阅读说明：本技术 一种适用于核酸检测试剂盒冻干的冻干保护剂及其冻干方法 (Freeze-drying protective agent suitable for freeze-drying of nucleic acid detection kit and freeze-drying method thereof ) 是由 王晓敏 王河清 冯礼群 张吉燕 赵慧 刘中华 王国强 于 2021-07-19 设计创作，主要内容包括：本发明公开了一种适用于核酸检测试剂盒冻干的冻干保护剂及其冻干方法,通过本发明提供的冻干工艺可以将各种液体核酸检测试剂盒制备成冻干制剂,使其可以完全摆脱冷链运输的限制,便于常温长途运输和常温长期储存,为各种液体核酸检测试剂盒提供了稳定的保存方法。冻干制剂具有稳定性高、使用方便的特点,冻干制剂含水量低,延长了核酸检测试剂的保质期。在本发明中对冻干所需的保护剂进行了优化,使冻干后的成品制剂性能更加稳定。(The invention discloses a freeze-drying protective agent suitable for freeze-drying of a nucleic acid detection kit and a freeze-drying method thereof. The freeze-dried preparation has the characteristics of high stability and convenience in use, has low water content, and prolongs the shelf life of the nucleic acid detection reagent. In the invention, the protective agent required by freeze-drying is optimized, so that the performance of the freeze-dried finished product preparation is more stable.)

1. A freeze-drying protective agent suitable for freeze-drying of a nucleic acid detection kit is characterized by comprising: a freeze-drying protective agent suitable for freeze-drying of a DNA detection kit and a freeze-drying protective agent suitable for freeze-drying of an RNA detection kit; the freeze-drying protective agent mixed liquor of the DNA detection kit comprises: lactose, trehalose, sorbitol, mannitol, bovine serum albumin, gelatin, arginine, glycine, ethylene diamine tetraacetic acid, polyvinylpyrrolidone (PVP), dextran, Tween20, and SE-15; the freeze-drying protective agent mixed liquor of the RNA detection kit comprises: lactose, trehalose, sorbitol, mannitol, bovine serum albumin, gelatin, arginine, glycine, ethylene diamine tetraacetic acid, polyvinylpyrrolidone (PVP), dextran, Tween20, and SE-15.

2. The freeze-drying protective agent suitable for freeze-drying of the nucleic acid detection kit according to claim 1, wherein the pH values of the freeze-drying protective agent mixed liquor of the DNA detection kit and the freeze-drying protective agent mixed liquor of the RNA detection kit are 6-10.

3. The lyoprotectant suitable for nucleic acid detection kit lyophilization according to claim 2, wherein the PH adjuster of PH of the lyoprotectant mixture is selected from one or more of acetate-acetate buffer, HEPES buffer, Tris-HCL buffer, and phosphate-sodium hydroxide buffer.

4. A freeze-drying method suitable for a nucleic acid detection kit, which is characterized by comprising the following steps: pre-freezing, sublimation drying and analysis drying;

the pre-freezing setting is as follows: the prefreezing temperature is between 20min and 60min and reaches-30 ℃ to-50 ℃, and the temperature is kept for 1 to 5 hours;

the sublimation drying is set as follows: the sublimation drying temperature reaches minus 45 ℃ to minus 20 ℃ within 20min to 60min, the temperature is kept for 5h to 25 h, and the vacuum degree is 0Pa to 30 Pa;

the analysis drying setting is as follows: the analysis drying temperature reaches 10-35 ℃ within 1-5 h, the temperature is kept for 2-10 h, and the vacuum degree is ultimate vacuum;

after the freeze drying is finished, inert gas is injected under a vacuum state, and the box is taken out after the box is plugged.

5. The freeze-drying protective agent suitable for freeze-drying of the nucleic acid detection kit and the freeze-drying method of the freeze-drying protective agent are characterized in that the water content of freeze-dried preparation finished products obtained by vacuum freeze-drying of various liquid nucleic acid detection kits is 0.1% -3%.

6. The lyoprotectant suitable for nucleic acid detection kit freeze-drying and the freeze-drying method thereof according to claim 4, wherein the inert gas is one of nitrogen, argon and carbon dioxide.

7. A preparation method of a freeze-dried preparation of an 8-linked PCR reaction tube for nucleic acid detection comprises the following steps:

(1) preparing a nucleic acid detection reaction reagent mixed solution;

(2) subpackaging the mixed solution into a single 8-linked PCR reaction tube by using an accurate liquid separation system;

(3) putting the 8-linked PCR reaction tube into a vacuum freeze dryer for freeze drying;

(4) and after the freeze drying is finished, vacuumizing and sealing the freeze-dried preparation of the 8-linked PCR reaction tube.

8. The preparation method of the 8-PCR reaction tube freeze-dried preparation for nucleic acid detection according to claim 7, wherein the water content of a freeze-dried preparation finished product obtained by vacuum freeze-drying of various liquid nucleic acid detection kits is 0.1% -3%.

9. A preparation method of a vial freeze-dried preparation for nucleic acid detection comprises the following steps:

(1) preparing a novel coronavirus nucleic acid detection reaction reagent mixed solution;

(2) subpackaging the mixed solution into penicillin bottles by using an accurate liquid separation system;

(3) putting the penicillin bottle into a vacuum freeze dryer for freeze drying;

(4) and after the freeze drying is finished, sealing the penicillin bottle.

10. The preparation method of the freeze-dried preparation for nucleic acid detection penicillin bottle according to claim 9, wherein the water content of the freeze-dried preparation finished product obtained by vacuum freeze-drying of various liquid nucleic acid detection kits is 0.1% -3%.

11. The freeze-drying protective agent suitable for freeze-drying of the nucleic acid detection kit and the freeze-drying method thereof are characterized in that the components of the freeze-drying protective agent are optimized, so that the performance of a freeze-dried finished preparation is more stable.

