Virus real heart rate detection method

Abstract

The invention provides a virus real heart rate detection method. The virus has a capsid and a nucleic acid molecule contained in the capsid, and the detection method comprises the following steps: marking the capsid of the virus in a sample containing the virus by using a first dye, and detecting the number of fluorescent positive events marked by the first dye; destroying the capsid of the virus, then marking the nucleic acid molecule by using a second dye, and detecting the number of fluorescent positive events marked by the second dye; and calculating the real heart rate of the virus according to the number of the events showing fluorescence positive after being marked by the first dye and the number of the events showing fluorescence positive after being marked by the second dye.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a method for detecting virus solid rate and virus empty shell rate. Background Art

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

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

[0004] The quality control technology of adeno-associated virus products is the most important factor restricting their clinical transformation and application. Internationally, in terms of the quality control of adeno-associated virus products, the Adenovirus Reference Material Working Group (ARMWG) first developed a standard sample guide. After that, the U.S. Food and Drug Administration (FDA), the National Institute of Health Recombinant DNA Advisory Committee (NIH-RAC), and the National Gene Vector Laboratory (FNGCL) jointly established the AAV Reference Standard Working Group (AAVRSWG). Based on this guideline, the standardization of AAV was further promoted, especially for the unified and standardized AAV titer determination and clinical dosage usage standards.

[0005] The study found that the requirement for virus production in clinical trials is 10 3 and 10 5 DRP / cell (DNase-Resistant Particles / cell). The dosage in clinical trials is generally 2×10 11 DRP / kg, the required single batch output is about 10 15DRP. Different tissues have different requirements for the titer of rAAV. In ophthalmic clinical trials, only about 2×10 8 DRP, while the treatment of muscle or liver diseases requires 1×10 14 Therefore, the titer control of adeno-associated virus products is considered to be an important factor affecting their clinical therapeutic effects.

[0006] Numerous studies have found that during the preparation and purification of viruses such as rAAV, in addition to amplifying and enriching solid AAV particles, additional empty AAV particles are inevitably produced. The solid fraction of AAV directly affects the titer of AAV and the effectiveness and safety of rAAV vectors. Only viral particles containing a complete genome (i.e., the solid fraction) can successfully deliver the target gene to the nucleus of the host cell and express it.

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

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

[0009] Optical density (OD) is a quantitative method for measuring the absorption of nucleic acids and proteins at 260 nm and 280 nm to quantify the titer of capsid protein and the content ratio of adeno-associated virus. This method requires a sample concentration higher than 5x10 11 VG / mL, the detection consumes a lot of sample, and the OD linear range is small and the coefficient of variation is high.

[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 and the equipment is expensive, which is not conducive to large-scale promotion and application, and the statistical representativeness is not high.

[0011] Analytical ultracentrifugation (AUC) combined with ultraviolet absorption or Raleigh interference assays can accurately characterize the 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 high sample consumption, low detection throughput, and high cost.

[0012] In summary, the field of gene therapy biotechnology still needs to develop a method for detecting parameters such as the virus solid rate and empty shell virus content. This method can detect the solid rate efficiently, accurately, easily, with small sample volume and good repeatability. Summary of the Invention

[0013] The inventors label the viral capsid and the nucleic acid within it, and remove free nucleic acid during the labeling step, eliminating interference from free nucleic acid on the results and improving the accuracy and reproducibility of the test. By removing impurities such as cell debris, floating cells, exosomes, miscellaneous proteins, lipids, and salts during the labeling step, background interference is reduced, improving the accuracy and reproducibility of the test. In particular, labeling the viral capsid with a fluorescent antibody dye makes the solidity rate detection method simpler and faster to operate.

[0014] The present invention provides a method for detecting the solidity rate of a virus. The virus has a capsid and a nucleic acid molecule contained within the capsid, and the method comprises:

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

[0016] (2) after destroying the viral capsid, labeling the nucleic acid molecule contained in the viral capsid with a second dye;

[0017] (3) detecting the number of fluorescence-positive events in the sample containing the virus mixture after being labeled with the first dye; detecting the number of fluorescence-positive events in the sample containing the virus mixture 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] The present invention further provides a method for detecting viral capsid titer and genomic titer. The virus has a capsid and a nucleic acid molecule contained within the capsid. The method comprises labeling the viral capsid in a sample containing the virus, calculating the viral capsid titer based on the number of fluorescence events that exhibit 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 molecule 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 fluorescence events that exhibit positive fluorescence after labeling with the second dye.

[0020] The method further comprises:

[0021] The viral capsid titer was calculated by formula 1,

[0022] Virus capsid titer = (N1 / V1)*α Formula 1,

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

[0024] In another aspect, the method further comprises:

[0025] The viral genome titer was calculated by formula 2:

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

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

[0028] Preferably, free nucleic acids in the sample are removed before labeling with the second dye, thereby eliminating the interference of free nucleic acids on the detection results of intracapsid nucleic acids, and the obtained results are highly accurate and reproducible.

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

[0030] In a first aspect of the present invention, a method for detecting the solidity rate of a virus is provided, wherein the virus has a capsid and a nucleic acid molecule contained within the capsid, the method comprising:

[0031] (1) labeling the capsid of the virus in the sample containing the virus with a first dye, wherein the first dye binds to a surface recognition molecule of the capsid of the virus and emits fluorescence;

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

[0033] (2) destroying the viral capsid, and then labeling the nucleic acid molecule contained in the viral capsid with a second dye, wherein the second dye can specifically bind to the nucleic acid molecule and emit fluorescence;

[0034] Optionally, removing free nucleic acid from the sample before disrupting the viral capsid;

[0035] (3) detecting the number of fluorescence events in which the sample to be tested containing the virus is labeled with the first dye and exhibits fluorescence within the flow volume per unit detection time, and detecting the number of fluorescence events in which the sample to be tested containing the virus is labeled with the second dye and exhibits fluorescence within the flow volume per unit detection time;

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

[0037] Optionally, in step (1), after or before labeling the viral capsid in the sample with a first dye, the sample is diluted to a detection working concentration at 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 a second dye, the sample is diluted to a detection working concentration at 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 exhibiting fluorescence after dye labeling comprises detecting the number of fluorescence events in a flow volume V of the diluted sample passing through the single molecule detection system within a unit detection time through the single molecule detection system using a single molecule detection system.

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

[0040] Virus solid rate (%) = (viral genome titer / viral capsid titer) * 100% Formula 4

[0041] The virus solidification rate in the sample to be tested is preferably calculated according to the following formula 3.

[0042] Virus solid rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... Formula 3

[0043] Wherein, N1 is the number of fluorescence events detected in the diluted sample I by the single molecule detection system within a unit detection time; V1 is the flow volume of the diluted sample I passing through the single molecule detection system within a unit time; α is as defined above;

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

[0045] The 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 a nucleic acid molecule contained inside the capsid. The method comprises calculating the empty shell rate of the virus in the sample to be tested according to Formula 5 or Formula 6 using the virus solid core rate calculated by the method of the first aspect or the number of fluorescence events A and the number of fluorescence events B detected by the method of the first aspect.

[0046] Virus empty shell ratio (%) = 100% - virus solid core ratio (%) ……………… Formula 5

[0047] Virus empty shell rate (%) = 100% - ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... Formula 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] In a third aspect of the present invention, a method for detecting the titer of a viral capsid is provided, wherein the virus has a capsid and a nucleic acid molecule contained within the capsid, the method comprising:

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

[0051] preparing a fluorescent microsphere standard containing a known particle concentration; detecting the number of fluorescent events in the fluorescent microsphere standard;

[0052] The viral capsid titer was calculated based on the detection data.

