CN115354068A - Inactivated virus preservation solution and preparation method thereof - Google Patents

Inactivated virus preservation solution and preparation method thereof Download PDF

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CN115354068A
CN115354068A CN202211127363.2A CN202211127363A CN115354068A CN 115354068 A CN115354068 A CN 115354068A CN 202211127363 A CN202211127363 A CN 202211127363A CN 115354068 A CN115354068 A CN 115354068A
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preservation solution
virus
polyethylene glycol
inactivated virus
phenol red
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李先强
姜昕
黄永伟
陈巨
陈佳
周向珍
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Wuhan Zhongzhi Biotechnologies Inc
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Wuhan Zhongzhi Biotechnologies Inc
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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
    • 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
    • C12Q1/701—Specific hybridization probes

Abstract

The invention discloses an inactivated virus preservation solution and a preparation method thereof, wherein the preservation solution comprises the following components in final concentration: 15-25 g/L of guanidinium isothiocyanate, 4-6 g/L of protease K, 1-2 mL/L of ethylphenyl polyethylene glycol, 1-10 mL/L of octylphenyl-polyethylene glycol, 1-10 mL/L of polyoxyethylene (20) hexadecyl ether, 0.1-0.2 g/L, proclin, 0.4mL/L of ethylenediamine tetraacetic acid, 9g/L of sodium chloride, 2.9g/L of disodium hydrogen phosphate, 0.27g/L of potassium dihydrogen phosphate, 0.25mL/L of 1M sodium hydroxide and 0.04g/L of phenol red. The inactivated virus preservation solution improves the stability of the inactivated virus preservation solution on the basis of not damaging virus nucleic acid, and further improves the in-vitro diagnosis and detection performance of downstream virus nucleic acid.

Description

Inactivated virus preservation solution and preparation method thereof
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to an inactivated virus preservation solution and a preparation method thereof.
Background
Viruses (viruses) belong to the smallest, simplest of the cell-free structures of microorganisms. Virosomes (virion) are intact mature viral particles composed of nucleic acids and proteins, with a core of only one nucleic acid (DNA or RNA), surrounded by a capsid of proteins forming a nucleocapsid, and some viruses are surrounded by an envelope. The virus must be parasitic within living cells and reproduce progeny in a replicative manner. In clinical microbial infection, the virus causes about 75%, and has the advantages of strong infectivity, rapid spread, wide epidemic, high death rate, serious sequelae, difficult clinical diagnosis and prevention, and some virus infections are closely related to tumors and autoimmune diseases. Therefore, the virus has important significance clinically. When the clinical detection is performed on infectious diseases caused by viruses, in order to prevent the infection of the viruses, particularly the infection of some highly pathogenic viruses, a sample needs to be inactivated, and the virus inactivation modes mainly comprise: physical inactivation (high temperature, ultraviolet irradiation, etc.), chemical inactivation (disinfectant, etc.). Generally, the principle of virus inactivation is that the virus is killed by destroying proteins and nucleic acids of the virus, the destruction of the proteins can cause the release of viral nucleic acids, and the destruction of viral nucleic acids (especially RNA viruses) can also result without any protective measures, which is not favorable for the subsequent detection of the molecular biology based on the viral nucleic acids.
Therefore, it is an urgent technical problem to develop an inactivated virus preservation solution that inactivates viruses and ensures the integrity of viral nucleic acids.
Disclosure of Invention
The invention aims to provide an inactivated virus preservation solution and a preparation method thereof, which can improve the stability of the inactivated virus preservation solution on the basis of not damaging virus nucleic acid, and further improve the in-vitro diagnosis and detection performance of downstream aiming at the virus nucleic acid.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided an inactivated virus preservation solution comprising the following components in final concentrations: 15-25 g/L of guanidinium isothiocyanate, 4-6 g/L of protease K, 1-2 mL/L of ethylphenyl polyethylene glycol, 1-10 mL/L of octylphenyl-polyethylene glycol, 1-10 mL/L of polyoxyethylene (20) hexadecyl ether, 0.1-0.2 g/L, proclin, 0.4mL/L of ethylenediamine tetraacetic acid, 9g/L of sodium chloride, 2.9g/L of disodium hydrogen phosphate, 0.27g/L of potassium dihydrogen phosphate, 0.25mL/L of 1M sodium hydroxide and 0.04g/L of phenol red.
