CN113801926A - Freeze-drying protective agent for molecular detection reagent and application thereof - Google Patents
Freeze-drying protective agent for molecular detection reagent and application thereof Download PDFInfo
- Publication number
- CN113801926A CN113801926A CN202111125384.6A CN202111125384A CN113801926A CN 113801926 A CN113801926 A CN 113801926A CN 202111125384 A CN202111125384 A CN 202111125384A CN 113801926 A CN113801926 A CN 113801926A
- Authority
- CN
- China
- Prior art keywords
- molecular detection
- freeze
- detection reagent
- vitamin
- reagent
- 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.)
- Granted
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 59
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 57
- 238000004108 freeze drying Methods 0.000 title claims abstract description 39
- 239000003223 protective agent Substances 0.000 title claims abstract description 16
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 19
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 19
- 229930195725 Mannitol Natural products 0.000 claims abstract description 19
- 108010039918 Polylysine Proteins 0.000 claims abstract description 19
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 19
- 229930006000 Sucrose Natural products 0.000 claims abstract description 19
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 19
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 19
- 229920001577 copolymer Polymers 0.000 claims abstract description 19
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims abstract description 19
- 239000000594 mannitol Substances 0.000 claims abstract description 19
- 235000010355 mannitol Nutrition 0.000 claims abstract description 19
- 239000005720 sucrose Substances 0.000 claims abstract description 19
- 229920002307 Dextran Polymers 0.000 claims abstract description 18
- 229920000656 polylysine Polymers 0.000 claims abstract description 18
- 229930003231 vitamin Natural products 0.000 claims abstract description 17
- 235000013343 vitamin Nutrition 0.000 claims abstract description 17
- 239000011782 vitamin Substances 0.000 claims abstract description 17
- 229940088594 vitamin Drugs 0.000 claims abstract description 17
- 150000003722 vitamin derivatives Chemical class 0.000 claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims description 21
- 108090000790 Enzymes Proteins 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 15
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 14
- 238000012408 PCR amplification Methods 0.000 claims description 14
- 229930003270 Vitamin B Natural products 0.000 claims description 9
- 235000019156 vitamin B Nutrition 0.000 claims description 9
- 239000011720 vitamin B Substances 0.000 claims description 9
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 7
- 229930003268 Vitamin C Natural products 0.000 claims description 7
- 235000019154 vitamin C Nutrition 0.000 claims description 7
- 239000011718 vitamin C Substances 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 6
- 229930003316 Vitamin D Natural products 0.000 claims description 5
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 claims description 5
- 238000007710 freezing Methods 0.000 claims description 5
- 230000008014 freezing Effects 0.000 claims description 5
- 239000013615 primer Substances 0.000 claims description 5
- 239000002987 primer (paints) Substances 0.000 claims description 5
- 235000019166 vitamin D Nutrition 0.000 claims description 5
- 239000011710 vitamin D Substances 0.000 claims description 5
- 150000003710 vitamin D derivatives Chemical class 0.000 claims description 5
- 229940046008 vitamin d Drugs 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 241000713869 Moloney murine leukemia virus Species 0.000 claims description 3
- 229930003448 Vitamin K Natural products 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 claims description 3
- 235000019168 vitamin K Nutrition 0.000 claims description 3
- 239000011712 vitamin K Substances 0.000 claims description 3
- 150000003721 vitamin K derivatives Chemical class 0.000 claims description 3
- 229940046010 vitamin k Drugs 0.000 claims description 3
- 102100034343 Integrase Human genes 0.000 claims description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 3
- 238000003752 polymerase chain reaction Methods 0.000 abstract description 9
- 230000000052 comparative effect Effects 0.000 description 18
- 150000007523 nucleic acids Chemical class 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 102000039446 nucleic acids Human genes 0.000 description 11
- 108020004707 nucleic acids Proteins 0.000 description 11
- 230000000694 effects Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 7
- 230000003321 amplification Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- 229920000136 polysorbate Polymers 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000009477 glass transition Effects 0.000 description 3
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 2
- 229930003571 Vitamin B5 Natural products 0.000 description 2
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 2
- 229960002079 calcium pantothenate Drugs 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 235000009492 vitamin B5 Nutrition 0.000 description 2
- 239000011675 vitamin B5 Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 208000005156 Dehydration Diseases 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000013213 extrapolation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- RMDJVOZETBHEAR-LQYWTLTGSA-N vitamin D5 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CC[C@@H](CC)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C RMDJVOZETBHEAR-LQYWTLTGSA-N 0.000 description 1
- 150000003718 vitamin K5 derivatives Chemical class 0.000 description 1
Images
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, 0.7-0.7 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 together with components required by fluorescent PCR (polymerase chain reaction), and is freeze-dried to prepare the molecular detection reagent, so that the molecular detection reagent can be transported and stored at normal temperature, and the application range is wide.
