A method for stable storage of pseudovirus at room temperature

Abstract

The application provides a method for stable storage of pseudo-virus at room temperature, a preservative solution prepared by using BSA, Tween 80, PEG-8000, Na2HPO4, NaH2PO4, KCl and Proclin-300, 500-800 mL of purified water is taken, the above reagents are added, fully stirred until completely dissolved, the pH value is adjusted to 7.2-7.6 by using dilute hydrochloric acid or 1M NaOH, the purified water is diluted to 1000 mL, and then 0.22 mu m filter membrane is used for filtering and sterilizing, so that the pseudo-virus can be stably stored in the preservative solution at 0-37 DEG C for 12 months, the storage period of the pseudo-virus at 0-37 DEG C is greatly prolonged, the transportation and the later related application of the pseudo-virus are more favorable, and the method has the advantages of low cost and convenient use.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of virus storage processing, in particular to a method for stable storage of pseudovirus at room temperature. BACKGROUND

[0002] Pseudovirus, also known as "pseudo virus", is a kind of chimeric virus particle, which is a chimeric virus particle expressing a recombinant glycoprotein of another virus on the surface of a replication-defective virus (viral vector). Its gene is usually changed or modified, and it is a virus-like particle with virus-mimicking physical structure and specific nucleic acid sequence. Its analysis characteristics are similar to those of real virus, but it does not have the ability of self-replication and infection, and can participate in the whole process from extraction to amplification of virus detection. It has biological safety and can be used as a measurement standard for verification and evaluation of virus nucleic acid qualitative and quantitative measurement methods, as well as quality control of laboratories and detection of neutralizing antibodies of vaccines and drugs.

[0003] The application field of pseudovirus is very wide, mainly including the following aspects:

[0004] 1. Gene expression regulation:

[0005] Pseudovirus has the same infection ability as real virus, can perform gene delivery, and realize the regulation of target genes in cells and animal organs - although the virus cannot replicate to produce new viruses after infecting cells, but the expression unit carried by it can be transcribed and translated in cells using the expression system of cells, so as to realize the regulation of gene expression.

[0006] 2. Antibody research:

[0007] By constructing a pseudovirus with a missing part of the amino acid of the viral protein, it is found that the protein truncation of the pseudovirus can improve the ability of the virus to enter the cell, and the virus is suitable for developing a pseudovirus neutralization test method.

[0008] 3. Nucleic acid diagnostic standard:

[0009] The development of molecular diagnosis is changing rapidly, and with the deepening understanding of pathogen molecular test projects such as viral infectious diseases and women's health in clinical practice, the detection demand is becoming more and more diverse. Not only the qualitative and typing detection of pathogen nucleic acid is needed, but also the accuracy and precision of pathogen ribonucleic acid quantification are required.

[0010] The application of pseudovirus in the detection field is mainly as a positive control standard and internal control standard in ribonucleic acid detection kit. The latest CFDA declaration guidance principle clearly proposes to use pseudovirus as a positive standard and internal control standard. Compared with artificial synthetic plasmid containing target gene, the pseudovirus as a positive standard and internal control standard has the following advantages:

[0011] 1) Amplification curve is more true

[0012] In PCR reaction, the amplification efficiency of different forms of gene templates in the reaction is inconsistent. Artificially synthesized plasmids are much simpler than gene templates in clinical samples, and have higher amplification efficiency than clinical samples. The nucleic acid in the pseudovirus is wrapped by the protein shell, just like the natural viral nucleic acid. Parallel processing with clinical samples can more truly reflect the amplification of viral nucleic acid templates in clinical samples.

[0013] 2) Performance is more stable

[0014] Compared with the traditional detection reagent using artificially synthesized plasmid as positive control standard, the nucleic acid molecules of the pseudovirus standard are wrapped by the protein shell, which can protect the DNA or RNA molecules from degradation, making the nucleic acid more stable and convenient for transportation and storage.

[0015] 3) Avoid false negative

[0016] Pseudovirus can be used as an internal control standard in nucleic acid detection reagents, participating in the parallel extraction of clinical sample nucleic acid, to monitor the sample nucleic acid release, separation, purification efficiency, reagent and instrument performance, amplification efficiency and other links. Artificially synthesized plasmid cannot simulate natural virus to detect these links.

[0017] 4) Clinical declaration and promotion are easier

[0018] The quality of positive standard is one of the most important components of detection reagent, and whether it has high-performance internal control standard has become an important indicator for declaration and promotion of detection kit. Compared with inactivated virus, pseudovirus as positive standard and internal control standard also has its unique advantages:

[0019] Using natural virus as raw material, it still has biological infectiousness to some extent. For example, HIV-1 RNA international standard material may be prohibited for transportation and use according to the laws of many countries. At the same time, the preparation of inactivated virus requires BLS3 or even BLS4 laboratory, only a part of professional research institutions have the corresponding conditions, while pseudovirus is safe, and can be prepared in a conventional BLS2 laboratory, which undoubtedly makes the research and development of nucleic acid detection kit more widely carried out.