Further, the preservation solution comprises the following components in final concentration: 9g/L of sodium chloride, 20g/L of guanidine isothiocyanate, 5g/L of protease K, 1.5mL/L of ethylphenylpolyethylene glycol, 2mL/L of octylphenyl-polyethylene glycol, 2mL/L of polyoxyethylene (20) hexadecyl ether, 0.15g/L, proclin 150.4 mL/L of ethylene diamine tetraacetic acid, 2.9g/L of disodium hydrogen phosphate, 0.27g/L of potassium dihydrogen phosphate, 0.25mL/L of 1M sodium hydroxide and 0.04g/L of phenol red.
In a second aspect of the invention, the invention provides a preparation method of the inactivated virus preservation solution, which is prepared by adopting the formula.
Further, adding phenol red into 1M sodium hydroxide to prepare a phenol red mother liquor;
and sequentially adding sodium chloride, guanidine isothiocyanate, proteinase K, ethylphenyl polyethylene glycol, octylphenyl-polyethylene glycol, polyoxyethylene (20) hexadecyl ether, ethylene diamine tetraacetic acid, proclin 150, disodium hydrogen phosphate, potassium dihydrogen phosphate and the phenol red mother liquor into the purified water, and uniformly mixing.
In a third aspect of the invention, the use of the inactivated virus preservation solution for preserving viruses is provided.
Further, in the application, the volume ratio of the virus stock solution to be preserved to the inactivated virus preservation solution is 1: (6-10).
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the components of the inactivated virus preservation solution are cooperatively matched, so that the stability of the inactivated virus preservation solution is improved on the basis of not damaging virus nucleic acid, and the in-vitro diagnosis and detection performance of downstream virus nucleic acid is improved. Particularly, the protective effect of the preservation solution on nucleic acid is greatly enhanced by adding the active agents of ethyl phenyl polyethylene glycol, octyl phenyl-polyethylene glycol and polyoxyethylene (20) hexadecyl ether. The proportion of 1-2 mL/L ethyl phenyl polyethylene glycol, 1-10 mL/L octyl phenyl polyethylene glycol and 1-10 mL/L polyoxyethylene (20) hexadecyl ether must be in the range, and the protective effect of the preservation solution on nucleic acid is not enhanced when the concentration is too large or too small.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 shows the results of verification of the preservation effect of influenza A virus nucleic acid;
FIG. 2 shows the results of the preservation of nucleic acid of respiratory syncytial virus.
Detailed Description
The present invention will be specifically explained below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by existing methods.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
according to an exemplary embodiment of the present invention, there is provided an inactivated virus preservation solution, comprising the following components in final concentrations:
15-25 g/L of guanidinium isothiocyanate, 4-6 g/L of protease K, 1-2 mL/L of ethylphenyl polyethylene glycol, 1-10 mL/L of octylphenyl-polyethylene glycol, 1-10 mL/L of polyoxyethylene (20) hexadecyl ether, 0.1-0.2 g/L, proclin, 0.4mL/L of ethylenediamine tetraacetic acid, 9g/L of sodium chloride, 2.9g/L of disodium hydrogen phosphate, 0.27g/L of potassium dihydrogen phosphate, 0.25mL/L of 1M sodium hydroxide and 0.04g/L of phenol red.
