Method for stably storing pseudovirus at normal temperature

Abstract

The invention provides a method for stably storing pseudoviruses at normal temperature, which comprises the following steps: measuring 500-800mL of purified water by using a preserving fluid prepared from BSA (Bovine Serum Albumin), Tween 80, PEG-8000, Na2HPO4, NaH2PO4, KCl and Proclin-300, adding the reagent components into the preserving fluid, fully stirring until the reagent components are completely dissolved, regulating the pH value to 7.2-7.6 by using diluted hydrochloric acid or 1M NaOH, fixing the volume to 1000mL by using purified water, and filtering and sterilizing by using a 0.22 mu m filter membrane. The preservation liquid can stably preserve the pseudovirus for 12 months under the condition of 0-37 DEG C, greatly prolongs the preservation period of the pseudovirus under the condition of 0-37 DEG C, is more beneficial to the transportation and later related application of the pseudovirus, and has the advantages of low cost and convenience in use.

Description

Technical Field

[0001] The invention relates to the technical field of virus storage and processing, and in particular to a method for stably storing pseudoviruses at room temperature. Background Art

[0002] Pseudovirus, also known as "pseudovirus", is a type of chimeric virus particle that expresses the recombinant glycoprotein of another virus on the surface of a replication-deficient virus (viral vector). Its genes are usually changed or modified, and it has a virus-like structure and specific nucleic acid sequence that simulates the virus. Its analytical characteristics are similar to those of real viruses, but it does not have the ability to replicate and infect itself, and can participate in the entire process of virus detection from extraction to amplification. It has biosafety and can be used as a measurement standard for the validation and evaluation of qualitative and quantitative measurement methods of viral nucleic acids, as well as laboratory quality control and neutralizing antibody detection of vaccines and drugs.

[0003] The application areas of pseudovirus are very wide, mainly in the following aspects:

[0004] 1. Regulation of gene expression:

[0005] Pseudoviruses have the same infectious ability as real viruses, and can be used for gene delivery to achieve target gene regulation in cells and animal organs. Although the virus cannot replicate and produce new viruses after infecting cells, the expression units it carries can be transcribed and translated in the cell using the cell's own expression system, thereby achieving gene expression regulation.

[0006] 2. Antibody research:

[0007] By constructing a pseudovirus in which some amino acids are missing from the viral protein, research has found that the ability of the pseudovirus to enter cells can be improved after the protein is truncate. It has been confirmed that the virus is suitable for developing pseudovirus neutralization test methods.

[0008] 3. Nucleic acid diagnostic standards:

[0009] Molecular diagnostics are developing rapidly. With the deepening of clinical understanding of molecular testing projects for pathogens such as viral infectious diseases and women's health, the demand for testing is becoming more diverse. Not only is it necessary to conduct qualitative and typing testing of pathogen nucleic acids, but the accuracy and precision of the quantification of pathogen RNA are also becoming increasingly higher.

[0010] The application of pseudovirus in the detection field is mainly as positive control standard and internal control standard in RNA detection kit. The latest CFDA application guidelines clearly propose the use of pseudovirus as positive standard and internal control standard. Compared with artificial synthetic plasmids containing target genes, pseudovirus as positive standard and internal control standard has the following advantages:

[0011] 1) Amplification curve is more realistic

[0012] In the PCR reaction, different forms of gene templates have different amplification efficiencies. Artificially synthesized plasmids are much simpler than gene templates in clinical samples, and their amplification efficiency is higher than that of clinical samples. The nucleic acid in the pseudovirus is wrapped in a protein shell like the natural viral nucleic acid, and can be processed in parallel with the clinical samples, which can more truly reflect the amplification of the viral nucleic acid template in the clinical samples.

[0013] 2) More stable performance

[0014] Compared with traditional detection reagents that use artificially synthesized plasmids as positive control standards, the nucleic acid molecules of pseudovirus standards are wrapped in a protein shell, which can protect the DNA or RNA molecules from degradation, making the nucleic acid more stable and easier to transport and store.

[0015] 3) Avoid false negatives

[0016] Pseudoviruses can be used as internal control standards in nucleic acid detection reagents and participate in the parallel extraction of nucleic acid from clinical samples to monitor the release, separation, purification efficiency, reagent and instrument performance, amplification efficiency, etc. However, artificially synthesized plasmids cannot detect these links by simulating natural viruses.