[0053] The method further comprises:

[0054] The viral capsid titer was calculated by formula 1,

[0055] Virus capsid titer = (N1 / V1)*α Formula 1,

[0056] Wherein, N1 is the number of fluorescence events in the diluted sample I; V1 is the flow volume of the 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 the titer of a viral genome, wherein the virus has a capsid and a nucleic acid molecule contained within the capsid, the method comprising:

[0058] removing free nucleic acids from the sample and destroying the viral capsid, then labeling the nucleic acid molecules contained within the viral capsid with a second dye, wherein the second dye is capable of specifically binding to the nucleic acid molecules and emitting fluorescence, thereby obtaining a test sample labeled with the second dye; and detecting the number of fluorescence events that are fluorescent-positive after labeling with the second dye;

[0059] preparing a fluorescent microsphere standard containing a known particle concentration; detecting the number of fluorescent events in the fluorescent microsphere standard;

[0060] The viral genome titer was calculated based on the detection data.

[0061] The method further comprises:

[0062] The viral genome titer was calculated by formula 2:

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

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

[0065] In the method of the present invention, the first dye is directly conjugated with the surface recognition molecule to emit fluorescence, or the first dye has a recognition group and is indirectly conjugated 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 an antibody or antibody fragment, antigen, ligand, ligand receptor or polysaccharide that specifically binds to the surface recognition molecule; more preferably, the recognition group is an antibody or antibody fragment that specifically binds to the surface recognition molecule, and further 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. Measuring viral filling rate can help determine the actual effective viral dose. For example, when calculating how much viral preparation needs to be injected to achieve the desired therapeutic effect, the filling rate must be considered.

[0071] If the dose is calculated based on the total number of viral particles, the presence of empty viral shells may result in an insufficient effective dose, failing to achieve the desired therapeutic goal. A low solid fraction of a viral product means fewer viral particles contain the target gene, and thus fewer viral particles are able to effectively transduce the target gene, significantly reducing the effectiveness of gene therapy.

[0072] Measuring the fill rate using the method of the present invention facilitates optimization of conditions to improve the accuracy of viral dosage, enhance product safety, optimize clinical treatment efficacy, and reduce the risk of immune response. The method of detecting viral fill rate of the present invention can be used to evaluate the effectiveness, dosage guidance, and medication safety of viruses, such as adeno-associated virus products.

[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 amount and good repeatability for detecting virus solid rate.

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

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

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

[0079] (5) In the method of the present invention, it is preferred to use fluorescent antibody dye to label the viral capsid, which is simple and quick to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is a graph of the empty shell rate and solid core rate detected by the AUC method of Comparative Example 1.

[0081] Figure 2 This is the detection result of the particle group with PC5 positive signal in the rAAV8 mixture 1e' in the fluorescence channel.

[0082] Figure 3 This is the detection result of the particle group with PC5 positive signal in the fluorescent microsphere standard solution in the fluorescence channel.

[0083] Figure 4 The graph shows the titer of rAAV8 sample 1 in the standard curve measured by ELISA.

[0084] Figure 5 This is the detection result of the particle group with FITC-positive signal in the fluorescence channel of rAAV2 mixture 3c.

[0085] Figure 6 This is the detection result of the particle group with FITC-positive signal in the rAAV8 mixture 3c' in the fluorescence channel.

[0086] Figure 7 This is the detection result diagram of the particle group with FITC positive signal in the fluorescence channel of the fluorescent standard solution.

[0087] Figure 8 The amplification curves of rAAV2 product 1 and rAAV8 sample 1 using fluorescence quantitative PCR method.

[0088] Figure 9 These are the melting curves of rAAV2 product 1 and rAAV8 sample 1 using the fluorescence quantitative PCR method.

[0089] Figure 10 4 is a data comparison chart of Example 4, Comparative Example 2, Example 5 and Comparative Example 3. The NanoAnalyzer method represents the method of the present invention. DETAILED DESCRIPTION

[0090] The present application will be further described in detail below through the accompanying drawings and examples, through which the features and advantages of the present application will become more clear and distinct.

[0091] The word "exemplary" is used exclusively herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. Although various aspects of the embodiments are shown in the drawings, the drawings are not necessarily drawn to scale unless otherwise indicated.

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

[0093] In the present invention, "room temperature" refers to ambient temperature, which may be 20°C-30°C; in some embodiments, 22°C-28°C; in some embodiments, 24°C-26°C; in some embodiments, 25°C.

[0094] In the foregoing text of the present invention, all numerical values ​​disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. Based on the disclosed numbers, the value of each numerical value may vary by less than ±10% or by a reasonable difference deemed by a person skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0095] The terms "optional", "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur. For example, "optional surfactant" means that a surfactant may or may not be present.

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

[0097] definition

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

[0099] Viruses having a capsid and a nucleic acid molecule contained within the capsid may include lentiviruses, adenoviruses, adeno-associated viruses, poxviruses, and the like.

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

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

[0102] The term "solid virus" refers to a virus having a capsid and a nucleic acid molecule inside the virus, including a virus having an incomplete nucleic acid inside and a virus having a complete nucleic acid inside.

[0103] The term "solidity rate" refers to the percentage of virus particles having a capsid and nucleic acid inside the virus (including viruses with incomplete nucleic acid inside (incompletely packaged viruses) and viruses with complete nucleic acid inside (completely packaged viruses)) to the total number of virus particles.

[0104] The term "empty capsid ratio" refers to the percentage of empty viral capsids (i.e., capsids that do not package the viral genome) to the total number of viral particles.

[0105] Viral genome titer refers to the number of viral genome copies per unit volume (such as per milliliter) of sample. For example, 10 6 viral genome copies, 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 (such as per milliliter) of sample, and its unit is, for example, particles / ml, including complete viral particles containing the viral genome and empty 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 viral vectors, is a physical unit.

[0108] GC / mL (Genomic Copies per mL): The number of genomic copies of a viral vector per unit volume. It is commonly used for titer determination of adeno-associated viral 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) passing through the laser detection area of ​​an instrument (such as a flow cytometer) during an assay. Each particle passing through the laser generates a signal, which is recorded by the instrument and is called an "event." Event counts are typically expressed in units of "numbers" (or "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 a combination of any 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 includes at least one fluorescence channel. The photodetector is used to detect fluorescence signals in the sample and convert them into electrical signals. The signal acquisition and analysis system is capable of collecting and analyzing the electrical signals.

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

[0115] (1) First dye labeling (capsid labeling): a first test sample containing the virus is mixed with a first dye, and the first dye specifically binds to and labels the surface recognition molecule of the capsid of the virus in the test sample, thereby obtaining a test sample labeled with the first dye for detection in step (3);

[0116] Optionally, before or after mixing, the sample is diluted to a detection working concentration at a dilution factor α relative to the sample 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 in the sample to be tested are removed. The removal method can be performed using the method described in the "Purification used in this method" section.

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

[0119] In one embodiment, the virus type having a capsid 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 an artificially edited segment inserted, a viral vaccine, etc., or a virus 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 one or a combination of two or more selected from proteins and carbohydrates. Preferably, the surface recognition molecule of the virus is a protein.

[0122] The first dye is directly conjugated with the surface recognition molecule to emit fluorescence, or the first dye has a recognition group and is indirectly conjugated with the surface recognition molecule to emit fluorescence.

[0123] In some embodiments, 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 that specifically binds to the surface recognition molecule; 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. Further preferably, the first dye is composed of a recognition group and a fluorescent dye that emits fluorescence.

[0124] Fluorescent dyes commonly used in the art can be used without particular limitation, and include, for example, organic fluorescent molecules, fluorescent proteins, nucleic acid dyes, lipid membrane dyes, quantum dots, polymer dots, and the like. Organic fluorescent molecules include, but are not limited to, YF488 and FITC. Fluorescent proteins include, but are not limited to, phycoerythrin, phycocyanin, red fluorescent protein, green fluorescent protein, and mCherry fluorescent protein.

