A method for detecting the titer of an adeno-associated viral vector genome

Abstract

The present application relates to the field of genetic engineering, and particularly relates to a detection method for the titer of an adeno-associated virus vector genome, which comprises the following steps: 1) mixing and reacting a system containing an adeno-associated virus with Benzonase to remove nucleic acid impurities in the system; 2) mixing and reacting the product obtained in step 1) with SDS and NaOH to lyse the protein shell of the adeno-associated virus; and 3) removing the qPCR inhibitor from the product obtained in step 2) and then performing qPCR to obtain the titer of the adeno-associated virus. The method of the present application can quickly and efficiently, accurately and widely detect the genome titer of rAAV, and can also be used for the determination of the vector genome titer in each production node and the final purified sample in the rAAV production process, and is cost-saving and has fewer operation steps.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a method for detecting the genome titer of an adeno-associated virus vector. Background Technology

[0002] Recombinant adeno-associated virus (rAAV) vectors have become promising gene therapy delivery vectors due to their safety and good transduction efficacy in clinical trials. As of June 2024, several rAAV vector drugs have been successfully marketed, including Upstaza (PMID:36103022) for treating aromatic L-amino acid decarboxylase (AADC) deficiency, Roctavian (PMID:36082993) for treating hemophilia A, EtranaDez (PMID:36490302) for treating hemophilia B, Luxturna (PMID:32175942) for treating inherited retinal diseases (IRD), and Zolgensma (PMID:33708361) for treating spinal muscular atrophy (SMA). Several other rAAV vector gene therapy drugs targeting ophthalmic and muscular system diseases are currently in clinical trials. Quality monitoring is crucial during rAAV vector production, with accurate titer determination being a key indicator. Clinical dosage of rAAV vector gene therapy drugs is usually based on vector genomic titer (PMID: 23987130).

[0003] Real-time quantitative polymerase chain reaction (qPCR) is considered a common method for determining the genomic titer of rAAV vectors. Sample pretreatment is required before qPCR detection (PMID: 20486768). Pretreatment steps include removing nucleic acid impurities, dissociating the protein coat, and removing qPCR inhibitors. Traditional methods use DNase I to degrade nucleic acid impurities and Proteinase K to lyse the protein coat. However, traditional methods are complex, time-consuming, and costly. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for detecting the genome titer of adeno-associated virus vectors to solve the problems in the prior art.

[0005] To achieve the above and other related objectives, the present invention provides a method for detecting the genome titer of an adeno-associated virus vector, the method comprising the following steps:

[0006] 1) The system containing adeno-associated virus was mixed with Benzonase and reacted to remove nucleic acid impurities from the system;

[0007] 2) The product obtained in step 1) was mixed with SDS and NaOH and reacted to lyse the protein coat of adeno-associated virus;

[0008] 3) After removing the qPCR inhibitor from the product obtained in step 2), perform qPCR to obtain the titer of the adeno-associated virus.

[0009] Preferably, based on the reaction volume of step 1), the reaction system includes Benzonase at a final concentration of 20–160 U / mL, a system containing adeno-associated virus at a volume fraction of 10%, and the remainder being PBS, culture medium, DNase buffer, or RB-TMS.

[0010] Preferably, based on the reaction volume of step 2), the reaction system includes SDS with a final volume concentration of 0.1-0.3%, NaOH with a final volume concentration of 0.05-0.2 mol / L, and the remainder is the product of step 1).

[0011] Preferably, the method for removing the qPCR inhibitor in step 3) is to dilute the product obtained in step 2) by 10 to 10,000 times.

[0012] As described above, the adeno-associated virus vector genome titer detection method of the present invention has the following beneficial effects: Benzosinase & SDS-NaOH combined with qPCR can rapidly, efficiently, accurately and widely detect rAAV vector genome titers. It can also be used to determine the vector genome titer in various production stages of rAAV vector production and in the final purified sample, and it saves costs and has fewer operation steps. Attached Figure Description

[0013] Figure 1 The diagram shows the rAAV vector genome titer detection process. The left side shows the process of this invention, and the right side shows the process of the prior art.

