A method for detecting residual DNA of sf9 host cells

Abstract

The application provides a detection method for residual DNA of Sf9 host cells, which comprises using a specific primer pair and a probe combination to perform a real-time fluorescent quantitative PCR reaction. The detection method of the application has high specificity, high sensitivity, good repeatability and simple operation, effectively solves the problems of cross-reaction, insufficient sensitivity and lack of standardization in the prior art, and provides key technical support for the development of production processes and quality control of biological products.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection, and specifically relates to a method for detecting residual DNA in Sf9 host cells. Background Technology

[0002] Insect cell-baculovirus expression systems (BEVS) are widely used in the production of recombinant proteins, particularly in vaccines and biopharmaceuticals. Sf9 cells (fall armyworm ovarian cells), as the core host of this system, possess strong protein expression capabilities and can perform eukaryotic post-translational modifications (such as glycosylation and phosphorylation). They are widely used in the production of therapeutic proteins, subunit vaccines, virus-like particles, and adeno-associated virus vectors.

[0003] Biological products produced using insect cell-baculovirus expression systems require simultaneous monitoring of residual Sf9 cell DNA and recombinant baculovirus (AcNPV) DNA. Currently, pharmacopoeias of various countries provide primer and probe sequences for detecting residual host DNA in several common expression systems, including CHO cells, but none provide primer and probe sequences suitable for detecting residual DNA in insect cell (e.g., Sf9 cell)-baculovirus systems (referred to as the "insect-baculovirus system"). The main reason is that insect cell genomes lack the abundance of highly repetitive sequences (such as SINE / LINE elements, multi-copy rRNA operons, or rDNA repeat units) found in other host cells. Although multi-copy genes (such as rDNA and mitochondrial DNA) exist, their repetitiveness and conservation still need further verification, making them difficult to directly serve as stable and reliable detection targets. Furthermore, although the Sf9 cell genome has been sequenced, its gene function annotation is still incomplete, the functions of many predicted genes are unknown, and specific sequence resources are scarce, which also poses challenges to primer and probe design. Although some literature reports on the detection standards for Sf9 residual DNA in products related to the kunzha expression system, none of them disclose the detection methods, sensitivity, and system validation data in detail, nor do they provide a sufficient evaluation of the broad spectrum and adaptability of primers and probes, which limits their practical application.

[0004] Currently, commercially available Sf9 residual DNA detection kits integrate both Sf9 and AcNPV detection reagents into a single kit. Users face the following challenges when using these kits for method validation: 1) Difficulty in specificity verification: All commercially available kits provide premixed Sf9 and AcNPV quantitative references, making it difficult for users to verify whether the Sf9 detection component is completely unaffected by AcNPV DNA interference, and vice versa, affecting the rigor of the validation; 2) Risk of homologous sequence interference: The exogenous gene sequence carried in recombinant baculoviruses may have potential unknown homology with the detection target region, leading to the detection signal possibly originating from both Sf9 residual DNA and baculovirus DNA, resulting in false positives; or, the exogenous gene sequence may inhibit PCR amplification of the target DNA due to partial homologous competition with primers and probes, leading to false negatives; 3) Limited instrument compatibility: Currently, commercially available kits generally employ multiplex qPCR strategies, requiring the simultaneous use of multiple fluorescence channels such as FAM, VIC, and CY5, placing high demands on the optical modules of qPCR instruments, thus hindering widespread application on conventional, economical equipment. Furthermore, due to potential differences in the residual levels of Sf9 DNA and baculovirus DNA, samples need to be diluted at different folds during detection, making simultaneous detection in the same reaction well impossible. This is not only cumbersome and inefficient, but also requires users to activate all fluorescence channels when detecting a single target. If only one channel is activated, the instrument will still acquire signals from all channels, leading to spectral overlap and signal crosstalk between channels, severely affecting the accuracy and reliability of the detection results.

