Probe primer combination for detecting AdV wild type virus residue and method and application thereof

Abstract

The invention belongs to the technical field of biological detection, and relates to a probe primer combination for detecting AdV wild type virus residues and a method and application thereof. According to the invention, two sets of primer probe combinations respectively targeting wild type adenovirus E1A-CR2 and E3 regions are designed, and dPCR is utilized to realize dual-target synchronous detection and signal distinguishing. By calculating the concentrations of linkages of two targets on the same DNA chain, false positive results caused by host cell DNA residues, incomplete E3 region knockout or single region non-specific amplification can be effectively eliminated, so that the specific recognition of the wild type adenovirus is realized. The method is convenient and rapid, can complete detection of AdV wild virus residues in a short time, and does not need expensive instruments and equipment; compared with a PCR (Polymerase Chain Reaction) method (about 1 / 107), the method has the advantages that the sensitivity is improved by 2 orders of magnitude, and the sensitivity can be improved to 0.5 / 109 or above.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a probe primer combination for detecting AdV wild-type virus residues, its method and application. Background Technology

[0002] Adenovirus (AdV) is a viral vector widely used in gene therapy and vaccine development. Its natural advantage lies in its ability to efficiently infect multiple cell types and simultaneously induce strong innate and adaptive immune responses in mammalian hosts. With technological advancements, AdV has evolved from a tool for gene replacement therapy into a true vaccine delivery vector.

[0003] To ensure the safety of its clinical application, AdV vectors are typically genetically modified to reduce their replication capacity or increase the loading capacity of exogenous genes. Currently, AdV modification can be achieved by manipulating the early 1 (E1) region or parts thereof, giving AdV-based vectors replication capacity or non-replication capacity. Common modification sites include E1A-CR2, E1B-55K, and E1B-19K. The E1A-CR2 region can bind to retinoblastoma mutant genes, prompting cell replication to enter the S phase, providing conditions for viral replication. Therefore, most AdV viruses are modified with E1A-CR2 to increase safety. Furthermore, since the E3 gene is not essential for viral replication, the early 3 (E3) gene is deleted to increase the capacity for transgene insertion.

[0004] During the production process, there is a risk of generating replicable wild-type AdV (RCA) due to homologous recombination or other genetic instabilities. Residual wild-type viruses of this type may pose a safety risk to test subjects; therefore, rigorous detection of wild-type AdV residues in the final product is a critical aspect of quality control.

[0005] Due to the lytic effect of AdV on cells, it cannot be detected using classic culture methods; traditional cell culture methods are not suitable for efficient and direct detection. Currently, the mainstream approach in the industry relies on molecular biology methods, especially polymerase chain reaction (PCR) technology. PCR methods utilize the modified characteristics of AdV. To avoid false positives caused by homologous recombination, long-fragment amplification primers are typically designed. Long-range PCR amplification is followed by electrophoresis detection, providing the possibility for detecting AdV wild-type virus residues. The strategy of "Long-range PCR combined with agarose gel electrophoresis" is based on the design principle of long-fragment amplification primers (usually greater than 7 kb in length) that span the deleted genomic regions specific to wild-type AdV but modified or deleted in recombinant vectors (such as the complete E1A-CR2 or E3 regions). Theoretically, only wild-type viral DNA containing the complete target sequence can be effectively amplified, thus allowing the observation of specific bands via electrophoresis. Recombinant vectors lacking this region cannot produce effective amplification.

[0006] However, the Long Range PCR detection method has drawbacks such as long processing time, relatively low sensitivity, and false positives due to non-specific amplification. Low sensitivity: Long-range PCR inherently has low amplification efficiency due to the long amplification fragments. Furthermore, to ensure specificity, reaction conditions are typically quite stringent, further limiting sensitivity. The typical detection limit of existing methods is approximately 1 wild-type viral genome / 10^7 vector genomes, which is insufficient for detecting extremely high-purity products.