[0014] Figure 2 This shows a comparison of the ability of Benzonase and DNase I to degrade nucleic acid impurities.

[0015] Figure 3 The results show the vector genome titer detection results for rAAV vector samples purified by different protein coat lysis methods.

[0016] Figure 4 The results show the genome titer detection of rAAV vector samples treated with different SDS-NaOH heating conditions.

[0017] Figure 5 The results show gene copy detection results when plasmids of known concentrations are incorporated into buffer solutions of different components.

[0018] Figure 6The results show the genomic titer of standard rAAV vector samples treated with different qPCR inhibitor removal methods.

[0019] Figure 7 The results show the genomic titer detection of the crude and purified rAAV vectors treated with the Benzoase & SDS-NaOH method.

[0020] Figure 8 The results show the rAAV vector genome titer detection results obtained from different categories and production nodes. Detailed Implementation

[0021] This invention provides a method for detecting adeno-associated virus (AAV) genomic titer, the method comprising the following steps:

[0022] 1) The system containing adeno-associated virus was mixed with Benzonase and reacted to remove nucleic acid impurities from the system;

[0023] 2) The product obtained in step 1) was mixed with SDS and NaOH and reacted to lyse the protein coat of adeno-associated virus;

[0024] 3) After removing the qPCR inhibitor from the product obtained in step 2), perform qPCR to obtain the titer of the adeno-associated virus.

[0025] In some embodiments of the present invention, the system containing adeno-associated virus (aAAV) is a system at any production stage after aAAV has been generated during the aAAV packaging process. The aAAV-containing system is selected from aAAV during or after purification, or from a culture system and / or cell lysate obtained after transfecting host cells with plasmids used for packaging aAAV and culturing them. The cell lysate includes rAAV released from the cells after lysis and rAAV released into the culture medium before lysis, as well as cell-derived proteins and small molecule compounds.

[0026] The serotype of the adeno-associated virus is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

[0027] The adeno-associated virus can exist in either a self-complementary or single-stranded form.

[0028] Pretreatment using Benzonase, a totipotent nuclease with stronger nucleic acid digestion capabilities than DNase I and less demanding requirements on digestion system components, for nucleic acid impurity removal can ensure the complete elimination of the impact of DNA not encapsulated by capsid proteins on the accuracy of vector genome titer determination.

[0029] In some embodiments of the present invention, based on the reaction volume of step 1), the reaction system includes Benzonase at a final concentration of 20-160 U / mL, a system containing adeno-associated virus at a volume fraction of 10%, and the remainder being PBS, culture medium, DNase buffer or RB-TMS.

[0030] The final concentration of the Benzonase is selected from any of the following ranges: 20–40 U / mL, 40–60 U / mL, 60–80 U / mL, 80–100 U / mL, 100–120 U / mL, 120–140 U / mL, and 140–160 U / mL.

[0031] In some embodiments of the present invention, the reaction time in step 1) is 0.5 to 2 hours, and the reaction temperature is 36.5 to 37.5°C. The reaction time, for example, is 0.5 to 1 hour, 1 to 1.5 hours, or 1.5 to 2 hours.

[0032] In step 2), the protein coat is lysed using the SDS-NaOH alkaline lysis method, which makes the lysis of the rAAV vector more efficient, saving time and making the measurement results more accurate.

[0033] In some embodiments of the present invention, based on the reaction volume of step 2), the reaction system includes SDS with a final volume concentration of 0.1 to 0.3%, NaOH with a final volume concentration of 0.05 to 0.2 mol / L, and the remainder is the product of step 1).

[0034] In some embodiments of the present invention, the reaction conditions for step 2) are 60–75°C and 20–35 minutes. The reaction temperature is, for example, 60–65°C, 65–70°C, or 70–75°C. The reaction time is, for example, 20–25 minutes, 25–30 minutes, 30–35 minutes, or 35–40 minutes.