Description
Technical Field
The invention belongs to the technical field of biomolecule 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 analyzing the functions of nucleic acids from the level of nucleic acid structure, replication, transcription or translation by directly probing the existence state or defects of nucleic acids using the theories and techniques of molecular biology. Its target molecule is DNA or RNA, reflecting the structure and function of nucleic acid. The detected gene includes endogenous gene for diagnosing the presence of pathological changes in the gene and exogenous gene for diagnosing the presence of pathogen infection.
The Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying specific DNA fragments, and can be regarded as the special replication of biomolecules in vitro, and the biggest characteristic of the PCR is that trace nucleic acid can be greatly increased. 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, primers, probes and the like of the PCR amplification reagent need to be stored in a freezing environment at the temperature of-20 ℃, repeated freezing and thawing has certain influence on the performance of the reagent, and the low-temperature storage of the reagent has higher requirements on the transportation, storage, use processes and the like. In addition, the liquid detection reagent needs cold chain transportation, the transportation cost is high, and the performance of the reagent is easily reduced due to improper storage temperature.
The freeze drying technology is a novel efficient drying technology which enables solid ice which is frozen in a mixture to be directly sublimated into a gas state without melting into liquid water, finally removes water and retains other effective components. Because the stability of the bioactive raw material in the solid dry powder state is far higher than that in the liquid state, the long-term storage and stable transportation of each component in the reaction reagent system at room temperature can be realized through freeze-drying dehydration treatment.
The defects of the current freeze-drying process of the diagnostic reagent are as follows: 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 at the later stage; 4) additives such as lyoprotectants inhibit subsequent amplification reactions and the like.
Therefore, there is a need for a lyoprotectant for molecular detection reagents and applications thereof.
Disclosure of Invention
The present invention is directed to a lyoprotectant for molecular detection reagents and applications thereof, which at least solve one of the problems described in the prior art.
The first purpose of the invention is to provide a molecular detection lyoprotectant, 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, 0.7 wt% of tween-800.3 and 0.05-0.25 mg/mL of dithiothreitol.
Further, the components are as follows: trehalose 100mg/mL, sucrose 100mg/mL, mannitol 4 wt%, dextran 5mmol/L, polylactic acid-glycolic acid copolymer 4mg/mL, polylysine 10mg/mL, tween-800.5 wt%, dithiothreitol 0.15 mg/mL.
Further, the components also comprise 5-20 mg/mL of vitamin combination, and the vitamin combination comprises one or more of vitamin B, vitamin C, vitamin D and vitamin K.
The second purpose of the invention is to provide a molecular detection reagent, which comprises a molecular detection system and the molecular detection lyoprotectant. Further, the molecular detection system comprises hot start Taq enzyme, UNG enzyme and dNTP. Preferably, the molecular detection system further comprises a reverse transcription MMLV enzyme, a primer, a probe and a buffer.