[0017] 4) Clinical application and promotion are easier

[0018] The quality of positive standards is one of the most important components of test reagents, and whether there are high-performance internal control standards has also become an important indicator for the application and promotion of test kits. Compared with the use of inactivated viruses, pseudoviruses as positive standards and internal control standards also have their unique advantages:

[0019] Using natural viruses as raw materials still has biological infectivity to a certain extent. For example, the international standard material of HIV-1RNA may be prohibited from transportation and use according to the laws of many countries. At the same time, the production of inactivated viruses requires BLS3 or even BLS4 laboratories, and only some professional research institutions have the corresponding conditions. Pseudoviruses are highly safe and can be prepared in conventional BLS2 laboratories, which undoubtedly enables the research and development of nucleic acid detection kits to be carried out more widely.

[0020] Pseudoviruses have played a huge role in basic research and practical applications, but the preservation of pseudoviruses has always been one of the difficulties in pseudovirus applications. Conventional pseudovirus preservation methods usually use buffers and ultra-low temperature preservation. Special buffer formulas are placed in ultra-low temperature environments at -70 or below, and repeated freezing and thawing must be avoided. Such harsh conditions can often only be preserved for 6-12 months, causing great inconvenience to scientific research and practical applications. In addition, ultra-low temperature refrigerators are expensive, consume huge energy, and have very high operating costs. Such low temperature conditions also bring huge challenges to the transportation of pseudoviruses.

[0021] Therefore, it is very necessary to develop a storage solution that can be used at room temperature and effectively stabilize pseudoviruses. Summary of the invention

[0022] In order to overcome the deficiencies in the prior art, the present invention provides a method for stably storing pseudoviruses at room temperature.

[0023] To achieve the above object, the present invention provides a pseudovirus preservation solution, which comprises the following components, wherein each unit of the pseudovirus preservation solution contains:

[0024] BSA 150-200 parts by mass Tween 80 0.5-1.0 volume parts PEG-8000 100-150 parts by mass <![CDATA[Na2HPO4]]> 2.0-3.0 parts by mass <![CDATA[NaH2PO4]]> 2.0-2.5 parts by mass KCl 1.0-1.5 parts by mass Proclin-300 0.1-0.5 volume parts

[0025] The unit of mass parts is gram or its proportional mass unit, the unit of volume parts is milliliter or its proportional volume unit, and the unit of mass-to-volume ratio is g / ml.

[0026] Preferably, the pH of the pseudovirus preservation solution is 7.2-7.6.

[0027] Preferably, water is also included.

[0028] The invention provides a method for preparing a pseudovirus preservation solution, which is characterized by taking a corresponding number of substances, measuring 500-800 mL of purified water, adding the above component reagents, fully stirring until completely dissolved, adjusting the pH value to 7.2-7.6 with dilute hydrochloric acid or 1M NaOH, settling the volume to 1000 mL with purified water, and then filtering and sterilizing with a 0.22 μm filter membrane.

[0029] The invention provides application of the pseudovirus preservation solution, wherein the pseudovirus preservation solution is used for preserving EB pseudovirus.

[0030] The beneficial effects of the present invention are as follows:

[0031] Traditional pseudovirus preservation solutions can usually only be preserved for 1-2 days at 0-37°C, which is different from traditional pseudovirus preservation solutions. Ultra-low temperature freezing requires corresponding equipment and is costly. The pseudovirus preservation solution prepared by the present invention adopts a special formula, and the preservation solution can stably preserve the pseudovirus for 12 months at 0-37°C, greatly extending the storage period of the pseudovirus at 0-37°C, which is more conducive to the transportation of the pseudovirus and related applications in the later stage, and has the advantages of low cost and convenient use. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the expansion curve of formula 1 at month 0;

[0033] Figure 2 This is the expansion curve of formula 1 in the first month;

[0034] Figure 3 This is the expansion curve of formula 2 in the first month;

[0035] Figure 4 This is the expansion curve of formula 3 in the first month;

[0036] Figure 5 This is the expansion curve of formula 1 in the second month;

[0037] Figure 6 This is the expansion curve of formula 1 in the third month;

[0038] Figure 7 This is the expansion curve of formula 1 in the 4th month;

[0039] Figure 8 This is the expansion curve of formula 1 in the 5th month;

[0040] Fig. 9 This is the expansion curve of formula 1 in the 6th month;

[0041] Fig.10 This is the expansion curve of formula 1 in the 7th month;

[0042] Fig.11 This is the expansion curve of formula 1 in the 8th month;

[0043] Fig.12 This is the expansion curve of formula 1 in the 9th month;

[0044] Fig.13 This is the expansion curve of formula 1 at month 10;

[0045] Fig.14 This is the expansion curve of formula 1 in the 11th month;

[0046] Fig.15 This is the expansion curve of formula 1 at month 12;

[0047] Fig.16This is the expansion curve of Formulation 1 at month 13. DETAILED DESCRIPTION

[0048] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present application will be further described below in conjunction with specific embodiments.

