CN113684253A

CN113684253A - Inactivated preservation solution for novel coronavirus

Info

Publication number: CN113684253A
Application number: CN202111059070.0A
Authority: CN
Inventors: 王棕岩
Original assignee: Shijiazhuang Kangweishi Medical Instrument Co ltd
Current assignee: Shijiazhuang Kangweishi Medical Instrument Co ltd
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2021-11-23

Abstract

An inactivated preservation solution for novel coronavirus belongs to the technical field of virus preservation solution. The virus preservation solution comprises the following components in percentage by mass: 4.5-5.0% of guanidinium isothiocyanate, 0.6-1.0% of sodium dodecyl sulfate, 0.4-0.7% of 2-mercaptoethanol, 1.0-1.3% of trisodium citrate, 0.3-0.7% of protease K, 0.07-0.09% of hydrochloric acid, 0.03-0.05% of triton X-100, 0.03-0.05% of DL-dithiothreitol-d 10, 0.03-0.05% of water and the balance of water. The preservative solution has the beneficial effects that the pH value of the preservative solution is 7.2-8 finally through the composition of the components and the proportion of the components, the activity of the inactivated components and the degradation-resistant components can be fully excited to reach the highest value in the pH value range, and therefore, the virus protein is effectively inactivated, and the perfect stability of nucleic acid is realized.

Description

Inactivated preservation solution for novel coronavirus

Technical Field

The invention belongs to the technical field of virus preservation solution, and relates to a formula of preservation solution for temporarily storing and transporting viruses, in particular to inactivated preservation solution for novel coronavirus aiming at inactivated viruses.

Background

The virus preservation solution is generally divided into an inactivated type and a non-inactivated type, the non-inactivated type virus preservation solution belongs to preservation and transportation by culturing or maintaining viruses due to the fact that no inactivated component exists, the preservation solution is relatively simple as long as the storage and the transportation are guaranteed without leakage, and the safety problem after leakage cannot be completely guaranteed. The inactivated virus preservation solution generally has no safety problem after leakage because it contains an inactivating component in the preservation solution to inactivate the protein cleavage of the virus, but has the following problems: 1. complete inactivation cannot be guaranteed, so that during storage or transportation, particularly in remote areas, the way is difficult due to relative remoteness, and if complete inactivation cannot be guaranteed, the risk of secondary infection is likely to occur midway. 2. On the basis of virus protein cleavage inactivation, the problem of how to effectively preserve the stable state of nucleic acid is solved, and once a nucleic acid sample is destroyed after inactivation, the nucleic acid detection error is finally caused, so that more infection risks are caused.

Disclosure of Invention

The invention aims to solve the problem of improving the stability of an inactivated virus preservation solution, further avoiding the technical problem that the virus cannot be always in an inactivated state in an effective period due to the instability of the preservation solution, and designs the inactivated preservation solution for the novel coronavirus, wherein the inactivation performance of the preservation solution can reach a stable state on the basis of not damaging nucleic acid through reasonable component proportion.

The technical scheme adopted by the invention is that the inactivated preserving fluid for the novel coronavirus comprises the following components in percentage by mass:

more preferably, the virus preservation solution comprises the following components in percentage by mass:

in the invention, guanidine isothiocyanate is a main inactivation component, so that the protein of the virus can be cracked to achieve the inactivation effect; simultaneously, triton X-100 is used as a surfactant to assist in matching with guanidine isothiocyanate to crack virus protein; the proteinase K has the capability of degrading proteins, the enzyme cutting site is at the carboxyl terminal peptide bond of aliphatic amino acid and aromatic amino acid, and can be used for degrading all proteins, and the combination of the three substances finally ensures the inactivation effect of viruses.

2-mercaptoethanol and DL-dithiothreitol-d 10 are main nucleic acid stabilizers, and the two substances are used in combination to prevent nucleic acid degradation; meanwhile, the trisodium citrate can generate a complex reaction with heavy metals in the liquid, and further assists two substances, namely 2-mercaptoethanol and DL-dithiothreitol-d 10, so that the aim of preventing nucleic acid degradation is fulfilled.

Sodium dodecyl sulfonate belongs to an anionic surfactant and has excellent functions of permeation, washing, wetting, decontamination and emulsification.

The hydrochloric acid is added to adjust the pH value of the preservation solution, so that the preservation solution is in a dark pink or light purple state. It has been found through experimentation that controlling the amount of hydrochloric acid added further maximizes the efficiency of the activity of the inactivating component and the nucleic acid degradation preventing component.

Water is the carrier for all the above ingredients.