12. The freeze-drying protective agent suitable for freeze-drying of the nucleic acid detection kit and the freeze-drying method thereof are characterized in that freeze-drying program parameters are optimized, the properties of the freeze-drying preparation are greatly improved, the stability of the freeze-drying preparation is ensured, and the obtained freeze-drying preparation has an attractive shape.

13. The freeze-drying protective agent suitable for freeze-drying of the nucleic acid detection kit and the freeze-drying method thereof are characterized in that after various liquid nucleic acid detection kits are prepared into freeze-dried preparations through the freeze-drying process provided by the invention, the cold chain transportation can be completely avoided, the long-distance transportation at normal temperature and the long-term storage at normal temperature are realized, and the technical problem that the low-temperature cold chain in remote areas is difficult to guarantee is particularly solved.

14. The freeze-drying protective agent suitable for freeze-drying of the nucleic acid detection kit and the freeze-drying method thereof are characterized in that various liquid nucleic acid detection kits are prepared into freeze-dried preparations through the freeze-drying process, the preparation method is simple, the reproducibility is good, the large-scale production is easy to realize, and the clinical use and the storage are more convenient.

15. The freeze-drying protective agent suitable for freeze-drying of the nucleic acid detection kit and the freeze-drying method thereof according to claim 1 are characterized in that a PCR amplification system for nucleic acid detection is uniformly mixed and then freeze-dried, and during later-stage nucleic acid detection, PCR amplification detection can be carried out only by redissolving a freeze-drying preparation and adding extracted nucleic acid;

compared with a liquid reagent, the method has the advantages of no complicated liquid mixing and subpackaging process, less operation error, simpler operation, capability of saving time fundamentally and increasing the detection amount.

The technical field is as follows:

the invention belongs to the technical field of biological agents, and particularly relates to a freeze-drying process suitable for vacuum freeze-drying of various nucleic acid detection kits.

Background art:

the vacuum freeze drying technology is a technology that water solution is frozen at low temperature, then the water in the water solution is directly sublimated and dried without being in a liquid-lifting state in a vacuum state, and the dried substance has basically unchanged physical and chemical properties, basically unchanged properties, small loss of effective components, good rehydration and long sealing and storage period. The vacuum freeze-drying technology provides a stable preservation method for various liquid nucleic acid detection kits which have poor stability and need cold chain transportation and storage.

During the freeze-drying of biologics, there are three main effects that affect the stability of the active components therein and even lead to inactivation: freezing effect: during the freezing process of the biological product, the continuous crystallization can lead the concentration of the solution to be rapidly increased, the ion concentration is increased, and the chemical reaction is promoted. During freezing of some bioproduct solutions, the PH of the solution may also change, resulting in physical aggregation and chemical denaturation of the proteins. (ii) dehydration effect: after the protein is fully hydrated, a hydration layer is attached to the surface of the protein molecule, a part of bound water is removed in the freeze drying process, and the removal of the bound water is likely to destroy the natural structure of the protein, and finally leads to the denaturation of the protein. ③ low temperature effect: the active components in the biological product can be denatured within a certain temperature range in the processes of temperature reduction and temperature rise. The freeze-drying process of biological products is a multi-step complex process, and in order to prevent the denaturation of key active components of the medicines and the biological products in the freeze-drying process, in most of the freeze-drying processes of the medicines and the biological products, a proper freeze-drying protective agent needs to be added to prepare a mixed solution, and then effective freeze-drying can be carried out.

Some commonly used protective agents in the freeze-drying process mainly include saccharides, polyols, surfactants, amino acids, other additives and the like. Saccharides: monosaccharides (glucose and galactose) cannot protect proteins during freeze drying, because monosaccharides only provide a very weak stabilizing effect during freezing, and proteins are irreversibly denatured before dehydration drying, so monosaccharides are not used alone as a protective agent in the freeze drying formula of current biological products. In the lyophilization process of many biological products, oligosaccharides (sucrose, trehalose) are often used, especially disaccharides, which function both as cryoprotectants during freezing and as dehydration protectors during drying and dehydration. Trehalose is a natural saccharide, has a relatively high glass transition temperature, and can effectively prevent the plasticizing effect of water on a glassy state. In the protein freeze-drying process, the protein can be denatured when losing water, and trehalose can fill in the vacancy generated by water loss, so that the protein denaturation in the freeze-drying process is effectively prevented.

Polyols (mannitol, sorbitol, glycerol) have the same effect as sugars. Mannitol is neutral and stable in sterile solution and is not easy to be oxidized. During the lyophilization process of biologics, mannitol generally provides a supporting structure, acts as a bulking agent, and does not react with the active ingredient. Sorbitol is an isomer of mannitol, is more soluble than mannitol, is hygroscopic, but is unstable at high temperatures, and is commonly used as a bulking agent in freeze-dried formulations. Glycerol (glycerin) is commonly used as a cryoprotectant in freeze-dried formulations.

Amino acids: amino acids are the basic building blocks of proteins, the most predominant of which are a-amino acids. Commonly used amino acid based protective agents are glycine, glutamic acid, arginine and histidine, with amino acids being good bulking agents. Because the amino acid ions have acid-base amphipathy, the pH value change of the solution can be inhibited in the processes of low-temperature preservation and freeze drying of biological products, thereby achieving the purpose of protecting active components. For example, low concentrations of glycine prevent protein denaturation by inhibiting the change in pH due to crystallization of 10 or 100mmol/L phosphate buffer, and can raise the collapse (disintegration) temperature of the finished product and prevent protein destruction due to collapse.

Polymers: the polymer can increase the glass transition temperature of mixed solutions of biological products, so that a plurality of polymer protective agents are often added into the formula of some biological products. Polymeric protectants generally function as both cryoprotectants and dehydration protectants, such as: polyvinylpyrrolidone, bovine serum albumin, dextran, polyethylene glycol, and the like. In general, the addition of polymers during the freeze-drying process in biologics can increase the viscosity of the solution; inhibiting crystallization of small molecule excipients (such as sucrose); inhibiting the pH value of the solution from decreasing; steric hindrance between protein molecules, etc.

