CN106591432A - Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent - Google Patents
Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent Download PDFInfo
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- CN106591432A CN106591432A CN201610911305.7A CN201610911305A CN106591432A CN 106591432 A CN106591432 A CN 106591432A CN 201610911305 A CN201610911305 A CN 201610911305A CN 106591432 A CN106591432 A CN 106591432A
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- 238000004108 freeze drying Methods 0.000 title claims abstract description 62
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 38
- 230000003321 amplification Effects 0.000 title claims abstract description 19
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 19
- 239000003223 protective agent Substances 0.000 title claims abstract description 7
- 239000003795 chemical substances by application Substances 0.000 title abstract 5
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 65
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- 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 25
- 238000003757 reverse transcription PCR Methods 0.000 claims abstract description 25
- 238000002156 mixing Methods 0.000 claims abstract description 14
- 239000000376 reactant Substances 0.000 claims description 34
- 229920001917 Ficoll Polymers 0.000 claims description 24
- 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 description 23
- 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 description 23
- 102000016911 Deoxyribonucleases Human genes 0.000 claims description 5
- 108010053770 Deoxyribonucleases Proteins 0.000 claims description 5
- 102000006382 Ribonucleases Human genes 0.000 claims description 5
- 108010083644 Ribonucleases Proteins 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 238000003753 real-time PCR Methods 0.000 abstract description 5
- 208000031888 Mycoses Diseases 0.000 abstract 2
- 239000012295 chemical reaction liquid Substances 0.000 abstract 2
- 238000003752 polymerase chain reaction Methods 0.000 description 29
- 230000035945 sensitivity Effects 0.000 description 22
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 17
- 239000000203 mixture Substances 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 9
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 239000012807 PCR reagent Substances 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 102100034343 Integrase Human genes 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 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
- 229930006000 Sucrose Natural products 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000013097 stability assessment Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- LRWZZZWJMFNZIK-UHFFFAOYSA-N 2-chloro-3-methyloxirane Chemical compound CC1OC1Cl LRWZZZWJMFNZIK-UHFFFAOYSA-N 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 108010001394 Disaccharidases Proteins 0.000 description 1
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 1
- -1 HCV nucleic acid Chemical class 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 230000009477 glass transition Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 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
- 238000007689 inspection Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000000859 sublimation Methods 0.000 description 1
- 230000008022 sublimation Effects 0.000 description 1
- 230000008542 thermal sensitivity Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000004017 vitrification Methods 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
- C12Q1/6851—Quantitative amplification
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Abstract
The invention discloses a freeze-drying protective agent and a freeze-drying method for an RNA amplification reaction agent, belonging to the field of biotechnologies. The freeze-drying protective agent contains mycose, polysucrose and water, and is prepared by mixing 6.4-16.0 g of mycose and 0.4-1.0 g of polysucrose and adding with water till the volume is 40ml. The invention further provides the freeze-drying method for the RNA amplification reaction agent. The freeze-drying method comprises the following steps: a, uniformly mixing the freeze-drying protective agent and a real-time fluorescent RT-PCR reaction agent, thus obtaining real-time fluorescent RT-PCR reaction liquid; and b, carrying out freeze drying on the RT-PCR reaction liquid, thus obtaining the RT-PCR freeze-dried reagent. With the adoption of the freeze-drying protective agent and the freeze-drying method provided by the invention, the RNA fluorescent quantitative PCR reaction agent can be stably stored for a long time at 2-8 DEG C or even at the room temperature.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of freeze drying protectant of RNA amplification reaction reagent and lyophilizing
Method.
Background technology
Real-Time Fluorescent Quantitative PCR Technique is to be released by Applied Biosystems companies of the U.S. for 1996, the technology
Refer to and add fluorophor in PCR reaction systems, using fluorescence signal the whole PCR processes of real-time monitoring are accumulated, finally by
Standard curve carries out quantitative analyses to unknown template.Real-time fluorescence quantitative PCR is a kind of in DNA amplification reaction, with Fluoresceinated
Learn the method that material detects product total amount after each polymerase chain reaction (PCR) circulation.By fluorescence signal, to PCR processes
Carry out real-time detection.Due to the exponential time base expanded in PCR, there is linear closing in the Ct values of template and the starting copy number of the template
System, so becoming quantitative foundation.The features such as Real-Time Fluorescent Quantitative PCR Technique has quick, special, sensitive.