[0007] Time-consuming and low-throughput: Long-range PCR requires a long extension time to ensure the complete synthesis of long fragments, and the cycle time of a single PCR is much longer than that of conventional PCR. From nucleic acid extraction and long-fragment PCR amplification to the final agarose gel electrophoresis analysis, the entire process usually takes a whole day or even longer, making rapid detection impossible.

[0008] Non-specific amplification: Long-fragment PCR is more likely to produce non-specific amplification products and primer dimers, resulting in multiple stray bands in the electrophoresis pattern, which interferes with interpretation and may even be misinterpreted as a positive signal. Summary of the Invention

[0009] To address the drawbacks of conventional PCR methods, such as long processing time, low sensitivity, poor specificity, and false positives, this invention develops a method for detecting wild-type AdV residues that overcomes these limitations. Based on two different modified regions of AdV, two pairs of primers with different labels are designed for the corresponding regions of wild-type AdV. Dual dPCR detection is performed, simultaneously detecting the amplification of the corresponding regions of wild-type AdV. By calculating the linkage relationship between the two modified regions, the presence of wild-type AdV can be detected.

[0010] To achieve the objectives of this invention, a first aspect of this invention discloses a method for detecting residual wild-type adenovirus, comprising the following steps: S1: The sample to be tested contains a modified adenovirus and / or wild-type adenovirus, the modification including at least the deletion or modification of the CR2 region of the early 1 (E1)A gene and / or the early 3 (E3) gene; S2: The sample to be tested is subjected to double amplification detection using a digital PCR (dPCR) reaction system, wherein the dPCR reaction system comprises: (1) The first set of primers and probes targets the E1A-CR2 region of wild-type adenovirus, wherein the 3' end of the reverse primer and / or the 5' end of the probe are located within the CR2 region or cross the boundary of the CR2 region, so that the adenovirus modified by the CR2 region cannot be effectively amplified or detected. (2) The second set of primers and probes targets the E3 gene region of wild-type adenovirus, so that adenoviruses modified by E3 gene deletion or modification cannot be effectively amplified or detected. S3: Perform dPCR amplification and detection, obtain the fluorescence signals corresponding to the E1A-CR2 and E3 targets in each droplet, and count the number of double-positive droplets, E1A-CR2-positive droplets only, E3-positive droplets only, and double-negative droplets; S4: Based on the Poisson distribution principle, calculate the concentration of linkage molecules in the E1A-CR2 region and E3 region of the sample to be tested that exist on the same adenovirus DNA strand according to the number of droplets counted in step S3. S5: Based on the calculated concentration of the linked molecules, determine whether wild-type adenovirus residue exists in the sample to be tested: when the concentration of the linked molecules is greater than 0, it is determined to be positive for wild-type adenovirus residue; when the concentration of the linked molecules is less than or equal to 0, it is determined to be negative for wild-type adenovirus residue.

[0011] In S2, the first group of probes and the second group of probes have distinguishable reporting labels. Preferably, the reporting label for the first group of probes is FAM, and the reporting label for the second group of probes is HEX.

[0012] In S1, the sample to be tested is not pretreated before dPCR detection, that is, it is not subjected to DNA digestion, or the sample is lysed before being added to the dPCR reaction system.

[0013] In S2, the sequences of the first set of primers and probes are shown in SEQ ID NO: 1-3, wherein the sequence of the forward primer is shown in SEQ ID NO: 1, the sequence of the reverse primer is shown in SEQ ID NO: 2, and the sequence of the probe is shown in SEQ ID NO: 3.

[0014] The sequences of the second set of primers and probes are shown in SEQ ID NO: 4-6, wherein the sequence of the forward primer is shown in SEQ ID NO: 4, the sequence of the reverse primer is shown in SEQ ID NO: 5, and the sequence of the probe is shown in SEQ ID NO: 6.

