阅读说明：本技术 一种冻干保护剂及其在核酸扩增试剂中的应用 (Freeze-drying protective agent and application thereof in nucleic acid amplification reagent ) 是由 孙刚 朱晓进 范春雷 李铭夫 于 2020-06-29 设计创作，主要内容包括：本发明公开了一种冻干保护剂及其在核酸扩增试剂中的应用,所述冻干保护剂含有以下物质：海藻糖、甘露醇、β-环糊精、聚乙二醇8000和水。本发明的冻干保护剂可显著提高核酸扩增冻干试剂在室温条件下的运输和保存期限。另外,本发明通过减小试剂体积,缩短了冷冻干燥的工艺耗时,提高了生产效率,具有良好的应用前景。(The invention discloses a freeze-drying protective agent and application thereof in a nucleic acid amplification reagent, wherein the freeze-drying protective agent contains the following substances: trehalose, mannitol, beta-cyclodextrin, polyethylene glycol 8000 and water. The freeze-drying protective agent can obviously improve the transportation and storage life of the nucleic acid amplification freeze-drying reagent at room temperature. In addition, the invention shortens the time consumption of the freeze drying process and improves the production efficiency by reducing the volume of the reagent, thereby having good application prospect.)

1. A lyoprotectant for a nucleic acid amplification reagent, said lyoprotectant comprising: trehalose, mannitol, beta-cyclodextrin and water.

2. The lyoprotectant for a nucleic acid amplification reagent according to claim 1, wherein said lyoprotectant comprises: trehalose, mannitol, beta-cyclodextrin, polyethylene glycol 8000 and water.

3. The lyoprotectant for nucleic acid amplification reagents according to claim 2, wherein the concentration of trehalose is 35 to 45% by mass, preferably 40% by mass; the mass concentration of the mannitol is 2-5%, preferably 5%; the mass concentration of the polyethylene glycol 8000 is 1-3%, preferably 2%; the mass concentration of the beta-cyclodextrin is 0.5-2%, preferably 1%.

4. The use of the lyoprotectant as claimed in any of claims 1 to 3 in a nucleic acid amplification reagent, wherein the lyoprotectant and the nucleic acid amplification reagent are uniformly mixed and then freeze-dried to obtain the nucleic acid amplification lyophilized reagent.

5. Use of a lyoprotectant according to claim 4 in a nucleic acid amplification reagent comprising the following components: HiScript II reverse transcriptase, Taq DNA polymerase, dNTP, MgSO4, primers and fluorescent probe.

6. The use of a lyoprotectant in a nucleic acid amplification reagent according to claim 5, wherein the nucleic acid amplification reagent does not comprise glycerol and the nucleic acid amplification reagent does not comprise HiScript II reverse transcriptase and Taq DNA polymerase.

7. Use of a lyoprotectant according to claim 4 in a nucleic acid amplification reagent, wherein the lyoprotectant is mixed with the nucleic acid amplification reagent in a volume ratio of 1:3 to 5, preferably 1: 4.

8. The use of a lyoprotectant in a nucleic acid amplification reagent according to claim 4, wherein said lyophilization process comprises:

s1 pre-freezing stage: the temperature is reduced to minus 50 ℃ to minus 30 ℃ within 30 to 60 minutes and kept for 1 to 3 hours;

s2 sublimation drying stage: firstly, vacuumizing for 20-40 minutes to 30-50Pa, then heating for 2-4 hours to-20 to-10 ℃ and keeping for 2-5 hours;

s3 analytic drying stage: further vacuum pumping is carried out for 30-50 minutes to 5-15Pa, and then the temperature is raised to 10-25 ℃ for 1-3 hours and kept for 1-4 hours.

9. The use of a lyoprotectant in a nucleic acid amplification reagent according to claim 8, wherein said lyophilization process comprises:

s1 pre-freezing stage: the temperature is reduced to-40 ℃ within 40 minutes and kept for 2 hours;

s2 sublimation drying stage: firstly, vacuumizing to 40Pa for 30 minutes, then heating to-15 ℃ for 3 hours and keeping for 3 hours;

s3 analytic drying stage: a further vacuum was first applied to 10Pa over 40 minutes and then allowed to warm to 20 ℃ over 2 hours and held for 2 hours.

Technical Field

The invention relates to the field of molecular biology, in particular to a freeze-drying protective agent and application thereof in a nucleic acid amplification reagent.

Background

Although the development of nucleic acid amplification technology has matured and large-scale industrial application in the field of biomedicine is realized, the transportation and storage of nucleic acid amplification reagents have not been able to get rid of the dependence on cold strands. The nucleic acid amplification reagent has complex components and contains a plurality of bioactive components, and the activity change of any one component can affect the performance of the reagent. In order to maintain the biological activity of the reagent, reagent manufacturers mostly store key components of the reagent, such as enzyme reagents, separately from other components, and then carry out cold chain transportation and cryopreservation. When the reagent is used by a user, the components of the reagent are mixed according to a specific ratio. The additional operation steps are added, which not only is inconvenient for the user to operate, but also increases the risk of cross-contamination of reagents.

The freeze-drying technology provides a new scheme for the transportation and storage of nucleic acid amplification reagents. Compared with a liquid reagent, the biological activity of the freeze-dried reagent is more stable, full-component freeze-drying can be realized, the use is convenient, a user can quickly redissolve by adding water, and the operation of mixing reagent components is not needed. However, freeze-drying requires the use of a lyoprotectant, which will undoubtedly introduce a new substance into the nucleic acid amplification reagent, affecting the working efficiency of the reagent. Furthermore, the lyophilization process itself can also result in the loss of some of the activity of the nucleic acid amplification reagents. Finally, the time consuming lyophilization process generally takes more than 20 hours, increasing the time cost of reagent production. Therefore, applying the freeze-drying technology to nucleic acid amplification reagents requires systematic research and optimization, and balances the performance stability, activity loss, cost effect and other aspects of the reagents.

