Primer combination and method for detecting residual DNA fragments in E1 region of host cell of non-replicative adenovirus vector

Abstract

The invention relates to the technical field of non-replicative adenovirus vectors, in particular to a primer combination and a method for detecting residual DNA fragments in an E1 region of a host cell of a non-replicative adenovirus vector. The primer combination comprises an E1A primer pair and an E1B primer pair, the E1A primer pair comprises a first primer as shown in SEQ ID NO. 1 and a second primer as shown in SEQ ID NO. 2, and the E1B primer pair comprises a third primer as shown in SEQ ID NO. 3 and a fourth primer as shown in SEQ ID NO. 4. The primer combination can be used for carrying out specific detection on E1 residual fragments in HEK293 cell host DNA (Deoxyribose Nucleic Acid); the detection method has the advantages of high efficiency, good effect, high specificity, good sensitivity and good repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of non-replicating adenovirus vectors, and in particular to a primer combination and a method for detecting residual DNA fragments in the E1 region of a host cell of a non-replicating adenovirus vector. Background Art

[0002] The HEK293 cell line was established by integrating a human adenovirus type 5 E1 region DNA fragment (including complete adenovirus E1A and E1B genes) into chromosome 19. It is commonly used in non-replicating adenovirus production processes. In the viral vector production process, not only residual host cell DNA and RNA impurities are inevitably produced, but also E1A and E1B residual DNA impurities are produced. These impurities will not only reduce the effectiveness of viral vector-related drugs, but may also bring safety issues such as infectivity or tumorigenicity.

[0003] The "Technical Guidelines for Pharmaceutical Research and Evaluation of In Vivo Gene Therapy Products (Trial)" and "Technical Guidelines for Pharmaceutical Research and Evaluation of In Vitro Gene Modification Systems (Trial)" recently issued by CDE (Center for Drug Evaluation of the National Medical Products Administration) in May 2022 both mention that cell banks used to package viral vectors, such as HEK293T cells, should be subject to residual control for the Adv E1A sequences and SV40 large T antigen sequences they carry, which have potential safety risks.

[0004] However, there is currently no method that can effectively detect E1 region DNA fragments. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a primer combination and a method for detecting the residual DNA fragments in the E1 region of a host cell of a non-replicating adenovirus vector.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The present invention provides a primer combination for detecting the residual DNA fragments in the E1 region of a host cell of a non-replicating adenovirus vector, comprising an E1A primer pair and an E1 B primer pair, wherein the E1A primer pair comprises a first primer as shown in SEQ ID NO.1 and a second primer as shown in SEQ ID NO.2, and the E1 B primer pair comprises a third primer as shown in SEQ ID NO.3 and a fourth primer as shown in SEQ ID NO.4.

[0008] The present invention also provides a kit, comprising the above primer combination.

[0009] The present invention also provides a use of the above-mentioned kit, wherein the kit is used to detect the residual DNA fragments in the host cell E1 region of the non-replicating adenovirus vector.

[0010] Furthermore, the host cell is a HEK293 cell line.

[0011] The present invention also provides a method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector, which is detected by using the primer combination as mentioned above or the kit as mentioned above.

[0012] Further, the following steps are included:

[0013] 1) Extract total DNA from samples;

[0014] 2) using the total DNA as a template and the primer combination for PCR amplification;

[0015] 3) determining whether the host cell E1 region DNA fragment exists in the sample according to the PCR amplification result;

[0016] Wherein, the host cell is HEK293 cell line.

[0017] Furthermore, in step 2), the annealing temperature of the PCR amplification is 62° C., the time is 15 s, and the number of cycles is 30.

[0018] Furthermore, in step 2), the extension temperature of the PCR amplification is 72° C., the number of cycles is 30, the extension time of the E1A primer pair is 1 min, and the extension time of the E1 B primer pair is 1 min 30 s.

[0019] Further, after the detection, the residual concentration of host cells in the sample is detected.

[0020] Furthermore, the sample is a non-replicating adenovirus vector product prepared using HEK293 cell line as host cell.

