CN117025553B - Virus preservation solution capable of keeping virus activity at non-ultralow temperature and preparation method thereof - Google Patents
Virus preservation solution capable of keeping virus activity at non-ultralow temperature and preparation method thereof Download PDFInfo
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- CN117025553B CN117025553B CN202311296290.4A CN202311296290A CN117025553B CN 117025553 B CN117025553 B CN 117025553B CN 202311296290 A CN202311296290 A CN 202311296290A CN 117025553 B CN117025553 B CN 117025553B
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- 241000700605 Viruses Species 0.000 title claims abstract description 150
- 239000003761 preservation solution Substances 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 40
- 230000000694 effects Effects 0.000 title claims abstract description 22
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 53
- 238000004321 preservation Methods 0.000 claims abstract description 31
- 239000000243 solution Substances 0.000 claims abstract description 29
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 28
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 28
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 28
- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 claims abstract description 27
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- 229960003531 phenolsulfonphthalein Drugs 0.000 claims abstract description 19
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- 230000000415 inactivating effect Effects 0.000 claims description 2
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 206010069767 H1N1 influenza Diseases 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241000712431 Influenza A virus Species 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 1
- 230000000531 effect on virus Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a virus preservation solution for keeping virus activity at non-ultralow temperature and a preparation method thereof, wherein the virus preservation solution comprises the following components: 6.5-9.5g/L KCl,0.4-0.6g/L NaH 2 PO 4 The method comprises the steps of carrying out a first treatment on the surface of the Na 2.4-3.4g/L 2 HPO 4 HEPES solution of 20-30mM, bovine serum albumin of 0.8-1.2g/L, glucose of 0.8-1.2g/L, gentamicin sulfate of 180-220mg/L, polymyxin of 3.2-4.8mg/L, nystatin of 8-12mg/L, phenol red of 20-30mg/L, glycerol of 85-115mL/L, and defoamer of 0.1-0.4mL/L. The virus preservation solution can maintain the activity of virus and the integrity of nucleic acid for a long time at 25 ℃ or 2-8 ℃ to a large extent, and reduce the preservation condition and cost in the process of collection and transportation.
Description
Technical Field
The invention relates to the technical field of biological reagents, in particular to a virus preservation solution capable of keeping virus activity at a non-ultralow temperature and a preparation method thereof.
Background
Most viruses are pathogenic microorganisms that are capable of causing the host to become pathogenic and to spread in the organism and are infectious, but viruses typically deactivate soon after leaving the host cell, the viral integrity is destroyed, the viral coat disintegrates, and the nucleic acid degrades. However, studies on virus morphology, pathogenesis, degree of variation, infectivity, etc. are required to ensure the activity and structural integrity of the virus. Therefore, a specific preservation solution is required to preserve the collected virus sample for subsequent research works such as virus culture, virus morphology, pathogenesis, genetic variation, infectivity, surface antigen, vaccine, and the like.
Chinese patent application publication No. CN113046412B discloses a high-safety non-inactivated virus preservation solution and a preparation method thereof, wherein after virus is added into the preservation solution, the preservation is carried out at-70 ℃, and after 6 days, the virus infectivity is reduced to about 60 percent. The virus preservation solution has poor effect of maintaining the virus activity; in addition, if the virus is kept in a temperature environment of-70 ℃ all the time during the collection and transportation, the condition is too severe, and the collection and transportation cost is greatly increased.
Chinese patent application CN 112501253A provides a virus preservation solution with preservation capability, comprising: 0.1-0.9g of magnesium sulfate, 2.0-3.6g of calcium chloride, 0.7-1.5g of potassium chloride, 6.0-10.0g of sodium chloride, 0.5-2.5g of D-glucose, 0.01-0.23g of monopotassium phosphate, 0.05-0.15g of disodium hydrogen phosphate dodecahydrate, 0.5-1.5g of bovine serum albumin, 3.0-8.0mL of diabase (penicillin (alkali) and streptomycin (alkali)), 50-90mL of glycerin, 5.0-7.0mg of phenol red, 0.1-0.3mL of defoaming agent, 0.28-2.95g of L-glutamic acid and 2.0-6.0g of HEPES-Na. Experiments prove that the virus sample preservation solution can also transport and preserve the virus sample at the temperature of 4 ℃ for about 3 days, preserve the virus sample at the room temperature of 25 ℃ for 2 days, and the measured concentration of the virus sample is reduced from 8.41 ng/mu L on the day 0 to about 1.43 on the day 3, so that the virus sample preservation solution has no capability of infecting cells.