The invention also provides a freeze-drying protection method of the molecular detection reagent, which comprises the following steps:
1) preparing a molecular detection system and a freeze-drying protective agent to obtain a molecular detection reagent;
2) placing the molecular detection reagent at a temperature of-30 to-20 ℃ for pre-freezing for 1 to 3 hours, and then cooling to-50 to-40 ℃ at a speed of 1.0 to 1.5 ℃/min for 1 to 3 hours;
3) and (3) placing the frozen molecular detection reagent in a vacuum environment of 5-50 Pa for processing for 1-3 h, raising the temperature to-40-35 ℃ at the speed of 0.3-0.8 ℃/min, keeping the temperature for 4-7 h, raising the temperature to-30-25 ℃ at the speed of 0.3-0.8 ℃/min, keeping the temperature for 3-5 h, and raising the temperature of the PCR amplification reagent to 20-30 ℃ to obtain the freeze-dried PCR amplification reagent.
The invention also aims to provide the molecular detection reagent prepared by the freeze-drying protection method.
Another object of the present invention is to provide the use of the above molecular detection reagent in fluorescence PCR detection. The method can perform fluorescent PCR amplification detection aiming at nucleic acids of different targets to obtain a molecular detection result.
The freeze-drying protective agent provided by the invention can be prepared together with components required by fluorescence PCR, and can also be matched with any necessary components required by PCR detection, and freeze-drying is carried out to prepare a PCR amplification reagent; the PCR amplification reagent can be transported and stored at normal temperature, and only needs ddH when in use2And O redissolving, and then adding the sample nucleic acid. Compared with the prior art, the PCR amplification reagent has the following advantages: 1) normal temperature storage 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 diagram showing the freeze-dried and protected appearance of the amplification reagent of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the present invention is further described in detail with reference to the following detailed description. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The freeze-drying protective agent is used for protecting the activity of specific enzymes and other components in a molecular detection reagent from being damaged, a good freeze-drying protective agent should have multiple protective characteristics, and any single protective agent cannot have all the protective characteristics based on the molecular structure and the performance of active substances such as the enzymes, so that the use of the protective agent with multiple components is considered to maintain the activity 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, 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 4 wt%, dextran 5mmol/L, polylactic acid-glycolic acid copolymer 4mg/mL, polylysine 10mg/mL, tween-800.5 wt%, dithiothreitol 0.15mg/mL, vitamin combination 15 mg/mL.
The principle of selecting the components is as follows:
trehalose and sucrose are non-reducing sugars that can prevent changes in the secondary structure of proteins, because they are rich in hydroxyl groups that can form a putative hydrated film with the enzyme surface, which can protect the bonding sites of hydrogen bonds from direct exposure to the surrounding environment, prevent denaturation of proteins due to lyophilization, and contribute significantly to the stretching and aggregation of protein polypeptide chains during lyophilization processing and during storage. Mannitol acts as a backbone, amorphous mannitol contributes to protein stability, and amorphous structures below 1% prevent aggregation of protein molecules. Dextran can raise the glass transition temperature of the product and prevent the enzyme activity induced by product collapse from being destroyed. The polylactic acid-glycolic acid copolymer as a cryoprotectant of the enzyme has higher glass transition temperature, and the amorphous structure of the polylactic acid-glycolic acid copolymer can also play a role in dispersing to prevent the aggregation of the enzyme. The polylysine is epsilon-polylysine, has good bactericidal ability and thermal stability, and can also improve the glass transition temperature. Dithiothreitol, as a small-molecule organic reducing agent, can reduce the redox potential of a reaction system in the freeze-drying process and after freeze-drying and thawing, prevent intra-molecular or intermolecular disulfide bonds of proteins formed between cysteines in the proteins, and can not reduce disulfide bonds embedded in the protein structure (inaccessible to a solvent), thereby well ensuring that the properties of the enzyme are not changed. Meanwhile, the vitamin combination can utilize the spatial structure of self small molecules to be matched with trehalose and sucrose, completely block the functional domain of zymoprotein, improve the stability of zymoprotein in the freeze-drying and storage processes, and protect the activity of zymoprotein serving as a main component through the synergistic effect of the components.
