CN113801926B - Freeze-drying protective agent of molecular detection reagent and application thereof - Google Patents
Freeze-drying protective agent of molecular detection reagent and application thereof Download PDFInfo
- Publication number
- CN113801926B CN113801926B CN202111125384.6A CN202111125384A CN113801926B CN 113801926 B CN113801926 B CN 113801926B CN 202111125384 A CN202111125384 A CN 202111125384A CN 113801926 B CN113801926 B CN 113801926B
- Authority
- CN
- China
- Prior art keywords
- freeze
- vitamin
- molecular detection
- detection reagent
- protective agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F26—DRYING
- F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
- F26B5/00—Drying solid materials or objects by processes not involving the application of heat
- F26B5/04—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
- F26B5/06—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Mechanical Engineering (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a freeze-drying protective agent of a molecular detection reagent and application thereof, wherein the freeze-drying protective agent comprises 80-120 mg/mL of trehalose, 80-120 mg/mL of sucrose, 2-5 wt% of mannitol, 3-7 mmol/L of dextran, 0.5-6 mg/mL of polylactic acid-glycolic acid copolymer, 5-20 mg/mL of polylysine, 800.3-0.7 wt% of tween-800.3, 0.05-0.25 mg/mL of dithiothreitol and 5-20 mg/mL of vitamin combination. The freeze-drying protective agent provided by the invention can be prepared with components required by fluorescence PCR, and is freeze-dried to prepare the molecular detection reagent, so that the normal-temperature transportation and storage can be realized, and the application range is wide.
Description
Technical Field
The invention belongs to the technical field of biological molecule detection, and particularly relates to a freeze-drying protective agent of a molecular detection reagent and application thereof.
Background
Nucleic acid diagnosis is a method for diagnosing human states and diseases by directly probing the presence state or defect of nucleic acids, analyzing the functions of nucleic acids from the level of nucleic acid structure, replication, transcription or translation using the theory and technology of molecular biology. Its target molecule is DNA or RNA, reflecting the structure and function of the nucleic acid. The detected genes are endogenous (i.e. the genes of the organism itself) and exogenous (such as viruses, bacteria, etc.), the former is used for diagnosing the presence or absence of lesions in the genes, and the latter is used for diagnosing the presence or absence of pathogen infection.
Polymerase Chain Reaction (PCR) is a molecular biological technique for amplifying specific DNA fragments, which can be regarded as a specific replication of biomolecules in vitro, and is characterized by a large increase in trace amounts of nucleic acids. Has the advantages of high sensitivity, strong specificity and the like, and is widely applied to the field of nucleic acid diagnosis at present.
The existing PCR amplification reagent is basically in a liquid state, components such as DNA polymerase, dNTP, primer, probe and the like are required to be stored in a freezing environment at the temperature of minus 20 ℃, repeated freezing and thawing have a certain influence on the performance of the reagent, and the low-temperature preservation of the reagent has higher requirements on transportation, storage, use processes and the like. In addition, liquid detection reagents require cold chain transport, are relatively costly to transport, and are prone to degradation of reagent performance due to improper storage temperatures.
Freeze-drying is a new and efficient drying technique that causes frozen solid ice in a mixture to sublimate directly into a gaseous state without melting into liquid water, eventually removing water and retaining other effective components. The stability of the bioactive raw materials in the solid dry powder state is far higher than that of the bioactive raw materials in the liquid state, so that the components in the reaction reagent system can be stored for a long time and stably transported at room temperature through freeze-drying and dehydration treatment.
The current lyophilization process of diagnostic reagents has the following drawbacks: 1) The freeze-drying time is too long, and generally more than 30 hours are needed; 2) The enzyme activity loss is serious after freeze-drying, the amplification efficiency is reduced, the detection sensitivity is reduced, and the like; 3) The freeze-dried product is easy to collapse in the later period; 4) Additives such as lyoprotectants can inhibit subsequent amplification reactions, etc.
Therefore, it is necessary to provide a lyoprotectant for molecular detection reagent and application thereof.