Disclosure of Invention
First, the technical problem to be solved
In view of the above-mentioned shortcomings and disadvantages of the prior art, the present invention provides a virus preservation solution for maintaining virus activity at a non-ultralow temperature and a preparation method thereof, wherein the virus preservation solution maintains the activity (infectivity) and the antigen/nucleic acid integrity of a virus sample for a long time to a large extent at a normal temperature (25 ℃) or a temperature of 2-8 ℃ when preserving viruses, thereby greatly reducing the requirements of the conditions for preserving viruses, optimizing the sample preservation conditions in the collection and transportation processes, reducing the cost of collection, preservation and transportation, and facilitating research work of research personnel for culturing, identification, sequencing, pathogenesis and the like of viruses. The invention also relates to a preparation method of the virus preservation solution.
(II) technical scheme
In a first aspect, the present invention provides a virus preservation solution that maintains virus viability at non-ultralow temperatures, consisting of an aqueous solution comprising: KCl and NaH 2 PO 4 、Na 2 HPO 4 HEPES solution, bovine serum albumin, glycerol, glucose, gentamicin sulfate, polymyxin, nystatin, defoamer, phenol red; wherein,
KCl concentration is 6.5-9.5g/L, naH 2 PO 4 The concentration of (2) is 0.4-0.6g/L; na (Na) 2 HPO 4 The concentration of (2.4-3.4 g/L), the concentration of HEPES solution is 20-30mM, the concentration of bovine serum albumin is 0.8-1.2g/L, the concentration of glucose is 0.8-1.2g/L, the concentration of gentamicin sulfate is 180-220mg/L, the concentration of polymyxin is 3.2-4.8mg/L, the concentration of nystatin is 8-12mg/L, the concentration of phenol red is 20-30mg/L, the addition of glycerol is 85-115mL/L,
the addition amount of the defoaming agent is 0.1-0.4mL/L.
As a preferred embodiment of the present invention, the concentration of KCl in the virus-preserving fluid is 7.5-8.5g/L, naH 2 PO 4 The concentration of (2) is 0.45-0.55g/L; na (Na) 2 HPO 4 The concentration of (2.8-3.2 g/L), the concentration of HEPES solution is 23-27mM, the concentration of bovine serum albumin is 0.9-1.1g/L, the concentration of glucose is 0.9-1.1g/L, the concentration of gentamicin sulfate is 195-205mg/L, the concentration of polymyxin is 3.8-4.2mg/L, the concentration of nystatin is 9-11mg/L, the concentration of phenol red is 23-27mg/L, the addition of glycerol is 90-110mL/L, and the addition of defoamer is 0.2-0.3mL/L.
As a means ofIn a preferred embodiment of the present invention, the concentration of KCl in the virus preservation solution is 8g/L, naH 2 PO 4 Is 0.5g/L; na (Na) 2 HPO 4 The concentration of (2) is 3g/L, the concentration of HEPES solution is 25mM, the concentration of bovine serum albumin is 1g/L, the concentration of glucose is 1g/L, the concentration of gentamicin sulfate is 200mg/L, the concentration of polymyxin is 4mg/L, the concentration of nystatin is 10mg/L, the concentration of phenol red is 25mg/L, the addition amount of glycerol is 100mL/L, and the addition amount of defoamer is 0.2-0.3mL/L.
As a preferred embodiment of the present invention, the pH of the virus preservation solution is=7.4±0.2.