In recent years, real-time fluorescence quantitative PCR reagent is widely used in food inspection, pathogen detection and oncogene diagnosis etc.
Aspect.But quantitative fluorescent PCR reagent requires harshness in storage and transport condition, generally requires and is stored in -20 DEG C and to molten
Liquid number of freezing and thawing is restricted, generally needs dry ice to transport.If these conditions are not being met, can be outstanding to the performance of PCR reagent
It is that sensitivity causes very big impact, even results in reagent failure.
PCR reagent to being applied to RNA, system domestic demand include reverse transcriptase to RNA using sequence reverse transcription into DNA as mould
Plate enters again performing PCR amplification, and the most suitable operating temperature of reverse transcriptase is about 37 DEG C～60 DEG C, gathers with respect to 94 DEG C of DNA of operating temperature
It is more sensitive to temperature for synthase, therefore RNA class PCR detectable is more sensitive to storage temperature.
Lyophilization is exactly, containing large quantity of moisture material, cooling to be carried out in advance and is frozen into solid, then in the bar of vacuum
Solid water is set directly to distil out, and in the left ice shelf when freezing of material itself under part, therefore constancy of volume after it is dried,
It is loose porous to absorb heat in distillation.Cause the decline of product self-temperature and slow down rate of sublimation, in order to increase distillation
Speed, shortens drying time, it is necessary to which product is suitably heated.Whole drying is carried out at a lower temperature.It is cold
Lyophilizing is dry to be carried out at low temperature, therefore particularly suitable for the material of many thermal sensitivitys.After reagent lyophilizing, 95% water is excluded
Part, and protein, microorganism etc will not occur degeneration or lose biologos, dried product can room temperature preserve and not
Mutagens matter, therefore Freeze Drying Technique pharmaceutically widely applied.
The content of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide a kind of lyophilizing of RNA amplification reaction reagent
Protective agent and freeze drying process, can enable RNA quantitative fluorescent PCRs reaction reagent in 2～8 DEG C or even ambient-temp-stable long storage time.
To solve the above problems, the technical solution adopted in the present invention is as follows：
A kind of freeze drying protectant of RNA amplification reaction reagent, it includes trehalose, ficoll and water.
Used as the preferred embodiment of the invention, the freeze drying protectant is by 6.4～16.0g of trehalose, ficoll 0.4
～1.0g mixes and adds water to made by 40ml.
Used as further preferred embodiment of the present invention, the trehalose is 16g with the proportioning of ficoll：1g, adds water to
40ml。
Specifically, the ficoll is Ficoll400.
Preferably, the water be ultra-pure water or without deoxyribonuclease and ribonuclease through DEPC process
Pure water.
Present invention also offers a kind of method for preparing freeze drying protectant as described above, its step is as follows：Weigh in proportion
Trehalose, ficoll and water, are placed in mixing in container, are subsequently placed in water-bath 5～15min of water-bath under the conditions of 50～60 DEG C
Dissolve it, mix homogeneously, ramuscule subpackage is put into 2～8 DEG C of Refrigerator stores.
As the preferred embodiment of the invention, trehalose, ficoll and water are weighed in proportion, be placed in centrifuge tube and mix
Close, being subsequently placed in the interior water-bath 10min under the conditions of 60 DEG C of water-bath dissolves it, and ramuscule subpackage after mix homogeneously is put into 2～8
DEG C Refrigerator store.
Present invention also offers a kind of freeze drying process of RNA amplification reaction reagent, it is comprised the following steps：
A, above-mentioned freeze drying protectant is mixed homogeneously with real-time fluorescence RT-PCR reaction reagent, obtain real-time fluorescence RT-
PCR reactant liquors；
B, RT-PCR reactant liquors are carried out into lyophilization, that is, obtain RT-PCR freeze-dried reagents.
