CN118086458A - Preparation method of premix liquid precast slab for amplification - Google Patents
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- 230000003321 amplification Effects 0.000 title claims abstract description 21
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 21
- 239000007788 liquid Substances 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 23
- 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 22
- 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 22
- 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 22
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 19
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052782 aluminium Inorganic materials 0.000 claims abstract description 10
- 238000007789 sealing Methods 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 239000000872 buffer Substances 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 51
- 238000010790 dilution Methods 0.000 claims description 19
- 239000012895 dilution Substances 0.000 claims description 19
- 238000004321 preservation Methods 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 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 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 19
- 102000004190 Enzymes Human genes 0.000 abstract description 19
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- 238000001704 evaporation Methods 0.000 abstract description 4
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- 230000007774 longterm Effects 0.000 abstract description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 27
- 229940098773 bovine serum albumin Drugs 0.000 description 27
- 238000003752 polymerase chain reaction Methods 0.000 description 11
- 239000000126 substance Substances 0.000 description 7
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- 102000004169 proteins and genes Human genes 0.000 description 3
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- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108091060592 XDNA Proteins 0.000 description 1
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- 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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Abstract
The invention discloses a preparation method of a premix liquid precast slab for amplification, which comprises the following steps: preparing a precast slab, and adding an amplification reagent required by a PCR reaction into micropores of the precast slab; adding BSA buffer and/or trehalose solution into the micropores; and sealing and preserving the prefabricated plate by adopting a pressure-sensitive aluminum film. The prefabricated plate prepared by the method can not reduce enzyme activity under the condition of long-term storage at the temperature of minus 15 ℃ to minus 25 ℃, can reduce the performance loss of Taq enzyme caused by repeated freezing and thawing, has better sealing and preserving effects of the pressure-sensitive aluminum film, and has lower evaporation loss.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a preparation method of a premix liquid precast slab for amplification.
Background
Polymerase Chain Reaction (PCR) is a molecular biological technique for amplifying specific DNA fragments, which can be regarded as specific DNA replication in vitro, and the greatest feature of PCR is the ability to greatly increase minute amounts of DNA.
The polymerase chain reaction requires a system consisting of buffer solution, template nucleic acid, dNTPs, DNA polymerase and primers, wherein the required preservation conditions of the reagents are different, and if the DNA polymerase is repeatedly frozen and thawed in multiple uses, the activity of the DNA polymerase can be influenced, so that the reaction can not be effectively carried out. However, the amounts of samples taken in each experiment were not uniform, resulting in unavoidable repeated freeze-thawing of the DNA polymerase. The preparation of the polymerase chain reaction system once is required for each experiment, which is time-consuming and labor-consuming, is unfavorable for the storage of the reagents and ensures the effectiveness of the reagents, and the risk of pollution is increased by one minute for each use of various reagents. And the enzyme activity of the system formed by mixing the DNA polymerase and the primer can be influenced by the storage time, and the whole shelf life can be shortened.
Therefore, in the field, it is necessary to develop a prefabricated plate for amplification, the prefabricated plate needs to be stored at-20 ℃ in order to preserve the activity of enzyme, so that the sealed preservation condition of the prefabricated plate is important, different sealing materials and modes can also influence the stability of enzyme, and different sealing modes of different materials can influence the evaporation of the reagent.
Disclosure of Invention
In order to solve at least one of the problems, the invention provides a preparation method of a premix liquid precast slab for amplification. The prefabricated plate prepared by the method can not reduce enzyme activity under the condition of long-term storage, and can reduce the performance loss of Taq enzyme caused by repeated freezing and thawing.
In order to achieve the above purpose, the invention adopts the following technical means:
the first aspect of the invention provides a method for preparing a premix liquid prefabricated plate for amplification, which comprises the following steps:
(1) Preparing a precast slab, and adding an amplification reagent required by a PCR reaction into micropores of the precast slab;
(2) Adding BSA buffer and/or trehalose solution into the micropores;
(3) And sealing and preserving the prefabricated plate by adopting a pressure-sensitive aluminum film.
Trehalose (Trehalose) is disaccharide, can form special protective film on the cell surface under severe conditions such as high temperature, high cold, drying and water loss, and can effectively protect the biological molecular structure from being damaged, thereby maintaining the life process and biological characteristics of a living body. Trehalose has heat stability and heat activation properties. Trehalose can maintain the skeleton of the protein during thermal denaturation of the protein, and maintain the natural structure of the protein. Trehalose may be used as a PCR enhancer. The stability of Taq DNA polymerase is maintained by reducing the DNA double-strand melting temperature, and the PCR reaction efficiency is improved.
