An aav vector integrity qualitative and quantitative detection method under a multi-primer itr design

Abstract

This invention discloses a qualitative and quantitative detection method for AAV vector integrity using a multi-primer ITR design. The detection method includes the following steps: S1, designing multiple primers in a conserved region of the AAV vector ITR sequence, wherein the primer length is 18-25 bp, the Tm value is 55-65℃, and the GC% is 40%-60%; S2, performing validity testing on the primers; S3, using the Multiplex LAM-PCR method, amplifying and sequencing with the primers to obtain chimeric sequences that are screened for ITR and host genome using bioanalysis methods, i.e., obtaining information on ITR breakpoints; S4, based on the information on ITR breakpoints, statistically analyzing the distribution of ITR breakpoints in the ITR region to obtain a statistical table; S5, based on the statistical table, obtaining qualitative and quantitative results of the ITR breakpoint locations. This invention comprehensively evaluates the integrity of the AAV vector by statistically analyzing the number of chimeric sequences in each primer region to qualitatively determine and quantitatively analyze ITR breakpoints.

Description

Technical Field

[0001] This invention relates to the fields of biological experiments and bioinformatics, specifically to a qualitative and quantitative detection method for the integrity of AAV vectors using a multi-primer ITR design. Background Technology

[0002] AAV (Adeno-Associated Virus) is a commonly used gene vector, widely used in gene therapy research. AAV is a single-stranded DNA virus with a unique T-shaped structure in its flanking ITR (Inverted Terminal Repeat) regions. These regions not only serve as the starting point for viral genome replication but also as key signals for viral packaging, deeply involved in viral replication, packaging, integration with the host genome, and escape. The integrity of the ITR sequence directly affects the production efficiency and gene expression of rAAV. Any mutation or truncation may trigger potential risks in gene therapy; therefore, detecting the integrity of the AAV insertion sequence is a crucial step in gene therapy research and application. Currently, solutions to the problem of incomplete ITR sequences in AAV vectors are significantly inadequate. Existing methods to improve sequencing efficiency, such as using truncated ITR sequences, specific E. coli strains and matching stabilizers, or the AAV-ITR sequencing method proposed by GENEWIZ, while optimizing sequencing results to some extent, cannot accurately locate the specific regions of ITR sequence mutations or truncations, nor can they comprehensively evaluate the rationality of ITR design. Technologies relying on whole-genome sequencing, such as next-generation sequencing and Oxford Nanopore Technologies (ONT) long-read sequencing, are costly, have scarce effective data, are difficult to quantify, and are constrained by sequencing data quality, making them unsuitable for practical applications. For example, Chinese patent (CN118609661B) provides a method for detecting adeno-associated virus integrity using a hidden Markov model, which involves directly trunculating the entire viral genome. This method increases noise in the data, requiring statistical methods to reduce noise and find ITR breakpoint information. When the proportion of AAVs with missing ITRs is extreme, the noise effect is difficult to eliminate, leading to misjudgments, and this method cannot output effective quantitative results. While third-generation sequencing methods can detect structural variations in the overall sequence with higher sensitivity and can achieve quantification, special long palindromic sequences in the ITR region can lead to sequencing failures. Moreover, compared to second-generation sequencing, third-generation sequencing has higher costs and sequencing errors, and the analysis process is more complex, resulting in high cost and low accuracy.

[0003] Given the limitations of existing technologies, there is an urgent need for a new method that can accurately detect the qualitative and quantitative integrity of the ITR region in AAV vectors, fundamentally improving the quality of sequencing data while ensuring efficient output of qualitative and quantitative results. Summary of the Invention

[0004] The main objective of this invention is to propose a qualitative and quantitative detection method for the integrity of AAV vectors using a multi-primer ITR design, aiming to solve the problems of high cost, limited effective data, and difficulty in quantifying results in existing methods for detecting the integrity of inserted sequences in AAV vectors.

