CN110923303A - Universal freeze-drying protective agent for multiplex fluorescence PCR and application thereof - Google Patents
Universal freeze-drying protective agent for multiplex fluorescence PCR and application thereof Download PDFInfo
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- 238000004108 freeze drying Methods 0.000 title claims abstract description 78
- 239000003223 protective agent Substances 0.000 title abstract description 33
- 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 14
- 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 12
- 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 12
- 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 10
- 229930195725 Mannitol Natural products 0.000 claims abstract description 10
- 239000000594 mannitol Substances 0.000 claims abstract description 10
- 235000010355 mannitol Nutrition 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000004353 Polyethylene glycol 8000 Substances 0.000 claims abstract description 9
- 229940085678 polyethylene glycol 8000 Drugs 0.000 claims abstract description 9
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 claims abstract description 9
- 239000008213 purified water Substances 0.000 claims abstract description 8
- 238000001514 detection method Methods 0.000 claims description 16
- 238000001035 drying Methods 0.000 claims description 8
- 238000007710 freezing Methods 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 239000002577 cryoprotective agent Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 14
- 239000012807 PCR reagent Substances 0.000 abstract description 12
- 230000003321 amplification Effects 0.000 abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 12
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 12
- 230000008859 change Effects 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 abstract description 6
- 238000010521 absorption reaction Methods 0.000 abstract description 6
- 239000000546 pharmaceutical excipient Substances 0.000 abstract description 4
- 238000006911 enzymatic reaction Methods 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 description 22
- 238000009472 formulation Methods 0.000 description 16
- 238000007403 mPCR Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 206010003694 Atrophy Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 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 description 2
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- 150000002016 disaccharides Chemical class 0.000 description 2
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- 239000002994 raw material Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000000959 cryoprotective effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 238000009792 diffusion process Methods 0.000 description 1
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- 238000006703 hydration reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
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- 235000000346 sugar Nutrition 0.000 description 1
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- 229910021642 ultra pure water Inorganic materials 0.000 description 1
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- 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]
Abstract
The invention discloses a multiple fluorescence PCR universal freeze-drying protective agent and application thereof, wherein the freeze-drying protective agent consists of the following components in parts by weight: 0.3-0.6M trehalose, 0.001-0.004M polyethylene glycol 8000, 0.02-0.08M mannitol, and the balance of purified water. The universal freeze-drying protective agent for the multiple fluorescence PCR disclosed by the invention not only can play a role of a protective agent and an excipient in the freeze-drying process of the multiple fluorescence PCR reagent, but also has simple components of the added protective agent, and does not influence the amplification reaction of the multiple fluorescence PCR. After the optimized freeze-drying protective agent is used for freeze-drying, the appearance is formed smoothly, the moisture absorption performance is low, the performance change of the reagent before and after freeze-drying is small, and the stability of a freeze-dried product is good. The optimized freeze-drying procedure has strong adaptability, and can be suitable for various enzyme reaction systems without glycerol under the condition of adopting the freeze-drying protective agent.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a multiple fluorescence PCR universal freeze-drying protective agent and application thereof.
Background
Multiplex PCR (multiplex PCR) is characterized in that more than two pairs of primers and probes are added into the same PCR reaction system, so that two or more different nucleic acid fragments are subjected to PCR amplification detection in one reaction system, the detection result of multiple targets can be rapidly obtained, and the detection time is remarkably shortened.
The freeze drying of PCR reagent is to freeze the compounded liquid PCR detecting system into solid after adding freeze drying protecting agent, and to dehydrate the material in vacuum condition to reach the aim of drying reagent. The heat-sensitive substance (enzyme) can not be denatured or inactivated in the freeze drying process, and the original activity can be kept; the volume of the reagent is almost unchanged before and after freeze-drying, the original structure is kept, and the concentration phenomenon cannot occur; the drying process is carried out in vacuum, so that little oxygen is generated, and substances which are easy to oxidize can be protected; the freeze drying can remove more than 95-99% of water, so that the dried product can be stored for a long time without deterioration. The whole process of freeze drying is carried out at low temperature, and the activity of the PCR reagent is not influenced. Therefore, compared with the conventional liquid PCR reagent, the freeze-dried reagent has the obvious advantages of stability at normal temperature, getting rid of cold chain, instant activation, coexistence of incompatible reagents, no cross contamination and the like.