Disclosure of Invention
The present invention is directed to a lyoprotectant for molecular detection reagents and application thereof, which at least solve one of the above-mentioned problems described in the prior art.
The first object of the invention is to provide a freeze-drying protective agent for molecular detection, which comprises the following components in concentration: 80-120 mg/mL of trehalose, 80-120 mg/mL of sucrose, 2-5 wt% of mannitol, 3-7 mmol/L of dextran, 0.5-6 mg/mL of polylactic acid-glycolic acid copolymer, 5-20 mg/mL of polylysine, 800.3-0.7 wt% of tween-and 0.05-0.25 mg/mL of dithiothreitol.
Further, the components are: 100mg/mL of trehalose, 100mg/mL of sucrose, 4wt% of mannitol, 5mmol/L of dextran, 4mg/mL of polylactic acid-glycolic acid copolymer, 10mg/mL of polylysine, 800.5wt% of tween-and 0.15mg/mL of dithiothreitol.
Further, the components also comprise 5-20 mg/mL of vitamin combination, wherein the vitamin combination comprises one or more of vitamin B, vitamin C, vitamin D and vitamin K.
A second object of the present invention is to provide a molecular detection reagent, including a molecular detection system, and further including a molecular detection lyoprotectant as described above. Further, the molecular detection system comprises a hot start Taq enzyme, a UNG enzyme and dNTPs. Preferably, the molecular detection system further comprises reverse transcribed MMLV enzyme, primers, probes and buffers.
The invention also provides a freeze-drying protection method of the molecular detection reagent, which comprises the following steps:
1) Taking a molecular detection system and a freeze-drying protective agent to prepare a molecular detection reagent;
2) After the molecular detection reagent is placed at the temperature of minus 30 ℃ to minus 20 ℃ for pre-freezing treatment for 1 to 3 hours, the molecular detection reagent is cooled to minus 50 ℃ to minus 40 ℃ at the speed of 1.0 to 1.5 ℃/min and then frozen for 1 to 3 hours;
3) And (3) placing the frozen molecular detection reagent in a vacuum environment of 5-50 Pa for 1-3 h, then raising the temperature to-40 to-35 ℃ at the speed of 0.3-0.8 ℃/min for 4-7 h, raising the temperature to-30 to-25 ℃ at the speed of 0.3-0.8 ℃/min for 3-5 h, and then raising the temperature of the PCR amplification reagent to 20-30 ℃ to obtain the freeze-dried PCR amplification reagent.
It is still another object of the present invention to provide a molecular assay reagent prepared by the lyoprotection method described above.
It is another object of the present invention to provide the use of the above-described molecular detection reagent in fluorescent PCR detection. The fluorescent PCR amplification detection can be carried out aiming at nucleic acids of different targets, and a molecular detection result is obtained.
The freeze-drying protective agent provided by the invention can be prepared with components required by fluorescent PCR, can be matched with any necessary components required by PCR detection, and can be freeze-dried to prepare a PCR amplification reagent; the PCR amplification reagent not only can realize normal-temperature transportation and preservation, but also can be prepared by only re-dissolving ddH 2 O and then adding sample nucleic acid when in use. Compared with the prior art, the PCR amplification reagent has the following advantages: 1) The normal temperature preservation and transportation are realized, and the transportation cost is reduced; 2) Can be matched with various primer probes for freeze-drying, and has strong applicability.
Drawings
FIG. 1 is a schematic view of the appearance of the amplification reagents of the present invention after lyoprotection.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention more apparent, the present invention will be described in further detail with reference to the following detailed description. It should be understood that the detailed description is intended to illustrate the invention, and not to limit the invention.
The lyoprotectant is used for protecting the activity of specific enzymes and other components in the molecular detection reagent from being destroyed, and the excellent lyoprotectant should have various protecting characteristics, and any single protecting agent cannot have all protecting characteristics based on the molecular structure and the performance of active substances such as enzymes, so that the activity of various components needs to be maintained by taking into consideration the protecting agents of various components.