In a second aspect, the present invention provides a method for preparing a virus preservation solution that maintains virus viability at a non-ultralow temperature, comprising:
s1, measuring HEPES solution into a liquid preparation container according to the composition of virus preservation liquid;
s2, accurately weighing NaH respectively 2 PO 4 、Na 2 HPO 4 Adding KCl, bovine serum albumin, glucose and phenol red into a liquid preparation container for dissolution;
s3, glycerol and an antifoaming agent are measured and added into a liquid preparation container, a purified water rinse measuring tool is adopted for 2-3 times, and rinse water of each time is added into the liquid preparation container;
s4, weighing gentamicin sulfate, polymyxin and nystatin, and adding the gentamicin sulfate, the polymyxin and the nystatin into a liquid preparation container;
s5, adding purified sterile water into a liquid preparation container for constant volume, shaking uniformly, and regulating the pH value to be pH value of 7.4+/-0.2 measured at 23-28 ℃;
s6, filtering by using a filtering device with the aperture of 0.22 micrometer, performing sterilization and impurity removal at the speed of 5L/min, and performing high-pressure sterilization for 30min under the condition of 121 ℃ and 0.1 Mpa; cooling, and standing at room temperature for storage.
In a third aspect, the present invention also provides a non-inactivating preservation method for a virus at a non-low temperature, where the virus is preserved in the virus preservation solution for maintaining the viability of the virus at a non-ultralow temperature according to any one of the first aspect, where the preservation temperature is as follows: sealing and preserving for less than or equal to 72 hours at room temperature; or sealing and preserving for less than or equal to 168 hours at the ambient temperature of 2-8 ℃.
(III) beneficial effects
(1) Compared with the prior technical scheme of preserving viruses at the temperature of less than or equal to-70 ℃, the invention has the most remarkable technical effects that: the virus preservation solution provided by the invention can realize long-time non-inactivation preservation of viruses in a room temperature environment or a temperature environment of 2-8 ℃; the virus still has the ability to infect normal cells when stored for 3 days in a room temperature environment, and has the ability to infect normal cells when stored for 7 days in a 2-8 ℃ temperature environment. The characteristic signals of the viruses can still be detected by PCR amplification detection of the virus sample stored for 7 days at normal temperature (25 ℃) in the virus preservation solution, which indicates that the nucleic acid of the viruses is not completely degraded and the complete nucleic acid of the viruses is still remained in the preservation solution. The PCR amplification detection shows that the virus sample stored at 2-8 ℃ for 7 days in the virus preservation solution has no difference between the Ct value at 7 days and the Ct value at 0 days, thus the integrity rate of the virus nucleic acid is not obviously reduced along with the increase of the preservation time.
(2) Compared with the prior art scheme that the virus sample is preserved for 2 days at normal temperature or only 3 days at 4 ℃, the virus preservation solution can maintain the infection activity of the virus sample and the integrity of antigen/nucleic acid for a longer time and to a greater extent.
The experimental results show that the virus preservation solution provided by the invention can realize good preservation effect on viruses under the non-ultralow temperature conditions such as normal temperature or 2-8 ℃, maintain the activity (infectivity) and the antigen/nucleic acid integrity of the viruses, greatly reduce the harsh conditions for preserving the viruses, optimize the sample preservation conditions in the collection and transportation processes, reduce the collection, preservation and transportation costs, and more facilitate the development, detection and research of researchers.
Drawings
FIG. 1 is a graph showing fluorescence PCR amplification by sampling on days 0, 3 and 7, respectively, when viruses were stored in a 25℃environment for 7 days using the virus storage solution of example 1.
FIG. 2 is a graph showing fluorescence PCR amplification by sampling on days 0, 3 and 7, respectively, when viruses were stored for 7 days at 2-8deg.C using the virus-preserving fluid of example 2.
FIG. 3 a is an illustration of the initial infection of cells by the use of the virus stock solution prepared in example 1 in the context of the preservation of infectious bronchitis virus in chickens at 25 ℃;
FIG. 3 b is a diagram showing the initial cell infection of a chicken infectious bronchitis virus preserved in an environment of 2-8deg.C using the virus preservation solution prepared in example 1;
FIG. 3 c is the infection of cells after preservation of chicken infectious bronchitis virus at 25℃for several days using the virus preservation solution prepared in example 1;
FIG. 3 d shows the infection of cells after the chicken infectious bronchitis virus was stored at 2-8deg.C for several days using the virus preservation solution prepared in example 1.
Detailed Description
The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.
Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not noted, according to conventional conditions or manufacturer's recommended conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
The virus preservation solution of the present embodiment is composed of an aqueous solution containing the following solutes: KCl and NaH 2 PO 4 、Na 2 HPO 4 HEPES solution, bovine serum albumin, glycerol, glucose, gentamicin sulfate, polymyxin, nystatin, defoamer, phenol red; wherein the concentration of KCl is 8g/L, naH 2 PO 4 Is 0.5g/L; na (Na) 2 HPO 4 The concentration of (2) was 3g/L, the concentration of HEPES solution was 25mM, the concentration of bovine serum albumin was 1g/L, the concentration of glucose was 1g/L, the concentration of gentamicin sulfate was 200mg/L, the concentration of polymyxin was 4mg/L, the concentration of nystatin was 10mg/L, the concentration of phenol red was 25mg/L, glycerolThe amount of the defoaming agent added was 100mL/L, and the amount of the defoaming agent added was 0.25mL/L.
The preparation method of the virus preservation solution comprises the following steps:
(1) according to the composition of the virus preservation solution, measuring HEPES solution into a solution preparation container;
(2) accurately weighed NaH respectively 2 PO 4 、Na 2 HPO 4 Adding KCl, bovine serum albumin, glucose and phenol red into a liquid preparation container for dissolution;
(3) adding glycerol and defoamer into a liquid preparation container, wetting the container with purified water for 2-3 times, and adding each time of wetting water into the liquid preparation container;
(4) weighing gentamicin sulfate, polymyxin and nystatin, and adding into a liquid preparation container;
(5) adding purified sterile water into a liquid preparation container for constant volume, shaking uniformly, and regulating pH to be 7.4+/-0.2 when the pH value is measured at 23-28 ℃;
(6) filtering with a 0.22 μm pore size filter, sterilizing at 5L/min, removing impurities, and autoclaving at 121deg.C under 0.1Mpa for 30min; cooling, and standing at room temperature for storage.
Example 2
The concentration of each component in the virus-retaining solution of this example was adjusted to a small extent as compared with that of example 1. Wherein the concentration of KCl is 8.5g/L, naH 2 PO 4 Is 0.55g/L; na (Na) 2 HPO 4 The concentration of (2) was 3.2g/L, the concentration of HEPES solution added was 27mM, the concentration of bovine serum albumin was 1.1g/L, the concentration of glucose was 1.1g/L, the concentration of gentamicin sulfate was 205mg/L, the concentration of polymyxin was 4.2mg/L, the concentration of nystatin was 11mg/L, the concentration of phenol red was 27mg/L, the amount of glycerol added was 110mL/L, and the amount of antifoaming agent added was 0.3mL/L. The preparation method is the same as in example 1.
Verification of the efficacy of preservation solution for preserving viruses
(1) The virus stock of example 1 was mixed with influenza A H1N1 virus at a volume ratio of 100:10, left at 25℃for 7 days, sampled on day 0, day 3, and day 7 (with equal sample volumes each time), and fluorescent PCR amplification was performed on the viral nucleic acid integrity in the stock, and the detection results were shown in FIG. 1. The fluorescent PCR amplification result shows that when influenza virus is added into the virus preservation solution and placed for 3 days at 25 ℃, the Ct value is about 22 and the difference between the Ct value and the day 0 is not great, which indicates that the virus antigen/nucleic acid integrity is very high when the influenza virus is placed and preserved for 2 days at 25 ℃. When left under 25℃for 7 days, the nucleic acid signal of influenza virus was detected although the CT value increased to about 28 as compared with the 0 th day, which indicates that the viral nucleic acid was not completely degraded when left under 25℃for 7 days.
(2) The virus preservation solution of example 2 and influenza A H1N1 virus are mixed according to the volume ratio of 100:10, placed for 7 days at the temperature of 2-8 ℃, sampled on the 0 th day, 3 rd day and 7 th day respectively (the sampling volumes are equal each time), and the integrity of the viral nucleic acid in the preservation solution is subjected to fluorescent PCR amplification, and the detection result is shown in figure 2. The CT value is not basically different from the Ct value at day 0 when the CT film is placed for 3 or 7 days under the environmental condition of 2-8 ℃. This shows that the viral nucleic acid in the virus preservation solution is not significantly degraded after preservation for 7 days, and does not affect the research works such as sequencing by researchers.