[0035] In some embodiments of the present invention, the method for removing the qPCR inhibitor in step 3) is to dilute the product obtained in step 2) by 10 to 10,000 times. The dilution factor is, for example, 10 to 100 times, 100 to 500 times, 500 to 1000 times, 1000 to 5000 times, or 5000 to 10000 times.

[0036] In some embodiments of the present invention, the diluent is water, preferably double-distilled water.

[0037] Diluting the product obtained in step 2) to remove qPCR inhibitors is more economical than the commonly used DNA column extraction method, and the detection results are stable between and within batches.

[0038] The steps involved in qPCR are standard procedures in this field.

[0039] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0040] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0041] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0042] Example 1: Comparison of the ability of Benzonase and DNase I to degrade nucleic acid impurities

[0043] Adeno-associated virus vector packaging and purification:

[0044] rAAV2, rAAV3, rAAV5, rAAV6, rAAV8, and rAAV9 were generated using a HEK293 cell triple transfection platform. Each 15cm cell culture plate contained 15μg pAAV-GOI (target gene), 15μg capsule plasmid (pACG2), and 30μg pAAV-Helper, with a mass ratio of 1:1:2 for the three plasmids. Transfected cells were harvested 72 hours post-transfection and resuspended in RB-TMS (50mM Tris-HCl + 150mM NaCl, pH 8.0, sterile). The resuspended cells were subjected to three freeze-thaw cycles to obtain cell lysates.

[0045] A quarter volume of PEG 8000 (MB2594, Meilun Biotechnology, China) was added to the supernatant of the culture medium, and the mixture was shaken overnight at 4°C to allow adsorption, resulting in a PEG 8000 precipitate. The resuspended cells were then added to RB-TMS, and the PEG 8000 precipitate was resuspended for purification to obtain the crude viral vector extract.

[0046] Subsequently, 1 μL of 4.8M MgCl2 and 2 μL of Benzonase (70664-4CN, Merck, USA) were added to 6 mL of crude viral vector extract and incubated in a 37°C water bath for 30 minutes. The mixture was purified by gradient ultracentrifugation with iodixanol, and the iodixanol-virus mixture was collected. Ion exchange column chromatography (5 mL HiTrap Q HP column, 17115301, GE Healthcare, US) was used for further purification of the rAAV viral vector. The column was washed sequentially with detergent buffer A (40 mM Tris, 30 mM NaCl, pH 8.5 aqueous solution), equilibration buffer B (20 mM Tris, 1000 mM NaCl, pH 8.5 aqueous solution), and enrichment buffer C (20 mM Tris, 350 mM NaCl, pH 8.5 aqueous solution), and then washed with the sample solution. The final diluted sample was concentrated by ultracentrifugation, with 1 mL of PBS as the eluent, and the purified product was collected in a clean test tube.

[0047] Virus titer detection:

[0048] The effects of different concentrations of Benzonase (70664-4CN, Merck, US) and DNase I (2270A, Takara, Japan) on each 100 μL reaction unit (containing 10 μL of the aforementioned three plasmid transfection cell lysate, with the remainder being DNase buffer premix NEB B7204S Cutsmart Buffer or 10X DNase I Buffer in Takara 2270A) were analyzed, and the unencapsulated ITR copy number was determined by qPCR.

[0049] The gradient of Benzonase additions in the samples is as follows (U enzyme units): 0, 2, 4, 8, 12, 16.

[0050] The addition gradient of DNase I in the samples was as follows (U enzyme units): 0, 5, 10, 15, 20, 25.

[0051] Both enzymes were incubated with the sample at 37°C for 1 hour.

[0052] A standard curve was prepared using the helical plasmid pTR-UF11 (157970, Addgene, US).

