CN113337572A - Formula and preparation method of virus preservation solution - Google Patents
Formula and preparation method of virus preservation solution Download PDFInfo
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- CN113337572A CN113337572A CN202110698952.5A CN202110698952A CN113337572A CN 113337572 A CN113337572 A CN 113337572A CN 202110698952 A CN202110698952 A CN 202110698952A CN 113337572 A CN113337572 A CN 113337572A
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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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
Abstract
The invention discloses a virus preservation solution formula and a preparation method thereof, wherein the virus preservation solution formula comprises the following components in parts by weight: basic preservation solution: 100 ml; buffer solution: 0.45-12.85g of citric acid, and 2.6-36.5g of 4-hydroxyethyl piperazine ethanesulfonic acid; BSA: 0.2-5.0 g; amino acids: 0.28-2.95 g; antibiotics: including but not limited to penicillin, vancomycin, gentamicin, polymyxin, amphotericin and nystatin, wherein penicillin 0.01-0.12g, vancomycin 0.50-2.82g, gentamicin 0.55-1.50g, polymyxin B0.01-0.10 g, amphotericin 0.10-2.95g and nystatin 0.10-0.70 g; the basic preservation solution can be used for constructing a neutral environment, is beneficial to increasing the survival time and infection stability of viruses, BSA can be used as a protein stabilizer, a protective film can be formed on the protein shell of the viruses, the protective film is not easy to decompose, the integrity of the viruses is ensured, then the antibiotics are combined by adopting a plurality of antibiotics, the protective film has broad-spectrum antibacterial and antifungal effects, and the liquid and the stable state can be maintained in a low-temperature environment through the antifreezing agent.
Description
Technical Field
The invention relates to the technical field of virus preservation solution preparation, in particular to a virus preservation solution formula and a preparation method thereof.
Background
Respiratory tract infections are the most common type of infectious diseases in humans and are one of the leading causes of morbidity and mortality in the population worldwide. The clinical symptoms and physical signs caused by respiratory tract infection are similar, the clinical manifestations mainly comprise cough, expectoration, fever, chest pain and other symptoms, and tracheitis, bronchitis, pneumonia and the like can be caused seriously. The severity of infection, infectivity and pathogenicity caused by different pathogens are different, the treatment method, the curative effect and the disease course are different, and differential diagnosis through pathogen detection is required.
Pathogen infection caused by respiratory viruses in respiratory infectious diseases is the most common, and methods for detecting the respiratory viruses comprise virus isolation culture, an enzyme-linked immunosorbent assay, an immunofluorescence assay, PCR detection and the like, wherein the PCR detection method has high sensitivity and specificity and short time, and becomes a common clinical detection method.
At present, a respiratory tract sample mostly adopts a throat swab, a nose swab, sputum and alveolar lavage fluid, and a throat swab and a nose swab are mostly adopted clinically, so that the sampling is non-traumatic and painless to a patient, convenient and rapid, and the swab needs to be put into a virus preservation solution for preservation after being collected, and then is transported to a laboratory for carrying out various treatments and detections on the sample; the virus is a microorganism with a simple structure and must be parasitic in living cells, the virus preservation solution needs to protect the integrity of the virus and reduce the virus degradation rate, otherwise, false negative detection is easily caused; although ordinary basic preservation solution such as Hank's solution can preserve cells to protect the integrity of viruses, the basic preservation solution cannot resist the growth of bacteria and fungi, is easy to cause pollution, cannot be preserved for a long time and influences the extraction rate and detection of the viruses; on the other hand, respiratory tract infection often occurs in autumn and winter, the temperature in most areas of China is reduced in autumn and winter, particularly the temperature in the north can reach minus dozens of degrees, and the existing virus preservation solution is easily frozen into a solid state in the environment, so that the sampling of clinical medical staff is influenced; therefore, it is necessary to improve the conventional virus preservation solution to solve the above problems.