(3) The virus stock solution of example 1 was used to preserve infectious bronchitis virus (IBV QXL87 strain), and the maintenance effect on viral activity (infectivity) was verified
1M infectious bronchitis Virus (IBV QXL87 strain) is respectively mixed with 1mL hank's solution and 1mL of the virus preservation solution, the mixture is respectively preserved for 7 days at the ambient temperature of 2-8 ℃ or 25 ℃ after being uniformly mixed, the specific grouping is shown in table 1, three repetitions are processed each time, then PEG (polyethylene glycol) solution is respectively used for precipitation, PBS solution is used for wetting, precipitation is resuspended, MDCK cells are infected by the resuspension, the culture is carried out for 3 days, and finally cytopathic effect is observed under a microscope.
Table 1:the experimental results are shown in fig. 3: FIG. 3 a is an initial preservation of infectious bronchitis viruses in chickens at 25℃using the virus preservation solution prepared in example 1Infection of cells; FIG. 3 b is a diagram showing the initial cell infection of a chicken infectious bronchitis virus preserved in an environment of 2-8deg.C using the virus preservation solution prepared in example 1; FIG. 3 c is the infection of cells after preservation of chicken infectious bronchitis virus at 25℃for several days using the virus preservation solution prepared in example 1; FIG. 3 d shows the infection of cells after the chicken infectious bronchitis virus was stored at 2-8deg.C for several days using the virus preservation solution prepared in example 1. As can be seen from FIG. 3 d, when the virus was stored in the above-mentioned preservation solution, the virus was preserved at an ambient temperature of 2-8deg.C for 7 days, and the cytopathic effect was demonstrated, indicating that the infectious bronchitis virus was still viable and infectious when the virus was preserved at an ambient temperature of 2-8deg.C for 7 days. As can be seen from the graph (c) of FIG. 3, the virus can be preserved at 25 ℃ for 3 days, and the cells are diseased, which shows that the virus has activity and infectivity, and after the cells are preserved for 3 days and 7 days, the normal growth cells can be obviously diseased, so that the virus preservation solution of the invention can preserve the virus, has better preservation capability on the virus under the environment of normal temperature (25 ℃) and the secondary low temperature (2-8 ℃) and maintains the infection activity of the virus.
Example 3
The concentration of each component in the virus-retaining solution of this example was adjusted to a small extent as compared with that of example 1. Wherein the concentration of KCl is 7.5g/L, naH 2 PO 4 Is 0.45g/L; na (Na) 2 HPO 4 The concentration of (2) 8g/L, the concentration of HEPES solution added was 23mM, the concentration of bovine serum albumin was 0.9g/L, the concentration of glucose was 0.9g/L, the concentration of gentamicin sulfate was 195mg/L, the concentration of polymyxin was 3.8mg/L, the concentration of nystatin was 9.5mg/L, the concentration of phenol red was 23mg/L, the amount of glycerol added was 90mL/L, and the amount of antifoaming agent added was 0.2mL/L. The preparation method is the same as in example 1.
Example 4
The concentration of each component in the virus-retaining solution of this example was adjusted as compared with that in example 3. Wherein the concentration of KCl is 9.5g/L, naH 2 PO 4 Is 0.6g/L; na (Na) 2 HPO 4 At a concentration of 3.4g/L, the HEPES solution was added at a concentration of 30mM, and bovine serum albumin was concentratedThe concentration of glucose is 1.2g/L, the concentration of gentamicin sulfate is 220mg/L, the concentration of polymyxin is 4.8mg/L, the concentration of nystatin is 12mg/L, the concentration of phenol red is 30mg/L, the addition amount of glycerol is 115mL/L, and the addition amount of defoamer is 0.4mL/L. The preparation method is the same as in example 1.
Example 5
The concentration of each component in the virus-retaining solution of this example was adjusted as compared with that in example 3. Wherein the concentration of KCl is 6.5g/L, naH 2 PO 4 Is 0.4g/L; na (Na) 2 HPO 4 The concentration of (2) 4g/L, the concentration of HEPES solution added was 20mM, the concentration of bovine serum albumin was 0.8g/L, the concentration of glucose was 0.8g/L, the concentration of gentamicin sulfate was 180mg/L, the concentration of polymyxin was 3.2mg/L, the concentration of nystatin was 8mg/L, the concentration of phenol red was 20mg/L, the amount of glycerol added was 85mL/L, and the amount of antifoaming agent added was 0.16mL/L. The preparation method is the same as in example 1.