[0021] The beneficial effects of the present invention are:

[0022] (1) The primer combination of the present invention can specifically detect the E1 residual fragment in the HEK293 cell host DNA;

[0023] (2) The method for detecting the residual DNA fragments of the E1 region of the host cell of the non-replicating adenovirus vector of the present invention adopts the primer combination or the kit of the present invention to detect the E1A region and the E1 B region respectively, thereby realizing the specific detection of the residual fragments of E1;

[0024] (3) In the method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector of the present invention, the PCR annealing temperature can make the detection result more specific;

[0025] (4) The method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector of the present invention has the advantages of high efficiency, good effect, high specificity, good sensitivity and good repeatability;

[0026] (5) The method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector of the invention can effectively detect the quality of the non-replicating adenovirus vector products, effectively control the safety of the use of the non-replicating adenovirus vector, and provide a good guarantee for the promotion and development of the non-replicating adenovirus vector products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The electrophoresis diagram of Example 1 is a method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector of the present invention;

[0028] Figure 2 The electrophoresis diagram of Example 2 is a method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector of the present invention;

[0029] Figure 3 The present invention is a method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector. In Example 3, the electrophoresis diagrams of the E1A region obtained by using different primer pairs at different annealing temperatures are shown;

[0030] Figure 4 The method for detecting the residual DNA fragments of the E1 region of the host cell of the non-replicating adenovirus vector of the present invention, in Example 3, the electrophoresis diagrams of the E1 B region obtained by using different primer pairs at different annealing temperatures;

[0031] Figure 5 The present invention is a method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector, and the electrophoresis diagram of the first repeated experiment in Example 4;

[0032] Figure 6 The present invention is a method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector, and the electrophoresis diagram of the second repeated experiment in Example 4;

[0033] Figure 7 The present invention is a method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector, and the electrophoresis diagram of the third repeated experiment in Example 4;

[0034] Figure 8 The present invention is a method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector, and the electrophoresis diagram of the specific experiment in Example 5;

[0035] Fig. 9This is the electrophoresis diagram of the accuracy experiment in Example 7 of the method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector of the present invention. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0037] The primer combination for detecting the residual DNA fragment of the host cell E1 region of a non-replicating adenovirus vector of the present invention comprises an E1A primer pair and an E1 B primer pair, the E1A primer pair comprises a first primer as shown in SEQ ID NO.1 and a second primer as shown in SEQ ID NO.2, and the E1 B primer pair comprises a third primer as shown in SEQ ID NO.3 and a fourth primer as shown in SEQ ID NO.4.

[0038] The primer combination of the present invention can specifically detect the E1 residual fragments in the HEK293 cell host DNA.

[0039] The kit of the present invention comprises the primer combination as described above.

[0040] The kit of the present invention can be used to detect the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector.

[0041] Specifically, the host cell is the HEK293 cell line.

[0042] The method for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector of the present invention uses the primer combination as mentioned above or uses the kit as mentioned above for detection.

[0043] The detection method of the present invention comprises the following steps:

[0044] 1) Extract total DNA from samples;

[0045] 2) Using total DNA as template, PCR amplification was performed using primer combinations.

[0046] Preferably, the reaction conditions for PCR amplification are: pre-denaturation at 95°C for 3 min, 1 cycle, denaturation at 95°C for 15 s, 30 cycles, annealing at 62°C for 15 s, 30 cycles, extension at 72°C, E1A for 1 min, E1 B for 1 min30 s, 30 cycles, and terminal extension at 72°C for 5 min, 1 cycle.

[0047] 3) Based on the PCR amplification results, determine whether the sample contains the host cell E1 region DNA fragment, and the host cell is the HEK293 cell line.

[0048] Preferably, the PCR product is subjected to 1% agarose gel electrophoresis to obtain an electrophoretogram. When a band appears at 1000 bp in the electrophoretogram, it indicates that the E1A segment is detected, and when a band appears at 1500 bp in the electrophoretogram, it indicates that the E1 B segment is detected.

[0049] Specifically, the nucleotide sequence of the E1A segment is shown in SEQ ID NO.5, the nucleotide sequence of the E1 B segment is shown in SEQ ID NO.6, and the full-length sequence of the E1 segment is shown in SEQ ID NO.7.

