CN111869658A - Inactivated virus preservation solution and preparation method thereof - Google Patents
Inactivated virus preservation solution and preparation method thereof Download PDFInfo
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- CN111869658A CN111869658A CN202010852816.2A CN202010852816A CN111869658A CN 111869658 A CN111869658 A CN 111869658A CN 202010852816 A CN202010852816 A CN 202010852816A CN 111869658 A CN111869658 A CN 111869658A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
Abstract
The application relates to an inactivated virus preservation solution and a preparation method thereof, relating to the field of virus detection, and the virus preservation solution comprises the following raw materials in parts by weight: 22-36 parts of tris (hydroxymethyl) aminomethane, 6-14 parts of oxalic acid tetraacetic acid, 5-11 parts of penicillin, 3-9 parts of streptomycin, 2-8 parts of polyhexamethylene biguanide, 3-9 parts of sodium dihydrogen phosphate, 2-8 parts of disodium hydrogen phosphate, 1-3 parts of phenolphthalein, 4-9 parts of sodium hydroxide, 60-120 parts of water and 2-7 parts of a fixing agent. According to the method, the inactivated virus preservation solution can be obtained by mixing the raw materials according to a specified mode, and the risk of virus secondary infection can be effectively reduced.
Description
Technical Field
The application relates to the field of virus detection, in particular to an inactivated virus preservation solution and a preparation method thereof.
Background
The virus is composed of a nucleic acid molecule and protein or only protein, the individual is tiny, the structure is simple, and the common epidemic infectious diseases are usually RNA virus, such as influenza virus, AIDS virus and influenza A virus. Without a cell structure, the virus itself cannot replicate, but rather invades a gene into a host cell, and replicates a new virus via the latter replication system. After the virus sample is collected, the sampling swab is put into a preservation solution for preservation and transportation in order to maintain the activity of the virus sample and prolong the survival time of the virus in the sample.
The prior Chinese patent with publication number CN111321123A discloses a virus preservation solution free of nucleic acid extraction, which comprises a component A and a component B, wherein the component A comprises: acid buffer solution, tween 20 and acid protease; wherein, the molarity of the acid buffer solution is 20mM-100mM, the pH is 1.0-5.0, the volume percentage of the Tween 20 is 0.1% -2%, and the concentration of the acid protease is 0.1-100 mg/mL; the component B comprises: neutral or alkaline buffer solution with concentration of 0.5-1M and pH of 7.0-9.0.
With respect to the related art among the above, the inventors consider that the following drawbacks exist: since human operation is required when detecting and testing viruses, it is easy for operators to infect if the viruses are in an active state.
Disclosure of Invention
In order to reduce secondary infection of viruses in the detection and experiment processes, the application provides an inactivated virus preservation solution and a preparation method thereof.
In a first aspect, the present application provides an inactivated virus preservation solution, which adopts the following technical scheme:
an inactivated virus preservation solution comprises the following raw materials in parts by weight: 22-36 parts of tris (hydroxymethyl) aminomethane, 6-14 parts of oxalic acid tetraacetic acid, 5-11 parts of penicillin, 3-9 parts of streptomycin, 2-8 parts of polyhexamethylene biguanide, 3-9 parts of sodium dihydrogen phosphate, 2-8 parts of disodium hydrogen phosphate, 1-3 parts of phenolphthalein, 4-9 parts of sodium hydroxide, 60-120 parts of water and 2-7 parts of a fixing agent.
By adopting the technical scheme, the tris (hydroxymethyl) aminomethane is used as a buffer base solution and can be used as a solvent for virus nucleic acid and protein, so that the stability of the virus is improved; meanwhile, the addition of the ethylene diamine tetraacetic acid can be used as an anticoagulant of the preservation solution, so that the influence of coagulation of the preservation solution on the preservation of the virus is reduced, and the penicillin and the streptomycin which are used as antibiotics are added into the virus preservation solution, so that the antibacterial property of the virus preservation solution can be improved, and the stability of the virus in the virus preservation solution is improved; sodium hydroxide is used as a pH value regulator, sodium dihydrogen phosphate and disodium hydrogen phosphate are used as pH value buffers, and phenolphthalein is used as a pH value indicator, so that the pH value of the virus preservation solution is stabilized in a specified range, and the decomposition of the virus is reduced; meanwhile, polyhexamethylene biguanide is added in the water-soluble antiviral liposome, the polyhexamethylene biguanide can generate ionization in water solution, a hydrophilic group part of the polyhexamethylene biguanide contains strong electropositivity, viruses which are usually electronegativity are adsorbed, and the synthesis of virus liposomes is inhibited, so that the viruses are inactivated, and the secondary infection of the viruses is effectively reduced; in addition, the fixing agent is added in the application, so that nucleic acid and protein of the virus can be fixed, and the integrity of the virus is improved.
