An inactivation preservation solution and a detection test tube for keeping virus particles intact

Abstract

The present invention belongs to the field of biomedical detection technology, and specifically relates to an inactivation preservation solution and a detection test tube that keep virus particles intact. An inactivation preservation solution that keeps virus particles intact, the preservation solution includes the following components by mass percentage: a buffer 20-100mM, a protein denaturant 1.00wt%-3.00wt%, a stabilizer 0.20wt%-2.00wt%, a free radical scavenger 0.50wt%-2.00wt%, a calcium and magnesium ion chelator 0.10wt%-0.50wt%; the protein denaturant is selected from one or more mixtures of ethyl p-hydroxybenzoate, diazolidinyl urea, and imidazolidinyl urea. This preservation solution can protect the integrity of virus particles at room temperature, avoid RNA release, degradation and contamination, and improve the accuracy of subsequent detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and in particular relates to an inactivation preservation solution and a detection test tube for keeping virus particles intact. Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), named by the World Health Organization (WHO) as Corona Virus Disease 2019 (COIVD-19), is a pathogen that causes respiratory diseases. This emerging infectious disease spreads rapidly, is highly contagious, and the general population is susceptible, so virus prevention and control is urgent.

[0003] The most commonly used method for diagnosing COVID-19 infection is the convenient and reliable detection technology of fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR). The quality control of nucleic acid testing involves many aspects, including sample collection, storage and transportation, detection reagents, and result interpretation. Sample preservation is a key but easily overlooked link that affects the detection effect. In clinical testing, various problems will arise if the virus samples are not properly preserved. The shell of RNA viruses is easily broken, which may release nucleic acids and cause nucleic acid degradation. If the virus is not inactivated in time, the risk of infection for medical staff will increase.

[0004] The existing disposable virus sampling tubes are mainly divided into non-inactivated virus preservation liquid, and inactivated virus preservation liquid is used to avoid infection in the sample collection and storage transportation links. These inactivated virus preservation liquids all use guanidine salts (CN113186251A, CN113444772A, CN113736755A, CN113684253A, CN114717292A, etc.), which denature proteins, denature and lyse viruses, inactivate viruses, and release nucleic acids into the preservation liquid for inspection. The protein shell of the virus is destroyed, which is not conducive to subsequent research on the morphology and structure of the virus.

[0005] Chinese invention patent application (CN108823286A) discloses that the protective agent for free DNA in blood is a composition, which includes imidazolidinyl urea, ethylenediaminetetraacetic acid tripotassium dihydrate and glycine. The patent discloses that imidazolidinyl urea plays a role in maintaining cell stability and preventing cell rupture and release of DNA in the protective agent. Similarly, in Chinese invention patents (CN103789202A, CN112322615A, etc.), imidazolidinyl urea plays a role in protecting cells from rupture. However, none of these documents discloses the role of inactivating viruses while keeping virus particles intact. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a preservation solution that does not contain guanidine salts and can inactivate virus samples. This preservation solution can protect the integrity of virus particles at room temperature, avoid RNA release, degradation and contamination, and improve the accuracy of subsequent detection.

[0007] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0008] An inactivation preservation solution for keeping virus particles intact, the preservation solution comprises the following components by mass percentage:

[0009]

[0010] The protein denaturant is selected from a mixture of one or more of ethyl p-hydroxybenzoate, diazolidinyl urea and imidazolidinyl urea.

[0011] Preferably, the preservation solution comprises the following components by mass percentage:

[0012]

[0013] Preferably, the protein denaturant is selected from imidazolidinyl urea.

[0014] Preferably, the stabilizer is selected from a mixture of one or more of L-glutamine, glycine, and glutaGRO. More preferably, the stabilizer is selected from a combination of L-glutamine and glycine.

[0015] Preferably, the free radical scavenger is selected from a mixture of one or more of reduced glutathione, vitamin C, and N-acetylcysteine. The free radical scavenger is selected from reduced glutathione.

[0016] As further preferred, the calcium and magnesium ion chelating agent is selected from one or more mixtures of EDTA, EDTA disodium salt, and EDTA tripotassium salt. As further preferred, the calcium and magnesium ion chelating agent is selected from EDTA tripotassium salt.

[0017] Furthermore, the present invention also discloses a virus detection test tube, which contains the inactivation preservation solution.