Surfactants: surfactants can reduce interfacial tension. In the process of freeze drying of biological products, the surfactant can reduce freezing and dehydration denaturation caused by ice water interfacial tension in the freezing and dehydration processes, and can also play a role of a wetting agent and a refolding agent on active components in the rehydration process. Other additive groups: other additive-based protectants include antioxidants, buffers, and lyophilization accelerators. Antioxidant: the antioxidant can prevent oxidation deterioration of biological product during freeze drying, such as vitamin E, sodium thiosulfate, and protein hydrolysate. The different antioxidants are used in combination and have higher efficacy than the antioxidants used alone, i.e. the antioxidant combination has a synergistic effect. Buffering agent: the concentration of the solution gradually increases during the freeze-drying process, and when the concentration is increased to a certain degree, the pH value of the solution can be changed, and the change of the pH value by 4 units can cause the denaturation of proteins, thereby inactivating biological products. Therefore, in the formulation of lyoprotectants, a suitable amount of buffer is added to adjust the pH of the biological product to the most stable region of the active substance, such as amino acids, phosphoric acid, sorbitol, disodium EDTA, etc. Freeze-drying accelerator: the freeze drying process is long in time consumption and high in energy consumption, and the freeze drying process needs to be optimized, so that the production cost is reduced. The tert-butyl alcohol is a small molecular alcohol, and can be added into the solution to reduce the resistance of the drying layer, so that the drying process is accelerated, the freeze-drying time is shortened, and meanwhile, for substances which are difficult to dissolve in water, the product has a higher specific surface area and a good appearance, is easy to rehydrate, improves the stability of the drug solution and the freeze-dried product, and has a certain antibacterial effect.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

The invention content is as follows:

the invention aims to provide a freeze-drying process suitable for vacuum freeze-drying of various nucleic acid detection kits, thereby overcoming the defects in the prior art.

In order to achieve the above object, the present invention provides a lyoprotectant suitable for lyophilization of a nucleic acid detection kit, comprising: a freeze-drying protective agent suitable for freeze-drying of a DNA detection kit and a freeze-drying protective agent suitable for freeze-drying of an RNA detection kit; the freeze-drying protective agent mixed liquor of the DNA detection kit comprises: lactose, trehalose, sorbitol, mannitol, bovine serum albumin, gelatin, arginine, glycine, ethylene diamine tetraacetic acid, polyvinylpyrrolidone (PVP), dextran, Tween20, and SE-15; the freeze-drying protective agent mixed liquor of the RNA detection kit comprises: lactose, trehalose, sorbitol, mannitol, bovine serum albumin, gelatin, arginine, glycine, ethylene diamine tetraacetic acid, polyvinylpyrrolidone (PVP), dextran, Tween20, and SE-15.

The freeze-drying protective agent and the freeze-drying method thereof suitable for freeze-drying of the nucleic acid detection kit can be used for preparing various liquid nucleic acid detection kits into freeze-dried preparations by the freeze-drying process provided by the invention, so that the freeze-drying preparation can completely get rid of the limitation of cold chain transportation, is convenient for long-distance transportation at normal temperature and long-term storage at normal temperature, and provides a stable storage method for various liquid nucleic acid detection kits. The freeze-dried preparation has the characteristics of high stability and convenience in use, has low water content, and prolongs the shelf life of the nucleic acid detection reagent. In the invention, the protective agent required by freeze-drying is optimized, so that the performance of the freeze-dried finished product preparation is more stable. The freeze-drying preparation method has the advantages of attractive appearance, high perfectness, stable enzyme activity, simplicity in operation and the like, and the freeze-drying process and the preparation method provided by the invention are simple and feasible, mild in conditions, good in reproducibility and strong in controllability, are suitable for large-scale production, and are more convenient for clinical use and storage.

Preferably, in the above technical solution, the lyoprotectant suitable for nucleic acid detection kit lyophilization according to claim 1, wherein a PH of the lyoprotectant mixture of the DNA detection kit and the lyoprotectant mixture of the RNA detection kit is 6 to 10.

Preferably, in the above technical solution, the PH adjusting agent for PH of the lyoprotectant mixture is selected from one or more of a buffer system consisting of acetate-acetate buffer, HEPES buffer, Tris-HCL buffer, and phosphate-sodium hydroxide.

A freeze-drying method suitable for a nucleic acid detection kit, wherein the freeze-drying method comprises the following steps: prefreezing, sublimation drying and desorption drying.

The pre-freezing setting is as follows: the prefreezing temperature is between 20min and 60min and reaches-30 ℃ to-50 ℃, and the temperature is kept for 1 to 5 hours;

the sublimation drying is set as follows: the sublimation drying temperature reaches minus 45 ℃ to minus 20 ℃ within 20min to 60min, the temperature is kept for 5h to 25 h, and the vacuum degree is 0Pa to 30 Pa;

the analysis drying setting is as follows: the analysis drying temperature reaches 10-35 ℃ within 1-5 h, the temperature is kept for 2-10 h, and the vacuum degree is ultimate vacuum;

after the freeze drying is finished, inert gas is injected under a vacuum state, and the box is taken out after the box is plugged.

Preferably, in the technical scheme, the water content of the finished freeze-dried preparation obtained by vacuum freeze-drying of various liquid nucleic acid detection kits is 0.1% -3%.

Preferably, in the above technical solution, the inert gas is one of nitrogen, argon and carbon dioxide.

A preparation method of a freeze-dried preparation of an 8-linked PCR reaction tube for nucleic acid detection comprises the following steps:

(1) preparing a nucleic acid detection reaction reagent mixed solution;

(2) subpackaging the mixed solution into a single 8-linked PCR reaction tube by using an accurate liquid separation system;

(3) and (3) putting the 8-joint PCR reaction tube into a vacuum freeze dryer for freeze drying.