[0005] Currently, several patent applications have proposed different methods for detecting residual DNA in Sf9 cells, but all have significant limitations. For example, patent CN114075594B amplifies the 16S rRNA region of Sf9 cells based on the SYBR Green dye method, which carries the risk of non-specific amplification; patent CN116716415A uses TaqMan... The MGB probe method detects the same region, but exhibits non-specific amplification in HF cells; patent CN117187412A uses the TaqMan probe method, claiming to simultaneously detect Hi-5 and Sf-9 cell DNA, but its probe design introduces degenerate bases, which may affect binding specificity and amplification efficiency; patents CN116716415A and CN117187412A have serious defects in standard design: both use recombinant plasmid DNA as quantitative standards, rather than the industry-recognized host cell genomic DNA (gDNA). The actual sample to be tested is complex genomic DNA obtained through extraction, and its molecular structure, size, and state are significantly different from small molecule, supercoiled plasmid standards, i.e., circular DNA. This leads to differences in PCR amplification efficiency and accessibility of primers / probes, failing to truly reflect the detection performance of the actual sample, ultimately affecting the accuracy and reliability of the quantitative results, and making it difficult to achieve absolute quantification that meets the requirements of the specifications. In addition, both patents CN115851969A and CN120425052A use the TaqMan probe method, but they are only used to analyze the distribution of DNA fragments in Sf9 cells. They have not verified whether they can accurately quantify the total residual DNA, and they also lack performance verification in actual biological products.

[0006] Based on the above-mentioned existing technology, it is clear that the detection methods for residual DNA in Sf9 host cells still urgently need to improve their specificity, sensitivity, and accuracy. At the same time, it is also necessary to enhance the versatility and convenience of the detection methods, and reduce the dependence on high-configuration equipment and usage costs, so as to be suitable for flexible application in routine laboratories and production quality control environments. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a real-time quantitative qPCR method for detecting residual DNA from Sf9 cells. This method features good reproducibility, high sensitivity, and strong specificity, enabling stable, accurate, and rapid detection of residual Sf9 cell DNA in samples. The technical solution adopted in this invention includes the following aspects.

[0008] In a first aspect, the present invention provides a primer pair for detecting residual DNA in Sf9 host cells, wherein the upstream primer binds to positions 468-485 of the Sf9 gene sequence shown in SEQ ID NO:1, and the downstream primer binds to positions 552-573 of the Sf9 gene sequence shown in SEQ ID NO:1, and the amplification product of the primer pair is 96-112 bp in length. Preferably, the amplification product is 106 bp in length. In some embodiments, the primers of the primer pair for detecting residual DNA in Sf9 host cells provided by the present invention are 18-22 bp in length. Preferably, the upstream primer is 18 bp in length, and the downstream primer is 22 bp in length. In a preferred embodiment, the primer pair used in the present invention has an upstream primer of 5'-AACCGACTCACACTTGGC-3' (SEQ ID NO:4) and a downstream primer of 5'-CCTAGACCACTTCCAATAGTCG-3' (SEQ ID NO:5).

[0009] Secondly, this invention provides a probe for detecting residual DNA in Sf9 host cells using the TaqMan probe method. Those skilled in the art, knowing the primer pairs, can design probes based on the template sequence between the binding sites of the upstream and downstream primers, and test the technical effectiveness of the probe and primer pairs. In some embodiments, the probe provided by this invention binds to positions 486-552 of the Sf9 gene sequence shown in SEQ ID NO:1. Preferably, this invention uses the following probe sequence: 5'-ACCTTGACCTAAAGACCTACCTCCATGC-3' (SEQ ID NO: 10). In some embodiments, the 5' end of the probe of this invention is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group. In some embodiments, the fluorescent reporter group used in the probe of this invention is selected from any one of FAM, JOE, HEX, or VIC, and the fluorescent quencher group is selected from any one of TAMRA, Eclipse, BHQ, or MGB. Preferably, the fluorescent reporter group used in the probe of this invention is FAM, and the fluorescent quencher group is TAMRA.