Chinese patents CN105274192A, CN105349529A, CN106591432A, etc. respectively introduce freeze-drying protective agents with different formulations and their applications in nucleic acid amplification reagents, which preliminarily prove the feasibility of freeze-drying technology in enhancing the stability of nucleic acid amplification reagents. However, the above patents generally have the disadvantage of large lyophilization volume, and the lyoprotectants thereof have much room for improvement in terms of shortening the process time.

Disclosure of Invention

Aiming at the technical problems in the prior art, the application aims to provide a freeze-drying protective agent and application thereof in a nucleic acid amplification reagent.

The freeze-drying protective agent for the nucleic acid amplification reagent is characterized by comprising the following substances: trehalose, mannitol, beta-cyclodextrin and water.

The freeze-drying protective agent for the nucleic acid amplification reagent is characterized by comprising the following substances: trehalose, mannitol, beta-cyclodextrin, polyethylene glycol 8000 and water.

The freeze-drying protective agent for the nucleic acid amplification reagent is characterized in that the mass concentration of trehalose in the freeze-drying protective agent is 35-45%, and preferably 40%; the mass concentration of the mannitol is 2-5%, preferably 5%; the mass concentration of the polyethylene glycol 8000 is 1-3%, preferably 2%; the mass concentration of the beta-cyclodextrin is 0.5-2%, preferably 1%.

The application of the freeze-drying protective agent in the nucleic acid amplification reagent is characterized in that the freeze-drying protective agent and the nucleic acid amplification reagent are uniformly mixed and then are subjected to freeze drying, and the nucleic acid amplification freeze-drying reagent is obtained.

The application of the lyoprotectant in nucleic acid amplification reagents is characterized in that the nucleic acid amplification reagents comprise the following components: HiScript II reverse transcriptase, Taq DNA polymerase, dNTP, MgSO4, primers and fluorescent probe.

The application of the freeze-drying protective agent in the nucleic acid amplification reagent is characterized in that the nucleic acid amplification reagent does not contain glycerol, and HiScript II reverse transcriptase and Taq DNA polymerase of the nucleic acid amplification reagent do not contain glycerol.

The application of the lyoprotectant in nucleic acid amplification reagents is characterized in that the volume ratio of the lyoprotectant to the nucleic acid amplification reagents is 1:3-5, preferably 1: 4.

The application of the freeze-drying protective agent in the nucleic acid amplification reagent is characterized in that the freeze-drying process comprises the following steps:

s1 pre-freezing stage: the temperature is reduced to minus 50 ℃ to minus 30 ℃ within 30 to 60 minutes and is kept for 1 to 3 hours;

s2 sublimation drying stage: firstly, vacuumizing to 30-50Pa for 20-40 minutes, then heating to-20 to-10 ℃ for 2-4 hours, and keeping for 2-5 hours;

s3 analytic drying stage: further vacuum pumping is carried out for 30-50 minutes to 5-15Pa, and then the temperature is raised to 10-25 ℃ for 1-3 hours and kept for 1-4 hours.

Further, the freeze-drying process is as follows:

s1 pre-freezing stage: the temperature is reduced to-40 ℃ within 40 minutes and kept for 2 hours;

s2 sublimation drying stage: firstly, vacuumizing to 40Pa for 30 minutes, then heating to-15 ℃ for 3 hours and keeping for 3 hours;

s3 analytic drying stage: a further vacuum was first applied to 10Pa over 40 minutes and then allowed to warm to 20 ℃ over 2 hours and held for 2 hours.

The invention discloses a freeze-drying protective agent suitable for a nucleic acid amplification reagent and freeze-drying process parameters suitable for the nucleic acid amplification reagent. The freeze-drying protective agent contains trehalose, mannitol, polyethylene glycol 8000 and beta-cyclodextrin, and can remarkably prolong the transportation and storage life of the freeze-drying agent at room temperature. In addition, the volume of the conventional nucleic acid detection reagent is usually more than 15 muL, but the volume of the nucleic acid detection reagent is reduced to be less than 5 muL by improving the component concentration of the nucleic acid detection reagent, so that the time consumption of the freeze drying process is greatly shortened, the production efficiency is improved, and the method has a good application prospect.

The invention uses trehalose as the main component of the freeze-drying protective agent, and the purpose of the trehalose is to reduce the activity loss of enzyme molecules in a nucleic acid amplification reagent in a freeze-drying process. The invention uses mannitol and polyethylene glycol 8000 as excipient, which can reduce the volatilization loss of effective components (such as primer, fluorescent probe, dNTP) in nucleic acid detecting reagent in the freeze drying process, and maintain the exterior of the nucleic acid amplifying reagent after freeze drying in certain physical structure and strength. The working concentration of mannitol is 2-4%, preferably, the concentration is 4%. The working concentration of the polyethylene glycol 8000 is 1-2%, preferably, the concentration is 1%.

According to the invention, the initial volume of the nucleic acid amplification reagent before freeze-drying is reduced by improving the component concentration of the nucleic acid amplification reagent, so that the duration of the freeze-drying process is shortened. To prevent certain components in the reagent, such as: the invention uses beta-cyclodextrin as a masking agent, protects enzyme molecules by utilizing the characteristics of hydrophobic outside and hydrophilic inside of the beta-cyclodextrin molecules, and enhances the biological activity of the enzyme molecules in the freeze drying process.

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.