(4) And after the freeze drying is finished, vacuumizing and sealing the freeze-dried preparation of the 8-linked PCR reaction tube.

Preferably, in the technical scheme, the water content of the finished freeze-dried preparation obtained by vacuum freeze-drying of various liquid nucleic acid detection kits is 0.1% -3%.

A preparation method of a vial freeze-dried preparation for nucleic acid detection comprises the following steps:

(1) preparing a novel coronavirus nucleic acid detection reaction reagent mixed solution;

(2) subpackaging the mixed solution into penicillin bottles by using an accurate liquid separation system;

(3) and (3) putting the penicillin bottle into a vacuum freeze dryer for freeze drying.

(4) And after the freeze drying is finished, sealing the penicillin bottle.

Preferably, in the technical scheme, the water content of the finished freeze-dried preparation obtained by vacuum freeze-drying of various liquid nucleic acid detection kits is 0.1% -3%.

Compared with the prior art, the invention has the following beneficial effects:

in the technical scheme, the components of the freeze-drying protective agent are optimized, so that the performance of the freeze-dried finished product preparation is more stable. The freeze-drying program parameters are optimized, the properties of the freeze-drying preparation are greatly improved, the stability of the freeze-drying preparation is ensured, and the obtained freeze-drying preparation has an attractive shape. After various liquid nucleic acid detection kits are prepared into freeze-dried preparations by the freeze-drying process provided by the invention, the transportation of cold chains can be completely avoided, the long-distance transportation at normal temperature and the long-term storage at normal temperature are realized, and the technical problem that the low-temperature cold chains in remote areas are difficult to guarantee is particularly solved. The various liquid nucleic acid detection kits are prepared into freeze-dried preparations by the freeze-drying process, the preparation method is simple, the reproducibility is good, the large-scale production is easy to realize, and the clinical use and the storage are more convenient. And (3) uniformly mixing the PCR amplification system for nucleic acid detection, and then freeze-drying, wherein in the later stage of nucleic acid detection, the PCR amplification detection can be carried out only by re-dissolving the freeze-dried preparation and adding the extracted nucleic acid. Compared with a liquid reagent, the method has the advantages of no complicated liquid mixing and subpackaging process, less operation error, simpler operation, capability of saving time fundamentally and increasing the detection amount.

Description of the drawings:

FIG. 1 is a diagram of a finished product of a 8-linked PCR reaction tube lyophilized preparation lyophilized by using a Klebsiella pneumoniae nucleic acid detection kit (fluorescence PCR method) (product number: JB 20105) produced by Jiangsu Shuicho Biotechnology GmbH in the example of the present invention.

FIG. 2 is a diagram of a sealed package of a freeze-dried preparation product freeze-dried by using a Klebsiella pneumoniae nucleic acid detection kit (fluorescence PCR method) (product number: JB 20105) produced by Jiangsu Shuicho Biotechnology GmbH in an embodiment of the present invention.

FIG. 3 is a graph showing the accelerated stability PCR amplification curve of a lyophilized preparation product of 8-linked PCR reaction tubes, which is lyophilized using a Klebsiella pneumoniae nucleic acid detection kit (fluorescence PCR method) (product number: JB 20105) manufactured by Jiangsu Shuicho Biotechnology GmbH and is placed at 45 ℃ for 3 months, according to the embodiment of the present invention.

FIG. 4 is a diagram of a vial lyophilized preparation that is lyophilized using a Klebsiella pneumoniae nucleic acid detection kit (fluorescence PCR method) (product number: JB 20105) produced by Jiangsu Shuicho Biotechnology GmbH in an embodiment of the present invention.

FIG. 5 is a graph showing the PCR amplification with accelerated stability when the lyophilized preparation product of 8-linked PCR reaction tube, which is lyophilized using the novel coronavirus nucleic acid detection kit (fluorescence PCR fluorescence method) manufactured by Jiangsu Shuicho Biotechnology Ltd, is left at 45 ℃ for 3 months, according to the present invention.

FIG. 6 is a graph showing the PCR amplification with accelerated stability when the lyophilized preparation product of 8-linked PCR reaction tube, which is lyophilized using the novel coronavirus nucleic acid detection kit (fluorescence PCR fluorescence method) manufactured by Jiangsu Shuicho Biotechnology GmbH and is placed at 37 ℃ for 6 months, according to the present invention.

FIG. 7 is a PCR amplification curve diagram of lyophilized preparation in vials, which was lyophilized and reconstituted using a novel coronavirus nucleic acid detection kit (fluorescence PCR fluorescence assay) manufactured by Jiangsu major Biotechnology Ltd, placed at-20 ℃ and frozen and thawed 10 times, according to an embodiment of the present invention.

The specific implementation mode is as follows:

the following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.

Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

Example 1

In this embodiment, a freeze-drying protective agent suitable for freeze-drying of a DNA detection kit and a freeze-drying method are provided, and a 8-linked PCR reaction tube freeze-dried preparation is prepared. The nucleic acid detection kit (fluorescence PCR method) for Klebsiella pneumoniae (cat # JB 20105) from Jiangsu Shuicho Biotech Co., Ltd was used for lyophilization. The optimized freeze-drying protective agent mixed liquid comprises: 25% trehalose, 16% mannitol, 4% glycine, 2.5mg/mL bovine serum albumin, 5% dextran, 0.5% Tween20, 0.1% SE-15, 100mmol/mL Tris-HCl, and diethyl pyrophosphate treated water.

The method comprises the following specific steps:

(1) and preparing optimized freeze-drying protective agent mixed liquor.

(2) Preparing a nucleic acid detection PCR reaction reagent mixed solution according to the instructions of a Klebsiella pneumoniae nucleic acid detection kit (fluorescent PCR method).

