Method for detecting the titer of a virus

Abstract

The present application provides a method for detecting a virus solid rate. The virus has a capsid and a nucleic acid molecule contained in the capsid, and the method comprises: using a first dye to label the capsid of the virus in a sample containing the virus, and detecting the number of events showing fluorescence positivity after being labeled by the first dye; destroying the capsid of the virus, and then using a second dye to label the nucleic acid molecule, and detecting the number of events showing fluorescence positivity after being labeled by the second dye; and calculating the solid rate of the virus according to the number of events showing fluorescence positivity after being labeled by the first dye and the number of events showing fluorescence positivity after being labeled by the second dye.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to methods for detecting viral solidity and viral empty shell rate. Background Technology

[0002] Viruses with capsids are widely used in the field of gene therapy. Examples of capsid-containing viruses include adeno-associated viruses, lentiviruses, adenoviruses, and poxviruses.

[0003] Adeno-associated virus (AAV) is a member of the helper virus-dependent parvovirus B19 family. It has a particle size of approximately 20 nm, a non-enveloped icosahedral structure, and contains dense single-stranded DNA. Recombinant adeno-associated virus (rAAV) vectors possess advantages such as high expression efficiency, long-term stable expression, low immunogenicity, no reported cases of pathogenicity or tumorigenicity, and potential for large-scale production, making them one of the most promising vectors in gene therapy. According to statistics, more than 100 clinical studies related to rAAV have been recorded worldwide, showing remarkable efficacy, particularly in the treatment of Leber's congenital cataracts and lipoprotein lipase deficiency.

[0004] Quality control technology for adeno-associated virus (AAV) products is the most significant factor restricting their clinical translation and application. Internationally, the Adenovirus Reference Material Working Group (ARMWG) was the first to develop guidelines for standard samples in AAV product quality control. Following this, the U.S. Food and Drug Administration (FDA), the National Institutes of Health Recombinant DNA Advisory Committee (NIH-RAC), and the National Gene Vector Laboratory (FNGCL) jointly established the AAV Reference Standard Working Group (AAVRSWG). Building upon these guidelines, they further advanced the standardization of AAV, particularly issuing guidelines for unified and standardized AAV titer determination and clinical dosage.

[0005] Studies have found that clinical trials require 10 units of virus production. 3 and 10 5 DRP / cell (DNase-resistant particles / cell). The dosage used in clinical trials is typically 2 × 10⁻⁶. 11 DRP / kg, the required batch yield is approximately 10 15DRP. Furthermore, different tissues have different requirements for the required titer of rAAV. In ophthalmic clinical trials, approximately 2 × 10⁻⁶ is sufficient. 8 DRP, however, requires 1×10 when treating muscle or liver diseases. 14 DRP. Therefore, titer control of adeno-associated virus (ARP) products is considered an important factor affecting their clinical treatment efficacy.

[0006] Numerous studies have found that during the preparation and purification of viruses such as rAAV, in addition to amplifying and enriching solid adeno-associated virus (AAV) particles, additional empty-shell AAV particles are inevitably generated. The solidity of the AAV particles directly affects the achievement of AAV titers, as well as the effectiveness and safety of the rAAV vector. Only viral particles containing the complete genome (i.e., the solid portion) can successfully deliver the target gene into the nucleus of the host cell and enable gene expression.

[0007] Currently, the following methods are mainly used to determine the virus, such as the titer of adeno-associated virus products:

[0008] Enzyme-linked immunosorbent assay (ELISA) is commonly used to determine the titer of adeno-associated virus capsid protein. This method involves numerous steps, has a relatively long timeframe, and the reproducibility of the results is not high.

[0009] Optical density (OD) quantifies capsid protein titers and adeno-associated virus (AAV) concentrations by measuring the absorbance of nucleic acids and proteins at 260 nm and 280 nm. This method requires sample concentrations higher than 5 x 10⁻⁶. 11 The detection rate of VG / mL is high, resulting in significant sample consumption, a small OD linear range, and a high coefficient of variation.

[0010] Transmission electron microscopy is a direct tool for measuring the relative number of empty capsids of adeno-associated virus, but this method is time-consuming, the equipment is expensive, which is not conducive to its widespread application, and the statistical representativeness is not high.

[0011] Analytical ultracentrifugation (AUC), combined with ultraviolet absorption or Raleigh interference assays, can accurately characterize various components of adeno-associated virus (AAV), including empty virus particles, solid virus particles, virus particle aggregates, viral fragments, and genomic fragments. However, this method suffers from drawbacks such as high sample consumption, low throughput, and high cost.

[0012] In summary, the field of gene therapy biotechnology still needs to develop detection methods for parameters such as viral solidity and empty shell viral content. Such methods should be efficient, accurate, easy to operate, require small sample amounts, and have good repeatability in detecting solidity. Summary of the Invention

[0013] The inventors improved the accuracy and reproducibility of the detection by labeling the viral capsid and intracapsid nucleic acid and removing free nucleic acid during the labeling step, thus eliminating interference from free nucleic acid. Furthermore, by removing impurities such as cell debris, floating cells, outer vesicles, extracellular proteins, lipids, and salts during the labeling step, background interference was reduced, further enhancing detection accuracy and reproducibility. In particular, the use of fluorescent antibody dyes to label the viral capsid makes the detection method for solidity more convenient and faster.

[0014] This invention provides a method for detecting the solidity of a virus. The virus has a capsid and nucleic acid molecules contained within the capsid. The method includes:

[0015] (1) The capsid of the virus in a sample containing the virus is labeled with a first dye;

[0016] (2) After destroying the capsid of the virus, a second dye is used to label the nucleic acid molecules contained in the capsid of the virus;

[0017] (3) Detect the number of events in a sample containing the virus mixture that show fluorescence positivity after being labeled with the first dye; detect the number of events in a sample containing the virus mixture that show fluorescence positivity after being labeled with the second dye;

[0018] (4) Calculate the solid rate and hollow rate of the virus based on the number of events.

[0019] This invention further provides a method for detecting viral capsid titer and genomic titer. The virus has a capsid and nucleic acid molecules contained within it. The method includes labeling the viral capsid in a sample containing the virus, calculating the viral capsid titer based on the number of fluorescent events showing positive fluorescence after labeling with a first dye, and / or removing free nucleic acid from the sample and destroying the viral capsid, then labeling the nucleic acid molecules contained within the viral capsid in the sample containing the virus with a second dye, and calculating the viral genomic titer based on the number of fluorescent events showing positive fluorescence after labeling with the second dye.

[0020] The method further includes:

[0021] The viral capsid titer is calculated using Equation 1.

[0022] Viral capsid titer = (N1 / V1) * α (Equation 1)

[0023] Wherein, N1 is the number of fluorescence events in diluted sample I; V1 is the flow rate volume of diluted sample I through the single-molecule detection system per unit time.

[0024] On the other hand, the method further includes:

[0025] The viral genome titer was calculated using Equation 2.

[0026] Viral genome titer = 1 × viral nucleic acid concentration = (N2 / V2) * β (Equation 2)

[0027] Wherein, N2 is the number of fluorescence events in diluted sample II; V2 is the flow rate volume of diluted sample II passing through the single-molecule detection system per unit time.

[0028] It is preferable to remove free nucleic acids in the sample before labeling with the second dye, thereby eliminating the interference of free nucleic acids on the detection results of intrashell nucleic acids, resulting in high accuracy and good repeatability.

[0029] Specifically, the present invention includes the following aspects.

[0030] A first aspect of the present invention provides a method for detecting the solidity of a virus, the virus having a capsid and nucleic acid molecules contained within the capsid, the method comprising:

[0031] (1) The capsid of a virus in a sample containing the virus is labeled with a first dye, wherein the first dye binds to surface recognition molecules on the capsid of the virus and emits fluorescence;

[0032] Optionally, after labeling with the first dye, the free dye and free nucleic acid in the sample containing the virus, i.e. the test sample, are removed;

[0033] (2) Destroy the capsid of the virus, and then use a second dye to label the nucleic acid molecules contained in the capsid of the virus, wherein the second dye can specifically bind to the nucleic acid molecules and emit fluorescence;

[0034] Optionally, free nucleic acids in the sample are removed before the capsid of the virus is destroyed;

[0035] (3) The number of fluorescence events in the test sample containing the virus within the flow volume of the detection unit within the detection time that exhibit fluorescence after being labeled with the first dye, and the number of fluorescence events in the test sample containing the virus within the flow volume of the detection unit within the detection time that exhibit fluorescence after being labeled with the second dye.

[0036] Optionally, the test sample containing the virus is purified to remove impurities before labeling;

[0037] Optionally, in step (1), after or before labeling the capsid of the virus in the sample with the first dye, the sample is diluted to the working concentration with a dilution factor α relative to the sample to obtain a labeled diluted sample I, wherein α is greater than or equal to 1; and in step (2), after or before labeling the nucleic acid molecule with the second dye, the sample is diluted to the working concentration with a dilution factor β relative to the sample to obtain a labeled diluted sample II, wherein β is greater than or equal to 1.

[0038] Preferably, in step (3), detecting the number of fluorescence events that exhibit fluorescence after dye labeling includes detecting the number of fluorescence events in the flow volume V of the diluted sample passing through the single-molecule detection system within a unit detection time using a single-molecule detection system.

[0039] (4) Calculate the virus solidity rate in the sample to be tested based on the test results.

[0040] Virus solidity (%) = (Viral genome titer / Viral capsid titer) * 100% Equation 4

[0041] Preferably, the virus solidity rate in the sample to be tested is calculated according to the following formula 3.

[0042] Virus solidity (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100%……Equation 3

[0043] Where N1 is the number of fluorescence events detected in diluted sample I by the single-molecule detection system per unit detection time; V1 is the flow rate volume of diluted sample I through the single-molecule detection system per unit time; α is as defined above;

[0044] N2 is the number of fluorescence events detected in the diluted sample II after being labeled with the second dye by the single-molecule detection system per unit detection time, V2 is the flow rate volume of the diluted sample II through the single-molecule detection system per unit time, and β is as defined above.

[0045] A second aspect of the present invention provides a method for detecting the empty shell rate of a virus, wherein the virus has a capsid and nucleic acid molecules contained within the capsid, the method comprising calculating the virus solidity using the method of the first aspect or detecting the number of fluorescence events A and B using the method of the first aspect, and calculating the empty shell rate of the virus in the sample to be tested according to Formula 5 or Formula 6.

[0046] Virus empty shell rate (%) = 100% - Virus solid shell rate (%) ... Equation 5

[0047] Virus empty shell rate (%) = 100% - ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... Equation 6

[0048] The definitions of the parameters in the second aspect of the present invention are the same as those in the first aspect.

[0049] A third aspect of the present invention provides a method for detecting viral capsid titer, wherein the virus has a capsid and nucleic acid molecules contained within the capsid, the method comprising:

[0050] The capsid of the virus in a sample containing the virus is labeled using a first dye, wherein the first dye specifically binds to surface recognition molecules on the capsid and emits fluorescence, thereby obtaining a test sample labeled with the first dye; after labeling with the first dye, free dye and free nucleic acid are removed from the sample; the number of fluorescence events that show positive fluorescence after labeling with the first dye is detected.

[0051] Prepare a fluorescent microsphere standard containing a known particle concentration; detect the number of fluorescence events in the fluorescent microsphere standard;

[0052] The viral capsid titer is calculated based on the test data.

[0053] The method further includes:

[0054] The viral capsid titer is calculated using Equation 1.

[0055] Viral capsid titer = (N1 / V1) * α (Equation 1)

[0056] Where N1 is the number of fluorescence events in diluted sample I; V1 is the flow rate volume of diluted sample I through the single-molecule detection system per unit time; and α is as defined above.

[0057] A fourth aspect of the present invention provides a method for detecting viral genome titers, wherein the virus has a capsid and nucleic acid molecules contained within the capsid, the method comprising:

[0058] Free nucleic acids in the sample are removed and the capsid of the virus is destroyed. Then, a second dye is used to label the nucleic acid molecules contained in the capsid of the virus. The second dye can specifically bind to the nucleic acid molecules and emit fluorescence to obtain the test sample labeled with the second dye. The number of fluorescent events that show positive fluorescence after being labeled with the second dye is detected.

[0059] Prepare a fluorescent microsphere standard containing a known particle concentration; detect the number of fluorescence events in the fluorescent microsphere standard;

[0060] The viral genome titer is calculated based on the test data.

[0061] The method further includes:

[0062] The viral genome titer was calculated using Equation 2.

[0063] Viral genome titer = 1 × viral nucleic acid concentration = (N2 / V2) * β (Equation 2)

[0064] Where N2 is the number of fluorescence events in diluted sample II; V2 is the flow volume of diluted sample II through the single-molecule detection system per unit time; and β is as defined above.