Preferably, the raw materials comprise the following components in parts by weight: 26-29 parts of trihydroxymethyl aminomethane, 9-11 parts of oxalic acid tetraacetic acid, 6-7 parts of penicillin, 4-6 parts of streptomycin, 3-6 parts of polyhexamethylene biguanide, 4-5 parts of sodium dihydrogen phosphate, 3-6 parts of disodium hydrogen phosphate, 1-2 parts of phenolphthalein, 5-7 parts of sodium hydroxide, 90-100 parts of water and 3-5 parts of a fixing agent.
By adopting the technical scheme, the content of each component in the preservation solution is further limited, so that the components can be better matched, the service performance of the virus preservation solution is further improved, and the secondary infection of viruses is reduced.
Preferably, the raw materials comprise the following components in parts by weight: 28 parts of trihydroxymethyl aminomethane, 10 parts of oxalic acid tetraacetic acid, 6 parts of penicillin, 5 parts of streptomycin, 4 parts of polyhexamethylene biguanide, 4 parts of sodium dihydrogen phosphate, 4 parts of disodium hydrogen phosphate, 2 parts of phenolphthalein, 5 parts of sodium hydroxide, 98 parts of water and 4 parts of a fixing agent.
By adopting the technical scheme, the content of each component in the preservation solution is further limited, so that the components can be optimally matched, the service performance of the virus preservation solution is further improved, and the secondary infection of viruses is further reduced.
Preferably, the fixing agent comprises the following components in parts by weight: 20-36 parts of tannic acid, 15-28 parts of phosphatidylcholine and 30-54 parts of ethanol.
Through adopting above-mentioned technical scheme, tannic acid can permeate the inside of virus, fix the nucleic acid and the protein of virus, phosphatidylcholine is an amphiprotic molecule simultaneously, constitute by hydrophilic head and hydrophobic afterbody, the principle that similar intermiscibility can be utilized in the addition of phosphatidylcholine, improve tannic acid to the infiltration rate of virus, can improve the fixed effect to the virus, ethanol also is regarded as a fixative solution simultaneously, can further fix the virus, thereby the fixative of this application can improve the integrality that keeps of virus.
Preferably, the preparation method of the fixing agent comprises the following steps: performing ball milling and mixing on tannic acid and three-quarter of phosphatidylcholine to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine to obtain a second mixture; and mixing the first mixture and the second mixture to obtain the fixing agent.
Through adopting above-mentioned technical scheme, mix tannin and phosphatidylcholine alone, then mix ethanol and phosphatidylcholine alone for the phosphatidylcholine can insert relatively between tannin and virus, ethanol and the virus, improves the leading-in effect of phosphatidylcholine, makes tannin and ethanol can fully fix the virus.
Preferably, the fixing agent further comprises the following components: 4-9 parts of saponin and 3-6 parts of polyether modified organic silicon; the preparation method of the fixing agent comprises the following steps: performing ball milling and mixing on tannic acid and three-quarter of phosphatidylcholine to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine to obtain a second mixture; mixing the first mixture with saponin and polyether modified silicone to obtain a third mixture; and then mixing the second mixture and the third mixture to obtain the fixing agent.
By adopting the technical scheme, the saponin is added into a fixing agent system, so that the penetration effect of the tannic acid can be further improved, the tannic acid can better fix the virus, meanwhile, the polyether modified organic silicon is also added to serve as an amphoteric surfactant, and the polyether modified organic silicon can be fully dissolved in virus preservation solution, so that the foaming of the saponin can be effectively inhibited, and the influence of the foaming of the saponin on the virus is reduced; and in the preparation process of the fixing agent, the first mixture is mixed with the saponin and the polyether modified organic silicon in advance, so that the saponin can fully act on the tannic acid, and the fixing effect of the tannic acid is improved.
Preferably, the raw materials also comprise the following components in parts by weight: 5-14 parts of L-cysteine hydrochloride.
