CN116287122A - Inactivated virus preservation solution and preparation method thereof - Google Patents
Inactivated virus preservation solution and preparation method thereof Download PDFInfo
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- CN116287122A CN116287122A CN202310379227.0A CN202310379227A CN116287122A CN 116287122 A CN116287122 A CN 116287122A CN 202310379227 A CN202310379227 A CN 202310379227A CN 116287122 A CN116287122 A CN 116287122A
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Images
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
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- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16161—Methods of inactivation or attenuation
- C12N2760/16163—Methods of inactivation or attenuation by chemical treatment
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
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- C12N2760/16263—Methods of inactivation or attenuation by chemical treatment
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
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- C12N2770/20011—Coronaviridae
- C12N2770/20061—Methods of inactivation or attenuation
- C12N2770/20063—Methods of inactivation or attenuation by chemical treatment
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- 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 belongs to the technical field of virus preservation, and particularly relates to an inactivated virus preservation solution and a preparation method thereof. The inactivated virus preservation solution comprises 20 g-210 g of guanidine hydrochloride, 1 g-3 g of tris (hydroxymethyl) aminomethane hydrochloride, 1 g-3 g of dithiothreitol, 1 g-10 g of triton X-100, 50 mL-70 mL of antibiotic additive, 0.1 g-0.2 g of ethylenediamine tetraacetic acid, 0.2 mL-0.3 mL of 1mol/L sodium hydroxide aqueous solution, 30 mg-50 mg of acid-base indicator and deionized water, wherein the mass or volume of the solution is equal to 1L. The inactivated virus preservation solution can realize rapid and complete inactivation of viruses, has good preservation effect, can maintain the stability of sample nucleic acid, and can be preserved for a long time at normal temperature without degradation.
Description
Technical Field
The invention belongs to the technical field of virus preservation, and particularly relates to an inactivated virus preservation solution and a preparation method thereof.
Background
Viruses are composed of a nucleic acid molecule and a protein or only a protein, and the individual is tiny and has a simple structure, and common epidemic diseases are usually RNA viruses such as influenza virus, AIDS virus, measles virus and novel coronavirus. Viruses can cause acute infection and persistent infection, and in severe cases, life can be endangered, so the viruses have important clinical significance.
In clinical detection of infectious diseases caused by viruses, in order to prevent virus infection, especially some highly pathogenic viruses, samples need to be inactivated firstly, and the virus inactivation method mainly comprises the following steps: physical inactivation (heating at 56 ℃ for 30min, ultraviolet irradiation, etc.), chemical inactivation (chlorine-containing disinfectant, peracetic acid, etc.). Common inactivation methods, such as heating at 56℃for 30min, result in detection of C T The value is larger, and the lowest detection limit of the virus is greatly influenced, so that false negative is caused. Whether the inactivated virus preservation solution can completely inactivate the collected sample is a risk, and the virus sample which is not completely inactivated has secondary infection risk to workers in the transportation and nucleic acid extraction processes. In addition, most of the collected samples need low-temperature transportation, otherwise, degradation can be caused to nucleic acid components of the samples, detection results are directly affected, and the preservation effect is poor.
Therefore, it is an urgent need to develop an inactivated virus preservation solution that can completely inactivate viruses and preserve viral nucleic acids for a long time at normal temperature without degradation.
Disclosure of Invention
In order to improve the preservation effect of the inactivated virus preservation solution, the application provides the inactivated virus preservation solution and a preparation method thereof.
In a first aspect, the present application provides an inactivated virus preservation solution, which is implemented by adopting the following technical scheme:
an inactivated virus preservation solution is prepared from the following raw materials in terms of mass or volume added per 1L of solution:
by adopting the technical scheme, the guanidine hydrochloride can lyse cells, so that nucleic acid can be released from protein, the guanidine hydrochloride can not only quickly destroy cell membranes, but also denature the protein, so that the protein denatures and precipitates, and the nucleic acid can be free from protein entanglement. The dithiothreitol plays a role in reducing in the preservation solution, so that the viral coat protein is broken, the damage of RNA viral nucleic acid is inhibited, and the preservation effect of the virus is improved. The tris hydrochloride is a biological buffer and has the function of maintaining the pH stability of the virus preservation solution. Triton X-100 is a nonionic surfactant that helps to break down proteases, can solubilize lipids to increase cell membrane permeability, and can also improve the stability of dithiothreitol and guanidine hydrochloride. The addition of ethylenediamine tetraacetic acid can be used as an anticoagulant of the preservation solution, so that the aggregation of the preservation solution is reduced to influence the preservation of viruses. Antibiotics interfere with the cell division process either inside or outside the bacterial cell, and exert antibacterial effects. The synergistic effect of the components can effectively inactivate pathogenic microorganisms in a sample, release and store nucleic acid efficiently, realize rapid and complete inactivation of viruses, maintain the stability of the sample nucleic acid, store the sample nucleic acid at normal temperature for a long time without degradation, and simultaneously facilitate the extraction of low-concentration virus nucleic acid, improve the detection sensitivity and improve the in-vitro diagnosis and detection performance of downstream on the virus nucleic acid.