Comparative example 1
This comparative example is based on example 1 by replacing "gentamicin sulphate, polymyxin, nystatin" with equal amounts of diabodies. The diabodies include penicillin and streptomycin, and each milliliter of diabodies contains 10000 units of penicillin (base) and 10000 units of streptomycin (base). The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
Comparative example 2
In this comparative example, gentamicin sulfate was removed on the basis of example 1, and the final total concentration of antibiotics (each increased by 100 mg/L) in the virus-preserving fluid was supplemented with polymyxin and nystatin to be consistent with the total concentration of example 1. The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
Comparative example 3
In this comparative example, polymyxin was removed on the basis of example 1, and the final total concentration of antibiotics in the virus preservation solution was supplemented with gentamicin sulfate so as to be identical to the total concentration of example 1. The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
Comparative example 4
This comparative example was based on example 1 with nystatin removed and the final total concentration of antibiotics in the virus stock was made up with gentamicin sulfate to be consistent with the total concentration of example 1. The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
The human herpesvirus EB virus is used as positive control, the virus preservation solution of the examples 1-5 and the virus 100:10 (volume ratio) are mixed to preserve the virus, meanwhile, 3 human herpesvirus EB virus samples are respectively added into the virus preservation solution corresponding to the comparative examples 1-4 (volume ratio 100:10), and the mixture is preserved at a constant temperature of 8 ℃. 200 mu L of preservation samples are taken at the 0 th, 24 th, 48 th and 72 th hours respectively, the change of the virus sample in the virus preservation solution is detected by adopting RT-qPCR, and the quantitative detection kit for human herpesvirus EB virus nucleic acid is selected for detection, so that the Ct value is measured. The experimental results are shown in table 2.
Table 2: sample detection Ct value preserved at constant temperature of 8 DEG C
From the above experimental results, it is found that when the virus preservation solutions prepared in examples 1 to 5 preserve viruses at 8℃for 72 hours, the Ct value detected by fluorescence PCR increases only slightly relative to the Ct value at time 0, indicating that the virus preservation solutions can maintain the integrity of viral nucleic acids to a large extent. In comparative example 1, if gentamicin sulfate, polymyxin and nystatin in the virus preservation solution are replaced by a double antibody composed of penicillin and streptomycin, ct value is obviously increased after EB virus is preserved for 72 hours at 8 ℃ in the virus preservation solution, which indicates that the virus nucleic acid is obviously degraded. The removal of gentamicin sulfate in comparative example 2, the removal of polymyxin in comparative example 3, and the removal of nystatin in comparative example 4, the increase in Ct value was more after the EB virus preservation solution was preserved at 8℃for 72 hours, indicating that the virus preservation solutions in comparative examples 2 to 4 were unfavorable for maintaining the structural integrity of viral nucleic acid. The above experiments show that any increase or decrease in the antibiotic component in the virus-preserving fluid of the present invention results in significant deterioration of the preserving ability of the virus-preserving fluid to viruses.
Meanwhile, as can be seen from the adjustment of the concentration of each component in the virus preservation solutions of examples 1 to 5, the virus preservation solution of example 1 has an optimal preservation effect on the integrity of the viral nucleic acid under the same preservation condition, while examples 2 to 3 are inferior to example 1, and examples 4 to 5 have a preservation effect on the integrity of the viral nucleic acid inferior to examples 2 to 3 after the concentration of each component is further adjusted. These experimental results demonstrate that even small adjustments in the concentration of the components of the virus-preserving fluid of the present invention can result in a decrease in the preserving performance of the virus-preserving fluid. And preferably, the concentration of KCl in the virus preservation solution is 7.5-8.5g/L, naH 2 PO 4 The concentration of (2) is 0.45-0.55g/L; na (Na) 2 HPO 4 The concentration of (2.8-3.2 g/L), the concentration of HEPES solution is 23-27mM, the concentration of bovine serum albumin is 0.9-1.1g/L, the concentration of glucose is 0.9-1.1g/L, the concentration of gentamicin sulfate is 195-205mg/L, the concentration of polymyxin is 3.8-4.2mg/L, the concentration of nystatin is 9-11mg/L, the concentration of phenol red is 23-27mg/L, the addition amount of glycerol is 90-110mL/L, and the addition amount of defoamer is 0.2-0.3mL/L; more preferably, the KCl concentration is 8g/L, naH 2 PO 4 Is 0.5g/L; na (Na) 2 HPO 4 The concentration of (2) is 3g/L, the concentration of HEPES solution is 25mM, the concentration of bovine serum albumin is 1g/L, the concentration of glucose is 1g/L, the concentration of gentamicin sulfate is 200mg/L, the concentration of polymyxin is 4mg/L, the concentration of nystatin is 10mg/L, the concentration of phenol red is 25mg/L, the addition amount of glycerol is 100mL/L, and the addition amount of defoamer is 0.2-0.3mL/L.
