CN117143975A - Stabilizer for air-drying preservation of PCR reagent and air-drying preservation method - Google Patents
Stabilizer for air-drying preservation of PCR reagent and air-drying preservation method Download PDFInfo
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- CN117143975A CN117143975A CN202310939264.2A CN202310939264A CN117143975A CN 117143975 A CN117143975 A CN 117143975A CN 202310939264 A CN202310939264 A CN 202310939264A CN 117143975 A CN117143975 A CN 117143975A
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- air
- stabilizer
- drying
- pcr reagent
- pcr
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- 239000003381 stabilizer Substances 0.000 title claims abstract description 28
- 239000012807 PCR reagent Substances 0.000 title claims abstract description 27
- 238000007605 air drying Methods 0.000 title claims abstract description 26
- 238000004321 preservation Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 9
- 235000011187 glycerol Nutrition 0.000 claims abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 5
- 229920001213 Polysorbate 20 Polymers 0.000 abstract description 2
- 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 abstract description 2
- 229930006000 Sucrose Natural products 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 abstract description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 abstract description 2
- 239000005720 sucrose Substances 0.000 abstract description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 abstract 3
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- 238000013112 stability test Methods 0.000 description 8
- 238000004108 freeze drying Methods 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000003223 protective agent Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 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 description 2
- 239000004471 Glycine Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 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 description 2
- 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 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- -1 defoamers Chemical compound 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- 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 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920001218 Pullulan Polymers 0.000 description 1
- 239000004373 Pullulan Substances 0.000 description 1
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 description 1
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 1
- 241000222355 Trametes versicolor Species 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- 239000013530 defoamer Substances 0.000 description 1
- 229940119744 dextran 40 Drugs 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229960002449 glycine Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 238000012792 lyophilization process Methods 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 235000019423 pullulan Nutrition 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 229960004025 sodium salicylate Drugs 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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]
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a stabilizer for air-drying preservation of a PCR reagent and application thereof, and an air-drying preservation method of the PCR reagent. The stabilizing agent for air-drying and preserving the PCR reagent comprises the following components in parts by mass: 1 to 5 parts of glycerin, 0.5 to 2 parts of sucrose, 1 to 4 parts of tween-20, 0.05 to 0.2 part of NP-40 and 0.5 to 2 parts of DMSO, and the beneficial effects of the invention are mainly shown as follows: (1) The stabilizer is adopted to air-dry and preserve the PCR reagent, so that cold chain transportation is not needed, and the transportation cost is reduced; (2) The storage can be carried out at room temperature or below, the lower the temperature is, the longer the effective period is, and the more reliable the stability is; (3) The storage can be realized under a looser condition, so that the storage cost of the kit is reduced; (4) The product can be made into single-tube single-person pre-assembly, and experimental error is smaller.
Description
Field of the art
The invention relates to a stabilizer for air-drying preservation of a PCR reagent and application thereof, and an air-drying preservation method of the PCR reagent.
(II) background art
The molecular diagnosis is a technology for diagnosing by detecting the structure or expression level of genetic material in a patient by using a molecular biological method, common molecular diagnosis methods include PCR (polymerase chain reaction), isothermal amplification, sequencing and the like, wherein the PCR is the molecular diagnosis method with the widest application range at present, and the molecular diagnosis method has the advantages of high detection sensitivity, high accuracy and strong specificity.
Some PCR reagents facilitate customer handling by preparing premix solutions, however, there are still some drawbacks in the stability of transportation and storage. The PCR reagents can be kept active in the dry state for a longer period of time by lyophilization (i.e., freeze drying). The lyophilization process is too long to be applicable to all reagents. Some lyoprotectant formulations include more than ten ingredients including trehalose, mannitol, pullulan, bovine serum albumin, surfactants, defoamers, sodium salicylate, chondroitin sulfate, coriolus versicolor polysaccharide, hyaluronic acid, and the like. The freeze-drying effect is improved, but the components are complex, the cost is high, and the surfactant and the defoamer in the formula can interfere with the PCR reaction result.
The freeze-drying protective agent formula of some PCR reagents comprises more than ten components of glycerol, trehalose, sucrose, tween-80, dextran-40, glycine, histidine, sodium glutamate, bacteriostat, tertiary butanol and the like. The freeze-drying protective agent has various components, complex composition and glycine, and has a certain inhibition effect on subsequent PCR reactions, and can influence subsequent detection.
Therefore, a pretreatment mode of the PCR premix is desired in the art, so that the treatment time can be reduced, the cost can be reduced, the use is more convenient, and the experimental error can be reduced.
(III) summary of the invention
The invention aims to solve the problems of long time consumption and high cost of the conventional freeze-drying treatment of PCR premix, and provides an air-drying protective agent scheme which is used for mixing Buffer and Taq enzyme in a PCR reagent and can be stably stored for 12 months.