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 a temperature of-30 to-20 ℃ for pre-freezing for 1 to 3 hours, and then cooling to-50 to-40 ℃ at a speed of 1.0 to 1.5 ℃/min 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 processing for 1-3 h, raising the temperature to-40-35 ℃ at the speed of 0.3-0.8 ℃/min, keeping the temperature for 4-7 h, raising the temperature to-30-25 ℃ at the speed of 0.3-0.8 ℃/min, keeping the temperature for 3-5 h, and 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 comprises reagents for fluorescent PCR detection of nucleic acid molecules, such as hot-start Taq enzyme, UNG enzyme, reverse-transcriptase MMLV enzyme, dNTP, and primers, probes and buffers.
The molecular detection reagent obtained by the freeze-drying protection method is subpackaged into eight connecting tubes, the appearance is shown in figure 1, and the freeze-drying shape is good.
Example 1
80mg/mL of trehalose, 120mg/mL of sucrose, 2 wt% of mannitol, 7mmol/L of dextran, 6mg/mL of polylactic acid-glycolic acid copolymer, 5mg/mL of polylysine, 800.7 wt% of tween, 0.15mg/mL of dithiothreitol and 5mg/mL of vitamin B.
Example 2
Trehalose 100mg/mL, sucrose 100mg/mL, mannitol 4 wt%, dextran 5mmol/L, polylactic acid-glycolic acid copolymer 4mg/mL, polylysine 10mg/mL, tween-800.5 wt%, dithiothreitol 0.15mg/mL, vitamin B5 mg/mL, vitamin C10 mg/mL.
Example 3
80mg/mL of trehalose, 80mg/mL of sucrose, 5 wt% of mannitol, 3mmol/L of dextran, 0.5mg/mL of polylactic acid-glycolic acid copolymer, 20mg/mL of polylysine, 800.3 wt% of tween, 0.15mg/mL of dithiothreitol, 5mg/mL of vitamin B, 10mg/mL of vitamin C and 5mg/mL of vitamin D.
Example 4
Trehalose 90mg/mL, sucrose 110mg/mL, mannitol 3 wt%, dextran 6mmol/L, polylactic acid-glycolic acid copolymer 5mg/mL, polylysine 8mg/mL, tween-800.5 wt%, dithiothreitol 0.15mg/mL, vitamin B5 mg/mL, vitamin C5 mg/mL, vitamin D5 mg/mL, vitamin K5 mg/mL.
Comparative example 1
80mg/mL of trehalose, 120mg/mL of sucrose, 2 wt% of mannitol, 7mmol/L of dextran, 6mg/mL of polylactic acid-glycolic acid copolymer, 0.15mg/mL of tween-800.7, 0.15mg/mL of dithiothreitol and 5mg/mL of vitamin B.
Comparative example 2
80mg/mL of trehalose, 120mg/mL of sucrose, 2 wt% of mannitol, 7mmol/L of dextran, 5mg/mL of polylysine, 78 wt% of tween-800.7, 0.15mg/mL of dithiothreitol, 5mg/mL of vitamin B and 10mg/mL of vitamin C.
Comparative example 3
80mg/mL of trehalose, 120mg/mL of sucrose, 2 wt% of mannitol, 7mmol/L of dextran, 800.7 wt% of tween, 0.15mg/mL of dithiothreitol, 5mg/mL of vitamin B, 10mg/mL of vitamin C and 5mg/mL of vitamin D.
Comparative example 4
Trehalose 90mg/mL, sucrose 110mg/mL, mannitol 3 wt%, dextran 6mmol/L, polylactic acid-glycolic acid copolymer 5mg/mL, polylysine 8mg/mL, tween-800.5 wt%, dithiothreitol 0.15mg/mL, vitamin B10 mg/mL, vitamin C10 mg/mL, vitamin D10 mg/mL, vitamin K10 mg/mL.
Comparative example 5
80mg/mL of trehalose, 120mg/mL of sucrose, 2 wt% of mannitol, 7mmol/L of dextran, 6mg/mL of polylactic acid-glycolic acid copolymer, 5mg/mL of polylysine, 78 wt% of tween-800.7 and 0.15mg/mL of dithiothreitol.