[0065] In the method of the present invention, the first dye directly conjugates with the surface recognition molecule to emit fluorescence, or the first dye has a recognition group and indirectly conjugates with the surface recognition molecule to emit fluorescence.

[0066] Preferably, the recognition group specifically binds to the surface recognition molecule.

[0067] Preferably, the recognition group is selected from one or more of antibodies or antibody fragments that specifically bind to the surface recognition molecule, antigens, ligands, ligand receptors, or polysaccharides; more preferably, the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule; and even more preferably, the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule of AVV virus.

[0068] The second dye can specifically bind to the nucleic acid molecule, and is preferably a nucleic acid fluorescent dye.

[0069] use

[0070] In the clinical application of gene therapy, accurate viral dosing is crucial. Detection of the viral load can help determine the actual effective viral dose. For example, the viral load must be considered when calculating how much viral agent needs to be injected to achieve the desired therapeutic effect.

[0071] Calculating the dosage based on the total number of viral particles may result in insufficient actual effective dosage due to the presence of empty viral shells, failing to achieve the intended therapeutic goal. A low solidity rate in viral products means fewer viral particles containing the target gene, resulting in fewer viral particles capable of effectively transducing the target gene, significantly reducing the effectiveness of gene therapy.

[0072] Measuring the viral solidity rate using the method of this invention helps optimize conditions to improve the accuracy of viral dosage, enhance product safety, optimize clinical treatment outcomes, and reduce the risk of immune responses. The viral solidity rate detection method of this invention can be used to evaluate viruses, such as the effectiveness of adeno-associated virus products, dosage guidance, and medication safety.

[0073] Advantages of the present invention

[0074] Compared with the prior art, the technical solution provided by the present invention has at least one of the following beneficial technical effects:

[0075] (1) The method provided by the present invention has the advantages of high efficiency, accuracy, easy operation, short time consumption, small sample volume, and good repeatability for detecting the solidity of viruses.

[0076] (2) The present invention removes free nucleic acid in the step of labeling with the first dye and / or the step of labeling with the second dye, thereby eliminating the interference of free nucleic acid on the results and improving the accuracy and reproducibility of detection.

[0077] (3) The present invention removes impurities such as cell debris, floating cells, outer vesicles, miscellaneous proteins, lipids, and salts, as well as free dyes and free nucleic acids in the steps of labeling with the first dye and / or labeling with the second dye, which helps to reduce background interference and improve detection accuracy and reproducibility.

[0078] (4) The detection method for virus solidity and virus empty shell content provided by the present invention has good detection linearity and high accuracy.

[0079] (5) In the method of the present invention, fluorescent antibody dye is preferably used to label the viral capsid, which is simple and quick to operate. Attached Figure Description

[0080] Figure 1 The graph shows the empty shell rate and solid shell rate detected by the AUC method in Comparative Example 1.

[0081] Figure 2 The image shows the detection results of particle groups with PC5 positive signals in the fluorescence channel for the rAAV8 mixture 1e'.

[0082] Figure 3 The image shows the detection results of a group of particles with a positive PC5 signal in the fluorescence channel of the fluorescent microsphere standard solution.

[0083] Figure 4 This is a graph showing the titer of sample 1 (rAAV8) in the standard curve obtained by ELISA.

[0084] Figure 5 The image shows the detection results of particle groups with FITC positive signals in the fluorescence channel of "rAAV2 mixture 3c".

[0085] Figure 6 The image shows the detection results of particle groups with FITC positive signals in the fluorescence channel for the rAAV8 mixture 3c'.

[0086] Figure 7 The image shows the detection results of particle groups with FITC positive signals in the fluorescent standard solution in the fluorescent channel.

[0087] Figure 8 The amplification curves of rAAV2 product 1 and rAAV8 sample 1 using real-time PCR are shown.

[0088] Figure 9 Melting curves of rAAV2 product 1 and rAAV8 sample 1 obtained by real-time PCR.

[0089] Figure 10 This is a data comparison chart for Example 4, Comparative Example 2, Example 5, and Comparative Example 3. The NanoAnalyzer method illustrates the method of the present invention. Detailed Implementation

[0090] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. Through these descriptions, the features and advantages of the present application will become clearer and more apparent.

[0091] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments. Although various aspects of embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless specifically indicated otherwise.

[0092] Furthermore, the technical features involved in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0093] In this invention, "room temperature" refers to the ambient temperature, which can be 20℃-30℃; in some embodiments, it is 22℃-28℃; in some embodiments, it is 24℃-26℃; and in some embodiments, it is 25℃.

[0094] In the foregoing description of this invention, all figures disclosed herein, whether or not the words “approximately” or “about” are used, are approximate values. Based on the disclosed figures, the value of each figure may vary by less than ±10% or by a difference that is considered reasonable by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0095] The terms "optional," "optional," or "optionally" refer to events or situations that may, but are not guaranteed to, occur. For example, "optional surfactant" means that the surfactant may or may not be present.

[0096] The term "wt%" indicates a percentage by mass.

[0097] definition

[0098] The viral capsid usually refers to the viral capsid.

[0099] Viruses, which have a capsid and nucleic acid molecules contained within it, can include lentiviruses, adenoviruses, adeno-associated viruses, poxviruses, etc.

[0100] The term "rAAV" refers to recombinant adeno-associated virus.

[0101] The term "AAV" stands for adeno-associated virus.

[0102] The term "solid virus" refers to a virus that has a capsid and contains nucleic acid molecules inside, including viruses with incomplete nucleic acid and viruses with complete nucleic acid.

[0103] The term "solidity" refers to the percentage of viral particles that have a capsid and contain nucleic acid (including viruses with incomplete nucleic acid (incomplete packaged viruses) and viruses with complete nucleic acid (complete packaged viruses)) out of the total number of viral particles.

[0104] The term "empty capsid ratio" refers to the percentage of viral particles with empty viral capsids (i.e., capsids without a packaged viral genome).

[0105] Viral genome titer refers to the number of copies of the viral genome in a unit volume (e.g., per milliliter) of sample. For example, 10 copies of the viral genome were detected in 1 ml of viral sample. 6 If there are 10 copies of the viral genome, then the viral genome titer of this sample is 10. 6 copies / ml or 10 6 vg / ml.

[0106] Viral capsid titer refers to the total number of viral capsid particles in a unit volume (e.g., per milliliter) of sample, and its unit is, for example, particles / ml. It includes complete viral particles containing the viral genome and empty capsid viral particles without the genome.

[0107] VG / mL (Vector Genomes per mL): The number of viral vector genomes per unit volume, commonly used for titer determination of adeno-associated virus vectors, and is a physical unit.

[0108] GC / mL (Genomic Copies per mL): The number of genome copies of a viral vector per unit volume. It is commonly used for titer determination of adeno-associated virus vectors and is a physical unit.

[0109] VP / mL (Virus Particles per mL): The number of virus particles per unit volume. It can be used to determine the titer of adenovirus vectors and is a physical unit.

[0110] The term "Event Count" refers to the number of particles (or cells) that pass through a laser detection area during the detection process of an instrument (such as flow cytometer). Each particle generates a signal when it passes through the laser, and this signal is recorded by the instrument; this is called an "event." The unit for the event count is usually "counts."

[0111] The term "dilution factor" refers to the ratio of the concentration before dilution to the concentration after dilution.

[0112] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0113] The single-molecule detection system includes an optical system, a photodetector, and a signal acquisition and analysis system. The optical system contains at least one fluorescence channel. The photodetector detects fluorescence signals in the sample and converts these signals into electrical signals. The signal acquisition and analysis system acquires and analyzes these electrical signals.

[0114] The first embodiment of the present invention is a method for detecting the solidity of a virus, wherein the virus has a capsid and nucleic acid molecules contained within the capsid, and the method includes:

[0115] (1) First dye labeling (capsid labeling): Take the first test sample containing the virus and mix it with the first dye. The first dye specifically binds to and labels the surface recognition molecules of the capsid of the virus in the test sample, and the test sample labeled with the first dye is used for detection in step (3).

[0116] Optionally, the sample is diluted to the working concentration for detection by a dilution factor α relative to the sample before or after mixing, to obtain a labeled diluted sample I for detection in step (3), wherein α is greater than or equal to 1.

[0117] Preferably, after the first dye labeling, free dye and free nucleic acid are removed from the sample to be tested. The removal method can be performed using the method described in the "Purification for this Method" section.

[0118] Preferably, the removal of free dye and free nucleic acid is performed using a separation column method. For example, the test sample labeled with the first dye can be passed through a dye and nucleic acid removal centrifuge column, centrifuged, and the eluent collected.

[0119] In one embodiment, the capsid virus type may include: lentivirus, adenovirus, adeno-associated virus, poxvirus, etc., preferably adeno-associated virus.

[0120] The adeno-associated virus can be a natural virus, a recombinant virus, an artificially modified virus such as a viral vector with inserted artificially edited fragments, a viral vaccine, or a viral strain with desired properties selected in cells or microorganisms.

[0121] In one embodiment, the first dye specifically binds to the surface recognition molecule. The surface recognition molecule of the virus is selected from one or more combinations of proteins and carbohydrates. Preferably, the surface recognition molecule of the virus is a protein.

[0122] The first dye directly binds to the surface recognition molecule and emits fluorescence, or the first dye has a recognition group and indirectly binds to the surface recognition molecule and emits fluorescence.

[0123] In some embodiments, the recognition group is selected from one or more of antibodies or antibody fragments that specifically bind to the surface recognition molecule, antigens, ligands, ligand receptors, or polysaccharides; more preferably, the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule; even more preferably, the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule of AVV virus. More preferably, the first dye consists of a recognition group and a fluorescent dye that emits fluorescence.

[0124] The fluorescent dye can be any commonly used fluorescent dye in the art, without particular limitation, including organic fluorescent molecules, fluorescent proteins, nucleic acid dyes, lipid membrane dyes, quantum dots, polymer dots, etc. Organic fluorescent molecules include, but are not limited to: YF488, FITC. Fluorescent proteins include, but are not limited to: phycoerythrin, phycocyanin, red fluorescent protein, green fluorescent protein, mcherry fluorescent protein, etc.

[0125] As the first dye, those described in the "Dyes for Marking - First Dyes" section may be used.

[0126] In one embodiment, the method may include purifying a sample containing the virus to remove impurities before or after labeling the capsid of the virus with a first dye.

[0127] In one embodiment, the method may include removing free dye and free nucleic acid after labeling the capsid of the virus with a first dye.

[0128] As purification methods to remove impurities and free dyes and free nucleic acids, those described in the "Purification for this Method" section may be used;

[0129] (2) Second marker (marker of intracapsular nucleic acid):

[0130] Take a second test sample containing the virus and destroy the capsid of the virus;

[0131] The second test sample is mixed with a second dye, which binds to and labels the nucleic acid molecules contained within the capsid of the virus, resulting in a test sample labeled with the second dye, which is used for detection in step (3).

[0132] Preferably, free nucleic acids in the sample are removed before the viral capsid is destroyed.

[0133] Optionally, after or before labeling the nucleic acid molecules with the second dye, the sample is diluted to the working concentration relative to the sample by a dilution factor β to obtain a labeled diluted sample II for detection in step (3), wherein β is greater than or equal to 1.

[0134] Preferably, purification is performed to remove impurities before the second dye labeling; more preferably, the purification is performed by magnetic separation, such as the SA magnetic bead method.

[0135] The second test sample may be the first test sample, the test sample labeled with the first dye, the diluted sample I mentioned above, or the solution obtained by diluting the first test sample, or the supernatant obtained by purifying the first test sample to remove impurities.

[0136] In one embodiment, the method for breaking the capsid is selected from nucleic acid extraction reagent method or thermal pyrolysis method. The nucleic acid extraction reagent method includes methods based on one or more combinations of pyrolysis reagents, divalent ion reagents, enzyme reagents, detergents, and solid-phase matrices. The heating temperature of the thermal pyrolysis method can be 50℃-130℃, preferably selected from 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃, 100℃, 105℃, 110℃, 115℃, 120℃, 125℃, or 130℃.

[0137] Optionally, the method includes removing the free dye and free nucleic acid after labeling the capsid of the virus in a sample containing the virus with a second dye.

[0138] In one embodiment, the method for removing free nucleic acid is selected from enzymatic digestion and column separation. Methods for removing impurities include those described in section (2) Purification for Removal of Free Dyes and Free Nucleic Acids.

[0139] In one embodiment, the second dye in the method of the present invention is a nucleic acid fluorescent dye.