[0015] In S4, the concentration of the linked molecules is calculated based on the following formula or equivalent algorithm: Poisson distribution statistics: , ( , , (for three variables) When k=0 )= )= ; Conc. = CPD / Vdrop (droplet volume nL) = copies / nL; = =Conc. × Vdrop droplet volume (Given that Bio-Rad QX200 is 0.85 nL) Assuming droplet a is a double-positive droplet, b and c are single-positive droplets, d is a negative droplet, and x is a non-linked double-positive droplet, since = = Through simple calculation, we can obtain x= ; = = ; It can be deduced that: = ;; The linkage concentration can be calculated based on the distribution of droplets with different labels and the droplet volume.

[0016] Where k is the number of positive droplets in the droplet, λ is the average number of molecules per droplet, Nnegative is the number of double-negative droplets, and Ntotal is the total number of effective droplets.

[0017] A second aspect of this invention discloses a probe-primer combination for detecting residual wild-type adenovirus, comprising a first set of primers and probes, and a second set of primers and probes; The first set of primers and probes targets the E1A-CR2 region of wild-type adenovirus, wherein the 3' end of the reverse primer and / or the 5' end of the probe are designed to be located within or cross the boundary of the CR2 region, so that the adenovirus modified with the CR2 region cannot be effectively amplified or detected; the sequences of the first set of primers and probes are shown in SEQ ID NO: 1-3. The second set of primers and probes targets the E3 gene region of wild-type adenovirus, making it impossible to effectively amplify or detect adenoviruses that have undergone E3 gene deletion or modification; the sequences of the second set of primers and probes are shown in SEQ ID NO: 4-6.

[0018] A third aspect of this invention discloses a kit for detecting residual wild-type adenovirus, comprising: (a) the first set of primers and probes, and the second set of primers and probes; (b) Reaction premix for digital PCR; (c) Oil phase reagents used to generate droplets; (d) Wild-type adenovirus standard or positive control.

[0019] The fourth aspect of this invention discloses the application of the above-described method, probe-primer combination, or kit for detecting wild-type adenovirus residues in the preparation of a detection agent for detecting wild-type virus residues in adenovirus vector products.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The method of this invention is convenient and quick, enabling the detection of AdV wild-type toxin residues in a short time without requiring expensive instruments or equipment; it has high sensitivity and good specificity, compared to PCR (approximately 1 / 10 the time). 7 This represents an improvement of two orders of magnitude, with sensitivity increased to 0.5 / 10. 9 The above. This invention has high reference value for the detection of AdV wild-type toxin residues and is suitable for widespread application.

[0021] 2. This invention designs two sets of primer-probe combinations targeting the E1A-CR2 and E3 regions of wild-type adenovirus respectively, and utilizes dPCR to achieve simultaneous detection and signal differentiation of dual targets. By calculating the concentration of linkage molecules on the same DNA strand for both targets, false positive results caused by residual host cell DNA, incomplete knockout of the E3 region, or non-specific amplification of a single region can be effectively eliminated, thereby achieving specific recognition of wild-type adenovirus.

[0022] 3. Compared to the traditional Long Range PCR method (detection limit 2.5), The method of this invention improves sensitivity by approximately two orders of magnitude, with a minimum detectability of 0.5. It is more suitable for detecting trace amounts of wild-type virus residues in high-purity adenovirus vector products.

[0023] 4. Experiments show that conventional DNA extraction or pre-lysis treatment easily leads to viral genome fragmentation, resulting in loss of detection signals. This invention allows for direct detection (extraction-free), with samples added directly to the reaction system, achieving a recovery rate of over 90%, significantly reducing the risk of false negatives introduced by sample processing.

[0024] 5. No long-fragment PCR amplification and gel electrophoresis are required. The entire detection process can be completed within hours, greatly improving detection throughput and efficiency, and is suitable for rapid quality control in industrial production processes.

[0025] 6. Based on the absolute quantification and Poisson statistical principle of droplet digital PCR, the detection results are independent of the standard curve, have good repeatability and accuracy, and can be automatically interpreted through dual fluorescence channel signals, reducing human error.