Comparative example 6
80mg/mL of trehalose, 120mg/mL of sucrose, 2 wt% of mannitol, 7mmol/L of dextran, 6mg/mL of polylactic acid-glycolic acid copolymer, 5mg/mL of polylysine, 800.7 wt% of tween, 5mg/mL of vitamin B and 10mg/mL of vitamin C.
Examples of the experiments
The freeze-drying effect of the molecular detection reagent prepared according to the freeze-drying protection method provided by the invention in the above examples 1-4 is tested by taking a Hepatitis B Virus (HBV) nucleic acid measurement system as an experimental example, and the test stages are respectively from appearance, re-solubility and accelerated stability: the test performance was examined at 45 ℃ after 3 days, 9 days, 15 days, and 21 days. And in addition, under the storage condition of 2-8 ℃, positive standard products are detected in 6, 12, 15, 18, 21, 24 and 27 months respectively, and the Ct value change of the positive standard products is observed by examining the real-time stability effect brought by the freeze-dried detection reagent.
According to the PCR amplification system: 1X amplification buffer, 0.05U/. mu. L, UNG enzyme, 0.0003U/. mu. L, dNTP 220. mu. mol/L hot start Taq enzyme lyophilized reagents were prepared as described in examples 1-4 and comparative examples 1-6, respectively. Positive standard substance: initial viral content 5.8X 103copy/ml, stepone plus instrument determination of Ct value 24.32.
1. Appearance and resolubility
The components of the freeze-drying agent in the components of examples 1-4 are freeze-dried with a PCR amplification system, two portions of the detection reagent obtained by freeze-drying are filled in eight connecting tubes in each example, the freeze-dried state is shown in figure 1, and it can be seen that the freeze-dried reagents provided in examples 1-4 are all in a better loose state. The lyophilized reagents of examples 1 to 4 and comparative examples 1 to 6 were taken in eight-connected tubes and added to ddH2And O, re-dissolving, and calculating the total dissolving time. The appearance and re-solubility indices are shown in Table 1.
Table 1:
2. accelerated stability test
The results of the tests of examples 1 to 4 and comparative examples 1 to 3 at 45 ℃ after 3 days, 9 days, 15 days and 21 days are shown in Table 2.
Table 2:
the acceptance criterion is determined by the Ct value of less than or equal to 1.60 (according to the stipulation of the standard nucleic acid amplification detection reagent (kit) YY/T1182-2010, the absolute deviation does not exceed 0.5 log-order when detecting the standard substance or the reference substance), as can be seen from Table 2, examples 1-4 all meet the requirements. However, it is preferable that the Ct value in example 2 is the smallest as a post-estimation value. Comparative examples 1-3 exhibited significant failures in Ct value post-push values after 15 days or 21 days, respectively. It can be seen that the freeze-dried agents of comparative examples 1 to 3 respectively show poor detection accuracy after being reconstituted under the storage condition of 45 ℃, and thus show poor high-temperature resistance in the absence of the polylactic acid-glycolic acid copolymer and/or the polylysine. Comparative example 4 when the vitamin combination content was increased relative to example 4, there was no significant change in Ct values; however, in comparative example 5, when the vitamin content is reduced compared with that in example 1, the Ct value is obviously increased after the prediction, and it can be seen that the vitamin is helpful for improving the stability of the enzyme in the processes of freeze-drying and storage. Comparative example 6 Ct values were significantly increased after extrapolation when dithiothreitol was not added, which is seen to help improve enzyme stability during lyophilization and storage.
3. Detection of Positive Standard
The test results of examples 1 to 4 and comparative examples 1 to 6 after 6, 12, 15, 18, 21, 24 and 27 months under the storage condition of 2 to 8 ℃ are shown in Table 3. And (5) determining that the Ct value is qualified within the range by the criterion that the Ct value is less than or equal to 1.60 after being deduced according to the acceptable standard.