By adopting the technical scheme, the L-cysteine hydrochloride is an important amino acid, and the L-cysteine hydrochloride is added into the virus preservation solution, so that the aging of the virus can be effectively inhibited, and the preservation time of the virus is prolonged.
In a second aspect, the present application provides a method for preparing an inactivated virus preservation solution, which adopts the following technical scheme:
a preparation method of an inactivated virus preservation solution comprises the following steps: and (3) uniformly mixing the components except the sodium hydroxide and the phenolphthalein, then adding the phenolphthalein, uniformly mixing, finally adding the sodium hydroxide, and adjusting the pH value of the solution to 8.4 +/-0.2 to obtain the inactivated virus preservation solution.
By adopting the technical scheme, the raw materials are mixed, and the pH value of the virus preservation solution is adjusted to 8.4 +/-0.2 by using sodium hydroxide, so that the preservation time and the integrity of the virus can be improved.
In summary, the present application includes at least one of the following beneficial technical effects:
1. according to the application, related components are added to maintain the normal use of the virus preservation solution, and polyhexamethylene biguanide is also added, so that the virus is inactivated, and the secondary infection of the virus is effectively reduced; in addition, the fixing agent is added, so that nucleic acid and protein of the virus can be fixed, and the integrity of the virus is improved;
2. in the fixing agent, not only are tannic acid and ethanol added to fix nucleic acid and protein of the virus, but also phosphatidylcholine is added to improve the penetration rate of the tannic acid to the virus and improve the fixing effect on the virus;
3. the saponin is added, so that the penetration effect of the tannic acid can be further improved, the tannic acid can better fix the virus, and meanwhile, the polyether modified organic silicon is added, so that the influence of saponin foaming on the virus can be reduced;
4. the application also adds the additive, so that the aging of the virus can be effectively inhibited, and the storage time of the virus is further prolonged.
Detailed Description
The present application will be described in further detail with reference to examples.
The first embodiment is as follows:
an inactivated virus preservation solution comprises the following raw materials in parts by weight: 22 parts of tris (hydroxymethyl) aminomethane, 6 parts of oxalic acid tetraacetic acid, 5 parts of penicillin, 3 parts of streptomycin, 2 parts of polyhexamethylene biguanide, 3 parts of sodium dihydrogen phosphate, 2 parts of disodium hydrogen phosphate, 1 part of phenolphthalein, 4 parts of sodium hydroxide, 60 parts of water and 2 parts of a fixing agent.
Wherein:
the fixing agent comprises the following components in parts by weight: 20 parts of tannic acid, 15 parts of phosphatidylcholine and 30 parts of ethanol;
the preparation method of the fixing agent comprises the following steps: ball-milling and mixing tannic acid and three-quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a second mixture; and mixing the first mixture and the second mixture at the rotating speed of 40 revolutions per minute for 12 minutes to obtain the fixing agent.
The preparation method of the inactivated virus preservation solution comprises the following steps: and (3) uniformly mixing the components except the sodium hydroxide and the phenolphthalein at the rotating speed of 60 revolutions per minute for 15 minutes, then adding the phenolphthalein, uniformly mixing, finally adding the sodium hydroxide, and adjusting the pH value of the solution to 8.4 to obtain the inactivated virus preservation solution.
Example two:
an inactivated virus preservation solution comprises the following raw materials in parts by weight: 26 parts of trihydroxymethyl aminomethane, 9 parts of oxalic acid tetraacetic acid, 6 parts of penicillin, 4 parts of streptomycin, 3 parts of polyhexamethylene biguanide, 4 parts of sodium dihydrogen phosphate, 3 parts of disodium hydrogen phosphate, 1 part of phenolphthalein, 5 parts of sodium hydroxide, 90 parts of water and 3 parts of a fixing agent.
Wherein:
the fixing agent comprises the following components in parts by weight: 23 parts of tannic acid, 20 parts of phosphatidylcholine and 36 parts of ethanol;
the preparation method of the fixing agent comprises the following steps: ball-milling and mixing tannic acid and three-quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a second mixture; and mixing the first mixture and the second mixture at the rotating speed of 40 revolutions per minute for 12 minutes to obtain the fixing agent.
The preparation method of the inactivated virus preservation solution comprises the following steps: and (3) uniformly mixing the components except the sodium hydroxide and the phenolphthalein at the rotating speed of 60 revolutions per minute for 15 minutes, then adding the phenolphthalein, uniformly mixing, finally adding the sodium hydroxide, and adjusting the pH value of the solution to 8.5 to obtain the inactivated virus preservation solution.