[0026] 7. This method is applicable to various adenovirus vector products modified with E1A-CR2 and / or E3 regions, and can be developed into standardized kits for monitoring wild-type virus residues in gene therapy products, vaccines and other biological products. Attached Figure Description

[0027] Figure 1 This is a flowchart of probe and primer design in the Long Range PCR method in Comparative Example 1; Figure 2 This is a diagram showing the results of electrophoretic separation of the Long Range PCR amplification products in Comparative Example 1. Figure 3 This is a flowchart illustrating the design of the probe primers for Example 1. Detailed Implementation

[0028] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to their scope. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0029] Adenovirus (AdV): A non-enveloped, double-stranded DNA virus commonly used as a vector for gene therapy and vaccine development. It is widely used due to its high infectivity and ability to accommodate large exogenous gene fragments. This invention refers to the virus itself as a vector or detection target.

[0030] Wild-type adenovirus (AdV): refers to natural adenovirus that has not been genetically modified and has full replication capacity. If it remains in a product, it may pose a safety risk.

[0031] The E1A-CR2 region is a conserved region 2 in the early adenovirus gene 1A (E1A). This region binds to retinoblastoma proteins in host cells, inducing cells to enter the S phase and creating conditions for viral replication. It is one of the common modification targets for adenovirus vectors, resulting in weakened or lost viral replication ability after modification. Detecting the integrity of this region can be used to determine whether the virus is wild-type.

[0032] The E3 gene region: Early gene 3 (E3) of adenoviruses encodes proteins primarily involved in evading the host immune response. This region is not essential for viral replication in cell culture. It is often deleted to make room for the insertion of foreign genes (such as therapeutic genes or antigen genes). Detecting the presence of this region can be used to differentiate between wild-type and modified viruses.

[0033] Droplet Digital PCR (ddPCR): A highly sensitive absolute nucleic acid quantification technique. The PCR reaction system is divided into tens of thousands of nanoliter-sized droplets, each of which undergoes independent PCR amplification. The number of positive droplets is counted based on fluorescence signals, and the copy number of the target molecule in the original sample is calculated using the Poisson distribution principle, eliminating the need for a standard curve.

[0034] Linkage: In this invention, linkage refers to the simultaneous presence of complete E1A-CR2 and E3 regions on the same adenovirus DNA molecule. This "co-localization" relationship is a characteristic of the wild-type adenovirus genome. Calculating the concentration of linkage molecules can specifically reflect the content of wild-type virus and eliminate false positives caused by host DNA contamination or non-specific amplification of single regions.

[0035] Poisson distribution: A statistical model describing the probability distribution of the number of rare events occurring within a fixed time or space. It forms the mathematical basis of the dPCR technology in this invention. By statistically analyzing the proportion of negative droplets, the average number of target molecules (λ) in each droplet can be deduced, thus calculating the absolute concentration of target molecules in the sample. This is used to ultimately calculate the concentration of linked molecules.

[0036] Replicating wild-type AdV (RCA): Adenoviruses capable of replication may be accidentally generated during the production process due to homologous recombination or other reasons. These are typically wild-type or partially reverted viruses. They pose a significant safety risk to gene therapy products. The method of this invention aims to detect RCA, which may exist at extremely low levels, with high sensitivity.

[0037] Long Range PCR (LMR): A PCR technique capable of amplifying DNA fragments up to several kb in length, typically using a specialized DNA polymerase with proofreading capabilities. It is a traditional detection method used in comparative studies. Its principle involves designing primers to amplify long-fragment regions specific to wild-type viruses; however, this method suffers from low sensitivity, long processing time, and a tendency to produce non-specific bands.

[0038] (50% Tissue Culture Infectious Dose): A unit for measuring viral virulence or infectivity, referring to the viral dilution that causes 50% of cell cultures to become diseased or infected. Used to express the concentration of wild-type adenovirus standards (e.g., ) and the sensitivity of the detection method (e.g., 0.5) ).

[0039] VP (Viral Particles): Refers to the physical number of viral particles, usually determined by methods such as optical density, and includes all particles, both infectious and non-infectious. It is used to express the concentration of recombinant adenovirus vector products (e.g., 1.8 × 10¹² VP / mL). Detection sensitivity is expressed as the number of infectious units of wild-type virus that can be detected per VP.