BSA, bovine serum albumin, is a stabilizer for enzymes, preventing decomposition and non-specific adsorption of enzymes; BSA reduces the denaturation of some enzymes and reduces the denaturation caused by adverse environmental factors such as heat, surface tension and chemical factors, probably because it has 17 disulfide bonds in its structure, and a thiol group which has a very active chemical reaction and has an anti-redox effect, thus being able to bind with various cations, anions and small molecules; BSA also prevents enzyme from adsorbing to the tube wall and losing. The preservation time can be prolonged, the enzyme activity can be enhanced, and the PCR reaction efficiency can be improved by adding a certain amount of BSA (bovine serum albumin) into the PCR premix.
In some embodiments of the invention, the BSA buffer is present at a concentration of 5-20mg/mL. Preferably, the concentration of BSA buffer is 10mg/mL.
In some embodiments of the invention, the trehalose solution has a concentration of 25-80mg/mL. Preferably, the concentration of trehalose solution is 50mg/mL.
In some embodiments of the invention, the volume ratio of BSA buffer to trehalose solution in the mixture of BSA buffer and trehalose solution is 1:1.
In some embodiments of the invention, the preservation conditions are: preserving at-15 to-25 ℃. Further, the preservation time is not longer than 12 months, and the effect is optimal.
In some embodiments of the invention, the amplification reagents include primers, primer dilution solutions, and 2 x DNA polymerase MIX.
In some embodiments of the invention, the primer dilution solution is a 10mM Tris-HCl solution.
Primer dilution using primer dilution solution: for the concentrated solution primer, transferring a certain volume of the original solution into a proper new container, and then adding a corresponding volume of diluted solution into the new container; for dry powder primers, a small amount of dilution solution is used for reconstitution, transferred to a suitable new container, and then the dilution solution is replenished.
In some embodiments of the invention, the primer concentration is 2.5 μm.
In some embodiments of the invention, the prefabricated plate prepared by the preparation method of the invention is applied to a PCR amplification kit.
The beneficial effects of the invention are that
Compared with the prior art, the invention has the following beneficial effects:
The reagent consumable used in the method is simple and easy to obtain, and special reagents and consumable materials are not required to be additionally prepared; the operation is simple, and redundant operation steps are not needed to be added in the experiment; the required reagent can be used according to the reaction quantity, and the activity of the reagent can not be influenced by repeated freezing and thawing of the reagent; and complicated amplification system preparation is not needed for each experiment, so that the risk of reagent pollution is reduced.
The addition of the BSA solution increases the preservation time of the premix and enhances the enzymatic activity. BSA is a stabilizer for enzymes, preventing decomposition and non-specific adsorption of enzymes; BSA reduces the denaturation of some enzymes and reduces the denaturation caused by adverse environmental factors such as heat, surface tension and chemical factors, probably because it has 17 disulfide bonds in its structure, and a thiol group which has a very active chemical reaction and has an anti-redox effect, thus being able to bind with various cations, anions and small molecules; BSA also prevents enzyme from adsorbing to the tube wall and losing.
The precast slab for storing the premix liquid has better storage effect when being stored at-15 to-25 ℃ than the thermosensitive film by using the pressure-sensitive film, and meanwhile, the precast slab added with 5 mu L of 10mg/mL BSA stabilizer has the longest storage time, and can keep 83% of effective rate after 12 months of storage.
Drawings
FIG. 1 shows the result of gel electrophoresis of PCR amplified products of the same sample by the prefabricated plate in example 1 of the present invention.
Detailed Description
The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.
The technical scheme of the patent is further described in detail below with reference to the specific embodiments.
Example 1
(1) Checking primer COA, confirming primer information, the confirmation method including but not limited to: primer name, amount of primer substance (nmol number), raw state (dry powder or solution concentration).
(2) A10 mg/mL BSA solution was prepared, 100mg of bovine serum albumin was added to 9.5mL water, the cap was closed, and the mixture was gently shaken until the bovine serum albumin was completely dissolved, without vortexing. Water was added to a constant volume of 10mL. If the required dilution is greater than 10mL, the dosage may be scaled up equally.
(3) A50 mg/mL trehalose solution was prepared, 500mg of bovine serum albumin was added to 9.5mL water, the cap was closed, and the mixture was gently shaken until the bovine serum albumin was completely dissolved, without vortexing. Water was added to a constant volume of 10mL. If the required dilution is greater than 10mL, the dosage may be scaled up equally.
(4) The dilution solution required to dilute each primer to a theoretical concentration of 2.5. Mu.M was calculated:
dry powder calculation mode: volume of dilution (μl) =400×amount of substance (nmol).
Solution calculation mode: dilution volume (μl) = [0.4 x concentration (μΜ) -1] ×primer volume (μl).
(5) Preparing primer dilution solution: 99mL nuclease-free water was measured with a measuring cylinder, 1mL of 1MTris-HCl (pH 8.0) was added thereto, and the mixture was thoroughly mixed. If the required dilution is greater than 100mL, the dosage may be scaled up equally.