Used as the preferred embodiment of the invention, trehalose is in real-time fluorescence RT-PCR reactant liquor in step a
Final concentration of 4～10%, ficoll in real-time fluorescence RT-PCR reactant liquor final concentration of 0.25～0.625%.
The wherein real-time fluorescence RT-PCR reaction reagent is a kind of aqueous solution, comprising following solute：Tris·HCl、
KCL、(NH4)2·SO4、Mn2+, Tth enzymes, dNTPs, primer and probe etc..
Used as the preferred embodiment of the invention, the lyophilisation condition in step b is：The pre-freeze stage is through 1h, dividing plate
Temperature drop to cryogenic hold-time when -45 ± 2 DEG C, pre-freeze is 2h；Primary drying phase is evacuated to first with 40min
0.16mbar, with 60min baffle temperature is risen to into -25 ± 2 DEG C again, then keep 4h；The parsing-desiccation stage first with 60～
120min is evacuated to 0.01mbar, then baffle temperature is risen to into 25 ± 2 DEG C with 60～90min and 2h is kept, and then uses 20min
Baffle temperature is risen to into 35 DEG C and 4h is kept, finally baffle temperature 25 DEG C is down to into 20min and is kept 2h.
In the present invention, trehalose is the non-reducing sugar being made up of special disaccharidase molecule, soluble in water, the dissolving in water
Degree varies with temperature more obvious；With very high glass transition temperature；Internal hydrogen bond is few, Stability Analysis of Structures, with extremely strong
Heat-resisting, acid resistance, is the stabilizer of many bioactive substances.Trehalose also has the constitutionally stable effect of protected protein matter, because
It is that it is rich in hydroxyl, the structure for being similar to hydration shell can be formed around protein, makes the structure of protein under the conditions of dehydration
Keep stable.Ficoll is sucrose and the polymer of the polymer, sucrose and chloromethyloxirane of epoxychloropropane, ficoll
The protective layer for being formed can be with vaccine to enzyme degraded it is more resistant, it is also possible to vitrification protect.
Compared to existing technology, the beneficial effects of the present invention is：
The lyophilizing filler that the freeze drying protectant of RNA amplification reaction reagent of the present invention is adopted has protection biological product
Active effect, also maintain the effect of the stability of reagent in freeze-drying process, additionally, this freeze drying protectant low cost, behaviour
Facilitate, can be used for large-scale production.Using the freeze drying protectant lyophilizing RNA amplification reaction reagent of the present invention, add at 37 DEG C
Speed examination 15 days, sensitivity can still keep stable；Reagent article after lyophilizing can for a long time be preserved under 2-8 DEG C or room temperature,
And performance is not affected, greatly reduce requirement and the cost of current existing RNA amplification reaction reagent storage and transport, lyophilizing
Reagent is not limited by number of freezing and thawing, improves the convenience that reagent is used.
Description of the drawings
Fig. 1 is the outward appearance in the embodiment of the present invention 1 after HCV-PCR freeze-dried reagent lyophilizing；
Fig. 2 is the sensitivity technique result figure of matched group HCV-PCR reactant liquors in the embodiment of the present invention 1；
Fig. 3 is the sensitivity technique result figure of HCV-PCR freeze-dried reagents in the embodiment of the present invention 1；
Fig. 4 is the outward appearance in the embodiment of the present invention 2 after HIV-1-PCR freeze-dried reagent lyophilizing；
Fig. 5 is the sensitivity technique result figure of matched group HIV-1-PCR reactant liquors in the embodiment of the present invention 2；
Fig. 6 is the sensitivity technique result figure of HIV-1-PCR freeze-dried reagents in the embodiment of the present invention 2.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and detailed description.
A kind of freeze drying protectant of RNA amplification reaction reagent, it includes trehalose, ficoll and water.
Above-mentioned freeze drying protectant is to mix and add water to 40ml systems by 6.4～16.0g of trehalose, 0.4～1.0g of ficoll
Into.