[0020] Pseudovirus plays a huge role in basic research and practical application, but the preservation of pseudovirus has been one of the difficulties in the application of pseudovirus. The conventional pseudovirus preservation method usually adopts the way of buffer plus ultra-low temperature preservation, and the special buffer formula is placed in the ultra-low temperature environment of-70 or below, and repeated freezing and thawing is also avoided. Such harsh conditions can only be preserved for 6-12 months, which causes great inconvenience to scientific research and practical application. Moreover, the ultra-low temperature refrigerator equipment is expensive, the energy consumption is huge, and the use cost is very high. Moreover, such low temperature conditions bring great challenges to the transportation of pseudovirus.

[0021] Therefore, it is very necessary to develop a pseudovirus preservation solution that can be used at room temperature and effectively stabilize the pseudovirus. SUMMARY

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

[0023] To achieve the above purpose, the present application provides a pseudovirus preservation solution, which comprises the following components, and each unit of the pseudovirus preservation solution,

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

[0025] Among them, the unit of mass fraction is gram or its equal proportion change mass unit, the unit of volume fraction is milliliter or its equal proportion change volume unit, and the unit of mass volume ratio is g / ml.

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

[0027] Preferably, it also includes water.

[0028] The present application provides a preparation method of the pseudovirus preservation solution, characterized in that: taking the corresponding parts of the substances, taking 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 by using dilute hydrochloric acid or 1M NaOH, and then diluting the purified water to 1000 mL, and then filtering and sterilizing by using 0.22 mu m filter membrane.

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

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

[0031] The conventional pseudovirus preservative solution can be stored for 1-2 days at 0-37 DEG C, and the ultra-low temperature freezing preservation needs corresponding equipment and is high in cost. The pseudovirus preservative solution prepared by the special formula can stably store the pseudovirus for 12 months at 0-37 DEG C, greatly prolongs the storage period of the pseudovirus at 0-37 DEG C, is more beneficial to the transportation and later application of the pseudovirus, and has the advantages of low cost and convenient use. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is the amplification curve of formula 1 in the 0th month;

[0033] Figure 2 is the amplification curve of formula 1 in the 1st month;

[0034] Figure 3 is the amplification curve of formula 2 in the 1st month;

[0035] Figure 4 is the amplification curve of formula 3 in the 1st month;

[0036] Figure 5 is the amplification curve of formula 1 in the 2nd month;

[0037] Figure 6 is the amplification curve of formula 1 in the 3rd month;

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

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

[0040] Figure 9 is the amplification curve of formula 1 in the 6th month;

[0041] Figure 10 is the amplification curve of formula 1 in the 7th month;

[0042] Figure 11 is the amplification curve of formula 1 in the 8th month;

[0043] Figure 12 is the amplification curve of formula 1 in the 9th month;

[0044] Figure 13 is the amplification curve of formula 1 in the 10th month;

[0045] Figure 14 is the amplification curve of formula 1 in the 11th month;

[0046] Figure 15 is the amplification curve of formula 1 in the 12th month;

[0047] Figure 16The amplification curve for Formula 1 at month 13. Detailed Implementation

[0048] To better illustrate the purpose, technical solution, and advantages of this invention, the following will provide further explanation of this application in conjunction with specific embodiments.

[0049] Terminology Explanation:

[0050] Quantitative real-time PCR (qPCR): A method for measuring the total amount of product after each polymerase chain reaction (PCR) cycle using fluorescent chemicals in nucleic acid amplification reactions. It involves quantitative analysis of specific nucleic acid sequences in the sample using internal or external controls.

[0051] Primers: Two artificially synthesized oligonucleotide sequences. One primer is complementary to one 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 that is complementary to the target gene, with a known sequence and a detection marker.

[0053] Pseudoviruses: Artificially constructed virus analogs. These are viral particles that resemble real viruses and are infectious, formed by the encapsulation of specific sequences of heterologous nucleic acids by viral capsid or envelope proteins. However, the encapsulated nucleic acid lacks the ability to replicate and form the complete nucleic acid sequence of a virus.

[0054] BSA: Bovine serum albumin.

[0055] Example 1:

[0056] 1. Based on the characteristics of pseudoviruses, a pseudovirus preservation solution is prepared, and its formula 1 is as follows:

[0057]

[0058]

[0059] Formula 2 is as follows:

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

[0061] Formula 3 is as follows:

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

[0063] Measure 500-800 mL of purified water, add the above-mentioned reagents, stir thoroughly until completely dissolved, adjust the pH to 7.2-7.6 with dilute hydrochloric acid or 1M NaOH, bring the purified water to a final volume of 1000 mL, and then filter through a 0.22 μm filter membrane for sterilization.

[0064] 2. The pseudovirus can be diluted to the required concentration using this preservation solution and stored at 0–37°C for 12 months.