Table 3:
the acceptance criterion is based on the Ct value of less than or equal to 1.60, and as can be seen from Table 3, the detection results of the examples 1 to 4 meet the requirements within 24 months under the storage condition of 2 to 8 ℃. However, the component in example 2 is preferable because the post-prediction value of the Ct value in the positive standard for detection after 27 months in example 2 is the smallest. Also, comparative examples 1 to 3 and comparative example 6 each exhibited a significant failure in the Ct value extrapolated after 21 months. It can be seen that the freeze-dried agents of comparative examples 1 to 3 and 6 can be stored for only 18 months under the storage condition of 2 to 8 ℃, which is obviously shorter than that of examples 1 to 4, particularly example 2, and therefore, the storage time of the freeze-dried agent can be obviously shortened even at a lower temperature in the absence of the polylactic acid-glycolic acid copolymer, the polylysine and the dithiothreitol.
The lyoprotectant has been described in detail with reference to specific embodiments thereof, but the description of the embodiments is illustrative and not restrictive, and several examples may be cited within the limits thereof, so that variations and modifications within the spirit and scope of the present technology should be considered within the scope of the present invention.
Claims (9)
1. A molecular detection freeze-drying protective agent is characterized by comprising 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, 0.7 wt% of tween-800.3 and 0.05-0.25 mg/mL of dithiothreitol.
2. The lyoprotectant of claim 1, wherein said component is: trehalose 100mg/mL, sucrose 100mg/mL, mannitol 4 wt%, dextran 5mmol/L, polylactic acid-glycolic acid copolymer 4mg/mL, polylysine 10mg/mL, tween-800.5 wt%, dithiothreitol 0.15 mg/mL.
3. The lyoprotectant according to claim 1, further comprising 5-20 mg/mL of a vitamin composition comprising one or more of vitamin B, vitamin C, vitamin D, and vitamin K.
4. A molecular assay reagent comprising a molecular assay system, further comprising the molecular assay lyoprotectant of any of claims 1-2.
5. The molecular detection reagent of claim 4, wherein the molecular detection system comprises a hot start Taq enzyme, a UNG enzyme and a dNTP.
6. The molecular detection reagent of claim 5, wherein the molecular detection system further comprises a reverse transcriptase MMLV enzyme, primers, probes, and buffers.
7. A method for protecting a molecular detection reagent from lyophilization according to any one of claims 4 to 6, comprising the steps of:
1) preparing a molecular detection system and a freeze-drying protective agent to obtain a molecular detection reagent;
2) placing the molecular detection reagent at a temperature of-30 to-20 ℃ for pre-freezing for 1 to 3 hours, and then cooling to-50 to-40 ℃ at a speed of 1.0 to 1.5 ℃/min for 1 to 3 hours;
3) and (3) placing the frozen molecular detection reagent in a vacuum environment of 5-50 Pa for processing for 1-3 h, raising the temperature to-40-35 ℃ at the speed of 0.3-0.8 ℃/min, keeping the temperature for 4-7 h, raising the temperature to-30-25 ℃ at the speed of 0.3-0.8 ℃/min, keeping the temperature for 3-5 h, and raising the temperature of the PCR amplification reagent to 20-30 ℃ to obtain the freeze-dried PCR amplification reagent.