The preservative solution has the beneficial effects that the pH value of the preservative solution is 7.2-8 finally through the composition of the components and the proportion of the components, the activity of the inactivated components and the degradation-resistant components can be fully excited to reach the highest value in the pH value range, and therefore, the virus protein is effectively inactivated, and the perfect stability of nucleic acid is realized.

Drawings

FIG. 1 shows an amplification curve of a sample stored in a virus storage solution.

FIG. 2 is an amplification curve of a sample stored in RNase-free water.

Detailed Description

The following describes the advantageous effects of the present invention with reference to specific examples and experimental data.

Firstly, a preservation solution inactivation and nucleic acid degradation prevention experiment:

the inactivated virus preservation solution is prepared according to the weight percentage in the table 1. The units for examples 1-7 are all grams.

TABLE 1 ingredient ratio List for specific examples 1-7

The experimental scheme is as follows:

all the components in table 1 are mixed according to the mass ratio of the storage solution of the examples 1-7, and the example numbers are marked respectively.

Avian infectious bronchitis virus (coronavirus) was used to verify the stability of the product to preservation of viral RNA in the samples.

(1) Mixing 100 μ L of avian infectious bronchitis virus with 2ml of virus collecting solution, dividing into 8 parts per tube according to 200 μ L, storing four parts at 37 deg.C, and storing the other four parts at-20 deg.C. Another 100. mu.L of virus was mixed with 2mL of RNase-free water. As shown in table 2.

(2) As shown in Table 2, 200. mu.L of each sample was taken from each treatment solution on days 0, 1, 2, 3 and 4 to extract RNA, and the RNA was stored at-20 ℃.

(3) One-step reverse transcription fluorescence quantitative detection is shown in table 3.

TABLE 2 sample treatment and nucleic acid extraction schedules

TABLE 3 one-step reverse transcription fluorescent quantitative detection procedure

The experimental results are as follows:

TABLE 4 RT-qPCR test results (Ct values) for samples from different storage solutions and storage conditions

In Table 4, the results of experiments based on the most preferred embodiment 4 of the present invention are shown, and the data of the results of the experiments of examples 1 to 3 and examples 5 to 7 are within the range of 0.02.

And (4) detection conclusion:

based on the above experiment, the amount of viral RNA in the sample preserved at 37 ℃ is not less than 1/2 of the RNA content in the sample preserved at-20 ℃ within 4 days after the sample added with the preservation solution is sampled, namely the CT difference is less than 1. Meets the technical requirements. In the sample without RNase water, from the 2 nd day after sampling, the amount of the viral RNA in the sample stored at 37 ℃ is lower than 1/2 of the amount of the RNA in the sample stored at-20 ℃, namely, the CT difference is greater than 1, and the inactivation performance is poor.

II, testing the preservation performance of the inactivated virus preservation solution at different pH values:

the experimental scheme is as follows:

experiments are carried out by adopting the components of the invention, and in addition to the above examples 1-7, the following examples 8-12 are also carried out, wherein the percentage content of the hydrochloric acid is controlled to be 0.07-0.09% in the examples 1-7, and the examples 8-12 are respectively based on the examples 1-5, except that the percentage content of the hydrochloric acid is adjusted to be larger or smaller, the specific gravity of the rest components is not changed, the content of water is finally adjusted to be 100%, and referring to the following table 5, the stability of the product on the preservation of the virus RNA in the sample is still verified by the avian infectious bronchitis virus (coronavirus). See the procedure of experiment one and tables 2 and 3, and the data of the results of examples 8-12 in table 6. In Table 5, the units of examples 8 to 12 are all in grams.

TABLE 5 ingredient ratio List for specific examples 8-12

TABLE 6 RT-qPCR test results (Ct values) for samples at different pH values

As can be seen from Table 6, in examples 8-12, the pH value adjusted by hydrochloric acid was not in the range of 7.2-8, and the content of hydrochloric acid was not in the range of 0.7-0.9%, and the final Ct difference was greater than 1 no matter whether the content of hydrochloric acid was higher or lower than 0.7-0.9% in the present invention, which indicates that the virus-preserving fluid of examples 8-12 was poor in performance and could not ensure the virus inactivation effect or the degradation prevention performance of nucleic acid.

Claims

1. An inactivated preservative fluid for a novel coronavirus, comprising: the virus preservation solution comprises the following components in percentage by mass:

the balance of water.

2. The inactivated preservation solution for a novel coronavirus according to claim 1, wherein: the virus preservation solution comprises the following components in percentage by mass:

3. the inactivated preservation solution for a novel coronavirus according to claim 1, wherein: the pH value of the preservation solution is 7.2-8.