[0050] The present invention is described below by means of specific examples.

[0051] The negative control used in the examples of the present invention is deionized water without enzyme.

[0052] The positive control used in the embodiment of the present invention is KD01, which was obtained by Wuhan Kaidejinuo Biotechnology Co., Ltd. KD01 is a conditionally replicative virus with human adenovirus type 5 as a vector, and its main feature is that 27bp of the E1A region of HEK2893 cells are deleted, and other regions of E1A and the complete E1 B region are retained, which can be effectively used as a positive control in the embodiment of this scheme.

[0053] In addition, HEK293 cells contain the complete E1 region sequence and can also be used as a positive control.

[0054] The sample used in the embodiments of the present invention is KDTV001, which was obtained by Wuhan Kaidejinuo Biotechnology Co., Ltd. KDTV001 is a non-replicating adenovirus vector product, the main feature of which is that it is prepared by Ad non-replicating adenovirus with E1 and E3 regions deleted, that is, it does not contain the HEK293 cell E1A region and E1 B region of the host cell. The KDTV001 samples used in the following embodiments are specifically divided into KDTV001 crude pure liquid, 20230818AH01-Core700, and KDTV001 stock solution (DS240822). The difference between these samples is that the purity is different and the host cell HCD residue is different. The specific examples can be seen. For the specific sequences of KDTV001 and KD01, please refer to patent CN202410543158 "A HPV16_18_52 therapeutic vaccine, preparation method and application", and for the specific sequence of KD01, please refer to CN202311530370 "A oncolytic adenovirus recombinant vector, construction method and application".

[0055] Example 1

[0056] In this example, the E1 region fragment inserted into HEK293 cells was used as a template to design primers. The upstream of the primers was located in the E1A region, and the downstream was located in the E1 B region. The specific primer information is shown in Table 1.

[0057] Table 1

[0058]

[0059] The viral DNA of this example was extracted according to conventional methods (such as "Viral DNA Extraction Operating Procedure SOP"), and the concentration was detected. The results are shown in Table 2.

[0060] Table 2

[0061] sample batch number Extraction volume Elution volume Concentration (ng / μl) HCD (ng / μl) KDTV001(crude pure liquid) 202305110 200 30 9.3 98 KD01 202112003 200 30 17.2 3.1 HEK293 cell pellet N / A 200 30 106.9 N / A

[0062] The PCR reaction system of this embodiment is shown in Table 3, and the reaction process is shown in Table 4.

[0063] Table 3

[0064] Reagents Blank control group Test group Positive control group Mix (Novozyme P520) (μl) 25 25 25 F primer (10 μM) (μl) 2 2 2 R primer (10 μM) (μl) 2 2 2 Test DNA (μg) —— 0.002 —— Positive control KD01 (μg) —— —— 0.002 Add enzyme-free water to the final volume (μl) 50 50 50

[0065] Table 4

[0066]

[0067] The agarose gel electrophoresis process of this embodiment is as follows:

[0068] Prepare 1% agarose: weigh 0.25g agarose and dissolve it in 25ml TAE solution. Heat in a microwave until melted, pour into the assembled gel plate, and wait for it to cool and form.

[0069] Place the agarose gel in an electrophoresis tank that has been added with electrophoresis buffer, and add 5 μl of DNA marker and 10 μl of PCR reaction product to the gel wells in sequence.

[0070] Set the electrophoresis instrument to 120V and run for 25-30 minutes. Then develop the gel on a gel imager. The resulting electrophoresis pattern is as follows: Figure 1 shown.

[0071] The PCR products of KD01 and KDTV001 were subjected to first-generation sequencing, and according to the sequencing results, the measured sequences were consistent with the E1 theoretical sequence alignment results.

[0072] according to Figure 1 It can be seen that after PCR, specific amplification products can be seen at 1500 bp for the three samples, KD01, KDTV001, and HEK293 cell precipitation, indicating that the primers and PCR method of this example can detect the E1 region.