The invention provides a freeze-drying protective agent of a molecular detection reagent, which comprises the following components in concentration: 80-120 mg/mL of trehalose, 80-120 mg/mL of sucrose, 2-5 wt% of mannitol, 3-7 mmol/L of dextran, 0.5-6 mg/mL of polylactic acid-glycolic acid copolymer, 5-20 mg/mL of polylysine, 800.3-0.7 wt% of tween-800.3, 0.05-0.25 mg/mL of dithiothreitol and 5-20 mg/mL of vitamin combination, wherein the vitamin combination comprises one or more of vitamin B, vitamin C, vitamin D and vitamin K.
Preferably, among the above components: trehalose 100mg/mL, sucrose 100mg/mL, mannitol 4wt%, dextran 5mmol/L, polylactic acid-glycolic acid copolymer 4mg/mL, polylysine 10mg/mL, tween-800.5 wt%, dithiothreitol 0.15mg/mL, and vitamin combination 15mg/mL.
The principle of the invention for selecting the components is as follows:
trehalose and sucrose are non-reducing sugars, which can prevent the secondary structure of protein from changing, and form a layer of putative hydration film with the enzyme surface due to the rich hydroxyl, thus protecting the bonding position of hydrogen bond from being directly exposed in the surrounding environment, preventing protein from denaturation due to freeze-drying, and playing a significant role in the extension and aggregation of protein polypeptide chains during freeze-drying treatment and storage period. Mannitol is used as a framework agent, amorphous mannitol plays a role in protein stability, and amorphous structures with concentration below 1% prevent aggregation of protein molecules. Dextran can raise the glass transition temperature of the product and prevent the activity of the induced enzyme from being destroyed due to collapse of the product. The polylactic acid-glycolic acid copolymer has higher glass transition temperature as a cryoprotectant of the enzyme, and the amorphous structure of the copolymer can play a role in dispersing to prevent the aggregation of the enzyme. The polylysine is epsilon-polylysine, has good sterilization capability and thermal stability, and can also improve the glass transition temperature. The dithiothreitol is used as a micromolecular organic reducing agent, can reduce the oxidation-reduction potential of a reaction system in the freeze-drying process and after freeze-drying and re-melting, prevents intra-molecular or intermolecular disulfide bonds of proteins formed between cysteines in the proteins, and can not reduce the disulfide bonds embedded in the inside of a protein structure (the solvent is inaccessible), so that the property of the enzyme is well ensured not to be changed. Meanwhile, the vitamin combination can utilize the space structure of small molecules of the vitamin combination, and is matched with trehalose and sucrose to completely block the functional domain of the enzyme protein, so that the stability of the enzyme in the freeze-drying and storage processes is improved, and the synergistic effect of the components protects the activity of the enzyme serving as a main component.
The invention also provides a freeze-drying protection method of the molecular detection reagent, which comprises the following steps:
S1, preparing a molecular detection system and a freeze-drying protective agent to obtain a molecular detection reagent;
S2, placing the molecular detection reagent at the temperature of minus 30 ℃ to minus 20 ℃ for pre-freezing treatment for 1 to 3 hours, and then cooling to minus 50 ℃ to minus 40 ℃ at the speed of 1.0 to 1.5 ℃/min for freezing for 1 to 3 hours;
S3, starting a vacuum pump, placing the frozen molecular detection reagent in a vacuum environment of 5-50 Pa for treatment for 1-3 hours, then raising the temperature to-40 to-35 ℃ at a speed of 0.3-0.8 ℃/min for 4-7 hours, then raising the temperature to-30 to-25 ℃ at a speed of 0.3-0.8 ℃/min for 3-5 hours, and then raising the temperature of the PCR amplification reagent to 20-30 ℃ to obtain the freeze-dried PCR amplification reagent.
In the present invention, the molecular detection system includes reagents for fluorescent PCR detection of nucleic acid molecules, such as hot start Taq enzyme, UNG enzyme, reverse transcription MMLV enzyme, dNTP, and primers, probes and buffers.
The molecular detection reagent obtained by the freeze-drying protection method is packaged in eight connecting pipes, and has the appearance shown in figure 1 and good freeze-drying form.