[0010] Thirdly, the present invention provides a reagent for detecting residual DNA in Sf9 host cells, the reagent comprising the primer pair described in the present invention. In some embodiments, the reagent for detecting residual DNA in Sf9 host cells provided by the present invention further comprises the probe described in the present invention. In some embodiments, the reagent for detecting residual DNA in Sf9 host cells provided by the present invention is used in a real-time quantitative PCR reaction. In some embodiments, the reagent for detecting residual DNA in Sf9 host cells provided by the present invention further comprises reagents for real-time quantitative PCR, such as conventional reagents like DNA templates and polymerases.

[0011] A fourth aspect of this invention is to provide a method for detecting residual DNA in Sf9 host cells, comprising detecting the sample to be tested using the reagents described in this invention. Preferably, the method for detecting residual DNA in Sf9 host cells provided by this invention is the TaqMan probe method. In some embodiments, the detection method of this invention includes the following steps: 1) processing the sample to be tested and extracting DNA as a template; 2) preparing a reaction system, wherein the reaction system includes: template, upstream primer, downstream primer, probe, and polymerase, etc.; 3) performing real-time quantitative PCR (q-PCR); 4) determining the results. In the detection method of this invention, the qPCR reaction conditions can be determined by those skilled in the art through routine experiments. In some embodiments, this invention uses the reaction procedure recommended in commercially available qPCR kits. In some embodiments, the qPCR reaction conditions of this invention are: Step 1: 95℃ for 30s-1h; Step 2: 95℃ for 5-15s, 60℃ for 30-60s, 40 cycles. In some embodiments, the detection method of the present invention further includes a step of establishing a standard curve, which includes: diluting a positive quantitative standard (insect cell Sf9 residue detection standard) in a 10-fold serial gradient, using it as a template for real-time quantitative PCR detection to establish a standard curve, and calculating the amount of Sf9 cell DNA in the sample to be tested based on the obtained standard curve. In some embodiments, the sample to be tested used in the present invention includes Sf9 cell genomic DNA of known concentration and biological products produced using Sf9 cells, such as recombinant proteins, vaccines, antibodies, and gene therapy vectors. In some embodiments, the sample DNA of the sample to be tested in the present invention is extracted using the method of patent application publication number CN120665857A. Preferably, the optimal lysis binding buffer composition and final concentration determined in Example 6 are used for sample DNA extraction.

[0012] A fifth aspect of the present invention is to provide a kit for detecting residual Sf9 host cell DNA, comprising the reagent for detecting residual Sf9 host cell DNA described herein. In some embodiments, the kit provided by the present invention further comprises conventional qPCR detection reagents. A sixth aspect of the present invention is to provide the use of the detection reagent for residual Sf9 host cell DNA described herein, including detecting the residual amount of Sf9 host cell DNA in biological products. In some embodiments, the biological products include recombinant proteins, vaccines, antibodies, and gene therapy vectors, etc. In some embodiments, the biological products include all sample types in the production process, such as intermediate products and final products. In some embodiments, the biological products include all sample types in the various processes of recombinant protein fermentation, purification, concentration, formulation, and bottling, such as fermentation filtrate, centrifugation supernatant, chromatography buffer, washing buffer, and eluent during purification. In some embodiments, the biological product is an influenza subunit vaccine.