[0053] Amplification was performed using the following qPCR system:

[0054]

[0055] (TSE203, Tsingke, China)

[0056] ddH2O 3.4μL

[0057] Total 20μL

[0058] The nucleotide sequences of the forward and reverse primers are as follows:

[0059] Forward primer sequence 5'-GGAACCCCTAGTGATGGAGTT-3'

[0060] Reverse primer sequence 5'-CGGCCTCAGTGAGCGA-3'

[0061] The reaction was performed according to a qPCR program of 95℃ pre-denaturation for 1 minute (95℃ for 10 seconds, 60℃ for 10 seconds, 72℃ for 10 seconds) × 40 cycles, and the fluorescence signal was read after each cycle. The melting curve was set by increasing the temperature from 60℃ to 95℃ by 0.5℃ every five seconds.

[0062] The results are as follows Figure 2 As shown in the figure, the standard curve prepared using the helical plasmid pTR-UF11 showed good linearity, good primer specificity, and the qPCR analysis met the standards and was reliable.

[0063] After plotting the standard curve based on the Ct values ​​obtained from qPCR and the standard concentrations, the sample genome copy number was calculated. The results showed that the number of uncoated ITR copies in samples degraded by Benzonase was lower than that degraded by DNase I, indicating that Benzonase has a stronger ability to degrade DNA impurities in samples than DNase I. Therefore, Benzonase was selected as the preferred nuclease for removing nucleic acid impurities in rAAV vector samples during processing in this method.

[0064] Example 2: Comparison of rAAV vector sample purification and vector genome titer detection results using different protein coat lysis treatment methods.

[0065] The preparation of rAAV is the same as in Example 1.

[0066] Each 100 μL reaction unit contained 10 μL of purified rAAV vector sample and 90 μL of DNase buffer premix. All reaction units were pre-mixed with 15 U Benzonase at 37°C for 1 hour to remove nucleic acid impurities. The purified rAAV vector sample was then processed using the following two methods:

[0067] (1) SDS-NaOH method, i.e. alkaline pyrolysis method: Add 2μL of 10% SDS stock solution and 1μL of 10M NaOH to every 100μL of reaction unit, and heat at 65℃ for 30 minutes.

[0068] (2) Proteinase K method: Add 12U Proteinase K (ST532, Beyotime Biotechnology, China) to every 100μL reaction unit and heat at 56℃ for 2 hours; then heat at 95℃ for 30 minutes to inactivate Proteinase K.

[0069] The treated sample was diluted 100 times with water to remove the qPCR inhibitor, and the ITR copy number was detected by qPCR according to the method in Example 1.

[0070] The measurement results are as follows Figure 3 As shown, two different batches of purified rAAV2 and rAAV8 vectors underwent protein coat lysis using both the Proteinase K and SDS-NaOH methods. There was no significant difference in the genomic titers of the rAAV vectors obtained by the two methods. However, the SDS-NaOH method was more cost-effective and time-efficient than the Proteinase K method, indicating that the SDS-NaOH method is superior for the protein coat lysis step of the rAAV vector samples.

[0071] Example 3: Comparison of rAAV vector sample genome titer detection results under different SDS-NaOH heating conditions

[0072] Crude extracts of rAAV2 and rAAV5 vectors were obtained according to the virus packaging method of Example 1. Each 100 μL reaction unit contained 10 μL of crude rAAV vector extract sample and 90 μL of DNase buffer premix. After adding 2 μL of 10% SDS and 1 μL of 10M NaOH to each 100 μL reaction unit treated with Benzonase, a series of heating conditions were set to lyse the rAAV vector protein coat sample. The heating conditions are as follows:

[0073] (1) 65℃, 20 minutes; (2) 65℃, 25 minutes; (3) 65℃, 30 minutes; (4) 65℃, 35 minutes;

[0074] (5) 70℃, 20 minutes; (6) 70℃, 25 minutes; (7) 70℃, 30 minutes; (8) 70℃, 35 minutes;

[0075] (9) 75℃, 20 minutes; (10) 75℃, 25 minutes; (11) 75℃, 30 minutes; (12) 75℃, 35 minutes;

[0076] The treated sample was diluted 100 times with water to remove the influence of the qPCR inhibitor, and then qPCR detection was performed according to the method in Example 1.