[0005] To achieve the above objectives, this invention proposes a qualitative and quantitative detection method for AAV vector integrity based on multi-primer ITR design, comprising the following steps: S1. Design multiple primers in the conserved region of the AAV vector ITR sequence. The primers are 18-25 bp in length, have a Tm value of 55-65℃, and a GC% of 40%-60%. S2. Perform validity testing on the primers; S3. Using the Multiplex LAM-PCR method and the primers, amplification and sequencing were performed to obtain chimeric sequences of ITR and host genome that were screened using biological analysis methods, thus obtaining information on ITR breakpoints. S4. Based on the information of the ITR breakpoints, statistically analyze the distribution of ITR breakpoints in the ITR region and obtain a statistical table. S5. Based on the statistical table, obtain the qualitative and quantitative results of the ITR fracture location.

[0006] Preferably, in step S1, the conserved region of the ITR sequence is the AA' region.

[0007] Preferably, the plurality of primers consists of three primers, and the nucleotide sequences of the three primers are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0008] Preferably, in step S2, the presence of bands in the target region is observed by gel electrophoresis to verify the effectiveness of the primers.

[0009] Preferably, in step S3, the amplification specifically includes the following steps: breaking the DNA into 500±50bp fragments by sonication, performing linear extension, ligating adapters, and PCR enrichment in sequence, and adding sequencing adapters.

[0010] Preferably, in step S4, the information of the ITR breakpoint includes the sample, primers, and sequence information.

[0011] Preferably, step S4 further includes: distinguishing samples by the barcode in the sequence, and statistically determining the number and relative proportion of ITR breakpoints in each primer region for each sample.

[0012] Preferably, the detection method further includes primer design and subsequent analysis of the promoter and enhancer functional elements to confirm the integrity of the remaining sequences of the AAV viral vector.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the technical solution provided by the present invention, multiple primers are first designed in the conserved region of the ITR sequence, and the effectiveness of the primers is verified by the ITR test to reduce the influence of special palindromic sequences of the ITR and improve the success rate of sequencing. Compared with traditional whole genome sequencing, this method eliminates the noise caused by interruption during whole genome sequencing, ensuring that the sequencing results more realistically reflect the integration of the AAV vector with the host genome, and providing a highly reliable data basis for subsequent breakpoint analysis.

[0014] (2) Based on the Multiplex LAM-PCR technology, this invention allows multiple primers to act simultaneously on a unified reaction system, enabling the simultaneous detection of multiple targets, reducing amplification and sequencing time, lowering operating costs, and achieving efficient and sensitive data sequencing.

[0015] (3) This invention utilizes known primer sequences to rapidly classify sequencing data, and combines bioinformatics analysis methods to accurately detect chimeric sequences. By statistically analyzing the number of chimeric sequences in each primer region, qualitative and quantitative analysis of ITR breakpoints is performed, thereby comprehensively assessing the integrity of the AAV vector. This method can not only locate ITR breakpoints but also quantify the frequency of different breakpoints, providing scientific and valuable analytical results for research on AAV vector integration mechanisms and gene therapy safety assessments. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a diagram of the end structure of the AAV carrier of the present invention.

[0018] Figure 2 This is a diagram showing the design region of the primers on the ITR of this invention.

[0019] Figure 3 This is a schematic diagram illustrating the primer design principle of this invention.

[0020] Figure 4 This is a schematic diagram of the Multiplex PCR experiment of the present invention.

[0021] Figure 5 This is a schematic diagram of the breakpoint identification principle of the present invention; Figure A shows the design of multiple primers in the ITR region of the AAV vector sequence; Figure B shows the ITR sequence of the vector with different degrees of breakage; Figure C shows the integration of the AAV vector into the host genome, in which part of the integrated sequence is the host genome (solid line) and the other part is the vector sequence (dashed line); Figure D shows that when the integrated sequence is unfolded for sequencing, if the ITR is intact or breaks before primer A, sequencing starts from primer A; if the break occurs between primer A and primer B, sequencing starts from primer B; if the break occurs between primer B and primer C, sequencing starts from primer C.

[0022] Figure 6 This is a schematic diagram illustrating the principle of ITR breakpoint quantification in this invention.

[0023] Figure 7 This is a diagram of the primer bands used in this invention.

[0024] Figure 8 Box plots showing the ITR breakpoint distribution of the two sets of data, AAV5 and AAV9, in this invention.

[0025] Figure 9 This is a bar chart showing the breakpoint distribution of groups AAV5 and AAV9 in this invention.