Preferably, the trehalose and the proportioning of ficoll are 16g：1g, adds water to 40ml.
Specifically, the ficoll is Ficoll400.
Preferably, the water be ultra-pure water or without deoxyribonuclease and ribonuclease through DEPC process
Pure water (i.e. DEPC water).
Present invention also offers a kind of method for preparing freeze drying protectant as described above, its step is as follows：Weigh in proportion
Trehalose, ficoll and water, are placed in mixing in container, are subsequently placed in water-bath 5～15min of water-bath under the conditions of 50～60 DEG C
Dissolve it, ramuscule subpackage after mix homogeneously is put into 2～8 DEG C of Refrigerator stores.
Preferably, mixed trehalose, ficoll and water are placed in water-bath into the water-bath 10min under the conditions of 60 DEG C
Dissolve it, ramuscule subpackage after mix homogeneously is put into 2～8 DEG C of Refrigerator stores, better.
Present invention also offers a kind of freeze drying process of RNA amplification reaction reagent, it is comprised the following steps：
A, above-mentioned freeze drying protectant is mixed homogeneously with real-time fluorescence RT-PCR reaction reagent, obtain real-time fluorescence RT-
PCR reactant liquors；Reaction volume on demand, subpackage is to without deoxyribonuclease (DNase) and ribonuclease (RNase)
The connecting legs of PCR eight in；
B, RT-PCR reactant liquors are dried, lyophilizing, dry air filling box is filled with toward in freeze drying box after the completion of lyophilizing
Body, outlet sealing preserve obtains RT-PCR freeze-dried reagent products.
Preferably, in the step a trehalose in real-time fluorescence RT-PCR reactant liquor final concentration of 4～10%,
Ficoll in real-time fluorescence RT-PCR reactant liquor final concentration of 0.25～0.625%.
Specifically, the wherein real-time fluorescence RT-PCR reaction reagent is a kind of aqueous solution, comprising following solute：Tris·
HCl、KCL、(NH4)2·SO4、Mn2+, Tth enzymes, dNTPs, primer and probe etc..
Specifically, the lyophilisation condition in step b is：The pre-freeze stage through 1h, baffle temperature drop to -45 ± 2 DEG C,
Cryogenic hold-time is 2h during pre-freeze；Primary drying phase is evacuated to 0.16mbar with 40min first, again with 60min by dividing plate
Temperature rises to -25 ± 2 DEG C, then keeps 4h；The parsing-desiccation stage is evacuated to 0.01mbar with 60～120min first, then uses
Baffle temperature is risen to 25 ± 2 DEG C and keeps 2h by 60～90min, then baffle temperature is risen to into 35 DEG C with 20min and is kept 4h,
Finally baffle temperature is down to into 25 DEG C with 20min and keeps 2h.
Carry out in detail with reference to the RNA class quantitative fluorescent PCR freeze-dried products techniques of embodiment 1 and 2 couples of present invention of embodiment
Thin description.Trehalose used and ficoll are bought in Sigma companies in embodiment, and it is biological that DEPC water is purchased from Korea Bioneer
Company.
Case study on implementation 1, HCV quantitative fluorescent PCR freeze-dried reagents
First, the preparation of 5 × RT-PCR freeze drying protectants
Trehalose 16g, Ficoll400 0.4g, plus DEPC water are weighed to 40ml, into the healthy and free from worry centrifuge tube that is placed in by more than
Bottle mixing, being placed in water-bath water-bath 10min at 60 DEG C dissolves it, mix homogeneously, ramuscule subpackage, is put into 2-8 DEG C of refrigerator and protects
Deposit standby.
2nd, the preparation of HCV quantitative fluorescent PCRs reaction reagent
HCV reaction reagents are formulated as follows：
Tth PCR Buffer：1×；
Tth enzymes：7.5U；
HCV pf：400nM；
HCV pr：400nM；
HCV pb：100nM；
RT-PCR freeze drying protectants：1×；50 μ l are supplied with water.