[0140] In some embodiments, the nucleic acid fluorescent dye includes one or more of cyanine dyes, non-permeable dyes, permeable dyes, intercalation dyes, and DNA double helix minor groove binding dyes. As the second dye, those described in the "Dyes for Labeling - Second Dye" section can be used;

[0141] (3) Detection:

[0142] The number of fluorescence events N1 in the fluorescent particle clusters of the test sample labeled with the first dye or the diluted sample I in a flow volume V1 per unit detection time, and the number of fluorescence events N2 in the fluorescent particle clusters of the test sample labeled with the second dye or the diluted sample II in a flow volume V2 per unit detection time are detected by a single-molecule detection system.

[0143] (4) Calculation:

[0144] The viral solidity rate in the sample to be tested is calculated according to Equation 3.

[0145] Virus solidity (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100%……Equation 3

[0146] Wherein, N1 is the number of fluorescence events detected in the fluorescent particle group of diluted sample I by the single-molecule detection system per unit detection time, V1 is the flow rate volume of diluted sample I through the single-molecule detection system per unit time, N2 is the number of fluorescence events detected in the fluorescent particle group of diluted sample II by the single-molecule detection system per unit detection time, and V2 is the flow rate volume of diluted sample II through the single-molecule detection system per unit time.

[0147] And / or, calculate the viral empty shell rate in the sample to be tested according to Equation 5 or Equation 6:

[0148] Virus empty shell rate (%) = 100% - Virus solid shell rate (%) Equation 5

[0149] Virus empty shell rate (%) = 100% - ((N2 / V2)*β) / ((N1 / V1)*α)*100% (Equation 6)

[0150] The parameters are defined as above.

[0151] In one embodiment, the method further includes a purification step, such as one or more of the following steps:

[0152] a) Before or after labeling the capsid of the virus in the sample containing the virus with the first dye, the sample containing the virus is purified to remove impurities;

[0153] b) After labeling the capsid of the virus in the sample containing the virus with the first dye, remove the free dye and free nucleic acid;

[0154] c) Before labeling the viral capsid in a sample containing the virus with the first dye, purify the sample containing the virus to remove impurities; after labeling with the first dye, remove free dye and free nucleic acid; or

[0155] d) Purify the sample containing the virus to remove impurities before or after destroying the capsid and labeling the nucleic acid molecules contained in the capsid of the virus with a second dye;

[0156] The impurities comprise at least one selected from free dyes and free nucleic acids, and preferably further comprise cell debris. Those described in the "Purification for this Method" section can be used as the above purification method to remove impurities and free dyes and free nucleic acids.

[0157] A second embodiment of the present invention provides a method for detecting viral capsid titer, wherein the virus has a capsid and nucleic acid molecules contained within the capsid, the method comprising:

[0158] As in the first embodiment, the sample is labeled with the first dye, and then the free first dye and free nucleic acid are removed from the sample after labeling with the first dye. The number of fluorescence events that show positive fluorescence after labeling with the first dye is detected to obtain the test sample labeled with the first dye.

[0159] This includes diluting the sample to the working detection concentration using a dilution factor α relative to the sample to obtain diluted sample I.

[0160] Preparation of standards: Prepare concentration standards containing standard fluorescent microspheres with known particle concentrations;

[0161] The concentration standard is diluted θ times to obtain a concentration standard solution;

[0162] The number of fluorescence events M1 in the diluted sample I at a flow rate volume V1 per unit detection time is detected; the number of fluorescence events M3 in the concentration standard solution at a flow rate volume V3 per unit detection time is detected.

[0163] Calculation: Calculate the viral capsid titer according to Formula 7 below.

[0164] Viral capsid titer = C × ((M1 / V1)*α) / ((M3 / V3)*θ) (Equation 7)

[0165] Where C is the particle concentration of the concentration standard solution; M1, V1, M3, V3, α, and θ are as defined above.

[0166] or

[0167] The viral capsid titer is calculated using Equation 1.

[0168] Viral capsid titer = (N1 / V1) * α (Equation 1)

[0169] Where N1 is the number of fluorescence events in diluted sample I; V1 is the flow rate volume of diluted sample I through the single-molecule detection system per unit time; and α is as defined above.

[0170] Regarding the concentration standard solutions and preparation methods, those described in "Detection - Concentration Standards" can be used.

[0171] Unless otherwise specified, the methods of using the virus, the first dye, the second dye, the labeling with the first dye, the labeling with the second dye, the dilution and purification of the sample to be tested are the same as in the first embodiment.

[0172] Another embodiment of the present invention is a method for detecting viral genome titers, wherein the virus has a capsid and nucleic acid molecules contained within the capsid, the method comprising:

[0173] As in the first embodiment, a second dye is applied to remove free nucleic acids from the sample before destroying the viral capsid.

[0174] Before or after labeling the nucleic acid molecules with the second dye, the sample is diluted to the detection working concentration by a dilution factor β relative to the sample to obtain a labeled diluted sample II, wherein β is greater than or equal to 1;

[0175] Preparation of standards: Prepare a concentration standard containing standard fluorescent microspheres with a known particle concentration; dilute the concentration standard by θ times to obtain a concentration standard solution;

[0176] The number of fluorescence events M2 in the diluted sample II at a flow rate volume V2 per unit detection time is detected, and the number of fluorescence events M3 in the concentration standard solution at a flow rate volume V3 per unit detection time is detected.

[0177] Calculation: Calculate the viral genome titer according to Equation 8, or according to Equation 2. Viral genome titer = 1 × viral nucleic acid concentration = C × ((M2 / V2)*β) / ((M3 / V3)*θ)…Equation 8

[0178] Where C is the particle concentration of the concentration standard solution; M2, V2, M3, V3, β and θ are as defined above.

[0179] Viral genome titer = 1 × viral nucleic acid concentration = (N2 / V2) * β (Equation 2)

[0180] Where N2 is the number of fluorescence events in diluted sample II; V2 is the flow volume of diluted sample II through the single-molecule detection system per unit time; and β is as defined above.

[0181] Regarding the concentration standard solutions and preparation methods, those described in "Detection - Concentration Standards" can be used.

[0182] Unless otherwise specified, the procedures for the virus, the first dye, the second dye, the labeling with the first dye, the labeling with the second dye, the dilution and purification of the sample to be tested are the same as in the first embodiment.

[0183] Virus

[0184] The method of this invention is applicable to viruses having a capsid and nucleic acid molecules contained within the capsid. The types of viruses that can be detected by the method of this invention include lentiviruses, adenoviruses, adeno-associated viruses, poxviruses, etc., preferably adeno-associated viruses.

[0185] In some embodiments, the virus in the sample to be tested is adeno-associated virus (AAV). The serotypes of AAV can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ18, AAV-PHPeB, AAV-PHP.S, AAV2-retro, AAV2-QuadYF, AAV2.7m8, etc.

[0186] In some implementations, the dilution of the test sample, such as the diluted sample, the test sample, the quantitative solution, etc., can be achieved using phosphate buffered saline (PBS) as a diluent.

[0187] In some embodiments, the detection working concentration of the virus in the steps of labeling with the first dye and / or labeling with the second dye is independently selected from 1×10⁻⁶. 5 -5×10 9 Genome titer / mL.

[0188] In some embodiments, the detection working concentration in the step of labeling with the first dye and / or the step of labeling with the second dye is independently selected from 1×10⁻⁶. 5 Genome titer / mL, 5×10 5 Genome titer / mL, 1×10 6 Genome titer / mL, 5×10 6 Genome titer / mL, 1×10 7 Genome titer / mL, 5×10 7 Genome titer / mL, 1×108 Genome titer / mL, 5×10 8 Genome titer / mL, 1×10 9 Genome titer / mL or 5×10 9 Genome titer / mL.

[0189] In some implementations, the test sample may come from: process intermediates, formulations, cell cultures, body fluids, tissues, cell lysates, etc., but is not limited to these.

[0190] Dyes for marking

[0191] -First dye

[0192] The first dye in the method of the present invention can specifically bind to the surface recognition molecule of the virus in the sample to be tested, which is a surface recognition molecule, and preferably the first dye is an optical dye.

[0193] In some embodiments, the optical dye comprises fluorescent molecules, fluorescent materials, or combinations thereof. In some embodiments, the optical dye comprises one or more of fluorescent molecules, fluorescent proteins, quantum dots, and polymer dots.

[0194] Preferably, the first dye carries a recognition group, which may include an antibody or antibody fragment, antigen, ligand, ligand receptor, or polysaccharide that specifically binds to a surface recognition molecule of the virus.

[0195] The surface recognition molecules of the virus can be one or more of proteins and sugars, which function as surface recognition molecules.

[0196] The first dye can bind specifically to the surface recognition molecule of the virus in the following ways: the first dye directly binds to the surface recognition molecule of the virus; or the first dye carries a recognition group that indirectly binds to the surface recognition molecule.

[0197] In some embodiments, the surface recognition molecule contains at least one of the functional groups such as hydroxyl, carboxyl, amino, and thiol.

[0198] The recognition group specifically binds to the surface recognition molecule, preferably the recognition group is selected from one or more of antibodies or antibody fragments, antigens, ligands, ligand receptors or polysaccharides; more preferably, the recognition group is an antibody or antibody fragment; even more preferably the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule of the AAV virus.

[0199] In some embodiments, the first dye comprises a fluorescent dye portion and a recognition group portion, preferably the recognition group portion comprising an AAV antibody, which may be a CaptureSelect antibody. TM Biotin anti-AAVX conjugate.

[0200] In one embodiment, the first dye is a fluorescent antibody dye comprising an AAVX antibody and a fluorescent dye portion that emits fluorescence.

[0201] CaptureSelect TM Biotin-anti-AAVX conjugate (ThermoFisher) binds specifically to different AAV serotypes, including AAV1, AAV2, AAV2_HSPG, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, and AAVrh10, with high affinity and selectivity.

[0202] As a preparation of fluorescent antibody dyes, for example, AAVX antibodies can be combined with... It is prepared by mixing optical dyes such as SE.

[0203] The first dye may not contain a recognition group portion. 647A SE is a class of fluorescent dyes with amino-reactive properties. The SE group in this class of dyes can react with amino groups to produce stable amide bonds. In this embodiment, only... 647A SE marks the proteins in the viral capsid, or may... 647ASE was used in combination with antibodies to prepare fluorescent antibody dyes.

[0204] In some preferred embodiments, the recognition group portion is an AAV antibody. In some embodiments, the AAV antibody includes at least one selected from clones ADK1a, A20, A20R, ADK4, ADK5a, ADK5b, ADK6, ADK8, and ADK8 / 9.

[0205] In some embodiments, the adeno-associated virus (AAV) serotype in the test sample is AAV1, and the AAV antibody clone number is ADK1a. In some embodiments, the AAV serotype in the test sample is AAV2, and the AAV antibody clone number is A20, A20R, or a combination thereof. In some embodiments, the AAV serotype in the test sample is AAV3, and the AAV antibody clone number is A20, A20R, or a combination thereof. In some embodiments, the AAV serotype in the test sample is AAV4, and the AAV antibody clone number is ADK4. In some embodiments, the AAV serotype in the test sample is AAV5, and the AAV antibody is selected from clone numbers ADK5a, ADK5b, or a combination thereof. In some embodiments, the AAV serotype in the test sample is AAV6, and the AAV antibody is ADK6. In some embodiments, the adeno-associated virus serotype in the test sample is AAV8, and the AAV antibody is selected from clones ADK8, ADK8 / 9, and combinations thereof. In some embodiments, the adeno-associated virus serotype in the test sample is AAV9, and the AAV antibody is selected from clones ADK9, ADK8 / 9, and combinations thereof.

[0206] The working concentration, incubation temperature, and incubation time of the first dye can be within the range commonly used in the art and are not particularly limited. For example, the working concentration can be 0.01 to 10 μg / μL, the incubation temperature can be in the range of 4°C to 37°C, and the incubation time can be 5 to 60 min.

[0207] In one embodiment, the method of the present invention includes detecting the number of fluorescently positive particles corresponding to a first dye in the sample after destroying the viral capsid. The quantity of undestroyed viral particles in the sample can be determined by the number of fluorescently positive particles corresponding to the first dye.

[0208] When the virus particles bound to the first dye disintegrate after undergoing capsid disruption treatment, the first dye will exhibit non-event-based diffuse fluorescence in the sample solution. Therefore, after capsid disruption treatment, the fluorescence channel for detecting the first dye can be used to detect the luminescence of the sample containing the first dye after capsid disruption treatment. Particles that still exhibit fluorescence corresponding to the first dye are virus particles whose capsids were not successfully disrupted.

[0209] Since the first dye and the second dye occupy different detection channels, the presence of the first dye in the sample does not affect the detection of the fluorescence exhibited by the second dye.