(6) Primer dilution using primer dilution solution: for the concentrated solution primer, transferring a certain volume of the original solution into a proper new container, and then adding a corresponding volume of diluted solution into the new container; for dry powder primers, a small amount of dilution solution is used for reconstitution, transferred to a suitable new container, and then the dilution solution is replenished.
(7) After the primer added with the diluted solution is subjected to short centrifugation, the primer is placed on a super constant temperature mixer, mixed uniformly for 2 minutes at a mixing speed of 1400rpm, and then centrifuged for a short time again, so that the primer solution is concentrated at the bottom of the container.
(8) Two special precast plates for inspection are manufactured, the inspection is submitted, the precast plates used for the special precast plates for inspection are 96-well plates, corresponding 64F-series primers and 66R-series primers are required to be added into the 96-well plates, 2.5 mu L of diluted solution is added into the holes of the diluted solution, and no liquid is added into the holes of the hollow holes.
Then 12.5. Mu.L of 2 XDNA polymerase was added to each non-empty site and the membrane was sealed. The serial names of the prefabricated panels are identified and the hole site areas loaded with reagents are indicated, and the prefabricated panel information is shown in table 1.
TABLE 1 primer examination of prefabricated plate information
(9) The same sample was tested using the above test-specific prefabricated panels: bacterial 16SDNA positive standard, 10 ng/. Mu.L, 50ng added; PCR amplification was performed.
(10) After completion of the library construction, 5. Mu.L of the amplified product was subjected to 1.5% agarose gel electrophoresis under conditions of 120V for 25 minutes using a Marker (e.g., DL 2000 produced by Takara Co., ltd.) capable of indicating a band of about 500 bp.
(11) After the electrophoresis, the film was photographed under the transmitted ultraviolet rays, and the agarose gel electrophoresis results are shown in FIG. 1.
The results show that: the library of each added primer had a significant target band in the corresponding region of interest, and the library corresponding to the "diluted solution" did not have that band.
(12) Barcode cross-contamination test:
(13) And mixing the amounts of the substances in the library obtained by constructing the library of the special precast slab for detection, and performing second generation sequencing.
(14) The results returned from sequencing were split according to 64X 66.
(15) The crossover rate of each primer was calculated as follows: (1-reasonable reads number/total reads number). Times.1000%. Taking the F01 primer as an example, if the number of reads of F01-R01 in the sequencing result is 20000, the number of reads of F01-R65 is 19000, and the total number of reads including the F01 sequence is 40000, the crossover rate is 2.5%.
(18) The crossover rate of each primer should not exceed 5.0%.
The results show that: the crossover rate of each primer was calculated to be in the range of 1-3% by the results returned from NGS sequencing.
Example 2
The primers required for preparing the preformed plate, the polymerase MIX, were removed from the freezer and allowed to equilibrate at room temperature for at least 30 minutes to ensure complete melting.
The materials were mixed by inversion, vortexing, etc., and then concentrated at the bottom of the vessel at 2000rpm in a snap-off manner.
And (3) carrying out primer packaging according to primer combinations corresponding to the prefabricated plates, wherein the prefabricated plates are divided into A, B, C, D identical prefabricated plates by polymerase MIX, and the divided prefabricated plates are respectively subjected to film packaging by adopting 4 modes of a pressure-sensitive plastic packaging film, a heat-sensitive plastic packaging film, a pressure-sensitive aluminum film and a heat-sensitive aluminum film.
And storing the prefabricated plate with the film sealed at the temperature of minus 20 ℃.
The enzyme activity was assayed for effectiveness after 3 months, 6 months, 9 months and 12 months of storage, respectively, by the following method:
(1) Taking 10 ng/. Mu.L and 50ng of bacterial 16SDNA positive standard substance for PCR amplification;
(2) After completion of the library construction, 5. Mu.L was subjected to 1.5% agarose gel electrophoresis under conditions of 120V for 25 minutes, using a Marker (DL 2,000 produced by Takara Co., ltd.) capable of indicating a band around 500 bp.
(3) After electrophoresis, shooting under the transmission ultraviolet, observing that the library added with the primers has obvious target strips in the corresponding target areas, and counting different treatment modes in each storage time period, wherein the effective conditions of the precast slabs are shown in the table 2.
TABLE 2 effectiveness of prefabricated panels with different sealing modes
The results show that: the pressure-sensitive film has better preservation effect than the heat-sensitive film when preserved at-15 to-25 ℃, and is supposed to be weakened in the film sealing effect at low temperature after the film sealing of the heat-sensitive film. The aluminum film has better preservation effect than the plastic packaging film and lower evaporation loss. Since the reagent evaporation loss can affect the effectiveness of PCR amplification, the preservation time of the plastic packaging film precast slab is preferably not more than 6 months, the preservation time of the aluminum film precast slab is preferably not more than 9 months, and the reagent preserved at low temperature is preferably not used as a thermosensitive film.