(3) And uniformly mixing the optimized freeze-drying protective agent mixed solution and the prepared nucleic acid amplification PCR reaction reagent mixed solution according to the ratio of 4: 16.

(4) And (3) subpackaging the uniformly mixed liquid reagent into 8-linked PCR reaction tubes by using an accurate liquid separation system.

(5) And (3) putting the 8-joint PCR reaction tube into a vacuum freeze dryer for freeze drying.

(6) Freeze-drying program parameter setting:

the pre-freezing setting is as follows:

the pre-freezing temperature of the first stage reaches 4 ℃ within 40min, and the temperature is kept for 0.5 h;

the pre-freezing temperature of the second stage reaches-45 ℃ within 30min, and the temperature is kept for 3 h.

The sublimation drying is set as follows:

the sublimation drying temperature of the first stage reaches-35 ℃ within 40min, the temperature is kept for 1h, and the vacuum degree is 10 Pa;

the sublimation drying temperature of the second stage reaches-30 ℃ within 30min, the temperature is kept for 10h, and the vacuum degree is 10 Pa.

The analysis drying setting is as follows:

in the first stage, the analysis drying temperature reaches 20 ℃ within 40min, the temperature is kept for 1h, and the vacuum degree is ultimate vacuum;

the second stage analysis drying temperature reaches 30 ℃ within 30min, the temperature is kept for 5h, and the vacuum degree is ultimate vacuum.

(7) And after the freeze-drying is finished, flushing nitrogen, pressing a plug in the box, opening the door of the freeze dryer after the temperature and the humidity of the environment meet the requirements, and taking out the 8-PCR freeze-drying preparation reaction tube for nucleic acid detection.

(8) And observing whether the appearance of the freeze-dried preparation is qualified.

(9) The moisture content of the lyophilized preparation was measured using a card-type moisture meter. (determination of the moisture content of the lyophilized preparation to 1.79% by means of a card-type moisture meter)

(10) Putting the nucleic acid detection 8-PCR freeze-drying preparation reaction tube and a drying agent into an aluminum foil bag together for vacuumizing and sealing.

(11) The 8-linked PCR reaction tube lyophilized preparation prepared by lyophilizing Klebsiella pneumoniae nucleic acid detection kit produced by Jiangsu Shuicho Biotechnology GmbH is shown in figure 1, and the 8-linked PCR reaction tube lyophilized preparation is sealed and packaged as shown in figure 2.

Example 2

In this embodiment, a freeze-drying protective agent suitable for freeze-drying of a DNA detection kit and a freeze-drying method are provided, wherein an annealing operation is added in a pre-freezing stage to prepare a 8-linked PCR reaction tube freeze-dried preparation. The nucleic acid detection kit (fluorescence PCR method) for Klebsiella pneumoniae (cat # JB 20105) from Jiangsu Shuicho Biotech Co., Ltd was used for lyophilization. The optimized protective agent mixed liquid comprises: 25% trehalose, 16% mannitol, 4% glycine, 2.5mg/mL bovine serum albumin, 5% dextran, 0.5% Tween20, 0.1% SE-15, 100mmol/mL Tris-HCl, and diethyl pyrophosphate treated water.

The method comprises the following specific steps:

(1) and preparing optimized freeze-drying protective agent mixed liquor.

(2) Preparing a nucleic acid detection PCR reaction reagent mixed solution according to the instructions of a Klebsiella pneumoniae nucleic acid detection kit (fluorescent PCR method).

(3) And uniformly mixing the optimized freeze-drying protective agent mixed solution and the prepared nucleic acid detection PCR reaction reagent mixed solution according to the ratio of 4: 16.

(4) And (3) subpackaging the uniformly mixed liquid reagent into 8-linked PCR reaction tubes by using an accurate liquid separation system.

(5) And (3) putting the 8-joint PCR reaction tube into a vacuum freeze dryer for freeze drying.

(6) Freeze-drying program parameter setting:

the pre-freezing setting is as follows:

the pre-freezing temperature of the first stage reaches 4 ℃ within 40min, and the temperature is kept for 0.5 h;

in the second stage, the pre-freezing temperature reaches-45 ℃ within 30min, and the temperature is kept for 2 h.

In the third stage, the pre-freezing temperature (annealing operation) reaches-30 ℃ within 30min, and is kept for 1 h;

the prefreezing temperature of the fourth stage reaches-45 deg.C within 40min, and is maintained for 3 h.

The sublimation drying comprises the following steps:

the sublimation drying temperature of the first stage reaches-35 ℃ within 40min, the temperature is kept for 1h, and the vacuum degree is 10 Pa;

the sublimation drying temperature of the second stage reaches-30 ℃ within 30min, the temperature is maintained for 7h, and the vacuum degree is 10 Pa.

The desorption drying comprises the following steps:

in the first stage, the analysis drying temperature reaches 20 ℃ within 40min, the temperature is kept for 1h, and the vacuum degree is ultimate vacuum;

the second stage analysis drying temperature reaches 30 ℃ within 30min, the temperature is kept for 5h, and the vacuum degree is ultimate vacuum.

(7) And after the freeze-drying is finished, flushing nitrogen, pressing a plug in the box, opening the door of the freeze dryer after the temperature and the humidity of the environment meet the requirements, and taking out the 8-PCR freeze-drying preparation reaction tube for nucleic acid detection.

(8) And observing whether the appearance of the freeze-dried preparation is qualified.

(9) The moisture content of the lyophilized preparation was measured using a card-type moisture meter. (determination of the moisture content of the lyophilized preparation to 1.64% by means of a card-type moisture meter)

(10) Putting the nucleic acid detection 8-PCR freeze-drying preparation reaction tube and a drying agent into an aluminum foil bag together for vacuumizing and sealing.