[0073] However, in theory, the DNA extracted from KDTV001 should not contain the E1 segment sequence, and the gel electrophoresis result of the PCR amplification product should be negative at 1500bp. After analysis, the KDTV001 sample used in this example is a crude pure liquid for process development, and the HCD content is high, and its detection result is 98ng / μl. Therefore, this example detected residual E1 fragments in the KDTV001 (batch number: 2023051101C) sample, and it is impossible to determine whether it is caused by residual host cell DNA or the E1 segment of the viral gene mutation. Therefore, the method needs to be optimized.

[0074] Example 2 Comparison of detection effects of different primer pairs

[0075] On the basis of Example 1, in order to optimize the detection effect, in this example, primers were designed for E1A and E1 B respectively, and the detection effects of different primer pairs were compared. At the same time, a KDTV001 sample with higher purity was used.

[0076] The specific primer pairs involved are shown in Table 5.

[0077] Table 5

[0078]

[0079] The sample types, extraction volumes, elution volumes, concentrations and residual host cell concentrations used in this example are shown in Table 6:

[0080] Table 6

[0081] sample Concentration ng / μl HCD (ng / μl) KDTV001(20230818AH01-Core700) 2.23 37.26 KD01(202204001) 9.86 0.73

[0082] The PCR reaction system of this embodiment is shown in Table 7:

[0083] Table 7

[0084]

[0085]

[0086] The PCR reaction system of this embodiment is shown in Table 8:

[0087] Table 8

[0088]

[0089] The agarose gel electrophoresis process of this embodiment is as follows:

[0090] Prepare 1% agarose: weigh 0.25g agarose and dissolve it in 25ml TAE solution. Heat in a microwave until melted, pour into the assembled gel plate, and wait for it to cool and form.

[0091] Place the agarose gel in an electrophoresis tank that has been added with electrophoresis buffer, and add 5 μl of DNA marker and 10 μl of PCR reaction product to the gel wells in sequence.

[0092] The electrophoresis apparatus was set to 120 V and run for 25-30 min before developing the gel on a gel imager.

[0093] Gel electrophoresis results Figure 2 As shown, Figure 2 In the figure, 1 is a blank sample, 2 is a positive control KD01 (batch number: 202204001), and 3 is a sample KDTV001.

[0094] According to the above experimental results, it can be seen that when the three primer pairs of E1A of this example are used for PCR amplification at an annealing temperature of 60°C, specific PCR products can be seen at about 1000 bp for the positive control (KD01) and the test sample KDTV001 (lot number: 20230818AH01-Core700), and multiple non-specific bands appear at greater than 2000 bp. When the three primer pairs of E1 B of this example are used, specific PCR products can be seen at about 1500 bp, and multiple non-specific bands appear at greater than 2000 bp.

[0095] Non-specific bands indicate poor test results and require adjustment of primers and PCR amplification conditions.

[0096] Example 3 Comparison of detection effects of different PCR annealing temperatures

[0097] On the basis of Example 2, this example adjusts the annealing temperature of PCR amplification to 62° C. and 65° C., and continues to use the primers of Example 2 to detect each experimental group.

[0098] The sample type, extraction volume, elution volume, concentration and residual host cell concentration used in this example are the same as those in Example 2, and the PCR reaction system is also the same as that in Example 2.

[0099] The PCR reaction conditions of this example are shown in Table 9.

[0100] Table 9

[0101]

[0102] The agarose gel electrophoresis process of this example is the same as that of Example 2. The gel electrophoresis results are as follows: Figure 3 and Figure 4 shown. Figure 3 The detection results of the three primer pairs of E1A at annealing temperatures of 62°C and 65°C are shown in Figure 2. Figure 4Results for the three primer pairs for E1 B at annealing temperatures of 62°C and 65°C.

[0103] According to the above test results, for the E1A segment of the host cell, under the annealing condition of 62°C, the PCR products of the three pairs of primers of the positive control KD01 (batch number: 202204001) can detect specific bands at 1000bp, and only the first pair of primers of the KDTV001 (batch number: 20230818AH01-Core700) sample can detect specific PCR products and no non-specific bands are generated, indicating that the detection sensitivity of primers 2 and 3 decreases after the annealing temperature is increased; after further increasing the annealing temperature to 65 degrees, the detection sensitivity of the three pairs of primers further decreases. Through this experiment, the E1A annealing temperature is determined to be 62°C, and the detection primer is determined to be E1A primer 1 (sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively).