[0210] -Second dye

[0211] The second dye in the method of the present invention can specifically bind to nucleic acid molecules in the sample to be tested, such as nucleic acid molecules contained in the capsid of the virus. Preferably, the second dye is a nucleic acid fluorescent dye.

[0212] In some embodiments, the nucleic acid fluorescent dye includes one or more combinations of cyanine dyes, non-permeable dyes, permeable dyes, intercalating dyes, and DNA double helix minor groove binding dyes.

[0213] In some embodiments, the nucleic acid fluorescent dye includes those selected from Acridine Orange, Actinomycin D, 7-AAD (7-aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodide, BOBO-3 Iodide, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), dihydroethidine, Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), Hexidium Iodide, Hoechst 33258, Pentahydrate (bis-Benzimide), Hoechst 33342, Trihydrochloride.Trihydrate, Trihydrate-FluoroPure Grade, Hoechst 34580, LDS 751, NeuroTrace Blue Fluorescent NisslStain, NeuroTrace Green Fluorescent Nissl Stain, NeuroTrace 530 / 615RedFluorescent Nissl Stain, NeuroTrace Deep-Red Fluorescent Nissl Stain, POPO-1Iodide, POPO-3Iodide, PO-PRO-1Iodide, Propidium Iodide, OliGreen, PicoGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR GreenII, SYBR Safe DNA Gel Stain, SYTO 40, SYTO 41, SYTO 42, SYTO 45, SYTO 9, SYTO 11, SYTO 12, SYTO 13.SYTO 14.SYTO 16.SYTO 21.SYTO 24.SYTO One or more of the following: BC Green, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTO17, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, TO-PRO-1Iodide, TO-PRO-3Iodide, TOTO-1Iodide, TOTO-3Iodide, YO-PRO-1Iodide, YO-PRO-3Iodide, YOYO-1Iodide, YOYO-3Iodide, HCS NuclearMask Deep Red Stain, HCS NuclearMask Blue Stain, HCS NuclearMask Red Stain, and ethidium bromide.

[0214] Preferably, the nucleic acid fluorescent dyes include, but are not limited to: SYTO9, SYTO16, SYTO21, SYTO24, SYTOXGreen, SYTO BC, SYTO RNASelect, and PicoGreen.

[0215] The working concentration, incubation temperature, and incubation time of the nucleic acid fluorescent dye can be within the range commonly used in the art and are not particularly limited. The working concentration can be 0.1 to 10 μM, the working temperature can be in the range of 4°C to 37°C, and the incubation time can be 1 to 60 min.

[0216] Purification used in this method

[0217] This method preferably includes a purification step. The purification steps used in this method may include: 1) purification to separate the target virus from impurities; and 2) purification to remove free dyes and free nucleic acids, which will be described separately below.

[0218] The impurities described in this invention are selected from cell debris, floating cells, extravesicles, miscellaneous proteins, lipids, salts, and combinations thereof.

[0219] By separating impurities such as cell debris, floating cells, outer vesicles, extracellular proteins, lipids, and salts from the target virus, and removing free optical dyes and free nucleic acids, background interference can be reduced, thereby improving detection accuracy and reproducibility.

[0220] 1) Purification of the target virus and impurities

[0221] In this invention, "separation" mainly refers to the purification of the virus from mixtures, liquid mixtures, cell fluids, cell lysates, etc., containing the virus.

[0222] In some embodiments, the step (1) of performing the first dye labeling or the step (2) of performing the second dye labeling includes purification to separate the target virus from impurities.

[0223] The purification methods used in step (1) or step (2) for separating the target virus from impurities can be independently selected from one or more combinations of magnetic separation, centrifugation, layering, precipitation, and solid-liquid separation. The solid-liquid separation method may include chromatography, ultrafiltration, or membrane filtration. The chromatography method may include liquid chromatography. The liquid chromatography method may include one or more combinations of affinity liquid chromatography, ion-exchange liquid chromatography, size exclusion liquid chromatography, hydrophobic liquid chromatography, and reversed-phase liquid chromatography.

[0224] In this invention, magnetic separation is preferred for the specific separation of target viruses and impurities.

[0225] The magnetic separation method may include magnetic bead separation and solid-phase chip separation. Magnetic bead separation may include surface-loaded functional group magnetic bead separation. Surface-loaded functional group magnetic bead separation may include antigen-antibody based magnetic bead separation, ligand-receptor based magnetic bead separation, enzyme-substrate based magnetic bead separation, protein-inhibitor based magnetic bead separation, electron donor-electron acceptor based magnetic bead separation, or structurally complementary magnetic bead separation.

[0226] In some embodiments, the antigen-antibody magnetic bead separation method includes, for example, the following steps: mixing magnetic beads carrying adeno-associated virus antibodies with the sample to be tested, and then adsorbing and separating the magnetic beads carrying adeno-associated virus antibodies by an external magnetic field; or including the following steps: mixing adeno-associated virus antibodies with the sample to be tested, adding modified magnetic beads that can bind to adeno-associated virus antibodies, and then adsorbing and separating the modified magnetic beads by an external magnetic field.

[0227] The antigen-antibody magnetic bead separation method is a separation technology based on antigen-antibody interaction. It achieves efficient separation and purification of target proteins through the specific binding between magnetic beads and target proteins.

[0228] In some embodiments, preferably, the antigen-antibody magnetic bead separation method uses micro-nano-sized magnetic beads for separation. The micro-nano-sized magnetic beads can be any commonly used in the art and are not particularly limited, such as carboxyl magnetic beads, amino magnetic beads, hydroxyl magnetic beads, thiol magnetic beads, toluenesulfonyl magnetic beads, protein A / G magnetic beads, Oligo(dT) magnetic beads, antibody-conjugated magnetic beads, SA magnetic beads, etc.

[0229] The term "titer" refers to the concentration of a specific component (rAAV genome, rAAV capsid, or free nucleic acid) in an rAAV virus suspension, including rAAV genome titer, rAAV capsid titer, or rAAV virus suspension free nucleic acid titer.

[0230] The term "SA magnetic beads" refers to streptavidin magnetic beads. The term "SA magnetic bead method" refers to the process of labeling a target substance (such as adeno-associated virus, protein, nucleic acid, etc.) with biotin (biotin and the target substance can be linked by antibodies and disulfide bonds), then contacting a mixed solution containing the biotin-labeled target substance with SA magnetic beads. Biotin specifically binds to streptavidin, thereby capturing the target substance onto the surface of the magnetic beads. Then, an external magnetic field is used to adsorb the SA magnetic beads, separating the beads with the bound target substance from unbound impurities, thus purifying the target substance.

[0231] A purification method using SA magnetic beads may include the following steps: mixing the test sample with biotin carrying antibodies and disulfide bonds, so that the virus is labeled with biotin carrying antibodies and disulfide bonds, obtaining a biotin-labeled test sample; contacting the obtained test sample with SA magnetic beads, so that biotin specifically binds to streptavidin, and the virus is captured on the surface of the SA magnetic beads; using an external magnetic field to adsorb the SA magnetic beads, separating the SA magnetic beads bound to the target substance.

[0232] In some embodiments, step (1) includes purifying the sample to be tested using the SA magnetic bead method prior to labeling with the first dye. Therefore, step (1) includes:

[0233] The sample to be tested was purified using the SA magnetic bead method to obtain a purified sample containing virus-SA magnetic beads ③, which is the first sample to be tested. The first sample to be tested was mixed with the first dye so that the first dye specifically binds to and labels the surface recognition molecule of the virus as a surface recognition molecule. A disulfide bond breaking reagent was added to break the disulfide bond between the SA magnetic beads ③ and the virus bound thereto. The supernatant A was separated by adsorption using a magnetic rack and used for detection in step (3).

[0234] Optionally, the supernatant A is diluted to the working concentration relative to the sample to be tested by a dilution factor of α (α is greater than or equal to 1) to obtain the diluted sample I for detection in step (3).

[0235] In some embodiments, in step (1) above, after the operation of "mixing the first sample to be tested with the first dye, so that the first dye specifically binds to and labels the surface recognition molecule of the virus as a surface recognition molecule", the operation of "incubation, magnetic rack adsorption, discarding supernatant, and resuspending" is repeated 0-5 times (0 times: i.e., the operation of "incubation, magnetic rack adsorption, discarding supernatant, and resuspending" is not performed).

[0236] In some embodiments, step (2) includes purifying the sample to be tested using the SA magnetic bead method before or after labeling with the second dye. The purification process is identical to that used in step (1) of purifying the sample to be tested using the SA magnetic bead method, except that after adsorption with a magnetic rack, supernatant B is separated instead of supernatant A, and supernatant B is diluted to obtain diluted sample II.

[0237] 2) Purification to remove free dyes and free nucleic acids

[0238] In this invention, "purification to remove free dyes and free nucleic acids" mainly refers to the purification process of removing free dyes and free nucleic acid components other than the virus and the nucleic acid molecules contained within the virus from mixtures, liquids, cell fluids, cell lysates, etc. containing the virus.

[0239] In some embodiments, step (1) further includes: removing the free dye and free nucleic acid after labeling the virus in the sample to be tested with a first dye.

[0240] In some embodiments, step (2) further includes removing the free dye and free nucleic acid before or after labeling the nucleic acid in the adeno-associated virus in the sample to be tested with a nucleic acid fluorescent dye.

[0241] In some embodiments, the method for removing free dye and free nucleic acid in step (1) or step (2) is selected from one or more of the following: ultracentrifugation, density gradient centrifugation, rate gradient centrifugation, ultrafiltration, size exclusion chromatography, dialysis, affinity capture, immunocapture, microfluidics, and dilution.

[0242] In some embodiments, the method for removing free nucleic acids in step (1) or step (2) is selected from enzyme digestion or separation column separation; for example, in separation column separation, a dye and nucleic acid removal centrifuge column are used for separation.

[0243] Examples of dye and nucleic acid removal centrifugation columns include, but are not limited to, the BeyoDesalt G-25 column.

[0244] Methods of breaking the capsid

[0245] In some embodiments, the method for destroying the viral capsid in the method of the present invention includes: nucleic acid extraction reagent method or thermal lysis method.

[0246] - Nucleic acid extraction reagent method

[0247] In some embodiments, the nucleic acid extraction reagent method includes a method based on one or more combinations of lysis reagents, divalent ion reagents, enzyme reagents, detergents, and solid-phase matrices.

[0248] In some embodiments, the pyrolysis reagent includes one or more of the following: hexadecyltriethylammonium bromide (CTAB), sodium dodecyl sulfonate (SDS), guanidine isothiocyanate, phenol, chloroform, isoamyl alcohol, cesium chloride-ethidium bromide (CsCl-EB), alkali, and water.

[0249] In some embodiments, the divalent ionic reagent comprises a transition metal divalent salt or an alkaline earth metal divalent salt. In some embodiments, the transition metal divalent salt is selected from manganese or zinc salts. In some embodiments, the alkaline earth metal divalent salt is selected from magnesium or calcium salts.

[0250] In some embodiments, the enzyme reagent comprises one or more of subtilisin, subtilisin protease, or basic serine protease. In some embodiments, the enzyme reagent further comprises one or more of proteinase K, proteinase R, proteinase T, subtilisin A, Nagarse, subtilisin B, and thermophilic protease.

[0251] In some embodiments, the detergent comprises one or more of the following: cetyltrimethylammonium bromide, Tween-type surfactants, Triton X-100, sodium dodecyl sulfate, nonylphenol polyoxyethylene ether, or polyoxyethylene nonionic detergents.

[0252] In some embodiments, the solid matrix includes magnetic microbeads, silica, diatomaceous earth, chromatography columns, glass particles, anion exchange resins, or a combination of two or more of these components, such as beads, membranes, chips, carbon nanotubes, pore plates, slides, chromatographic matrices, or any combination thereof.

[0253] -Thermal decomposition method

[0254] In some embodiments, the heating temperature of the pyrolysis method is 50℃-130℃. In some embodiments, the heating temperature of the pyrolysis method is 0℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃, 100℃, 105℃, 110℃, 115℃, 120℃, 125℃, or 130℃.

[0255] In some embodiments, the heating time of the pyrolysis method is 1 minute to 60 minutes. In some embodiments, the heating time of the pyrolysis method is 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes.

[0256] Detection

[0257] In some implementations, the sample to be tested is labeled with the first dye and the second dye, and then detected using a single-molecule detection system.

[0258] In some embodiments, the single-molecule detection system includes an optical system, a photodetector, and a signal acquisition and analysis system. The optical system includes at least one fluorescence channel. The photodetector detects fluorescence signals in the sample and converts these signals into electrical signals. The signal acquisition and analysis system is capable of acquiring and analyzing the electrical signals.

[0259] In some embodiments, the optical system includes a light source emitter and at least one fluorescence channel.