[0077] The measurement results are as follows Figure 4 As shown, when the heating temperature was increased from 65℃ to 75℃ and the heating time was extended to 35 minutes, the rAAV5 vector genome titer increased, indicating that the SDS-NaOH method requires appropriate adjustment of heating conditions when processing the thermostable rAAV5. However, for serotypes with moderate thermostability, such as rAAV2, there was no significant difference in the genome titers of the vectors across different heating groups, indicating that the SDS-NaOH method is generally applicable to the protein coat cleavage of rAAV vectors of all serotypes.

[0078] Example 4: Comparison of gene copy detection results when plasmids of known concentrations are incorporated into buffer solutions of different components.

[0079] pTR-UF11 plasmids of known concentrations were diluted with buffers of different compositions. The composition of the buffers for each group is as follows (all percentages below are volume fractions):

[0080] The components of each buffer in the first group are: ddH2O, 0.2% SDS, 0.1M NaOH + 0.2% SDS, 0.02% SDS, 0.002% SDS, and 0.0002% SDS.

[0081] The buffer components in the second group are: ddH2O, 10% RB-TMS, and 1% RB-TMS.

[0082] The samples were tested by qPCR according to the method in Example 1.

[0083] The measurement results are as follows Figure 5 As shown, when the SDS concentration in the sample is 0.2%, qPCR detection is severely inhibited. This SDS concentration is consistent with the SDS concentration in the undiluted SDS-NaOH treated sample.

[0084] When the SDS concentration in the sample is 0.02%, the SDS component inhibits qPCR detection, while when the SDS concentration is 0.002%, qPCR detection is not inhibited. This suggests that samples treated with the SDS-NaOH method need to be diluted at least 100 times before subsequent qPCR detection can be performed.

[0085] Example 5: Comparison of vector genome titer detection results for standard rAAV vector samples treated with different qPCR inhibitor removal methods

[0086] The standard rAAV2-RSS vector purchased from ATCC was treated with Benzonase and SDS-NaOH according to the method in Example 2, and then the qPCR inhibitors were removed using the following two methods:

[0087] (1) Dilute the sample 100 times with ddH2O;

[0088] (2) Extract DNA components from the sample using a DNA extraction column (DNAClean & Concentrator-25, ZYMO Research, US) according to the manufacturer’s instructions.

[0089] The treated samples were subjected to qPCR detection according to the method in Example 1.

[0090] The measurement results are as follows Figure 6 As shown, the 100-fold dilution method yielded no difference in genomic titer of the standard vector obtained by DNA column extraction. Given that the former is less expensive and does not result in DNA loss, the 100-fold dilution method is superior in the qPCR inhibitor removal step for rAAV vector genomic titer determination.

[0091] Example 6: Overview of results for detecting the genomic titer of rAAV vector crude and purified extracts treated with the Benzonase & SDS-NaOH method.

[0092] The crude rAAV2 extract and purified sample carrier prepared in Example 1 were serially diluted with ddH2O in the following dilution groups: 1, 1:10, 1:100, 1:1000, and 1:10000.

[0093] The steps for treating the rAAV carrier sample using the Benzonase & SDS-NaOH method are described in Example 2, which covers Benzonase treatment and the SDS-NaOH method.

[0094] The steps for treating rAAV vector samples using the DNase I & Proteinase K method are as follows: Add 40 U of DNase I to every 100 μL reaction unit (containing 10 μL of stock solution or diluted rAAV vector sample), heat at 37°C for 15 hours, and then heat-inactivate at 75°C for 30 minutes. The Proteinase K treatment steps are described in Example 2.

[0095] Flowcharts for the Benzonase & SDS-NaOH method and the DNase I & Proteinase K method are shown below. Figure 1 .

[0096] After the treated sample was diluted 100 times with water to remove the qPCR inhibitor, qPCR detection was performed according to the method in Example 1.