Saccharides are an important class of excipients in the formulation of lyoprotectants for biological products. Different sugars have different cryoprotective effects on drugs, disaccharides are probably the most effective protective agents, and sucrose and trehalose are most commonly used among disaccharides, but trehalose has a much higher protective effect than sucrose. Trehalose has a high glass transition temperature (Tg), excellent hydrolytic stability, does not hydrolyze during freezing, and has high hydration, a lower water diffusion coefficient and a higher viscosity. And are therefore commonly used as lyoprotectants.
The establishment of the multiplex fluorescent PCR system is complicated, and the change of the exogenous additive can cause the change of the multiplex PCR performance. Currently, many enzyme systems are available on the market for performing multiplex PCR reaction systems, but few multiplex PCR enzyme systems are available that can be directly subjected to lyophilization. The main problems are as follows: 1) the absence of a suitable lyoprotectant results in reduced biological activity in the PCR system during lyophilization. 2) The added freeze-drying protective agent has great influence on the multiplex amplification performance of the PCR reagent, and the whole performance of the freeze-dried reagent is reduced.
At present, the main process for constructing the multiple fluorescence PCR detection freeze-drying system comprises the steps of firstly constructing the multiple PCR liquid reaction system, secondly screening raw materials of the freeze-drying protective agent, secondly optimizing the multiple PCR liquid reaction system containing the freeze-drying protective agent, and finally optimizing a freeze-drying program. The whole process has long time period, large workload and higher cost.
Therefore, there is an urgent need to develop a freeze-drying protection system which has wide applicability, less influence on the performance of a multiplex fluorescence PCR reaction system and better freeze-drying effect.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the universal freeze-drying protective agent for the multiple fluorescent PCR, which has the advantages of simple formula components, small influence on the multiple PCR amplification and wide suitability of freeze-drying procedures.
The second purpose of the invention is to provide the application of the universal cryoprotectant for multiplex fluorescence PCR in multiplex fluorescence PCR detection, and the cryoprotectant can exert the optimal effect in multiplex fluorescence PCR detection through an optimized freeze drying program.
In order to achieve the first object, the invention provides the following technical scheme: a universal lyoprotectant for multiplex fluorescent PCR, said lyoprotectant consisting of: 0.3-0.6M trehalose, 0.001-0.004M polyethylene glycol 8000, 0.02-0.08M mannitol, and purified water as solvent.
As a preferred embodiment, the lyoprotectant consists of: 0.3M trehalose, 0.001M polyethylene glycol 8000, 0.02M mannitol, and the balance of purified water.
In order to achieve the second object, the invention provides the following technical scheme: the application of the universal freeze-drying protective agent for multiplex fluorescence PCR in multiplex fluorescence PCR detection comprises the following freeze-drying conditions:
(1) a pre-freezing stage: setting the temperature of the laminate to be 2-4 ℃, and keeping the temperature for 0.5-1 h; secondly, reducing the temperature of the laminate to-50 ℃ to-55 ℃ within 0.5h to 1h, keeping the temperature at-50 ℃ to-55 ℃ for 1h to 2h, quickly heating the temperature of the laminate to-25 ℃ to-30 ℃ again, and keeping the temperature for 1h to 2 h; finally, reducing the temperature of the laminate to minus 50 ℃ to minus 55 ℃, and keeping the temperature for 1h to 2 h;
(2) a primary drying stage: vacuumizing to 150 mTorr-200 mTorr, and keeping for 2-3 hours at-50 ℃ to-55 ℃; then, the temperature of the laminate is increased to-25 ℃ to-30 ℃ within 1h to 2h, the laminate is vacuumized to 100mTorr to 80mTorr, and the laminate is kept for 6h to 10h under the condition; keeping the temperature of the laminate at-25 ℃ to-30 ℃, vacuumizing to 50mTorr to 70mTorr, and keeping for 6h to 10h under the condition.
(3) And (3) secondary drying stage: keeping the vacuum at 50 mTorr-70 mTorr, raising the temperature of the laminate to 20-25 ℃ within 1-2 h, and keeping for 3-4 h.