[0026] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The technical terms involved in this invention are explained as follows: AAV: Adeno-Associated Virus. AAV is a commonly used gene vector, widely used in gene therapy research.

[0029] Primers: also known as primers, are short DNA sequences used as the starting point for amplification in molecular biology experiments such as PCR.

[0030] Barcode: A short and unique DNA sequence tag used to identify different samples or experimental conditions.

[0031] ITR stands for Inverted Terminal Repeat. ITRs are repetitive sequences located at both ends of the AAV genome, typically around 145 base pairs, forming a T-shaped hairpin structure. An ITR is a palindromic sequence of 125 nucleotides, including AA', BB', and CC' regions, followed by a 20-nucleotide D or D' region. ITRs play a crucial role in the replication, encapsulation, and integration of the AAV genome.

[0032] Multiplex LAM-PCR: Multiplex linear amplification PCR. Based on LTA-PCR, it uses multiple sets of primers for amplification, enabling the simultaneous amplification of multiple DNA target sequences in the same reaction. Then, next-generation sequencing is used to detect or quantify different base sequences separately.

[0033] Chimeric sequence: When an AAV vector integrates into the host genome, it produces a sequence in which one part is the AAV vector genome sequence and the other part is the host genome sequence.

[0034] ITR breakpoint: In a chimeric sequence, the part where the ITR region of the AAV vector connects to the host genome is called the ITR breakpoint.

[0035] Given the problems of high cost, limited effective data, and difficulty in quantifying results in existing AAV genome integrity detection methods, this invention proposes a qualitative and quantitative detection method for AAV vector integrity based on multi-primer ITR design, comprising the following steps: S1. Design multiple primers in the conserved region of the AAV vector ITR sequence. The primers are 18-25 bp in length, have a Tm value of 55-65℃, and a GC% of 40%-60%. Preferably, in this step, the conserved region of the ITR sequence is the AA' region; the primer design includes the following steps: the ITR sequence of the AAV vector is known ( Figure 1 Based on literature review, conserved regions on the ITR sequence were identified, and appropriate ranges were selected as the primer design regions. Figure 2Design multiple primers in the conserved region (primer design principles are as follows). Figure 3 As shown in the diagram, the primers can amplify the ITR region. Primer design strategy: Multiple primers are designed in the ITR region of the AAV vector sequence. These primers do not interfere with each other and can all effectively amplify the region. When the vector sequence integrates into the host genome, the ITR region has a probability of random mutations or breaks, resulting in different primers in the integrated sequence. During sequencing, amplification is initiated from the primer with the closest ITR breakpoint. The multiple primers consist of three primers, and the nucleotide sequences of the three primers are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0036] SEQ ID NO.1: GCTATGAACTAATGACCCCGTAA SEQ ID NO.2: GGAACCCCTAGTGATGGAGTTG SEQ ID NO.3: TAGTGATGGAGTTGGCCACTCC S2. Perform validity testing on the primers; In this step, HETR93 cells were infected using an AAV vector with multiple primers, and cell lysate containing AAV was obtained. PCR fragment amplification was performed using specific primers designed in the ITR. Multiple sets of primers were used to simultaneously amplify multiple DNA target sequences. The amplified products were then observed by gel electrophoresis to check for the presence of target region bands. Agarose gel electrophoresis was performed using a 2000bp marker. If scattered bands were observed, it proved that the primers in the ITR sequence were functioning correctly; otherwise, primers needed to be redesigned. Multiplex PCR experimental principle: Multiplex linear amplification PCR is a technique that combines linear amplification and multiplex PCR. Based on LTA-PCR, multiple sets of primers are used for amplification, enabling the simultaneous amplification of multiple DNA target sequences in the same reaction. Then, real-time PCR or digital PCR methods are used to detect or quantify the base sequences labeled with different fluorescent markers.

[0037] S3. Using the Multiplex LAM-PCR method and the primers, amplification and sequencing were performed to obtain chimeric sequences of ITR and host genome that were screened using biological analysis methods, thus obtaining information on ITR breakpoints. In this step, the amplified fragments obtained using the Multiplex LAM-PCR method specifically include: breaking the DNA into 500±50 bp fragments using sonication; performing linear extension of the fragments using ITR-F1 from the above primers; ligating the obtained product into a double-linked primer; performing 35 cycles of PCR enrichment of the target fragment using ITR-F2 and adapter primer 1; and continuing enrichment using ITR-F3 and adapter primer 2. Sequencing adapters need to be added during this PCR step to obtain the final library.