HCV-PCR freeze-dried reagents containing freeze drying protectant are formulated as follows：
After the reverse mixing of above each component, by the often μ l subpackages of pipe 50 to the connecting legs of PCR eight.
3rd, the lyophilizing of HCV quantitative fluorescent PCRs reaction reagent：
Eight connecting legs that will be equipped with HCV quantitative fluorescent PCR lyophilizing reaction reagents are put on freeze dryer flaggy, according to following procedure
Carry out lyophilizing：
Eight connecting legs are taken out after the completion of lyophilizing, is covered stand-by.The outward appearance of HCV-PCR freeze-dried reagents is as shown in Figure 1.
4th, will as a control group, it be formulated as follows without protectant HCV-PCR reactant liquors (25 μ l/T)：
| Composition | ×1 | ×110 |
| 5×Tth PCR bufffer | 10 | 1100 |
| Tth enzymes (4.5U/ μ l) | 1.67 | 183.7 |
| UNG enzymes (5U/ μ l) | 0.1 | 11 |
| HCV-pf(50μM) | 0.4 | 44 |
| HCV-pr(50μM) | 0.4 | 44 |
| HCV-pb(50μM) | 0.1 | 11 |
| DEPC water | 12.33 | 1356 |
Above reactant liquor is packed as 6 pipes using centrifuge tube after mixing by the often μ l of pipe 25 × 17T (425 μ l), stand-by.
5th, stability assessment：
HCV-PCR freeze-dried reagents are divided into into every 16T for one group, totally 6 groups；6 pipe HCV-PCR reactant liquors are often managed as 1 group：37℃
It is lower to place 0 day, 3 days, 7 days, 10 days, 15 days and 20 days respectively；Respectively in each time point test HCV-PCR reactant liquor and HCV-
The sensitivity of PCR freeze-dried reagents, compares HCV-PCR reactant liquors and has added the HCV-PCR freeze-dried reagents of freeze drying protectant stable
Difference in property.
Detection process is as follows：
Found according to previous experiments the result, HCV liquid PCR reagents sensitivity about 25IU/ml, therefore examination every time is set
Test per group of detection tri- concentration HCV standard substance of 25IU/ml, 50IU/ml and 100IU/ml, 5 parts of each concentration Parallel testing.
1st, HCV standard substance are extracted：The HCV standard substance of over-richness will be demarcated with WHO HCV nucleic acid standards, use negative human body
Diluted plasma carries out the purification of nucleic acid, 0.2ml samples, 60 μ l eluting with extracts kit to desired concn.
2nd, it is loaded：In by the often μ l subpackages of pipe HCV-PCR reactant liquors 25 to the connecting legs of PCR eight, then add the μ l of sample of nucleic acid 25, concussion
Mix and upper machine after moment centrifugation；Often pipe HCV-PCR freeze-dried reagents add 25 μ l DEPC water, then add the μ l of sample of nucleic acid 25, and concussion is mixed
Upper machine after the centrifugation of even and moment；
3rd, upper machine：It is as follows that response procedures are set on quantitative fluorescent PCR machine：
Channel selecting：FAM
After having run, relative method automatically analyzes result.As a result distinguish as shown in Table 1 and Table 2：
The stability result of the HCV-PCR reactant liquor sensitivity of table 1
| HCV specimen | 0 day | 37 DEG C 3 days | 37 DEG C 7 days | 37 DEG C 10 days | 37 DEG C 15 days | 37 DEG C 20 days |
| 25IU/ml | 5/5 | 0/5 | 0/5 | 0/5 | 0/5 | 0/5 |
| 50IU/ml | 5/5 | 2/5 | 0/5 | 0/5 | 0/5 | 0/5 |
| 100IU/ml | 5/5 | 5/5 | 0/5 | 0/5 | 0/5 | 0/5 |
The stability result of the HCV-PCR freeze-dried reagent sensitivity of table 2
From Fig. 2～3 and table 1～2, at the 0th day, the HCV-PCR freeze-dried reagents containing freeze drying protectant were initial
Sensitivity is consistent with the initial sensitivity of HCV-PCR reactant liquors；As time goes on, HCV-PCR reactant liquors spirit at the 3rd day
Sensitivity is significantly affected, its 37 DEG C of stability ＜ 3 days；And the HCV-PCR freeze-dried reagents containing freeze drying protectant were at the 15th day
Sensitivity does not have a significant effect, 37 DEG C of stability >=15 day.