[0040] Recovery rate: In analytical chemistry or detection experiments, it refers to the percentage of the actual measured amount of the target substance relative to the known amount added, used to evaluate the efficiency of sample pretreatment or detection methods. In Example 2 of this invention, it is used to compare the extraction or detection efficiency of target DNA by different sample pretreatment methods (column extraction, magnetic bead method, lysis method, and direct detection). A high recovery rate indicates minimal method loss.

[0041] Comparative Example 1 AdV wild-type toxin residue detection is mainly performed using molecular biology methods (PCR technology). The primer and probe design process is as follows: Figure 1 As shown, in order to reduce homologous recombination, long-fragment amplification primers are usually involved. After PCR amplification, electrophoresis is performed for identification. In order to avoid false negative results, the limit method is generally used for simultaneous spiking and detection. On the basis of meeting the applicability of the system, the authenticity of the detection result is determined, which provides the possibility for the detection of wild-type adv. However, the Long Range PCR detection method has the disadvantages of being time-consuming, having relatively low sensitivity, and being prone to false positives due to non-specific amplification.

[0042] 1. Primer information:

[0043] 2. Sample handling Sample Name Sample preparation Equivalence Limit LOD-01 The sample was mixed with STD6 (3.2E4 copies / mL) at a ratio of 9:1. 1.0 copies / E8 VG LOD-02 The sample was diluted 5-fold and mixed with STD6 (3.2E4 copies / mL) at a 9:1 ratio. 5.0 copies / E8 VG LOD-03 The sample was diluted 25-fold and mixed with STD6 (3.2E4 copies / mL) at a 9:1 ratio. 2.5 copies / E7 VG 3. Test results as follows Figure 2 As shown.

[0044] 4. Method Sensitivity: The detection limit is 2.5 copies / E7 VG. Summary: Due to the specific nature of the modification sites in the AdV wild-type virus detection method, to avoid false positives caused by homologous recombination, low-sensitivity long-fragment PCR amplification is usually used, followed by qualitative detection using agarose gel electrophoresis. Therefore, the sensitivity is only 2.5 copies / E7 VG. Furthermore, the use of long-fragment PCR cannot avoid non-specific amplification, resulting in more mixed bands (see...). Figure 2 ).

[0045] Example 1 Currently, the modification of AdV can be achieved by manipulating the early 1 (E1) region or a portion thereof, which can make AdV-based vectors have replication capabilities or no replication capabilities. In addition, since the E3 gene is not essential for viral replication, the capacity for transgene insertion can be increased by deleting the early 3 (E3) gene.

[0046] AdV viruses typically involve knocking out the CR2 region of the E1A gene and the E3 gene. Based on the AdV5 wild-type virus standard sequence (NCBI Reference Sequence: AC_000008.1), primers and probes were designed for these two regions respectively. The first primer / probe pair, with the 3' end of the reverse primer and the 5' end of the probe designed in the CR2 region, prevents the modified AdV from amplifying and can only amplify wild-type virus and host cell HEK293. The second primer / probe pair was designed in the missing E3 gene, preventing the amplification of E3 knockout modified AdV and HEK293 host cell DNA, and can only specifically amplify wild-type virus. Using the Poisson distribution principle, after dPCR detects the number of double-positive, double-negative, and single-positive droplets, the number of linkages where E1A and E3 coexist on the same DNA strand (identified as wild-type virus) can be calculated based on the distribution of positive and negative droplets. This can eliminate false positives caused by host cell residues and false positives caused by incomplete E3 knockout, thus determining whether wild-type adenovirus remains in the test sample. The design process is as follows: Figure 3 As shown.

[0047] Poisson distribution statistics: , ( , , (for three variables) When k=0 )= )= Based on the distribution of droplets with different labels, the linkage concentration can be derived.

[0048] 1. Primer information:

[0049] Note: The underlined base sequences in the table are located in the deletion region of the CR2 modified sequence.