(11) High temperature accelerated stability test

The finished product of the 8-unit PCR reaction tube freeze-dried preparation for detecting the Klebsiella pneumoniae nucleic acid is subjected to high-temperature accelerated test at 45 ℃, and the PCR amplification result shows that the difference of the 8-unit PCR reaction tube freeze-dried preparation for detecting the Klebsiella pneumoniae nucleic acid in a control liquid kit is not large when the 8-unit PCR reaction tube freeze-dried preparation is placed at 45 ℃ for 3 months, as shown in figure 3.

Example 3

In this embodiment, a freeze-drying protective agent suitable for freeze-drying of a DNA detection kit and a freeze-drying method are provided, and a vial freeze-dried preparation is prepared. The nucleic acid detection kit (fluorescence PCR method) for Klebsiella pneumoniae (cat # JB 20105) from Jiangsu Shuicho Biotech Co., Ltd was used for lyophilization. The optimized protective agent mixed liquid comprises: 25% trehalose, 16% mannitol, 4% glycine, 2.5mg/mL bovine serum albumin, 5% dextran, 0.5% Tween20, 0.1% SE-15, 100mmol/mL Tris-HCl, and diethyl pyrophosphate treated water.

The method comprises the following specific steps:

(1) and preparing optimized freeze-drying protective agent mixed liquor.

(2) Preparing a nucleic acid detection PCR reaction reagent mixed solution according to the instructions of a Klebsiella pneumoniae nucleic acid detection kit (fluorescent PCR method).

(3) And uniformly mixing the optimized freeze-drying protective agent mixed solution and the prepared nucleic acid detection PCR reaction reagent mixed solution according to the ratio of 4: 16.

(4) And (3) subpackaging the uniformly mixed liquid reagent into penicillin bottles by using an accurate liquid separation system.

(5) And (5) putting the penicillin bottle into a vacuum freeze dryer for freeze drying.

(6) Freeze-drying program parameter setting:

the pre-freezing setting is as follows:

in the first stage, the pre-freezing temperature reaches 0 ℃ within 40min, and the temperature is kept for 1 h;

the pre-freezing temperature of the second stage reaches-45 ℃ within 50min, and the temperature is kept for 5 h.

The sublimation drying comprises the following steps:

the sublimation drying temperature of the first stage reaches-40 ℃ within 40min, the temperature is kept for 2h, and the vacuum degree is 10 Pa;

the sublimation drying temperature of the second stage reaches-35 ℃ within 50min, the temperature is kept for 17h, and the vacuum degree is 10 Pa.

The desorption drying comprises the following steps:

in the first stage, the analysis drying temperature reaches 20 ℃ within 40min, the temperature is kept for 2h, and the vacuum degree is ultimate vacuum;

the second stage analysis drying temperature reaches 25 ℃ within 50min, the temperature is kept for 6h, and the vacuum degree is ultimate vacuum.

(7) And after the freeze-drying is finished, flushing nitrogen, pressing a plug in the box, opening the door of the freeze-drying machine after the temperature and the humidity of the environment meet the requirements, and taking out the freeze-drying preparation reaction tube of the nucleic acid detection penicillin bottle.

(8) And observing whether the appearance of the freeze-dried preparation is qualified.

(9) The moisture content of the lyophilized preparation was measured using a card-type moisture meter. (determination of the moisture content of the lyophilized preparation to 2.24% by means of a card-type moisture meter)

(10) And sealing the nucleic acid detection penicillin bottle.

(11) The product of lyophilized preparation in penicillin bottle, which is lyophilized by using the nucleic acid detection kit for Klebsiella pneumoniae manufactured by Jiangsu Shuicho Biotechnology GmbH, is shown in figure 4.

Example 4

In this embodiment, a freeze-drying protective agent suitable for freeze-drying of an RNA detection kit and a freeze-drying method are provided, and the freeze-dried preparation of the 8-linked PCR reaction tube is prepared. The novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR fluorescence method) produced by Jiangsu Shuicheng Biotechnology GmbH was used for freeze-drying. The optimized mixture of the protective agent and the excipient comprises: 20% trehalose, 15% mannitol, 5% glycine, 3mg/mL bovine serum albumin, 6% polyvinylpyrrolidone (PVP), 0.5% Tween20, 0.1% SE-15, 100mmol/mL Tris-HCl, and diethyl pyrophosphate treated water.

The method comprises the following specific steps:

(1) and preparing optimized freeze-drying protective agent mixed liquor.

(2) And preparing a nucleic acid detection PCR reaction reagent mixed solution according to the instructions of the novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR fluorescence method).

(3) And uniformly mixing the optimized freeze-drying protective agent mixed solution and the prepared novel coronavirus 2019-nCoV nucleic acid amplification PCR reaction reagent mixed solution according to the proportion of 3.5: 16.5.

(4) And (3) subpackaging the uniformly mixed liquid reagent into 8-linked PCR reaction tubes by using an accurate liquid separation system.

(5) And (3) putting the 8-joint PCR reaction tube into a vacuum freeze dryer for freeze drying.

(6) Freeze-drying program parameter setting:

the pre-freezing setting is as follows:

the pre-freezing temperature of the first stage reaches 4 ℃ within 40min, and the temperature is kept for 0.5 h;

the pre-freezing temperature of the second stage reaches-45 ℃ within 30min, and the temperature is kept for 3 h.

The sublimation drying comprises the following steps:

the sublimation drying temperature of the first stage reaches-35 ℃ within 40min, the temperature is kept for 1h, and the vacuum degree is 10 Pa;

the sublimation drying temperature of the second stage reaches-30 ℃ within 30min, the temperature is kept for 10h, and the vacuum degree is 10 Pa.

The desorption drying comprises the following steps:

in the first stage, the analysis drying temperature reaches 20 ℃ within 40min, the temperature is kept for 1h, and the vacuum degree is ultimate vacuum;

the second stage analysis drying temperature reaches 30 ℃ within 30min, the temperature is kept for 5h, and the vacuum degree is ultimate vacuum.