Disclosure of Invention
The invention aims to solve the problems that although the common basic preservation solution such as Hank's solution can preserve cells to protect the integrity of viruses, the common basic preservation solution cannot resist the growth of bacterial fungi, is easy to cause pollution, cannot be preserved for a long time and influences the extraction rate and detection of the viruses; on the other hand, respiratory tract infection often occurs in autumn and winter, the temperature in most areas of China is reduced in autumn and winter, particularly the temperature in the north can reach minus dozens of degrees, and the conventional virus preservative fluid formula is easily frozen into a solid state in the environment, so that the sampling of clinical medical staff is influenced; the formula and the preparation method of the virus preservation solution realize that the preservation solution is safe and nontoxic, can keep a liquid state at a low temperature, and is convenient to sample.
In order to achieve the purpose, the invention adopts the technical scheme that:
a formula of a virus preservation solution comprises the following components in parts by weight:
basic preservation solution: 100 ml;
buffer solution: 0.45-12.85g of citric acid, and 2.6-36.5g of 4-hydroxyethyl piperazine ethanesulfonic acid;
BSA：0.2-5.0g；
amino acids: 0.28-2.95 g;
antibiotics: including but not limited to penicillin, vancomycin, gentamicin, polymyxin, amphotericin and nystatin, wherein penicillin 0.01-0.12g, vancomycin 0.50-2.82g, gentamicin 0.55-1.50g, polymyxin B0.01-0.10 g, amphotericin 0.10-2.95g and nystatin 0.10-0.70 g;
acid-base indicator: 0.01-0.66 g;
an antifreezing agent: 1.0-10.0 ml.
The foregoing virus preservation solution formulation, the base preservation solution includes, but is not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate and glucose, wherein the sodium chloride is 0.5-15.6g, the potassium chloride is 0.5-5.5g, the calcium chloride is 0.15-3.6g, the magnesium chloride is 0.1-2.4g, the magnesium sulfate is 0.1-2.8g, the disodium hydrogen phosphate is 0.3-7.0g, the potassium dihydrogen phosphate is 0.1-6.3g and the glucose is 1.0-10.0 g.
In the formula of the virus preservation solution, the amino acid is L-glutamic acid.
In the formula of the virus preservation solution, the acid-base indicator adopts phenol red.
In the formula of the virus preservation solution, the antifreezing agent is one or a combination of more of glycerol, dimethyl sulfoxide and polyvinylpyrrolidone.
The formula of the virus preservation solution comprises the following components in parts by weight:
7.6g of sodium chloride, 3.5g of potassium chloride, 1.8g of calcium chloride, 1.3g of magnesium chloride, 2.1g of magnesium sulfate, 3.2g of disodium hydrogen phosphate, 4.5g of potassium dihydrogen phosphate and 6.3g of glucose;
5.75g of citric acid, and 22.3g of 4-hydroxyethyl piperazine ethanesulfonic acid;
BSA2.8g；
1.92g of L-glutamic acid;
penicillin 0.07g, vancomycin 1.73g, gentamicin 0.85g, polymyxin B0.06 g, amphotericin 2.14g and nystatin 0.58 g;
0.55g of phenol red;
glycerol, dimethyl sulfoxide and polyvinylpyrrolidone were combined in a ratio of 1:1:2 to 8.0 ml.