[0050] 2. Instruments and equipment:

[0051] 3. Reagents and consumables: name factory Item number ddPCR Supermix for Probes Bio-Rad 1863010 Special oil for microdroplet generation Bio-Rad 1863005 Microdroplet generating card / sealing pad Bio-Rad 1864007 Special oil for microdroplet analysis Bio-Rad 1863004 96-well plate heat-sealing film (dPCR) Bio-Rad 1814040 AdV5 Wild Poison Standard ATCC VR-5 Primers / Probes Biotechnology N / A 96-hole plate Bio-Rad 12001925 4. Preparation of Standards: Label the 8-tube PCR kits as STD1, STD2, STD3, STD4, and STD5, respectively. Use AdV5 wild-type virus standard (Stock concentration 1.1 × 10⁻⁶). 9 TCID 50 Serial dilutions were performed using the method ( / mL).

[0052]

[0053] 5. Preparation of dPCR reaction solution: Prepare the dPCR reaction solution according to the table below, and vortex to mix well;

[0054] Aliquot 17.6 µL / well into 8-tube PCR kits, add 4.4 µL of the sample, vortex to mix, and centrifuge briefly.

[0055] 6. Droplet generation: Place the droplet generating card into the droplet generating card slot, add the mixture to the middle row of the droplet generating card, add 70 µL of droplet generating oil to the bottom row, and place it in the droplet generator to generate droplets.

[0056] 7. PCR reaction: Place the 96-well plate containing the droplets into a PCR instrument for amplification.

[0057]

[0058] 8. Droplet reading: After the PCR reaction is completed, the sample is read in the droplet reader.

[0059] 9. Results Analysis: When the linkage value > 0 linkage copies / 20 μL well, the result is considered positive. When the linkage value is <0 linkage copies / 20 μL well, the result is considered negative.

[0060] Example 2: Examination of Pretreatment Methods Different pretreatment methods (column extraction, magnetic bead extraction, lysis method, and direct detection method) were investigated, and the results are as follows:

[0061] Results showed that: Sample pretreatment: Adenovirus (AdV) is a non-enveloped, double-stranded DNA virus with a diameter of approximately 70-90 nm. Comparisons of different pretreatment methods revealed that both rapid viral lysis buffers and commonly used commercial extraction reagents resulted in sample fragmentation, potentially leading to false negatives (a problem also present in PCR electrophoresis). In contrast, the direct detection method achieved a recovery rate of 50%–150%. This method is not only effective for spiked DNA but also confirmed in genomic titer detection for intracapsular viral samples.

[0062] Example 3 Performance Indicators (Sensitivity) Preparation of test sample 10 μL STD5 (1.1×10 4 TCID50 ( / mL) + 90 μL sample (sample concentration is 1.8 × 10⁻⁶) 12 VP / mL), i.e., 0.5 TCID 50 / 10 9 VP. Testing is performed using 6 parallel wells and 2 duplicate wells.

[0063] Detection results: For the spiked sample group, there were 6 parallel samples and 2 replicates, totaling 12 data points. All results met expectations: linkage value > 0 linkage copies / 20 μL well, indicating a positive result. Therefore, the sensitivity of this method for detecting AdV wild-type toxicity is 0.5 TCID. 50 / 10 9 VP.