When the freeze-drying protective agent is applied to multiplex fluorescence PCR detection, the freeze-drying condition can be selected according to instruments, and the preferable freeze-drying program is suitable for conventional instruments such as VirTis Advantage ProTM.
The concentration of the freeze-drying protective agent is prepared into 2.5 times of the concentration of the working solution in advance, and when the PCR reaction solution is used, the concentration of the corresponding amount of 2.5 times of the concentration of the working solution is added according to the volume of the prepared PCR reaction solution, so that the concentration of the freeze-drying protective agent in a final system is 1 x.
2.5 times of working solution concentration freeze-drying protective agent:
| components | Dosage of |
| Trehalose | 28.38g~56.75g |
| Polyethylene glycol 8000 | 2g~8g |
| Mannitol | 0.91g~3.64g |
| Sterilized ultrapure water | The volume is up to 100mL |
The invention has the following beneficial effects: the universal freeze-drying protective agent for the multiple fluorescence PCR disclosed by the invention not only can play a role of a protective agent and an excipient in the freeze-drying process of the multiple fluorescence PCR reagent, but also has simple components of the added protective agent, and does not influence the amplification reaction of the multiple fluorescence PCR. After the optimized freeze-drying protective agent is used for freeze-drying, the appearance is formed smoothly, the moisture absorption performance is low, the performance change of the reagent before and after freeze-drying is small, and the stability of a freeze-dried product is good. The optimized freeze-drying procedure has strong adaptability, and can be suitable for various enzyme reaction systems without glycerol under the condition of adopting the freeze-drying protective agent.
Drawings
Figure 1 is an appearance of the formulation lyophilization of eight groups of lyoprotectants.
Fig. 2 shows the hygroscopicity of the products of the six sets of lyoprotectant formulations after lyophilization.
FIG. 3 shows a comparison of multiplex PCR performance before and after lyophilization of multiplex fluorescent PCR reagents.
FIG. 4 shows the stability of the multiplex fluorescent PCR reagents at 37 ℃ before and after lyophilization.
Detailed Description
Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention. Materials and reagents used in the following examples are commercially available unless otherwise specified.
Example 1 comparison of lyoprotectant Performance
Raw materials of the freeze-drying protective agent: trehalose was purchased from sigma aldrich (shanghai) trade, PEG8000 from dow chemical, mannitol from bio-engineering (shanghai) gmbh. Glycerol-free multiplex PCR enzyme system: One-StepRT-PCR Premix was purchased from Korea BioAssay. The One-Step RT-PCR Premix comprises 5 × RT-PCR buffer mix, 20 × RT-PCR Enzyme mix.
2.5 × preparation of lyoprotectant (sterilized purified water to 100 mL):
| trehalose (g) | Mannitol (g) | Polyethylene glycol 8000 (g) | |
| |
40.24 | 2.72 | 0 |
| Formulation 2 | 40.24 | 0 | 4.12 |
| |
40.24 | 2.72 | 4.12 |
| Formulation 4 | 28.38 | 2.18 | 4.12 |
| |
30.56 | 3.5 | 7.5 |
| Formulation 6 | 5 | 1.45 | 1.28 |
| Formulation 7 | 20 | 2 | 2.56 |
| Formulation 8 | 35.32 | 0.5 | 0.8 |
Preparing a 30-times multiplex primer probe mixed solution:
| primer probe | Dosage (Unit: mu L) |
| FluA-F(100μM) | 7μL |
| FluA-R(100μM) | 7μL |
| FluA-P(100μM)-FAM | 3.5μL |
| FluB-F(100μM) | 7μL |
| FluB-R(100μM) | 7μL |
| FluB-P(100μM)-ROX | 3.5μL |
| RSV-F(100μM) | 8μL |
| RSV-R(100μM) | 8μL |
| RSV-P(100μM)-VIC | 5μL |
| IC-F (100μM) | 7μL |
| IC-F (100μM) | 7μL |
| IC-F (100μM)-CY5 | 3.5μL |
| Sterilized purified water | 26.5μL |
| Total | 100μL |
Preparation of 30 μ L multiplex fluorescence PCR lyophilization reaction system:
| 30 μ L system | 1T (Unit: μ L) | |
| 5×RT-PCRBuffer | 6μL | |
| 20×RT-PCR Enzyme mix | 1.5μL | |
| 30X multiple primer probe mixture | 1μL | |
| 2.5X Freeze-drying protective agent ( |
12μL | |
| Sterilized purified water | 9.5μL | |
| Total | 30μL |
And subpackaging the prepared multiple PCR freeze-drying reaction system into PCR eight-connected tubes according to the volume ratio of 30 mu L/T, putting the PCR into an eight-connected tube frame and putting the PCR into a freeze-drying tray.