[0125] As the first dye, those described in the section "Dye for labeling - first dye" can be used.

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

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

[0128] As the purification method for removing impurities and removing free dyes and free nucleic acids, those described in the "Purification used in the present method" section can be used;

[0129] (2) Second labeling (labeling of intracapsid nucleic acid):

[0130] taking a second sample to be tested containing the virus, and destroying the viral capsid;

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

[0132] Preferably, free nucleic acid is removed from the sample before disrupting the viral capsid.

[0133] Optionally, after or before labeling the nucleic acid molecule with a second dye, the sample is diluted to a detection working concentration at a dilution factor β relative to the sample 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 is performed, more preferably the purification is performed by magnetic separation, such as SA magnetic beads method,

[0135] Wherein, the second sample to be tested can be the first sample to be tested, the sample to be tested labeled with the first dye, the diluted sample I or the solution obtained by diluting the first sample to be tested, or the supernatant obtained by purifying the first sample to be tested to remove impurities;

[0136] In one embodiment, the method for destroying the capsid is selected from a nucleic acid extraction reagent method or a thermal lysis method. The nucleic acid extraction reagent method includes a method based on a lysis reagent, a divalent ion reagent, an enzyme reagent, a detergent, and a solid phase matrix, or a combination of two or more. The heating temperature of the thermal lysis method can be 50°C-130°C. Preferably, the heating temperature is selected from 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C, 125°C or 130°C.

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

[0138] In one embodiment, the method for removing free nucleic acids is selected from the group consisting of enzyme digestion and separation column separation. As the method for removing impurities, those described in the section "(2) Removal of free dyes and purification of free nucleic acids" are included.

[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 comprises one or a combination of two or more of a cyanine dye, a non-permeable dye, a permeable dye, an intercalating dye, or a DNA double helix minor groove binding dye. As the second dye, those described in the "Dyes for Labeling - Second Dye" section can be used;

[0141] (3) Detection:

[0142] The single molecule detection system is used to detect the number of fluorescence events N1 in the fluorescent particle group in the flow volume V1 of the first dye-labeled sample to be tested or the diluted sample I within the unit detection time, and the number of fluorescence events N2 in the fluorescent particle group in the flow volume V2 of the second dye-labeled sample to be tested or the diluted sample II within the unit detection time;

[0143] (4) Calculation:

[0144] The virus solid rate in the sample to be tested is calculated according to formula 3:

[0145] Virus solid rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... Formula 3

[0146] Wherein, N1 is the number of fluorescence events in the fluorescent particle population in the diluted sample I detected by the single molecule detection system within a unit detection time, V1 is the flow volume of the diluted sample I passing through the single molecule detection system within a unit time, N2 is the number of fluorescence events in the fluorescent particle population in the diluted sample II detected by the single molecule detection system within a unit detection time, and V2 is the flow volume of the diluted sample II passing through the single molecule detection system within a unit time;

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

[0148] Virus empty shell rate (%) = 100% - virus solid core rate (%) Formula 5

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

[0150] The parameters are as defined above.

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

[0152] a) purifying the sample containing the virus to remove impurities before or after labeling the capsid of the virus in the sample containing the virus with a first dye;

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

[0154] c) purifying the virus-containing sample to remove impurities before labeling the virus capsid in the virus-containing sample with the first dye, and removing free dye and free nucleic acid after labeling with the first dye; or

[0155] d) purifying a sample containing the virus to remove impurities before or after disrupting the capsid and labeling the nucleic acid molecules contained within the viral capsid with a second dye;

[0156] Wherein, the impurities at least comprise at least one selected from free dye and free nucleic acid, preferably further comprise cell debris.As the above-mentioned purification to remove impurities and remove free dye and free nucleic acid, those described in the "Purification for this method" section can be used.

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

[0158] The first dye labeling is performed as in the first embodiment, and free first dye and free nucleic acid in the sample are removed after the first dye labeling; the number of fluorescence events showing positive fluorescence after the first dye labeling is detected to obtain the sample to be tested labeled with the first dye,

[0159] The method comprises diluting the sample to a detection working concentration at a dilution factor α relative to the sample to obtain a diluted sample I,

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

[0161] Diluting the concentration standard by θ times to obtain a concentration standard solution;

[0162] Detecting the number of fluorescence events M1 of the diluted sample I in the flow volume V1 within the unit detection time; detecting the number of fluorescence events M3 of the concentration standard solution in the flow volume V3 within the unit detection time;

[0163] Calculation: Calculate the viral capsid titer according to the following formula 7:

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

[0165] Wherein, 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 was calculated by formula 1,

[0168] Virus capsid titer = (N1 / V1)*α Formula 1,

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

[0170] Regarding the concentration standard solution and preparation method, those described in "Assay - Concentration Standard" can be used.

[0171] Unless otherwise specified, the viruses, the first dye, the second dye, the first dye marker, the second dye marker, the dilution and purification of the test sample are the same as those in the first embodiment.

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

[0173] The second dye labeling is performed as in the first embodiment, and free nucleic acid in the sample is removed before destroying the viral capsid.

[0174] wherein after or before labeling the nucleic acid molecule with a second dye, the sample is diluted to a detection working concentration at 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 a standard: preparing a concentration standard containing standard fluorescent microspheres of known particle concentration; diluting the concentration standard by θ times to obtain a concentration standard solution;

[0176] Detecting the number of fluorescence events M2 of the diluted sample II in the flow volume V2 within the unit detection time, and detecting the number of fluorescence events M3 of the concentration standard solution in the flow volume V3 within the unit detection time;

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

[0178] Wherein, 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 Formula 2,

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

[0181] Regarding the concentration standard solution and preparation method, those described in "Assay - Concentration Standard" can be used.

[0182] The virus, the first dye, the second dye, the first dye label, the second dye label, the dilution and purification of the sample to be tested are the same as those in the first embodiment unless otherwise specified.

[0183] Virus

[0184] The method of the present invention can be applied to viruses having a capsid and a nucleic acid molecule contained within the capsid. The types of viruses that can be detected by the method of the present invention may include: lentivirus, adenovirus, adeno-associated virus, poxvirus, etc., preferably adeno-associated virus.

[0185] In some embodiments, the virus in the test sample is an adeno-associated virus. The serotype of the adeno-associated virus 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 embodiments, the dilution of the test sample, such as the diluted sample, the test sample, the quantitative solution, etc., can use phosphate buffered saline (PBS) as a diluent.

[0187] In some embodiments, the detection working concentration of the virus 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 5 -5×10 9 Genome titer / mL.

[0188] In some embodiments, the detection working concentrations in the step of labeling with the first dye and / or the step of labeling with the second dye are independently selected from 1×10 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 embodiments, the test sample can be derived from: process intermediates, preparations, cell cultures, body fluids, tissues, cell lysates, etc., but is not limited thereto.

[0190] Dyes used for labeling

[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, and preferably the first dye is an optical dye.

[0193] In some embodiments, the optical dye comprises a fluorescent molecule, a fluorescent material, or a combination thereof. In some embodiments, the optical dye comprises one or a combination of two or more of a fluorescent molecule, a fluorescent protein, a quantum dot, or a polymer dot.

[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 molecule of the virus can be one of proteins and sugars or a combination of two or more, and can function as a surface recognition molecule.

[0196] The first dye can specifically bind to the surface recognition molecule of the virus by: direct conjugation of the first dye to the surface recognition molecule of the virus; or indirect conjugation of the first dye with the surface recognition molecule by a recognition group carried in the first dye.