Example three:
an inactivated virus preservation solution comprises the following raw materials in parts by weight: 28 parts of trihydroxymethyl aminomethane, 10 parts of oxalic acid tetraacetic acid, 6 parts of penicillin, 5 parts of streptomycin, 4 parts of polyhexamethylene biguanide, 4 parts of sodium dihydrogen phosphate, 4 parts of disodium hydrogen phosphate, 2 parts of phenolphthalein, 5 parts of sodium hydroxide, 98 parts of water and 4 parts of a fixing agent.
Wherein:
the fixing agent comprises the following components in parts by weight: 30 parts of tannic acid, 23 parts of phosphatidylcholine and 42 parts of ethanol;
the preparation method of the fixing agent comprises the following steps: ball-milling and mixing tannic acid and three-quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a second mixture; and mixing the first mixture and the second mixture at the rotating speed of 40 revolutions per minute for 12 minutes to obtain the fixing agent.
The preparation method of the inactivated virus preservation solution comprises the following steps: and (3) uniformly mixing the components except the sodium hydroxide and the phenolphthalein at the rotating speed of 60 revolutions per minute for 15 minutes, then adding the phenolphthalein, uniformly mixing, finally adding the sodium hydroxide, and adjusting the pH value of the solution to 8.3 to obtain the inactivated virus preservation solution.
Example four:
an inactivated virus preservation solution comprises the following raw materials in parts by weight: 29 parts of trihydroxymethyl aminomethane, 11 parts of oxalic acid tetraacetic acid, 7 parts of penicillin, 6 parts of streptomycin, 6 parts of polyhexamethylene biguanide, 5 parts of sodium dihydrogen phosphate, 6 parts of disodium hydrogen phosphate, 2 parts of phenolphthalein, 7 parts of sodium hydroxide, 100 parts of water and 5 parts of a fixing agent.
Wherein:
the fixing agent comprises the following components in parts by weight: 35 parts of tannic acid, 26 parts of phosphatidylcholine and 50 parts of ethanol;
the preparation method of the fixing agent comprises the following steps: ball-milling and mixing tannic acid and three-quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a second mixture; and mixing the first mixture and the second mixture at the rotating speed of 40 revolutions per minute for 12 minutes to obtain the fixing agent.
The preparation method of the inactivated virus preservation solution comprises the following steps: and (3) uniformly mixing the components except the sodium hydroxide and the phenolphthalein at the rotating speed of 60 revolutions per minute for 15 minutes, then adding the phenolphthalein, uniformly mixing, finally adding the sodium hydroxide, and adjusting the pH value of the solution to 8.6 to obtain the inactivated virus preservation solution.
Example five:
an inactivated virus preservation solution comprises the following raw materials in parts by weight: 36 parts of tris (hydroxymethyl) aminomethane, 14 parts of oxalic acid tetraacetic acid, 11 parts of penicillin, 9 parts of streptomycin, 8 parts of polyhexamethylene biguanide, 9 parts of sodium dihydrogen phosphate, 8 parts of disodium hydrogen phosphate, 3 parts of phenolphthalein, 9 parts of sodium hydroxide, 120 parts of water and 7 parts of a fixing agent.
Wherein:
the fixing agent comprises the following components in parts by weight: 36 parts of tannic acid, 28 parts of phosphatidylcholine and 54 parts of ethanol;
the preparation method of the fixing agent comprises the following steps: ball-milling and mixing tannic acid and three-quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a second mixture; and mixing the first mixture and the second mixture at the rotating speed of 40 revolutions per minute for 12 minutes to obtain the fixing agent.
The preparation method of the inactivated virus preservation solution comprises the following steps: and (3) uniformly mixing the components except the sodium hydroxide and the phenolphthalein at the rotating speed of 60 revolutions per minute for 15 minutes, then adding the phenolphthalein, uniformly mixing, finally adding the sodium hydroxide, and adjusting the pH value of the solution to 8.5 to obtain the inactivated virus preservation solution.