[0104] For the E1B segment of the host cell, the PCR products of the three pairs of primers of the positive control KD01 (batch number: 202204001) and KDTV001 (batch number: 20230818AH01-Core700) can detect specific bands at 1500bp, and no non-specific bands are generated; after further increasing the annealing temperature to 65 degrees, no specific bands were detected by the three pairs of primers, indicating that the detection sensitivity is significantly reduced when the annealing temperature is 65°C. Through this experiment, the annealing temperature of the E1B segment was determined to be 62°C, and the detection primer was determined to be E1B primer 2 (sequences are SEQ ID NO.3 and SEQ ID NO.4, respectively).

[0105] Example 4 Repeatability Verification

[0106] This example uses the primer pairs and PCR annealing temperature selected in Example 3 to detect samples with lower residual host cell DNA concentration and higher purity to verify the repeatability of the present invention.

[0107] The sample information of this example is shown in Table 10.

[0108] Table 10

[0109]

[0110] The PCR reaction system of this example is the same as that of Example 2. The primers used are shown in Table 11, and the PCR reaction conditions are shown in Table 12.

[0111] Table 11

[0112]

[0113] Table 12

[0114]

[0115]

[0116] The agarose gel electrophoresis process of this example is the same as that of Example 1, and the experiment is repeated three times. The gel electrophoresis results are as follows: Figures 5 to 7 shown. Figure 5 In the figure, bands 1, 5, and 9 are DNA marker bands, bands 2 and 6 are negative control groups, bands 3 and 7 are positive controls KD01 (202204001), and bands 4 and 8 are sample KDTV001 stock solutions (DS240822). Figure 6 In the figure, bands 1 and 8 are DNA markers, bands 2 and 5 are negative controls, bands 3 and 6 are positive controls KD01 (202204001), and bands 4 and 7 are sample KDTV001 stock solution (DS240822). Figure 7 In the figure, bands 4 and 5 are DNA markers, bands 1 and 6 are negative controls, bands 2 and 7 are positive controls KD01 (202204001), and bands 3 and 8 are KDTV001 stock solution (DS240822).

[0117] according to Figures 5 to 7 It can be seen that the method of this embodiment has good repeatability and high accuracy.

[0118] Example 5 Specificity Verification

[0119] In this embodiment, before the sample DNA extraction process, a control test group treated with a nuclease reagent was set up to eliminate the interference of exogenous DNA and verify the specificity of the method.

[0120] The nuclease reagent treatment method of this embodiment is to mix 1 ml of KDTV001 (crude pure solution) with 10 U nuclease (nearshore protein) and 2 mM MgCl2 for enzymatic hydrolysis, and the enzymatic hydrolysis reaction conditions are: 37°C, 2 hours. DNA extraction was performed on each experimental group, and the specific information is shown in Table 13.

[0121] Table 13

[0122] sample batch number Extraction volume Elution volume Concentration ng / μl HCD KDTV001 20230818AH01-Core700 200 30 20.8 0.79 KD01 202204001 200 30 9.8 0.26 KDTV001+nuclease treatment 20230818AH01-Core700 200 30 16.5 N / A

[0123] The PCR reaction system, reaction conditions and electrophoresis conditions of this example are the same as those of Example 1. The loading volume of agarose gel electrophoresis is 5 μl, and the loading volume of DNA Marker is 3 μl.

[0124] The electrophoresis diagram of this example is as follows Figure 8In the figure, 1 is a negative control (deionized water without enzyme), 2 is a positive control KD01 (batch number 202204001), 3 is KDTV001 (20230818AH01-Core700) + nuclease treatment, and 4 is KDTV001 (batch number 20230818AH01-Core700).

[0125] According to the above electrophoresis diagram, it can be seen that the E1 segment residues can be detected in the KDTV001 intermediate sample (20230818AH01-Core700), and no E1 segment residues were detected in the sample extracted after adding nuclease treatment, indicating that the E1 segment residues in the KDTV001 intermediate sample (20230818AH01-Core700) are derived from host cell DNA.