Example 1
80Mg/mL of trehalose, 120mg/mL of sucrose, 2wt% of mannitol, 7mmol/L of dextran, 6mg/mL of polylactic acid-glycolic acid copolymer, 5mg/mL of polylysine, 800.7wt% of tween-dithiothreitol, 0.15mg/mL of vitamin B and 5mg/mL.
Example 2
100Mg/mL of trehalose, 100mg/mL of sucrose, 4wt% of mannitol, 5mmol/L of dextran, 4mg/mL of polylactic acid-glycolic acid copolymer, 10mg/mL of polylysine, 800.5wt% of tween-35, 0.15mg/mL of dithiothreitol, 5mg/mL of vitamin B and 10mg/mL of vitamin C.
Example 3
Trehalose 80mg/mL, sucrose 80mg/mL, mannitol 5wt%, dextran 3mmol/L, polylactic acid-glycolic acid copolymer 0.5mg/mL, polylysine 20mg/mL, tween-800.3 wt%, dithiothreitol 0.15mg/mL, vitamin B5 mg/mL, vitamin C10 mg/mL, and vitamin D5 mg/mL.
Example 4
90Mg/mL of trehalose, 110mg/mL of sucrose, 3wt% of mannitol, 6mmol/L of dextran, 5mg/mL of polylactic acid-glycolic acid copolymer, 8mg/mL of polylysine, 800.5wt% of tween-35, 0.15mg/mL of dithiothreitol, 5mg/mL of vitamin B, 5mg/mL of vitamin C, 5mg/mL of vitamin D and 5mg/mL of vitamin K.
Comparative example 1
Trehalose 80mg/mL, sucrose 120mg/mL, mannitol 2wt%, dextran 7mmol/L, polylactic acid-glycolic acid copolymer 6mg/mL, tween-800.7 wt%, dithiothreitol 0.15mg/mL, and vitamin B5 mg/mL.
Comparative example 2
Trehalose 80mg/mL, sucrose 120mg/mL, mannitol 2wt%, dextran 7mmol/L, polylysine 5mg/mL, tween-800.7 wt%, dithiothreitol 0.15mg/mL, vitamin B5 mg/mL, and vitamin C10 mg/mL.
Comparative example 3
Trehalose 80mg/mL, sucrose 120mg/mL, mannitol 2wt%, dextran 7mmol/L, tween-800.7 wt%, dithiothreitol 0.15mg/mL, vitamin B5 mg/mL, vitamin C10 mg/mL, and vitamin D5 mg/mL.
Comparative example 4
90Mg/mL of trehalose, 110mg/mL of sucrose, 3wt% of mannitol, 6mmol/L of dextran, 5mg/mL of polylactic acid-glycolic acid copolymer, 8mg/mL of polylysine, 800.5wt% of tween-35, 0.15mg/mL of dithiothreitol, 10mg/mL of vitamin B, 10mg/mL of vitamin C, 10mg/mL of vitamin D and 10mg/mL of vitamin K.
Comparative example 5
Trehalose 80mg/mL, sucrose 120mg/mL, mannitol 2wt%, dextran 7mmol/L, polylactic acid-glycolic acid copolymer 6mg/mL, polylysine 5mg/mL, tween-800.7 wt%, dithiothreitol 0.15mg/mL.
Comparative example 6
80Mg/mL of trehalose, 120mg/mL of sucrose, 2wt% of mannitol, 7mmol/L of dextran, 6mg/mL of polylactic acid-glycolic acid copolymer, 5mg/mL of polylysine, 800.7wt% of tween-5, 5mg/mL of vitamin B and 10mg/mL of vitamin C.
Experimental example
Taking a Hepatitis B Virus (HBV) nucleic acid determination system as an experimental example, the freeze-drying effects of the molecular detection reagents prepared by the freeze-drying protection method provided by the invention in the above examples 1-4 are respectively tested, and the test stages are respectively from the aspects of appearance, resolubility and accelerated stability: the test performance after 3 days, 9 days, 15 days and 21 days was examined at 45 ℃. In addition, under the preservation condition of 2-8 ℃, positive standard substances are detected in 6, 12, 15, 18, 21, 24 and 27 months respectively, and the Ct value change is observed by checking the real-time stability effect brought by the freeze-dried detection reagent.