(7) And after the freeze-drying is finished, flushing nitrogen, pressing a plug in the box, opening the box door of the freeze dryer after the temperature and the humidity of the environment meet the requirements, and taking out the freeze-dried preparation of the nucleic acid amplification 8-linked PCR reaction tube.

(8) And observing whether the appearance of the freeze-dried preparation is qualified.

(9) The moisture content of the lyophilized preparation was measured using a card-type moisture meter. (the moisture content of the lyophilized preparation was 1.74% by using a card-type moisture meter).

(10) Putting the freeze-dried preparation of the nucleic acid amplification 8-linked PCR reaction tube and a drying agent into an aluminum foil bag together for vacuumizing and sealing.

Example 5

In this embodiment, a freeze-drying protective agent suitable for freeze-drying of an RNA detection kit and a freeze-drying method are provided, wherein an annealing operation is added in a pre-freezing stage to prepare a 8-linked PCR reaction tube freeze-dried preparation. The novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR fluorescence method) produced by Jiangsu Shuicheng Biotechnology GmbH was used for freeze-drying. The optimized protective agent mixed liquid comprises: 20% trehalose, 15% mannitol, 5% glycine, 3mg/mL bovine serum albumin, 6% polyvinylpyrrolidone (PVP), 0.5% Tween20, 0.1% SE-15, 100mmol/mL Tris-HCl, and diethyl pyrophosphate treated water.

The method comprises the following specific steps:

(1) and preparing optimized freeze-drying protective agent mixed liquor.

(2) And preparing a nucleic acid detection PCR reaction reagent mixed solution according to the instructions of the novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR fluorescence method).

(3) And uniformly mixing the optimized freeze-drying protective agent mixed solution and the prepared novel coronavirus 2019-nCoV nucleic acid detection PCR reaction reagent mixed solution according to the proportion of 3.5: 16.5.

(4) And (3) subpackaging the uniformly mixed liquid reagent into 8-linked PCR reaction tubes by using an accurate liquid separation system.

(5) And (3) putting the 8-joint PCR reaction tube into a vacuum freeze dryer for freeze drying.

(6) Freeze-drying program parameter setting:

the pre-freezing setting is as follows:

the pre-freezing temperature of the first stage reaches 4 ℃ within 40min, and the temperature is kept for 0.5 h;

in the second stage, the pre-freezing temperature reaches-45 ℃ within 30min, and the temperature is kept for 2 h;

in the third stage, the pre-freezing temperature (annealing operation) reaches-30 ℃ within 30min, and is kept for 1 h;

the prefreezing temperature of the fourth stage reaches-45 deg.C within 40min, and is maintained for 3 h.

The sublimation drying comprises the following steps:

in the first stage, the oil guiding temperature reaches-35 ℃ within 40min, the temperature is maintained for 1h, and the vacuum degree is 10 Pa;

the oil guiding temperature of the second stage reaches-30 ℃ within 30min, the second stage is kept for 8h, and the vacuum degree is 10 Pa.

The desorption drying comprises the following steps:

in the first stage, the oil guiding temperature reaches 20 ℃ within 40min, the oil guiding temperature is kept for 1h, and the vacuum degree is ultimate vacuum;

the oil guiding temperature of the second stage reaches 30 ℃ within 30min, the second stage is kept for 5h, and the vacuum degree is ultimate vacuum.

(7) And after the freeze-drying is finished, flushing nitrogen, pressing a plug in the box, opening the door of the freeze dryer after the temperature and the humidity of the environment meet the requirements, and taking out the freeze-dried preparation of the 8-PCR reaction tube for nucleic acid detection.

(8) And observing whether the appearance of the freeze-dried preparation is qualified.

(9) The moisture content of the lyophilized preparation was measured using a card-type moisture meter. (moisture content of the lyophilized preparation was 1.68% by using a card-type moisture meter)

(10) Putting the freeze-dried preparation of the 8-linked PCR reaction tube for nucleic acid detection and a drying agent into an aluminum foil bag together for vacuum pumping and sealing.

(11) High temperature accelerated stability testing.

The performance difference of the freeze-dried 8-linked PCR reaction tube freeze-dried preparation finished product which is subjected to freeze drying by the novel coronavirus nucleic acid detection kit (fluorescence PCR fluorescence method) is small compared with that of the liquid novel coronavirus nucleic acid detection kit when the freeze-dried preparation finished product is placed for 3 months at 45 ℃, and the accelerated stability PCR amplification curve chart of the freeze-dried preparation finished product when the freeze-dried preparation finished product is placed for 3 months at 45 ℃ is shown in attached figure 5.

The performance difference of the freeze-dried 8-linked PCR reaction tube freeze-dried preparation finished product which is subjected to freeze drying by the novel coronavirus nucleic acid detection kit (fluorescence PCR fluorescence method) is small compared with that of the liquid novel coronavirus nucleic acid detection kit when the freeze-dried preparation finished product is placed at 37 ℃ for 6 months, and the accelerated stability PCR amplification curve chart of the freeze-dried preparation finished product when the freeze-dried preparation finished product is placed at 37 ℃ for 6 months is shown in attached figure 6.

Example 6

In this embodiment, a freeze-drying protective agent suitable for freeze-drying of an RNA detection kit and a freeze-drying method are provided, and a vial freeze-dried preparation is prepared. The novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR fluorescence method) produced by Jiangsu Shuicheng Biotechnology GmbH was used for freeze-drying. The optimized protective agent mixed liquid comprises: 20% trehalose, 15% mannitol, 5% glycine, 3mg/mL bovine serum albumin, 6% polyvinylpyrrolidone (PVP), 0.5% Tween20, 0.1% SE-15, 100mmol/mL Tris-HCl, and diethyl pyrophosphate treated water.

The method comprises the following specific steps:

(1) and preparing optimized freeze-drying protective agent mixed liquor.