A method for preparing virus preservation solution comprises the following steps:
preparing a basic preservation solution, sequentially measuring 0.5-15.6g of sodium chloride, 0.5-5.5g of potassium chloride, 0.15-3.6g of calcium chloride, 0.1-2.4g of magnesium chloride, 0.1-2.8g of magnesium sulfate, 0.3-7.0g of disodium hydrogen phosphate, 0.1-6.3g of potassium dihydrogen phosphate and 1.0-10.0g of glucose into a quantitative container, and adding physiological saline to fix the volume to 100 ml;
adding a buffer solution into the basic preservation solution, sequentially measuring and adding 0.45-12.85g of citric acid and 2.6-36.5g of 4-hydroxyethyl piperazine ethanesulfonic acid into the basic preservation solution, and stirring for 3-5min to be uniform to prepare a mixed solution A;
adding BSA and amino acid into the mixed solution A, sequentially measuring and adding 0.2-5.0g of BSA and 0.28-2.95g of L-glutamic acid into the mixed solution A, and stirring for 1-2min to be uniform to prepare a mixed solution B;
adding antibiotics into the mixed solution B, sequentially measuring and placing 0.01-0.12g of penicillin, 0.50-2.82g of vancomycin, 0.55-1.50g of gentamicin, 0.01-0.10g of polymyxin B, 0.10-2.95g of amphotericin and 0.10-0.70g of nystatin into a glass container for mixing, adding the antibiotics mixed in the glass container into the mixed solution B, and stirring for 3-5min to be uniform to prepare mixed solution C;
adding an antifreezing agent into the mixed solution C, measuring and adding 1.0-10.0ml of one or a combination of more of glycerol, dimethyl sulfoxide and polyvinylpyrrolidone into the mixed solution C, and stirring for 1-2min to be uniform to prepare a preservation solution;
and (F) adding an acid-base indicator into the preservation solution, measuring and adding 0.01-0.66g of phenol red into the preservation solution, and stirring for 3-5min to be uniform.
The preparation method of the virus preservation solution comprises the following steps of (F), adding an acid-base indicator into the preservation solution, measuring and adding 0.01-0.66g of phenol red into the preservation solution, and stirring for 3-5min until the mixture is uniform, wherein the specific process comprises the following steps:
(F1) weighing 0.01-0.66g of phenol red into a glass container, and shaking for 1-2min to be uniform;
(F2) adding phenol red in a glass container into the prepared preservation solution, and stirring for 3-5min to be uniform;
(F3) and the pH value of the virus preservation solution is 6.0-8.0, the color of the preservation solution is observed during stirring, the preservation solution is packaged if the preservation solution is orange, normal saline is slowly dropped and stirred if the preservation solution is red or yellow, the normal saline is stopped adding when the preservation solution is just changed from red or yellow to orange, and the preservation solution is packaged after stirring for 1min after no obvious color change of the preservation solution.
In the method for preparing the virus preservation solution, the basic preservation solution in the step (A) contains 7.6g of sodium chloride, 3.5g of potassium chloride, 1.8g of calcium chloride, 1.3g of magnesium chloride, 2.1g of magnesium sulfate, 3.2g of disodium hydrogen phosphate, 4.5g of potassium dihydrogen phosphate and 6.3g of glucose; the buffer solution in the step (B) contains 5.75g of citric acid and 22.3g of 4-hydroxyethyl piperazine ethanesulfonic acid; step (C) is BSA2.8g, and L-glutamic acid 1.92 g; the antibiotics in the step (D) comprise 0.07g of penicillin, 1.73g of vancomycin, 0.85g of gentamicin, 0.06g of polymyxin B, 2.14g of amphotericin and 0.58g of nystatin; combining 8.0ml of glycerol, dimethyl sulfoxide and polyvinylpyrrolidone in the antifreeze agent according to the proportion of 1:1: 2; the acid-base indicator in the step (F) contains 0.55g of phenol red.
The invention has the beneficial effects that:
the virus preservation solution formula and the preparation method thereof of the invention can be used for constructing a neutral environment through a basic preservation solution, are beneficial to increasing the survival time and the infection stability of the virus, can buffer the constructed neutral environment through a buffer solution, are also beneficial to increasing the survival time and the infection stability of the virus, can form a protective film on a protein shell of the virus through BSA (bovine serum albumin), ensure that the protective film is not easy to decompose, ensure the integrity of the virus, can provide necessary nutrient substances for the survival of the virus through amino acid, has broad-spectrum antibacterial and fungal effects through combining antibiotics by adopting a plurality of antibiotics, can keep the liquid and stable state under a low-temperature environment through an antifreezing agent, and can keep the preservation solution within a certain pH range under the action of an acid-base indicator, thereby ensuring the stability of the virus protein, the preservation solution is safe and nontoxic, can be kept in a liquid state at low temperature, is convenient to sample, and has wide application prospect.