Preferably, the preparation raw materials comprise, by mass or volume, per 1L of solution:
by adopting the technical scheme, the synergistic effect of the components can be further improved by adjusting the content of the components, pathogenic microorganisms in a sample can be effectively inactivated, nucleic acid can be efficiently released and stored, the inhibition effect on the subsequent PCR amplification process can be reduced, the storage effect can be improved, and the reduction of the nucleic acid can be realizedLow C T Values.
Preferably, the preparation raw materials comprise, by mass or volume, per 1L of solution:
by adopting the technical scheme, the collected viruses are quickly inactivated, the proteins of the viruses are strongly degraded, the nucleic acids of the viruses are efficiently stored, the inactivation treatment before detection is avoided, the detection efficiency is improved, the storage effect is further improved, and the C is reduced T Values.
Preferably, the antibiotic additive is formed by mixing gentamicin and tetracycline according to the volume ratio of 1 (0.1-0.2).
By adopting the technical scheme, gentamicin is an aminoglycoside antibiotic with heat stability, can be combined with 30s subunits of a bacterial ribosome, blocks the synthesis of bacterial proteins and damages the integrity of bacterial cell membranes; tetracyclines are broad-spectrum antibiotics with phenanthridine parent nucleus, and inhibit protein synthesis by forming reversible combination with bacterial ribosome 30s subunit, so as to play an antibacterial effect; the combination of gentamicin and tetracycline obviously improves the antibacterial rate of the inactivated virus preservation solution, and also ensures that the effective components in the preservation solution do not adversely affect the subsequent PCR nucleic acid detection, thereby improving the preservation effect and reducing C T Values.
Preferably, the volume ratio of gentamicin to tetracycline is 1:0.15.
By adopting the technical scheme, when the volume ratio of gentamicin to tetracycline is 1:0.15, the antibacterial rate of the inactivated virus preservation solution is higher, and the preservation effect on viruses is better.
Preferably, the preparation raw materials also comprise 0.15 to 0.25mg of amino acid surfactant according to the mass or volume of each 1L of solution.
By adopting the technical scheme, the amino acid surfactant has excellent surface activity, improves the stability of the inactivated virus preservation solution, especially can improve the defects that dithiothreitol is easy to be oxidized by air and has poor stability, improves the preservation effect of the inactivated virus preservation solution, and reduces C T The value also has antibacterial effect, and improves the antibacterial rate of the inactivated virus preservation solution.
Preferably, the amino acid surfactant is formed by mixing sodium dodecyl sarcosinate and N-acyl-L-lysine according to the mass ratio of 1 (0.2-0.3).
By adopting the technical scheme, the co-action of the sodium dodecyl sarcosinate and the N-acyl-L-lysine can overcome the defects that dithiothreitol is easy to oxidize by air and has poor stability, and further improve the preservation effect and the antibacterial effect of the inactivated virus preservation solution.
In a second aspect, the present application provides a method for preparing an inactivated virus preservation solution, which is implemented by adopting the following technical scheme: a preparation method of an inactivated virus preservation solution comprises the following steps:
mixing guanidine hydrochloride, tris hydrochloride, dithiothreitol, triton X-100, an antibiotic additive, ethylenediamine tetraacetic acid, 1mol/L sodium hydroxide aqueous solution, an acid-base indicator, an amino acid surfactant and deionized water, and uniformly dissolving to obtain an inactivated virus preservation solution.
In summary, the present application has the following beneficial effects:
1. the synergistic effect of the components can effectively inactivate pathogenic microorganisms in a sample, release and store nucleic acid efficiently, realize rapid and complete inactivation of viruses, maintain the stability of the sample nucleic acid, store the sample nucleic acid at normal temperature for a long time without degradation, and simultaneously facilitate the extraction of low-concentration virus nucleic acid, improve the detection sensitivity and improve the in-vitro diagnosis and detection performance of downstream on the virus nucleic acid.