[0097] The measurement results are as follows Figure 7 As shown, for both crude and purified rAAV2 vector samples, there was no significant difference in vector genomic titer between the two treatment groups at dilutions from 1 to 1:10000. The crude extract reached the detection limit at a dilution of 1:10000, at which point the vector genomic titer was 10. 9 The converted original rAAV vector genome titer is 10. 4 The detection limit is consistent with that of the qPCR method. This suggests that the Benzonase & SDS-NaOH method is suitable for original vector genomic titers greater than 10. 4 Detection of crude extract and purified extract of rAAV vector.

[0098] Example 7: Detection of genomic titers of rAAV vectors obtained from different categories and production nodes.

[0099] Crude extracts were obtained by packaging self-complementary (sc) AAV vectors of serotypes 2, 3, 5, 6, 8, and 9, as well as single-stranded (ss) AAV2 vectors, according to the method in Example 1. Samples containing rAAV vectors were collected from each stage of the rAAV vector production process, as follows: culture medium after triple plasmid transformation, cell lysates after triple plasmid transformation, Buffer A containing purified rAAV vectors during the purification step, and the final purified product diluted in PBS.

[0100] The steps for treating the rAAV carrier sample using the Benzonase & SDS-NaOH method are described in Example 2, which covers Benzonase treatment and the SDS-NaOH method.

[0101] The treated sample was diluted 100 times with water to remove the qPCR inhibitor, and then qPCR detection was performed according to the method in Example 1.

[0102] The measurement results are as follows Figure 8 As shown, the Benzonase & SDS-NaOH combined qPCR method is generally applicable to the detection of genomic titers of AAV serotype vectors and the monitoring of product quality at various production stages.

[0103] In summary, the Benzonase & SDS-NaOH method proposed in this invention is superior to DNase I (PMID: 12349826) in terms of nucleic acid impurity removal. SDS-NaOH alkaline lysis of the protein shell is less costly than enzymatic lysis, and the sample processing steps are simpler. Furthermore, SDS-NaOH can fully lyse the protein shell at 65°C for 30 minutes, while Proteinase K requires 2 hours. For qPCR inhibitors introduced during the production and pretreatment processes, the dilution method used in this invention is more cost-effective and involves fewer steps compared to traditional DNA column extraction.

[0104] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for detecting adeno-associated virus (AAV) genomic titer, characterized in that, The detection method includes the following steps: 1) The system containing adeno-associated virus is mixed with Benzonase and reacted to remove nucleic acid impurities from the system; based on the reaction volume of step 1), the reaction system includes Benzonase at a final concentration of 20-160 U / mL, 10% of the system containing adeno-associated virus by volume, and the remainder is PBS, culture medium, DNase buffer or RB-TMS; the reaction time of step 1) is 0.5-2 hours, and the reaction temperature is 36.5-37.5℃; 2) The product obtained in step 1) is mixed with SDS and NaOH and reacted to lyse the protein coat of adeno-associated virus; based on the reaction volume of step 2), the reaction system includes SDS with a final volume concentration of 0.2-0.3%, NaOH with a final volume concentration of 0.05-0.2 mol / L, and the remainder is the product of step 1); the reaction conditions are 60-75℃ and the reaction time is 20-35 minutes. 3) After removing the qPCR inhibitor from the product obtained in step 2), perform qPCR to obtain the titer of the adeno-associated virus; the method for removing the qPCR inhibitor in step 3) is to dilute the product obtained in step 2) with water 10 to 10,000 times.

2. The detection method according to claim 1, characterized in that, The system containing adeno-associated virus is selected from adeno-associated virus during or after purification, or from a culture system and / or cell lysate obtained by transfecting host cells with plasmids used to package adeno-associated virus and culturing them.

3. The detection method according to claim 1, characterized in that, The serotype of the adeno-associated virus is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

4. The detection method according to claim 1, characterized in that, The adeno-associated virus exists in either a self-complementary or single-stranded form.

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