[0126] Example 6 Sensitivity Verification

[0127] In this example, samples with high purity and low HCD residue were used to verify the sensitivity of the method.

[0128] In this example, KDTV001 stock solution (DS240822) was selected, and the HPLC purity test report was 100%, and the HCD residue was 0.46 ng / μl. The positive control selected KD01 finished preparation (202204001), and the HPLC purity test report was 100%, and the HCD residue was 0.37 ng / μl.

[0129] Example 7 Accuracy Verification

[0130] In this example, DNA from KDTV001 stock solution, KDTV001 virus storage solution, HEK293 cells, and HEK293 cells + KDTV001 (volume 1:1) was extracted as templates, and PCR experiments were performed using specific primers. The amplified products were observed by agarose gel electrophoresis. The PCR reaction system, reaction conditions, and electrophoresis conditions of this example are the same as those of Example 4.

[0131] The experimental results of this embodiment are shown in Table 14 and Fig. 9 As shown. According to the experimental results, the PCR products of KDTV001 stock solution, KDTV001 virus preservation solution and negative control have no bands. The PCR product of HEK293 cells + KDTV001 (volume 1:1) has two bands, which are 1017bp of the E1A segment and 1784bp of the E1B segment; the PCR product of HEK293 cells has two bands, which are 1017bp of the E1A segment and 1784bp of the E1B segment. The results are consistent with the theory and meet the standard requirements.

[0132] Table 14

[0133]

[0134]

[0135] In the above embodiments of the present invention, the main reagents are as follows:

[0136]

[0137] In the above embodiment of the present invention, the main consumables are as follows:

[0138]

[0139] In the above embodiment of the present invention, the main equipment is as follows:

[0140]

[0141] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer combination, characterized in that: The method is used to detect the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector, comprising an E1A primer pair and an E1 B primer pair, wherein the E1A primer pair comprises a first primer as shown in SEQ ID NO.1 and a second primer as shown in SEQ ID NO.2, and the E1 B primer pair comprises a third primer as shown in SEQ ID NO.3 and a fourth primer as shown in SEQ ID NO.

4.

2. A kit, characterized in that: Comprising the primer combination as claimed in claim 1.

3. A use of the kit as claimed in claim 2, characterized in that: The kit is used for detecting the residual DNA fragments in the E1 region of the host cell of the non-replicating adenovirus vector.

4. The use according to claim 3, characterized in that: The host cell is HEK293 cell line.

5. A method for detecting the residual DNA fragments in the E1 region of a host cell of a non-replicating adenovirus vector, characterized in that: The detection is performed using the primer combination as described in claim 1 or the kit as described in claim 2.

6. The method for detecting the residual DNA fragments in the E1 region of the host cell of a non-replicating adenovirus vector according to claim 5, characterized in that: The following steps are involved: 1) Extract total DNA from samples; 2) using the total DNA as a template and the primer combination for PCR amplification; 3) determining whether the host cell E1 region DNA fragment exists in the sample according to the PCR amplification result; Wherein, the host cell is HEK293 cell line.

7. The method for detecting the residual DNA fragments in the E1 region of the host cell of a non-replicating adenovirus vector according to claim 6, characterized in that: In step 2), the annealing temperature of the PCR amplification is 62° C., the time is 15 s, and the number of cycles is 30.

8. The method for detecting the residual DNA fragments in the E1 region of the host cell of a non-replicating adenovirus vector according to claim 6, characterized in that: In step 2), the extension temperature of the PCR amplification is 72° C., the number of cycles is 30, the extension time of the E1A primer pair is 1 min, and the extension time of the E1 B primer pair is 1 min 30 s.

9. The method for detecting the residual DNA fragments in the E1 region of a host cell of a non-replicating adenovirus vector according to claim 6, characterized in that: After the detection, the residual concentration of host cells in the sample is then detected.

10. The method for detecting the residual DNA fragments in the E1 region of the host cell of a non-replicating adenovirus vector according to claim 6, characterized in that: The sample is a non-replicating adenovirus vector product prepared using HEK293 cell line as host cell.

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