The technical scheme adopted by the invention is as follows:
a stabilizer for air-drying and preserving PCR reagent comprises the following components in parts by mass:
preferably, the stabilizer comprises the following components in parts by mass:
the invention also relates to application of the stabilizer in air-drying preservation of PCR reagents.
Specifically, the application is as follows: adding the stabilizer into sterilized water to prepare a stabilizer solution with the mass concentration of glycerin of 1-5%, adding the stabilizer solution into a PCR reagent according to the volume ratio of 40-70%, air-drying to prepare an air-drying reagent for preservation, and adding the sterilized water for full dissolution for later use when in use.
The invention also relates to a method for air-drying and preserving the PCR reagent, which comprises the following steps: adding a stabilizer into a PCR reagent, air-drying to prepare an air-dried reagent for preservation, and adding sterilized water for full dissolution for later use when in use, wherein the stabilizer comprises the following components in the PCR reagent:
preferably, the stabilizer composition and its final concentration in the PCR reagents are as follows:
the beneficial effects of the invention are mainly as follows: (1) The stabilizer is adopted to air-dry and preserve the PCR reagent, so that cold chain transportation is not needed, and the transportation cost is reduced; (2) The storage can be carried out at room temperature or below, and the lower the temperature is, the longer the effective period is; (3) The storage can be realized under a looser condition, so that the storage cost of the kit is reduced; (4) The product can be made into single tube single-person pre-package, and can be used immediately without a separate reagent preparation laboratory, and the experimental error is smaller.
(IV) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
stabilizer formulation (formulated in sterilized water):
buffer formulation (prepared with sterilized water):
| name of the name | Concentration (w/w) |
| dATP | 0.4％ |
| dCTP | 0.4％ |
| dGTP | 0.4％ |
| dUTP | 0.8％ |
| Tris-HCl pH8.8 | 9.0％ |
| MgCl2 | 1.8％ |
| Tween-20 | 2.0％ |
| Hot start Taq enzyme | 5.0％ |
| UDG enzyme | 2％ |
| Primer(s) | 0.5％ |
| Probe with a probe tip | 0.25％ |
And adding 11.8 microliters of stabilizer into each PCR reaction buffer (8.2 microliters), and performing air drying (about 2 hours at 45 ℃) by an air dryer to prepare a finished product air drying reagent, wherein sterilizing water is directly added for full dissolution during use for later use.
And (3) detecting the air-drying preservation stability of the reagent:
the prepared finished product air-dried reagent is placed at room temperature, and the lowest detection limit and the lowest precision of the reagent are tested in 0, 3, 6, 9, 11, 12 and 13 months respectively, and the test results are as follows:
results of 0 month stability test:
results of 3 month stability test:
6 months stability test results:
results of 9 month stability test:
results of 11 month stability test:
12 months stability test results:
13 month stability test results:
conclusion: the stability test result shows that the invention has qualified stability at 12 months and unqualified stability at 13 months, so the stabilizer can be used for air-drying preservation and can be effectively preserved for 12 months at room temperature.
Claims (6)
1. A stabilizer for air-drying and preserving PCR reagent comprises the following components in parts by mass:
2. the stabilizer as claimed in claim 1, wherein the following components are present in the mass proportions:
3. use of a stabilizer according to claim 1 or 2 in the air-drying preservation of PCR reagents.
4. A use according to claim 3, characterized in that the use is: adding the stabilizer into sterilized water to prepare a stabilizer solution with the mass concentration of glycerin of 1-5%, adding the stabilizer solution into a PCR reagent according to the volume ratio of 40-70%, air-drying to prepare an air-drying reagent for preservation, and adding the sterilized water for full dissolution for later use when in use.
5. A method for air-drying and preserving a PCR reagent, which comprises the following steps: adding a stabilizer into a PCR reagent, air-drying to prepare an air-dried reagent for preservation, and adding sterilized water for full dissolution for later use when in use, wherein the stabilizer comprises the following components in the PCR reagent:
6. the method of claim 5, wherein the stabilizer composition and its final concentration in the PCR reagent is as follows:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310939264.2A CN117143975A (en) | 2023-07-28 | 2023-07-28 | Stabilizer for air-drying preservation of PCR reagent and air-drying preservation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310939264.2A CN117143975A (en) | 2023-07-28 | 2023-07-28 | Stabilizer for air-drying preservation of PCR reagent and air-drying preservation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117143975A true CN117143975A (en) | 2023-12-01 |
Family
ID=88906981
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310939264.2A Pending CN117143975A (en) | 2023-07-28 | 2023-07-28 | Stabilizer for air-drying preservation of PCR reagent and air-drying preservation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117143975A (en) |
-
2023
- 2023-07-28 CN CN202310939264.2A patent/CN117143975A/en active Pending
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