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

[0198] The recognition group specifically binds to the surface recognition molecule, and preferably 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; more preferably, the recognition group is an antibody or an antibody fragment; further preferably, the recognition group is an antibody or an 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 comprises an AAV antibody, and the AAV antibody can be CaptureSelect 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 comprise a recognition group moiety. 647A SE is a type of fluorescent dye with amino-reactive activity. The SE group of this type of dye can react with the amino group to produce a stable amide bond. In this embodiment, only 647A SE marks the protein of the virus capsid, or 647ASE is used in conjunction 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 comprises at least one selected from clone numbers ADK1a, A20, A20R, ADK4, ADK5a, ADK5b, ADK6, ADK8, and ADK8 / 9.

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

[0206] The working concentration, incubation temperature, incubation time, etc. of the first dye can be within the range commonly used in the art without special limitation. The working concentration can be, for example, 0.01 to 10 μg / μL, the incubation temperature can be, for example, in the range of 4°C to 37°C, and the incubation time can be, for example, 5 to 60 minutes.

[0207] In one embodiment, the method of the present invention comprises, after disrupting the viral capsid, detecting the number of particles in the sample that fluoresce positively to a first dye. The number of particles in the sample that fluoresce positively to the first dye can be used to determine the amount of intact viral capsid present in the sample.

[0208] When viral particles bound to the first dye undergo capsid disruption and disintegrate, the first dye will exhibit diffuse, non-event fluorescence in the sample solution. Therefore, after capsid disruption, the fluorescence channel detecting the first dye can be used to monitor the luminescence of the sample containing the first dye after capsid disruption. Particles that still exhibit fluorescence corresponding to the first dye indicate that the capsid has not been 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 presented 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 comprises one or a combination of two or more of a cyanine dye, a non-permeable dye, a permeable dye, an intercalating dye, and a DNA double helix minor groove binding dye.

[0213] In some embodiments, the nucleic acid fluorescent dye includes a member selected from the group consisting of 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 (Hydroethidine), 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 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 RedStain, ethidium bromide, or a combination of two or more.

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

[0215] The working concentration, incubation temperature, incubation time, etc. of the nucleic acid fluorescent dye can be within the commonly used ranges 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] The method preferably includes a purification step, which may include: 1) purification to separate the target virus from impurities; and 2) purification to remove free dyes and free nucleic acids, each of which is described below.

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

[0219] By separating impurities such as cell debris, floating cells, extracellular vesicles, miscellaneous proteins, lipids, salts, etc. from the target virus, and removing free optical dyes and free nucleic acids, it is helpful to reduce background interference and improve detection accuracy and reproducibility.

[0220] 1) Separation of target virus and purification of impurities

[0221] In the present invention, "isolation" mainly refers to the purification of the virus from a mixture, mixed solution, cell fluid, cell lysate, etc. containing the virus.

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

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

[0224] In the present invention, it is preferred to specifically separate the target virus from impurities by magnetic separation.

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

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

[0227] The antigen-antibody magnetic bead separation method is a separation technology based on antigen-antibody interaction, which 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. Micro-nano-sized magnetic beads can be those commonly used in the art, without particular limitation, and can be selected from, for example, carboxyl magnetic beads, amino magnetic beads, hydroxyl magnetic beads, thiol magnetic beads, tosyl magnetic beads, protein A / G magnetic beads, Oligo(dT) magnetic beads, antibody-coupled magnetic beads, SA magnetic beads, etc.

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

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

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

[0232] In some embodiments, step (1) comprises purifying the sample to be tested using SA magnetic beads before labeling with the first dye. Thus, step (1) comprises:

[0233] The test sample is purified using the SA magnetic bead method to obtain a purified sample containing virus-SA magnetic beads ③, i.e., the first test sample; the first test sample is mixed with a 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 cleavage reagent is added to destroy the disulfide bond between the SA magnetic beads ③ and the virus bound thereto; and the supernatant A is separated by adsorption using a magnetic stand for detection in step (3).

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

[0235] In some embodiments, in the above step (1), after the operation of "mixing the first test sample with the first dye, allowing the first dye to specifically bind to and label the surface recognition molecule of the virus as a surface recognition molecule", the operation of "incubating, adsorbing on a magnetic stand, discarding the supernatant, and resuspending" is repeated 0-5 times (0 times: i.e., the operation of "incubating, adsorbing on a magnetic stand, discarding the supernatant, and resuspending" is not performed).

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

[0237] 2) Removal of free dyes and purification of free nucleic acids

[0238] In the present invention, "purification by removing free dyes and free nucleic acids" mainly refers to the purification by removing free dyes and free nucleic acid components other than the virus and the nucleic acid molecules contained inside the virus from a mixture, mixed solution, cell fluid, cell lysate, etc. containing the virus.

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

[0240] In some embodiments, the step (2) further comprises: removing 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 dyes and removing free nucleic acids in step (1) or step (2) is selected from the group consisting of: ultracentrifugation, density gradient centrifugation, rate gradient centrifugation, ultrafiltration, size exclusion chromatography, dialysis, affinity capture, immunocapture, microfluidics, and dilution methods, or a combination of two or more thereof.

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

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

[0244] Methods for destroying the capsid

[0245] In some embodiments, the method of destroying the viral capsid in the method of the present invention comprises: a nucleic acid extraction reagent method or a 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 a combination of two or more of a lysis reagent, a divalent ion reagent, an enzyme reagent, a detergent, and a solid matrix.

[0248] In some embodiments, the lysis reagent includes one or a combination of two or more of cetyltriethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), guanidine isothiocyanate, phenol, chloroform, isoamyl alcohol, cesium chloride-ethidium bromide (CsCl-EB), alkali, and water.

[0249] In some embodiments, the divalent ion 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 a manganese salt or a zinc salt. In some embodiments, the alkaline earth metal divalent salt is selected from a magnesium salt or a calcium salt.

[0250] In some embodiments, the enzyme reagent comprises one or a combination of two or more of subtilisin, subtilisin, or alkaline serine protease. In some embodiments, the enzyme reagent further comprises one or a combination of two or more of proteinase K, proteinase R, proteinase T, subtilisin A, Nagarse, subtilisin B, or thermolysin.

[0251] In some embodiments, the detergent comprises one or a combination of two or more of cetyltrimethylammonium bromide, Tween-type surfactant, Triton X-100, sodium lauryl sulfate, nonylphenol polyoxyethylene ether, or polyoxyethylene nonionic detergent.

[0252] In some embodiments, the solid phase matrix comprises beads composed of one or a combination of two or more of magnetic microbeads, silica, diatomaceous earth, chromatography columns, glass particles, anion exchange resins, membranes, chips, carbon nanotubes, well plates, slides, chromatography matrices, or any combination thereof.

[0253] - Thermal cracking method

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

[0255] In some embodiments, the heat time of the thermal cracking method is 1 minute to 60 minutes. In some embodiments, the heat time of the thermal cracking 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 embodiments, 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 is configured to detect fluorescence signals in a sample and convert the fluorescence signals into electrical signals. The signal acquisition and analysis system is configured to acquire and analyze the electrical signals.

[0259] In some embodiments, the optical system comprises 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 conversion of the electrical signals to obtain the number of fluorescence events in the fluorescent particle population.

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

[0262] FITC and PC5 are fluorescent channels that can be used in the present method. These two channels can be used on commonly used detection instruments in the art, such as the Flow NanoAnalyzer from NanoFCM. The FITC fluorescent channel can detect fluorescence from at least AF488 and SYTO BC fluorescent nucleic acid dyes; the PC5 fluorescent channel can detect fluorescence from at least AF647 and YF647 dyes.

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

[0264] -Concentration standard solution

[0265] There is no special limitation on the fluorescent microspheres in the concentration standard solution, and 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 5 -5×10 9 In some embodiments, the concentration of the standard fluorescent microspheres in the concentration standard solution is 1×10 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 with a common diluent, such as phosphate buffered saline (PBS).

[0268] use

[0269] Taking the adeno-associated virus (AAV) filling rate as an example, the important uses of the virus filling rate in several aspects of product quality and clinical use are explained:

[0270] (1) Viral activity, potency, and therapeutic effects

[0271] A high fill rate generally indicates a greater number of viral particles with intact structure and function. Fully capsid AAV is more likely to carry the correct therapeutic gene and effectively deliver and express the gene upon entry into target cells. In contrast, a low fill rate may result in reduced viral activity, compromising therapeutic efficacy.

[0272] (2) Purity and safety

[0273] A higher solid fraction helps improve product purity. Impure AAV preparations may contain unassembled viral proteins, empty shells, or other impurities that may trigger an immune response or cause adverse effects in patients.

[0274] Increasing the solid fraction can reduce the presence of non-functional particles and lower the potential risk of immunogenicity. The immune system may respond to incomplete or abnormal viral particles, thus affecting the safety and efficacy of treatment.

[0275] (3) Dosage regimen and dosage determination

[0276] Accurate determination of the fill rate can help optimize dosing regimens and determine appropriate dosages. Understanding the fill rate allows for a more accurate assessment of viral activity and potency, allowing for personalized treatment plans tailored to the patient's specific circumstances.

[0277] Therefore, the solid rate of the virus detected by the method of the present invention has an important impact on product quality and clinical use.

[0278] Improving the piling rate can enhance the activity and potency of the virus, improve the purity and safety of the product, optimize clinical treatment effects, reduce the risk of immune response, and provide a basis for drug administration and dosage determination. In the research and development, production, and clinical application of viruses such as AAV, the control and monitoring of the piling rate should be highly valued to ensure product quality and clinical treatment success.

[0279] Example

[0280] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0281] The term "M" stands for mol / L, i.e., moles per liter. "vg / ml" stands for genome copies per ml. PBS or PBS buffer stands for phosphate buffered saline, 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 of the rAAV viral suspension (rAAV viral genome, rAAV viral capsid or free nucleic acid), including the titer of rAAV viral genome, the titer of rAAV viral capsid or the titer of free nucleic acid of the rAAV viral suspension.

[0283] The term "OD" means absorbance, such as "OD230nm" 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 succinimidyl ester) was purchased from BIORIGIN;

[0288] SYTO BC Green Fluorescent Nucleic Acid Stains (SYTO BC fluorescent nucleic acid stains) were purchased from Invitrogen.

[0289] CaptureSelect TM Biotin anti-AAVX conjugate antibody: purchased from ThermoFisher. This conjugate exhibits binding reactivity to 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) Fluorescent Antibody Dyes: The procedure for using the YF647A Antibody Labeling Kits is provided on the IN website:

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

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

[0295] c. Separate the labeled antibody by running the solution through a dialysis column.

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

[0297] Preparation of YF647-AAVX antibody (according to the procedure provided on the BIORIGIN official website "YF647A Antibody Labeling Kits (YF647A Antibody Labeling Kit) Instructions"):

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

[0299] b. AAVX antibody (CaptureSelect TM Biotin anti-AAVX conjugate) (2.5 mg / mL) was transferred to a Add the SE dye to the vial. Mix well and incubate with agitation at room temperature for 1 hour.

[0300] c. Separate the labeled antibody by running the solution through a dialysis column.

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

[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 of Biotin-Disulfide Bond-AAV8 Antibody (according to the procedure provided on the ThermoFisherScientific official website, "User Guide: EZ-Link NHS-SS-Biotin"):

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

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

[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 spin column: BeyoDesalt G-25 column (Superfine, 5 ml); Shanghai Beyotime Biotechnology Co., Ltd.

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

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

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

[0313] A Tecnai G2 Spirit transmission electron microscope was purchased from FEI Czech Ltd.

[0314] Comparative Example 1: Detection of Empty Shell Rate and Solid Core Rate by AUC Method

[0315] Sample: rAAV8 sample 1 (obtained after dilution of the stock solution, stock solution: manufacturer: Paigen Bio, AAV8 [scAAV.CAG.EGFP.WPRE.SV40pA], product number PGS08001).

[0316] The AUC method is currently considered the standard for evaluating rAAV filling rate and product-specific impurity analysis in the field. The AUC method separates empty, full, partially filled, and overfilled rAAV capsids based on the size and weight of rAAV capsids, as well as the sedimentation coefficient unique to the AUC process. The AUC can be used to determine the relative abundance of empty capsids, incompletely packaged virus, full rAAV capsids, and viral aggregates.

[0317] Sample preparation: Solutions were measured by optical density measurement at 260 nm with OD values ​​ranging from 0.1 to 1.0. rAAV8 sample 1 was adjusted to a target concentration greater than 5 x 10 by direct dilution or further concentration using an Amicon Ultra-0.5 / 30K MWCO centrifugal filter device. 11vg / mL.

[0318] Sedimentation velocity AUC data acquisition:

[0319] Sedimentation velocity analytical ultracentrifugation (AUC) analysis was performed on a Beckman Coulter XL-I. 400 μL of sample was loaded into the sample sector of the two sector 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 at 20°C and full vacuum for 1 hour.

[0320] Sedimentation velocity centrifugation was performed at 20,000 RPM, 20°C, and a 0.003 cm radius step setting, without delay and without repetition. Radial concentration was recorded synchronously with absorbance (230 nm) and Raleigh interference optics as a function of time until the smallest sedimenting component passed through the optical window (1.2 hours). The assay throughput was limited to a single sample per run, with parameters specified based on an absorbance scan collection time greater than 1 minute, and the size and rapid sedimentation of the AAV.

[0321] AUC data analysis:

[0322] The solid fraction 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, Smin = 1, Smax = 200, and friction ratio = 1.0. RI and TI noise reduction were applied, and the meniscus position was allowed to float, with the optimal position automatically selected by the software.

[0323] The model fits the Lamm equation to the data and the resulting size distribution is the "distribution of sedimentation coefficients", with the area under each peak being expressed in units of fringes or OD. 230nm Determine the sedimentation coefficient and relative concentration of each component in the distribution, where the units of sedimentation coefficient are Svedberg units and the relative concentration is expressed in OD units.

[0324] Each AUC run was an independent assay, and the following properties of each assay were monitored: goodness of fit (rmsd), OD of each peak 230nm The A230 / IF ratio, the consistency of the sedimentation coefficient for each species between runs, and the overall quality of the scan were tested to ensure the quality of the results.

[0325] Absorbance Optical (230 nm): The molar concentration and actual percentage of the intact vector peak were calculated from the absorbance data using the extinction coefficient. The molar absorptivity extinction coefficients for empty capsids and intact viral particles (intact vector) were calculated based on published formulas. Extinction coefficients were applied to both empty capsids and intact AAV peaks. The SEDFIT algorithm was used to determine C(S) values. The molar concentrations of both intact vector and empty capsids were calculated using Beer's law and the results are shown in Table 1. Figure 1 .

[0326] The detection time of the method is more than 6 hours from the preparation of the test sample to the acquisition of the test result.

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

[0328] Example 1: Detection of virus solid rate and empty shell rate ( 647ASE-labeled capsid)

[0329] 647ASE is a type of fluorescent dye with amino-reactive activity. The SE group of this type of dye can react with the amino group to produce a stable amide bond. In this example, 647ASE is used to label the capsid protein in the viral capsid.

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

[0331] Step (1):

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

[0333] Take BeyoDesalt G-25 column, add rAAV mixture 1a, centrifuge to remove free 647ASE and free nucleic acid, and the liquid flowing out is collected as rAAV mixture 1b.

[0334] The rAAV mixture 1b was diluted 62.5-fold with PBS (ie, diluted 70-fold relative to the labeled rAAV test solution) to obtain the rAAV mixture 1c (ie, diluted sample I).

[0335] Step (2):

[0336] 1) Dilute a portion of the aforementioned rAAV mixture 1b to 4.5-fold 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-fold with PBS buffer, add 1 μl of 100 μM SYTO BCGreen Fluorescent Nucleic Acid Stains aqueous solution for labeling (i.e., diluted 50.904-fold relative to the labeled rAAV test solution), and incubate for 5 minutes to obtain rAAV mixture 2b (i.e., diluted sample II).

[0339] Step (3): Detection

[0340] Flow NanoAnalyzer was used to detect rAAV mixture 1c and rAAV mixture 2b respectively.

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

[0342] According to the above detection parameters, the number of fluorescence events N1 in the fluorescent particle group in the flow volume V1 of the mixed liquid 1c within the unit detection time, and the number of fluorescence events N2 in the fluorescent particle group in the flow volume V2 of the mixed liquid 2b within the unit detection time are detected; and the detection results are recorded.

[0343] Step (4): Calculate:

[0344] The virus solidification rate in the test sample was calculated according to the following formula 3, and the results are shown in Table 1.

[0345] Virus solid rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... Formula 3;

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

[0347] The virus-filled rate in the sample to be tested was further calculated according to the following formula 5, and the results are shown in Table 1:

[0348] Adeno-associated virus empty shell ratio (%) = 100% - virus solid core ratio (%) ... ... Formula 5

[0349] Table 1 Solid ratio and hollow ratio calculated according to formula 1

[0350]

[0351] This method takes time to detect: 1h

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

[0353] Example 2: Detection of virus solid and empty capsid rates (fluorescent antibody dye-labeled capsids)

[0354] Step (1):

[0355] 200 μL of the labeled rAAV test solution (same as in Example 1) was added to a centrifuge for removing dyes and nucleic acids. Free nucleic acids were removed by centrifugation, and the effluent liquid was collected to obtain a rAAV mixed solution 1 d-1.

[0356] 100 μL of rAAV mixture 1d-1 was taken, 20 μL of YF647-AAVX antibody was added, and incubated for 10 min as rAAV mixture 1d-2.

[0357] The labeled rAAV mixture 1d-2 was diluted 43.5 times with PBS (ie, diluted 52.2 times relative to the labeled rAAV test solution) to obtain the rAAV mixture 1e (ie, diluted sample I).

[0358] Step (2):

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

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

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

[0362] Step (3): Detection

[0363] Flow NanoAnalyzer was used to detect rAAV mixture 1e and rAAV mixture 2b-2, respectively.

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

[0365] According to the above detection parameters, the number of fluorescence events N1 in the fluorescent particle group in the flow volume V1 of the mixed liquid 1e within the unit detection time, and the number of fluorescence events N2 in the fluorescent particle group in the flow volume V2 of the mixed liquid 2b-2 within the unit detection time are detected; and the detection results are recorded.

[0366] Step (4): Calculate:

[0367] The virus solidification rate in the test sample was calculated according to the following formula 3, and the results are shown in Table 2.

[0368] Virus solid rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... Formula 3;

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

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

[0371] Adeno-associated virus empty shell ratio (%) = 100% - virus solid core ratio (%) ... ... ... Formula 5

[0372] Table 2: Solid ratio and hollow ratio

[0373] N1 V1 α N2 V2 β Solidity rate Shell rate 5979 30nL 52.2 6840 30nL 50.904 97.1％ 2.9％

[0374] This method takes time to detect: 1h

[0375] Result analysis: The empty shell rate detected in Example 2 is consistent with that in Example 1. This result shows that the use of fluorescent antibody dye in the method of the present invention can also accurately detect the solid shell rate and empty shell rate of the virus.

[0376] Example 3: Detection of the solid and empty rAAV ratios in cell lysates

[0377] Step (1)

[0378] 1) Preparation of rAAV simulation samples:

[0379] Dilute the stock with known solid fraction to a titer of 2.6×10 11 The rAAV8 sample 2 (obtained by dilution of the stock solution; the manufacturer and product number of the stock solution were the same as in Example 1, but with a different batch number) at a concentration of 500 μg / ml was mixed with the supernatant of the HEK-293T cell lysate to obtain an rAAV mock sample with a known solid fraction of 44.5%. This rAAV mock sample was used as the test sample and tested as follows.

[0380] 2) Sample purification:

[0381] Take 30 μl 2×10 10100 μl of SA magnetic beads (0.1 wt% BSA / ml) were added to 500 μl of PBS containing 0.1 wt% BSA (bovine serum albumin), adsorbed on a magnetic stand for 1 min, and the supernatant was discarded. The above steps (adding 500 μl of PBS containing 0.1 wt% BSA, adsorbed on a magnetic stand for 1 min, and the supernatant was discarded) were repeated 3 times to obtain SA magnetic beads ①;

[0382] Resuspend the SA magnetic beads ① with 200 μl of PBS containing 0.2 wt% BSA, add 10 μl of Biotin-SS-AAV8 antibody, incubate with rotation mixing at 4°C for 5 min, adsorb on a magnetic stand for 1 min, discard the supernatant, and resuspend with 500 μl of PBS containing 0.2 wt% BSA. Repeat the above steps (incubate with rotation mixing at 4°C for 5 min, adsorb on a magnetic stand for 1 min, discard the supernatant, and resuspend with PBS containing 0.2% BSA) 3 times to obtain 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 rAAV simulation sample with a known solid ratio, and the mixture was rotated and incubated at 4°C for 8 min, adsorbed on a magnetic stand for 1 min, the supernatant was discarded, and the mixture was resuspended in 400 μl of PBS. The above steps (rotated and incubated at 4°C for 8 min, adsorbed on a magnetic stand for 1 min, the supernatant was discarded, and the mixture was resuspended in PBS) were repeated 4 times to obtain SA magnetic beads ③.

[0385] Among them, 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) Labeling of sample capsids:

[0387] Take 50 μl of SA magnetic beads ③, add 20 μl of YF647-AAV8 antibody to the SA magnetic beads ③, incubate at room temperature for 8 minutes. Adsorb on the magnetic stand for 1 minute, discard the supernatant, and resuspend with 200 μl PBS. Repeat the above steps (incubate at room temperature for 8 minutes. Adsorb on the magnetic stand for 1 minute, discard the supernatant, and resuspend with PBS) 3 times.

[0388] For the first two repetitions, resuspend the sample in 200 μl of PBS. For the final repetition, discard the supernatant and resuspend the sample in 50 μl of PBS. Add 4 μl of 2.5 M DTT (a disulfide bond cleavage reagent) and react at room temperature for 1.5 h to obtain reaction solution A. Adsorb the sample on a magnetic stand to obtain the supernatant of reaction solution A. A portion of the supernatant of reaction solution A was diluted 43.5-fold with PBS to obtain mixed solution 1f (i.e., diluted sample I).

[0389] Step (2)

[0390] Labeling of nucleic acids in sample shells:

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

[0392] Step (3)

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

[0394] Step (4) Calculation:

[0395] The virus solidification rate in the sample to be tested was calculated according to Formula 3.

[0396] Virus solid rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% ... Formula 3;

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

[0398] Furthermore, the virus filling rate in the sample to be tested was calculated according to the following formula 5:

[0399] Adeno-associated virus empty shell ratio (%) = 100% - virus solid core ratio (%) …………………… Formula 5

[0400] Table 3 Solid ratio and hollow ratio

[0401] N1 V1 α N2 V2 β Solidity rate Shell rate 4969 30nL 70 8095 30nL 50.904 44.6％ 55.4％

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

[0403] Comparative Example 2: ELISA method to detect viral capsid titer

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

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

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

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

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

[0411] 7) Add 100 μl of the stop solution, read the OD value at 650 nm and 450 nm respectively, and calculate the final OD value as the OD value at 650 nm - the OD value at 450 nm.

[0412] 8) Positive quantitative determination:

[0413] The standard curve was drawn based on the 7 concentrations of standard controls obtained in step 2) of this comparative example (see Figure 4 ), and the test results of the diluted rAAV8 sample 1 were substituted for quantitative determination.

[0414] By substituting the standard curve into the titer, the capsid titer of the diluted rAAV8 sample 1 measured by ELISA was 4.6×10 8 vp / mL (see Figure 4 ), and the final viral capsid titer of rAAV8 sample 1 was 4.6×10 11 vp / mL.

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

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

[0417] (i) Fluorescent antibody labeling of the capsid of rAAV8 sample 1:

[0418] 100 μL of rAAV8 sample 1 (same as in Example 1) was taken as the test sample, 20 μL of YF647-AAVX antibody was added, and the mixture was incubated for 10 min to obtain rAAV8 mixture 1d'. rAAV8 mixture 1d' was diluted 10-fold with PBS buffer to obtain rAAV8 mixture 1e'. rAAV8 mixture 1e' was diluted 120-fold relative to rAAV8 sample 1.

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

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

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

[0422] (iii) Capsid titer calculation

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

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

[0425] Virus capsid titer = C × ((M1 / V1)*α) / ((M3 / V3)*θ) ... Formula 7

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

[0427] Virus capsid titer = 2.14 × 10 10 ×5893×120 / 6743×100

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

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

[0430] Furthermore, the standard error of the capsid titer of AAV8 sample 1 detected by the method provided in Example 4 of the present invention is significantly lower than that of the ELISA method provided in Comparative Example 2 (see Figure 10 ). It is suggested that the method provided in Example 4 of the present invention uses a specific antibody to bind to the AAV capsid protein, thereby avoiding nonspecific binding to 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 rAAV2 and rAAV8 viral genome titers

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

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

[0434] according to Titration Kit (for Real Time PCR) Ver.2 (Takara, 6233) was used according to the instructions:

[0435] 1) 2 μl of each rAAV2 product 1 and rAAV8 sample 1 (same as in Example 1) were added to 1 μl of DNase I, 2 μl of 10× DNase I buffer, and 15 μl of deionized water to form mixed solutions v1 and v2, respectively. The mixtures were reacted at 37°C for at least 15 minutes. The mixed solutions v1 and v2 were then heat-treated at 95°C for 10 minutes to inactivate the DNase I. This was done to remove free nucleic acids and surface-adsorbed nucleic acids and to release nucleic acid material encapsidated by the rAAV2 product capsid.

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

[0437] 3) Dilution

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

[0439] Take the positive control stock solution (known genome titer is 2×10 10 vg / ml, from the same kit), and serially diluted with EASYDilution (the same kit) in 6 dilution 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] 5 μl of each of the above serial dilution solutions was taken as a template to perform Real Time PCR reaction. The data obtained from the positive control was used to draw a standard curve.

[0441] 4) Prepare 50× Primer mix solution

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

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

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

[0445] 7) PCR reaction conditions: Pre-denaturation at 95°C for 2 minutes, followed by 35 cycles of a two-step PCR reaction at 95°C for 5 seconds and 60°C for 30 seconds. Fluorescence detection using FAM was then performed, and melting curve analysis was performed after the reaction.

[0446] 8) Positive quantitative determination:

[0447] The positive control standard curve was drawn based on the positive control data of the six gradients in step 3). The test results of the 100-fold diluted AAV 2 genome extract a and the 100-fold diluted AAV 8 genome extract b were substituted into the calculation, and the results were as follows: Figure 8 shown.

[0448] Results: As Figure 8 As shown in Figure 2, the Ct value obtained by testing the 100-fold diluted AAV 2 genome extract a was 17.7 ± 0.064, and the Ct value obtained by testing the 100-fold diluted AAV 8 genome extract b was 17.2 ± 0.086. According to the standard curve, the genome titer of the 100-fold diluted AAV 2 genome extract a was 1.04 × 10 8 vg / ml, the genome titer of AAV 8 genome extract b diluted 100 times was 1.53×10 8 vg / ml.

[0449] After conversion based on the dilution ratio, the final measured genome titer of rAAV2 product 1 was 2.08×10 11 vg / ml, and the genome titer of rAAV8 sample 1 was 3.16×10 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: Fluorescent labeling to detect rAAV2 and rAAV8 viral genome titers

[0452] rAAV2 product 1: same as Example 4.

[0453] rAAV8 sample 1: same as Example 1.

[0454] Enzymatic digestion to remove nucleic acids: Take 100 μL of the sample to be tested, add 10 μL of 1 M NaHCO₃ aqueous solution, mix thoroughly, and let stand at room temperature for 5 minutes. Then add 2 μL of 10 μg / μL Alexa Fluor 6475-TFP, mix thoroughly, and incubate at room temperature in the dark for 15-30 minutes. Centrifuge at 100,000 g at 4°C for 80 minutes, discard the supernatant, and resuspend the pellet in PBS. Centrifuge at 100,000 g at 4°C for 20 minutes, discard the supernatant, and resuspend the pellet in 100 μL of 10 mM PBS containing 3 μM SYTO BC Green Fluorescent Nucleic Acid Stains 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. Incubate at 37°C for 30 minutes. This will yield rAAV mixture 3C.

[0455] According to the above-mentioned nucleic acid removal method, the rAAV2 product 1 was used as the test sample to remove the free nucleic acid of the rAAV2 product 1, thereby obtaining the rAAV2 mixed solution 3c'; the rAAV8 sample 1 was used as the test sample to remove the free nucleic acid of the rAAV8 sample 1, thereby obtaining the rAAV8 mixed solution 3c".

[0456] According to the method for obtaining rAAV mixture 2b from rAAV mixture 1b in step 2 of Example 1, rAAV2 mixture 3c' and rAAV8 mixture 3c" are subjected to nucleic acid labeling to obtain rAAV2 mixture 2b' and rAAV8 mixture 2b";

[0457] The concentration is C = 2.19 × 10 10 The quality control fluorescent microsphere standard (same as above) with a concentration of 100 particles / mL was diluted 50 times to obtain the concentration standard solution f';

[0458] Flow NanoAnalyzer was used to detect rAAV2 mixture 2b', rAAV8 mixture 2b", and concentration standard solution f'.

[0459] Laser detector 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal. FITC fluorescence channel is used. The FITC fluorescence channel is the SYTO BC Green Fluorescent Nucleic Acid Stains excitation signal for the number of particles containing nucleic acid in the capsid of rAAV2 product 1. Analyze the particle population with FITC-positive signal in the fluorescence channel detection results. Record the test results ( 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 the 2-min measurement.

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

[0462] The number of particles in the concentration standard solution was calculated to be 7423 after 2 minutes of measurement.

[0463] calculate:

[0464] Calculate the nucleic acid concentration of rAAV2 Product 1 using the dilution factor, concentration standard curve, and the nucleic acid test 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) ... Formula 8

[0466] Wherein, 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 = 2.19 × 10 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 10 particles / mL. The nucleic acid concentration of rAAV8 sample 1 was 1.01×10 11 particles / mL.

[0470] Since each FITC-positive signal particle detected in the Flow NanoAnalyzer is equivalent to one viral genome copy or one event, the genome titer of rAAV2 product 1 measured in this example is 1×nucleic acid concentration of rAAV2 product 1=6.25×10 10 vg / ml,

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

[0472] analyze:

[0473] Compared with the genome titer of AAV8 sample 1 detected by the method of Example 5 of the present invention and the genome titer detected by the method of Comparative Example 3, the genome titer of AAV8 sample 1 detected by the method of Example 5 of the present invention is lower.

[0474] The results suggest that the traditional fluorescence quantitative PCR method can non-specifically amplify non-AAV nucleic acid targets in the sample that can bind to the detection primers, which may lead to an overestimation of the AAV sample genome titer; the method of the present invention can eliminate the interference of non-AAV nucleic acid targets in the sample on the results, thereby avoiding the overestimation of the AAV sample genome titer.

[0475] discuss

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

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

[0478] The capsid titer of AAV2 Product 1 of Example 5 was compared with the genome titer of AAV2 Product 1 of Comparative Example 3. The results showed that the measured capsid titer (particles / ml) of the sample 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-shell AAV in the test samples.

[0480] Comparative Example 4: No removal of free nucleic acids and free dyes

[0481] The preparation and detection were carried out according to the method of Example 1, except that After 647ASE, no spin column was used to remove free 647A SE and free nucleic acid were tested four times in parallel, with the remaining procedures being the same as in Example 1. The results are shown in Table 4.

[0482] Table 4 Test results (n=4)

[0483]

[0484] SD (Standard Deviation): A statistical indicator used to measure the degree of dispersion of data. It indicates the degree of deviation of each value in a data set from the mean of the data set.

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

[0486] In summary, according to the results of the above embodiments and comparative examples, the detection method provided by the present invention has good accuracy and repeatability, is easy to operate, saves time, requires a small amount of sample, has high detection efficiency, avoids overestimation of the solid rate, and has excellent and unexpected technical effects.

[0487] Example 6: Incompletely packaged virus content

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

[0489] The AAV virus solid rate calculated by the present invention is combined with the determination of the virus full shell rate and AAV aggregate content, and the incompletely packaged virus content can be calculated by the following formula 9:

[0490] Virus incomplete packaging virus content % = virus solid rate % - virus full shell rate % - virus aggregate content % ... ... Formula 9.

[0491] The present application has been described above in conjunction with preferred embodiments, but these embodiments are merely exemplary and serve only as an illustrative example. On this basis, various replacements and improvements can be made to the present application, all of which fall within the scope of protection of the present application.

Claims

1. A method for detecting the solidity rate of a virus, wherein the virus has a capsid and a nucleic acid molecule contained within the capsid, the method comprising: (1) labeling the capsid of the virus in the sample containing the virus with a first dye, wherein the first dye specifically binds to the surface recognition molecule of the capsid and emits fluorescence; (2) destroying the viral capsid, and then labeling the nucleic acid molecule contained in the viral capsid with a second dye, wherein the second dye can specifically bind to the nucleic acid molecule and emit fluorescence; (3) detecting the number of fluorescence events that are positive after being labeled with the first dye, and detecting the number of fluorescence events that are positive after being labeled with the second dye; The virus solidification rate was calculated based on the number of fluorescence events showing positive fluorescence after labeling with the first dye and the number of fluorescence events showing positive fluorescence after labeling with the second dye.

2. The method according to claim 1, wherein the method comprises: In step (1), after or before labeling the viral capsid in the sample with the first dye, the sample is diluted to a detection working concentration at 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 a second dye, the sample is diluted to a detection working concentration at 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 fluorescence events that are fluorescence-positive 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 through 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 comprises at least one fluorescence channel; the photodetector is used to detect the fluorescence signal in the sample and convert the fluorescence signal into an electrical signal; the signal acquisition and analysis system is capable of acquiring and analyzing the electrical signal.

5. The method according to claim 3, further comprising: The viral capsid titer was calculated by formula 1, Virus capsid titer = (N1 / V1)*α Formula 1, Wherein, N1 is the number of fluorescence events in the diluted sample I; V1 is the flow volume of the diluted sample I passing through the single molecule detection system per unit time.

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

7. The method according to claim 3, further comprising: The virus solid rate is calculated by formula 3, Virus solid rate (%) = ((N2 / V2)*β) / ((N1 / V1)*α)*100% Formula 3 Wherein, N1 is the number of fluorescence events in the diluted sample I; V1 is the flow volume of the diluted sample I through the single molecule detection system per unit time; N2 is the number of fluorescence events in the diluted sample II; V2 is the flow volume of the diluted sample II passing through the single molecule detection system per unit time.

8. The method according to any one of claims 1 to 7, comprising one or more of the following steps: a) purifying a sample containing the virus to remove impurities before or after labeling with the first dye; b) removing free dye and free nucleic acid from the sample containing the virus after labeling with the first dye; c) purifying the sample containing the virus to remove impurities before labeling with the first dye, and after labeling with the first dye, removing free dye and free nucleic acid; and d) purifying 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, extracellular vesicles, miscellaneous proteins, lipids, salts and combinations thereof, Preferably, the method comprises step a) and step b).

9. The method according to any one of claims 1 to 8, wherein The virus is selected from adeno-associated virus, lentivirus, adenovirus, and poxvirus, and preferably the virus is adeno-associated virus.

10. The method according to any one of claims 1 to 9, wherein The first dye is directly conjugated with the surface recognition molecule to emit fluorescence; or the first dye has a recognition group and is indirectly conjugated with the surface recognition molecule to emit fluorescence, The recognition group specifically binds to the surface recognition molecule, Preferably, 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; more preferably, the recognition group is an antibody or an antibody fragment; further preferably, the recognition group is an antibody or an antibody fragment that specifically binds to the surface recognition molecule of the adeno-associated virus, Further preferably, the first dye consists of a recognition group and a fluorescent dye.

11. The method according to any one of claims 1 to 10, wherein The second dye is a nucleic acid fluorescent dye, Preferably, the second dye is one or a combination of two or more selected from cyanine dyes, non-permeable dyes, permeable dyes, intercalating dyes, and DNA double helix minor groove binding dyes.

12. The method according to any one of claims 1 to 11, wherein The method for destroying the capsid is selected from the nucleic acid extraction reagent method or the thermal lysis method.

13. The method according to claim 12, wherein: The heating temperature of the thermal cracking method is 50°C-130°C. Preferably, the heating temperature is selected from 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C, 125°C or 130°C. The heating time of the thermal cracking method is 1 minute to 60 minutes, preferably, the heating time of the thermal cracking 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.

14. The method according to claim 12, wherein the nucleic acid extraction reagent method comprises a method based on one or a combination of two or more of a lysis reagent, a divalent ion reagent, an enzyme reagent, a detergent, and a solid phase matrix.

15. The method according to any one of claims 8 to 14, wherein: In different steps, the method for removing free dye and free nucleic acid is independently selected from: ultracentrifugation, density gradient centrifugation, rate gradient centrifugation, ultrafiltration, size exclusion chromatography, dialysis, affinity capture, immunocapture, microfluidics, dilution method, or a combination of two or more thereof; or The method for removing free nucleic acids is preferably selected from the group consisting of: enzymatic digestion and separation column separation, preferably separation column separation.

16. The method according to any one of claims 8 to 14, wherein: In different steps, the purification method for removing impurities is independently selected from: magnetic separation, centrifugation, delamination, precipitation, solid-liquid separation, or a combination of two or more thereof; preferably magnetic separation.

17. The method according to claim 16, wherein: The magnetic separation method is selected from: magnetic bead separation method and solid phase chip separation method, or a combination of two or more thereof. The magnetic bead separation method includes a surface-loaded functional group magnetic bead separation method; The surface-loaded functional group magnetic bead separation method is selected from: 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 structural complementary magnetic bead separation method, or a combination of two or more thereof.

18. The method according to claim 17, wherein The antigen-antibody magnetic bead separation method comprises: After mixing the magnetic beads carrying virus antibodies with a sample containing the virus, the magnetic beads carrying virus antibodies are adsorbed and separated by an external magnetic field; or After the viral antibodies are mixed with a sample containing the virus, modified magnetic beads that can bind to the viral antibodies are added, and the modified magnetic beads are adsorbed and separated by an external magnetic field.

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