Detailed Description
The formula of the virus preservation solution comprises the following components in parts by weight:
basic preservation solution: 100 ml;
buffer solution: 0.45-12.85g of citric acid, and 2.6-36.5g of 4-hydroxyethyl piperazine ethanesulfonic acid;
BSA：0.2-5.0g；
amino acids: 0.28-2.95 g;
antibiotics: including but not limited to penicillin, vancomycin, gentamicin, polymyxin, amphotericin and nystatin, wherein penicillin 0.01-0.12g, vancomycin 0.50-2.82g, gentamicin 0.55-1.50g, polymyxin B0.01-0.10 g, amphotericin 0.10-2.95g and nystatin 0.10-0.70 g;
acid-base indicator: 0.01-0.66 g;
an antifreezing agent: 1.0-10.0 ml.
Wherein, the basic preservation solution can be used for constructing a neutral environment; the buffer solution can buffer the constructed neutral environment; BSA can be used as a protein stabilizer, and can form a protective film on the protein shell of the virus, so that the BSA is not easy to decompose and the integrity of the virus is ensured; amino acids can provide essential nutrients for virus survival; the antibiotic has broad-spectrum antibacterial and antifungal effects; the antifreezing agent can keep liquid and stable state in low temperature environment; the acid-base indicator allows the preservation solution to be maintained within a certain pH range.
Preferably, the basic preservation solution includes, but is not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and glucose, wherein the sodium chloride is 0.5-15.6g, the potassium chloride is 0.5-5.5g, the calcium chloride is 0.15-3.6g, the magnesium chloride is 0.1-2.4g, the magnesium sulfate is 0.1-2.8g, the disodium hydrogen phosphate is 0.3-7.0g, the potassium dihydrogen phosphate is 0.1-6.3g, and the glucose is 1.0-10.0 g; the amino acid is L-glutamic acid; the acid-base indicator adopts phenol red; the antifreezing agent is one or a combination of more of glycerol, dimethyl sulfoxide and polyvinylpyrrolidone.
One embodiment of the formula of the virus preservation solution comprises the following components in parts by weight:
7.6g of sodium chloride, 3.5g of potassium chloride, 1.8g of calcium chloride, 1.3g of magnesium chloride, 2.1g of magnesium sulfate, 3.2g of disodium hydrogen phosphate, 4.5g of potassium dihydrogen phosphate and 6.3g of glucose;
5.75g of citric acid, and 22.3g of 4-hydroxyethyl piperazine ethanesulfonic acid;
BSA2.8g；
1.92g of L-glutamic acid;
penicillin 0.07g, vancomycin 1.73g, gentamicin 0.85g, polymyxin B0.06 g, amphotericin 2.14g and nystatin 0.58 g;
0.55g of phenol red;
glycerol, dimethyl sulfoxide and polyvinylpyrrolidone were combined in a ratio of 1:1:2 to 8.0 ml.
The preferred embodiment is safe and non-toxic, can be kept in a liquid state at low temperature, and is convenient for sampling.