[0260] In some embodiments, the signal acquisition and analysis system is capable of acquiring and analyzing electrical signals, and performing calculations based on the electrical signal conversion to obtain the number of fluorescence events in the fluorescent particle swarm.

[0261] In some embodiments, a viral quantitative solution with a known solidity can be labeled using the steps (1) and / or (2) and detected using a single-molecule detection system to locate the number of events of solid viral particles in the sample to be tested.

[0262] FITC and PC5 are fluorescence channels that can be used in the method of this invention. Both channels can be found on corresponding fluorescence channels of commonly used detection instruments in the field, such as the fluorescence channels on the Flow NanoAnalyzer from NanoFCM. Specifically, the FITC fluorescence channel can detect at least the fluorescence of AF488 dye and SYTO BC fluorescent nucleic acid dye; the PC5 fluorescence channel can detect at least the fluorescence of AF647 and YF647 dyes.

[0263] In one implementation, the detection channel parameters can be: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488.

[0264] -Concentration standard solution

[0265] There are no special restrictions on the concentration of fluorescent microspheres in the standard solution. Fluorescent microsphere standards commonly used in virus quality control can be used, such as NBD&Cy5 quality control fluorescent microsphere standards.

[0266] In some embodiments, the concentration of the standard fluorescent microspheres in the concentration standard solution is 1 × 10⁻⁶. 5 -5×10 9 Particles / mL. In some embodiments, the concentration of standard fluorescent microspheres in the concentration standard solution is 1 × 10⁻⁶. 5 Particles / mL, 5×10 5 Particles / mL, 1×10 6 Particles / mL, 5×10 6 Particles / mL, 1×10 7 Particles / mL, 5×10 7 Particles / mL, 1×10 8 Particles / mL, 5×10 8 Particles / mL, 1×10 9 Particles / mL or 5×10 9 Particles / mL.

[0267] The concentration standard solution can be obtained by diluting it with a commonly used diluent, such as phosphate buffered saline (PBS).

[0268] use

[0269] Using adeno-associated virus (AAV) solidity as an example, this illustrates several important uses of viral solidity for product quality and clinical use:

[0270] (1) Viral activity, efficacy and therapeutic effect

[0271] A high solidity rate typically indicates more viral particles with complete structure and function. Fully encapsulated AAVs are more likely to carry the correct therapeutic genes and efficiently deliver and express them after entering target cells. In contrast, a low solidity rate may lead to reduced viral activity, affecting treatment efficacy.

[0272] (2) Purity and safety

[0273] A higher solidity rate helps improve the purity of the product. Impure AAV formulations may contain unassembled viral proteins, empty shells, or other impurities, which may trigger an immune response or have adverse effects on patients.

[0274] An increased solidity rate can reduce the presence of non-functional particles and lower the potential risk of immunogenicity. The immune system may mount an immune response to incomplete or abnormal viral particles, thereby affecting the safety and efficacy of treatment.

[0275] (3) Determination of dosing regimen and dosage

[0276] Accurate determination of heart rate can help optimize dosing regimens and determine appropriate dosages. Understanding heart rate allows for a more accurate assessment of viral activity and efficacy, enabling the development of personalized treatment plans based on the patient's specific situation.

[0277] Therefore, the solidity of the virus detected by the method of the present invention has a significant impact on product quality and clinical use.

[0278] Increasing the core fraction can enhance viral activity and potency, improve product purity and safety, optimize clinical treatment outcomes, reduce the risk of immune responses, and provide a basis for determining dosing regimens and dosages. In the research, development, production, and clinical application of viruses such as AAV, the control and monitoring of the core fraction should be given high priority to ensure product quality and the success of clinical treatment.

[0279] Example

[0280] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0281] The term "M" stands for mol / L, meaning moles per liter. "vg / ml" means genome copies per ml. PBS or PBS buffer means phosphate buffer, which is an aqueous solution containing 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4.

[0282] The term "titer" refers to the concentration of a specific component (rAAV genome, rAAV capsid, or free nucleic acid) in an rAAV virus suspension, including the titer of the rAAV genome, the titer of the rAAV capsid, or the titer of the free nucleic acid in the rAAV virus suspension.

[0283] The term "OD" refers to absorbance, such as "OD230nm" which means absorbance at 230nm.

[0284] Reagents and instruments used in the examples or comparative examples

[0285] 1) Reagents:

[0286] 1.1) Fluorescent labeling reagents

[0287] SE( 647A succinimide ester was purchased from BIORIGIN.

[0288] SYTO BC Green Fluorescent Nucleic Acid Stains were purchased from Invitrogen.

[0289] CaptureSelect TM Biotin-anti-AAVX conjugate antibody: purchased from ThermoFisher. This conjugate exhibits binding activity against AAV serotypes including AAV1, AAV2, AAV2_HSPG, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, and AAVrh10.

[0290] AAV8 antibody: Purchased from Genscript, MonoRab rm AAV8 (intact particle) Antibody (72E8)

[0291] Preparation method of YF647-AAV8 antibody (according to BIORIG)

[0292] 1.2) The operating procedure for the fluorescent antibody dye YF647A, as provided on the official website of IN, is as follows: 《YF647A Antibody Labeling Kit Instruction Manual》

[0293] a. Prepare the antibody in the reaction buffer: Dilute the antibody with 0.1M NaHCO3 solution (pH ~ 8.3).

[0294] b. Transfer the AAV8 antibody (Genscript, MonoRab rm AAV8 (intact particle) Antibody (72E8)) (2.5 mg / mL) to a container containing Place the SE dye in a vial. Mix well and incubate with stirring at room temperature for 1 hour.

[0295] c. Labeled antibodies are separated from the solution using a dialysis column.

[0296] d. Store the labeled antibody in the dark at 2℃-6℃.

[0297] Preparation method of YF647-AAVX antibody (following the operating procedure provided on the BIORIGIN website, "YF647A Antibody Labeling Kit Instruction Manual"):

[0298] a. Prepare the antibody in the reaction buffer: Dilute the antibody with 0.1M NaHCO3 solution (pH ~ 8.3).

[0299] b. AAVX antibody (Capture Select) TM Biotin-anti-AAVX conjugate (2.5 mg / mL) was transferred to a container containing Place the SE dye in a vial. Mix well and incubate with stirring at room temperature for 1 hour.

[0300] c. Labeled antibodies are separated from the solution using a dialysis column.

[0301] d. Store the labeled antibody in the dark at 2℃-6℃.

[0302] 1.3) Other

[0303] EZ-Link TM NHS-SS-Biotin: Biotin disulfide active ester was purchased from ThermoFisher Scientific.

[0304] Biotin-SS-AAV8 antibody: Preparation method of biotin-disulfide-AAV8 antibody (following the operating procedure provided by Thermo Fisher Scientific website "User Guide: EZ-Link NHS-SS-Biotin"):

[0305] a. Before use, prepare a 10mM NHS-SS-Biotin solution.

[0306] b. Add 100 μL of 10 mM NHS SS biotin to each milliliter reaction volume of AF647-AAV8 antibody.

[0307] c. Incubate the reaction at room temperature for 30 minutes to obtain the biotin-disulfide bond-AAV8 antibody.

[0308] 2) Instruments or equipment:

[0309] Dye and nucleic acid removal centrifugation column: BeyoDesalt G-25 column (Superfine, 5ml); Shanghai Beyotime Biotechnology Co., Ltd.

[0310] SA magnetic beads: using Streptavidin Magnetic Beads: MCE, product number: HY-K0208;

[0311] Single-molecule detection system: Flow NanoAnalyzer (manufacturer: NanoFCM Inc);

[0312] The Centrifuge 5810R refrigerated centrifuge was purchased from Eppendorf.

[0313] The Tecnai G2 Spirit transmission electron microscope was purchased from FEI Czech GmbH.

[0314] Comparative Example 1: AUC method for detecting empty shell rate and solid shell rate

[0315] Sample: rAAV8 sample 1 (obtained after dilution of stock solution; stock solution: manufacturer: Paizhen Biotechnology, AAV8 [scAAV.CAG.EGFP.WPRE.SV40pA], product code PGS08001).

[0316] The AUC method is currently considered the standard in the field for evaluating rAAV fill factor and analyzing product-specific impurities. The AUC method separates empty, full, partially filled, and overfilled rAAV capsids from each other based on the size and weight of the rAAV capsid and the sedimentation coefficient specific to the AUC process. AUC can be used to determine the relative quantities of empty capsids, incompletely packaged viruses, fully packaged adeno-associated viruses, and viral aggregates.

[0317] Sample preparation: The OD values ​​of solutions ranging from 0.1 to 1.0 were measured at 260 nm using densitometric methods. The solution could be directly diluted or further concentrated using an Amicon Ultra-0.5 / 30K MWCO centrifuge filter to adjust the rAAV8 sample 1 to a target concentration greater than 5 x 10⁻⁶. 11vg / mL.

[0318] Settlement velocity AUC data acquisition:

[0319] Sedimentation velocity was analyzed using an analytical ultracentrifugation (AUC) instrument with a Beckman Coulter XL-I. 400 μL of sample was loaded into the sample sector of each of the two velocity chambers, and 400 μL of PBS buffer was loaded into the corresponding reference sector. The sample was placed in a four-well rotor and allowed to equilibrate in the instrument until it was maintained at 20 °C and a complete vacuum for 1 hour.

[0320] Sedimentation was performed at a setpoint of 20,000 RPM, 20°C, and a 0.003 cm radius, with no delay and no repeatability. Radial concentration as a function of time was synchronously recorded using absorbance (230 nm) and Raleigh interferometric optics until the smallest sedimenting component passed through the optical window (1.2 h). The assay throughput was limited to a single sample per run, with parameters specified based on absorbance scan collection times greater than 1 minute, as well as the size of the AAV and rapid sedimentation.

[0321] AUC data analysis:

[0322] Solidity was determined by analyzing approximately 75 scans from each detection method using the SEDFIT continuous-size C(S) distribution model. Second-order (2nd) derivative regularization was applied to the fit, with a confidence level of 0.68 for the F-statistic. The following C(S) parameters were kept constant: resolution = 200S, minimum S = 1, maximum S = 200, and friction ratio = 1.0. RI and TI noise reduction were applied, and the meniscus position was allowed to fluctuate, with the optimal position automatically selected by software.

[0323] The model fits the data from the Lamm equation and yields a size distribution that is a "distribution of the settling coefficient," with the area under each peak expressed in units of fringes or OD. 230nm The concentrations expressed in units are proportional. Determine the sedimentation coefficient and relative concentration of each component in the distribution, where the sedimentation coefficient is in Svedberg units and the relative concentration is expressed in OD units.

[0324] Each AUC run is an independent determination, and the following properties are monitored for each analysis: goodness of fit (rmsd), OD of each peak. 230nm The ratio of the interferometric signal (represented by fringes) (A230 / IF ratio), the consistency of the sedimentation coefficient for each type between runs, and the overall quality of the scans are all considered. These properties are examined to ensure the quality of the results.

[0325] Absorbance optics (230 nm): The molar concentration and actual percentage of the intact carrier peak were calculated from absorbance data using extinction coefficients. The molar absorbance extinction coefficients for empty capsids and intact viral particles (intact carriers) were calculated based on publicly available formulas. Extinction coefficients were used for both empty capsids and intact AAV peaks. The C(S) value was determined using the SEDFIT algorithm. The molar concentrations of both intact carriers and empty capsids were calculated using Beer's Law, and the results are presented below. Figure 1 .

[0326] From sample preparation to obtaining test results, the detection time of the method is more than 6 hours.

[0327] Results analysis: Based on these values, the empty shell rate of sample 1 (rAAV8) was calculated to be 2.89%, and the solid shell rate was 97.11%. The AUC method has drawbacks such as long detection time and cumbersome operation.

[0328] Example 1: Detection of virus solidity and empty shell rate ( 647ASE marked cap)

[0329] 647ASE is a class of fluorescent dyes with amino reactivity. The SE group of this class of dyes can react with the amino group to produce a stable amide bond. In this embodiment, 647ASE was used. 647ASE is used to label capsid proteins in the viral capsid.

[0330] Sample: rAAV8 sample 1 (same as comparative example 1, stock solution: manufacturer: Paizhen Biotechnology, AAV8 [scAAV.CAG.EGFP.WPRE.SV40pA], product code PGS08001).

[0331] Step (1):

[0332] Take 100 μL of the rAAV test solution to be labeled (AAV8 sample 1), add 5 μL of 1M NaHCO3 aqueous solution, and mix thoroughly. Then add 7 μL of 10 μg / μL [amount missing] solution. Mix 647A SE thoroughly and react for 30 min to obtain rAAV mixture 1a.

[0333] Take a BeyoDesalt G-25 column, add rAAV mixture 1a, and centrifuge to remove free radicals. 647ASE and free nucleic acids were collected, and the effluent was the rAAV mixture 1b.