The formula 1-formula 8 freeze-drying procedures are set as follows:
(1) a pre-freezing stage: firstly, setting the temperature of a laminated board to be 4 ℃, and preserving heat for 0.5 h; secondly, reducing the temperature of the laminate to-50 ℃ within 0.5h, keeping the temperature at-50 ℃ for 1h, raising the temperature of the laminate to-25 ℃ at the highest speed, and keeping the temperature for 1 h; finally, the temperature of the laminate is reduced to-50 ℃ and kept for 1 h.
(2) A primary drying stage: vacuumizing to 200mTorr, and keeping at-50 ℃ for 2 hours; then the temperature of the laminate is raised to-25 ℃ in 1h, and the laminate is vacuumized to 100mTorr and kept for 6h under the condition; the laminate temperature was maintained at-25 ℃ and a vacuum was applied to 70mTorr for 6h under these conditions.
(3) And (3) secondary drying stage: the vacuum was maintained at 70mTorr, and the temperature of the laminate was raised to 20 ℃ over 1h for 3 h.
After the freeze-drying is finished, the freeze-drying effect of the freeze-drying protective agent with the 8 groups of formulas is evaluated respectively according to appearance, hygroscopicity, multiple amplification performance after freeze-drying and accelerated stability of a freeze-drying reagent.
(1) Freeze-dried appearance:
as shown in fig. 1: the formula 1 is poor in freeze-drying forming, and the phenomenon of serious exfoliation of a freeze-dried product is caused; according to the formula 2, the freeze-drying forming is slightly good, but the appearance forming consistency of each freeze-drying product is poor; the freeze-dried products of the formula 3, the formula 4 and the formula 5 have smooth surfaces and consistent appearances. Formula 6, the freeze-dried product collapsed, incomplete freeze-drying, failed freeze-drying, formula 7 and formula 8, the freeze-dried molding was poor, the consistency was poor, and the individual freeze-dried products collapsed due to atrophy.
Formula 6, formula 7, and formula 8 failed in lyophilization, and subsequent hygroscopicity and fluorescent PCR detection were not required. Only subsequent moisture absorption and fluorescence PCR performance detection is needed to be carried out on the formulas 1-5.
(2) Moisture absorption:
as shown in fig. 2: the formula 1 is poor in freeze-drying forming, and after the product is placed in a room-temperature environment for 2 hours, the product is obviously damped and reduced; the freeze-drying forming of the formula 2 is slightly good, but the product still shows obviously reduced damp appearance after being placed in a room temperature environment for 2 hours; the moisture absorption of the formula 3, the formula 4 and the formula 5 is obviously smaller than that of the formula 1 and the formula 2, and the appearance of the product is basically kept intact after the product is placed in a room temperature environment for 2 hours.
(3) Comparison of multiple PCR performances before and after freeze-drying of multiple fluorescent PCR reagent
As shown in fig. 3: compared with a liquid multiplex PCR reaction system, when the multiple templates are simultaneously amplified after the freeze-drying of the formula 1, the multiplex amplification performance is poor, and particularly the detection performance of the low-concentration multiple templates is poor; formula 2, the multiplex amplification performance is good, but the amplification detection curve is poor in linearity; the difference between the amplification performance of the multiple templates and the liquid control group is smaller in the formula 3, the formula 4 and the formula 5.
(4) Stability of multiplex fluorescence PCR reagent at 37 ℃ before and after freeze-drying
As shown in fig. 4: placing the multiple fluorescent PCR liquid form reagent for 10 days at 37 ℃, and failing to detect all targets; in both formula 1 and formula 2, the performance change is obvious after the composition is placed for 10 days at 37 ℃; in the formulas 3, 4 and 5, the freeze-dried reagent is placed at 37 ℃ for 10 days, and the performance change is small.