The preparation method of the virus preservation solution of the invention comprises the following steps,
step (A), preparing a basic preservation solution, sequentially measuring and adding 0.5-15.6g of sodium chloride, 0.5-5.5g of potassium chloride, 0.15-3.6g of calcium chloride, 0.1-2.4g of magnesium chloride, 0.1-2.8g of magnesium sulfate, 0.3-7.0g of disodium hydrogen phosphate, 0.1-6.3g of potassium dihydrogen phosphate and 1.0-10.0g of glucose into a quantitative container, adding physiological saline to fix the volume to 100ml,
the optimal basic preservation solution comprises 7.6g of sodium chloride, 3.5g of potassium chloride, 1.8g of calcium chloride, 1.3g of magnesium chloride, 2.1g of magnesium sulfate, 3.2g of disodium hydrogen phosphate, 4.5g of potassium dihydrogen phosphate and 6.3g of glucose;
adding buffer solution into the basic preservation solution, sequentially measuring and adding 0.45-12.85g of citric acid and 2.6-36.5g of 4-hydroxyethyl piperazine ethanesulfonic acid into the basic preservation solution, stirring for 3-5min to be uniform to prepare mixed solution A,
the optimal buffer solution comprises 5.75g of citric acid and 22.3g of 4-hydroxyethyl piperazine ethanesulfonic acid;
adding BSA and amino acid into the mixed solution A, sequentially measuring and adding 0.2-5.0g of BSA and 0.28-2.95g of L-glutamic acid into the mixed solution A, stirring for 1-2min to be uniform to prepare a mixed solution B,
the optimal BSA2.8g and the optimal L-glutamic acid 1.92 g;
step (D), adding antibiotics into the mixed solution B, sequentially measuring and placing 0.01-0.12g of penicillin, 0.50-2.82g of vancomycin, 0.55-1.50g of gentamicin, 0.01-0.10g of polymyxin B, 0.10-2.95g of amphotericin and 0.10-0.70g of nystatin into a glass container for mixing, adding the antibiotics mixed in the glass container into the mixed solution B, stirring for 3-5min to be uniform to prepare mixed solution C,
the optimal antibiotics comprise 0.07g of penicillin, 1.73g of vancomycin, 0.85g of gentamicin, 0.06g of polymyxin B, 2.14g of amphotericin and 0.58g of nystatin;
step (E), adding an antifreezing agent into the mixed solution C, measuring and adding 1.0-10.0ml of one or more of glycerol, dimethyl sulfoxide and polyvinylpyrrolidone into the mixed solution C, stirring for 1-2min to be uniform, preparing a preservation solution,
the optimal antifreeze agent is 8.0ml of glycerol, dimethyl sulfoxide and polyvinylpyrrolidone which are combined according to the proportion of 1:1: 2;
step (F), adding an acid-base indicator into the preservation solution, measuring and adding 0.01-0.66g of phenol red into the preservation solution, stirring for 3-5min until the mixture is uniform,
the optimal acid-base indicator comprises 0.55g of phenol red;
the specific process is as follows:
(F1) weighing 0.01-0.66g of phenol red into a glass container, and shaking for 1-2min to be uniform;
(F2) adding phenol red in a glass container into the prepared preservation solution, and stirring for 3-5min to be uniform;
(F3) and the pH value of the virus preservation solution is 6.0-8.0, the color of the preservation solution is observed during stirring, the preservation solution is packaged if the preservation solution is orange, normal saline is slowly dropped and stirred if the preservation solution is red or yellow, the normal saline is stopped adding when the preservation solution is just changed from red or yellow to orange, and the preservation solution is packaged after stirring for 1min after no obvious color change of the preservation solution.
The virus preservation solution prepared by the preparation method has the following specific effects:
(1) contributes to the increase of the survival time and infection stability of the virus; (2) a protective film can be formed on the protein shell of the virus, so that the protein shell is not easy to decompose and the integrity of the virus is ensured; (3) can provide necessary nutrient substances for the survival of the virus; (4) so that the product has broad-spectrum antibacterial and antifungal effects; (5) the liquid and the stable state can be kept in a low-temperature environment; (6) can be kept in a certain pH range, thereby ensuring the stability of the virus protein.