To further investigate the effect of the concentrations of the three antibiotics in the virus-preserving fluid on the virus-preserving efficacy of the virus-preserving fluid, the concentrations of a portion of the permeation resistant proteins were also adjusted during the experiment, see comparative examples 5-6:
comparative example 5
In the comparative example, the concentration of gentamicin sulfate was changed to 120mg/L based on example 1, and the concentration of polymyxin was increased by 80mg/L to make the final total concentration of antibiotics consistent with that of example 1. The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
Comparative example 6
In this comparative example, the polymyxin concentration was changed to the same concentration value as nystatin on the basis of example 1, and the gentamicin sulfate concentration was increased so that the final total concentration of the antibiotic was identical to the total concentration of example 1. The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
The experiment uses influenza virus as a positive control, the virus preservation solution of the example 1 and the influenza virus 100:10 (volume ratio) are mixed to preserve the virus, 3 influenza virus positive samples are respectively added into the virus preservation solution corresponding to the comparative examples 5-6 (volume ratio of 100:10), and the mixture is preserved at the constant temperature of 4 ℃. 200 mu L of preservation samples are taken at the 0 th, 24 th, 48 th and 72 th hours respectively, the change of the virus sample in the virus preservation solution is detected by adopting RT-qPCR, and the quantitative detection kit of influenza virus nucleic acid is used for detection, so that the Ct value is measured. The experimental results are shown in table 3:
table 3: sample detection Ct value is preserved at constant temperature of 4 ℃ (larger Ct means lower virus infectivity)
From the above experimental results, it was found that the integrity maintenance ability of the virus-preserving fluid against influenza virus nucleic acid was reduced under the same conditions and the same preservation period after changing the concentration/ratio of the antibiotics in the virus-preserving fluid, although the same antibiotics were still maintained. This shows that if the concentration of the components in the virus preservation solution provided by the invention is adjusted at will, the preservation capacity of the virus preservation solution on virus nucleic acid is easily reduced, and the detection and research of viruses by subsequent researchers are affected.
To further investigate the effect of the concentration of other non-antibiotic components in the virus-preserving fluid on the virus-preserving efficacy of the virus-preserving fluid, the concentration of the non-antibiotic components was also adjusted during the experiment, see comparative examples 7-10:
comparative example 7
This comparative example was based on example 1 by increasing the concentration of the "bovine serum albumin" component to 2g/L. The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
Comparative example 8
This comparative example was based on example 1 by increasing the concentration of the "glucose" component to 2g/L. The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
Comparative example 9
This comparative example was based on example 1 with "bovine serum albumin" removed and the "glucose" component concentration was increased to 2g/L to be consistent with the total concentration of example 1. The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
Comparative example 10
This comparative example was based on example 1 with "glucose" removed and the concentration of the "bovine serum albumin" component was increased to 2g/L to bring it to the total concentration of example 1. The concentrations, addition amounts and preparation methods of the other components were the same as in example 1.
Virus stock solutions were prepared according to the formulations of example 1 and comparative examples 7 to 10, and were prepared with H1N1 influenza a virus 100:10, mixing, standing at 4deg.C for 7 days, precipitating with PEG (polyethylene glycol) solution, respectively, washing with PBS solution, and suspending to precipitate; the cell sap containing MDCK cells was inoculated into a 12-well plate, MDCK cells were infected with the virus resuspension, cultured for 3 days, and finally the cell infection was observed under a microscope. The infection rate of each comparative example is shown in table 4.