Case study on implementation 2, HIV-1 quantitative fluorescent PCR freeze-dried reagents
First, the preparation of 5 × RT-PCR freeze drying protectants
Trehalose 16g, Ficoll400 0.4g, plus DEPC water are weighed to 40ml, into the healthy and free from worry centrifuge tube that is placed in by more than
Bottle mixing, being placed in water-bath water-bath 10min at 60 DEG C dissolves it, mix homogeneously, ramuscule subpackage, is put into 2-8 DEG C of refrigerator and protects
Deposit standby.
2nd, the preparation of HIV-1 quantitative fluorescent PCRs reaction reagent
HIV-1 reaction reagents are formulated as follows：
Tth PCR Buffer：1×；
Tth enzymes：7.5U；
HIV-1 pf1：400nM；
HIV-1 pr1：400nM；
HIV-1 pf2：400nM；
HIV-1 pr2：400nM；
HIV-1 pb1：100nM；
HIV-1 pb2：100nM；
RT-PCR freeze drying protectants：1×；50 μ l are supplied with water.
HIV-1-PCR freeze-dried reagents containing freeze drying protectant are formulated as follows：
After by the reverse mixing of above each component, by the often μ l subpackages of pipe 50 to the connecting legs of PCR eight.
3rd, the lyophilizing of HIV-1 quantitative fluorescent PCRs reaction reagent：
Eight connecting legs that will be equipped with HIV-1 quantitative fluorescent PCR lyophilizing reaction reagents are put on freeze dryer flaggy, according to following journey
Sequence carries out lyophilizing：
After the completion of lyophilizing, eight connecting legs are taken out, covered stand-by.Outward appearance such as Fig. 4 institutes after HIV-1-PCR freeze-dried reagent lyophilizing
Show.
4th, by the HIV-1-PCR reactant liquors (25 μ l/T) without freeze drying protectant, as a control group, it is formulated as follows：
Above reactant liquor is packed as 6 pipes using centrifuge tube after mixing by the often μ l of pipe 25 × 17T (425 μ l), stand-by.
5th, stability assessment：
HIV-1-PCR freeze-dried reagents are divided into into every 16T for one group, totally 6 groups；6 pipe HIV-1-PCR reactant liquors are often managed as 1 group：
Place 0 day, 3 days, 7 days, 10 days, 15 days and 20 days at 37 DEG C respectively；Respectively in each time point test HIV-1-PCR reactant liquor
With the sensitivity of HIV-1-PCR freeze-dried reagents, compare HIV-1-PCR reactant liquors and added the HIV-1-PCR of freeze drying protectant to freeze
Difference of the dry reagent in stability.
6th, detect
Found according to previous experiments the result, HIV liquid PCR reagents sensitivity about 100IU/ml, therefore arrange each
Per group of detection tri- concentration HIV-1 standard substance of 100IU/ml, 400IU/ml and 2000IU/ml of test, each concentration Parallel testing 5
Part.
1st, HIV-1 standard substance are extracted：The HIV-1 nucleic acid standards of over-richness will be demarcated with WHO HIV-1 nucleic acid standards,
Desired concn is diluted to negative human plasma, the purification of nucleic acid, 0.2ml samples, 60 μ l eluting are carried out with extracts kit.