[0049] Terminology explanation:

[0050] Fluorescence quantitative PCR: A method that uses fluorescent chemicals to measure the total amount of products after each polymerase chain reaction (PCR) cycle in a nucleic acid amplification reaction. A method that uses internal or external reference methods to quantitatively analyze specific nucleic acid sequences in the sample to be tested.

[0051] Primer: Two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target gene, and the other primer is complementary to the other DNA template strand at the other end of the target gene.

[0052] Probe: A nucleic acid sequence with a known sequence and a detectable label that is complementary to the target gene.

[0053] Pseudovirus: An artificially constructed virus analog. It is a virus particle similar to a real virus and infectious, formed by the capsid protein or envelope protein of the virus encapsulating a heterologous nucleic acid carrying a specific sequence, but the encapsulated nucleic acid does not have the ability to replicate to form the entire nucleic acid sequence of the virus.

[0054] BSA: bovine serum albumin.

[0055] Embodiment 1:

[0056] 1. According to the characteristics of pseudovirus, a pseudovirus preservation solution was prepared, and its formula 1 is as follows:

[0057]

[0058]

[0059] Recipe 2 is as follows:

[0060] Reagent name Mass / Volume <![CDATA[Na2HPO4]]> 3g <![CDATA[NaH2PO4]]> 2.5g NaCl 2g Proclin-300 0.5mL pH 7.2-7.6 Water to volume 1000mL

[0061] Recipe 3 is as follows:

[0062] Reagent name Mass / Volume BSA 50g Tween 80 1mL PEG-8000 50g Tris 10g NaCl 1.5g Proclin-300 0.5mL pH 7.2-7.6 Water to volume 1000mL

[0063] Measure 500-800 mL of purified water, add the above reagent components, stir thoroughly until completely dissolved, adjust the pH value to 7.2-7.6 with dilute hydrochloric acid or 1M NaOH, make up to 1000 mL with purified water, and then filter and sterilize with a 0.22μm filter membrane.

[0064] 2. Use this preservation solution to dilute the pseudovirus to the required concentration and store it at 0-37°C for 12 months.

[0065] 3. Take EB pseudovirus as an example:

[0066] 3.1 Introduction to EB virus

[0067] Epstein-Barr Virus (EBV) is a double-stranded DNA virus belonging to the family Herpesviridae, subfamily γ. After being infected with EBV, people remain latently infected for life, and more than 90% of adults have positive serum EBV antibodies. EBV is an important tumor-associated virus, closely related to the occurrence of various tumors such as nasopharyngeal carcinoma, lymphoma, gastric cancer, and post-transplant lymphoproliferation. Studies have shown that 1% of the world's population is affected by EBV-related tumors. Several tumor diagnosis and treatment guidelines also suggest that detecting the EBV DNA load in patients with nasopharyngeal carcinoma and some lymphoma patients may reflect differences in tumor prognosis and treatment efficacy. Non-neoplastic EBV infection diseases mainly include infectious mononucleosis, chronic active EBV infection, EBV-associated hemophagocytic lymphohistiocytosis and other symptoms.

[0068] 3.2 Preparation of EB virus pseudovirus minimum detection limit reference material

[0069] Epstein-Barr virus pseudovirus was synthesized by Sangon Biotech (Shanghai) Co., Ltd. with a concentration of 1.0E+08 copies / mL. The minimum detection limit of the Epstein-Barr virus nucleic acid detection kit (fluorescent quantitative PCR method) (Jiangsu Merle Biotech Co., Ltd.) is 50 copies / mL. The preservation solution was used to prepare the Epstein-Barr virus pseudovirus to 50 copies / mL as a minimum detection limit reference. The minimum detection limit reference was tested using the Epstein-Barr virus nucleic acid detection kit (fluorescent quantitative PCR method) (Jiangsu Merle Biotech Co., Ltd., National Medical Device Registration No. 20223401598) and repeated 8 times. The test results were used as the data for month 0. The reference was stored in an environment of 33-37°C. The minimum detection limit reference was tested using the Epstein-Barr virus nucleic acid detection kit (fluorescent quantitative PCR method) (Jiangsu Merle Biotech Co., Ltd.) every month. The test results were compared with the data for month 0. If the absolute value of the difference in the Ct values ​​of the two tests was less than 1, it was considered that the minimum detection limit reference was in a stable state until the end of the 13th month.