(2) And preparing a nucleic acid detection PCR reaction reagent mixed solution according to the instructions of the novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR fluorescence method).

(3) And uniformly mixing the optimized freeze-drying protective agent mixed solution and the prepared novel coronavirus 2019-nCoV nucleic acid detection PCR reaction reagent mixed solution according to the proportion of 3.5: 16.5.

(4) And (3) subpackaging the uniformly mixed liquid reagent into penicillin bottles by using an accurate liquid separation system.

(5) And (5) putting the penicillin bottle into a vacuum freeze dryer for freeze drying.

(6) Freeze-drying program parameter setting:

the pre-freezing setting is as follows:

in the first stage, the pre-freezing temperature reaches 0 ℃ within 40min, and the temperature is kept for 1 h;

the pre-freezing temperature of the second stage reaches-45 ℃ within 50min, and the temperature is kept for 3 h.

The sublimation drying is set as follows:

the sublimation drying temperature of the first stage reaches-40 ℃ within 40min, the temperature is kept for 2h, and the vacuum degree is 10 Pa;

the sublimation drying temperature of the second stage reaches-35 ℃ within 50min, the temperature is kept for 17h, and the vacuum degree is 10 Pa.

The analysis drying setting is as follows:

in the first stage, the analysis drying temperature reaches 20 ℃ within 40min, the temperature is kept for 2h, and the vacuum degree is ultimate vacuum;

the second stage analysis drying temperature reaches 25 ℃ within 50min, the temperature is kept for 6h, and the vacuum degree is ultimate vacuum.

(7) And after the freeze-drying is finished, flushing nitrogen, pressing a plug in the box, opening the door of the freeze-drying machine after the temperature and the humidity of the environment meet the requirements, and taking out the freeze-drying preparation reaction tube of the nucleic acid detection penicillin bottle.

(8) And observing whether the appearance of the freeze-dried preparation is qualified.

(9) The moisture content of the lyophilized preparation was measured using a card-type moisture meter. (determination of the moisture content of the lyophilized preparation to 2.47% by means of a card-type moisture meter)

(10) And sealing the freeze-dried preparation of the nucleic acid detection penicillin bottle.

(11) Test of freeze-thaw times of lyophilized preparation in penicillin bottle after redissolution

The freeze-drying preparation of the novel coronavirus nucleic acid detection penicillin bottle is subjected to freeze-thaw times after redissolution, the performance of the preparation is not greatly different from that of a liquid novel coronavirus nucleic acid detection box when the freeze-drying preparation is subjected to freeze-thaw for 10 times, and a PCR amplification curve graph 7 when the freeze-thaw is performed for 10 times is shown.

Example 7

In this embodiment, a freeze-drying protective agent suitable for freeze-drying of an RNA detection kit and a freeze-drying method are provided, and an annealing operation is added in a pre-freezing stage to prepare a vial freeze-dried preparation. The nucleic acid detection kit (fluorescent PCR method) for influenza A/B virus (cat # JC 10202N) from Jiangsu Shuiches Biotech Ltd was used for lyophilization. The optimized protective agent mixed liquid comprises: water was treated with 25% trehalose, 12% mannitol, 4% glycine, 3mg/mL bovine serum albumin, 8% polyvinylpyrrolidone (PVP), 0.5% Tween20, 0.1% SE-15, 100mmol/mL Tris-HCl and diethyl pyrophosphate.

The method comprises the following specific steps:

(1) and preparing optimized freeze-drying protective agent mixed liquor.

(2) Preparing a nucleic acid detection PCR reaction reagent mixed solution according to the instruction of a nucleic acid detection kit (fluorescence PCR method) for the influenza A/B virus.

(3) And uniformly mixing the optimized freeze-drying protective agent mixed solution and the prepared PCR reaction reagent mixed solution for detecting the influenza A/B virus nucleic acid according to the proportion of 3.5: 16.5.

(4) And (3) subpackaging the uniformly mixed liquid reagent into penicillin bottles by using an accurate liquid separation system.

(5) And (5) putting the penicillin bottle into a vacuum freeze dryer for freeze drying.

(6) Freeze-drying program parameter setting:

the pre-freezing setting is as follows:

in the first stage, the pre-freezing temperature reaches 0 ℃ within 40min, and the temperature is kept for 1 h;

the pre-freezing temperature of the second stage reaches-45 ℃ within 50min, and the temperature is kept for 3 h.

In the third stage, the pre-freezing temperature (annealing operation) reaches-30 ℃ within 40min, and is kept for 1 h;

the prefreezing temperature of the fourth stage reaches-45 deg.C within 50min, and is maintained for 4 h.

The sublimation drying is set as follows:

the sublimation drying temperature of the first stage reaches-40 ℃ within 40min, the temperature is kept for 1h, and the vacuum degree is 10 Pa;

the sublimation drying temperature of the second stage reaches-35 ℃ within 50min, the temperature is kept for 15h, and the vacuum degree is 10 Pa.

The analysis drying setting is as follows:

in the first stage, the analysis drying temperature reaches 20 ℃ within 40min, the temperature is kept for 1h, and the vacuum degree is ultimate vacuum;

the second stage analysis drying temperature reaches 25 ℃ within 50min, the temperature is kept for 5h, and the vacuum degree is ultimate vacuum.

(7) And after the freeze-drying is finished, flushing nitrogen, pressing a plug in the box, opening the door of the freeze-drying machine after the temperature and the humidity of the environment meet the requirements, and taking out the freeze-dried preparation in the nucleic acid detection penicillin bottle.

(8) And observing whether the appearance of the freeze-dried preparation is qualified.

(9) The moisture content of the lyophilized preparation was measured using a card-type moisture meter. (determination of the moisture content of the lyophilized preparation to 2.41% by means of a card-type moisture meter)

(10) And sealing the freeze-dried preparation in the nucleic acid detection vial.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.