Experiments show that the protective effect of the preservation solution on nucleic acid is greatly enhanced by adding the active agents of ethyl phenyl polyethylene glycol, octyl phenyl-polyethylene glycol and polyoxyethylene (20) hexadecyl ether. The proportion of 1-2 mL/L ethyl phenyl polyethylene glycol, 1-10 mL/L octyl phenyl polyethylene glycol and 1-10 mL/L polyoxyethylene (20) hexadecyl ether must be in the range, and the protective effect of the preservation solution on nucleic acid is not enhanced when the concentration is too large or too small.
In addition, the components cooperate with each other: guanidine isothiocyanate is a main inactivation component, can crack proteins of viruses to achieve an inactivation effect, and can inhibit the activity of nuclease; the proteinase K has the capability of degrading proteins, and enzyme cutting sites are positioned at carboxyl-terminal peptide bonds of aliphatic amino acids and aromatic amino acids, so that the proteinase K can be used for degrading all proteins; the addition of the three activators greatly enhances the protective effect of the preservation solution on nucleic acid; the ethylene diamine tetraacetic acid is a metal chelating agent and plays a role in inhibiting nuclease; proclin 150 is used as a conventional bacteriostatic agent for inhibiting the production of mixed bacteria; sodium chloride, disodium hydrogen phosphate and disodium hydrogen phosphate are used as a buffer system of the basic solution; adjusting the pH value of the solution by 1M sodium hydroxide; phenol red is a color developing agent.
As a preferable embodiment, the following formula of the preservation solution has been found through experiments to have longer preservation time.
9g/L of sodium chloride, 20g/L of guanidine isothiocyanate, 5g/L of protease K, 1.5mL/L of ethylphenylpolyethylene glycol, 2mL/L of octylphenyl-polyethylene glycol, 2mL/L of polyoxyethylene (20) hexadecyl ether, 0.15g/L, proclin 150.4 mL/L of ethylene diamine tetraacetic acid, 2.9g/L of disodium hydrogen phosphate, 0.27g/L of potassium dihydrogen phosphate, 0.25mL/L of 1M sodium hydroxide and 0.04g/L of phenol red.
According to a typical embodiment of the present invention, there is provided a method for preparing an inactivated virus preservation solution, which is prepared by using the above formulation.
The use method of the inactivated virus preservation solution comprises the following steps:
adding a virus stock solution to be stored into an inactivated virus preservation solution, wherein the volume ratio of the virus stock solution to be stored to the inactivated virus preservation solution is 1: (6-10).
The inactivated virus preservation solution of the present application will be described in detail below with reference to examples, comparative examples, and experimental data.
Example 1
1. Preparation of Virus preserving fluid
The virus preservation solution is prepared according to the following formula
Figure BDA0003848767720000041
When preparing, phenol red is added to 1M sodium hydroxide to prepare phenol red mother liquor. Then adding sodium chloride, guanidine isothiocyanate, proteinase K, ethylphenyl polyethylene glycol, octylphenyl-polyethylene glycol, polyoxyethylene (20) hexadecyl ether, ethylene diamine tetraacetic acid, proclin 150, disodium hydrogen phosphate, potassium dihydrogen phosphate and phenol red mother liquor into the purified water in sequence, and mixing uniformly.