8. A molecular detection reagent prepared by the lyophilization protection method according to claim 7.
9. Use of the molecular detection reagent according to any one of claims 4 to 6 or 8 in fluorescence PCR detection.
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 true CN113801926A (en) | 2021-12-17 |
| CN113801926B 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) |
Cited By (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 (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2154164A1 (en) * | 1993-02-23 | 1994-09-01 | Jeffrey L. Cleland | Excipient stabilization of polypeptides treated with organic solvents |
| 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 |
| US20170135333A1 (en) * | 2015-11-12 | 2017-05-18 | Regents Of The University Of Minnesota | Compositions and method for storing liquid biospecimens |
| 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 |
| 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 |
| 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 |
-
2021
- 2021-09-23 CN CN202111125384.6A patent/CN113801926B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2154164A1 (en) * | 1993-02-23 | 1994-09-01 | Jeffrey L. Cleland | Excipient stabilization of polypeptides treated with organic solvents |
| 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 |
| US20170135333A1 (en) * | 2015-11-12 | 2017-05-18 | Regents Of The University Of Minnesota | Compositions and method for storing liquid biospecimens |
| 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 |
| 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 |
| 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 (4)
| Title |
|---|
| PEDRO FONTE等: "Co-encapsulation of lyoprotectants improves the stability of protein-loaded PLGA nanoparticles upon lyophilization", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 496, no. 2, pages 850 - 862, XP029344049, DOI: 10.1016/j.ijpharm.2015.10.032 * |
| 何黎黎等: "PEG-PLGA嵌段共聚物空白纳米粒的冻干工艺筛选", 西南民族大学学报(自然科学版), vol. 41, no. 05, pages 566 - 570 * |
| 钟原等: "ε-聚赖氨酸在牛胚胎玻璃化冷冻中的保护作用", 中国兽医学报, vol. 37, no. 03, pages 554 - 558 * |
| 高妍等: "ε-聚赖氨酸对小鼠胚胎玻璃化冷冻的影响", 中国兽医学报, vol. 40, no. 06, pages 1237 - 1240 * |
Cited By (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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113801926B (en) | 2024-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mitchell et al. | Ice-recrystallization inhibiting polymers protect proteins against freeze-stress and enable glycerol-free cryostorage | |
| CN112048545B (en) | Freeze-drying protective agent, PCR amplification reagent, freeze-drying method and application thereof | |
| 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 | |
| US10905113B2 (en) | Compositions and method for storing liquid biospecimens | |
| Fayter et al. | Ice recrystallisation inhibiting polymers prevent irreversible protein aggregation during solvent-free cryopreservation as additives and as covalent polymer-protein conjugates | |
| CN107467013A (en) | A method for preparing a universal plasma matrix for quality control products that can be stored for a long time | |
| WO2024055479A1 (en) | Microsphere formulation for nucleic acid amplification, amplification method, and use in joint detection | |
| KR102021605B1 (en) | Lyophilization method of the composition for multi-target nucleic acid sequence amplification reaction | |
| CN113801926B (en) | Freeze-drying protective agent of molecular detection reagent and application thereof | |
| CN108070583B (en) | Application of hydroxypropyl-beta-cyclodextrin in preparation of nucleic acid freeze-drying protective agent | |
| US20200345000A1 (en) | Compositions and methods for storing liquid biospecimens | |
| WO2024221933A1 (en) | Protective agent and use thereof in preparation of air drying reagent for reverse transcription pcr | |
| CN114317700B (en) | Freeze-drying protective agent for fluorescent PCR (polymerase chain reaction) reagent, freeze-drying method and application | |
| CN116676422A (en) | A primer-probe combination for detecting bovine viral diarrhea virus, freeze-dried pellets and application thereof | |
| CN111961762A (en) | Fluorescent RT-PCR primer, probe, reagent and method for detecting coronavirus SARS-CoV-2 | |
| CN106701940A (en) | Method for detecting DNA of schistosoma through fluorescent probe | |
| WO2024164676A1 (en) | Composition for improving stability of pcr air-dried reagent, and use thereof | |
| CN109988857A (en) | For detecting two kinds of cryptococcal LAMP primer compositions and its application | |
| CN104611319A (en) | Method for improving thermal stability of liquid fructosaminase | |
| CN117587104A (en) | Freeze-drying protective agent applicable to fluorescent quantitative PCR (polymerase chain reaction) reagent, freeze-drying method and application | |
| CN114231605B (en) | Freeze-dried composition for nucleic acid amplification | |
| 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 | |
| CN116327961A (en) | Freeze-drying protective agent, NGAL antigen freeze-drying powder and preparation method 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 |