[0070] 3.3PCR detection:

[0071] 3.3.1 Preparation of PCR reaction solution:

[0072] Reagent name Amount per person (μL) <![CDATA[10×PCR Buffer(Mg 2+ plus)]]> 2.5 dNTPs

including dUTP

[0073] 3.3.2PCR reaction procedure:

[0074]

[0075] 3.4PCR test data statistics:

[0076] Recipe 1 Month 1:

[0077]

[0078] Recipe 2 Month 1:

[0079]

[0080] Recipe 3 Month 1:

[0081]

[0082]

[0083] Recipe 1 for month 2:

[0084]

[0085] Recipe 1 for month 3:

[0086]

[0087] Recipe 1 for month 4:

[0088]

[0089]

[0090] Recipe 1 for month 5:

[0091]

[0092] Recipe 1 for month 6:

[0093]

[0094] Recipe 1 for month 7:

[0095]

[0096]

[0097] Recipe 1 for month 8:

[0098]

[0099] Recipe 1 for month 9:

[0100]

[0101] Recipe 1 for month 10:

[0102]

[0103]

[0104] Recipe 1 for month 11:

[0105]

[0106] Recipe 1 for month 12:

[0107]

[0108] Recipe 1 for month 13:

[0109]

[0110]

[0111] In the EB virus nucleic acid detection kit (fluorescence quantitative PCR method) (Jiangsu Mole Biotechnology Co., Ltd.), the FAM fluorescence channel indicates EB virus, and the VIC fluorescence channel indicates the human internal standard. Since the EB pseudovirus does not contain a human internal standard, the VIC fluorescence channel is NoCt. From the above 13-month test data, it can be seen that the absolute value of the fluctuation of the detection Ct value of the preservation solution of formula 1 from the 1st to the 12th month and the Ct value of the 0th month are both less than 1, while in the 13th month, the absolute value of the fluctuation of the Ct value of some data is greater than 1. Therefore, it can be considered that the reference product of the minimum detection limit of the preservation solution has good stability within 12 months. After more than 12 months, the reference product has a decrease in stability, so the preservation solution can stably preserve the pseudovirus at 0-37°C for 12 months. The Ct of the other two formulas fluctuated greatly in the first month, and the pseudovirus could not be preserved for a long time. The pseudovirus simulates the structure of the natural virus for assembly, and the outer shell is a structural protein that wraps the internal nucleic acid. The stability of the pseudovirus is mainly the stability of the external structural protein. The stability of proteins is mainly related to temperature, freeze-thaw, pH, ionic strength, addition of irrelevant proteins as protective agents, etc. The present invention uses high-concentration BSA as an irrelevant protein protective agent, the optimal ionic strength buffer maintains the optimal pH environment of the protein, and the room temperature is stored to avoid repeated freeze-thaw. The preservative prevents the growth of bacteria in the buffer to destroy the protein structure. After PEG is covalently linked to the protein, it can reduce the thermal shock of the internal groups of the molecule, thereby increasing the thermal stability of the protein. The PEG molecules can also form a protective layer on the surface of the protein, blocking the attack of the inactivation factor and reducing the interaction between the protein and the inactivation factor. Therefore, the factors that are conducive to the stability of the protein are all reflected in the preservation solution of the present invention, and the final experimental results also prove the rationality of the formula of the present invention.

[0112] This preservation solution formula can be used to preserve pseudoviruses at room temperature for 12 months, facilitating basic virology research, vaccine and drug development, etc.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A pseudovirus preservation solution, characterized in that: The pseudovirus preservation solution comprises the following components, each unit of which contains: The unit of mass part is gram or its proportional mass unit, the unit of volume part is milliliter or its proportional volume unit, and the unit of mass-to-volume ratio is g / ml.

2. The pseudovirus preservation solution according to claim 1, characterized in that: The pH of the pseudovirus preservation solution is 7.2-7.

6.

3. The pseudovirus preservation solution according to claim 1, characterized in that: Also includes water.

4. The method for preparing the pseudovirus preservation solution according to claim 1, characterized in that: Take the corresponding number of the substance in claim 1, measure 500-800 mL of purified water, add the above components and reagents, stir thoroughly until completely dissolved, adjust the pH value to 7.2-7.6 with dilute hydrochloric acid or 1M NaOH, make the volume to 1000 mL with purified water, and then filter and sterilize with a 0.22μm filter membrane.

5. The use of the pseudovirus preservation solution according to claim 1, characterized in that: The pseudovirus preservation solution is used to preserve EB pseudovirus.

Images