Example 2
The virus preservation solution is prepared according to the following formula
Figure BDA0003848767720000042
Figure BDA0003848767720000051
Example 3
The virus preservation solution is prepared according to the following formula
Figure BDA0003848767720000052
Comparative example 1
This comparative example does not contain octylphenyl-polyethylene glycol and polyoxyethylene (20) cetyl ether, and is otherwise as in example 1, formulated specifically as follows:
Figure BDA0003848767720000053
Figure BDA0003848767720000061
comparative example 2
This comparative example does not contain ethylphenylpolyethylene glycol and polyoxyethylene (20) cetyl ether, and is otherwise as in example 1, formulated specifically as follows:
Figure BDA0003848767720000062
comparative example 3
This comparative example does not contain ethylphenylpolyethylene glycol and octylphenyl-polyethylene glycol, and is otherwise as in example 1, formulated specifically as follows:
Figure BDA0003848767720000063
Figure BDA0003848767720000071
comparative example 4
This comparative example does not contain ethylphenylpolyethylene glycol, octylphenyl-polyethylene glycol and polyoxyethylene (20) cetyl ether, and is otherwise as in example 1, formulated specifically as follows:
Figure BDA0003848767720000072
experimental example 1 verification of preservation Effect of different preserving solutions
The influenza A virus and the respiratory syncytial virus are selected, treated by the virus preservation solution of the invention, certain commercial virus preservation solution, the virus preservation solution of comparative example 1, the virus preservation solution of comparative example 2, the virus preservation solution of comparative example 3 and the virus preservation solution of comparative example 4, then respectively placed in an environment at 37 ℃, the detection is carried out by a fluorescence PCR method after 24 hours, 0 hour is taken as a positive control, and the CT value (total cycle number 45) result of the fluorescence PCR is as follows:
TABLE 1 verification of the preservation Effect of influenza A Virus nucleic acid
Figure BDA0003848767720000073
TABLE 2 verification of the preservation Effect of respiratory syncytial Virus nucleic acids
Figure BDA0003848767720000074
Figure BDA0003848767720000081
From the results of tables 1 and 2, it can be seen that:
in comparative example 1, the surfactant contained ethylphenylpolyethylene glycol alone, contained octylphenyl-polyethylene glycol and polyoxyethylene (20) cetyl ether, and the other examples were the same as in example 1, and the preservation effects of influenza a virus nucleic acid and respiratory syncytial virus were inferior to those of example 1;
in comparative example 2, the surfactant contained only octylphenyl-polyethylene glycol and no ethylphenyl-polyethylene glycol and polyoxyethylene (20) cetyl ether, and the other examples were the same as example 1, and the preservation effects of influenza a virus nucleic acid and respiratory syncytial virus were inferior to that of example 1;
in comparative example 3, the surfactant contained only polyoxyethylene (20) cetyl ether and no ethylphenylpolyethylene glycol or octylphenyl-polyethylene glycol, and the other examples were the same as example 1, and the preservation effect of influenza a virus nucleic acid and respiratory syncytial virus was inferior to that of example 1;
in comparative example 4, which does not contain ethylphenylpolyethylene glycol, octylphenyl-polyethylene glycol and polyoxyethylene (20) cetyl ether, the preservation effects of influenza A virus nucleic acid and respiratory syncytial virus were inferior to those of example 1, as in example 1;
the virus preservation solution of example 1 of the present invention has a higher effect of protecting nucleic acids than a commercially available preservation solution.
Experimental example 2 verification of Virus inactivation Effect
Adding the prepared seed virus of the avian infectious bronchitis virus IBV strain into the virus preservation solution of the example 1, and inactivating the seed virus at room temperature of 18-26 ℃ for 5min according to the proportion of 1 (virus stock solution) to 9 (virus preservation solution); inoculating the inactivated virus solution to SPF (specific pathogen free) chick embryos for inactivation test, culturing in an incubator at 37 ℃ for 168h after inoculation, observing and recording the death condition of the chick embryos every day, performing autopsy for 168h after inoculation, observing the lesion condition of the chick embryos, and harvesting chick embryo allantoic fluid.
Performing nucleic acid detection on the harvested chick embryo allantoic fluid by using avian infectious bronchitis virus, wherein the detection method refers to GB/T23197-2008 avian infectious bronchitis diagnosis technology, RNA is extracted by using an RNA extraction kit, and virus detection is performed by using a fluorescence PCR method. See table 3 for details.