The experiments show that the universal freeze-drying protective agent for the multiplex fluorescence PCR not only can play a role of a protective agent and an excipient in the freeze-drying process of the multiplex fluorescence PCR reagent, but also has simple components of the added protective agent, is combined with an optimal freeze-drying program, and does not influence the amplification reaction of the multiplex fluorescence PCR. After the optimized freeze-drying protective agent is used for freeze-drying, the appearance is formed smoothly, the moisture absorption performance is low, the performance change of the reagent before and after freeze-drying is small, and the stability of a freeze-dried product is good. The addition amounts of trehalose, polyethylene glycol 8000 and mannitol play a key role in the multiple PCR performance of the multiple fluorescent PCR reagent before and after freeze-drying, polyethylene glycol 8000 is not added in formula 1, the freeze-drying molding is poor, the phenomenon of serious exfoliation of a freeze-dried product occurs, when multiple templates are simultaneously amplified after freeze-drying, the performance of multiple amplification performance is poor, and particularly the detection performance of the multiple templates with low concentration is poor; formula 2 does not add mannitol, and the multiplex amplification performance is still good, but the amplification detection curve is poor in linearity. In the optimized combined formula (formula 3, formula 4 and formula 5), the appearance performance of the freeze-dried product is ideal, and the freeze-dried product has good hygroscopicity, multiple fluorescence PCR detection performance and thermal stability. Formulations which are not in the optimized combination (formulation 6, formulation 7, formulation 8) cannot be lyophilized or have poor lyophilized appearance, and the lyophilized product suffers collapse and atrophy and is not suitable for use as a lyoprotectant.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. The universal cryoprotectant for multiplex fluorescence PCR is characterized by consisting of the following components in percentage by weight: 0.3-0.6M trehalose, 0.001-0.004M polyethylene glycol 8000, 0.02-0.08M mannitol, and the balance of purified water.
2. The universal lyoprotectant for multiplex fluorescence PCR according to claim 1, wherein said lyoprotectant consists of: 0.3M trehalose, 0.001M polyethylene glycol 8000, 0.02M mannitol, and the balance of purified water.
3. The application of the universal lyophilization protectant for multiplex fluorescence PCR as claimed in claim 1 in multiplex fluorescence PCR detection is characterized in that the lyophilization conditions are as follows:
(1) a pre-freezing stage: setting the temperature of the laminate to be 2-4 ℃, and keeping the temperature for 0.5-1 h; secondly, reducing the temperature of the laminate to-50 ℃ to-55 ℃ within 0.5h to 1h, keeping the temperature at-50 ℃ to-55 ℃ for 1h to 2h, quickly heating the temperature of the laminate to-25 ℃ to-30 ℃ again, and keeping the temperature for 1h to 2 h; finally, reducing the temperature of the laminate to minus 50 ℃ to minus 55 ℃, and keeping the temperature for 1h to 2 h;
(2) a primary drying stage: vacuumizing to 150 mTorr-200 mTorr, and keeping for 2-3 hours at-50 ℃ to-55 ℃; then, the temperature of the laminate is increased to-25 ℃ to-30 ℃ within 1h to 2h, the laminate is vacuumized to 100mTorr to 80mTorr, and the laminate is kept for 6h to 10h under the condition; keeping the temperature of the laminate within-25 ℃ to-30 ℃, vacuumizing to 50mTorr to 70mTorr, and keeping for 6h to 10h under the condition;
(3) and (3) secondary drying stage: keeping the vacuum at 50 mTorr-70 mTorr, raising the temperature of the laminate to 20-25 ℃ within 1-2 h, and keeping for 3-4 h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114214434A (en) * | 2022-02-22 | 2022-03-22 | 中国肉类食品综合研究中心 | Multiple RT-PCR premixed reagent freeze-dried ball for synchronously identifying various animal-derived components in food and preparation method and application thereof |
| CN114934107A (en) * | 2022-06-29 | 2022-08-23 | 广州生凌医疗科技有限公司 | Multiple reverse transcription fluorescence PCR premixed reaction solution capable of being preserved in frozen mode |
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| CN114934107A (en) * | 2022-06-29 | 2022-08-23 | 广州生凌医疗科技有限公司 | Multiple reverse transcription fluorescence PCR premixed reaction solution capable of being preserved in frozen mode |
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