Table 4: preserving the relative infection rate of the sample to the cells at a constant temperature of 4 DEG C
From the above experimental results, it was found that the preservation efficiency of the virus preservation solution in example 1 was the highest. In comparative example 7, when the concentration of bovine serum albumin in the virus-preserving fluid was increased to 2 times that of glucose, the infection rate was significantly reduced after the virus-preserving fluid was preserved for 7 days at 4 ℃ for the type a H1N1 virus; in comparative example 8, after the glucose concentration in the virus-preserving fluid was increased to 2 times that of bovine serum albumin, the infection rate was also significantly reduced after the virus-preserving fluid was preserved for 7 days at 4 ℃ for the type a H1N1 virus; the infection rate was further decreased after the bovine serum albumin was removed in comparative example 9 and the glucose was removed in comparative example 10, and the virus-preserving fluid was preserved for 7 days at 4℃for the type A H1N1 virus. The above experiments show that the virus preservation solution in comparative examples 7-10 affects the integrity of the virus, and is not beneficial to subsequent research on the characteristics such as virus structure. Therefore, bovine serum albumin and glucose are taken as nutrient substances, so that the protective effect on viruses can be achieved, the integrity of the viruses is ensured, but the dosage of the bovine serum albumin and the glucose can not be increased or decreased at will; any increase or decrease in the bovine serum albumin and glucose components in the virus-preserving fluid of the present invention may cause significant deterioration in the preserving ability of the virus-preserving fluid to viruses.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (3)
1. A virus preservation solution for maintaining virus viability at a non-ultralow temperature, comprising an aqueous solution of: KCl and NaH 2 PO 4 、Na 2 HPO 4 HEPES solution, bovine serum albumin, glycerol, glucose, gentamicin sulfate, polymyxin, nystatin, defoamer, phenol red;
wherein the concentration of KCl is 8g/L, naH 2 PO 4 Is 0.5g/L; na (Na) 2 HPO 4 The concentration of HEPES solution was 3g/L, the concentration of bovine serum albumin was 25mM, the concentration of glucose was 1g/L, the concentration of gentamicin sulfate was 200mg/L, the concentration of polymyxin was 4mg/L, the concentration of nystatin was 10mg/L, the concentration of phenol red was 25mg/L, and the addition amount of glycerol was 100mL/L, the adding amount of the defoaming agent is 0.25mL/L;
the pH of the virus preservation solution=7.4±0.2.
2. A method for preparing the virus preservation solution for maintaining the virus activity at a non-ultralow temperature according to claim 1, comprising the steps of:
s1, measuring HEPES solution into a liquid preparation container according to the composition of virus preservation liquid;
s2, accurately weighing NaH respectively 2 PO 4 、Na 2 HPO 4 Adding KCl, bovine serum albumin, glucose and phenol red into a liquid preparation container for dissolution;
s3, glycerol and an antifoaming agent are measured and added into a liquid preparation container, a purified water rinse measuring tool is adopted for 2-3 times, and rinse water of each time is added into the liquid preparation container;
s4, weighing gentamicin sulfate, polymyxin and nystatin, and adding the gentamicin sulfate, the polymyxin and the nystatin into a liquid preparation container;
s5, adding purified sterile water into a liquid preparation container for constant volume, shaking uniformly, and regulating the pH value to be pH value of 7.4+/-0.2 measured at 23-28 ℃;
s6, filtering by using a filtering device with the aperture of 0.22 micrometer, performing sterilization and impurity removal at the speed of 5L/min, and performing high-pressure sterilization for 30min under the condition of 121 ℃ and 0.1 Mpa; cooling, and standing at room temperature for storage.
3. A method for preserving a virus in a non-inactivating manner, comprising the steps of placing the virus in the virus preservation solution for preserving the virus activity at a non-ultralow temperature according to claim 1, wherein the preservation temperature is as follows: sealing and preserving for less than or equal to 72 hours at room temperature; or sealing and preserving for less than or equal to 168 hours at the ambient temperature of 2-8 ℃.
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