2. The application adopts the combination of gentamicin and tetracycline, which obviously improves the antibacterial rate of the inactivated virus preservation solution and can also lead the preservation solution to be provided withThe effective components do not adversely affect the subsequent PCR nucleic acid detection, thereby improving preservation effect and reducing C T Values.
3. The amino acid surfactant is added, so that the antibacterial rate and the preservation effect of the inactivated virus preservation solution are improved.
4. The application adopts the combination of the sarcosyl and the N-acyl-L-lysine as the amino acid surfactant, and the combination of the sarcosyl and the N-acyl-L-lysine further improves the preservation effect and the antibacterial effect of the inactivated virus preservation solution.
Drawings
FIG. 1 is a graph showing the amplification results of the inactivated virus holding solution of example 1 after 3 days of holding influenza B virus at different temperatures;
FIG. 2 is a graph showing the amplification results of the inactivated virus holding solution of example 1 before and after being left at 65℃for 45 minutes;
FIG. 3 is a graph showing the amplification results of the inactivated virus holding solution of example 1 on 500copies/mL of novel coronavirus;
FIG. 4 is a graph showing the amplification results of the inactivated virus storage solution of example 1 compared with other commercially available virus storage solutions and physiological saline.
Detailed Description
The present application is described in further detail below with reference to examples.
Examples
Example 1 provides an inactivated virus preservation solution, which is prepared by the following steps:
20g of guanidine hydrochloride, 1g of tris hydrochloride, 1g of dithiothreitol, 2g of triton X-100, 50mL of gentamicin, 0.1g of ethylenediamine tetraacetic acid, 0.2mL of 1mol/L aqueous sodium hydroxide solution, 30mg of methyl orange indicator solution and 200mL of deionized water are mixed, uniformly dissolved, and then the deionized water is supplemented to 1L, so that the inactivated virus preservation solution is obtained.
The following performance test was performed on the inactivated virus storage solution prepared in example 1.
Test a: the storage stability at different temperatures is tested, and the testing steps are as follows:
s1, diluting influenza B virus to 1000copies/mL by using the inactivated virus preservation solution prepared in the embodiment 1, and uniformly mixing for later use;
s2, taking 4 parts of 5mL diluted influenza B virus, subpackaging the influenza B virus into 45 mL sterilizing centrifuge tubes, and respectively placing the influenza B virus in an ultralow temperature preservation box at-80 ℃, a low temperature preservation box at-20 ℃, a constant temperature preservation box at 25 ℃ and a constant temperature preservation box at 37 ℃ for 3 days;
s3, taking out and uniformly mixing the stored samples, and extracting 200 mu L of each sample by using an A-type nucleic acid extraction reagent (from Shenzhen catalpa health biotechnology Co., ltd.);
s4, loading the extracted sample by using an influenza A virus nucleic acid detection kit (derived from catalpa ovata), and amplifying by using an ABI7500 quantitative amplification instrument, wherein the amplification result is shown in figure 1.
As can be seen from FIG. 1, the nucleic acid of influenza B virus detects C corresponding to the signal value T The values are shown in Table 1.
TABLE 1 test results of storage stability at different temperatures example 1
| Preservation conditions | Preserving at-80 ℃ for 3 days | Preserving at-20deg.C for 3 days | Preserving at 25deg.C for 3 days | Storing at 37deg.C for 3 days |
| C T Value of | 34.52 | 34.91 | 35.12 | 35.47 |
As can be seen from Table 1, C T The difference between the maximum value and the minimum value is less than 1, which indicates that the inactivated preservation solution prepared in the embodiment 1 can stably preserve influenza virus nucleic acid for at least 3 days at-80 ℃, -20 ℃, 25 ℃ and 37 ℃, and indicates that the inactivated virus preservation solution provided by the application can maintain the stability of sample nucleic acid, can be preserved for a long time at normal temperature without degradation, does not influence the detection result, i.e. does not need low-temperature transportation, reduces the transportation cost, and improves the in vitro diagnosis detection performance of downstream aiming at the virus nucleic acid.