TABLE 3 inactivation efficacy test group for inactivation of preservation fluid
Figure BDA0003848767720000082
Figure BDA0003848767720000091
As can be seen from Table 3, after the inactivated sample is inoculated with the chick embryos, the conditions of the death of the chick are counted by photographing the chick embryos every day, no abnormality occurs after the chick embryos are inoculated with the samples of the test group, the negative control group and the virus control group in the observation period, and the conditions of the death of the chick embryos of each group are shown in Table 4;
TABLE 4 inactivation of preservative fluid efficacy of inactivation test results
Figure BDA0003848767720000092
As can be seen from the data in table 4, the cell preservation solution (inactivated type) according to the example of the present invention can completely inactivate the I BV virus.
Experimental example 3 verification of protective Effect of a preserving solution on nucleic acids under different conditions
The influenza A virus and the respiratory syncytial virus are selected and treated by using the virus preservation solution of the embodiment 1, then are respectively placed in the environments with the temperature of 4 ℃, 25 ℃ and 37 ℃, then the fluorescence PCR method detection is respectively carried out at the time points of 0h, 2h, 4h, 6h and 24h, and the influenza A virus and the respiratory syncytial virus are set to be stored in physiological saline as the reference.
TABLE 5 Experimental settings for each group
Figure BDA0003848767720000093
TABLE 6 verification of the preservation Effect of nucleic acid of respiratory syncytial Virus
Figure BDA0003848767720000094
Figure BDA0003848767720000101
The test results in Table 6 show that the virus preservation solution of the present invention can ensure stable preservation of pathogens at 4 deg.C, 25 deg.C, and 37 deg.C for 24 hours without affecting the subsequent nucleic acid detection.
Finally, it should be further noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (6)

1. An inactivated virus preservation solution, which is characterized by comprising the following components in final concentration:
15-25 g/L of guanidinium isothiocyanate, 4-6 g/L of protease K, 1-2 mL/L of ethylphenyl polyethylene glycol, 1-10 mL/L of octylphenyl-polyethylene glycol, 1-10 mL/L of polyoxyethylene (20) hexadecyl ether, 0.1-0.2 g/L, proclin, 0.4mL/L of ethylenediamine tetraacetic acid, 9g/L of sodium chloride, 2.9g/L of disodium hydrogen phosphate, 0.27g/L of potassium dihydrogen phosphate, 0.25mL/L of 1M sodium hydroxide and 0.04g/L of phenol red.
2. The inactivated virus preservation solution according to claim 1, wherein the preservation solution comprises the following components in final concentrations: 20g/L of guanidinium isothiocyanate, 5g/L of protease K, 1.5mL/L of ethylphenylpolyethylene glycol, 2mL/L of octylphenyl-polyethylene glycol, 2mL/L of polyoxyethylene (20) cetyl ether, 0.15g/L, proclin 0.4mL/L of ethylenediamine tetraacetic acid, 9g/L of sodium chloride, 2.9g/L of disodium hydrogen phosphate, 0.27g/L of potassium dihydrogen phosphate, 0.25mL/L of 1M sodium hydroxide and 0.04g/L of phenol red.
3. A method for preparing an inactivated virus preservation solution, which is prepared by using the formulation according to claim 1 or 2.
4. The method of claim 3, wherein the formulating comprises the steps of:
adding phenol red into 1M sodium hydroxide to prepare phenol red mother liquor;
and sequentially adding sodium chloride, guanidine isothiocyanate, proteinase K, ethylphenyl polyethylene glycol, octylphenyl-polyethylene glycol, polyoxyethylene (20) hexadecyl ether, ethylene diamine tetraacetic acid, proclin 150, disodium hydrogen phosphate, potassium dihydrogen phosphate and the phenol red mother liquor into the purified water, and uniformly mixing.
5. Use of the inactivated virus preservation solution according to claim 1 or 2 for preserving viruses.
6. The use according to claim 5, wherein the volume ratio of the virus stock solution to be preserved to the inactivated virus preservation solution is 1: (6-10).
CN202211127363.2A 2022-09-16 2022-09-16 Inactivated virus preservation solution and preparation method thereof Pending CN115354068A (en)

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