According to a PCR amplification system: a lyophilized reagent was prepared from the amplification buffer 1X, hot start Taq enzyme 0.05U/. Mu. L, UNG enzyme 0.0003U/. Mu. L, dNTP. Mu.mol/L and examples 1-4 and comparative examples 1-6, respectively. Positive standard: initial viral content 5.8X10 3 copy/ml, stepone plus instrument determines Ct value 24.32.
1. Appearance and resolubility
The lyophilized preparation components and the PCR amplification system of examples 1-4 were lyophilized, and each example was filled with two lyophilized test reagents in eight-tube, and the lyophilized state was shown in FIG. 1, which revealed that the lyophilized reagents provided in examples 1-4 all had a good loose state. The lyophilized reagents of examples 1 to 4 and comparative examples 1 to 6 were taken in eight-tube and ddH 2 O was added for reconstitution, and the total dissolution time was calculated. The appearance and the re-solubility index are shown in Table 1.
Table 1:
2. stability acceleration test
The results of the measurements after 3 days, 9 days, 15 days and 21 days at 45℃in examples 1 to 4 and comparative examples 1 to 3 are shown in Table 2.
Table 2:
The acceptable standard is based on Ct value being less than or equal to 1.60 (absolute deviation is not more than 0.5 logarithmic order of magnitude when detecting standard or reference according to the specification of standard kit YY/T1182-2010 for nucleic acid amplification detection), as can be seen from Table 2, examples 1-4 all meet the requirements. However, the Ct value in example 2 is preferably the smallest. Comparative examples 1-3 exhibited Ct value post-roll values that were significantly unacceptable after 15 days or 21 days, respectively. From this, the freeze-dried agent of comparative examples 1 to 3 showed poor detection accuracy after reconstitution at 45℃storage conditions, respectively, and thus the freeze-dried agent showed poor high temperature resistance in the absence of polylactic acid-glycolic acid copolymer and/or polylysine. The Ct value does not change significantly when the vitamin combination content of comparative example 4 is increased relative to example 4; however, when the vitamin content of comparative example 5 is reduced relative to example 1, the Ct value is significantly increased after the push, and it is seen that the vitamin helps to improve the stability of the enzyme during lyophilization and storage. Comparative example 6 when dithiothreitol was not added, the Ct value was significantly increased after the push, and it was found that dithiothreitol helped to promote the stability of the enzyme during lyophilization and preservation.
3. Positive standard detection
The results of the tests after 6, 12, 15, 18, 21, 24 and 27 months under the conditions of 2-8deg.C for examples 1-4 and comparative examples 1-6 are shown in Table 3. And (3) according to the criterion that the Ct value is less than or equal to 1.60 after the acceptance criterion, detecting that the Ct value is qualified in the range in real time.
Table 3:
The acceptable standard is based on Ct value being less than or equal to 1.60, and it is clear from Table 3 that the test results of examples 1-4 are all satisfactory within 24 months under the preservation condition of 2-8 ℃. However, the component of example 2 is preferable because the post-detection Ct value in the positive standard is the smallest after 27 months in example 2. Also comparative examples 1-3 and comparative example 6, respectively, exhibited Ct values after 21 months that were significantly disqualified. As can be seen, the lyophilization agents of comparative examples 1-3 and comparative example 6 can be stored for only 18 months at 2-8deg.C, which is significantly shorter than examples 1-4, and particularly example 2, and thus the storage time of the lyophilization agent can be significantly shortened even at lower temperatures in the absence of polylactic acid-glycolic acid copolymer, polylysine, and dithiothreitol.
While the lyoprotectant has been described in detail and with reference to specific embodiments thereof, the description of the embodiments is illustrative and not restrictive, and several examples can be enumerated in the limited scope, so that variations and optimization of the present invention will be within the scope of the present invention, without departing from the spirit and scope of the present invention.