Test b: and testing the sample preservation effect under the high temperature condition, wherein the testing steps are as follows:
s1, diluting influenza A virus to 1000copies/mL by using the inactivated virus preservation solution prepared in the embodiment 1, uniformly mixing, taking 2 parts of 1mL of diluted influenza A virus, subpackaging into 21 mL centrifuge tubes, placing the 1 tube in a refrigerator with the temperature of 2-8 ℃ for 45min (marked as before heating), placing the 1 tube in a super micro-thermostat with the temperature of 65 ℃ for 45min (marked as after heating at 65 ℃ for 45 min);
s2, uniformly mixing samples after 45min, and extracting 200 mu L of each sample by using an A-type nucleic acid extraction reagent (from Shenzhen catalpa health biotechnology Co., ltd.);
s3, loading the extracted sample by using an influenza A virus nucleic acid detection kit (derived from catalpa ovata), and amplifying by using an ABI7500 quantitative amplification instrument, wherein the amplification result is shown in figure 2.
As can be seen from FIG. 2, the nucleic acid of influenza A virus detects C corresponding to the signal value T The values are shown in Table 2.
TABLE 2 test results of temperature resistance of example 1
| Treatment conditions | Before heating | Heating at 65deg.C for 45min |
| C T Value of | 28.25 | 28.18 |
As is clear from Table 2, C was heated at 65℃for 45 minutes T Value and C before heating T Values differ by 0.07, and C before and after 45min of treatment at 65 DEG C T The values have no obvious difference, which indicates that the inactivated preservation solution prepared in the embodiment 1 has good high temperature resistance and excellent sample preservation effect on influenza A virus under high temperature condition.
Test c: the sensitivity is tested, and the testing steps are as follows:
s1, diluting a freeze-dried ball of the novel coronavirus of Cyania with the inactivated preservation solution prepared in the embodiment 1, calculating an average value, diluting to 500copies/mL, and uniformly mixing;
s2, extracting a sample by using an A-type nucleic acid extraction reagent (from Shenzhen catalpa biological science and technology Co., ltd.) and 200 mu L of each hole site;
s3, loading the extracted sample by using a novel coronal 2019-nCoV virus nucleic acid detection kit (from Shenzhen catalpa health biotechnology Co., ltd.), and amplifying the N gene and orf1ab gene of the Sinomenii novel coronavirus by using an ABI7500 quantitative amplification instrument, wherein the amplification result is shown in figure 3.
From FIG. 3, it can be seen that the corresponding C of the signal value can be detected from the internal standard, the N gene and orf1ab gene of the novel coronavirus T The values are shown in Table 3.
TABLE 3 test results of sensitivity for example 1
| Detection conditions | Internal standard | N gene | orf1ab gene |
| C T Value of | 37.80 | 37.10 | 37.74 |
As is clear from Table 3, the internal standard, the N gene and the C of orf1ab gene T The values are all smaller than 38, which indicates that the inactivated preservation solution prepared in example 1 has high sensitivity to virus preservation of not higher than 500 copies/mL.
Test d: example 1 sample preservation effect of inactivated virus preservation solution and other commercial virus preservation solutions and physiological saline is compared and tested, and the test steps are as follows:
s1, diluting influenza B virus to 1000copies/mL by using an inactivated virus preservation solution (marked as catalpa) prepared in the embodiment 1, a comma virus sampling tube solution (marked as comma) produced by Shenzhen comma biotechnology limited company and physiological saline (marked as water) respectively, and uniformly mixing;
s2, extracting samples with an A-type nucleic acid extraction reagent (from Shenzhen catalpa biological technology Co., ltd.) with 200 mu L of each sample;
s3, loading the extracted sample by using an influenza A virus nucleic acid detection kit (from Shenzhen catalpa biological technology Co., ltd.), and amplifying by using an ABI7500 quantitative amplification instrument, wherein the amplification result is shown in figure 4.
As can be seen from FIG. 4, the inactivated virus preservation solution prepared in example 1, the comma virus sampling tube solution produced by Shenzhen comma biotechnology Co., ltd, and the viral nucleic acid corresponding to physiological saline can detect C corresponding to the signal value T The values are shown in Table 4.
Table 4 test results comparing preservation effects
As is clear from Table 4, the inactivated virus preservation solution prepared in example 1 of the present application is extracted to obtain C T The value is 29.44, and the comma corresponds to C after extraction T The value is 30.60, which indicates that the inactivated virus preservation solution prepared in the example 1 of the application is superior to other similar products on the market.
Test e: the antibacterial ability of the inactivated virus preservation solution prepared in example 1 is tested, and the test steps are as follows: s1, adding 4mL of sterilized LB liquid medium into a 5mL sterilizing centrifuge tube, and adding 40 mu L of escherichia coli strain according to the proportion of 1:100;
s2, placing the materials into a shaking table, setting the constant temperature of 37 ℃ for 12 hours, and taking out the materials after finishing;
s3, uniformly mixing after finishing, and carrying out gradient dilution by sterilized pure water, namely 10 -3 ～10 -7 Mixing, adding 500 μl of diluted sample into each plate, coating with coating rod, sealing with sealing film, placing into a constant temperature incubator at 37deg.C, and observing colony number under microscope after 1 day;
s4, taking 1 tube of inactivated preservation solution (3 mL, experimental group) and 1 tube of sterilized normal saline (3 mL, contrast), respectively adding 30 mu L of bacterial culture solution according to the proportion of 1:100, uniformly mixing, putting into a shaking table, setting the constant temperature of 37 ℃ for 1 day, and taking out after finishing;
s5, taking 6 sterilized LB culture mediums, wherein each tube corresponds to 3 culture mediums, the operation steps are the same as the step 3, the reverse buckling culture is carried out, the constant temperature culture is carried out at 37 ℃ for 1 day, and then the colony number is observed under a microscope;
according to the bacteriostasis rate X= (A) 0 -A 1 )/A 0 X 100%, where A 0 Mean colony count of the control group is expressed, and the unit is cfu/mL; a is that 1 The average colony number of the experimental group is expressed as cfu/mL, the bacteriostasis rate is calculated, and the bacteriostasis rate of the inactivated virus preservation solution prepared in example 1 is 95.2%.
Examples 2-7 provide an inactivated virus preservation solution, which differs from example 1 only in that: the compositions of the preparation raw materials are different, and are shown in table 5.
TABLE 5 examples 1-7 preparation of the compositions of the raw materials
The antibacterial rate of the inactivated virus preservation solutions prepared in examples 2 to 7 was tested according to the test e described above, and the test results are shown in Table 6;
the inactivated virus-preserving fluid prepared in examples 2 to 7 was tested for C corresponding to the detectable signal value according to the above test d T Values, test results are shown in Table 6.
TABLE 6 bacteriostasis rates and C for examples 1-7 T Test results of values
| Bacteriostasis rate (%) | C T Value of | |
| Example 1 | 96.9 | 29.44 |
| Example 2 | 98.0 | 31.46 |
| Example 3 | 96.1 | 29.25 |
| Example 4 | 97.8 | 29.38 |
| Example 5 | 97.7 | 28.97 |
| Example 6 | 97.5 | 29.31 |
| Example 7 | 97.1 | 29.14 |
As is clear from the test data in Table 6, the inactivated virus-preserving fluid according to example 5 can strongly degrade viral proteins and efficiently preserve viral nucleic acids, C T The value is lower, and the preservation effect is better.
Example 8 provides an inactivated virus preservation solution, which differs from example 5 only in that: the equal volume of gentamicin is replaced by tetracycline.
Example 9 provides an inactivated virus preservation solution, which differs from example 5 only in that: the equal volume of gentamicin is replaced by a mixture of gentamicin and tetracycline, and the volume ratio of gentamicin to tetracycline is 1:0.1.
Example 10 provides an inactivated virus preservation solution, which differs from example 9 only in that: the volume ratio of gentamicin to tetracycline is 1:0.2.
Example 11 provides an inactivated virus preservation solution, which differs from example 9 only in that: the volume ratio of gentamicin to tetracycline is 1:0.15.
The antibacterial ratio of the inactivated virus preservation solutions prepared in examples 8 to 11 was tested according to the test e described above, and the test results are shown in Table 7; the inactivated virus-preserving fluid prepared in examples 8 to 11 was tested for C corresponding to the detectable signal value according to test d above T Values, test results are shown in Table 7.
TABLE 7 bacteriostasis rates and C for examples 5, 8-11 T Test results of values
From the test data in Table 7, it can be seen that the combination of gentamicin and tetracycline significantly improves the antibacterial rate of the inactivated virus preservation solution and reduces C T The value is better when the volume ratio of gentamicin to tetracycline is 1:0.15, and the antibacterial rate is higher.
Example 12 provides an inactivated virus preservation solution, which is prepared by the following steps:
50g of guanidine hydrochloride, 2g of tris hydrochloride, 2.1g of dithiothreitol, 6.8g of triton X-100, 51.3mL of gentamicin, 7.7mL of tetracycline, 0.17g of ethylenediamine tetraacetic acid, 0.25mL of 1mol/L aqueous sodium hydroxide solution, 35mg of methyl orange indicator solution, 0.15mg of sodium dodecyl sarcosinate and 200mL of deionized water are mixed, dissolved uniformly, and deionized water is supplemented to 1L to obtain an inactivated virus preservation solution.