[0038] S4. Based on the information of the ITR breakpoints, statistically analyze the distribution of ITR breakpoints in the ITR region and obtain a statistical table. Preferably, the information of the ITR breakpoint includes sample, primer, and sequence information.

[0039] ITR breakpoint identification specifically includes: obtaining amplification product fragments using the Multiplex LAM-PCR method (the experimental principle of Multiplex PCR is as follows...). Figure 4 As shown in the figure, second-generation sequencing was performed using a MiSeq instrument to obtain paired-end Fastq files. The principle of ITR breakpoint identification is as follows: Figure 5 As shown, when the AAV vector integrates into the host genome and the ITR region breaks, amplification and sequencing can still be initiated from the primers retained within the ITR region due to the presence of multiple primers. Therefore, the location of the breakpoint can be determined based on the primer sequences retained in the sequencing sequence. If a sequence contains primer A, the breakpoint of the corresponding AAV vector ITR sequence is located between the ITR end and primer A. Similarly, if a sequence contains primer B, its ITR breakpoint is located between primer A and primer B. After obtaining the paired-end Fastq files, they are divided into multiple smaller Fastq files according to primers, with each primer resulting in a smaller Fastq file. For all the smaller Fastq files, chimeric sequences, i.e., sequencing sequences that simultaneously contain AAV and the host genome, are screened using bioinformatics analysis. In chimeric sequences, the point where the ITR region of the AAV vector connects to the host genome is the ITR breakpoint. Through the above steps, an ITR breakpoint identification table for each primer is finally obtained. Each row in the table records one ITR breakpoint and its annotation information, providing accurate data support for subsequent research.

[0040] S5. Based on the statistical table, obtain the qualitative and quantitative results of the ITR fracture location (the quantitative principle of ITR fracture point is as follows). Figure 6 (As shown).

[0041] In this step, based on the existing ITR breakpoint identification table for each primer, the total number of sequences supporting ITR breakpoints in each primer region is directly counted. If multiple samples exist, they can be distinguished by the barcode sequence in the sequence, and then the number and relative proportion of ITR breakpoints in each primer region for each sample can be counted.

[0042] Preferably, the qualitative and quantitative detection method for AAV vector integrity under the multi-primer ITR design further includes primer design and subsequent analysis of promoter and enhancer functional elements to confirm the integrity of the remaining sequences of the AAV viral vector.

[0043] This invention first designs multiple primers in conserved regions of the ITR sequence and verifies their effectiveness using an ITR test, reducing the impact of ITR palindromic sequences and improving sequencing success rates. Compared to traditional whole-genome sequencing, this method eliminates noise caused by interruptions during whole-genome sequencing, ensuring that sequencing results more accurately reflect the integration of the AAV vector with the host genome, providing a highly reliable data foundation for subsequent breakpoint analysis. Based on Multiplex LAM-PCR technology, this invention allows multiple primers to act simultaneously on a unified reaction system, enabling simultaneous detection of multiple targets, reducing amplification and sequencing time, lowering operating costs, and achieving efficient and sensitive data sequencing. This invention utilizes known primer sequences to rapidly classify sequencing data, combines bioinformatics analysis methods to accurately detect chimeric sequences, and by statistically analyzing the number of chimeric sequences in each primer region, qualitative and quantitative analysis of ITR breakpoints is performed, thereby comprehensively assessing the integrity of the AAV vector.

[0044] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0045] Example 1: Feasibility Verification of Primer Design Method for ITR Region of AAV Vector 1. In this embodiment, two types of AAV vectors, namely AAV5 and AAV9, are used.

[0046] 2. The primer sequences designed in the ATIR region in this embodiment are shown in the table below:

[0047] 3. Primer design specifically includes the following steps: (1) Search for conserved regions on the ITR sequence: The ITR sequence of the known AAV vector ( Figure 1 Based on literature review, conserved regions on the ITR sequence are identified, and appropriate ranges are selected as the primer design regions (e.g., Figure 2As shown in the diagram, in this embodiment, three different primers are designed within the AA' region, enabling the primers to amplify the ITR region. When the AAV vector integrates into the host genome, even if the ITR region is broken, amplification can still be initiated using primers located near the ITR breakpoint.

[0048] (2) HETR93 cells were infected with an AAV vector with multiple primers and cell lysate containing AAV was obtained. PCR fragment amplification was performed using specific primers designed in the ITR. Multiple sets of primers were used to amplify multiple DNA target sequences simultaneously. The amplification products were then observed by gel electrophoresis to check for the presence of target region bands. Agarose gel electrophoresis was performed using a 2000bp marker. If scattered bands were observed, it proved that the primers in the ITR sequence could function normally; otherwise, primers needed to be redesigned.

[0049] (3) Multiplex LAM-PCR to obtain amplified fragments: DNA was broken into 500±50 bp fragments by sonication, and the fragments were linearly extended using ITR-F1 from the above primers; the obtained products were ligated with double-linked primers; the target fragments were enriched by PCR for 35 cycles using ITR-F2 and adapter primer 1; the enrichment was continued using ITR-F3 and adapter primer 2. Sequencing adapters need to be added during this PCR step to obtain the final library.

[0050] Gel electrophoresis results as follows Figure 7 As shown, multiple clear bands appear in the gel image, and these bands are scattered, proving the presence of the target sequence and meeting the design expectations. This demonstrates that the AAV vector with designed primers can be amplified normally starting from the designed primers.

[0051] Example 2: Integrity Assessment of AAV Vector Integration In this embodiment, the experimental materials include: (1) AAV5 sample group 1, a total of 10 samples, were infected with HETR93 somatic cells and cell fluid containing AAV was extracted; (2) AAV9 sample 1 group, a total of 10 samples, were infected with HETR93 somatic cells and the cell fluid containing AAV was extracted.

[0052] The above AAV5 and AAV9 samples were processed simultaneously using the following methods: (1) Sample sequencing: Mulptiplex LAM-PCR was used. Figure 4Based on LTA-PCR, multiple sets of primers are used for amplification, which can amplify multiple DNA target sequences simultaneously in the same reaction. The PCR products are then used for next-generation sequencing using a MiSeq instrument (Illumina). DNA barcodes are used to label the samples, allowing multiple samples to be sequenced in parallel in one sequencing run, while minimizing sample cross-contamination, resulting in next-generation sequencing results, i.e., a pair of paired-end Fastq files.

[0053] (2) ITR breakpoint identification: The principle of ITR breakpoint identification is as follows Figure 5 As shown. For the paired-end Fastq files obtained in step (1), sequence splitting is performed based on the known primer sequences. If a sequence contains primer A, it is classified as fqA; if a sequence contains primer B, it is classified as fqB; if a sequence contains primer C, it is classified as fqC; if no primers are found in a sequence, it is filtered out. Three pairs of classified Fastq files are obtained, namely fqA, fqB, and fqC.

[0054] (3) For fqA, fqB, and fqC, bioinformatics analysis was performed to identify and screen chimeric sequences. If a sequence contains both sequences from the AAV genome and sequences from the host genome, it is identified as a chimeric sequence, and the splice point between the two sequences is the ITR breakpoint. Simultaneously, based on the barcode information, the sample source of each sequence was identified. If a sequence contains a barcode sequence corresponding to a sample, the corresponding sample annotation information was added to that sequence. If no valid barcode was identified in the sequence, it was filtered out. Based on the screening process of bioinformatics analysis, three ITR breakpoint annotation tables were obtained: tableA, tableB, and tableC.

[0055] (4) Qualitative and quantitative analysis of ITR breakpoints: Figure 6 This is a schematic diagram of the ITR breakpoint quantification principle. For the tables A, B and C obtained in step (3), count the number of rows to obtain the total number of chimeric sequences in the regions between each primer and the number of chimeric sequences in each sample in the region. Combine the three ITR breakpoint information annotation tables to calculate the relative proportion of ITR breakpoints in each region for each sample and obtain the statistical table TableFinal.