Example six:
the difference from the first embodiment is that: the fixing agent also comprises the following components: 4 parts of saponin and 4 parts of polyether modified organic silicon; the preparation method of the fixing agent comprises the following steps: ball-milling and mixing tannic acid and three-quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a second mixture; mixing the first mixture with saponin and polyether modified organic silicon at the rotating speed of 40 revolutions per minute for 10 minutes to obtain a third mixture; and then mixing the second mixture and the third mixture at the rotating speed of 40 revolutions per minute for 12 minutes to obtain the fixing agent.
Example seven:
the difference from the first embodiment is that: the fixing agent also comprises the following components: 4 parts of saponin and 6 parts of polyether modified organic silicon; the preparation method of the fixing agent comprises the following steps: ball-milling and mixing tannic acid and three-quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a second mixture; mixing the first mixture with saponin and polyether modified organic silicon at the rotating speed of 40 revolutions per minute for 10 minutes to obtain a third mixture; and then mixing the second mixture and the third mixture at the rotating speed of 40 revolutions per minute for 12 minutes to obtain the fixing agent.
Example eight:
the difference from the first embodiment is that: the fixing agent also comprises the following components: 9 parts of saponin and 5 parts of polyether modified organic silicon; the preparation method of the fixing agent comprises the following steps: ball-milling and mixing tannic acid and three-quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine, wherein the ball-milling radius is 40mm, the rotating speed is 50 r/min, and the time is 10 min, so as to obtain a second mixture; mixing the first mixture with saponin and polyether modified organic silicon at the rotating speed of 40 revolutions per minute for 10 minutes to obtain a third mixture; and then mixing the second mixture and the third mixture at the rotating speed of 40 revolutions per minute for 12 minutes to obtain the fixing agent.
Example nine:
the difference from the first embodiment is that the inactivated virus preservation solution raw material also comprises the following components: 5 parts of L-cysteine hydrochloride.
Example ten:
the difference from the first embodiment is that the inactivated virus preservation solution raw material also comprises the following components: 8 parts of L-cysteine hydrochloride.
Example eleven:
the difference from the first embodiment is that the inactivated virus preservation solution raw material also comprises the following components: 14 parts of L-cysteine hydrochloride.
Example twelve:
the difference from the sixth embodiment is that the inactivated virus preservation solution raw material also comprises the following components: 5 parts of L-cysteine hydrochloride.
Example thirteen:
the difference from the sixth embodiment is that the inactivated virus preservation solution raw material also comprises the following components: 8 parts of L-cysteine hydrochloride.
Example fourteen:
the difference from the sixth embodiment is that the inactivated virus preservation solution raw material also comprises the following components: 14 parts of L-cysteine hydrochloride.
Comparative example one:
in contrast to example one, no fixative was added.
Comparative example two:
in contrast to example one, polyhexamethylene biguanide was not added.
Comparative example three:
in comparison to example one, polyhexamethylene biguanide was replaced with an equivalent amount of imidazolidinyl urea.
Comparative example four:
in contrast to example six, no phosphatidylcholine was added.
Comparative example five:
compared with the ninth embodiment, polyether modified organic silicon is not added.
Comparative example six:
in comparison with example nine, the polyether modified silicone was replaced with an equal amount of polydimethylsiloxane.
Comparative example seven:
compared with the sixth embodiment, the preparation method of the fixing agent comprises the following steps: all components of the fixing agent are mixed together, the rotating speed is 40 r/min, and the time is 12 min, so that the fixing agent can be obtained.
And (3) performance detection:
PRC detection was performed by taking the virus preservation solutions prepared in examples one to five and comparative examples one to seven, and taking pure Hanks buffer as a control virus preservation solution:
the detection method comprises the following steps: respectively collecting H7N9 virus and measles virus with sampling swabs, adding each virus sample into the virus preservation solution, diluting the samples by 10 times, 100 times, 1000 times and 10000 times with physiological saline, performing PCR nucleic acid sensitivity test on the samples, and determining CT value.
TABLE 1
In order to test whether the virus preservation solution can inactivate viruses, we also performed virus activity detection:
step one, setting an experimental group and a control group, wherein the experimental group comprises: mu.l of lentivirus with a titer of 2X10^8TU/ml were taken and placed in 500. mu.l of the preservative solution of the examples and comparative examples of the present application. Control group: mu.l of lentivirus with a titer of 2X10^8TU/ml was taken in 500. mu.l PBS (phosphate buffered saline). The experimental and control groups were placed at room temperature and 4 ℃ for 1 day, respectively.