[0334] The rAAV mixture 1b was diluted with PBS to a ratio of 62.5 (i.e., 70 times dilution relative to the rAAV test solution to be labeled) to obtain rAAV mixture 1c (i.e., diluted sample I).

[0335] Step (2):

[0336] 1) Dilute part of the aforementioned rAAV mixture 1b to 4.5 times with PBS buffer, and take 100 μl of the diluted rAAV product to obtain rAAV mixture 2a.

[0337] 2) Heat the rAAV mixture 2a at 95°C for 10 minutes to obtain the rAAV DNA sample.

[0338] 3) Take 10 μl of rAAV DNA sample, dilute it 10 times with PBS buffer, add 1 μl of 100 μM SYTO BCGreen Fluorescent Nucleic Acid Stains aqueous solution for labeling (i.e., diluted 50.904 times relative to the rAAV test solution to be labeled), incubate for 5 min to obtain rAAV mixture 2b (i.e., diluted sample II).

[0339] Step (3): Detection

[0340] The rAAV mixture 1c and rAAV mixture 2b were analyzed using the Flow NanoAnalyzer.

[0341] The detection parameters are as follows: laser detector 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488nm; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using the PC5 fluorescence channel. The PC5 fluorescence channel is... The 647 excitation signal is used to characterize the number of capsid events in rAAV and to analyze the presence of... The detection results of particle groups with 647 positive signals in the PC5 fluorescence channel. The fluorescence of SYTO fluorescent nucleic acid dye was detected using the FITC fluorescence channel to characterize the number of nucleic acid events in rAAV. The detection results of particle groups with SYTO positive signals in the FITC fluorescence channel were analyzed and recorded.

[0342] The number of fluorescence events N1 in the fluorescent particle group of mixture 1c within a flow volume V1 per unit detection time and the number of fluorescence events N2 in the fluorescent particle group of mixture 2b within a flow volume V2 per unit detection time were detected according to the above detection parameters; the detection results were recorded.

[0343] Step (4): Calculate:

[0344] The virus solidity in the sample was calculated according to Formula 3 below, and the results are shown in Table 1.

[0345] Virus solidity (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100%……Equation 3;

[0346] Wherein, N1 is the number of fluorescence events in the fluorescent particle group detected in mixture 1c by the single-molecule detection system per unit detection time, V1 is the flow rate volume of mixture 1c through the single-molecule detection system per unit time, α is the dilution factor of mixture 1c relative to the sample to be tested, N2 is the number of fluorescence events in the fluorescent particle group detected in mixture 2b by the single-molecule detection system per unit detection time, V2 is the flow rate volume of mixture 2b through the single-molecule detection system per unit time, and β is the dilution factor of mixture 2b relative to the sample to be tested;

[0347] The viral solidity in the test sample was further calculated using Formula 5, and the results are shown in Table 1:

[0348] Adeno-associated virus empty shell rate (%) = 100% - virus solid shell rate (%) ... Equation 5

[0349] Table 1 shows the solid fraction and hollow fraction calculated according to Equation 1.

[0350]

[0351] The detection time for this method is 1 hour.

[0352] Results analysis: Compared with Comparative Example 1, the detection method provided by the present invention has the same empty shell rate results, and the method of the present invention takes less time and saves more sample, and has excellent and unexpected technical effects.

[0353] Example 2: Detection of viral solidity and empty shell rate (fluorescent antibody dye labeling of capsid)

[0354] Step (1):

[0355] Take 200 μL of the rAAV test solution to be labeled (same as in Example 1), add it to a centrifuge to remove dye and nucleic acid, centrifuge to remove free nucleic acid, collect the effluent liquid to obtain rAAV mixture 1d-1.

[0356] Take 100 μL of rAAV mixture 1d-1, add 20 μL of YF647-AAVX antibody, and incubate for 10 min to make rAAV mixture 1d-2.

[0357] The labeled rAAV mixture 1d-2 was diluted with PBS to a 43.5-fold dilution (i.e., a 52.2-fold dilution relative to the rAAV test solution to be labeled), resulting in rAAV mixture 1e (i.e., diluted sample I).

[0358] Step (2):

[0359] 1) Take 100 μL of the aforementioned rAAV mixture 1d-1, add 20 μL of PBS buffer, and then dilute with PBS buffer to 4.5 times. Take 100 μL of the diluted rAAV product to obtain 2g of rAAV mixture.

[0360] 2) Heat 2g of rAAV mixture at 95℃ for 10 minutes to obtain the rAAV DNA sample.

[0361] Take 10 μl of rAAV DNA sample, dilute it 10 times with PBS buffer, add 1 μl of 100 μM SYTO BC Green Fluorescent Nucleic Acid Stains aqueous solution for labeling (i.e., diluted 50.904 times relative to the rAAV test solution to be labeled), incubate for 5 min to obtain the in-shell nucleic acid labeling solution, and denot it as rAAV mixture 2b-2 (i.e., diluted sample II).

[0362] Step (3): Detection

[0363] The rAAV mixture 1e and rAAV mixture 2b-2 were detected using the Flow NanoAnalyzer.

[0364] The detection parameters are as follows: laser detector 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488nm; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using the PC5 fluorescence channel. The PC5 fluorescence channel is... The 647 excitation signal is used to characterize the number of capsid events in rAAV and to analyze the presence of... The detection results of particle groups with 647 positive signals in the PC5 fluorescence channel were recorded. The fluorescence of the SYTO fluorescent nucleic acid dye was detected using the FITC fluorescence channel to characterize the number of nucleic acid events in rAAV. The detection results of particle groups with SYTO positive signals in the FITC fluorescence channel were analyzed and recorded.

[0365] The number of fluorescence events N1 in the fluorescent particle group of mixture 1e in the flow volume V1 of the mixture within a unit detection time and the number of fluorescence events N2 in the fluorescent particle group of mixture 2b-2 in the flow volume V2 of the mixture within a unit detection time were detected according to the above detection parameters; the detection results were recorded.

[0366] Step (4): Calculate:

[0367] The virus solidity in the sample was calculated according to Formula 3 below, and the results are shown in Table 2.

[0368] Virus solidity (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100%……Equation 3;

[0369] Wherein, N1 is the number of fluorescence events in the fluorescent particle group detected in mixture 1e by the single-molecule detection system per unit detection time, V1 is the flow rate volume of mixture 1e through the single-molecule detection system per unit time, α is the dilution factor of mixture 1e relative to the sample to be tested, N2 is the number of fluorescence events in the fluorescent particle group detected in mixture 2b-2 by the single-molecule detection system per unit detection time, V2 is the flow rate volume of mixture 2b-2 through the single-molecule detection system per unit time, and β is the dilution factor of mixture 2b-2 relative to the sample to be tested;

[0370] Furthermore, the empty shell rate of adeno-associated virus in the sample was calculated according to Equation 5 below, as shown in Table 2:

[0371] Adeno-associated virus empty shell rate (%) = 100% - Virus solid shell rate (%) ... Equation 5

[0372] Table 2: Solid Content Ratio and Empty Content Ratio

[0373] N1 V1 α N2 V2 β solid rate Empty shell rate 5979 30nL 52.2 6840 30nL 50.904 97.1％ 2.9％

[0374] The detection time for this method is 1 hour.

[0375] Results Analysis: The empty shell rate detected in Example 2 was consistent with that in Example 1. This result demonstrates that the method of the present invention, using fluorescent antibody dyes, can also accurately detect the solid shell rate and empty shell rate of the virus.

[0376] Example 3: Detection of rAAV solid fraction and empty fraction in cell lysate

[0377] Step (1)

[0378] 1) Preparation of rAAV simulation samples:

[0379] Dilute the known solid fraction to a genomic titer of 2.6 × 10⁻⁶. 11 A sample of rAAV8 at a concentration of vg / ml (obtained by dilution of the stock solution, the manufacturer and catalog number of the stock solution are the same as in Example 1, but different batch numbers) was mixed with the supernatant of HEK-293T cell lysis buffer to obtain a simulated rAAV sample containing a known solidity of 44.5%. This simulated rAAV sample was then used as the test sample and analyzed as follows.

[0380] 2) Sample purification:

[0381] Take 30 μl 2×10 10SA magnetic beads per ml were added to 500 μl of PBS containing 0.1 wt% BSA (bovine serum albumin), and the beads were magnetically adsorbed for 1 min. The supernatant was discarded. The above steps (adding 500 μl of PBS containing 0.1 wt% BSA, magnetically adsorbing for 1 min, and discarding the supernatant) were repeated 3 times to obtain SA magnetic beads ①.

[0382] The SA magnetic beads ① were resuspended in 200 μl of PBS containing 0.2 wt% BSA, and 10 μl of Biotin-SS-AAV8 antibody was added. The mixture was incubated at 4°C with rotation for 5 min, followed by magnetic adsorption for 1 min. The supernatant was discarded, and the mixture was resuspended in 500 μl of PBS containing 0.2 wt% BSA. This process was repeated three times to obtain the SA magnetic bead mixture ②.

[0383] In the first two repetitions, the volume of PBS containing 0.2 wt% BSA used for resuspension was 500 μl. In the third repetition, after discarding the supernatant, the volume of PBS containing 0.2 wt% BSA used for resuspension was 200 μl.

[0384] The SA magnetic bead mixture ② was divided into two tubes, each containing 100 μl. The following steps were performed on each tube: 100 μl of the SA magnetic bead mixture ② was added to 1 ml of the aforementioned rAAV simulated sample with a known solidity. The mixture was incubated at 4°C with rotation for 8 min, followed by magnetic adsorption for 1 min. The supernatant was discarded, and the mixture was resuspended in 400 μl of PBS. This process was repeated four times to obtain SA magnetic beads ③.

[0385] In the first three repeated operations, the volume of PBS used for resuspension was 400 μl. In the last repeated operation, after discarding the supernatant, the two tubes of SA magnetic beads were combined into one tube, and then the SA magnetic beads were resuspended with 200 μl of PBS.

[0386] 3) Marking of the sample coating:

[0387] Take 50 μl of SA magnetic beads ③, add 20 μl of YF647-AAV8 antibody to the SA magnetic beads ③, and incubate at room temperature for 8 min. Adsorb on a magnetic rack for 1 min, discard the supernatant, resuspend in 200 μl of PBS, and repeat the above steps (incubate at room temperature for 8 min, adsorb on a magnetic rack for 1 min, discard the supernatant, resuspend in PBS) 3 times.

[0388] In the first two repetitions, 200 μl of PBS was used for resuspension. In the final repetition, after discarding the supernatant, 50 μl of PBS was used for resuspension, and 4 μl of 2.5 M DTT (disulfide bond cleaving reagent) was added. The mixture was reacted at room temperature for 1.5 h to obtain reaction solution A. The supernatant of reaction solution A was obtained by magnetic adsorption. A portion of the supernatant of reaction solution A was diluted 43.5 times with PBS to obtain mixture 1f (i.e., diluted sample I).

[0389] Step (2)

[0390] Labeling of nucleic acids within the sample shell:

[0391] A portion of the supernatant of reaction solution A was used to replace rAAV mixture 1d-2 in step (2) of Example 2. The remaining operations were the same as in step (2) of Example 2, and the in-shell nucleic acid labeling solution was obtained, which was denoted as rAAV mixture 4b (i.e., diluted sample II).

[0392] Step (3)

[0393] Sample testing: Mixture 1f and rAAV mixture 4b were tested according to the testing parameters in step (3) of Example 2.

[0394] Step (4) Calculation:

[0395] The virus solidity rate in the sample to be tested is calculated according to Equation 3.

[0396] Virus solidity (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100%……Equation 3;

[0397] Wherein, N1 is the number of fluorescence events in the fluorescent particle group detected in the mixture 1f by the single-molecule detection system per unit detection time, V1 is the flow rate volume of the mixture 1f through the single-molecule detection system per unit time, α is the dilution factor of the mixture 1f relative to the sample to be tested, N2 is the number of fluorescence events in the fluorescent particle group detected in the mixture 4b by the single-molecule detection system per unit detection time, V2 is the flow rate volume of the mixture 4b through the single-molecule detection system per unit time, and β is the dilution factor of the mixture 4b relative to the sample to be tested;

[0398] Furthermore, the viral solidity in the sample is calculated using the following formula 5:

[0399] Adeno-associated virus empty shell rate (%) = 100% - Virus solid shell rate (%) ……………………Equation 5

[0400] Table 3. Solid content and empty shell content

[0401] N1 V1 α N2 V2 β solid rate Empty shell rate 4969 30nL 70 8095 30nL 50.904 44.6％ 55.4％

[0402] Results analysis: For a simulated rAAV sample with a known solidity of 44.5%, the solidity measured by the method of the present invention was 44.6%, indicating that the method of the present invention can effectively measure the solidity of the virus in cell lysate.

[0403] Comparative Example 2: Detection of viral capsid titer using ELISA method

[0404] Sample to be tested: rAAV8 sample 1 (same as in Example 1). Follow the instructions for the AAV8 Titration ELISA kit (Progen, Cat. No. PRAAV8):

[0405] 1) Bring all reagent kits (unless otherwise specified, from the kits described above) to room temperature;

[0406] 2) Solution preparation: Place reagent ASSB (Assay Buffer, from the kit) in a 37°C water bath and dilute with ultrapure water to 1×ASSB (20-fold dilution); dilute reagent Kit control with 750 μL of 1×ASSB, incubate at room temperature for 5 min with continuous mixing to prepare reagent KC dilutions; serially dilute KC dilutions with 1×ASSB to obtain 7 concentrations of standard controls; dilute reagent biotin conc with 750 μL of 1×ASSB, incubate at room temperature for 5 min with continuous mixing to prepare reagent 1×biotin conc; dilute reagent strep-HRP with ASSB at a ratio of 1:20 to prepare reagent 1×strep-HRP before use; dilute rAAV8 sample 1 1000-fold with 1×ASSB to prepare diluted rAAV8 sample 1.

[0407] 3) Add 100 μl of KC dilutions at 7 different concentrations and 100 μl of diluted rAAV8 quantitative standard sample to a 96-well plate, incubate at 37°C for 1 h, and then wash 3 times with 200 μl of 1×ASSB.

[0408] 4) Add 100 μl of 1×biotin to a 96-well plate, incubate at 37°C for 1 h, and then wash 3 times with 200 μl of 1×ASSB.

[0409] 5) Add 100 μl of 1×strep-HRP to a 96-well plate, incubate at 37°C for 1 h, and then wash 3 times with 200 μl of 1×ASSB.

[0410] 6) Add 100 μl of reagent TMB and incubate at room temperature for 15 min;

[0411] 7) Add 100 μl of reagent stop solution, and read the OD values ​​at 650 nm and 450 nm respectively. The final OD reading is calculated as the OD value at 650 nm minus the OD value at 450 nm.

[0412] 8) Positive quantitative determination:

[0413] Based on the seven concentrations of standard controls obtained in step 2) of this comparative example, a standard curve was plotted (see...). Figure 4 The results of the diluted rAAV8 sample 1 were then substituted into the data for quantitative determination.

[0414] By substituting into the standard curve, the coating titer of the diluted rAAV8 sample 1, as measured by the ELISA method, was 4.6 × 10⁻⁶. 8 vp / mL (see vp / mL) Figure 4 The final viral capsid titer of rAAV8 sample 1 was determined to be 4.6 × 10⁻⁶. 11 vp / mL.

[0415] Example 4: Detection of viral capsid titer using fluorescent antibody labeling

[0416] Sample to be tested: rAAV8 sample 1 (same as Example 1)

[0417] (i) The capsid of rAAV8 sample 1 was labeled with a fluorescent antibody:

[0418] Take 100 μL of rAAV8 sample 1 (same as in Example 1) as the test sample, add 20 μL of YF647-AAVX antibody, incubate for 10 min, and obtain rAAV8 mixture 1d'. Dilute rAAV8 mixture 1d' to 10 times with PBS buffer to obtain rAAV8 mixture 1e'. rAAV8 mixture 1e' is a total dilution of 120 times relative to rAAV8 sample 1.

[0419] The concentration was 2.14 × 10 10 The NBD&Cy5 quality control fluorescent microsphere standard (manufacturer NanoFCM, catalog number QS2503) was diluted 100 times to obtain the concentration standard solution f.

[0420] (ii) The rAAV8 mixture 1e' and concentration standard solution f were detected using a Flow NanoAnalyzer with a laser detector of 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kpa; signal type: small signal, using the PC5 fluorescence channel.

[0421] The PC5 fluorescence channel, using the Alexa Fluor 647 excitation signal, was used to characterize the number of rAAV8 particles. The detection results of particle groups with PC5 positive signals in the PC5 fluorescence channel were analyzed and recorded.

[0422] (iii) Capsid titer calculation

[0423] The particle count of the rAAV8 mixture 1e' was calculated to be 5893 particles after 2 minutes of measurement. The particle count of the concentration standard solution was calculated to be 6743 after 2 minutes of measurement. The results are shown in... Figure 2 , Figure 3 .

[0424] Based on the dilution factor, concentration standard curve, and the detection results of rAAV8 mixture 1e', the capsid titer of rAAV8 sample 1 was calculated using the following formula.

[0425] Viral capsid titer = C × ((M1 / V1)*α) / ((M3 / V3)*θ)……Equation 7

[0426] Where C is the particle concentration of the concentration standard solution; M1, V1, M3, V3, α, and θ are as defined above.

[0427] Viral capsid titer = 2.14 × 10⁻⁶ 10 ×5893×120 / 6743×100

[0428] The calculated capsid titer of rAAV8 sample 1 was 2.24 × 10⁻⁶. 11 vp / mL.

[0429] Discussion: The capsid titer of AAV8 sample 1 obtained by the method in Example 4 was lower than that obtained by the ELISA method in Comparative Example 2, confirming that the traditional ELISA method overestimates the capsid titer of AAV samples. This suggests that the ELISA method may be responsible for the non-specific detection of non-capsid-related proteins and viral fragments in the sample.

[0430] Furthermore, the standard error of the method provided in Example 4 of this invention for detecting the capsid titer of AAV8 sample 1 is significantly lower than that of the ELISA method provided in Comparative Example 2 (see Example 2). Figure 10 The method provided in Embodiment 4 of this invention uses a specific antibody to bind to the AAV capsid protein, avoiding non-specific binding of non-capsid impurities in the AAV sample, thereby improving the signal-to-noise ratio and accuracy of the detection method.

[0431] Comparative Example 3: Real-Time PCR Method for Detecting the Genomic Titer of rAAV2 and rAAV8 Viruses

[0432] Sample: rAAV2 product 1 (obtained by dilution of stock solution, stock solution: manufacturer: Paizhen Biotechnology, AAV2 [SCAAV.CAG.EGFP.WPRE SV40pA], serotype: AAV2, product code PGS02001, total amount: 5E+12GC, titer: 1E+13GC / mL).

[0433] Sample: rAAV8 sample 1 (same as Example 1).

[0434] according to Follow the instructions in the Titration Kit (for Real Time PCR) Ver.2 kit (Takara, 6233) manual:

[0435] 1) Take 2 μl each of rAAV2 product 1 and rAAV8 sample 1 (same as in Example 1), add 1 μl of DNase I, 2 μl of 10×DNase I buffer, and 15 μl of deionized water to prepare mixtures v1 and v2 respectively. React at 37°C for at least 15 minutes, and then heat-treat mixtures v1 and v2 at 95°C for 10 minutes to inactivate DNase I. The purpose is to remove free nucleic acids and surface-adsorbed nucleic acids, and to release the nucleic acid material encapsulated in the rAAV2 product capsid.

[0436] 2) Add equal amounts of Lysis Buffer (from the kit) to 20 μl each of mixtures v1 and v2. Heat treat at 70°C for 10 minutes to obtain AAV 2 genome extraction solution a and AAV 8 genome extraction solution b.

[0437] 3) Dilution

[0438] The obtained AAV 2 genome extract a and AAV 8 genome extract b were diluted 100-fold using EASY Dilution (from the kit) for later use.

[0439] Take the positive control stock solution (known genomic titer is 2×10). 10 vg / ml (from the same kit as above), serially diluted using EASYDilution (same kit as above) to 6 gradients: 2 × 10 10 vg / ml (undiluted positive control stock solution), 2×10 9 vg / ml, 2×10 8 vg / ml, 2×10 7 vg / ml, 2×10 6 vg / ml, 2×10 5 vg / ml.

[0440] Take 5 μl of each of the above serially diluted solutions as templates and perform Real-Time PCR. The data obtained from the positive control will be used to plot the standard curve.

[0441] 4) Prepare 50×Primer mix solution

[0442] A 50X Primer mix solution was prepared according to the following components: 5 μl AAV Forward Titer Primer, 5 μl AAV Reverse Titer Primer, and 15 μl deionized water.

[0443] 5) Prepare the PCR reaction solution (per tube) according to the following composition: 12.5 μl of TB Green Premix Ex Taq 11 (2×conc.), 0.5 μl of 50×Primer mix, 0.5 μl of ROX Reference Dye, and 6.5 μl of deionized water;

[0444] 6) Add 5 μl of 6 gradient positive controls, AAV 2 genome extract a diluted 100-fold, or AAV 8 genome extract b diluted 100-fold to each reaction tube, mix well, and place on a Real-Time PCR instrument.

[0445] 7) PCR reaction conditions: pre-denaturation at 95℃ for 2 min, 2-step PCR reaction for a total of 35 cycles, cycling conditions: 95℃ for 5 seconds; 60℃ for 30 seconds. Then perform fluorescence detection: FAM, and perform melting curve analysis after the reaction.

[0446] 8) Quantitative determination of positive results:

[0447] Based on the positive control data from the six gradients in step 3) above, a standard curve for the positive controls was plotted. The detection results from AAV 2 genome extract a diluted 100-fold and AAV 8 genome extract b diluted 100-fold were substituted into the calculation, and the results are as follows: Figure 8 As shown.

[0448] Result: As Figure 8 As shown, the Ct value obtained by detecting AAV 2 genome extract a diluted 100-fold was 17.7 ± 0.064, and the Ct value obtained by detecting AAV 8 genome extract b diluted 100-fold was 17.2 ± 0.086. Based on the standard curve, the genome titer of AAV 2 genome extract a diluted 100-fold was calculated to be 1.04 × 10⁻⁶. 8 The genomic titer of AAV 8 genomic extract b, diluted 100 times with vg / ml, was 1.53 × 10⁻⁶. 8 vg / ml.

[0449] After dilution conversion, the final measured genomic titer of rAAV2 product 1 was 2.08 × 10⁻⁶. 11 The genomic titer of rAAV8 sample 1 was 3.16 × 10⁻⁶ vg / ml. 11 vg / ml.

[0450] Analysis: The peak positions of the melting curves of rAAV2 product 1, rAAV8 sample 1, and the positive control are consistent, indicating that there is no nonspecific amplification and the quantitative results are accurate (see...). Figure 9 ).

[0451] Example 5: Detection of rAAV2 and rAAV8 viral genome titers using fluorescent labeling

[0452] rAAV2 Product 1: Same as Example 4.

[0453] rAAV8 Sample 1: Same as Example 1.

[0454] Enzymatic digestion method for removing nucleic acids: Take 100 μL of the sample to be tested, add 10 μL of 1M NaHCO3 aqueous solution, mix thoroughly, and let stand at room temperature for 5 min. Then add 2 μL of 10 μg / μL Alexa Fluor 6475-TFP, mix thoroughly, and react at room temperature in the dark for 15-30 min. Centrifuge at 100,000g for 80 min at 4℃, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 100,000g for 20 min at 4℃, discard the supernatant, and resuspend the precipitate in 10mM PBS buffer containing 100 μL of 3 μM SYTO BC Green Fluorescent Nucleic Acid Stains aqueous solution to obtain rAAV mixture 3A. Take 50 μl of rAAV mixture 3A and follow the instructions for Recombinant DNase I (RNase-free, Takara, 2270): add 10 μl of 10×DNase I Buffer, 4 μl of Recombinant DNase I (RNase-free), and 36 μl of water sequentially. Incubate at 37°C for 30 minutes to obtain rAAV mixture 3C.

[0455] Following the above method for removing nucleic acids, rAAV2 product 1 was used as the test sample to remove the free nucleic acids of rAAV2 product 1, resulting in rAAV2 mixture 3c'; rAAV8 sample 1 was used as the test sample to remove the free nucleic acids of rAAV8 sample 1, resulting in rAAV8 mixture 3c”.

[0456] Following the method of obtaining rAAV mixture 2b from rAAV mixture 1b in step 2 of Example 1, nucleic acid labeling was performed on rAAV2 mixture 3c' and rAAV8 mixture 3c” to obtain rAAV2 mixture 2b' and rAAV8 mixture 2b”;

[0457] The concentration is C = 2.19 × 10 10 The quality control fluorescent microsphere standard (particles / mL) was diluted 50 times to obtain the concentration standard solution f';

[0458] The rAAV2 mixture 2b', rAAV8 mixture 2b”, and concentration standard solution f' were detected using the Flow NanoAnalyzer.

[0459] Laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal. Under these conditions, the FITC fluorescence channel was used. The FITC fluorescence channel was excited by SYTO BC Green Fluorescent Nucleic Acid Stains to count the number of nucleic acid particles contained within the capsid of rAAV2 product 1. The detection results of particle groups with FITC positive signals in the fluorescence channel were analyzed. The detection results were recorded. Figure 5 , Figure 6 , Figure 7 ).

[0460] The number of particles in the rAAV2 mixture 2b' was calculated, and the result was 2098 particles in 2 minutes of measurement.

[0461] The number of particles in the rAAV8 mixture 2b” was calculated, and the result was 3390 particles in 2 minutes of measurement.

[0462] The particle count of the concentration standard solution was 7423 after 2 minutes of measurement.

[0463] calculate:

[0464] The nucleic acid concentration of rAAV2 product 1 was calculated using the following formula based on the dilution factor, concentration standard curve, and nucleic acid detection results of rAAV2 mixture 2b' and rAAV8 mixture 2b".

[0465] Viral genome titer = 1 × viral nucleic acid concentration = C × ((M2 / V2)*β) / ((M3 / V3)*θ)... Equation 8

[0466] Where C is the particle concentration of the concentration standard solution; M2, V2, M3, V3, β and θ are as defined above.

[0467] The nucleic acid concentration of rAAV2 product 1 is 2.19 × 10⁻⁶. 10 *(2098 / 7423)*(dilution factor of rAAV2 mixture 2b' / 50)

[0468] The nucleic acid concentration of rAAV8 sample 1 was calculated using the same formula.

[0469] Results: The nucleic acid concentration of rAAV2 product 1 was 6.25 × 10⁻⁶. 10 Particles / mL. The nucleic acid concentration of rAAV8 sample 1 was 1.01 × 10⁻⁶. 11 Particles / mL.

[0470] Since each FITC-positive signal particle detected in the Flow NanoAnalyzer corresponds to one viral genome copy and is equal to one event, the genomic titer of rAAV2 product 1 measured in this embodiment is 1 × rAAV2 product 1 nucleic acid concentration = 6.25 × 10⁻⁶. 10 vg / ml,

[0471] Similarly, the genomic titer of rAAV8 sample 1 was calculated to be 1.01 × 10⁻⁶. 11 vg / ml.

[0472] analyze:

[0473] The genomic titer of AAV8 sample 1 detected by the method of Example 5 of the present invention is lower than that detected by the method of Comparative Example 3.

[0474] Results suggest that traditional quantitative PCR methods can lead to overestimation of AAV genomic titers because they can non-specifically amplify all non-AAV nucleic acid targets in the sample that can bind to the detection primers. The method of this invention can eliminate the interference of non-AAV nucleic acid targets in the sample on the results, thereby avoiding overestimation of AAV genomic titers.

[0475] discuss

[0476] like Figure 10 As shown, the capsid titer of AAV8 sample 1 in Example 4 was compared with the genome titer of AAV8 sample 1 in Example 5. The results showed that the measured capsid titer (particles / ml) was higher than the genome titer (vg / ml).

[0477] Compared with the genome titer of AAV8 sample 1 in Comparative Example 2, the capsid titer of AAV8 sample 1 in Comparative Example 3 showed that the measured capsid titer (particles / ml) was higher than the genome titer (vg / ml).

[0478] Compared with the AAV2 product 1 genome titer of Comparative Example 3, the capsid titer of AAV2 product 1 in Example 5 showed that the measured sample capsid titer (particles / ml) was higher than the genome titer (vg / ml).

[0479] The detected capsid titers were all higher than the genome titers, indicating the presence of empty capsid AAVs in the tested samples.

[0480] Comparative Example 4: Without removing free nucleic acids and free dyes

[0481] The preparation and testing were carried out according to the operation method of Example 1, the difference being that in the addition After 647ASE, centrifuge column is not used to remove free radicals. 647A SE and free nucleic acid were used, and the remaining procedures were the same as in Example 1, with four parallel tests performed. The results are shown in Table 4.

[0482] Table 4. Detection results (n=4)

[0483]

[0484] SD (Standard Deviation): A statistical metric used to measure the dispersion of data. It represents the degree to which each value in a dataset deviates from the mean of the dataset.

[0485] Result: Without removing free... In the case of 647A SE and free nucleic acid, solid rate and empty shell rate cannot be detected.

[0486] In summary, based on the results of the above embodiments and comparative examples, the detection method provided by the present invention has good accuracy, repeatability, simple operation, short time consumption, small sample volume, and high detection efficiency, avoiding overestimation of solidity, and has excellent and unexpected technical effects.

[0487] Example 6: Virus content in incomplete packaging

[0488] Adeno-associated virus (AAV) aggregates refer to cluster structures formed when multiple AAV virus particles aggregate together through physical or chemical interactions during the preparation, purification, or storage of AAV virus particles.

[0489] The solidity rate of AAV virus detected and calculated by this invention, combined with the determination of the full-shell rate of virus and the content of AAV aggregates, can be used to calculate the content of incompletely packaged virus using the following formula 9:

[0490] Incomplete viral packaging viral content % = Virus solidity % - Virus full shell % - Virus aggregate content % ... Equation 9.

[0491] The present application has been described above with reference to preferred embodiments; however, these embodiments are merely exemplary and illustrative. Various substitutions and modifications can be made to the present application based on these embodiments, all of which fall within the protection scope of the present application.

Claims

1. A method for detecting the solidity of a virus, wherein the virus has a capsid and nucleic acid molecules contained within the capsid, the method comprising: (1) The capsid of a virus in a sample containing the virus is labeled with a first dye, wherein the first dye specifically binds to surface recognition molecules of the capsid and emits fluorescence; (2) Destroy the capsid of the virus, and then use a second dye to label the nucleic acid molecules contained in the capsid of the virus, wherein the second dye can specifically bind to the nucleic acid molecules and emit fluorescence; (3) Detect the number of fluorescent events that show positive fluorescence after being labeled with the first dye, and detect the number of fluorescent events that show positive fluorescence after being labeled with the second dye; The viral solidity rate was calculated based on the number of fluorescent events that showed positive fluorescence after being labeled with the first dye and the number of fluorescent events that showed positive fluorescence after being labeled with the second dye.

2. The method according to claim 1, wherein the method comprises: In step (1), after or before labeling the capsid of the virus in the sample with the first dye, the sample is diluted to the detection working concentration by a dilution factor α relative to the sample to obtain a labeled diluted sample I, wherein α is greater than or equal to 1. and In step (2), after or before labeling the nucleic acid molecules with the second dye, the sample is diluted to the detection working concentration by a dilution factor β relative to the sample to obtain a labeled diluted sample II, wherein β is greater than or equal to 1.

3. The method according to claim 1 or 2, wherein in step (3), detecting the number of fluorescent events that are fluorescently positive after being labeled with dye includes detecting the number of fluorescent events in the flow volume V of the diluted sample passing through the single-molecule detection system within a unit detection time using a single-molecule detection system.

4. The method according to claim 3, wherein the single-molecule detection system comprises an optical system, a photodetector, and a signal acquisition and analysis system. The optical system includes at least one fluorescence channel; the photodetector is used to detect fluorescence signals in the sample and convert the fluorescence signals into electrical signals; the signal acquisition and analysis system is capable of acquiring and analyzing electrical signals.

5. The method according to claim 3, wherein the method further comprises: The viral capsid titer is calculated using Equation 1. Viral capsid titer = (N1 / V1) * α (Equation 1) Wherein, N1 is the number of fluorescence events in diluted sample I; V1 is the flow rate volume of diluted sample I through the single-molecule detection system per unit time.

6. The method according to claim 3, wherein the method further comprises: The viral genome titer was calculated using Equation 2. Viral genome titer = 1 × viral nucleic acid concentration = (N2 / V2) * β (Equation 2) Wherein, N2 is the number of fluorescence events in diluted sample II; V2 is the flow rate volume of diluted sample II passing through the single-molecule detection system per unit time.

7. The method according to claim 3, wherein the method further comprises: The solidity of the virus is calculated using Equation 3. Virus solidity (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% Equation 3 Where N1 is the number of fluorescence events in diluted sample I; V1 is the flow rate volume of diluted sample I through the single-molecule detection system per unit time; N2 is the number of fluorescence events in diluted sample II; V2 is the flow rate volume of diluted sample II passing through the single-molecule detection system per unit time.

8. The method according to claim 1 or 2, wherein the method comprises one or more of the following steps: a) The sample containing the virus is purified to remove impurities before or after labeling with the first dye; b) After labeling with the first dye, remove the free dye and free nucleic acid from the sample containing the virus; c) Before labeling with the first dye, the sample containing the virus is purified to remove impurities; after labeling with the first dye, free dye and free nucleic acid are removed; and d) Purify the sample containing the virus to remove impurities before or after labeling with the second dye; in, The impurities are selected from cell debris, floating cells, external vesicles, miscellaneous proteins, lipids, salts, and combinations thereof.

9. The method according to claim 8, wherein the method comprises step a) and step b): a) The sample containing the virus is purified to remove impurities before or after labeling with the first dye; b) After labeling with the first dye, the free dye and free nucleic acid are removed from the sample containing the virus.

10. The method according to claim 1 or 2, wherein, The viruses are selected from adeno-associated viruses, lentiviruses, adenoviruses, and poxviruses.

11. The method according to claim 10, wherein the virus is an adeno-associated virus.

12. The method according to claim 1 or 2, wherein, The first dye directly conjugates with the surface recognition molecule and emits fluorescence; or the first dye has a recognition group and indirectly conjugates with the surface recognition molecule and emits fluorescence. The recognition group specifically binds to the surface recognition molecule.

13. The method according to claim 12, wherein the recognition group is selected from one or more of an antibody or antibody fragment, an antigen, a ligand, a ligand receptor, or a polysaccharide.

14. The method according to claim 13, wherein the recognition group is an antibody or an antibody fragment.

15. The method of claim 14, wherein the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule of the adeno-associated virus.

16. The method according to claim 12, wherein the first dye comprises a recognition group and a fluorescent dye.

17. The method according to claim 1 or 2, wherein, The second dye is a nucleic acid fluorescent dye.

18. The method according to claim 17, wherein, The second dye is selected from one or more of cyanine dyes, non-permeable dyes, permeable dyes, intercalating dyes, and DNA double helix minor groove binding dyes.

19. The method according to claim 1 or 2, wherein, The method for destroying the capsid is selected from nucleic acid extraction reagent method or thermal pyrolysis method.

20. The method according to claim 19, wherein, The heating temperature of the pyrolysis method is 50℃-130℃, and the heating time of the pyrolysis method is 1 minute to 60 minutes.

21. The method according to claim 20, wherein, The heating temperature is selected from 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃, 100℃, 105℃, 110℃, 115℃, 120℃, 125℃ or 130℃.

22. The method according to claim 20, wherein, The heating time for the pyrolysis method is selected from 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes.

23. The method according to claim 19, wherein the nucleic acid extraction reagent method comprises a method based on one or more combinations of lysis reagents, divalent ion reagents, enzyme reagents, detergents, and solid-phase matrices.

24. The method according to claim 8, wherein, In different steps, the method for removing free dye and free nucleic acid is independently selected from one or more combinations of: ultracentrifugation, density gradient centrifugation, rate gradient centrifugation, ultrafiltration, size exclusion chromatography, dialysis, affinity capture, immunocapture, microfluidics, and dilution; or Methods for removing free nucleic acids include: enzyme digestion and separation column separation.

25. The method according to claim 24, wherein, The method for removing free nucleic acids is column separation.

26. The method according to claim 8, wherein, In different steps, the purification method for removing impurities is independently selected from one or more combinations of magnetic separation, centrifugation, layering, precipitation, and solid-liquid separation.

27. The method according to claim 26, wherein, In different steps, the purification method for removing impurities is magnetic separation.

28. The method according to claim 26, wherein, The magnetic separation method is selected from one or more of the following: magnetic bead separation method and solid-state chip separation method. The magnetic bead separation method includes the magnetic bead separation method with surface-loaded functional groups; The surface-loaded functional group magnetic bead separation method is selected from one or more of the following: antigen-antibody magnetic bead separation method, ligand-receptor magnetic bead separation method, enzyme-substrate magnetic bead separation method, protein-inhibitor magnetic bead separation method, electron donor-electron acceptor magnetic bead separation method, or structurally complementary magnetic bead separation method.

29. The method of claim 28, wherein, The antigen-antibody magnetic bead separation method includes: After mixing magnetic beads carrying viral antibodies with a sample containing the virus, the magnetic beads carrying viral antibodies are adsorbed and separated by an external magnetic field; or After mixing the viral antibody with a sample containing the virus, modified magnetic beads that can bind to the viral antibody are added, and the modified magnetic beads are adsorbed and separated by an external magnetic field.

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