[0056] After completing the statistical analysis of all samples of AAV5 and AAV9, two statistical tables were obtained: Table Final5 for the AAV5 group and Table Final9 for the AAV9 group.

[0057] By merging the two statistical tables, we obtain the distribution of ITR breakpoints for all samples in the two AAV groups (Table 1) and the statistical table of breakpoint percentage test results (Table 2), including the number of ITR breakpoints for each sample between each two primers (Table 1: number of reads column) and the relative percentage of the number of ITR breakpoints (Table 2: percentage of reads column).

[0058] Table 1. Statistics on the distribution of ITR breakpoints in AAV5 and AAV9

[0059] Table 2. Statistical table of the percentage of ITR breakpoints for AAV5 and AAV9 in each region using Wilcoxon test results.

[0060] For each AAV group, plot a box plot of the ITR breakpoint distribution of all its samples in the ITR primer region. Figure 8 The scatter plot represents the percentage of reads for each sample, each bin represents the quartile range of that batch of samples, and the horizontal line within each bin represents the median value of the data. Figure 8 It is evident that in the AAV5 group, the ITR is more likely to break in the region between primer A and primer B; while in the AAV9 group, the ITR breaks are more distributed between the end of the ITR and primer A. Figure 9 A bar chart comparing the total number of ITR breakpoints in each region for the AAV5 and AAV9 groups. The horizontal axis represents the distribution area of ​​primers on the ITR, and the vertical axis represents the actual number of ITR breakpoints within that region. The bar chart distribution of the AAV5 and AAV9 groups is shown below. Figure 8 Consistent.

[0061] Wilcoxon test was performed on the relative proportion of ITR breakpoints in AAV5 and AAV9 samples within each region. It was found that the frequency of ITR breakpoints in the AAV5 group was significantly lower than that in the AAV9 group between the end of the ITR and Primer A. However, in the other two regions, the frequency of ITR breakpoints in the AAV5 group was significantly higher than that in the AAV9 group. Therefore, it is believed that under the experimental conditions of this embodiment, the serotype of AAV has a certain influence on the location of ITR breakpoints.

[0062] The results of this implementation show that by designing multiple primers in the conserved region of the ITR sequence, this invention can quickly and accurately qualitatively and quantitatively assess the ITR breakpoints after integration of different samples, and ultimately achieve the integrity assessment of the AAV vector after integration.

[0063] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A qualitative and quantitative detection method for AAV vector integrity based on multi-primer ITR design, characterized in that, Includes the following steps: S1. Design multiple primers in the conserved region of the AAV vector ITR sequence. The primers are 18-25 bp in length, have a Tm value of 55-65℃, and a GC% of 40%-60%. S2. Perform validity testing on the primers; S3. Using the Multiplex LAM-PCR method and the primers, amplification and sequencing were performed to obtain chimeric sequences of ITR and host genome that were screened using biological analysis methods, thus obtaining information on ITR breakpoints. S4. Based on the information of the ITR breakpoints, statistically analyze the distribution of ITR breakpoints in the ITR region and obtain a statistical table. S5. Based on the statistical table, obtain the qualitative and quantitative results of the ITR fracture location; In step S1, the conserved region of the ITR sequence is the AA' region; The multiple primers consist of three primers, and the nucleotide sequences of the three primers are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

2. The detection method according to claim 1, characterized in that, In step S2, the presence of bands in the target region is observed by gel electrophoresis to verify the effectiveness of the primers.

3. The detection method according to claim 1, characterized in that, In step S3, the amplification specifically includes the following steps: breaking the DNA into 500±50bp fragments by sonication, performing linear extension, ligating adapters, and PCR enrichment in sequence, and adding sequencing adapters.

4. The detection method according to claim 1, characterized in that, In step S4, the information of the ITR breakpoint includes the sample, primers, and sequence information.

5. The detection method according to claim 1, characterized in that, Step S4 also includes: distinguishing samples by the barcode in the sequence, and statistically determining the number and relative proportion of ITR breakpoints in each primer region for each sample.

6. The detection method according to any one of claims 1-5, characterized in that, The detection method also includes primer design and subsequent analysis of promoter and enhancer functional elements to confirm the integrity of the remaining sequences of the AAV viral vector.

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