And secondly, diluting the experimental group and the control group, respectively taking the virus liquid of 8x10^2TU to infect 293T cells (the 293T cells are planted in a 96-well plate one day in advance), and observing the number of fluorescent proteins (GFP) under a fluorescence microscope after infecting for 2 days so as to reflect the virus infection activity.
TABLE 2
TABLE 3
According to the detection results of the first to fifth examples and the first and third comparative examples, the virus preservation solution can successfully inactivate viruses and improve the preservation integrity of the viruses.
According to the detection results of the six examples to the nine examples, and the fourth comparative example and the seventh comparative example, the addition of the fixing agent can effectively improve the storage integrity of the virus.
From the test results of the nine to fourteen examples and the five and six comparative examples, it can be seen that the storage integrity of the virus can be further improved by adding the polyether modified silicone and the saponin together.
The above embodiments are preferred embodiments of the present application, and the protection scope of the present application is not limited by the above embodiments, so: all equivalent changes made according to the structure, shape and principle of the present application shall be covered by the protection scope of the present application.
Claims (8)
1. The inactivated virus preservation solution is characterized by comprising the following raw materials in parts by weight: 22-36 parts of tris (hydroxymethyl) aminomethane, 6-14 parts of oxalic acid tetraacetic acid, 5-11 parts of penicillin, 3-9 parts of streptomycin, 2-8 parts of polyhexamethylene biguanide, 3-9 parts of sodium dihydrogen phosphate, 2-8 parts of disodium hydrogen phosphate, 1-3 parts of phenolphthalein, 4-9 parts of sodium hydroxide, 60-120 parts of water and 2-7 parts of a fixing agent.
2. The inactivated virus preservation solution according to claim 1, wherein the raw materials comprise the following components in parts by weight: 26-29 parts of trihydroxymethyl aminomethane, 9-11 parts of oxalic acid tetraacetic acid, 6-7 parts of penicillin, 4-6 parts of streptomycin, 3-6 parts of polyhexamethylene biguanide, 4-5 parts of sodium dihydrogen phosphate, 3-6 parts of disodium hydrogen phosphate, 1-2 parts of phenolphthalein, 5-7 parts of sodium hydroxide, 90-100 parts of water and 3-5 parts of a fixing agent.
3. The inactivated virus preservation solution according to claim 2, wherein the raw materials comprise the following components in parts by weight: 28 parts of trihydroxymethyl aminomethane, 10 parts of oxalic acid tetraacetic acid, 6 parts of penicillin, 5 parts of streptomycin, 4 parts of polyhexamethylene biguanide, 4 parts of sodium dihydrogen phosphate, 4 parts of disodium hydrogen phosphate, 2 parts of phenolphthalein, 5 parts of sodium hydroxide, 98 parts of water and 4 parts of a fixing agent.
4. The inactivated virus preservation solution according to claim 1, wherein: the fixing agent comprises the following components in parts by weight: 20-36 parts of tannic acid, 15-28 parts of phosphatidylcholine and 30-54 parts of ethanol.
5. The inactivated virus preservation solution according to claim 4, wherein: the preparation method of the fixing agent comprises the following steps: performing ball milling and mixing on tannic acid and three-quarter of phosphatidylcholine to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine to obtain a second mixture; and mixing the first mixture and the second mixture to obtain the fixing agent.
6. The inactivated virus preservation solution according to claim 4, wherein: the fixing agent also comprises the following components: 4-9 parts of saponin and 3-6 parts of polyether modified organic silicon; the preparation method of the fixing agent comprises the following steps: performing ball milling and mixing on tannic acid and three-quarter of phosphatidylcholine to obtain a first mixture; ball-milling and mixing ethanol and one quarter of phosphatidylcholine to obtain a second mixture; mixing the first mixture with saponin and polyether modified silicone to obtain a third mixture; and then mixing the second mixture and the third mixture to obtain the fixing agent.
7. The inactivated virus preservation solution according to claim 1, wherein the raw materials further comprise the following components in parts by weight: 5-14 parts of L-cysteine hydrochloride.
8. The method for producing an inactivated virus preservation solution according to any one of claims 1 to 7, comprising the steps of: and (3) uniformly mixing the components except the sodium hydroxide and the phenolphthalein, then adding the phenolphthalein, uniformly mixing, finally adding the sodium hydroxide, and adjusting the pH value of the solution to 8.4 +/-0.2 to obtain the inactivated virus preservation solution.
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