2nd, it is loaded：In by the often μ l subpackages of pipe HIV-1-PCR reactant liquors 25 to the connecting legs of PCR eight, then add the μ l of sample of nucleic acid 25, shake
Swing upper machine after mixing and moment centrifugation；Often pipe HIV-1-PCR freeze-dried reagents add 25 μ l DEPC water, then add the μ l of sample of nucleic acid 25, shake
Swing upper machine after mixing and moment centrifugation；
3rd, upper machine：It is as follows that response procedures are set on quantitative fluorescent PCR machine：
Channel selecting：FAM
After having run, relative method automatically analyzes result.As a result as shown in table 3, table 4：
The stability result of the HIV-1-PCR reactant liquor sensitivity of table 3
| HIV-1 specimen | 0 day | 37 DEG C 3 days | 37 DEG C 7 days | 37 DEG C 10 days | 37 DEG C 15 days | 37 DEG C 20 days |
| 100IU/ml | 5/5 | 1/5 | 0/5 | 0/5 | 0/5 | 0/5 |
| 400IU/ml | 5/5 | 3/5 | 0/5 | 0/5 | 0/5 | 0/5 |
| 2000IU/ml | 5/5 | 5/5 | 1/5 | 0/5 | 0/5 | 0/5 |
The stability result of the HIV-1-PCR reactant liquor sensitivity of table 4
| HIV-1 specimen | After lyophilizing | 37 DEG C 3 days | 37 DEG C 7 days | 37 DEG C 10 days | 37 DEG C 15 days | 37 DEG C 20 days |
| 100IU/ml | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 |
| 400IU/ml | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 |
| 2000IU/ml | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 |
From Fig. 5～6 and table 3～4, at the 0th day, the HIV-1-PCR freeze-dried reagents containing freeze drying protectant
Initial sensitivity is consistent with the initial sensitivity of HIV-1-PCR reactant liquors；As time goes on, HIV-1-PCR reactant liquors the 3rd
Its sensitivity is significantly affected, its 37 DEG C of stability ＜ 3 days, and 37 DEG C all can not detect after 10 days；And HIV-1-PCR lyophilizing
The sensitivity in 20 days at 37 DEG C of reagent does not have a significant effect, and 37 DEG C of stability are >=20 days, and product is uninfluenced.
By embodiment 1 and embodiment 2, two groups of RNAPCR reactant liquors without freeze drying protectant of the present invention,
The condition stability inferior that accelerates the failure at 37 DEG C is equal less than 3 days, and contains this protectant corresponding freeze-dried reagent and accelerate the failure at 37 DEG C
Under the conditions of stability substantially can >=15 days, significantly extend.Therefore freeze drying protectant of the present invention and freeze drying process can be with
Significantly improve the stability of RNA class PCR reaction reagents.
Above-mentioned embodiment is only the preferred embodiment of the present invention, it is impossible to limit the scope of protection of the invention with this,
The change and replacement of any unsubstantiality that those skilled in the art is done on the basis of the present invention belongs to institute of the present invention
Claimed scope.
Claims (10)
1. a kind of freeze drying protectant of RNA amplification reaction reagent, it is characterised in that：Including trehalose, ficoll and water.
2. the freeze drying protectant of RNA amplification reaction reagent according to claim 1, it is characterised in that：The frozen-dried protective
Agent is that 6.4～16.0g of trehalose, 0.4～1.0g of ficoll are mixed and added water to made by 40ml.
3. the freeze drying protectant of RNA amplification reaction reagent according to claim 2, it is characterised in that：The trehalose with
The proportioning of ficoll is 16g：1g, adds water to 40ml.
4. the freeze drying protectant of the RNA amplification reaction reagent according to any one of claims 1 to 3, it is characterised in that：Institute
Ficoll is stated for Ficoll400.
5. the freeze drying protectant of the RNA amplification reaction reagent according to any one of claims 1 to 3, it is characterised in that：Institute
It is ultra-pure water or the pure water through DEPC process without deoxyribonuclease and ribonuclease to state water.
6. a kind of method for preparing the freeze drying protectant as any one of claims 1 to 3, it is characterised in that：Claim in proportion
Take trehalose, ficoll and water, be placed in mixing in container, be subsequently placed in water-bath under the conditions of 50～60 DEG C water-bath 5～
15min dissolves it, mix homogeneously, ramuscule subpackage, is put into 2～8 DEG C of Refrigerator stores.
7. method according to claim 6, it is characterised in that：Trehalose, ficoll and water are weighed in proportion, are placed in centrifugation
Mixing in pipe, being subsequently placed in the interior water-bath 10min under the conditions of 60 DEG C of water-bath dissolves it, and ramuscule subpackage after mix homogeneously is put
Enter 2～8 DEG C of Refrigerator stores.
8. a kind of freeze drying process of RNA amplification reaction reagent, it is characterised in that：Comprise the following steps：
A, the freeze drying protectant any one of claims 1 to 3 is mixed homogeneously with real-time fluorescence RT-PCR reaction reagent,
Obtain real-time fluorescence RT-PCR reactant liquor；
B, RT-PCR reactant liquors are carried out into lyophilization, that is, obtain RT-PCR freeze-dried reagents.
9. freeze drying process according to claim 8, it is characterised in that：Trehalose is in real-time fluorescence RT- in step a
In PCR reactant liquors final concentration of 4～10%, ficoll in real-time fluorescence RT-PCR reactant liquor final concentration of 0.25～
0.625%.
10. freeze drying process according to claim 8, it is characterised in that：Lyophilisation condition in step b is：Pre-freeze rank
Section through 1h, baffle temperature drop to -45 ± 2 DEG C, pre-freeze when cryogenic hold-time be 2h；Primary drying phase uses first 40min
It is evacuated to 0.16mbar, with 60min baffle temperature risen to into -25 ± 2 DEG C again, then keeps 4h；The parsing-desiccation stage is first
0.01mbar is evacuated to 60～120min, then baffle temperature is risen to into 25 ± 2 DEG C with 60～90min and 2h is kept, then
Baffle temperature is risen to into 35 DEG C with 20min and 4h is kept, finally baffle temperature 25 DEG C is down to into 20min and is kept 2h.
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| CN108411037A (en) * | 2018-03-26 | 2018-08-17 | 郑州安图生物工程股份有限公司 | A kind of freeze-dried reagent that can distinguish three kinds of pathogen nucleic acids simultaneously |
| CN108913758A (en) * | 2018-07-18 | 2018-11-30 | 贝南生物科技（厦门）有限公司 | A kind of freeze drying process and its application for fluorescent PCR amplifing reagent |
| CN109735655A (en) * | 2019-01-21 | 2019-05-10 | 上海科华生物工程股份有限公司 | It is a kind of to detect DNA simultaneously and the multiple fluorescence PCR of RNA prestores freeze-dried reagent |
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| CN110452956B (en) * | 2018-05-08 | 2023-11-03 | 北京中科生仪科技有限公司 | Freeze-dried microsphere of PCR reaction reagent and preparation method thereof |
| CN108913758B (en) * | 2018-07-18 | 2022-06-14 | 贝南生物科技（厦门）有限公司 | Freeze-drying method for fluorescent PCR amplification reagent and application thereof |
| CN108913758A (en) * | 2018-07-18 | 2018-11-30 | 贝南生物科技（厦门）有限公司 | A kind of freeze drying process and its application for fluorescent PCR amplifing reagent |
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| CN111118222A (en) * | 2020-02-26 | 2020-05-08 | 珠海丽珠试剂股份有限公司 | HBV detection kit and using method and application thereof |
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| CN114277104A (en) * | 2021-04-01 | 2022-04-05 | 北京电子科技职业学院 | Freeze-drying protective agent and application thereof |
| CN113621251A (en) * | 2021-08-18 | 2021-11-09 | 深圳市博锐德生物科技有限公司 | Mixed fluorescent dye reagent and freeze-drying method thereof |
| CN113621251B (en) * | 2021-08-18 | 2022-04-05 | 深圳市博锐德生物科技有限公司 | Mixed fluorescent dye reagent and freeze-drying method thereof |
| CN113801926A (en) * | 2021-09-23 | 2021-12-17 | 湖北海卓生物科技有限公司 | Freeze-drying protective agent for molecular detection reagent and application thereof |
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