[0065] 3. Taking the 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 Herpesviridae family, Gamma subfamily. Humans infected with EBV remain latent for life, and approximately 90% of adults test positive for EBV antibodies. EBV is an important tumor-associated virus, closely related to the development of various cancers, including nasopharyngeal carcinoma, lymphoma, gastric cancer, and post-transplant lymphoproliferative disorder. Studies show that up to 1% of the world's population is affected by EBV-related cancers. Several cancer treatment guidelines also suggest that detecting EBV DNA load in nasopharyngeal carcinoma patients and some lymphoma patients may reflect differences in tumor prognosis and treatment efficacy. Non-tumor-related EBV infections mainly include infectious mononucleosis, chronic active EBV infection, and EBV-associated hemophagocytic lymphohistiocytosis.

[0068] 3.2 Preparation of reference material for the lowest detection limit of EB virus pseudovirus

[0069] EB virus pseudovirus was synthesized by Sangon Biotech (Shanghai) Co., Ltd. at a concentration of 1.0E+08 copies / mL. The limit of detection (LOD) of the EB virus nucleic acid detection kit (quantitative PCR method) (Jiangsu Mole Biotechnology Co., Ltd.) is 50 copies / mL. Using this preservation solution, the EB virus pseudovirus was reduced to 50 copies / mL to prepare a LOD reference sample. The LOD reference sample was tested using the EB virus nucleic acid detection kit (quantitative PCR method) (Jiangsu Mole Biotechnology Co., Ltd., National Medical Device Registration Certificate No. 20223401598), repeated 8 times. The test results were used as the data for month 0. The reference sample was stored at 33-37℃. Every month, the LOD reference sample was tested using the EB virus nucleic acid detection kit (quantitative PCR method) (Jiangsu Mole Biotechnology Co., Ltd.). The test results were compared with the data for month 0. The absolute value of the difference between the two Ct values ​​was less than 1, indicating that the LOD reference sample was in a stable state, until the end of month 13.

[0070] 3.3 PCR detection:

[0071] 3.3.1 Preparation of PCR reaction solution:

[0072] Reagent name Amount per person (μL) 10x PCR Buffer (Mg 2+ plus) 2.5 dNTPs

with dUTP

[0073] 3.3.2 PCR reaction procedure:

[0074]

[0075] 3.4 PCR detection data statistics:

[0076] Formula 1, Month 1:

[0077]

[0078] Formula 2, Month 1:

[0079]

[0080] Formula 3, Month 1:

[0081]

[0082]

[0083] Formula 1, Month 2:

[0084]

[0085] Formula 1, Month 3:

[0086]

[0087] Formula 1, 4th month:

[0088]

[0089]

[0090] Formula 1, Month 5:

[0091]

[0092] Formula 1, Month 6:

[0093]

[0094] Formula 1, 7th month:

[0095]

[0096]

[0097] Formula 1, Month 8:

[0098]

[0099] Formula 1, Month 9:

[0100]

[0101] Formula 1, 10th month:

[0102]

[0103]

[0104] Formula 1, 11th month:

[0105]

[0106] Formula 1, 12th month:

[0107]

[0108] Formula 1, 13th month:

[0109]

[0110]

[0111] In the EB virus nucleic acid detection kit (quantitative real-time PCR method) (Jiangsu Mole Biotechnology Co., Ltd.), the FAM fluorescent channel indicates EB virus, and the VIC fluorescent channel indicates human internal standard. Since EB pseudovirus does not contain human internal standard, the VIC fluorescent channel is NoCt. From the above 13 months of detection data, it can be seen that the absolute value of the Ct value fluctuation in the preservation solution of Formula 1 from month 1 to 12 is less than 1, while in month 13, some data show an absolute value of Ct value fluctuation greater than 1. Therefore, it can be considered that the reference standard of the lowest detection limit of this preservation solution has good stability within 12 months. After 12 months, the stability of the reference standard decreased. Therefore, this preservation solution can stably preserve pseudovirus for 12 months at 0–37℃. The other two formulas showed significant fluctuations in Ct in month 1 and cannot preserve pseudovirus for a long time. Pseudoviruses are assembled by mimicking the structure of natural viruses. The outer shell is a structural protein that encapsulates the internal nucleic acid. The stability of pseudoviruses is mainly due to the stability of the external structural protein. Protein stability is mainly affected by temperature, freeze-thaw cycles, pH, ionic strength, and the addition of irrelevant proteins as preservatives. This invention uses a high concentration of BSA as an irrelevant protein preservative, an optimal ionic strength buffer to maintain the optimal pH environment for the protein, room temperature storage to avoid repeated freeze-thaw cycles, a preservative to prevent bacterial growth in the buffer and damage to the protein structure, and PEG, after covalently linking to the protein, to reduce thermal vibrations of internal molecular groups, thereby increasing the protein's thermal stability. PEG molecules can also form a protective layer on the protein surface, blocking the attack of inactivating factors and reducing the interaction between the protein and these factors. Therefore, factors beneficial to protein stability are all reflected in the preservation solution of this invention, and the final experimental results also prove the rationality of the formulation of this invention.

[0112] This preservation solution formula can preserve pseudoviruses for 12 months at room temperature, 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 solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A pseudovirus preservation solution, characterized in that: The following components are included in each liter of the pseudovirus preservation solution:

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: It also includes water.

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

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

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