 20

[0100] Table 11 is a PCR lyophilized reagent containing no lyophilization protectant of the present application, three protectant combinations (raffinose, trehalose, dextran):

[0101] Table 11

[0102] Component Final Concentration 20 μL System (μL) 10 x PCR buffer 1× 2 Syncytial virus upstream primer 0.4 μM 0.08 Syncytial virus downstream primer 0.4 μM 0.08 Syncytial virus probe 0.2 μM 0.04 dNTP 200 μM 0.2 KAPA 3G HotStart DNA Polymerase (glycerol free) 1 U / μL 0.4 NxtScript 2G RT enzyme (glycerol free) 1 U / μL 0.4 Raffinose 3％ 2 Trehalose 2％ 2 Dextran 3％ 2 ddH2O 10.8 Total 20

[0103] Table 12 is a PCR lyophilized reagent containing the lyophilization protectant combination of the present application, 2% t-butanol:

[0104] Table 12

[0105] Component Final Concentration 20 μL System (μL) 10 x PCR buffer 1× 2 Syncytial virus upstream primer 0.4 μM 0.08 Syncytial virus downstream primer 0.4 μM 0.08 Syncytial virus probe 0.2 μM 0.04 dNTP 200 μM 0.2 KAPA 3G HotStart DNA Polymerase (glycerol free) 1 U / μL 0.4 NxtScript 2G RT enzyme (glycerol free) 1 U / μL 0.4 Lactose 3％ 1.5 Inulin 3％ 1.5 Mannitol 2％ 1.3 PEG 1％ 0.5 PVP 0.5％ 0.25 Hydrolyzed gelatin 0.5％ 0.25 Tert-butanol 2％ 4 Brij-35 0.5％ 1 SE-15 0.2％ 0.4 ddH2O 6.1 Total 20

[0106] 2. Preparation of lyophilized microspheres

[0107] The PCR reaction reagent was prepared according to the above formulation, filled into a beading machine, and the beading machine was used to drop 20 μL / drop into liquid nitrogen. The pre-frozen microspheres were quickly transferred to a pre-frozen vial, half-lid was pressed, and then transferred to a pre-frozen vacuum freeze dryer. Vacuum freeze drying was performed according to Table 13.

[0108] Table 13

[0109]

[0110]

[0111] 3. Experimental results

[0112] 3.1. Performance test

[0113] After lyophilization, the lyophilized microspheres of different combinations were exposed to an environment with a relative humidity of 30% for 0 hours, 3 hours, 6 hours, 12 hours, and 24 hours, respectively, and the performance of the lyophilized microspheres was detected. The results are shown in Table 14. Figures 4-6 .

[0114] The results show that the CT values of the freeze-dried microspheres shown in Table 10 are delayed after 3 hours of exposure in an environment with a relative humidity of 30%, the performance decreases, and the performance tends to 0 after 6 hours, 12 hours and 24 hours of exposure Figure 4 ); the CT values of the freeze-dried microspheres shown in Table 11 are delayed after 3 hours of exposure in an environment with a relative humidity of 30%, the performance decreases, and the performance tends to 0 after 6 hours, 12 hours and 24 hours of exposure Figure 5 ); and the performance of the freeze-dried microspheres shown in Table 12 is consistent with that of the freeze-dried microspheres without exposure in an environment with a relative humidity of 30% for 24 hours Figure 6 , indicating that the freeze-dried microspheres added with the freeze-drying protective agent combination of the present application are good in moisture resistance and do not affect the activity of the reagent.

[0115] 3.2, stability detection

[0116] After freeze-drying, the freeze-dried microspheres of different combinations were divided into eight rows of tubes in an environment with a humidity of 30%, sealed and stored, and placed in an oven at 55°C for accelerated 0 days, 3 days, 5 days, 7 days, 10 days to detect the performance of the freeze-dried microspheres, and the results are shown in Figures 7-9 .

[0117] The results show that the CT values of the freeze-dried microspheres shown in Table 10 are delayed with time and the performance gradually decreases Figure 7 ), the CT values of the freeze-dried microspheres shown in Table 11 are delayed with time and the performance gradually decreases Figure 8 ), and the performance of the freeze-dried microspheres shown in Table 12 is consistent with that of the freeze-dried microspheres without exposure after 10 days of accelerated stability detection at 55°C Figure 9 ), indicating that the freeze-dried microspheres added with the freeze-drying protective agent combination of the present application are good in performance.

[0118] The above is only a preferred embodiment of the present application, and it should be noted that for ordinary skilled persons in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A lyoprotectant characterized in that, It is composed of lactose, inulin, mannitol, PEG20000, PVP, hydrolyzed gelatin, tert-butanol, dodecyl polyethylene glycol ether, and a defoamer; the defoamer is SE-15. The mass ratio of each component is (0.03~0.25): (0.02~0.07): (0.01~0.15): (0.005~0.05): (0.002~0.05): (0.001~0.01): (0.001~0.02): (0.001~0.01): (0.001~0.005).

2. The lyoprotectant according to claim 1, characterized in that, The mass ratio of lactose, inulin, mannitol, PEG20000, PVP, hydrolyzed gelatin, tert-butanol, dodecyl polyethylene glycol ether, and defoamer is 0.03:0.03:0.02:0.01:0.005:0.005:0.005:0.005:0.

002.

3. The use of the lyophilization protectant according to claim 1 or 2 in the preparation of PCR lyophilization reagents.

4. A PCR lyophilizate, characterized in that It includes PCR amplification reagents and the lyophilization protectant as described in claim 1 or 2; The concentrations of each component in the PCR amplification reagent are as follows: 20 mmol / L to 200 mmol / L buffer system, 10 mmol / L to 100 mmol / L cation, 30 μmol / L to 400 μmol / L deoxyribonucleotide, 0.5 U / μL to 10 U / μL DNA polymerase or isothermal amplification enzyme, 0.5 U / μL to 5 U / μL reverse transcriptase, 0.3 μmol / L to 1.0 μmol / L upstream and downstream primers and 0.15 μmol / L to 0.5 μmol / L probe.

5. The PCR lyophilized reagent according to claim 4, characterized in that, In the PCR lyophilization reagent, the mass fraction of the lyophilization protectant is 10wt% to 30wt%.

6. The PCR lyophilized reagent according to claim 4, characterized in that, The buffer system includes at least one of Tris-HCl, HEPES, and Tricine.

7. The PCR lyophilized reagent according to claim 4, characterized in that, The cations include at least one of K + , Mg 2+ , Mn 2+ .

8. The PCR lyophilized reagent according to claim 4, characterized in that, The DNA polymerase or isothermal amplification enzyme includes at least one of Taq polymerase, Pfu polymerase, KOD polymerase, Bst polymerase, and Phi29 polymerase; the reverse transcriptase includes at least one of MMLV reverse transcriptase and T7 reverse transcriptase.

9. The method for preparing the PCR lyophilized reagent according to any one of claims 4 to 8, characterized in that, This involves pre-freezing the prepared PCR reagents in liquid nitrogen and then freeze-drying them under vacuum to obtain lyophilized PCR reagents.

10. The preparation method according to claim 9, characterized in that, The vacuum freeze-drying process includes the following steps: Pre-freeze at -60℃±2℃ for 120~360min; The temperature is increased to -40℃±2℃ at a rate of 0.5℃±0.1℃ / minute; -40℃±2℃, freeze for 720~1000min; The temperature is increased to 40℃±2℃ at a rate of 0.5℃±0.1℃ / min; Dry at 40℃±2℃ for 120~600min.

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