In conclusion, the formula and the preparation method of the virus preservation solution can be used for constructing a neutral environment through a basic preservation solution, are beneficial to increasing the survival time and the infection stability of the virus, can buffer the constructed neutral environment through a buffer solution, is also beneficial to increasing the survival time and the infection stability of the virus, can form a protective film on a protein shell of the virus through BSA (bovine serum albumin) serving as a protein stabilizer, are not easy to decompose, ensure the integrity of the virus, can provide necessary nutrient substances for the survival of the virus through amino acid, has broad-spectrum antibacterial and antifungal effects through combining antibiotics by adopting a plurality of antibiotics, can keep the liquid and the stable state under a low-temperature environment through an antifreezing agent, and can keep the preservation solution in a certain pH range under the action of an acid-base indicator, thereby ensuring the stability of the virus protein.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (9)
1. The formula of the virus preservation solution is characterized by comprising the following components in parts by weight:
basic preservation solution: 100 ml;
buffer solution: 0.45-12.85g of citric acid, and 2.6-36.5g of 4-hydroxyethyl piperazine ethanesulfonic acid;
BSA：0.2-5.0g；
amino acids: 0.28-2.95 g;
antibiotics: including but not limited to penicillin, vancomycin, gentamicin, polymyxin, amphotericin and nystatin, wherein penicillin 0.01-0.12g, vancomycin 0.50-2.82g, gentamicin 0.55-1.50g, polymyxin B0.01-0.10 g, amphotericin 0.10-2.95g and nystatin 0.10-0.70 g;
acid-base indicator: 0.01-0.66 g;
an antifreezing agent: 1.0-10.0 ml.
2. The virus preservative fluid formulation of claim 1, wherein the base preservative fluid includes, but is not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate and glucose, wherein the sodium chloride is 0.5-15.6g, the potassium chloride is 0.5-5.5g, the calcium chloride is 0.15-3.6g, the magnesium chloride is 0.1-2.4g, the magnesium sulfate is 0.1-2.8g, the disodium hydrogen phosphate is 0.3-7.0g, the potassium dihydrogen phosphate is 0.1-6.3g and the glucose is 1.0-10.0 g.
3. The formulation for virus preservation solution according to claim 1, wherein L-glutamic acid is used as the amino acid.
4. The virus preservation solution formulation according to claim 1, wherein phenol red is used as the acid-base indicator.
5. The virus preservation solution formulation according to claim 1, wherein the anti-freezing agent is one or a combination of glycerol, dimethyl sulfoxide and polyvinylpyrrolidone.
6. The virus preservation fluid formulation according to claims 1 to 5, comprising the following components in amounts by weight:
7.6g of sodium chloride, 3.5g of potassium chloride, 1.8g of calcium chloride, 1.3g of magnesium chloride, 2.1g of magnesium sulfate, 3.2g of disodium hydrogen phosphate, 4.5g of potassium dihydrogen phosphate and 6.3g of glucose;
5.75g of citric acid, and 22.3g of 4-hydroxyethyl piperazine ethanesulfonic acid;
BSA2.8g；
1.92g of L-glutamic acid;
penicillin 0.07g, vancomycin 1.73g, gentamicin 0.85g, polymyxin B0.06 g, amphotericin 2.14g and nystatin 0.58 g;
0.55g of phenol red;
glycerol, dimethyl sulfoxide and polyvinylpyrrolidone were combined in a ratio of 1:1:2 to 8.0 ml.
7. A method for preparing a virus preservation solution is characterized by comprising the following steps:
preparing a basic preservation solution, sequentially measuring 0.5-15.6g of sodium chloride, 0.5-5.5g of potassium chloride, 0.15-3.6g of calcium chloride, 0.1-2.4g of magnesium chloride, 0.1-2.8g of magnesium sulfate, 0.3-7.0g of disodium hydrogen phosphate, 0.1-6.3g of potassium dihydrogen phosphate and 1.0-10.0g of glucose into a quantitative container, and adding physiological saline to fix the volume to 100 ml;
adding a buffer solution into the basic preservation solution, sequentially measuring and adding 0.45-12.85g of citric acid and 2.6-36.5g of 4-hydroxyethyl piperazine ethanesulfonic acid into the basic preservation solution, and stirring for 3-5min to be uniform to prepare a mixed solution A;
adding BSA and amino acid into the mixed solution A, sequentially measuring and adding 0.2-5.0g of BSA and 0.28-2.95g of L-glutamic acid into the mixed solution A, and stirring for 1-2min to be uniform to prepare a mixed solution B;
adding antibiotics into the mixed solution B, sequentially measuring and placing 0.01-0.12g of penicillin, 0.50-2.82g of vancomycin, 0.55-1.50g of gentamicin, 0.01-0.10g of polymyxin B, 0.10-2.95g of amphotericin and 0.10-0.70g of nystatin into a glass container for mixing, adding the antibiotics mixed in the glass container into the mixed solution B, and stirring for 3-5min to be uniform to prepare mixed solution C;
adding an antifreezing agent into the mixed solution C, measuring and adding 1.0-10.0ml of one or a combination of more of glycerol, dimethyl sulfoxide and polyvinylpyrrolidone into the mixed solution C, and stirring for 1-2min to be uniform to prepare a preservation solution;
and (F) adding an acid-base indicator into the preservation solution, measuring and adding 0.01-0.66g of phenol red into the preservation solution, and stirring for 3-5min to be uniform.