Claims (1)
1. The application of the molecular detection lyoprotectant in preparing fluorescent PCR amplification reagents is characterized in that the molecular detection lyoprotectant consists of the following components in concentration: 100mg/mL of trehalose, 100mg/mL of sucrose, 4wt% of mannitol, 5mmol/L of dextran, 4mg/mL of polylactic acid-glycolic acid copolymer, 10mg/mL of polylysine, 0.5wt% of tween-80, 0.15 mg/mL of dithiothreitol, 5 mg/mL of vitamin B and 10mg/mL of vitamin C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111125384.6A CN113801926B (en) | 2021-09-23 | 2021-09-23 | Freeze-drying protective agent of molecular detection reagent and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111125384.6A CN113801926B (en) | 2021-09-23 | 2021-09-23 | Freeze-drying protective agent of molecular detection reagent and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113801926A CN113801926A (en) | 2021-12-17 |
| CN113801926B true CN113801926B (en) | 2024-07-23 |
Family
ID=78896561
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111125384.6A Active CN113801926B (en) | 2021-09-23 | 2021-09-23 | Freeze-drying protective agent of molecular detection reagent and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113801926B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118600056A (en) * | 2024-06-21 | 2024-09-06 | 郑州人民医院(郑州人民医院医疗管理中心) | A multiplex fluorescence PCR kit and method for simultaneously detecting multiple pathogenic microorganisms and drug-resistant genes |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103893133A (en) * | 2012-12-26 | 2014-07-02 | 李和伟 | Formula and preparing method of freeze-dried excipient preparation |
| CN103911367A (en) * | 2014-03-20 | 2014-07-09 | 中国农业大学 | Freeze-drying protective agent of nucleic acid amplification reaction reagents and freeze-drying method |
| CN105535981A (en) * | 2015-12-29 | 2016-05-04 | 瑞普（保定）生物药业有限公司 | Heat-resisting protective agent, poultry heat-resisting protective agent live vaccine and preparation method of poultry heat-resisting protective agent live vaccine |
| CN106591432A (en) * | 2016-10-19 | 2017-04-26 | 珠海丽珠试剂股份有限公司 | Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent |
| CN110747263A (en) * | 2019-11-12 | 2020-02-04 | 南京黎明生物制品有限公司 | Freeze-drying additive, fluorescent PCR reaction mixture dry powder and preparation method |
| CN112048545A (en) * | 2020-09-10 | 2020-12-08 | 上海创宏生物科技有限公司 | Freeze-drying protective agent, PCR amplification reagent, freeze-drying method and application thereof |
| CN112106765A (en) * | 2020-10-22 | 2020-12-22 | 郑州佐爵生物科技有限公司 | Stem cell protection solution and stem cell preservation method |
| CN112626175A (en) * | 2020-12-26 | 2021-04-09 | 杭州百迈生物股份有限公司 | Freeze-drying protective agent of SNP detection reagent and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2154164C (en) * | 1993-02-23 | 2005-11-22 | Jeffrey L. Cleland | Excipient stabilization of polypeptides treated with organic solvents |
| US10905113B2 (en) * | 2015-11-12 | 2021-02-02 | Regents Of The University Of Minnesota | Compositions and method for storing liquid biospecimens |
| CN111662902A (en) * | 2020-06-29 | 2020-09-15 | 诺迦(杭州)生物工程有限公司 | Freeze-drying protective agent and application thereof in nucleic acid amplification reagent |
-
2021
- 2021-09-23 CN CN202111125384.6A patent/CN113801926B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103893133A (en) * | 2012-12-26 | 2014-07-02 | 李和伟 | Formula and preparing method of freeze-dried excipient preparation |
| CN103911367A (en) * | 2014-03-20 | 2014-07-09 | 中国农业大学 | Freeze-drying protective agent of nucleic acid amplification reaction reagents and freeze-drying method |
| CN105535981A (en) * | 2015-12-29 | 2016-05-04 | 瑞普（保定）生物药业有限公司 | Heat-resisting protective agent, poultry heat-resisting protective agent live vaccine and preparation method of poultry heat-resisting protective agent live vaccine |