Example 3
The primers required for preparation of the preformed plate, the polymerase MIX, the BSA solution and the trehalose solution were removed from the freezer and allowed to equilibrate for at least 30 minutes at room temperature to ensure complete melting.
The materials were mixed by inversion, vortexing, etc., and then concentrated at the bottom of the vessel at 2000rpm in a snap-off manner.
And (3) carrying out primer filling according to the primer combination corresponding to the prefabricated plates, wherein the prefabricated plates are divided into A, B, C, D identical prefabricated plates by polymerase MIX, 5 mu L of BSA solution is added into the prefabricated plates B, 5 mu L of trehalose solution is added into the prefabricated plates C, and 5 mu L of mixed solution of the trehalose solution and the BSA solution according to a ratio of 1:1 is added into the prefabricated plates D.
And sealing the films by adopting a pressure-sensitive aluminum film mode respectively.
And storing the prefabricated plate with the film sealed at the temperature of minus 20 ℃.
The enzyme activity was assayed for effectiveness after 3 months, 6 months, 9 months and 12 months of storage, respectively, by the following method:
(1) Taking 10 ng/. Mu.L and 50ng of bacterial 16SDNA positive standard substance for PCR amplification;
(2) After completion of the library construction, 5. Mu.L was subjected to 1.5% agarose gel electrophoresis under conditions of 120V for 25 minutes, using a Marker (DL 2,000 produced by Takara Co., ltd.) capable of indicating a band around 500 bp.
(3) After electrophoresis, shooting under the transmission ultraviolet, observing that the library added with the primers has obvious target bands in the corresponding target areas, and counting different treatment modes in each storage time period, wherein the effective conditions of the precast slabs are shown in the table 3.
TABLE 3 effectiveness of prefabricated panels with different additives
The results show that: the precast slab added with 5 mu L of 10mg/mL BSA solution can maintain 83% of effective rate after 12 months of storage, and the mixed solution of 5 mu L of trehalose solution and BSA solution according to the ratio of 1:1 can maintain 75% of effective rate after 12 months of storage, and the storage effect of the added trehalose solution is poor and is only 67%, but is significantly higher than 54% of effective rate without adding enzyme stabilizer.
Under the condition of selecting the pressure-sensitive plastic packaging film, the BSA solution can be stored for 12 months at the temperature of minus 15 to minus 25 ℃, and the trehalose solution can be stored for 9 months at the temperature of minus 15 to minus 25 ℃.
In summary, the prefabricated plate prepared by adopting the sealing mode of adding 5 mu L of 10mg/mL BSA and simultaneously using the pressure-sensitive aluminum film has longer storage time under the storage condition of minus 15 ℃ to minus 25 ℃.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be understood that various changes and modifications may be made by those skilled in the art after reading the above teachings of the application, and such equivalents are intended to fall within the scope of the application as defined by the claims.
Claims (8)
1. The preparation method of the premix liquid precast slab for amplification is characterized by comprising the following steps:
(1) Preparing a precast slab, and adding an amplification reagent required by a PCR reaction into micropores of the precast slab;
(2) Adding BSA buffer and/or trehalose solution into the micropores;
(3) And sealing and preserving the prefabricated plate by adopting a pressure-sensitive aluminum film.
2. The method for preparing a premix liquid prefabricated plate for amplification according to claim 1, wherein the method comprises the following steps: the concentration of the BSA buffer is 5-20mg/mL.
3. The method for preparing a premix liquid prefabricated plate for amplification according to claim 1, wherein the method comprises the following steps: the concentration of the trehalose solution is 25-80mg/mL.
4. The method for preparing a premix liquid prefabricated plate for amplification according to claim 1, wherein the method comprises the following steps: in the mixed solution of the BSA buffer solution and the trehalose solution, the volume ratio of the BSA buffer solution to the trehalose solution is 1:1.
5. The method for preparing a premix liquid prefabricated plate for amplification according to claim 1, wherein the method comprises the following steps: the preservation conditions are as follows: preserving at-15 to-25 ℃.
6. The method for preparing a premix liquid prefabricated plate for amplification according to claim 1, wherein the method comprises the following steps: the amplification reagents include primers, primer dilution solution, and 2 x DNA polymerase MIX.
7. The method for preparing a pre-mixed liquid precast slab for amplification according to claim 6, wherein: the primer dilution solution was 10mM Tris-HCl solution.
8. The method for preparing a pre-mixed liquid precast slab for amplification according to claim 6, wherein: the primer concentration was 2.5. Mu.M.
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