Example 13 provides an inactivated virus preservation solution, which differs from example 12 only in that: the mass of sarcosyl is 0.25mg.
Example 14 provides an inactivated virus preservation solution, which differs from example 13 only in that: the mass of the sarcosyl is replaced by N-acyl-L-lysine.
Example 15 provides an inactivated virus preservation solution, which differs from example 13 only in that: the equal mass of the sarcosyl is replaced by a mixture of the sarcosyl and the N-acyl-L-lysine, and the mass ratio of the sarcosyl to the N-acyl-L-lysine is 1:0.2.
Example 16 provides an inactivated virus preservation solution, which differs from example 15 only in that: the mass ratio of the sarcosyl to the N-acyl-L-lysine is 1:0.3.
The antibacterial ratio of the inactivated virus preservation solutions prepared in examples 12 to 16 was tested according to the test e, and the test results are shown in Table 8; the inactivated virus-preserving fluid prepared in examples 12 to 16 was tested for C corresponding to the detectable signal value according to test d above T Values, test results are shown in Table 8.
TABLE 8 bacteriostasis rates and C for examples 11-16 T Test results of values
As can be seen from the test data in Table 8, in example 12, the addition of sarcosyl improves the antibacterial rate of the inactivated virus preservation solution, and reduces C T A value; example 15 the combination of sarcosyl and N-acyl-L-lysine further improved the preservation effect and bacteriostatic action of the inactivated virus preservation solution.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (8)
1. An inactivated virus preservation solution is characterized in that the preparation raw materials comprise the following components in terms of mass or volume added per 1L of solution:
20 g-210 g of guanidine hydrochloride;
1-3 g of tris hydrochloride;
1-3 g of dithiothreitol;
2 g-10 g of triton X-100;
50-70 mL of antibiotic additive;
0.1 g-0.2 g of ethylenediamine tetraacetic acid;
0.2 mL-0.3 mL of 1mol/L sodium hydroxide aqueous solution;
30-50 mg of acid-base indicator;
deionized water was added to the solution volume to 1L.
2. The inactivated virus holding fluid according to claim 1, wherein the preparation raw materials comprise, per 1L of the solution, by mass or volume:
20 g-100 g of guanidine hydrochloride;
1.5 g-2.5 g of tris hydrochloride;
1.5 g-2.5 g of dithiothreitol;
6 g-7 g of triton X-100;
55-65 mL of antibiotic additive;
0.15 g-0.2 g of ethylenediamine tetraacetic acid;
0.23-0.27 mL of 1mol/L sodium hydroxide aqueous solution;
30-40 mg of acid-base indicator;
deionized water was added to the solution volume to 1L.
3. An inactivated virus preservation solution according to claim 2, wherein the preparation materials comprise, per 1L of solution, by mass or volume:
50g of guanidine hydrochloride;
2g of tris hydrochloride;
dithiothreitol 2.1g;
triton X-100.8 g;
59mL of antibiotic additive;
0.17g of ethylenediamine tetraacetic acid;
0.25mL of 1mol/L sodium hydroxide aqueous solution;
35mg of acid-base indicator;
deionized water was added to the solution volume to 1L.
4. The inactivated virus preservation solution according to claim 1, wherein the antibiotic additive is formed by mixing gentamicin and tetracycline according to a volume ratio of 1 (0.1-0.2).
5. The inactivated virus holding fluid according to claim 4, wherein the gentamicin and the tetracycline have a volume ratio of 1:0.15.
6. The inactivated virus storage solution according to any one of claims 1 to 5, wherein the preparation raw material further comprises 0.15mg to 0.25mg of an amino acid surfactant per 1L of the solution by mass or volume.
7. The inactivated virus preservation solution according to claim 6, wherein the amino acid surfactant is formed by mixing sodium dodecyl sarcosinate and N-acyl-L-lysine according to a mass ratio of 1 (0.2-0.3).
8. A method of preparing the inactivated virus preservation solution of claim 6, comprising the steps of:
mixing guanidine hydrochloride, tris hydrochloride, dithiothreitol, triton X-100, an antibiotic additive, ethylenediamine tetraacetic acid, 1mol/L sodium hydroxide aqueous solution, an acid-base indicator, an amino acid surfactant and deionized water, and uniformly dissolving to obtain an inactivated virus preservation solution.
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