| CN106591432A (en) * | 2016-10-19 | 2017-04-26 | 珠海丽珠试剂股份有限公司 | Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent |
| CN110747263A (en) * | 2019-11-12 | 2020-02-04 | 南京黎明生物制品有限公司 | Freeze-drying additive, fluorescent PCR reaction mixture dry powder and preparation method |
| CN112048545A (en) * | 2020-09-10 | 2020-12-08 | 上海创宏生物科技有限公司 | Freeze-drying protective agent, PCR amplification reagent, freeze-drying method and application thereof |
| CN112106765A (en) * | 2020-10-22 | 2020-12-22 | 郑州佐爵生物科技有限公司 | Stem cell protection solution and stem cell preservation method |
| CN112626175A (en) * | 2020-12-26 | 2021-04-09 | 杭州百迈生物股份有限公司 | Freeze-drying protective agent of SNP detection reagent and application |
Non-Patent Citations (2)
| Title |
|---|
| ε-聚赖氨酸在牛胚胎玻璃化冷冻中的保护作用;钟原等;中国兽医学报;第37卷(第03期);摘要、第554-558页 * |
| ε-聚赖氨酸对小鼠胚胎玻璃化冷冻的影响;高妍等;中国兽医学报;第40卷(第06期);摘要、第1237-1240页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113801926A (en) | 2021-12-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112048545B (en) | Freeze-drying protective agent, PCR amplification reagent, freeze-drying method and application thereof | |
| Mitchell et al. | Ice-recrystallization inhibiting polymers protect proteins against freeze-stress and enable glycerol-free cryostorage | |
| JP3282819B2 (en) | Stabilized enzyme composition for nucleic acid amplification | |
| EP2294222B1 (en) | Freeze-dried compositions for pcr and rt-pcr | |
| CN108913758B (en) | A freeze-drying method for fluorescent PCR amplification reagent and its application | |
| CN114015758B (en) | Freeze-drying protective agent, fluorescent PCR detection kit and freeze-drying process | |
| US20230227927A1 (en) | Freeze-Dried Composition | |
| CN113801926B (en) | Freeze-drying protective agent of molecular detection reagent and application thereof | |
| KR102021605B1 (en) | Lyophilization method of the composition for multi-target nucleic acid sequence amplification reaction | |
| MX2011011616A (en) | A dried and stabilized ready-to-use composition containing nucleic acid polymerization enzymes for molecular biology applications. | |
| WO2024055479A1 (en) | Microsphere formulation for nucleic acid amplification, amplification method, and use in joint detection | |
| CN110923303A (en) | Universal freeze-drying protective agent for multiplex fluorescence PCR and application thereof | |
| JP2010529850A (en) | Composition | |
| CN114317700B (en) | Freeze-drying protective agent for fluorescent PCR (polymerase chain reaction) reagent, freeze-drying method and application | |
| CN116121339A (en) | Lyoprotectant and its application in the preparation of PCR freeze-dried reagent | |
| JP2015092870A (en) | Nucleic acid amplification reagent which can be preserved for long term | |
| WO2024164676A1 (en) | Composition for improving stability of pcr air-dried reagent, and use thereof | |
| CN116004780A (en) | Drying method of PCR reagent and application thereof | |
| CN117535389A (en) | PCR detection reagent freeze-drying protection composition | |
| CN119020460A (en) | Freeze-dried protective agent, freeze-dried nucleic acid reaction reagent and freeze-drying method thereof | |
| CN117625758A (en) | Freeze-drying protective agent for PCR (polymerase chain reaction) reagent, application of freeze-drying protective agent, mixed reagent, and freeze-drying method and product of mixed reagent | |
| CN118360367A (en) | Nucleic acid detection reagent freeze-drying protection composition | |
| WO2023142130A1 (en) | Application of improved enzyme pellet in target nucleic acid detection | |
| CN117887807A (en) | Freeze-drying protective agent capable of improving full premix performance of RNA detection reagent and application thereof | |
| CN119082276A (en) | Freeze-dried protective agent for PCR premix, freeze-dried reagent, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |