Single-particle analysis method for a viral nucleic acid vector

Abstract

The present invention relates to a method for detecting one or more indicators of a viral nucleic acid vector, belonging to the field of molecular biotechnology. The method may optionally include making a particle size standard curve for the viral nucleic acid vector and / or measuring its particle size and concentration using a concentration standard solution; the method includes steps such as preparing a specific anti-target reagent conjugated with an optical labeling reagent for each parameter in the indicators of the viral nucleic acid vector; detecting the sample using a flow particle detection device; the method can achieve a more in-depth analysis of the "virus titer" obtained by traditional protocols, obtain more accurate detection results, and is of great significance for downstream release decisions, process control, and evaluation of clinical safety.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for single-particle analysis of viral nucleic acid vectors. Background Art

[0002] Viral nucleic acid vectors are a type of viral vectors that can carry foreign genes after genetic modification of their own elements. Among them, the four most representative viral nucleic acid vectors are: lentiviral vectors, retroviral vectors, herpes simplex virus vectors, and recombinant coronavirus vectors. The above vectors have the advantages of carrying large fragments of exogenous target genes, stably and continuously expressing the target nucleic acid, and having a small immune response. Therefore, they have become one of the most widely used gene therapy delivery tools, and it has been proven that such products have achieved clear clinical efficacy. For example, in targeted tumor immunotherapy, chimeric antigen receptor T-cell (CAR-T) therapy has shown superior response rates, so the FDA has approved CAR-T products based on lentiviral and retroviral vectors (Novartis: Kymriah, Kite Pharma: Yescarta) for market. As an important oncolytic virus product, the first oncolytic HSV-1 (orienX010) virus was approved for clinical trials in China in 2009; in 2018, the oncolytic HSV-2 (rHSV2hGM-CSF) virus was approved for clinical trials. The recently intensive clinical trials of non-replicating recombinant coronavirus vaccines (CN111218459A and WO2021184560) are of great significance for the current and future epidemic prevention and control caused by coronaviruses.

[0003] In the prior art, taking the determination of lentivirus titer as an example, the main methods include TCID 50 -GFP fluorescence microscopy method, enzyme-linked immunosorbent assay (ELISA) method, and quantitative PCR method. Among them, TCID 50- The GFP fluorescence protein fluorescence microscopy method requires labeling the lentiviral vector with the green fluorescent protein gene, collecting the supernatant after transfection of cells, diluting it in gradients, observing and counting the virus titer under a fluorescence microscope. This method is rather cumbersome in operation. Moreover, due to the complexity of the genetic expression system, the evaluation of lentiviral titer by this method is often on the low side. The ELISA method evaluates the packaging efficiency by detecting the content of HIV-1 p24 protein, which is more accurate and sensitive than the previous methods. However, this method only measures the particle number of lentivirus and does not measure the actual infectivity of lentivirus to target cells. The conversion of the actual titer measurement of lentivirus by this method is still based on empirical numerical judgment and cannot cover the individual differences of real products. And since there must be a certain amount of free p24 protein in the virus suspension, the ELISA method is considered to overestimate the actual virus titer. The absolute quantitative PCR method can measure the copy number of viral genomes in the genomes of target cells infected by the virus per cell on average, and can reflect the titer of lentivirus, especially the lentivirus without a labeled gene, more accurately to a certain extent. However, it is powerless for the detection of the titer of other non-nucleic acid biological functional indicators of lentivirus, and this method is very sensitive to contamination, and the false positive results measured after contamination are extremely harmful. The above methods are the main methods for evaluating other viral nucleic acid vectors.

[0004] During the preparation of viral nucleic acid vectors, the quality of the virus is usually measured by the virus titer. At present, the above case methods only measure the partial titer of lentivirus, and the formed detection data cannot comprehensively and truly reflect the real titer differences caused by the biological functional differences of different compositions of lentivirus. The accurate determination of the quality index of viral nucleic acid vectors with the virus titer as the core has become a limiting link in the technological development of the gene therapy field. The gene therapy biotechnology field still needs to develop a method for measuring the quality index of lentivirus with high efficiency, accuracy, easy operation, small sample consumption and good repeatability. Summary of the Invention

[0005] An object of the present invention is to provide a method for detecting the biological functional titer of viral nucleic acid, which can realize the simultaneous detection of multiple parameters of a single particle of viral nucleic acid vector, and generate multiple technical and / or product quality indicators at the same time, so as to help solve the optimization of the research and development process of viral nucleic acid vectors and carry out the quality control of gene therapy biotechnology.

[0006] In the first aspect, the present invention provides a method for detecting one or more indicators of a viral nucleic acid vector.

[0007] A method for detecting one or more indicators of a viral nucleic acid vector, comprising:

[0008] Step (1): Prepare optical reagents for each parameter in the virus nucleic acid vector index, and mix the optical reagents with a sample containing the virus nucleic acid vector to specifically bind the optical reagents to the virus nucleic acid vector; the optical reagents include nucleic acid probes or nucleic acid dye reagents, and at least one specific anti-target reagent selected from liposome dye reagents or conjugated fluorescent dye reagents, and the parameters include virus capsid protein marker expression signals and / or virus envelope marker expression signals, as well as nucleic acid signals or target nucleic acid signals; preferably, the parameters include nucleic acid signals or target nucleic acid signals, and envelope protein marker expression signals.

[0009] Step (2): Use a flow particle detection device to detect the sample solution containing the virus nucleic acid vector, and record the scattered light channel detection data and / or fluorescence channel detection data of the sample containing the virus nucleic acid vector; and

[0010] Step (3): Data analysis, calculate the results of each index.

[0011] Among them, the index includes the biological functional titer of the virus nucleic acid vector; the biological functional titer of the virus nucleic acid vector includes: the titer of the virus nucleic acid vector containing nucleic acid, virus capsid and envelope, the titer of the virus nucleic acid vector containing nucleic acid and envelope, the titer of the virus nucleic acid vector containing nucleic acid and virus capsid, the titer of the virus nucleic acid vector containing target nucleic acid, virus capsid and envelope, the titer of the virus nucleic acid vector containing target nucleic acid and envelope, the titer of the virus nucleic acid vector containing target nucleic acid and virus capsid, and the titer of the empty capsid virus nucleic acid vector.

[0012] In some embodiments, the virus nucleic acid vector includes a lentiviral vector, a retroviral vector, a herpes simplex virus vector or a recombinant coronavirus vector.

[0013] In some embodiments, the metrics include the biological functional titer of a lentiviral vector, and the biological functional titer of the lentiviral vector includes: the titer of a lentiviral vector containing nucleic acid, a lentiviral capsid, and an envelope; the titer of a lentiviral vector containing a target nucleic acid, a lentiviral capsid, and an envelope; the titer of a lentiviral vector of nucleic acid and VSV-G; the titer of a lentiviral vector containing a target nucleic acid and VSV-G; the titer of a lentiviral vector containing nucleic acid and an envelope; the titer of a lentiviral vector containing a target nucleic acid and an envelope; the titer of an empty capsid lentiviral vector; the titer of a lentiviral vector lacking VSV-G and containing nucleic acid; the titer of a lentiviral vector lacking VSV-G and containing a target nucleic acid; the titer of a lentiviral vector lacking an envelope and containing nucleic acid; the titer of a lentiviral vector lacking an envelope and containing a target nucleic acid; the titer of a lentiviral vector containing nucleic acid and a lentiviral capsid; the titer of a lentiviral vector containing a target nucleic acid and a lentiviral capsid; the titer of a lentiviral vector lacking a lentiviral capsid and containing nucleic acid; the titer of a lentiviral vector lacking a lentiviral capsid and containing a target nucleic acid; the titer of a lentiviral vector containing nucleic acid and p24 protein; the titer of a lentiviral vector containing a target nucleic acid and p24 protein; the titer of a lentiviral vector lacking p24 protein and containing nucleic acid; the titer of a lentiviral vector lacking p24 protein and containing a target nucleic acid; the titer of a lentiviral vector lacking a lentiviral capsid and containing nucleic acid and an envelope; the titer of a lentiviral vector lacking a lentiviral capsid and containing a target nucleic acid and an envelope; the titer of a lentiviral vector lacking an envelope and containing nucleic acid and a lentiviral capsid; the titer of a lentiviral vector lacking an envelope and containing a target nucleic acid and a lentiviral capsid; the titer of a lentiviral vector lacking a lentiviral capsid and an envelope and containing nucleic acid; or the titer of a lentiviral vector lacking a lentiviral capsid and an envelope and containing a target nucleic acid.

[0014] In some embodiments, the metrics include the biological functional titer of a retroviral vector, and the biological functional titer of the retroviral vector includes: the titer of a retroviral vector containing nucleic acid, a gag capsid protein, and an env envelope protein; the titer of a retroviral vector containing a target nucleic acid, a gag capsid protein, and an env envelope protein; the titer of a retrovirus of nucleic acid and an env envelope protein; the titer of a retroviral vector containing a target nucleic acid and an env envelope protein; the titer of an empty capsid retroviral vector; the titer of a retroviral vector lacking an env envelope protein and containing nucleic acid; the titer of a retroviral vector lacking an env envelope protein and containing a target nucleic acid; the titer of a retroviral vector containing nucleic acid and a gag capsid protein; the titer of a retroviral vector containing a target nucleic acid and a gag capsid protein; the titer of a retroviral vector lacking a gag capsid protein and containing nucleic acid; the retroviral vector lacking a gag capsid protein and containing a target nucleic acid.

[0015] In some embodiments, the indicators include the biological functional titer of a herpes simplex virus vector, and the biological functional titer of the herpes simplex virus vector includes: the titer of a herpes simplex virus vector containing nucleic acid and g envelope glycoprotein, the titer of a herpes simplex virus vector containing target nucleic acid and g envelope glycoprotein, the titer of an empty-shell herpes simplex virus vector, the titer of a herpes simplex virus vector lacking g envelope glycoprotein and containing nucleic acid, and the titer of a herpes simplex virus vector lacking g envelope glycoprotein and containing target nucleic acid.

[0016] In some embodiments, the g envelope glycoprotein can be any one or a combination of envelope glycoprotein gB, envelope glycoprotein gC, envelope glycoprotein gD, envelope glycoprotein gE, envelope glycoprotein gG, envelope glycoprotein gH, envelope glycoprotein gI, envelope glycoprotein gJ, envelope glycoprotein gL, envelope glycoprotein gM, and envelope glycoprotein gN.

[0017] In some embodiments, the indicators include the biological functional titer of a recombinant coronavirus vector, and the biological functional titer of the recombinant coronavirus vector includes: the titer of a recombinant coronavirus vector containing nucleic acid and SPIKE protein, the titer of a recombinant coronavirus vector containing target nucleic acid and SPIKE protein, the titer of an empty-shell recombinant coronavirus vector, the titer of a recombinant coronavirus vector lacking SPIKE protein and containing nucleic acid, and the titer of a recombinant coronavirus vector lacking SPIKE protein and containing target nucleic acid.

[0018] In some embodiments, the detection method further includes the following steps:

[0019] Step (a): Before step (2), prepare a concentration standard solution, detect the concentration standard solution using a flow particle detection device, record the detection data of the scattered light channel and / or fluorescence channel of the concentration standard solution and / or the injection flow rate of the flow particle detection device, and measure the particle count concentration of the sample containing the virus nucleic acid vector; and / or

[0020] Step (b): Before step (2), prepare a particle size standard solution of particle size standards with different particle sizes, detect the particle size standard solution using a flow particle detection device, record the detection data of the scattered light channel of the particle size standard solution, and measure the particle size and particle size distribution of the virus nucleic acid vector.

[0021] In some embodiments, the operation of mixing the optical reagent with the sample containing the virus nucleic acid vector in step (1) may further include adding a surfactant and mixing it with the sample containing the virus nucleic acid vector.

[0022] In some embodiments, in the operation of mixing the optical reagent with the sample containing the virus nucleic acid carrier in step (1), it may further include adding a surfactant and mixing it with the sample containing the virus nucleic acid carrier; the antibody capable of specifically binding to the lentiviral envelope expressed protein is selected from p24 antibody.

[0023] In some embodiments, the surfactant may include at least one of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants. In some embodiments, the surfactant includes at least one selected from Tween-type surfactants, Triton-X100, SDS (sodium dodecyl sulfate), NP40 (nonylphenol polyoxyethylene ether), or polyoxyethylene-type nonionic surfactants.

[0024] In some embodiments, step (a) may include: preparing beads with a known absolute quantity as a concentration standard and preparing a concentration standard solution.

[0025] In some embodiments, step (b) may include: preparing a bead type with the same or similar refractive index as the virus composition as a particle size standard, and using electron microscopy to characterize the median particle size of this particle size standard as the accurate particle size of the particle size standard, and preparing a particle size standard solution containing particle size standards with different particle sizes.

[0026] In some embodiments, the anti-target reagent may include at least one selected from antibodies, antibody fragments, antibody analogs, lectins, aptamers, peptides, growth factors, glycolipids, polysaccharides, ligands, or receptors.

[0027] In some embodiments, the anti-target reagent may be an antibody, antibody fragment or antibody analog, ligand or receptor.

[0028] In some embodiments, the antibody may include at least one selected from VSV-G antibody, gB antibody, gC antibody, gD antibody, gE antibody, gG antibody, gH antibody, gI antibody, gJ antibody, gL antibody, gM antibody, gN antibody, env antibody, SPIKE antibody, an antibody capable of specifically binding to the lentiviral envelope expressed protein, an antibody capable of specifically binding to the retroviral envelope expressed protein, an antibody capable of specifically binding to the herpes simplex virus envelope expressed protein, and an antibody capable of specifically binding to the recombinant coronavirus envelope expressed protein.

[0029] In some embodiments, the antibody capable of specifically binding to the lentiviral envelope expressed protein may include at least one selected from p24 antibody.

[0030] In some embodiments, the antibody capable of specifically binding to the retroviral envelope expressed protein may include at least one selected from gag antibody.

[0031] In some embodiments, the nucleic acid probe may comprise a sequence complementary to the target nucleic acid or the bases of the target nucleic acid, and the 5'-end or 3'-end or middle of the sequence is modified with a fluorescent dye.

[0032] In some embodiments, the nucleic acid dye reagent comprises a cyanine dye, a non-permeable dye, a permeable dye, an intercalating dye, a DNA minor groove binding dye.

[0033] In some embodiments, the lipid membrane dye reagent may comprise a lipophilic fluorescent dye selected from those capable of binding to cell membranes and other lipid-soluble membrane structures.

[0034] In some embodiments, the fluorescent dye is a fluorescent molecule, a fluorescent material or a combination thereof, including at least one selected from organic fluorescent molecules, fluorescent proteins, nucleic acid dyes, lipid membrane dyes, quantum dots.

[0035] In some embodiments, the lipid membrane dye in the lipid membrane dye reagent or the fluorescent dye is independently selected from at least one of DiD dye, DiO dye, DiI dye, DiR dye, DiA dye, Di-8-Anepps dye, Di-4-ANEPPS dye, PHK26 dye, PKH67 dye, CellMask Green dye, CellMask Orange dye, CellMask Red dye, CellVue Lavender dye, CellVue Plum dye, CellVue NIR780 dye, carboxyfluorescein diacetate succinimidyl ester.

[0036] In some embodiments, the nucleic acid dyes in the nucleic acid dye reagent or the fluorescent dye are each independently selected from Acridine Orange, Acridine Orange, Actinomycin D, 7-AAD (7-Aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodide (462 / 481), BOBO-3 Iodide(570 / 602), 1 mM solution in DMSO, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), FluoroPure grade, dihydroethidium (hydroethidine), dihydroethidium (hydroethidine), dihydroethidium (hydroethidine), Ethidium Homodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium Monoazide Bromide (EMA), Hexidium Iodide, Hoechst 33258, Pentahydrate (bis-Benzimide), Hoechst 33258, Pentahydrate (bis-Benzimide), Hoechst 33258, Pentahydrate (bis-Benzimide) FluoroPureGrade, Hoechst 33342, Trihydrochloride, Trihydrate, Hoechst 33342, Trihydrochloride, Trihydrate, Hoechst 33342, Trihydrochloride, Trihydrate - FluoroPure Grade, Hoechst 34580, LDS 751, NeuroTrace 435 / 455 Blue Fluorescent Nissl Stain, NeuroTrace 500 / 525 Green Fluorescent Nissl Stain, NeuroTrace 530 / 615 Red Fluorescent Nissl Stain, NeuroTrace 640 / 660 Deep-Red Fluorescent Nissl Stain, POPO-1 Iodide (434 / 456), POPO-3 Iodide (534 / 570), PO-PRO-1 Iodide(435 / 455), Propidium Iodide, Propidium Iodide - FluoroPure Grade, Propidium Iodide, Quant-iT OliGreen ssDNA Assay Kit, Quant-iT OliGreen ssDNA Reagent, Quant-iT PicoGreen dsDNA Assay Kit, Quant-iT PicoGreen dsDNA Reagent, Quant-iT RiboGreen RNA Assay Kit, Quant-iT RiboGreen RNA Reagent, RediPlate 96 RiboGreen RNA Quantitation Kit, SYBR Gold Nucleic Acid Gel Stain, SYBR Green I Nucleic Acid Gel Stain, SYBR Green I, SYBR Green II RNA Gel Stain, SYBR Safe DNA Gel Stain, SYTO 40 Blue Fluorescent Nucleic Acid Stain, SYTO 41 Blue Fluorescent Nucleic Acid Stain, SYTO Blue Fluorescent Nucleic Acid Stain Sampler Kit (SYTO dyes 40, 41, 42, 45), SYTO 9 Green Fluorescent Nucleic Acid Stain, SYTO 11 Green Fluorescent Nucleic Acid Stain, SYTO 12 Green Fluorescent Nucleic Acid Stain, SYTO 13 Green Fluorescent Nucleic Acid Stain, SYTO 14 Green Fluorescent Nucleic Acid Stain, SYTO 16 Green Fluorescent Nucleic Acid Stain, SYTO 21 Green Fluorescent Nucleic Acid Stain, SYTO 24 Green Fluorescent Nucleic Acid Stain, SYTO BC Green Fluorescent Nucleic Acid Stain, SYTO GreenFluorescent Nucleic Acid Stain Sampler Kit #1 - SYTO dyes 11 - 16, SYTO 82 Orange Fluorescent Nucleic Acid Stain, SYTO 83 Orange Fluorescent Nucleic Acid Stain, SYTO 84 Orange Fluorescent Nucleic Acid Stain, SYTO 85 Orange Fluorescent Nucleic Acid Stain, SYTO Orange Fluorescent Nucleic Acid Stain Sampler Kit - SYTO dyes, SYTO 17 Red Fluorescent Nucleic Acid Stain, SYTO 59 Red Fluorescent Nucleic Acid Stain, SYTO 60 Red Fluorescent Nucleic Acid Stain, SYTO 61 Red Fluorescent Nucleic Acid Stain, SYTO 62 Red Fluorescent Nucleic Acid Stain, SYTO 63 Red Fluorescent Nucleic Acid Stain, SYTO 64 Red Fluorescent Nucleic Acid Stain, SYTO Red Fluorescent Nucleic Acid Stain Sampler Kit - SYTO dyes 17 and 59 - 64, SYTO RNASelect Green Fluorescent cell Stain, SYTOX Blue Dead Cell Stain, SYTOX Blue Nucleic Acid Stain, SYTOX Green Dead Cell Dye, SYTOX Green Nucleic Acid Stain, SYTOX Orange Nucleic Acid Stain, SYTOX Orange Dead Cell Dye, SYTOX Red Dead Cell Stain, SYTOX Dead Cell Multiplex Staining Kit, TO - PRO - 1 Iodide (515 / 531), TO - PRO - 3 Iodide (642 / 661), TOTO - 1 Iodide (514 / 533), TOTO - 3 IodideAt least one of (642 / 660), YO-PRO-1 Iodide (491 / 509), YO-PRO-3 Iodide (612 / 631), YOYO-1 Iodide (491 / 509), YOYO-3 Iodide (612 / 631), HCS NuclearMask Deep Red Stain, HCS NuclearMask Blue Stain, HCS NuclearMask Red Stain, and UltraPure ethidium bromide.

[0037] In some embodiments, the anti-target reagent is an antibody, and the antibody includes at least one of an antibody against VSV-G, an antibody against env envelope protein, an antibody against g envelope glycoprotein, an antibody against SPIKE protein, an antibody against gag, an antibody against p24, or an antibody against viral capsid protein or an antibody against envelope-expressed protein.

[0038] In some embodiments, the anti-target reagent is a ligand and / or a receptor, and the ligand and / or the receptor is at least one of a ligand and / or a receptor for VSV-G, a ligand and / or a receptor for env envelope protein, a ligand and / or a receptor for g envelope glycoprotein, a ligand and / or a receptor for SPIKE protein, a ligand and / or a receptor for gag, a ligand and / or a receptor for p24, a ligand and / or a receptor for viral capsid protein, or a ligand and / or a receptor for envelope-expressed protein.

[0039] In some embodiments, the fluorescent dye includes a fluorescent molecule, a fluorescent material, or a combination thereof, and the fluorescent dye is directly conjugated to the anti-target reagent; or the fluorescent dye is indirectly conjugated to the anti-target reagent via a recognition group, and the recognition group includes an antibody, an antigen, a receptor, or a polysaccharide, or any other particle or molecule, or any part thereof, and the recognition group can specifically recognize the anti-target reagent.

[0040] In some embodiments, mixing the optical reagent with the sample containing the viral nucleic acid carrier in step (1) includes mixing the optical reagent and a surfactant with the sample containing the viral nucleic acid carrier.

[0041] In some embodiments, step (2) may include photographing, counting, and measuring the signal intensity value of particles having specific optical characteristics in the sample using a flow particle detection device.

[0042] In some embodiments, step (2) may include detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels or a flow particle detection device with at least two fluorescence channels. One fluorescence channel is used to characterize the nucleic acid or target nucleic acid signal, and the other fluorescence channel is used to characterize the viral envelope marker expression signal. Analyze the fluorescence channel detection results of the particle population with event positive signals, record the detection result scatter plot, and gate the obtained result scatter plot, where,

[0043] The particle population with event positive signals, nucleic acid positive signals, and viral envelope marker expression positive signals is the particle of the viral nucleic acid vector containing nucleic acid and envelope, or the particle population with event positive signals, target nucleic acid positive signals, and viral envelope marker expression positive signals is the particle of the viral nucleic acid vector containing target nucleic acid and envelope;

[0044] The particle population with event positive signals, viral envelope marker expression positive signals, and nucleic acid negative signals is the particle of the empty capsid viral nucleic acid vector;

[0045] The particle population with event positive signals, nucleic acid positive signals, and viral envelope marker expression negative signals is the particle of the viral nucleic acid vector with a missing envelope containing nucleic acid, or the particle population with event positive signals, target nucleic acid positive signals, and viral envelope marker expression negative signals is the particle of the viral nucleic acid vector with a missing envelope containing target nucleic acid.

[0046] In some embodiments, the viral nucleic acid vector is a lentiviral vector. Step (2) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels or a flow particle detection device with at least two fluorescence channels. One fluorescence channel is used to characterize the nucleic acid or target nucleic acid signal, and the other fluorescence channel is used to characterize the VSV-G expression signal. Analyze the fluorescence channel detection results of the particle population with event positive signals, record the detection result scatter plot, and gate the obtained result scatter plot, where,

[0047] The particle population with event positive signals, nucleic acid positive signals, and VSV-G expression positive signals is the particle of the lentiviral vector containing nucleic acid and VSV-G, or the particle population with event positive signals, target nucleic acid positive signals, and VSV-G expression positive signals is the particle of the lentiviral vector containing target nucleic acid and VSV-G;

[0048] The particle population with event positive signals, VSV-G expression positive signals, and nucleic acid negative signals is the particle of the empty capsid lentiviral vector;

[0049] Particles with a positive event signal, a positive nucleic acid signal, and a negative VSV-G expression signal are particles of a lentiviral vector lacking VSV-G containing nucleic acid, or a population of particles with a positive event signal, a positive target nucleic acid signal, and a negative VSV-G expression signal is a population of particles of a lentiviral vector lacking VSV-G containing the target nucleic acid.

[0050] In some embodiments, the viral nucleic acid vector is a retroviral vector, and step (2) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels or a flow particle detection device with at least two fluorescence channels. One fluorescence channel is used to characterize the nucleic acid or target nucleic acid signal, and the other fluorescence channel is used to characterize the viral envelope marker expression signal. Analyze the fluorescence channel detection results of the population of particles with a positive event signal, record the scatter plot of the detection results, and set a gate for the obtained scatter plot of the results. Among them,

[0051] A population of particles with a positive event signal, a positive nucleic acid signal, and a positive env envelope protein expression signal is a population of particles of a retroviral vector containing nucleic acid and the env envelope protein, or a population of particles with a positive event signal, a positive target nucleic acid signal, and an env envelope protein expression signal is a population of particles of a retroviral vector containing the target nucleic acid and the env envelope protein;

[0052] A population of particles with a positive event signal, a positive env envelope protein expression signal, and a negative nucleic acid signal is a population of empty retroviral vector particles;

[0053] Particles with a positive event signal, a positive nucleic acid signal, and a negative env envelope protein expression signal are particles of a retroviral vector lacking the env envelope protein containing nucleic acid, or a population of particles with a positive event signal, a positive target nucleic acid signal, and a negative env envelope protein expression signal is a population of particles of a retroviral vector lacking the env envelope protein containing the target nucleic acid.

[0054] In some embodiments, the viral nucleic acid vector is a herpes simplex virus vector, and step (2) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels or a flow particle detection device with at least two fluorescence channels. One fluorescence channel is used to characterize the nucleic acid or target nucleic acid signal, and the other fluorescence channel is used to characterize the viral envelope marker expression signal. Analyze the fluorescence channel detection results of the population of particles with a positive event signal, record the scatter plot of the detection results, and set a gate for the obtained scatter plot of the results. Among them,

[0055] A population of particles with an event positive signal, a nucleic acid positive signal, and a positive signal for g envelope glycoprotein expression is a particle of a herpes simplex virus vector containing nucleic acid and g envelope glycoprotein, or a population of particles with an event positive signal, a target nucleic acid positive signal, and a g envelope glycoprotein expression signal is a particle of a herpes simplex virus vector containing target nucleic acid and g envelope glycoprotein;

[0056] A population of particles with an event positive signal, a positive signal for g envelope glycoprotein expression, and a nucleic acid negative signal is a particle of an empty-shell herpes simplex virus vector;

[0057] A population of particles with an event positive signal, a nucleic acid positive signal, and a negative signal for g envelope glycoprotein expression is a particle of a herpes simplex virus vector lacking g envelope glycoprotein and containing nucleic acid, or a population of particles with an event positive signal, a target nucleic acid positive signal, and a negative signal for g envelope glycoprotein expression is a particle of a herpes simplex virus vector lacking g envelope glycoprotein and containing target nucleic acid.

[0058] In some embodiments, the viral nucleic acid vector is a recombinant coronavirus vector, and step (2) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels or a flow particle detection device with at least two fluorescence channels. One fluorescence channel is used to characterize the nucleic acid or target nucleic acid signal, and the other fluorescence channel is used to characterize the viral envelope marker expression signal. Analyze the fluorescence channel detection results of the population of particles with an event positive signal, record the detection result scatter plot, and gate the obtained result scatter plot, wherein,

[0059] A population of particles with an event positive signal, a nucleic acid positive signal, and a positive signal for SPIKE protein expression is a particle of a recombinant coronavirus vector containing nucleic acid and SPIKE protein, or a population of particles with an event positive signal, a target nucleic acid positive signal, and a SPIKE protein expression signal is a particle of a recombinant coronavirus vector containing target nucleic acid and SPIKE protein;

[0060] A population of particles with an event positive signal, a positive signal for SPIKE protein expression, and a nucleic acid negative signal is a particle of an empty-shell recombinant coronavirus vector;

[0061] A population of particles with an event positive signal, a nucleic acid positive signal, and a negative signal for SPIKE protein expression is a particle of a recombinant coronavirus vector lacking SPIKE protein and containing nucleic acid, or a population of particles with an event positive signal, a target nucleic acid positive signal, and a negative signal for SPIKE protein expression is a particle of a recombinant coronavirus vector lacking SPIKE protein and containing target nucleic acid.

[0062] In some embodiments, step (2) may include detecting using a flow cytometry particle detection device with a scattered light channel and at least two fluorescence channels or a flow cytometry particle detection device with at least two fluorescence channels. One fluorescence channel is used to characterize the nucleic acid signal or target nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral capsid protein marker. Analyze the fluorescence channel detection results of the particle population with event positive signals, record the detection result scatter plot, and gate the obtained result scatter plot. Among them,

[0063] The particle population with event positive signals, nucleic acid positive signals, and viral capsid protein marker expression positive signals is the particle population of the viral nucleic acid vector with nucleic acid and lentiviral capsid, or the particle population with event positive signals, target nucleic acid positive signals, and viral capsid protein marker expression positive signals is the particle population of the viral nucleic acid vector containing the target nucleic acid and viral capsid;

[0064] The particle population with event positive signals, viral capsid protein marker expression positive signals, and nucleic acid negative signals is the particle population of the empty capsid viral nucleic acid vector;

[0065] The particle population with event positive signals, nucleic acid positive signals, and viral capsid protein marker expression negative signals is the particle population of the viral nucleic acid vector with nucleic acid but lacking the viral capsid, or the particle population with event positive signals, target nucleic acid positive signals, and viral capsid protein marker expression negative signals is the particle population of the viral nucleic acid vector containing the target nucleic acid but lacking the viral capsid.

[0066] In some embodiments, the viral nucleic acid vector is a lentiviral vector. Step (2) includes detecting using a flow cytometry particle detection device with a scattered light channel and at least two fluorescence channels or a flow cytometry particle detection device with at least two fluorescence channels. In the detection of lentiviral vectors, preferably, one fluorescence channel is used to characterize the nucleic acid signal or target nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the lentiviral p24 protein. Analyze the fluorescence channel detection results of the particle population with event positive signals, record the detection result scatter plot, and gate the obtained result scatter plot. Among them,

[0067] The particle population with event positive signals, nucleic acid positive signals, and lentiviral p24 protein expression positive signals is the particle population of the lentiviral vector containing nucleic acid and lentiviral p24, or the particle population with event positive signals, target nucleic acid positive signals, and lentiviral p24 protein expression positive signals is the particle population of the lentiviral vector containing the target nucleic acid and p24 protein;

[0068] The particle population with event positive signals, lentiviral p24 protein expression positive signals, and nucleic acid negative signals is the particle population of the empty capsid lentiviral vector;

[0069] A population of particles having an event positive signal, a nucleic acid positive signal, and a negative signal for lentiviral p24 protein expression is a population of particles of a lentiviral vector lacking p24 protein containing nucleic acid, or a population of particles having an event positive signal, a target nucleic acid positive signal, and a negative signal for p24 protein expression is a population of particles of a lentiviral vector lacking p24 protein containing the target nucleic acid.

[0070] In some embodiments, the viral nucleic acid vector is a retroviral vector, and step (2) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels or a flow particle detection device with at least two fluorescence channels. In the retroviral vector detection, preferably, one fluorescence channel is used to characterize the nucleic acid signal or the target nucleic acid signal, and another fluorescence channel is used to characterize the gag coat protein expression signal. Analyze the fluorescence channel detection results of the population of particles having an event positive signal, record the detection result scatter plot, and gate the obtained result scatter plot, wherein,

[0071] A population of particles having an event positive signal, a nucleic acid positive signal, and a positive signal for gag coat protein expression is a population of particles of a retroviral vector containing nucleic acid and gag coat protein, or a population of particles having an event positive signal, a target nucleic acid positive signal, and a positive signal for gag coat protein expression is a population of particles of a retroviral vector containing the target nucleic acid and gag coat protein;

[0072] A population of particles having an event positive signal, a positive signal for gag coat protein expression, and a negative nucleic acid signal is a population of particles of an empty capsid retroviral vector;

[0073] A population of particles having an event positive signal, a nucleic acid positive signal, and a negative signal for gag coat protein expression is a population of particles of a retroviral vector lacking gag coat protein containing nucleic acid, or a population of particles having an event positive signal, a target nucleic acid positive signal, and a negative signal for gag coat protein expression is a population of particles of a retroviral vector lacking gag coat protein containing the target nucleic acid.

[0074] In some embodiments, step (2) may include performing multi-parameter detection using a flow particle detection device with a scattered light channel and at least one or at least two fluorescence channels or a flow particle detection device with at least one or at least two fluorescence channels. At least one fluorescence channel is used to characterize the nucleic acid signal or the target nucleic acid signal, and other fluorescence channels are used to characterize the expression signal of at least one viral coat protein marker or the expression signal of at least one viral envelope marker. Analyze the fluorescence channel detection results of the population of particles having an event positive signal, record the detection results on a single or multiple scatter plots, and gate the obtained result scatter plots, wherein,

[0075] A population of particles with positive event signals, positive nucleic acid signals, positive expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector containing nucleic acid, viral capsid, and envelope; or a population of particles with positive event signals, positive target nucleic acid signals, positive expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector containing target nucleic acid, viral capsid, and envelope;

[0076] A population of particles with positive event signals and negative nucleic acid signals is a population of particles of an empty capsid viral nucleic acid vector, and the population of particles of the empty capsid viral nucleic acid vector includes at least one of the following populations of particles: a) a population of particles of an empty capsid viral nucleic acid vector with positive event signals, negative nucleic acid signals, negative expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers; b) a population of particles of an empty capsid viral nucleic acid vector with positive event signals, negative nucleic acid signals, positive expression signals of viral capsid protein markers, and negative expression signals of viral envelope markers; c) a population of particles of an empty capsid viral nucleic acid vector with positive event signals, negative nucleic acid signals, positive expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers; d) a population of particles of an empty capsid viral nucleic acid vector with positive event signals, negative nucleic acid signals, negative expression signals of viral capsid protein markers, and negative expression signals of viral envelope markers;

[0077] A population of particles with positive event signals, positive nucleic acid signals, negative expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector lacking a viral capsid containing nucleic acid and envelope; or a population of particles with positive event signals, positive target nucleic acid signals, negative expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector lacking a viral capsid containing target nucleic acid and envelope;

[0078] A population of particles with positive event signals, positive nucleic acid signals, positive expression signals of viral capsid protein markers, and negative expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector lacking an envelope containing nucleic acid and capsid, or a population of particles with positive event signals, positive target nucleic acid signals, positive expression signals of viral capsid protein markers, and negative expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector lacking an envelope containing target nucleic acid and viral capsid;

[0079] A population of particles having a positive event signal, a positive nucleic acid signal, a negative signal for the expression of a viral capsid protein marker, and a negative signal for the expression of a viral envelope marker is a population of particles of a viral nucleic acid vector containing a nucleic acid and lacking a viral capsid and envelope; or a population of particles having a positive event signal, a positive target nucleic acid signal, a negative signal for the expression of a viral capsid protein marker, and a negative signal for the expression of a viral envelope marker is a population of particles of a viral nucleic acid vector containing a target nucleic acid and lacking a viral capsid and envelope. The fluorescence channel is a device that uses incident light to enable a target to be measured to emit a fluorescence signal after absorbing light waves of a specific wavelength. Different types of fluorescence channels can be obtained through different spectral splitting means.

[0080] In some embodiments, step (a) may include using a flow particle detection device to detect and record a concentration standard solution and a sample solution containing a viral nucleic acid vector under the same injection pressure and detection time.

[0081] The calculation method for the particle count concentration in the viral nucleic acid vector is as follows:

[0082] Particle count concentration in the viral nucleic acid vector (particles / mL) = c × A1 ÷ A2;

[0083] Where,

[0084] c is the concentration of the concentration standard solution, particles / mL;

[0085] A1 is the number of particles with a positive event signal in the sample solution containing the viral nucleic acid vector obtained by detecting the gate using the scattered light channel within the same unit time as A2;

[0086] A2 is the number of particles with a positive event signal in the concentration standard solution obtained by detecting the gate using the scattered light channel within the same unit time as A1.

[0087] In some embodiments, step (a) may include using a flow particle detection device to detect and record a concentration standard solution and a sample solution containing a viral nucleic acid vector under the same injection pressure and detection time.

[0088] The calculation method for the particle count concentration in the viral nucleic acid vector is

[0089] Particle count concentration in the viral nucleic acid vector (particles / mL) = A1 / K;

[0090] Where,

[0091] K is the volume flow rate per unit time of the flow particle detection device;

[0092] A1 is the number of particles with a positive event signal in the sample solution containing the viral nucleic acid vector obtained by detecting the gate using the scattered light channel, converted to the number of particles within the unit time.

[0093] In some embodiments, step (b) may include using a flow particle detection device to detect a particle size standard solution containing particle size standard products with different particle sizes and a sample solution containing a viral nucleic acid vector under the same detection setting parameters and detection time. The detection setting parameters include laser power and scattering channel attenuation coefficient. The calculation method for the particle size and particle size distribution of the viral nucleic acid vector is as follows: based on the detection results of the scattering light channel detection gate of the particle size standard solution containing particle size standard products with different particle sizes, a relationship curve between the average single-particle scattering light intensity and the particle size is constructed, and the detection results of the scattering light channel detection gate of the sample solution containing the viral nucleic acid vector are calculated through the relationship curve between the average single-particle scattering light intensity and the particle size to obtain the particle size and particle size distribution of the virus in the sample solution containing the viral nucleic acid vector.

[0094] In some embodiments, the calculation method for the titer of the viral nucleic acid vector containing nucleic acid and envelope protein may be: the product of P1 and the particle count concentration in the viral nucleic acid vector, where P1 is the percentage of the number of particles in the particle population with event positive signal, nucleic acid positive signal, and envelope protein expression positive signal to the total number of particles in all particle populations with event positive signal.

[0095] In some embodiments, the calculation method for the titer of the viral nucleic acid vector containing nucleic acid and viral envelope may be: the product of P1a and the particle count concentration in the viral nucleic acid vector, where P1a is the percentage of the number of particles in the particle population with event positive signal, nucleic acid positive signal, and viral envelope marker expression positive signal to the total number of particles in all particle populations with event positive signal.

[0096] In some embodiments, the calculation method of the titer of the empty capsid lentiviral vector can be: the product of P2 and the particle counting concentration in the viral nucleic acid vector, where P2 is the percentage of the number of particles in the particle population with positive event signal, positive envelope protein expression signal, and negative nucleic acid signal in the total number of particles in all particle populations with positive event signal; or P2 is the percentage of the number of particles in the particle population with positive event signal, positive viral capsid protein marker expression signal, and negative nucleic acid signal in the total number of particles in all particle populations with positive event signal; or P2 is the percentage of the number of particles in the particle population of the empty capsid lentiviral vector with positive event signal, negative nucleic acid signal, negative viral capsid protein marker expression signal, and positive viral envelope marker expression signal in the total number of particles in all particle populations with positive event signal; or P2 is the percentage of the number of particles in the particle population of the empty capsid viral nucleic acid vector with positive event signal, negative nucleic acid signal, positive viral capsid protein marker expression signal, and negative viral envelope marker expression signal in the total number of particles in all particle populations with positive event signal; or P2 is the percentage of the number of particles in the particle population of the empty capsid viral nucleic acid vector with positive event signal, negative nucleic acid signal, positive viral capsid protein marker expression signal, and positive viral envelope marker expression signal in the total number of particles in all particle populations with positive event signal; or P2 is the percentage of the number of particles in the particle population of the empty capsid viral nucleic acid vector with positive event signal, negative nucleic acid signal, negative viral capsid protein marker expression signal, and negative viral envelope marker expression signal in the total number of particles in all particle populations with positive event signal.

[0097] In some embodiments, the calculation method of the titer of the nucleic acid-containing virus nucleic acid vector lacking an envelope can be: the product of P3 and the particle counting concentration in the viral nucleic acid vector, where P3 is the percentage of the total number of points with positive event signal, positive nucleic acid signal, and negative envelope expression signal in the total number of particles in all particle populations with positive event signal.

[0098] In some embodiments, the calculation method of the titer of the nucleic acid-containing virus nucleic acid vector lacking an envelope can be: the product of P4 and the particle counting concentration in the viral nucleic acid vector, where P4 is the percentage of the total number of points with positive event signal, positive nucleic acid signal, and negative viral envelope marker expression signal in the total number of particles in all particle populations with positive event signal.

[0099] In some embodiments, the calculation method of the titer of the nucleic acid-containing virus nucleic acid vector lacking a viral capsid can be: the product of P5 and the particle counting concentration in the viral nucleic acid vector, where P5 is the percentage of the total number of points with positive event signal, positive nucleic acid signal, and negative viral capsid protein marker expression signal in the total number of particles in all particle populations with positive event signal.

[0100] In some embodiments, the method for calculating the titer of the nucleic acid-containing virus nucleic acid vector lacking the capsid protein may be: the product of P6 and the particle counting concentration in the virus nucleic acid vector, where P6 is the percentage of the total number of points with event positive signal, nucleic acid positive signal, and negative virus capsid protein expression signal in the total number of particles in all particle populations with event positive signal.

[0101] In some embodiments, the method for calculating the titer of the lentiviral vector lacking the virus capsid and containing nucleic acid and envelope may be: the product of P7 and the particle counting concentration in the virus vector, where P7 is the percentage of the particle population with event positive signal, nucleic acid positive signal, negative virus capsid protein expression signal, and positive virus envelope expression signal in the total number of particles in all particle populations with event positive signal.

[0102] In some embodiments, the method for calculating the titer of the virus nucleic acid vector lacking the virus envelope and containing nucleic acid and virus capsid may be: the product of P8 and the particle counting concentration in the virus nucleic acid vector, where P8 is the percentage of the particle population with event positive signal, nucleic acid positive signal, positive virus capsid protein expression signal, and negative virus envelope expression signal in the total number of particles in all particle populations with event positive signal.

[0103] In some embodiments, the method for calculating the titer of the virus nucleic acid vector lacking the virus capsid and envelope and containing nucleic acid may be: the product of P9 and the particle counting concentration in the virus vector, where P9 is the percentage of the particle population with event positive signal, nucleic acid positive signal, negative virus capsid protein expression signal, and negative virus envelope expression signal in the total number of particles in all particle populations with event positive signal.

[0104] In some embodiments, the method for calculating the titer of the virus nucleic acid vector containing the target nucleic acid and envelope protein may be: the product of P1b and the particle counting concentration in the virus nucleic acid vector, where P1b is the percentage of the number of particles in the particle population with event positive signal, target nucleic acid positive signal, and positive envelope protein expression signal in the total number of particles in all particle populations with event positive signal.

[0105] In some embodiments, the method for calculating the titer of the virus nucleic acid vector containing the target nucleic acid and envelope may be: the product of P1c and the particle counting concentration in the virus nucleic acid vector, where P1c is the percentage of the number of particles in the particle population with event positive signal, target nucleic acid positive signal, and positive virus envelope marker expression signal in the total number of particles in all particle populations with event positive signal.

[0106] In some embodiments, the calculation method of the titer of the viral nucleic acid vector lacking envelope protein and containing the target nucleic acid may be: the product of P3a and the particle count concentration in the viral nucleic acid vector, where P3a is the percentage of the total number of points with event positive signal, target nucleic acid positive signal, and negative envelope protein expression signal in the total number of particles in all particle populations with event positive signal.

[0107] In some embodiments, the calculation method of the titer of the viral nucleic acid vector lacking envelope and containing the target nucleic acid may be: the product of P4a and the particle count concentration in the viral nucleic acid vector, where P4a is the percentage of the total number of points with event positive signal, target nucleic acid positive signal, and negative viral envelope marker expression signal in the total number of particles in all particle populations with event positive signal.

[0108] In some embodiments, the calculation method of the titer of the viral nucleic acid vector lacking viral capsid and containing the target nucleic acid may be: the product of P5a and the particle count concentration in the viral nucleic acid vector, where P5a is the percentage of the total number of points with event positive signal, target nucleic acid positive signal, and negative viral capsid protein marker expression signal in the total number of particles in all particle populations with event positive signal.

[0109] In some embodiments, the calculation method of the titer of the viral nucleic acid vector lacking viral capsid protein and containing the target nucleic acid is: the product of P6a and the particle count concentration in the viral nucleic acid vector, where P6a is the percentage of the total number of points with event positive signal, target nucleic acid positive signal, and negative viral capsid protein expression signal in the total number of particles in all particle populations with event positive signal.

[0110] In some embodiments, the calculation method of the titer of the viral nucleic acid vector lacking viral capsid protein and containing the target nucleic acid and envelope may be: the product of P7a and the particle count concentration in the viral nucleic acid vector, where P7a is the percentage of the particle population with event positive signal, target nucleic acid positive signal, negative viral capsid protein expression signal, and positive viral envelope expression signal in the total number of particles in all particle populations with event positive signal.

[0111] In some embodiments, the calculation method of the titer of the viral nucleic acid vector lacking viral envelope and containing the target nucleic acid and viral capsid may be: the product of P8a and the particle count concentration in the viral nucleic acid vector, where P8a is the percentage of the particle population with event positive signal, target nucleic acid positive signal, positive viral capsid protein expression signal, and negative viral envelope expression signal in the total number of particles in all particle populations with event positive signal.

[0112] In some embodiments, the calculation method of the titer of the viral nucleic acid vector containing the deletion viral envelope and capsid of the target nucleic acid can be: the product of P9a and the particle count concentration in the viral nucleic acid vector, where P9a is the percentage of the particle population with positive event signal, positive target nucleic acid signal, negative viral capsid protein expression signal, and negative viral envelope expression signal in the total number of particles in all particle populations with positive event signal.

[0113] In some embodiments, the flow cytometry particle detection device is a particle analysis and detection device capable of realizing the directional flow of the sample flow.

[0114] In some embodiments, the directional fluid system is composed of a sampling unit and a flow unit.

[0115] In some embodiments, the particle analysis and detection device includes an optical system and a particle detector; the optical system is used to provide a light source for irradiating the sample particle population, collect the light emitted by the light source, or transmit or block the light based on the light correlation emission angle.

[0116] In some embodiments, the particle detector is composed of a photoelectric sensor and a signal conditioning circuit with a function of limited-band filtering high-frequency noise; the signal conditioning circuit with the function of limited-band filtering high-frequency noise is used to obtain the information of the particle population after being irradiated by the light source.

[0117] In some embodiments, the nucleic acid positive signal, the target nucleic acid positive signal, the positive signal of the expression of the viral envelope protein marker, the positive signal of the expression of the lentiviral vector VSV-G, the positive signal of the env envelope protein of the retroviral vector, the positive signal of the g capsid glycoprotein of the herpes simplex virus vector, or the positive signal of the recombinant coronavirus SPIKE protein indicates the particle signal located on the right side or the upper side of the dividing line of the gate set in the scatter plot of the results obtained for the corresponding index. The nucleic acid negative signal, the target nucleic acid negative signal, the negative signal of the expression of the lentiviral envelope protein marker, the negative signal of the expression of the lentiviral vector VSV-G, the negative signal of the env envelope protein of the retroviral vector, the negative signal of the g capsid glycoprotein of the herpes simplex virus vector, or the negative signal of the recombinant coronavirus SPIKE protein indicates the particle signal located on the left side or the lower side of the dividing line of the gate set in the scatter plot of the results obtained for the corresponding index.

[0118] In some embodiments, the scattered light positive signal refers to the particle signal value of the scattered light signal generated by particles with a particle size not less than 30 nm and not more than 600 nm during the determination of the concentration and particle size distribution of the viral nucleic acid vector sample in the flow cytometry particle population.

[0119] In some embodiments, the scattered light negative signal refers to the particle signal value that is lower than the scattered light signal generated by particles with a diameter smaller than 30 nm during the measurement of the concentration and particle size distribution of the viral nucleic acid carrier sample in the flow cytometry particle population.

[0120] In some embodiments, the event positive signal refers to including the scattered light positive signal or, after replacing the scattered light signal with a fluorescence signal through fluorescence labeling technology, using the event as the output signal to represent particles with a diameter not less than 30 nm and not more than 600 nm.

[0121] In some embodiments, the event negative signal refers to including the scattered light positive signal or, after replacing the scattered light signal with a fluorescence signal through fluorescence labeling technology, using the event as the output signal to represent particles with a diameter smaller than 30 nm.

[0122] In some embodiments, a viral nucleic acid carrier containing nucleic acid and envelope protein, or a viral nucleic acid carrier having nucleic acid and an envelope, or a viral nucleic acid carrier having nucleic acid, a viral capsid, and an envelope is a completely packaged structural viral nucleic acid carrier.

[0123] In some embodiments, the detection parameters for the detection in step (2) are as follows: laser detectors 488 nm + 638 nm; single laser channel detection Laser: 20 / 50 mW, 488 nm; scattered light attenuation: 0.2%; detection pressure: 1 kPa; signal type: Large signal, using the scattered light channel; the detection parameters are used to detect the concentration of retroviruses or lentiviral vectors in the sample solution.

[0124] In some embodiments, the detection parameters for the detection in step (2) are as follows: laser detectors 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, and under this condition, using the scattered light channel, FITC, and PC5 fluorescence dual channels; the detection parameters are used to detect particles of lentiviral vectors containing nucleic acid and VSV-G or retroviruses containing nucleic acid and VSV-G, particles of empty capsid lentiviral vectors or empty capsid retroviruses, particles of lentiviral vectors lacking VSV-G containing nucleic acid or retroviruses lacking VSV-G containing nucleic acid, and / or particles of lentiviral vectors lacking VSV-G containing the target nucleic acid or retroviruses lacking VSV-G containing the target nucleic acid.

[0125] In some embodiments, the detection parameters for the detection in step (2) are as follows: laser detector 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1 kPa; signal type: small signal. Under these conditions, the scattered light channel is used; the fixed Sampling pressure is 1.0 kPa; the detection parameters are used to detect the particle size distribution of retroviruses or lentiviral vectors in the sample solution.

[0126] In some embodiments, the detection parameters for the detection in step (2) are as follows: laser detector 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1 kPa; signal type: small signal. Under these conditions, the FITC and PC5 fluorescence dual channels are used; the detection parameters are used to detect the particles of lentiviral vectors containing capsids and VSV-G or retroviruses containing capsids and VSV-G, particles of lentiviral vectors lacking capsids but containing VSV-G or retroviruses lacking capsids but containing VSV-G, and / or particles of lentiviral vectors containing capsids but lacking VSV-G or retroviruses containing capsids but lacking VSV-G in the sample solution.

[0127] In some embodiments, the detection parameters for the detection in step (2) are as follows: laser detector 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1 kPa; signal type: small signal. Under these conditions, the scattered light channel and the PC5 fluorescence channel are used; the detection parameters are used to detect the particles of lentiviral vectors containing envelopes or retroviruses containing envelopes, and / or particles of lentiviral vectors lacking envelopes or retroviruses lacking envelopes in the sample solution.

[0128] In some embodiments, the detection parameters for the detection in step (2) are as follows: laser detector 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1 kPa; signal type: small signal. Under these conditions, the FITC and PC5 fluorescence dual channels are used; the detection parameters are used to detect the particles of herpes simplex virus vectors containing nucleic acids and envelopes, the particles of empty herpes simplex virus vectors, and / or the particles of herpes simplex virus vectors lacking envelopes but containing nucleic acids in the sample solution.

[0129] In some embodiments, the detection parameters for the detection in step (2) are: laser detector 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1 kPa; signal type: small signal. Under these conditions, the scattered light channel, FITC, and PC5 fluorescence dual channels are used. The detection parameters are used to detect particles of herpes simplex virus vectors containing nucleic acid and envelope, particles of empty herpes simplex virus vectors, and / or particles of herpes simplex virus vectors lacking envelope and containing nucleic acid in a sample solution.

[0130] Beneficial effects

[0131] Compared with the prior art, a certain embodiment of the present invention has at least one of the following beneficial effects:

[0132] (1) By using the method provided by the present invention to detect the biological functional titer in a group of virus nucleic acid vector particles, it is possible to achieve a more in-depth analysis of the "total virus titer" obtained by traditional methods. Single-particle analysis of each sub-population of virus nucleic acid vectors can be carried out according to markers such as the envelope, outer shell protein, and glycoprotein of the virus, obtaining more accurate detection results. Moreover, the operation is simple, the speed is fast, which is of great significance for downstream release decisions, process control, and evaluation of clinical safety.

[0133] (2) Compared with the TCID 50 -GFP fluorescence protein fluorescence microscopy method, the method provided by the present invention is simple to operate, fast, and can perform single-particle analysis on each sub-population of lentiviral vectors. Brief description of the drawings

[0134] Figure 1 It is the concentration particle size distribution diagram of the concentrated solution of the lentiviral vector product in Example 1 in the flow cytometry particle population.

[0135] Figure 2 It is the detection result of the scattered light channel and the scatter plot of the fluorescence signal particle population with positive scattered light signal in Example 1.

[0136] Figure 3 It is the standard curve of the HIV-1 p24 concentration and absorbance and the detection result diagram of the lentiviral vector product in Comparative Example 1.

[0137] Figure 4 It is the fluorescence microscopy observation diagram of the concentrated solution of the lentiviral vector product in Comparative Example 2.

[0138] Figure 5 It is the comparison diagram of the results obtained in Example 1, Comparative Example 1, and Comparative Example 2 in Example 2.

[0139] Figure 6It is the scatter plot of the fluorescence signal particle groups in the FITC and PC5 fluorescence channels in Example 3.

[0140] Figure 7 It is the detection result of the scatter light channel in Comparative Example 4 and the scatter plot of the fluorescence signal particle groups with positive scatter light signals.

[0141] Figure 8 It is the comparison chart of the positive ratios after capsule dye labeling after treatment with different concentrations of membrane permeabilizers in Example 4. Among them, 0 on the abscissa represents 0× membrane permeabilizer, 0.5 represents 0.5× membrane permeabilizer, 1 represents 1× membrane permeabilizer, and 2 represents 2× membrane permeabilizer.

[0142] Figure 9 It is the scatter plot of the fluorescence signal particle groups in the FITC and PC5 fluorescence channels in Example 5.

[0143] Figure 10 It is the comparison chart of the detection result of the scatter light channel after labeling with antibody reagents from different manufacturers in Comparative Example 5 and the scatter plot of the fluorescence signal particle groups with positive scatter light signals. Among them, P1 represents the proportion of particles containing herpes simplex virus vector particles with positive expression of the capsule specific marker; P2 represents the proportion of particles containing herpes simplex virus vector particles with negative expression of the capsule specific marker.

[0144] Figure 11 It is the detection result of the scatter light channel in Example 6 and the scatter plot of the fluorescence signal particle groups with positive scatter light signals.

[0145] Figure 12 It is the detection result of the scatter light channel in Example 7 and the scatter plot of the fluorescence signal particle groups with positive scatter light signals.

[0146] Figure 13 It is the scatter plot of the fluorescence signal particle groups in the FITC and PC5 fluorescence channels in Example 8.

[0147] Figure 14 It is the scatter plot of the fluorescence signal particle groups in the FITC and PC5 fluorescence channels in Example 9.

[0148] Term Explanation

[0149] In the present invention, "room temperature" refers to the ambient temperature, which can be 10°C - 40°C, and can be 20°C - 30°C; in some embodiments, it is 22°C - 28°C; in some embodiments, it is 24°C - 26°C; in some embodiments, it is 25°C.

[0150] In the above text of the present invention, regardless of whether the words "about" or "approximately" are used, all the numbers disclosed herein are approximate values. Based on the disclosed numbers, the value of each number may vary by less than ±10% or reasonable differences considered by those skilled in the art, such as ±1%, ±2%, ±3%, ±4% or ±5%.

[0151] The term "plurality" refers to quantities of 2 or more.

[0152] The term "titer" refers to the number of biologically active viral particles per milliliter.

[0153] The term "TU" is the abbreviation of "transducing units", which means the number of viral genomes that can infect and enter target cells. "Encapsulated viral nucleic acid vector" refers to a type of viral nucleic acid vector that has a lipid layer-based membrane structure and is attached to the outside of the nucleocapsid.

[0154] "Gating" means setting a circle gate, which means defining a range or an area in the flow particle distribution diagram, and then performing single-parameter or multi-parameter analysis on the particle group in it. The shapes of the gates include linear gates, cross gates, rectangular gates, circular gates, polygonal gates, arbitrary-shaped gates, and four-quadrant gates.

[0155] The term "optional", "optional" or "optionally" means 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.

[0156] The term "VSV-G" refers to the fusogenic coat G glycoprotein of a lentiviral vector.

[0157] The term "g envelope glycoprotein" refers to a glycoprotein on the envelope of a herpes simplex virus vector, including at least one of envelope glycoprotein gB, envelope glycoprotein gC, envelope glycoprotein gD, envelope glycoprotein gE, envelope glycoprotein gG, envelope glycoprotein gH, envelope glycoprotein gI, envelope glycoprotein gJ, envelope glycoprotein gL, envelope glycoprotein gM or envelope glycoprotein gN.

[0158] The term "env envelope protein" refers to the envelope glycoprotein of a retroviral vector.

[0159] The term "SPIKE protein" refers to the spike glycoprotein of the recombinant coronavirus.

[0160] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0161] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples. Detailed implementation manners

[0162] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further elaborate on the present invention.

[0163] All the reagents used in the present invention can be purchased from the market or can be prepared by the methods described in the present invention.

[0164] Specimen sources: A company provides the concentrated solution of the lentiviral vector product, a company provides the concentrated solution of the herpes simplex virus vector product, a company provides the concentrated solution of the retroviral vector product, 293T cells are purchased from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, and the concentrated solution of the SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector is purchased from Beijing Sino Biological Inc.

[0165] Unless otherwise specified, the buffers, solutions, or reagents described in the present invention all use water as the solvent. For example, 3 μM SYTO9 represents an aqueous solution of 3 μM SYTO9; PBS buffer represents an aqueous PBS buffer solution; the PBS buffer solution of 3 μM SYTO9 represents an aqueous PBS buffer solution of 3 μM SYTO9.

[0166] Unless otherwise specified, the preparation method of each antibody conjugated with a fluorescent dye in the embodiments or comparative examples of the present invention includes conjugating the fluorescent dye with the antibody according to the method described in the EZ-Link™ Maleimide Protein Labeling Kit instruction manual.

[0167] Reagents and instruments used:

[0168] 1) Reagents:

[0169] Unless otherwise specified, the following reagents are selected from the following manufacturers:

[0170] SYTO Green (SYTO BC Green - Fluorescent Nucleic Acid Stains), SYTO16, SYTO21, SYTO24, PicoGreen, SYTOX Green, SYTO BC, RNA Select, SYTO60, SYTO62, SYTO63 were purchased from Invitrogen;

[0171] p24 ELISA (QuickTiter HIV Lentivirus Quantitation Kit) was purchased from CellBiolabs;

[0172] Polybrene, PKH67 lipid membrane dye, DID membrane dye were purchased from Sigma - Aldrich;

[0173] 293Pro series serum - free medium was purchased from Shanghai Yuanpei Company;

[0174] Membrane permeabilizer (product number: P2320, stock solution (20×, active ingredient is Tween 20, 5 ml)), FITC Anti - HIV1p24 antibody (product number: NHAP24 - FITC - 50T), SYTO9 (100 μM, product number: NHATO9 - 488 - 50T), PE - Cy5 - VSVG antibody (PE - Cy5 anti - human VSVG, product number: NHAVSVG - PECy5 - 50T), AF647 - Anti - Glycoprotein B of HSV (gB) (product number: NHAGB - AF647 - 50T), fluorescent microsphere standard products are from NanoFCM;

[0175] Anti - HSV1 + HSV2 gB antibody [10B7] and Anti - HSV1 + HSV2 gD antibody were purchased from Abcam;

[0176] HIV - 1 env Antibody, HIV - 1 gag - pol antibody were purchased from LS bio;

[0177] AF488 - Anti - Spike protein [4A8], Human IgG1, Kappa antibody was purchased from Absoluteantibody;

[0178] Herpes Simplex Virus Type 1 / 2 gB antibody and EZ-Link™ Maleimide Protein Labeling Kit were purchased from Thermo Fisher;

[0179] PBS buffer: an aqueous solution containing 136.89 mM of NaCl, 2.67 mM of KCl, 8.1 mM of Na2HPO4 and 1.76 mM of KH2PO4;

[0180] The concentration of Herpes Simplex Virus vector products (HSV-1, HSV-2) is about 1×10 8 PFU / ml;

[0181] The concentration of SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector product is about 1×10 10 virus copies / mL;

[0182] The concentration of retrovirus vector product is about 1×10 9 TU / mL.

[0183] 2) Instruments:

[0184] Flow NanoAnalyzer was purchased from NanoFCM Inc;

[0185] Centrifuge 5810R refrigerated centrifuge was purchased from Eppendorf;

[0186] Optima Max-XP Tabletop Ultracentrifuge was purchased from Beckman coulter;

[0187] X81 Universal Control BOX IX2 UBC 2 microscope fluorescence microscope was purchased from Olympus;

[0188] Feyond-A300 microplate reader was purchased from Hangzhou Ausheng Instruments Co., Ltd.

[0189] Example 1: Detection by nano-flow cytometer after labeling with nucleic acid dye and VSVG antibody conjugated with fluorescent dye

[0190] 1) Detection of the concentration of lentivirus vector product

[0191] Flow NanoAnalyzer detection parameters: Laser detectors 488nm + 638nm; Single laser channel detection Laser: 20 / 50mW, 488nm; Scattered light attenuation: 0.2%; Detection pressure: 1kpa; Signal type: Large signal, using the scattered light channel.

[0192] Concentration detection of lentiviral vector products: Dilute the fluorescent microsphere standard product with a concentration of 2.19×10 10 particles / mL by 100 times to obtain the concentration standard product solution. Dilute the concentrated solution of the lentiviral vector product by 3 times to obtain the sample solution. Perform flow particle detection under the same detection parameters, and record the number of particles in the same injection pressure and the same detection time using the concentration standard solution. During the detection time, the number of particles in the sample solution is 2742, and the number of particles in the concentration standard product solution is 5683; Calculate the concentration of the lentiviral vector product through the dilution multiple, the concentration standard curve and the detection results of the lentiviral vector product, and the result is 3.17×10 8 particles / mL (see Figure 1 ).

[0193] 2) Dual optically labeled lentiviral vector

[0194] Dilute the concentrated solution of the lentiviral vector product 10 times with a buffer containing PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4) and 0.5% Tween 20 to obtain Solution 1. Take 50 μL of Solution 1 and incubate it with 20 μL of PE-Cy5-VSVG antibody (VSVG antibody conjugated with PE-Cy5) and 3 μM SYTO Green at 37°C for 30 min. Centrifuge at 100,000 g at 4°C for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 100,000 g at 4°C for 20 min, discard the supernatant, and add 100 μL of PBS buffer to resuspend the precipitate to obtain the dual-labeled lentiviral vector.

[0195] 3) Flow NanoAnalyzer detection of the dual-labeled lentiviral vector

[0196] Flow NanoAnalyzer detection parameters: Laser detectors 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 scattered light channel, FITC and PC5 fluorescence dual channels.

[0197] Detection of lentiviral vectors after dual labeling: Flow NanoAnalyzer was used to detect lentiviral vectors after dual labeling. FITC fluorescence was the excitation signal of nucleic acid dye to characterize the nucleic acids in the lentiviral vectors, and PC5 fluorescence characterized the fusogenic envelope G glycoprotein in the lentiviral vectors. The detection results of the FITC fluorescence channel and the PC5 fluorescence channel of the particle population with positive scattered light signals were analyzed, and the scatter plot of the detection results was recorded. The obtained scatter plot of the results was set with four quadrant gates. Among them,

[0198] The particle population with positive scattered light signals, positive nucleic acid signals, and positive VSV-G expression signals was the particles of lentiviral vectors containing nucleic acids and VSV-G, or the particle population with positive scattered light signals, positive target nucleic acid signals, and positive VSV-G expression signals was the particles of lentiviral vectors containing target nucleic acids and VSV-G;

[0199] The particle population with positive scattered light signals, positive VSV-G expression signals, and negative nucleic acid signals was the particles of empty capsid lentiviral vectors;

[0200] The particle population with positive scattered light signals, positive nucleic acid signals, and negative VSV-G expression signals was the particles of lentiviral vectors lacking VSV-G containing nucleic acids, or the particle population with positive scattered light signals, positive target nucleic acid signals, and negative VSV-G expression signals was the particles of lentiviral vectors lacking VSV-G containing target nucleic acids.

[0201] The detection results of the scattered light channel and the results of the particle population of fluorescent signals with positive scattered light signals were as Figure 2 shown.

[0202] 4) Calculation of the biological functional titer of the lentiviral vector product

[0203] The particle population with positive scattered light signals, positive nucleic acid signals, and positive VSV-G expression signals was the particles of lentiviral vectors containing nucleic acids and VSV-G; the titer of the particles of lentiviral vectors containing nucleic acids and VSV-G was the product of P1 and the concentration of the lentiviral vector product obtained in step 1) of Example 1, and the result was 1.34×10 8 TU / mL, where P1 was the percentage of the number of particles in the particle population with positive scattered light signals, positive nucleic acid signals, and positive VSV-G expression signals to the total number of particles in all particle populations with positive scattered light signals.

[0204] The particle population with positive scattered light signals, positive VSV-G expression signals, and negative nucleic acid signals was the particles of empty capsid lentiviral vectors; the calculation method for the titer of the empty capsid lentiviral vectors was the product of P2 and the concentration of the lentiviral vector product obtained in step 1) of Example 1, and the result was 1.4×10 7TU / mL, where P2 is the percentage of the number of particles in the particle population with positive scattered light signal, positive VSV-G expression signal and negative nucleic acid signal to the total number of particles in all particle populations with positive scattered light signal.

[0205] The particle population with positive scattered light signal, positive nucleic acid signal and negative VSV-G expression signal is the particle of the lentiviral vector lacking VSV-G containing nucleic acid; the calculation method of the titer of the lentiviral vector lacking VSV-G containing nucleic acid is the product of P3 and the concentration of the lentiviral vector product obtained in step 1), and the result is 1.15×10 8 TU / mL, where P3 is the percentage of the total number of points with positive scattered light signal, positive nucleic acid signal and negative VSV-G expression signal to the total number of particles in all particle populations with positive scattered light signal.

[0206] 5) Detection of the particle size distribution of the lentiviral vector product: Set the detection parameters as: laser detector 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. Under this condition, use the scattered light channel; Fix the Sampling pressure at 1.0 kPa. Based on the data of the 68±2 nm, 91±3 nm, 113±3 nm, 155±3 nm particle size standard mixture tested under this condition, generate a fitting curve according to the Median value and Events value of the peaks shown by the particle size standards. For the data of the concentrated solution of the lentiviral vector product tested under the same detection parameters after dilution by 10 times, use the gating tool to define the target particle size distribution range, record the number of particles within the gate, the percentage of the number of particles within the gate, and the median value, average value and standard deviation of the particle size of the particles within the gate, and convert the single particle size value of the lentiviral vector product in combination with the fitting curve; After statistics, a histogram of the particle size distribution of the lentiviral vector product can be generated ( Figure 1 )

[0207] Comparative Example 1: Detection by p24 ELISA

[0208] Operate according to the p24 ELISA kit instructions:

[0209] 1) Gradient dilution of the HIV-1 p24 quantitative standard: Take the positive control containing 320 pg / mL of recombinant HIV-1 p24 antigen and serially dilute it in multiples with the diluent to obtain five concentrations of positive controls. Number the corresponding wells of the above samples in sequence. Each plate should have three negative control wells, two positive control wells and one blank well. Add 25 μL of lysis buffer to each reaction well.

[0210] 2) Dilute the concentrated solution of the lentiviral vector product by 105 Multiply by this factor to obtain Sample Solution 2. Add Sample Solution 2 and the reference substance described in Step 1) of Comparative Example 1 to the reaction wells containing the lysis solution, shake for 30 - 60 seconds to mix evenly, and incubate at 37°C for 1 hour; wash the plate 5 times, pat dry, then add 125 μL / well of the enzyme conjugate to each well (do not add to the blank well), and incubate at 37°C for 1 hour; wash the plate 5 times, pat dry, and then add 125 μL / well of the substrate (substrate solution: chromogenic solution = 1:1 mixture) to each well. Incubate at 37°C for 30 minutes, and then add 50 μL / well of the stop solution.

[0211] 3) Read the values using an enzyme - linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0212] 4) Positive quantitative determination: Based on the five - concentration positive controls in Step 1) of Comparative Example 1, plot a standard curve and substitute the test results of the lentiviral vector product for quantitative determination. The results are as Figure 3 shown.

[0213] 5) Calculate the p24 content (ng / mL) of the concentrated solution of the lentiviral vector product diluted 10 5 times and the lentiviral vector titer (based on 1 ng p24 = 1.25x10 5 TU) according to Step 4) of Comparative Example 1. The results show that the p24 content of the concentrated solution of the lentiviral vector product diluted 10 5 times is 19.3 pg / mL; that is, the titer of the lentiviral vector product is 19.3×10 2 ng / mL×1.25×10 5 TU / mL = 2.4×10 8 TU / mL.

[0214] Comparative Example 2: TCID 50 -GFP fluorescence protein fluorescence microscopy method

[0215] 1) Take the lentiviral vector product and dilute it starting from a 10 3 -fold dilution, for a total of 8 dilution gradients (with a 10 - fold difference between each gradient).

[0216] 2) Add 0.05 mL of each dilution gradient from Step 1) to the culture medium in a 96 - well plate containing 293T cells (2.5×10 6 cells / well) without serum and containing 8 mg / L Polybrene (for promoting infection). Add 10 wells for each dilution gradient, and allow the lentivirus to infect the 293T cells and culture the cells at 37°C for 2 days.

[0217] 3) After 2 days, observe under an inverted fluorescence microscope and calculate the number of wells showing green fluorescence in each row (10 wells). The results of the fluorescence microscope for the concentrated solution of the lentiviral vector product are as Figure 4As shown

[0218] 4) Calculate the titer of the lentiviral vector: It was found that green fluorescence was visible in all the wells in the row corresponding to the dilution before the 10-fold dilution, and no green fluorescence was visible in all the wells in the row corresponding to the dilution after the 10-fold dilution. At the 10-fold dilution, 3 positive wells were visible, and at the 10-fold dilution, 1 positive well was visible. The calculation formula is: 3 Before the 10-fold dilution, green fluorescence was visible in all the wells in the row corresponding to the dilution. After the 10-fold dilution, no green fluorescence was visible in all the wells in the row corresponding to the dilution. At the 10-fold dilution, 3 positive wells were visible, and at the 10-fold dilution, 1 positive well was visible. The calculation formula is: 6 After the 10-fold dilution, no green fluorescence was visible in all the wells in the row corresponding to the dilution. At the 10-fold dilution, 3 positive wells were visible, and at the 10-fold dilution, 1 positive well was visible. The calculation formula is: 4 At the 10-fold dilution, 3 positive wells were visible, and at the 10-fold dilution, 1 positive well was visible. The calculation formula is: 5 At the 10-fold dilution, 1 positive well was visible. The calculation formula is:

[0219] Infection titer T = 10 1 + d( s-0.5) / V.

[0220] Where d is the logarithm of the dilution ratio (i.e., when diluted 10-fold, d = 1); s is the sum of the logarithm of the initial dilution and the proportion of positive wells in each test dilution, i.e., 3 + 1 + 0.3 + 0.1 = 4.4; V is the inoculation volume of the lentiviral vector product diluent (i.e., V = 0.05 mL).

[0221] After calculation, the infection titer T of the lentiviral vector product = 1.6×10 6 TU / mL.

[0222] Example 2: Comparison of the results obtained in Example 1, Comparative Example 1, and Comparative Example 2

[0223] As Figure 5 shown. From the results, it can be seen that the virus titer of the concentrated solution of the lentiviral vector product measured by the p24 ELISA method is often overestimated due to the interference of free p24 and the fact that virus particles with p24 protein expression do not represent the specific ability of the virus particles to infect and integrate the genome into the host. In Example 1, Comparative Example 1, and Comparative Example 2 of this example, the virus titer of the concentrated solution of the lentiviral vector product measured by the p24 ELISA method was 2.4×10 8 TU / mL, which is similar to the sum of the titer of the lentiviral vector with nucleic acid and VSV-G and the titer of the lentiviral vector lacking VSV-G with nucleic acid in the lentiviral vector product (1.34×10 8 TU / mL + 1.15×10 8 TU / mL equals 2.49×10 8 TU / mL). Since the titer of the lentiviral vector lacking VSV-G with nucleic acid lacks infectivity, it also proves from the data level that the p24 ELISA method overestimates the actual virus titer. The result measured by the TCID 50 -GFP fluorescence microscopy method was 1.6×10 6 TU / mL, which is the same as the titer of the lentiviral vector with nucleic acid and VSV-G, which is 1.34×108 The viral titer of the concentrated solution of the lentiviral vector product measured by TU / mL and p24 ELISA was about 2 orders of magnitude lower than 2.4×10 8 TU / mL, indicating its underestimation of the true titer. It shows that the accuracy of using the titer of the lentiviral vector with nucleic acid and VSV-G as the measurement index of the true viral titer of the lentiviral vector composition is high.

[0224] Example 3: After double-labeling the VSVG antibody and P24 antibody conjugated with a fluorescent dye after treatment with a membrane permeabilizer, it was detected by a Nano Flow Cytometer

[0225] 1) Membrane permeabilizer treatment and dual optical labeling of lentiviral vectors

[0226] Take 50 μL of 1×10 10 TU / mL lentivirus, add 50 μL of 0.5× membrane permeabilizer (take the membrane permeabilizer (stock solution (20×)) diluted with water, and the membrane permeabilizer of other concentrations below is prepared in the same way), mix well, and incubate on ice for 30 min. Add 5 μL of FITC-P24 antibody (manufacturer: NanoFCM) and 20 μL of PE-Cy5-VSVG antibody (manufacturer: NanoFCM), mix well, and incubate in the dark on ice for 60 min. Resuspend the lentivirus with 1 ml of 0.5× membrane permeabilizer, centrifuge at 80000g for 30 min, and discard the supernatant. Resuspend the lentivirus with 1 ml of 0.5× membrane permeabilizer again, centrifuge at 80000g for 30 min, and resuspend the precipitate with 100 μL of PBS buffer to obtain the lentiviral vector after double labeling.

[0227] 2) Detection of the lentiviral vector after double labeling by Flow NanoAnalyzer

[0228] Flow NanoAnalyzer detection parameters: laser detector 488 nm + 638 nm; single laser channel detection Laser: 10 / 50mW 488; light scattering attenuation: 10%; detection pressure: 1 kPa; signal type: under the condition of small signal, use the FITC and PC5 fluorescence double channels.

[0229] Detection of the lentiviral vector after double labeling: The lentiviral vector after double labeling was detected by Flow NanoAnalyzer. The FITC fluorescence was used as the excitation signal of the P24 fluorescent antibody to characterize the capsid protein in the lentiviral vector, and the PC5 fluorescence characterized the fusogenic envelope G glycoprotein in the lentiviral vector. Analyze the detection results of the FITC fluorescence channel and the PC5 fluorescence channel, record the scatter plot of the detection results, and set the four-quadrant gate for the obtained scatter plot of the results. Among them,

[0230] The particle population with positive P24 expression signal and positive VSV-G expression signal is the particle of the lentiviral vector containing the capsid and VSV-G;

[0231] The particle population with positive VSV-G expression signal and P24 expression signal is the particle of the lentiviral vector lacking the capsid and containing VSV-G;

[0232] The particle with positive P24 expression signal and negative VSV-G expression is the particle of the lentiviral vector lacking VSV-G and containing the capsid.

[0233] The results of the fluorescent signal particle population are as Figure 6 shown in Table 1.

[0234] Comparative Example 3: After treatment with different concentrations of membrane permeabilizer, the VSVG antibody and P24 antibody conjugated with fluorescent dye were labeled and detected by a nano flow cytometer

[0235] According to the method provided in Example 3, the concentrations of the membrane permeabilizer were set to 0×, 1×, and 2× respectively.

[0236] The comparison results of the proportion of particles of the lentiviral vector containing the capsid and VSV-G obtained after treatment with different concentrations of the membrane permeabilizer are shown in Table 1.

[0237]

[0238] Comparative Example 4: After treatment with a membrane permeabilizer, the P24 antibody conjugated with fluorescent dye was labeled and detected by a nano flow cytometer

[0239] 1) Membrane permeabilizer treatment and optical labeling of the lentiviral vector

[0240] Take 50 μL of 1×10 10 TU / mL lentiviral vector, add 50 μL of 0.5× membrane permeabilizer, mix well, and incubate on ice for 30 min. Add 5 μL of FITC-P24 antibody (manufacturer: NanoFCM), mix well, and incubate in the dark on ice for 60 min. Resuspend the lentivirus with 1 ml of 0.5× membrane permeabilizer, centrifuge at 80000 g for 30 min, and discard the supernatant. Resuspend the lentivirus again with 1 ml of 0.5× membrane permeabilizer, centrifuge at 80000 g for 30 min, and resuspend the precipitate with 100 μL of PBS buffer to obtain the labeled lentiviral vector.

[0241] 2) Detection of the lentiviral vector after double labeling by Flow NanoAnalyzer

[0242] Detection parameters of Flow NanoAnalyzer: Laser detectors 488 nm + 638 nm; Single laser channel detection Laser: 10 / 50 mW 488; Scattered light attenuation: 10%; Detection pressure: 1 kpa; Signal type: Under the condition of small signal, the scattered light channel and the FITC fluorescence channel are used.

[0243] Detection of the labeled lentiviral vector: The labeled lentiviral vector was detected by Flow NanoAnalyzer. The FITC fluorescence was used as the excitation signal of the P24 fluorescent antibody to characterize the capsid protein in the lentiviral vector. The detection results of the FITC fluorescence channel were analyzed, and the scatter plot of the detection results was recorded. The obtained scatter plot of the results was set with a two-quadrant gate, where

[0244] The particle population with positive scattered light signal and positive P24 expression signal is the particle of the lentiviral vector containing the capsid;

[0245] The particle population with positive scattered light signal and negative P24 expression signal is the particle of the lentiviral vector lacking the capsid;

[0246] The results of the fluorescent signal particle population with positive scattered light signal are as Figure 7 shown.

[0247] Conclusion: In Example 3, Comparative Example 3, and Comparative Example 4, the particles of the lentiviral vector containing the capsid and VSV-G measured in Example 3 accounted for 41.5% of the total number of lentiviral vector particles; while in Comparative Example 3, the proportion of the particles of the lentiviral vector containing the capsid and VSV-G obtained by the treatment method with a membrane permeabilizer concentration of 0 accounted for only 14% of the total number of lentiviral vector particles, indicating that the membrane permeabilizer plays a key role in the detection of the capsid protein by penetrating the fluorescent dye into the lentiviral envelope. The particles of the lentiviral vector containing the capsid measured in Comparative Example 4 accounted for 62.3% of the total number of lentiviral vector particles. This data is significantly higher than the proportion of the lentiviral particles with both capsid and envelope in Example 3: 41.5% of the total number of lentiviral vector particles. Since the lentiviral vector lacking VSV-G is considered to be non-infectious, it is further proved from the data level that using p24 as a single index for determination overestimates the actual virus titer.

[0248] Example 4: After treatment with different concentrations of membrane permeabilizer, the lentiviral vector was labeled with an envelope dye and then detected by a nano-flow cytometer.

[0249] 1) Labeling of the lentiviral vector envelope with an envelope dye after treatment with a membrane permeabilizer

[0250] Take 1 μL of the DID membrane dye, dilute it 10 times, and prepare the DID membrane dye stock solution (prepared and used immediately). Add 50 μL of membrane permeabilizers with different concentrations and 10 μL of the DID membrane dye stock solution respectively, mix well, and incubate on ice for 30 min. Take 1 μL of the DID membrane dye stock solution and add it to 100 μl of the lentiviral vector with a concentration of 1×10 8 TU / mL, and mix well. Incubate in the dark at 37 °C for 15 min to obtain the labeled lentiviral vector treated with the membrane permeabilizer. Resuspend the lentiviral vector with 1 ml of the membrane permeabilizer, centrifuge at 80,000 g for 30 min, and discard the supernatant. According to the above method, set the membrane permeabilizer concentrations to 0×, 0.5×, 1×, and 2× to obtain the labeled lentiviral vectors treated with different concentrations of the membrane permeabilizer (the labeled lentiviral vector treated with 0× membrane permeabilizer, the labeled lentiviral vector treated with 0.5× membrane permeabilizer, the labeled lentiviral vector treated with 1× membrane permeabilizer, and the labeled lentiviral vector treated with 2× membrane permeabilizer).

[0251] 2) Detection of the lentiviral vector labeled with the envelope dye by Flow NanoAnalyzer

[0252] Flow NanoAnalyzer detection parameters: laser detectors 488 nm + 638 nm; single laser channel detection Laser: 10 / 50 mW 488; scattered light attenuation: 10 %; detection pressure: 1 kPa; signal type: under the condition of small signal, use the scattered light channel and the PC5 fluorescence channel.

[0253] Detection of the labeled lentiviral vector: Use Flow NanoAnalyzer to detect the labeled lentiviral vectors treated with different concentrations of the membrane permeabilizer obtained in step 1) of Example 4 respectively. The PC5 fluorescence is the excitation signal of the DID membrane dye, which is used to characterize the envelope in the lentiviral vector. Analyze the detection results of the PC5 fluorescence channel, record the scatter plot of the detection results, and set a two-quadrant gate for the obtained scatter plot of the results. Among them,

[0254] The particle population with positive scattered light signal and positive DID membrane dye signal is the particle of the lentiviral vector containing the envelope;

[0255] The particle population with positive scattered light signal and negative DID membrane dye signal is the particle of the lentiviral vector lacking the envelope;

[0256] The comparison results of the fluorescence signal particle populations with positive scattered light signals after treatment with different concentrations of the membrane permeabilizer are as Figure 8 shown.

[0257] Conclusion: In Example 4 of this embodiment, compared with the membrane permeabilizer concentrations of 0 and 0.5×, increasing the membrane permeabilizer concentration to 1× and 2× will significantly reduce the positive rate of DID membrane dye. It indicates that the membrane permeabilizer may cause a small part of the lentivirus to rupture; as the concentration of the membrane permeabilizer increases, the labeling ratio of the DID membrane dye slightly decreases, which further proves that using an appropriate concentration of the membrane permeabilizer in Example 3 is a key step to obtain accurate measurement results of lentiviral vector particles containing capsids and VSV-G.

[0258] Example 5: Detection with a Nano Flow Analyzer after labeling with a nucleic acid dye and a gB antibody conjugated with a fluorescent dye

[0259] 1) Dual optical labeling of herpes simplex virus vectors

[0260] Dilute the concentrated solution of the herpes simplex virus vector product 10-fold with PBS buffer to obtain Solution 1. Take 50 μL of Solution 1 and incubate it with 20 μL of AF647-Anti-Glycoprotein B of HSV (gB) antibody at 37 °C for 30 min, centrifuge at 100,000 g at 4 °C for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 100,000 g at 4 °C for 20 min, discard the supernatant, and add 100 μL of PBS buffer containing 3 μM SYTO9 to resuspend the precipitate to obtain the dual-labeled herpes simplex virus vector.

[0261] 2) Detection of the dual-labeled herpes simplex virus vector by Flow NanoAnalyzer

[0262] Flow NanoAnalyzer detection parameters: Laser detectors 488 nm + 638 nm; single laser channel detection Laser: 10 / 50 mW 488; scattered light attenuation: 10%; detection pressure: 1 kPa; signal type: under the condition of small signal, use the FITC and PC5 fluorescence dual channels.

[0263] Detection of the dual-labeled herpes simplex virus vector: Use Flow NanoAnalyzer to detect the dual-labeled herpes simplex virus vector obtained in Step 1) of Example 5. The PC5 fluorescence is the excitation signal of the nucleic acid dye used to characterize the g capsid glycoprotein in the slow herpes simplex virus vector, and the FITC fluorescence characterizes the nucleic acid of the herpes simplex virus vector in the slow herpes simplex virus vector. Analyze the detection results of the FITC fluorescence channel and the PC5 fluorescence channel, record the scatter plot of the detection results, and set a quadrant gate for the obtained scatter plot of the results. Among them,

[0264] The particle population with positive nucleic acid signals and positive gB expression signals is the herpes simplex virus vector particles containing nucleic acid and capsid;

[0265] The particle population with positive gB expression signal and negative nucleic acid signal is the particle of the empty capsid herpes simplex virus vector;

[0266] The particle population with positive nucleic acid signal and negative gB expression signal is the particle of the herpes simplex virus vector lacking an envelope and containing nucleic acid.

[0267] The results of the fluorescent signal particle population are as Figure 9 shown.

[0268] Conclusion: After detecting the herpes simplex virus vector after double labeling with Flow NanoAnalyzer, it was found that the proportion of herpes simplex virus vector particles containing nucleic acid and envelope in the herpes simplex virus vector product was 35.4%.

[0269] Comparative Example 5: Detection was performed using a nano-flow detector after labeling with antibodies conjugated with fluorescent dyes from different sources

[0270] 1) Optical labeling of herpes simplex virus vector

[0271] Dilute the concentrated solution of herpes simplex virus vector products HSV-1 or HSV-2 10-fold with PBS buffer to obtain Solution 1. Take 50 μL of Solution 1 and incubate it with 20 μL of antibodies conjugated with fluorescent dyes from different sources, namely AF647-Anti-HSV1 + HSV2 gB antibody [10B7] (Anti-HSV1 + HSV2 gB antibody [10B7] conjugated with AF647 fluorescent dye, manufacturer of Anti-HSV1 + HSV2 gB antibody [10B7]: Abcam), AF647-Herpes Simplex Virus Type 1 / 2 gB antibody (Herpes Simplex Virus Type 1 / 2 gB antibody conjugated with AF647 fluorescent dye, manufacturer of Herpes Simplex Virus Type 1 / 2 gB antibody: Thermo Fisher), AF647-Anti-Glycoprotein B of HSV (gB) (herpes simplex virus envelope glycoprotein gB conjugated with AF647 fluorescent dye, antibody manufacturer: Nano FCM), AF647-Anti-HSV1 + HSV2 gD antibody (Anti-HSV1 + HSV2 gD antibody conjugated with AF647 fluorescent dye, manufacturer of Anti-HSV1 + HSV2 gD antibody: Abcam)) at 37°C for 30 min, then centrifuge at 100,000 g at 4°C for 80 min. Discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 100,000 g at 4°C for 20 min, discard the supernatant, and add 100 μL of PBS buffer to resuspend the precipitate to obtain the double-labeled herpes simplex virus vector.

[0272] The preparation method of the above-mentioned Anti-HSV1 + HSV2 gB antibody conjugated with AF647 fluorescent dye includes conjugating the AF647 fluorescent dye to the Anti-HSV1 + HSV2 gB antibody according to the instructions of the EZ-Link™ Maleimide Protein Labeling Kit to obtain the Anti-HSV1 + HSV2 gB antibody conjugated with AF647 fluorescent dye.

[0273] The preparation method of the above-mentioned Herpes Simplex Virus Type 1 / 2 gB antibody conjugated with AF647 fluorescent dye includes conjugating the AF647 fluorescent dye to the Herpes Simplex Virus Type 1 / 2 gB antibody according to the instructions of the EZ-Link™ Maleimide Protein Labeling Kit to obtain the Herpes Simplex Virus Type 1 / 2 gB antibody conjugated with AF647 fluorescent dye.

[0274] The manufacturer of the Anti-HSV1 + HSV2 gD antibody conjugated with the AF647 fluorescent dye is NanoFCM.

[0275] The preparation method of the Anti-HSV1 + HSV2 gD antibody conjugated with the AF647 fluorescent dye includes conjugating the AF647 fluorescent dye to the Anti-HSV1 + HSV2 gD antibody according to the method described in the EZ-Link™ Maleimide Protein Labeling Kit instructions to obtain the Anti-HSV1 + HSV2 gD antibody conjugated with the AF647 fluorescent dye.

[0276] 2) Detection of herpes simplex virus vector by Flow NanoAnalyzer

[0277] Take the herpes simplex virus vector after double labeling obtained in step 1) of Comparative Example 5 and perform detection according to the following detection parameters: Flow NanoAnalyzer detection parameters: laser detectors 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kpa; signal type: under the condition of small signal, use the scattered light channel and the FITC fluorescence channel / PC5 fluorescence channel. Record the scatter plot of the detection results, and set a two-quadrant gate for the obtained scatter plot of the results, where

[0278] The particle population with positive scattered light signal and positive fluorescence antibody expression signal is the particle containing the herpes simplex virus vector with positive expression of the specific envelope marker;

[0279] The particle population with positive scattered light signal and negative fluorescence antibody expression signal is the particle of the herpes simplex virus vector lacking the expression of the specific envelope marker;

[0280] Obtain the comparison result of the proportion of particles containing the herpes simplex virus vector particles with positive expression of the specific envelope marker as Figure 10 shown.

[0281] Conclusion: In Example 5 and Comparative Example 5, compare the proportions of particles containing herpes simplex virus vector particles expressing specific envelope markers obtained after treatment with AF647-Anti-HSV1 + HSV2 gB antibody [10B7] (Anti-HSV1 + HSV2 gB antibody [10B7] conjugated with AF647 fluorescent dye, manufacturer of Anti-HSV1 + HSV2 gB antibody [10B7]: Abcam), AF647-Herpes Simplex Virus Type 1 / 2 gB antibody (Herpes Simplex Virus Type 1 / 2 gB antibody conjugated with AF647 fluorescent dye, manufacturer of Herpes Simplex Virus Type 1 / 2 gB antibody: Thermo Fisher), AF647-Anti-Glycoprotein B of HSV (gB) (herpes simplex virus envelope glycoprotein gB conjugated with AF647 fluorescent dye, manufacturer of Anti-Glycoprotein B of HSV (gB): Nano FCM), AF647-Anti-HSV1 + HSV2 gD antibody (Anti-HSV1 + HSV2 gD antibody conjugated with AF647 fluorescent dye, manufacturer of Anti-HSV1 + HSV2 gD antibody: Abcam). It was found that the AF647-Anti-Glycoprotein B of HSV (gB) antibody and SYTO9 could obtain the optimal result for simultaneous labeling of herpes simplex virus vector nucleic acid and envelope.

[0282] Example 6: Detection by Nano Flow Cytometer after labeling with nucleic acid dye and lipid membrane dye

[0283] 1) Concentration detection of herpes simplex virus vector

[0284] Flow NanoAnalyzer detection parameters: Laser detectors 488nm + 638nm; Single laser channel detection Laser: 20 / 50mW, 488nm; Scattered light attenuation: 0.2%; Detection pressure: 1kpa; Signal type: Large, using the scattered light channel.

[0285] Concentration detection of herpes simplex virus vector product: The concentration is 2.17×10 10The fluorescence microsphere standard product at a concentration of 8 particles / mL was diluted 100-fold to obtain a concentration standard solution. The concentrated solution of the herpes simplex virus vector product was diluted 3-fold to obtain a sample solution. Flow particle detection was performed under the same detection parameters. The number of particles in the concentration standard solution was recorded at the same injection pressure and within the same detection time. During the detection time, the number of particles in the sample solution was 3,863, and the number of particles in the concentration standard solution was 7,863. The concentration of the herpes simplex virus vector product was calculated based on the dilution factor, the concentration standard curve, and the detection results of the herpes simplex virus vector product. The result was 3.2×10

[0286] 2) Dual-optical-labeled herpes simplex virus vector

[0287] The concentrated solution of the herpes simplex virus vector product was diluted 10-fold with a buffer containing PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, and 1.76 mM KH2PO4) and 0.5% Tween 20 to obtain Solution 1. 50 μL of Solution 1 was incubated with 20 μL of PKH67 lipid membrane dye (manufacturer: sigma) at 37 °C for 30 min, centrifuged at 100,000 g at 4 °C for 80 min, the supernatant was discarded, the precipitate was resuspended with PBS buffer, centrifuged at 100,000 g at 4 °C for 20 min, the supernatant was discarded, and the precipitate was resuspended with 100 μL of PBS buffer containing 5 μM SYTO62 Red nucleic acid dye to obtain the dual-labeled herpes simplex virus vector.

[0288] 3) Detection of the dual-labeled herpes simplex virus vector by Flow NanoAnalyzer

[0289] Flow NanoAnalyzer detection parameters: Laser detectors 488 nm + 638 nm; single-laser channel detection Laser: 10 / 50 mW 488; scattered light attenuation: 10%; detection pressure: 1 kPa; signal type: under the condition of small signal, the scattered light channel, FITC, and PC5 fluorescence dual channels were used.

[0290] Detection of the dual-labeled herpes simplex virus vector: The dual-labeled herpes simplex virus vector was detected by Flow NanoAnalyzer. The FITC fluorescence was the excitation signal of the nucleic acid dye to characterize PKH67 in the herpes simplex virus vector, and the PC5 fluorescence characterized the excitation signal of the nucleic acid dye to characterize the nucleic acid of the herpes simplex virus vector. The detection results of the FITC fluorescence channel and the PC5 fluorescence channel of the particle population with positive scattered light signals were analyzed, and the scatter plot of the detection results was recorded. The obtained scatter plot of the results was set with four quadrant gates, where

[0291] The population of particles with positive scattered light signal, SYTO62 positive signal, and PKH67 positive signal are particles of herpes simplex virus vector containing nucleic acid and envelope;

[0292] The population of particles with positive scattered light signal, SYTO62 negative signal, and PKH67 positive signal are particles of empty shell herpes simplex virus vector;

[0293] The population of particles with positive scattered light signal, SYTO62 positive signal, and PKH67 negative signal are particles of herpes simplex virus vector lacking envelope and containing nucleic acid.

[0294] Results: The detection results of the scattered light channel and the results of the population of fluorescent signal particles with positive scattered light signal are as Figure 11 shown.

[0295] Conclusion: The calculation of the biological functional titer ratio of the herpes simplex virus vector product was completed. The percentage of the population of particles with positive scattered light signal, positive nucleic acid signal, and positive lipid membrane signal, which are particles of herpes simplex virus vector containing nucleic acid and lipid membrane, in the total number of particles of all populations of particles with positive scattered light signal was 46.8%.

[0296] Example 7: Detection with a nano flow cytometer after labeling with a nucleic acid dye and a Spike antibody conjugated with a fluorescent dye

[0297] 1) Dual optical labeling of SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector

[0298] Dilute the concentrate of the SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector product (1×10 10 virus copies / mL) 10-fold with PBS buffer to obtain Solution 1. Take 50 μL of Solution 1 and incubate it with 20 μL of AF488-Anti-Spikeprotein [4A8], Human IgG1, Kappa antibody (antibody against the Spike protein of SARS-CoV-2 conjugated with AF488) at 37 °C for 30 min, centrifuge at 100,000 g at 4 °C for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 100,000 g at 4 °C for 20 min, discard the supernatant, and add 100 μL of PBS buffer containing 5 μM SYTO62 Red nucleic acid dye to resuspend the precipitate to obtain the dual-labeled SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector.

[0299] The preparation method of the Anti-Spike protein [4A8], Human IgG1, Kappa antibody conjugated with AF488 fluorescent dye includes conjugating the AF488 fluorescent dye to the Anti-Spike protein [4A8], Human IgG1, Kappa antibody according to the method described in the EZ-Link™ Maleimide Protein Labeling Kit instruction manual to obtain the Anti-Spike protein [4A8], Human IgG1, Kappa antibody conjugated with AF488 fluorescent dye.

[0300] 2) Detection of the SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector after dual labeling by Flow NanoAnalyzer

[0301] Flow NanoAnalyzer detection parameters: Laser detectors 488nm + 638nm; Single laser channel detection Laser: 10 / 50mW 488; Scattered light attenuation: 10%; Detection pressure: 1kpa; Signal type: Under the condition of small signal, use the scattered light channel, FITC and PC5 fluorescence dual channels.

[0302] Detection of the SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector after dual labeling: Use Flow NanoAnalyzer to detect the SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector obtained in step 1) of Example 7 after dual labeling. The FITC fluorescence is the excitation signal of the AF488-Anti-Spike protein [4A8], Human IgG1, Kappa antibody, which is used to characterize the Spike in the SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector. The PC5 fluorescence characterizes the excitation signal of the nucleic acid dye, which is used to characterize the nucleic acid of the SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector. Analyze the detection results of the FITC fluorescence channel and the PC5 fluorescence channel of the particle population with positive scattered light signals, record the scatter plot of the detection results, and set a quadrant gate for the obtained scatter plot of the results. Among them,

[0303] The particle population with positive scattered light signals, SYTO62 positive signals, and Spike protein positive signals is the particle of the SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector containing nucleic acid and Spike protein;

[0304] A population of particles with positive scattered light signal, negative SYTO62 signal, and positive Spike protein signal are particles of an empty-shell SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector;

[0305] A population of particles with positive scattered light signal, positive SYTO62 signal, and negative Spike protein signal are particles of a SARS-CoV-2 (2019-nCoV) Spike Pseudovirus vector lacking the Spike protein and containing nucleic acid.

[0306] Results: The detection results of the scattered light channel and the results of the population of fluorescent signal particles with positive scattered light signal are as Figure 12 shown.

[0307] Conclusion: The calculation of the biological functional titer ratio of SARS-CoV-2 (2019-nCoV) Spike Pseudovirus was completed. The percentage of the population of particles with positive scattered light signal, positive nucleic acid signal, and positive Spike protein expression signal, which are particles of SARS-CoV-2 (2019-nCoV) Spike Pseudovirus containing nucleic acid and Spike protein, in the total number of particles of all populations of particles with positive scattered light signal is 22.2%.

[0308] Example 8: Detection with a nano-flow cytometer after labeling with a nucleic acid dye and a gag antibody conjugated with a fluorescent dye

[0309] 1) Dual-optical labeling of retroviral vectors

[0310] Dilute the concentrated solution of the retroviral vector product 10-fold with 10 mM PBS buffer to obtain Solution 1. Take 50 μL of Solution 1 and incubate it with 20 μL of AF488 HIV-1 gag-pol antibody (antibody against the gag protein of retrovirus conjugated with AF488) at 37 °C for 30 min, centrifuge at 100,000 g at 4 °C for 80 min, discard the supernatant, resuspend the precipitate with PBS, centrifuge at 100,000 g at 4 °C for 20 min, discard the supernatant, and resuspend the precipitate with 100 μL of 10 mM PBS buffer containing 3 μM SYTO62 to obtain the retroviral vector after dual labeling.

[0311] The method for preparing the HIV-1 gag-pol antibody conjugated with AF488 fluorescent dye includes conjugating the AF488 fluorescent dye to the HIV-1 gag-pol antibody according to the method described in the EZ-Link™ Maleimide Protein Labeling Kit instruction manual to obtain the HIV-1 gag-pol antibody conjugated with AF488 fluorescent dye.

[0312] 2) Detection of the retroviral vector after dual labeling by Flow NanoAnalyzer

[0313] Flow NanoAnalyzer detection conditions: Laser detectors 488nm + 638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1 kpa; signal type: under the condition of small signal, use the FITC and PC5 fluorescence dual channels.

[0314] Detection of the retroviral vector after dual labeling: Use Flow NanoAnalyzer to detect the retroviral vector after dual labeling. The PC5 fluorescence is the excitation signal of the nucleic acid dye to characterize the nucleic acid of the retroviral vector, and the FITC fluorescence characterizes the gag protein in the retroviral vector. Analyze the detection results of the FITC fluorescence channel and the PC5 fluorescence channel, record the scatter plot of the detection results, and set a quadrant gate for the obtained scatter plot of the results. Among them,

[0315] The particle population with positive nucleic acid signal and positive gag expression signal is the retroviral vector particle containing nucleic acid and capsid;

[0316] The particle population with positive gag expression signal and negative nucleic acid signal is the particle of the empty capsid retroviral vector;

[0317] The particle population with positive nucleic acid signal and negative gag expression signal is the particle of the retroviral vector lacking capsid containing nucleic acid.

[0318] The results of the fluorescent signal particle population are as Figure 13 shown.

[0319] Conclusion: The retroviral vector after dual labeling was detected by Flow NanoAnalyzer, and it was found that the proportion of retroviral vector particles containing nucleic acid and capsid in the retroviral vector product was 41.5%.

[0320] Example 9: Detection by a nano-flow cytometer after labeling with a nucleic acid dye and an env antibody conjugated with a fluorescent dye

[0321] 1) Dual optical labeling of the retroviral vector

[0322] Dilute the concentrated retroviral vector product 10-fold with 10 mM PBS buffer to obtain Solution 1. Take 50 μL of Solution 1 and incubate it with 20 μL of AF488 HIV-1 env Antibody (antibody conjugated to AF488-labeled retroviral env protein) at 37 °C for 30 min. Centrifuge at 100,000 g at 4 °C for 80 min, discard the supernatant, resuspend the precipitate with PBS, centrifuge at 100,000 g at 4 °C for 20 min, discard the supernatant, and add 100 μL of 10 mM PBS buffer containing 3 μM SYTO62 to resuspend the precipitate to obtain the doubly labeled retroviral vector.

[0323] The preparation method of the above-mentioned HIV-1 env Antibody conjugated with AF488 fluorescent dye includes conjugating the AF488 fluorescent dye to the HIV-1 env Antibody according to the method described in the EZ-Link™ Maleimide Protein Labeling Kit instructions to obtain the HIV-1 env Antibody conjugated with AF488 fluorescent dye.

[0324] 2) Detection of the doubly labeled retroviral vector by Flow NanoAnalyzer

[0325] Flow NanoAnalyzer detection conditions: Laser detectors 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. Under these conditions, use the FITC and PC5 fluorescence dual channels.

[0326] Detection of the doubly labeled retroviral vector: Use Flow NanoAnalyzer to detect the doubly labeled retroviral vector. The PC5 fluorescence is the excitation signal of the nucleic acid dye used to characterize the nucleic acid of the retroviral vector, and the FITC fluorescence characterizes the env protein in the retroviral vector. Analyze the detection results of the FITC fluorescence channel and the PC5 fluorescence channel, record the scatter plot of the detection results, and set a quadrant gate for the obtained scatter plot of the results. Among them,

[0327] The particle population with positive nucleic acid signal and positive env expression signal is the retroviral vector particle containing nucleic acid and envelope;

[0328] The particle population with positive env expression signal and negative nucleic acid signal is the particle of the empty-shell retroviral vector;

[0329] The particle population with positive nucleic acid signal and negative env expression signal is the particle of the retroviral vector lacking envelope and containing nucleic acid.

[0330] The results of the fluorescent signal particle population are as Figure 14 shown.

[0331] Conclusion: After detecting the retroviral vector after dual labeling using Flow NanoAnalyzer, it was found that the proportion of retroviral vector particles containing nucleic acid and envelope in the retroviral vector product was 29%.

[0332] The method of the present invention has been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters for implementation. It should be particularly pointed out that all such similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention.

Claims

1. A method for detecting one or more indicators of a viral nucleic acid vector, comprising: Step (1): Prepare optical reagents for each parameter in the indicators of the viral nucleic acid vector, and mix the optical reagents with a sample containing the viral nucleic acid vector to specifically bind the optical reagents to the viral nucleic acid vector; the optical reagents include nucleic acid probes or nucleic acid dye reagents, and at least one selected from lipid membrane dye reagents or specific anti-target reagents conjugated with fluorescent dyes, and the parameters include viral capsid protein marker expression signals and / or viral envelope marker expression signals and nucleic acid signals; Step (2): Prepare a concentration standard solution, detect the concentration standard solution using a flow particle detection device, record the detection data of the scattered light channel and the fluorescence channel of the concentration standard solution and the injection flow rate of the flow particle detection device, and measure the particle count concentration of the sample solution containing the viral nucleic acid vector; the injection pressure and detection time of the concentration standard solution and the sample solution containing the viral nucleic acid vector are the same; Step (3): Detect the sample solution containing the viral nucleic acid vector using a flow particle detection device, and record the detection data of the scattered light channel and the fluorescence channel of the sample containing the viral nucleic acid vector; and Step (4): Data analysis to calculate the results of each indicator; wherein, the viral nucleic acid vector includes a retroviral vector, a herpes simplex virus vector, and a recombinant coronavirus vector; the indicators include the biological functional titer of the viral nucleic acid vector; the biological functional titer of the viral nucleic acid vector includes: the titer of the viral nucleic acid vector containing nucleic acid, viral capsid, and envelope, the titer of the viral nucleic acid vector containing nucleic acid and envelope, the titer of the viral nucleic acid vector containing nucleic acid and viral capsid, and the titer of the empty capsid viral nucleic acid vector; The calculation formula for the biological functional titer of the viral nucleic acid vector is: biological functional titer of the viral nucleic acid vector = P × particle count concentration in the viral nucleic acid vector; wherein, P is the percentage of the particle population with an event positive signal and a positive signal for the biological function to be detected in the total number of particles in all particle populations with an event positive signal.

2. The detection method according to claim 1, wherein the viral nucleic acid vector includes a lentiviral vector.

3. The detection method according to claim 2, wherein the nucleic acid signal in step (1) includes a target nucleic acid signal.

4. The detection method according to claim 3, wherein the indicators include the titer of the viral nucleic acid vector containing target nucleic acid, viral capsid, and envelope, the titer of the viral nucleic acid vector containing target nucleic acid and envelope, and the titer of the viral nucleic acid vector containing target nucleic acid and viral capsid.

5. The detection method according to claim 2, wherein the index includes the biological functional titer of the lentiviral vector, and the biological functional titer of the lentiviral vector includes: Titers of lentiviral vectors containing nucleic acid, lentiviral capsid, and envelope; titers of lentiviral vectors containing nucleic acid and envelope; titers of empty capsid lentiviral vectors; titers of lentiviral vectors containing nucleic acid and lacking envelope; titers of lentiviral vectors containing nucleic acid and lentiviral capsid; titers of lentiviral vectors containing nucleic acid and lacking lentiviral capsid; titers of lentiviral vectors containing nucleic acid and envelope and lacking lentiviral capsid; titers of lentiviral vectors containing nucleic acid, lentiviral capsid, and lacking envelope; titers of lentiviral vectors containing nucleic acid and lacking both lentiviral capsid and envelope.

6. According to the detection method described in claim 2, the index includes the biological functional titer of the lentiviral vector, and the biological functional titer of the lentiviral vector includes: Titers of lentiviral vectors containing nucleic acid and VSV-G; titers of lentiviral vectors containing nucleic acid and lacking VSV-G; titers of lentiviral vectors containing nucleic acid and p24 protein; titers of lentiviral vectors containing nucleic acid and lacking p24 protein.

7. According to the detection method described in claim 1, the index includes the biological functional titer of the retroviral vector, and the biological functional titer of the retroviral vector includes: Titers of retroviral vectors containing nucleic acid, gag capsid protein, and env envelope protein; titers of retroviruses containing nucleic acid and env envelope protein; titers of empty capsid retroviral vectors; titers of retroviral vectors containing nucleic acid and lacking env envelope protein; titers of retroviral vectors containing nucleic acid and gag capsid protein; titers of retroviral vectors containing nucleic acid and lacking gag capsid protein; and / or The indicators include the biological functional titer of a herpes simplex virus vector, and the biological functional titer of the herpes simplex virus vector includes: the titer of a herpes simplex virus vector containing nucleic acid and g envelope glycoprotein, the titer of an empty capsid herpes simplex virus vector, and the titer of a herpes simplex virus vector containing nucleic acid and lacking g envelope glycoprotein; The g envelope glycoprotein includes any one or a combination of envelope glycoprotein gB, envelope glycoprotein gC, envelope glycoprotein gD, envelope glycoprotein gE, envelope glycoprotein gG, envelope glycoprotein gH, envelope glycoprotein gI, envelope glycoprotein gJ, envelope glycoprotein gL, envelope glycoprotein gM, and envelope glycoprotein gN; and / or The indicators include the biological functional titer of a recombinant coronavirus vector, and the biological functional titer of the recombinant coronavirus vector includes: the titer of a recombinant coronavirus vector containing nucleic acid and SPIKE protein, the titer of an empty capsid recombinant coronavirus vector, and the titer of a recombinant coronavirus vector containing nucleic acid and lacking SPIKE protein.

8. The detection method according to claim 3, wherein the index includes the biological functional titer of the lentiviral vector, and the biological functional titer of the lentiviral vector includes: Titers of lentiviral vectors containing target nucleic acid, lentiviral capsid, and envelope; titers of lentiviral vectors containing target nucleic acid and envelope; titers of lentiviral vectors containing target nucleic acid and lacking envelope; titers of lentiviral vectors containing target nucleic acid and lentiviral capsid; titers of lentiviral vectors containing target nucleic acid and lacking lentiviral capsid; titers of lentiviral vectors containing target nucleic acid and envelope and lacking lentiviral capsid; titers of lentiviral vectors containing target nucleic acid, lentiviral capsid, and lacking envelope; or titers of lentiviral vectors containing target nucleic acid and lacking both lentiviral capsid and envelope.

9. The detection method according to claim 3, wherein the index includes the biological functional titer of the lentiviral vector, and the biological functional titer of the lentiviral vector includes: The titers of lentiviral vectors containing the target nucleic acid and VSV-G, the titers of lentiviral vectors lacking VSV-G and containing the target nucleic acid, the titers of lentiviral vectors containing the target nucleic acid and p24 protein, and the titers of lentiviral vectors lacking p24 protein and containing the target nucleic acid.

10. The detection method according to claim 3, wherein the index includes the biological functional titer of the retroviral vector, and the biological functional titer of the retroviral vector includes: The titers of retroviral vectors containing the target nucleic acid, gag capsid protein, and env envelope protein, the titers of retroviral vectors containing the target nucleic acid and env envelope protein, the titers of retroviral vectors lacking env envelope protein and containing the target nucleic acid, the titers of retroviral vectors containing the target nucleic acid and gag capsid protein, and retroviral vectors lacking gag capsid protein and containing the target nucleic acid; and / or The said indicators include the biological functional titer of herpes simplex virus vectors, and the biological functional titer of herpes simplex virus vectors includes: the titer of herpes simplex virus vectors containing the target nucleic acid and g envelope glycoprotein, and the titer of herpes simplex virus vectors lacking g envelope glycoprotein and containing the target nucleic acid; The said g envelope glycoprotein includes any one or a combination of envelope glycoprotein gB, envelope glycoprotein gC, envelope glycoprotein gD, envelope glycoprotein gE, envelope glycoprotein gG, envelope glycoprotein gH, envelope glycoprotein gI, envelope glycoprotein gJ, envelope glycoprotein gL, envelope glycoprotein gM, and envelope glycoprotein gN; and / or The said indicators include the biological functional titer of recombinant coronavirus vectors, and the biological functional titer of recombinant coronavirus vectors includes: the titer of recombinant coronavirus vectors containing the target nucleic acid and SPIKE protein, and the titer of recombinant coronavirus vectors lacking SPIKE protein and containing the target nucleic acid.

11. According to the method described in claim 1, the said parameters include nucleic acid signals and envelope protein marker expression signals.

12. According to the method described in claim 3, the said parameters include target nucleic acid signals and envelope protein marker expression signals.

13. According to the method described in claim 1, in the operation of mixing the said optical reagent with the sample containing the viral nucleic acid vector in step (1), adding a surfactant and mixing it with the sample containing the viral nucleic acid vector is also included.

14. According to the method described in claim 13, the said surfactant includes at least one of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants.

15. According to the method described in claim 13, the said surfactant includes at least one of Tween-type surfactants, Triton X-100, sodium dodecyl sulfate, nonylphenol polyoxyethylene ether, or polyoxyethylene-type nonionic surfactants.

16. The method according to claim 1, wherein the step (2) comprises: Prepare beads with an absolute known quantity as a concentration standard and prepare a concentration standard solution.

17. According to the method described in claim 1, the said anti-target reagent includes at least one of antibodies, antibody fragments, lectins, aptamers, peptides, growth factors, glycolipids, polysaccharides, ligands, or receptors.

18. According to the method described in claim 1, the said anti-target reagent is an antibody, antibody fragment, ligand, or receptor.

19. The method according to claim 17 or 18, wherein the antibody comprises at least one of a VSV-G antibody, a gB antibody, a gC antibody, a gD antibody, a gE antibody, a gG antibody, a gH antibody, a gI antibody, a gJ antibody, a gL antibody, a gM antibody, a gN antibody, an env antibody, and a SPIKE antibody.

20. According to claim 17 or 18 The method, wherein the antibody comprises an antibody that can specifically bind to a retrovirus envelope-expressed protein specifically, an antibody that can specifically bind to a herpes simplex virus envelope-expressed protein, and at least one of an antibody that can specifically bind to a recombinant coronavirus envelope-expressed protein.

21. The method according to claim 17 or 18, wherein the viral nucleic acid vector comprises a lentiviral vector; and the antibody comprises an antibody that can specifically bind to a lentivirus envelope-expressed protein.

22. The method according to claim 21, wherein the antibody that can specifically bind to a lentivirus envelope-expressed protein comprises a p24 antibody.

23. The method according to claim 20, wherein the antibody that can specifically bind to a retrovirus envelope-expressed protein comprises a gag antibody.

24. The method according to claim 3, wherein the nucleic acid probe comprises a sequence complementary to the target nucleic acid, and the 5'-end or 3'-end or middle of the sequence is modified with a fluorescent dye; and / or the nucleic acid dye reagent comprises a cyanine dye, a non-permeable dye, a permeable dye, an intercalating dye, or a DNA minor groove-binding dye; and / or the lipid membrane dye reagent comprises a lipophilic fluorescent dye selected from those capable of binding to cell membranes and other lipid-soluble membrane structures; and / or the fluorescent dye comprises a fluorescent molecule, a fluorescent material, or a combination thereof.

25. The method according to claim 1, wherein the fluorescent dye comprises at least one of an organic fluorescent molecule, a fluorescent protein, a nucleic acid dye, a lipid membrane dye, a quantum dot, and a Polymer Dot.

26. The method according to claim 1, wherein the lipid membrane dye reagent is selected from at least one of a DiD dye, a DiO dye, a DiI dye, a DiR dye, a DiA dye, a Di-8-Anepps dye, a Di-4-ANEPPS dye, a PHK26 dye, a PKH67 dye, a CellMask Green dye, a CellMask Orange dye, a CellMask Red dye, a CellVue Lavender dye, a CellVue Plum dye, and a CellVue NIR780 dye; and / or The nucleic acid dye reagents include Acridine Orange, Actinomycin D, 7-AAD (7-Aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodid, BOBO-3 Iodide, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), dihydroethidium (hydroethidine), EthidiumHomodimer-1 (EthD-1), Ethidium Homodimer-2 (EthD-2), Ethidium MonoazideBromide (EMA), Hexidium Iodide, Hoechst 33258, Pentahydrate (bis-Benzimide), Hoechst 33342, Trihydrochloride,At least one of Trihydrate, Trihydrate - FluoroPure Grade, Hoechst 34580, LDS 751, NeuroTrace Blue Fluorescent Nissl Stain, NeuroTrace Green Fluorescent Nissl Stain, NeuroTrace 530 / 615 Red Fluorescent Nissl Stain, NeuroTrace Deep - Red Fluorescent Nissl Stain, POPO - 1 Iodide, POPO - 3 Iodide, PO - PRO - 1 Iodide, Propidium Iodide, OliGreen, PicoGreen, RiboGreen, SYBRGold, SYBR Green I, SYBR Green II, SYBR Safe DNA Gel Stain, SYTO 40, SYTO 41, SYTO42, 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, SYTO 17, SYTO 59, SYTO 60, SYTO61, SYTO 62, SYTO 63, SYTO 64, SYTO RNASelect, SYTOX Blue, SYTOX Green, SYTOXOrange, SYTOX Red, TO - PRO - 1 Iodide, TO - PRO - 3 Iodide, TOTO - 1 Iodide, TOTO - 3 Iodide, YO - PRO - 1 Iodid, YO - PRO - 3 Iodide, YOYO - 1 Iodide, YOYO - 3 Iodide, HCS NuclearMaskDeep Red Stain, HCS NuclearMask Blue Stain, HCS NuclearMask Red Stain, Ethidium Bromide; and / or, the anti-target reagent is an antibody, and the antibody comprises at least one of an antibody against VSV-G, an antibody against an env envelope protein, an antibody against a g envelope glycoprotein, an antibody against a SPIKE protein, an antibody against gag, and an antibody against p24.

27. The method according to claim 1, wherein the anti-target reagent is an antibody, and the antibody comprises at least one of an antibody against a viral capsid protein or an antibody against an envelope-expressed protein.

28. The method according to claim 1, wherein the anti-target reagent is a ligand or a receptor, and the ligand or receptor is at least one of a ligand or receptor of VSV-G, a ligand or receptor of env envelope protein, a ligand or receptor of g envelope glycoprotein, a ligand or receptor of SPIKE protein, a ligand or receptor of gag, and a ligand or receptor of p24.

29. The method according to claim 1, wherein the anti-target reagent is a ligand or a receptor, and the ligand or receptor is at least one of a ligand or receptor of a viral capsid protein or a ligand or receptor of an envelope-expressed protein.

30. The method according to claim 25, wherein the lipid membrane dyes in the fluorescent dyes are each independently selected from at least one of DiD dye, DiO dye, DiI dye, DiR dye, DiA dye, Di-8-Anepps dye, Di-4-ANEPPS dye, PHK26 dye, PKH67 dye, CellMask Green dye, CellMask Orange dye, CellMask Red dye, CellVueLavender dye, CellVue Plum dye, and CellVue NIR780 dye; and / or The nucleic acid dyes in the fluorescent dyes each independently include Acridine Orange, Actinomycin D, 7-AAD (7-Aminoactinomycin D), ACMA (9-Amino-6-Chloro-2-Methoxyacridine), BOBO-1 Iodid, BOBO-3 Iodide, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), dihydroethidium (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,At least one of Trihydrate, Trihydrate - FluoroPure Grade, Hoechst 34580, LDS 751, NeuroTrace Blue Fluorescent Nissl Stain, NeuroTrace Green Fluorescent Nissl Stain, NeuroTrace 530 / 615 Red Fluorescent Nissl Stain, NeuroTrace Deep - Red Fluorescent Nissl Stain, POPO - 1 Iodide, POPO - 3 Iodide, PO - PRO - 1 Iodide, Propidium Iodide, OliGreen, PicoGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, 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, SYTOXBlue, SYTOX Green, SYTOX Orange, SYTOX Red, TO - PRO - 1 Iodide, TO - PRO - 3 Iodide, TOTO - 1 Iodide, TOTO - 3 Iodide, YO - PRO - 1 Iodid, YO - PRO - 3 Iodide, YOYO - 1 Iodide, YOYO - 3 Iodide, HCS NuclearMask Deep Red Stain, HCS NuclearMask Blue Stain, HCS NuclearMask Red Stain, Ethidium Bromide., 31. The method according to claim 1, wherein the fluorescent dye comprises a fluorescent molecule, a fluorescent material or a combination thereof, and the fluorescent dye is directly conjugated to the anti-target reagent; or the fluorescent dye is indirectly conjugated to the anti-target reagent via a recognition group, and the recognition group comprises an antibody, an antigen, a receptor or a polysaccharide, and the recognition group can specifically recognize the anti-target reagent; and / or The mixing of the optical reagent with the sample containing the viral nucleic acid carrier in step (1) comprises mixing the optical reagent and a surfactant with the sample containing the viral nucleic acid carrier; and / or Step (3) comprises photographing, counting and measuring the signal intensity value of particles with specific optical characteristics on the sample by using a flow particle detection device.

32. The method according to claim 1, wherein step (3) comprises detecting by using a flow particle detection device with a scattered light channel and at least two fluorescence channels, one fluorescence channel is used to characterize the nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker, analyzing the fluorescence channel detection results of the particle population with event positive signals, recording the detection result scatter plot, and gating the obtained result scatter plot, wherein The particle population with event positive signals, nucleic acid positive signals and viral envelope marker expression positive signals is the particle of the viral nucleic acid carrier containing nucleic acid and envelope; The particle population with event positive signals, viral envelope marker expression positive signals and nucleic acid negative signals is the particle of the empty capsid viral nucleic acid carrier; The particle population with event positive signals, nucleic acid positive signals and viral envelope marker expression negative signals is the particle of the viral nucleic acid carrier lacking an envelope containing nucleic acid.

33. The method according to claim 1, wherein the fluorescence channel is used to collect the fluorescence signal in a specific wavelength range emitted after the target to be measured absorbs light waves in a specific wavelength range.

34. The method according to claim 3, wherein step (3) includes detecting by using a flow cytometry particle detection device with a scattered light channel and at least two fluorescence channels, one fluorescence channel is used to characterize the target nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker, analyzing the fluorescence channel detection results of the particle population with event positive signals, recording the scatter plot of the detection results, and gating the obtained scatter plot of the results, wherein, The particle population with event positive signals, target nucleic acid positive signals and viral envelope marker expression positive signals is the particle of the viral nucleic acid carrier containing the target nucleic acid and the envelope; The particle population with event positive signals, viral envelope marker expression positive signals and target nucleic acid negative signals is the particle of the empty capsid viral nucleic acid carrier; The particle population with event positive signals, target nucleic acid positive signals and viral envelope marker expression negative signals is the particle of the viral nucleic acid carrier lacking the envelope and containing the target nucleic acid.

35. The method according to claim 2, wherein the viral nucleic acid carrier is a lentiviral vector, and step (3) includes detecting by using a flow cytometry particle detection device with a scattered light channel and at least two fluorescence channels, one fluorescence channel is used to characterize the nucleic acid, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker, analyzing the fluorescence channel detection results of the particle population with event positive signals, recording the scatter plot of the detection results, and gating the obtained scatter plot of the results, wherein, The particle population with event positive signals, nucleic acid positive signals and VSV-G expression positive signals is the particle of the lentiviral vector containing the nucleic acid and VSV-G; The particle population with event positive signals, VSV-G expression positive signals and nucleic acid negative signals is the particle of the empty capsid lentiviral vector; The particle with event positive signals, nucleic acid positive signals and VSV-G expression negative signals is the particle of the lentiviral vector lacking VSV-G and containing the nucleic acid.

36. The method according to claim 1, wherein the viral nucleic acid carrier is a retroviral vector, and step (3) includes detecting by using a flow cytometry particle detection device with a scattered light channel and at least two fluorescence channels, one fluorescence channel is used to characterize the nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker, analyzing the fluorescence channel detection results of the particle population with event positive signals, recording the scatter plot of the detection results, and gating the obtained scatter plot of the results, wherein, The particle population with event positive signals, nucleic acid positive signals and env envelope protein expression positive signals is the particle of the retroviral vector containing the nucleic acid and the env envelope protein; The particle population with event positive signals, env envelope protein expression positive signals and nucleic acid negative signals is the particle of the empty capsid retroviral vector; The particle with event positive signals, nucleic acid positive signals and env envelope protein expression negative signals is the particle of the retroviral vector lacking the env envelope protein and containing the nucleic acid; and / or The viral nucleic acid vector is a herpes simplex virus vector. Step (3) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels. One fluorescence channel is used to characterize the nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker. Analyze the fluorescence channel detection results of the particle population with an event positive signal, record the scatter plot of the detection results, and gate the obtained scatter plot of the results. Among them, The particle population with an event positive signal, a nucleic acid positive signal, and a positive signal for the expression of the g envelope glycoprotein is the particle of the herpes simplex virus vector containing nucleic acid and the g envelope glycoprotein; The particle population with an event positive signal, a positive signal for the expression of the g envelope glycoprotein, and a nucleic acid negative signal is the particle of the empty-shell herpes simplex virus vector; The particle population with an event positive signal, a nucleic acid positive signal, and a negative signal for the expression of the g envelope glycoprotein is the particle of the herpes simplex virus vector lacking the g envelope glycoprotein and containing nucleic acid; and / or The viral nucleic acid vector is a recombinant coronavirus vector. Step (3) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels. One fluorescence channel is used to characterize the nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker. Analyze the fluorescence channel detection results of the particle population with an event positive signal, record the scatter plot of the detection results, and gate the obtained scatter plot of the results. Among them, The particle population with an event positive signal, a nucleic acid positive signal, and a positive signal for the expression of the SPIKE protein is the particle of the recombinant coronavirus vector containing nucleic acid and the SPIKE protein; The particle population with an event positive signal, a positive signal for the expression of the SPIKE protein, and a nucleic acid negative signal is the particle of the empty-shell recombinant coronavirus vector; The particle population with an event positive signal, a nucleic acid positive signal, and a negative signal for the expression of the SPIKE protein is the particle of the recombinant coronavirus vector lacking the SPIKE protein and containing nucleic acid.

37. According to the method described in claim 1, step (3) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels. One fluorescence channel is used to characterize the nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral capsid protein marker. Analyze the dual-fluorescence channel detection results of the particle population with an event positive signal, record the scatter plot of the detection results, and gate the obtained scatter plot of the results. Among them, The particle population with an event positive signal, a nucleic acid positive signal, and a positive signal for the expression of the viral capsid protein marker is the particle population of the viral nucleic acid vector with nucleic acid and a viral capsid; The particle population with an event positive signal, a positive signal for the expression of the viral capsid protein marker, and a nucleic acid negative signal is the particle population of the empty-shell viral nucleic acid vector; The particle population with an event positive signal, a nucleic acid positive signal, and a negative signal for the expression of the viral capsid protein marker is the particle population of the viral nucleic acid vector lacking a viral capsid and containing nucleic acid.

38. The method according to claim 2, wherein the viral nucleic acid vector is a lentiviral vector, and step (3) includes detecting by using a flow particle detection device with a light scattering channel and at least two fluorescence channels. In the detection of the lentiviral vector, analyze the fluorescence channel detection results of the particle population with an event positive signal, record the detection result scatter plot, and gate the obtained result scatter plot, wherein, The particle population with an event positive signal, a nucleic acid positive signal, and a positive signal for the expression of lentiviral p24 protein is the particle population of the lentiviral vector containing nucleic acid and lentiviral p24; The particle population with an event positive signal, a positive signal for the expression of lentiviral p24 protein, and a nucleic acid negative signal is the particle population of the empty capsid lentiviral vector; The particle population with an event positive signal, a nucleic acid positive signal, and a negative signal for the expression of lentiviral p24 protein is the particle population of the lentiviral vector lacking p24 protein and containing nucleic acid.

39. The method according to claim 1, wherein the viral nucleic acid vector is a retroviral vector, and step (3) includes detecting by using a flow particle detection device with a light scattering channel and at least two fluorescence channels. In the detection of the retroviral vector, analyze the fluorescence channel detection results of the particle population with an event positive signal, record the detection result scatter plot, and gate the obtained result scatter plot, wherein, The particle population with an event positive signal, a nucleic acid positive signal, and a positive signal for the expression of gag capsid protein is the particle population of the retroviral vector containing nucleic acid and gag capsid protein; The particle population with an event positive signal, a positive signal for the expression of gag capsid protein, and a nucleic acid negative signal is the particle population of the empty capsid retroviral vector; The particle population with an event positive signal, a nucleic acid positive signal, and a negative signal for the expression of gag capsid protein is the particle population of the retroviral vector lacking gag capsid protein and containing nucleic acid.

40. The method according to claim 3, wherein the viral nucleic acid vector is a lentiviral vector, and step (3) includes detecting by using a flow particle detection device with a light scattering channel and at least two fluorescence channels. One fluorescence channel is used to characterize the target nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker. Analyze the fluorescence channel detection results of the particle population with an event positive signal, record the detection result scatter plot, and gate the obtained result scatter plot, wherein, The particle population with an event positive signal, a target nucleic acid positive signal, and a positive signal for the expression of VSV-G is the particle of the lentiviral vector containing the target nucleic acid and VSV-G; The particle population with an event positive signal, a positive signal for the expression of VSV-G, and a target nucleic acid negative signal is the particle of the empty capsid lentiviral vector; The particle population with an event positive signal, a target nucleic acid positive signal, and a negative signal for the expression of VSV-G is the particle of the lentiviral vector lacking VSV-G and containing the target nucleic acid.

41. According to the method described in claim 3, the viral nucleic acid vector is a lentiviral vector, and step (3) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels. In the detection of the lentiviral vector, analyze the fluorescence channel detection results of the particle population with an event positive signal, record the detection result scatter plot, and gate the obtained result scatter plot. Among them, The particle population with an event positive signal, a target nucleic acid positive signal, and a lentiviral p24 protein expression positive signal is the particle population of the lentiviral vector containing the target nucleic acid and p24 protein; The particle population with an event positive signal, a lentiviral p24 protein expression positive signal, and a target nucleic acid negative signal is the particle population of the empty capsid lentiviral vector; The particle population with an event positive signal, a target nucleic acid positive signal, and a p24 protein expression negative signal is the particle population of the lentiviral vector lacking p24 protein and containing the target nucleic acid.

42. According to the method described in claim 3, the viral nucleic acid vector is a retroviral vector, and step (3) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels. One fluorescence channel is used to characterize the target nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker. Analyze the fluorescence channel detection results of the particle population with an event positive signal, record the detection result scatter plot, and gate the obtained result scatter plot. Among them, The particle population with an event positive signal, a target nucleic acid positive signal, and an env envelope protein expression positive signal is the particle of the retroviral vector containing the target nucleic acid and env envelope protein; The particle population with an event positive signal, an env envelope protein expression positive signal, and a target nucleic acid negative signal is the particle of the empty capsid retroviral vector; The particle population with an event positive signal, a target nucleic acid positive signal, and an env envelope protein expression negative signal is the particle of the retroviral vector lacking env envelope protein and containing the target nucleic acid; and / or The viral nucleic acid vector is a herpes simplex virus vector, and step (3) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels. One fluorescence channel is used to characterize the target nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker. Analyze the fluorescence channel detection results of the particle population with an event positive signal, record the detection result scatter plot, and gate the obtained result scatter plot. Among them, The particle population with an event positive signal, a target nucleic acid positive signal, and a g envelope glycoprotein expression positive signal is the particle of the herpes simplex virus vector containing the target nucleic acid and g envelope glycoprotein; The particle population with an event positive signal, a g envelope glycoprotein expression positive signal, and a target nucleic acid negative signal is the particle of the empty capsid herpes simplex virus vector; The particle population with an event positive signal, a target nucleic acid positive signal, and a g envelope glycoprotein expression negative signal is the particle of the herpes simplex virus vector lacking g envelope glycoprotein and containing the target nucleic acid; and / or The viral nucleic acid vector is a recombinant coronavirus vector. Step (3) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels. One fluorescence channel is used to characterize the target nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral envelope marker. Analyze the fluorescence channel detection results of the particle population with event positive signals, record the detection result scatter plot, and gate the obtained result scatter plot. Among them, The particle population with event positive signals, target nucleic acid positive signals, and SPIKE protein expression positive signals is the particle of the recombinant coronavirus vector containing the target nucleic acid and SPIKE protein; The particle population with event positive signals, SPIKE protein expression positive signals, and target nucleic acid negative signals is the particle of the empty-shell recombinant coronavirus vector; The particle population with event positive signals, target nucleic acid positive signals, and SPIKE protein expression negative signals is the particle of the recombinant coronavirus vector lacking SPIKE protein and containing the target nucleic acid.

43. According to the method described in claim 3, step (3) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels. One fluorescence channel is used to characterize the target nucleic acid signal, and the other fluorescence channel is used to characterize the expression signal of the viral capsid protein marker. Analyze the dual-fluorescence channel detection results of the particle population with event positive signals, record the detection result scatter plot, and gate the obtained result scatter plot. Among them, The particle population with event positive signals, target nucleic acid positive signals, and viral capsid protein marker expression positive signals is the particle population of the viral nucleic acid vector containing the target nucleic acid and the viral capsid; The particle population with event positive signals, viral capsid protein marker expression positive signals, and target nucleic acid negative signals is the particle population of the empty-shell viral nucleic acid vector; The particle population with event positive signals, target nucleic acid positive signals, and viral capsid protein marker expression negative signals is the particle population of the viral nucleic acid vector lacking the viral capsid and containing the target nucleic acid.

44. According to the method described in claim 3, the viral nucleic acid vector is a retroviral vector. Step (3) includes detecting using a flow particle detection device with a scattered light channel and at least two fluorescence channels. In the detection of the retroviral vector, analyze the fluorescence channel detection results of the particle population with event positive signals, record the detection result scatter plot, and gate the obtained result scatter plot. Among them, The particle population with event positive signals, target nucleic acid positive signals, and gag capsid protein expression positive signals is the particle population of the retroviral vector containing the target nucleic acid and the gag capsid protein; The particle population with event positive signals, gag capsid protein expression positive signals, and target nucleic acid negative signals is the particle population of the empty-shell retroviral vector; The particle population with event positive signals, target nucleic acid positive signals, and gag capsid protein expression negative signals is the particle population of the retroviral vector lacking the gag capsid protein and containing the target nucleic acid.

45. The method according to claim 1, wherein step (3) comprises performing multi-parameter detection using a flow particle detection device with a scattered light channel and at least one or at least two fluorescence channels. At least one fluorescence channel is used to characterize nucleic acid signals, and other channels are used to characterize the expression signals of at least one viral capsid protein marker or at least one viral envelope marker. Analyze the fluorescence channel detection results of the particle population with event positive signals, record the detection results in one or more scatter plots, and gate the obtained scatter plots of the results, wherein, The particle population with event positive signals, nucleic acid positive signals, viral capsid protein marker expression positive signals, and viral envelope marker expression positive signals is the particle population of viral nucleic acid carriers containing nucleic acids, viral capsids, and envelopes; The particle population with event positive signals and nucleic acid negative signals is the particle population of empty capsid viral nucleic acid carriers, and the particle population of empty capsid viral nucleic acid carriers includes at least one of the following particle populations: a) the particle population of empty capsid viral nucleic acid carriers with event positive signals, nucleic acid negative signals, viral capsid protein marker expression negative signals, and viral envelope marker expression positive signals; b) the particle population of empty capsid viral nucleic acid carriers with event positive signals, nucleic acid negative signals, viral capsid protein marker expression positive signals, and viral envelope marker expression negative signals; c) the particle population of empty capsid viral nucleic acid carriers with event positive signals, nucleic acid negative signals, viral capsid protein marker expression positive signals, and viral envelope marker expression positive signals; d) the particle population of empty capsid viral nucleic acid carriers with event positive signals, nucleic acid negative signals, viral capsid protein marker expression negative signals, and viral envelope marker expression negative signals; The particle population with event positive signals, nucleic acid positive signals, viral capsid protein marker expression negative signals, and viral envelope marker expression positive signals is the particle population of viral nucleic acid carriers lacking viral capsids and containing nucleic acids and envelopes; The particle population with event positive signals, nucleic acid positive signals, viral capsid protein marker expression positive signals, and viral envelope marker expression negative signals is the particle population of viral nucleic acid carriers lacking envelopes and containing nucleic acids and capsids; The particle population with event positive signals, nucleic acid positive signals, viral capsid protein marker expression negative signals, and viral envelope marker expression negative signals is the particle population of viral nucleic acid carriers lacking viral capsids and envelopes and containing nucleic acids.

46. The method according to claim 3, wherein step (3) comprises performing multi-parameter detection using a flow particle detection device with a scattered light channel and at least one or at least two fluorescence channels. At least one fluorescence channel is used to characterize target nucleic acid signals, and other channels are used to characterize the expression signals of at least one viral capsid protein marker or at least one viral envelope marker. Analyze the fluorescence channel detection results of the particle population with event positive signals, record the detection results in one or more scatter plots, and gate the obtained scatter plots of the results, wherein, A population of particles with positive event signals, positive target nucleic acid signals, positive expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector containing the target nucleic acid, viral capsid, and envelope; A population of particles with positive event signals and negative target nucleic acid signals is a population of particles of an empty capsid viral nucleic acid vector, and the population of particles of the empty capsid viral nucleic acid vector includes at least one of the following populations of particles: a) A population of particles of an empty capsid viral nucleic acid vector with positive event signals, negative target nucleic acid signals, negative expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers; b) A population of particles of an empty capsid viral nucleic acid vector with positive event signals, negative target nucleic acid signals, positive expression signals of viral capsid protein markers, and negative expression signals of viral envelope markers; c) A population of particles of an empty capsid viral nucleic acid vector with positive event signals, negative target nucleic acid signals, positive expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers; d) A population of particles of an empty capsid viral nucleic acid vector with positive event signals, negative target nucleic acid signals, negative expression signals of viral capsid protein markers, and negative expression signals of viral envelope markers; A population of particles with positive event signals, positive target nucleic acid signals, negative expression signals of viral capsid protein markers, and positive expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector lacking a viral capsid containing the target nucleic acid and envelope; A population of particles with positive event signals, positive target nucleic acid signals, positive expression signals of viral capsid protein markers, and negative expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector lacking an envelope containing the target nucleic acid and viral capsid; A population of particles with positive event signals, positive target nucleic acid signals, negative expression signals of viral capsid protein markers, and negative expression signals of viral envelope markers is a population of particles of a viral nucleic acid vector lacking a viral capsid and envelope containing the target nucleic acid.

47. According to the method described in claim 1, step (2) includes using a flow particle detection device to detect and record a concentration standard solution and a sample solution containing a viral nucleic acid vector under the same injection pressure and detection time. The calculation method for the particle count concentration in the viral nucleic acid vector is as follows: The particle count concentration in the viral nucleic acid vector = c × A1 ÷ A2, with the unit of particles / mL; Wherein, c is the concentration of the concentration standard solution, in particles / mL; A1 is the number of particles with positive event signals in the sample solution containing the viral nucleic acid vector obtained by detecting the gate in the scattered light channel within the same unit time as A2; A2 is the number of particles with positive event signals in the concentration standard solution obtained by detecting the gate in the scattered light channel within the same unit time as A1; Or The calculation method for the particle count concentration in the viral nucleic acid vector is as follows: The particle count concentration in the viral nucleic acid vector = A1 / K, with the unit of particles / mL; Wherein, K is the volume flow rate per unit time of the flow particle detection device. A1 is the number of particles with positive event signals per unit time after conversion in the sample solution of virus nucleic acid carriers obtained by detecting the gate using a scattered light channel.

48. The calculation method for the titer of the viral nucleic acid vector containing nucleic acid and envelope protein according to claim 1 is: the product of P1 and the particle count concentration in the viral nucleic acid vector, wherein, P1 is the percentage of the number of particles in the particle population with positive event signals, positive nucleic acid signals, and positive envelope protein expression signals among the total number of particles in all particle populations with positive event signals; and / or The calculation method for the titer of a virus nucleic acid carrier containing nucleic acid and an envelope is: the product of P1a and the particle counting concentration in the virus nucleic acid carrier, where P1a is the percentage of the number of particles in the particle population with positive event signals, positive nucleic acid signals, and positive virus envelope marker expression signals among the total number of particles in all particle populations with positive event signals.

49. The method for calculating the titer of the empty-shell lentiviral vector according to claim 2 is: the product of P2 and the particle counting concentration in the viral nucleic acid vector, where P2 is the percentage of the number of particles in the particle population with positive event signals, positive envelope protein expression signals, and negative nucleic acid signals among the total number of particles in all particle populations with positive event signals; or P2 is the percentage of the number of particles in the particle population with positive event signals, positive virus coat protein marker expression signals, and negative nucleic acid signals among the total number of particles in all particle populations with positive event signals; or P2 is the percentage of the number of particles in the particle population of empty capsid lentiviral vectors with positive event signals, negative nucleic acid signals, negative virus coat protein marker expression signals, and positive virus envelope marker expression signals among the total number of particles in all particle populations with positive event signals; or P2 is the percentage of the number of particles in the particle population of empty capsid virus nucleic acid carriers with positive event signals, negative nucleic acid signals, positive virus coat protein marker expression signals, and negative virus envelope marker expression signals among the total number of particles in all particle populations with positive event signals; or P2 is the percentage of the number of particles in the particle population of empty capsid virus nucleic acid carriers with positive event signals, negative nucleic acid signals, positive virus coat protein marker expression signals, and positive virus envelope marker expression signals among the total number of particles in all particle populations with positive event signals; or P2 is the percentage of the number of particles in the particle population of empty capsid virus nucleic acid carriers with positive event signals, negative nucleic acid signals, negative virus coat protein marker expression signals, and negative virus envelope marker expression signals among the total number of particles in all particle populations with positive event signals.

50. The calculation method for the titer of the nucleic acid-containing virus nucleic acid vector lacking an envelope is: the product of P3 and the particle counting concentration in the virus nucleic acid vector, where P3 is the percentage of the total number of points with positive event signals, positive nucleic acid signals, and negative envelope expression signals among the total number of particles in all particle populations with positive event signals.

51. The calculation method for the titer of the nucleic acid-containing virus nucleic acid vector lacking an envelope is: the product of P4 and the particle counting concentration in the virus nucleic acid vector, wherein, P4 is the percentage of the total number of points with positive event signals, positive nucleic acid signals, and negative virus envelope marker expression signals among the total number of particles in all particle populations with positive event signals.

52. The method for calculating the titer of the viral nucleic acid vector lacking a viral capsid containing nucleic acid according to claim 1 is: the product of P5 and the particle count concentration in the viral nucleic acid vector, wherein, P5 is the percentage of the total number of points with positive event signals, positive nucleic acid signals, and negative virus coat protein marker expression signals among the total number of particles in all particle populations with positive event signals.

53. For the method according to claim 1, the calculation method for the titer of the nucleic acid-containing virus nucleic acid vector lacking a capsid protein is: the product of P6 and the particle counting concentration in the virus nucleic acid vector, wherein, P6 is the percentage of the total number of points with positive event signals, positive nucleic acid signals, and negative virus coat protein expression signals among the total number of particles in all particle populations with positive event signals.

54. The method for calculating the titer of the lentiviral vector lacking a viral capsid and containing nucleic acid and an envelope according to claim 2 is: the product of P7 and the particle counting concentration in the viral vector, wherein, P7 is the percentage of the total number of particles in the particle population with positive event signal, positive nucleic acid signal, negative virus capsid protein expression signal, and positive virus envelope expression signal among all the particle populations with positive event signal.

55. For the method according to claim 1, the calculation method for the titer of a viral nucleic acid vector lacking a viral envelope and containing nucleic acid and a viral capsid is: the product of P8 and the particle counting concentration in the viral nucleic acid vector, wherein, P8 is the percentage of the total number of particles in the particle population with positive event signal, positive nucleic acid signal, positive virus capsid protein expression signal, and negative virus envelope expression signal among all the particle populations with positive event signal.

56. The calculation method for the titer of a viral nucleic acid vector containing a nucleic acid and lacking a viral outer shell and envelope according to claim 1 is: the product of P9 and the particle count concentration in the viral vector, wherein, P9 is the percentage of the total number of particles in the particle population with positive event signal, positive nucleic acid signal, negative virus capsid protein expression signal, and negative virus envelope expression signal among all the particle populations with positive event signal.

57. For the method according to claim 3, the calculation method for the titer of the viral nucleic acid vector containing the target nucleic acid and the envelope protein is: the product of P1b and the particle counting concentration in the viral nucleic acid vector, wherein, P1b is the percentage of the number of particles in the particle population with positive event signal, positive target nucleic acid signal, and positive envelope protein expression signal among all the particle populations with positive event signal.

58. The calculation method for the titer of the viral nucleic acid vector containing the target nucleic acid and the envelope in the method according to claim 3 is: the product of P1c and the particle counting concentration in the viral nucleic acid vector, wherein, P1c is the percentage of the number of particles in the particle population with positive event signal, positive target nucleic acid signal, and positive virus envelope marker expression signal among all the particle populations with positive event signal.

59. The calculation method for the titer of the virus nucleic acid vector lacking an envelope protein and containing a target nucleic acid according to claim 3 is: the product of P3a and the particle count concentration in the virus nucleic acid vector, wherein, P3a is the percentage of the total number of points with positive event signal, positive target nucleic acid signal, and negative envelope protein expression signal among all the particle populations with positive event signal.

60. The calculation method for the titer of the virus nucleic acid vector lacking an envelope and containing the target nucleic acid according to claim 3 is: the product of P4a and the particle counting concentration in the virus nucleic acid vector, wherein, P4a is the percentage of the total number of points with positive event signal, positive target nucleic acid signal, and negative virus envelope marker expression signal among all the particle populations with positive event signal.

61. The calculation method for the titer of the viral nucleic acid vector lacking a viral capsid containing the target nucleic acid according to claim 3 is: the product of P5a and the particle count concentration in the viral nucleic acid vector, where P5a is the percentage of the total number of points with event signal, positive target nucleic acid signal, and negative virus capsid protein marker expression signal among all the particle populations with positive event signal.

62. The method for calculating the titer of the viral nucleic acid vector lacking the viral coat protein containing the target nucleic acid according to claim 3 is: the product of P6a and the particle counting concentration in the viral nucleic acid vector, wherein, P6a is the percentage of the total number of points with positive event signal, positive target nucleic acid signal, and negative virus capsid protein expression signal among all the particle populations with positive event signal.

63. The calculation method for the titer of the viral nucleic acid vector lacking the viral capsid protein and containing the target nucleic acid and the envelope is: the product of P7a and the particle counting concentration in the viral nucleic acid vector, where, P7a is the percentage of the total number of particles in the particle population with positive event signal, positive target nucleic acid signal, negative virus capsid protein expression signal, and positive virus envelope expression signal among all the particle populations with positive event signal.

64. For the method according to claim 3, the calculation method for the titer of the viral nucleic acid vector lacking a viral envelope and containing a target nucleic acid and a viral capsid is: the product of P8a and the particle count concentration in the viral nucleic acid vector, wherein, P8a is the percentage of the total number of particles in the particle population with positive event signal, positive target nucleic acid signal, positive virus capsid protein expression signal, and negative virus envelope expression signal among all the particle populations with positive event signal.

65. According to the method described in claim 3, the method for calculating the titer of a viral nucleic acid vector containing a deletion virus coat and envelope of a target nucleic acid is: the product of P9a and the particle counting concentration in the viral nucleic acid vector, wherein, P9a is the percentage of the total number of particles in the particle population with positive event signal, positive target nucleic acid signal, negative virus capsid protein expression signal, and negative virus envelope expression signal among all the particle populations with positive event signal.

66. The method according to any one of claims 1-18, 22-65, wherein the flow cytometry particle detection device is a particle analysis and detection device capable of realizing the directional flow of the sample flow.

67. The method according to claim 66, wherein the particle analysis and detection device comprises an optical system and a particle detector.

68. The method according to claim 67, wherein the particle detector is composed of a photoelectric sensor and a signal conditioning circuit with a function of limited-band filtering high-frequency noise.

69. According to the method described in claim 19, the flow particle detection device is a particle analysis and detection device capable of realizing the directional flow of the sample flow.

70. According to the method described in claim 69, the particle analysis and detection device includes an optical system and a particle detector.

71. According to the method described in claim 70, the particle detector consists of a photoelectric sensor and a signal conditioning circuit with a function of band-limiting and filtering high-frequency noise.

72. According to the method described in claim 20, the flow particle detection device is a particle analysis and detection device capable of realizing the directional flow of the sample flow.

73. According to the method described in claim 72, the particle analysis and detection device includes an optical system and a particle detector.

74. According to the method described in claim 73, the particle detector consists of a photoelectric sensor and a signal conditioning circuit with a function of band-limiting and filtering high-frequency noise.

75. According to the method described in claim 21, the flow particle detection device is a particle analysis and detection device capable of realizing the directional flow of the sample flow.

76. According to the method described in claim 75, the particle analysis and detection device includes an optical system and a particle detector.

77. According to the method described in claim 76, the particle detector consists of a photoelectric sensor and a signal conditioning circuit with a function of band-limiting and filtering high-frequency noise.

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