[0018] Due to the adoption of the above technical solution, the PBS buffer makes the pH range of the storage solution between 6.50-7.50, providing a stable pH environment for the entire virus. The protein denaturant makes the outer layer of the virus composed of lipids and glycoproteins lose its ability to infect. The stabilizer forms a protective film outside the viral protein capsid, making the protein difficult to decompose. The free radical scavenger removes the free radicals generated during metabolism, maintains the redox environment, and prevents the virus from rupturing during storage. The calcium and magnesium ion chelator can chelate the divalent cations (Ca 2+ With Mg 2+ ), so that the nuclease activity is blocked. The preservation solution effectively prevents the virus from rupturing and releasing nucleic acids to be degraded by nucleases in the surrounding environment, greatly increasing the storage time of virus samples at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Electron micrographs of cells in the infection control group.

[0020] Figure 2 This is a graph showing the results of the cell infection experiment in Example 1-5.

[0021] Figure 3 This is a graph showing the results of the cell infection experiment in Comparative Examples 1-5.

[0022] Figure 4 The SDS-PAGE electrophoresis diagram of the viral proteins in Examples 1-5.

[0023] Figure 5 The SDS-PAGE electrophoresis diagram of the virus proteins in Comparative Examples 1-5. DETAILED DESCRIPTION

[0024] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described in detail. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Unless otherwise specified in the examples of the present invention, all reagents and consumables used are commercially available products.

[0026] The formulas of the embodiments of the present invention and the comparative examples are as follows:

[0027]

[0028] 1. Test on inactivation ability of preservation solution

[0029] Lentivirus GV115 was suspended in the storage solution of different examples and comparative examples at room temperature for 15 minutes and infected HEK293T cells at MOI=10. PBS was used as a control group. Enhanced green fluorescent protein (eGFP) fluorescence was determined by electron microscopy at 48 hours after infection. Microscope magnification 100×. Figure 1 Electron microscope photos of the control group. Figure 2 This is a diagram showing the results of the cell infection experiment in Example 1-5. Figure 3 The results of the cell infection experiment of Comparative Example 1-5 are shown in FIG. Figure 1 , Figure 2 , Figure 3 It can be seen that the preservation solution of the present application has the inactivation ability.

[0030] 2. Particle integrity test

[0031] The lentiviral GV115 protein was stored in the storage solution of different examples and comparative examples for 7 days. Figure 4 , Figure 5 The SDS-PAGE electrophoresis diagram of viral proteins. Figure 4 , Figure 5 It can be seen that the technical solutions of Examples 1-5 adopted in the present invention significantly improve the integrity of viral protein particles compared with Comparative Examples 1-5.

[0032] 3. Nucleic acid preservation effect test

[0033] Nucleic acid test results of the lentivirus stored in the storage solutions of different examples and comparative examples for 7 days. The Ct values ​​of the fluorescent PCR of the examples of the present invention and the comparative examples are as follows:

[0034] Save for 3 days Save for 7 days Example 1 23.21 23.89 Example 2 23.12 23.68 Example 3 23.03 23.34 Example 4 24.52 26.35 Example 5 25.36 26.98 Comparative Example 1 23.15 30.39 Comparative Example 2 23.67 27.65 Comparative Example 3 25.59 31.52 Comparative Example 4 23.31 26.03 Comparative Example 5 23.63 27.36

[0035] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. An inactivation preservation solution for keeping virus particles intact, characterized in that: The preservation solution is composed of the following components by mass percentage: Buffer 20-100mM Protein denaturant 1.00wt%-2.50wt% Stabilizer 0.20wt%-2.00wt% Free radical scavenger 0.50wt%-2.00wt% Calcium and magnesium ion chelating agent 0.10wt%-0.50wt%; The protein denaturant is selected from imidazolidinyl urea; the stabilizer is selected from a mixture of one or more of L-glutamine, glycine, and glutaGRO; the free radical scavenger is selected from reduced glutathione; and the calcium and magnesium ion chelating agent is selected from a mixture of one or more of EDTA, disodium EDTA, and tripotassium EDTA.

2. The inactivation preservation solution for keeping virus particles intact according to claim 1, characterized in that: The preservation solution is composed of the following components by mass percentage: Buffer 40-80mM Protein denaturant 1.50wt%-2.50wt% Stabilizer 0.50wt%-1.50wt% Free radical scavenger 0.60wt%-1.50wt% Calcium and magnesium ion chelating agent 0.15wt%-0.30wt%.

3. The inactivation preservation solution for keeping virus particles intact according to claim 1, characterized in that: The stabilizer is selected from a combination of L-glutamine and glycine.

4. The inactivation preservation solution for keeping virus particles intact according to claim 1, characterized in that: The calcium and magnesium ion chelating agent is selected from EDTA tripotassium salt.

5. A virus detection test tube, characterized in that: The test tube contains the inactivation preservation solution according to any one of claims 1 to 4.

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