[0064] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting residual wild-type adenovirus, characterized in that, Includes the following steps: S1: The sample to be tested contains a modified adenovirus and / or wild-type adenovirus, the modification including at least the deletion or modification of the CR2 region of the early 1 (E1)A gene and / or the early 3 (E3) gene; S2: The sample to be tested is subjected to double amplification detection using a digital PCR (dPCR) reaction system, wherein the dPCR reaction system comprises: (1) The first set of primers and probes targets the E1A-CR2 region of wild-type adenovirus, wherein the 3' end of the reverse primer and / or the 5' end of the probe are located within the CR2 region or cross the boundary of the CR2 region, so that the adenovirus modified by the CR2 region cannot be effectively amplified or detected. (2) The second set of primers and probes targets the E3 gene region of wild-type adenovirus, so that adenoviruses modified by E3 gene deletion or modification cannot be effectively amplified or detected. S3: Perform dPCR amplification and detection, obtain the fluorescence signals corresponding to the E1A-CR2 and E3 targets in each droplet, and count the number of double-positive droplets, E1A-CR2-positive droplets only, E3-positive droplets only, and double-negative droplets; S4: Based on the Poisson distribution principle, calculate the concentration of linkage molecules in the E1A-CR2 region and E3 region of the sample to be tested that exist on the same adenovirus DNA strand according to the number of droplets counted in step S3. S5: Based on the calculated concentration of the linked molecules, determine whether wild-type adenovirus residue exists in the sample to be tested: when the concentration of the linked molecules is greater than 0, it is determined to be positive for wild-type adenovirus residue. When the concentration of the linked molecules is less than or equal to 0, it is determined to be negative for wild-type adenovirus residue.

2. The method for detecting residual wild-type adenovirus according to claim 1, characterized in that, In S2, the first group of probes and the second group of probes are marked with distinguishable reporting tags.

3. The method for detecting residual wild-type adenovirus according to claim 2, characterized in that, In S2, the report label for the first group of probes is FAM, and the report label for the second group of probes is HEX.

4. The method for detecting residual wild-type adenovirus according to claim 1, characterized in that, In S1, the sample to be tested is not pretreated before dPCR detection.

5. The method for detecting residual wild-type adenovirus according to claim 1, characterized in that, The sequences of the first set of primers and probes are shown in SEQ ID NO: 1-3, and the sequences of the second set of primers and probes are shown in SEQ ID NO: 4-6.

6. The method for detecting residual wild-type adenovirus according to claim 1, characterized in that, In S4, the concentration of the linked molecules is calculated based on the following formula or equivalent algorithm: Poisson distribution statistics: , ( , , (for three variables) When k=0 )= )= ; Conc. = CPD / V drop Droplet volume = copies / nL; )= =Conc.×V drop microdroplet volume Assuming droplet a is a double-positive droplet, b and c are single-positive droplets, d is a negative droplet, and x is a non-linked double-positive droplet, since = = Through simple calculation, we can obtain x= ; = = ； It can be deduced that: = ) The linkage concentration can be calculated based on the distribution of droplets with different labels and the droplet volume. Where k is the number of positive molecules in a droplet, λ is the average number of molecules per droplet, and N is the number of positive molecules in a droplet. negative The number of double-negative droplets, N total This represents the total number of effective droplets.

7. The method for detecting residual wild-type adenovirus according to claim 1, characterized in that, In S3, the dPCR is droplet digital PCR (dPCR).

8. A probe-primer combination for detecting residual wild-type adenovirus, characterized in that, Includes the first set of primers and probes, and the second set of primers and probes; The first set of primers and probes targets the E1A-CR2 region of wild-type adenovirus, wherein the 3' end of the reverse primer and / or the 5' end of the probe are designed to be located within or cross the boundary of the CR2 region, so that the adenovirus modified with the CR2 region cannot be effectively amplified or detected; the sequences of the first set of primers and probes are shown in SEQ ID NO: 1-3. The second set of primers and probes targets the E3 gene region of wild-type adenovirus, making it impossible to effectively amplify or detect adenoviruses that have undergone E3 gene deletion or modification; the sequences of the second set of primers and probes are shown in SEQ ID NO: 4-6.

9. A kit for detecting residual wild-type adenovirus, characterized in that, Include: (a) The first set of primers and probes as described in claim 8, and the second set of primers and probes; (b) Reaction premix for digital PCR; (c) Oil phase reagents used to generate droplets; (d) Wild-type adenovirus standard or positive control.

10. The use of the method of any one of claims 1-7, the probe-primer combination of claim 8, or the kit of claim 9 in the preparation of a detection agent for detecting wild-type virus residues in adenovirus vector products.

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