8. The method for preparing a virus preservation solution according to claim 7, wherein in the step (F), the acid-base indicator is added into the preservation solution, 0.01 to 0.66g of phenol red is measured and added into the preservation solution, and then the mixture is stirred for 3 to 5min until the mixture is uniform, and the specific process is as follows:
(F1) weighing 0.01-0.66g of phenol red into a glass container, and shaking for 1-2min to be uniform;
(F2) adding phenol red in a glass container into the prepared preservation solution, and stirring for 3-5min to be uniform;
(F3) and the pH value of the virus preservation solution is 6.0-8.0, the color of the preservation solution is observed during stirring, the preservation solution is packaged if the preservation solution is orange, normal saline is slowly dropped and stirred if the preservation solution is red or yellow, the normal saline is stopped adding when the preservation solution is just changed from red or yellow to orange, and the preservation solution is packaged after stirring for 1min after no obvious color change of the preservation solution.
9. The method for producing a virus preservation solution according to claim 7, wherein the base preservation solution in the step (A) contains 7.6g of sodium chloride, 3.5g of potassium chloride, 1.8g of calcium chloride, 1.3g of magnesium chloride, 2.1g of magnesium sulfate, 3.2g of disodium hydrogen phosphate, 4.5g of potassium dihydrogen phosphate, and 6.3g of glucose; the buffer solution in the step (B) contains 5.75g of citric acid and 22.3g of 4-hydroxyethyl piperazine ethanesulfonic acid; step (C) is BSA2.8g, and L-glutamic acid 1.92 g; the antibiotics in the step (D) comprise 0.07g of penicillin, 1.73g of vancomycin, 0.85g of gentamicin, 0.06g of polymyxin B, 2.14g of amphotericin and 0.58g of nystatin; combining 8.0ml of glycerol, dimethyl sulfoxide and polyvinylpyrrolidone in the antifreeze agent according to the proportion of 1:1: 2; the acid-base indicator in the step (F) contains 0.55g of phenol red.
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2021
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020135390A1 (en) * | 2018-12-26 | 2020-07-02 | 上海元宋生物技术有限公司 | Oncolytic virus expressing interferon and application therefor |
| CN110438089A (en) * | 2019-07-08 | 2019-11-12 | 深圳市华晨阳科技有限公司 | A kind of virus preservation liquid that can effectively save viral equal samples for a long time |
| CN111808827A (en) * | 2020-08-12 | 2020-10-23 | 泰州鑫联诚润生物技术有限公司 | Virus preserving fluid capable of effectively preserving samples such as viruses for long time |
| CN112314593A (en) * | 2020-11-03 | 2021-02-05 | 武汉尚飞生物科技有限公司 | Preservation solution suitable for normal-temperature preservation of virus samples and preparation method thereof |
| CN112501253A (en) * | 2021-01-25 | 2021-03-16 | 青岛汉唐生物科技有限公司 | Virus preserving fluid with preserving capability and preparation method thereof |
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