[0013] The specific primers, probes, and qPCR detection method for detecting residual DNA in Sf9 cells provided by this invention have the following significant advantages: 1) High specificity and strong anti-interference ability: By screening high-copy gene regions specific to Sf9 cells through whole-genome alignment, the detection targets are fundamentally avoided from homology cross-reaction with mammalian cell (such as CHO, Vero), prokaryotic cell (such as E. coli) and recombinant baculovirus (AcNPV) DNA. Experiments have confirmed that even in recombinant baculovirus samples containing different exogenous genes, the primer pairs of this invention can accurately and specifically detect Sf9 host DNA, completely solving the problem of false positives or false negatives caused by homologous sequence competition; 2) High sensitivity and wide linear range: The detection limit of this method can reach 0.0006 pg / μL, the quantitative linear range is wide (0.003 pg / μL–300 pg / μL), and the amplification efficiency is high, which can meet the accurate quantification requirements of extremely low residual DNA in biological products, especially suitable for monitoring trace residues in the later stage of purification process and product release inspection; 3) Good repeatability and robustness: In the repeatability test of samples with different concentrations, the relative standard deviation (RSD) is less than 20%; intermediate precision verification shows that different individuals The RSD of different operations at different times is less than 20%, indicating that the method has good reproducibility and high robustness, and is suitable for stable application under different experimental conditions; 4) Simple operation and strong instrument compatibility: Developed based on the TaqMan probe single qPCR method, it can be run on most economical qPCR instruments, which significantly reduces the equipment threshold and usage cost, and is conducive to widespread promotion; 5) Validated by actual production samples, with strong practicality: The present invention was tested on actual production samples of various subunit vaccines (such as cell harvesting fluid and chromatography collection fluid), and the spiked recovery rate was stable between 86.6% and 113.0%, indicating that the entire detection method system is accurate and reliable, and is suitable for quality control of the entire production process of Sf9 insect cell-baculovirus expression system bioproducts.

[0014] In summary, this invention establishes a highly specific, sensitive, reproducible, and easy-to-operate method for detecting Sf9 residual DNA, effectively solving the problems of cross-reactivity, insufficient sensitivity, and lack of standardization in existing technologies, and providing key technical support for the safe production and compliant release of such biological products. Attached Figure Description

[0015] Figure 1 The standard curve of combination 1 in Example 1.

[0016] Figure 2 : Specificity result diagram of Example 2. Detailed Implementation

[0017] Experimental methods not specified with particular conditions or parameters in the examples are conventional methods and conditions well known in the field, or are performed according to the manufacturer's recommended conditions or parameters; all chemical reagents used in the examples are commercially available, and all primers used are outsourced for synthesis.

[0018] Example 1 Primer Design and Screening

[0019] 1.1 Primer design: The gene sequence shown in SEQ ID NO:1 was obtained through whole-genome alignment and whole-genome analysis. Primers and probes were designed for SEQ ID NO:1 using software such as NCBI and Primer Express 3, resulting in the following primer pairs.

[0020] Table 1 Candidate primer pairs

[0021]

[0022] 1.2 Primer Screening Experiment: The amplification specificity, amplification efficiency, detection sensitivity, and background signal of candidate primers at different annealing temperatures were systematically evaluated using the SYBR Green method. Primer pairs capable of achieving efficient, specific, and stable amplification were screened, and their optimal annealing temperatures were preliminarily determined. Three annealing temperature gradients (60℃, 62℃, 64℃) were set for each primer pair to evaluate the effect of temperature on reaction specificity. Under each temperature condition, a known concentration of target template (Sf9 cell genomic DNA) was used for sequential 10-fold serial dilutions (300 pg / μL, 30 pg / μL, 3 pg / μL) to construct standard curves and calculate amplification efficiency and linear range. Template-free controls (NTCs) were included in all reactions to monitor primer dimer formation and reagent contamination. By analyzing the amplification curves, melting curves, and standard curve parameters under each condition, the optimal primer pairs and their reaction conditions were preliminarily screened through comprehensive comparison.

[0023] 1.2.1 Materials and Methods: The template was an insect cell Sf9 DNA residue detection standard (purchased from the China National Institutes for Food and Drug Control). The main reagent was TB Green® Premix Ex Taq™ II (Tli RNaseH Plus). The PCR reaction program was 95℃, 30s; 95℃, 5s, 60℃ / 62℃ / 64℃, 34s ​​(fluorescence acquisition), 40 cycles. The melting curve program was 95℃, 15s; 60℃, 1min; 95℃, 1s (image acquisition). The PCR reaction was then initiated.

[0024] Table 2. Preparation of qPCR Mix (single reaction system)

[0025]

[0026] 1.2.2 Results and Conclusions: The L1 primer pair produced significant non-specific amplification products at both 60℃ and 62℃; however, no amplification was observed at 64℃. This indicates a design flaw in the L1 primer pair, making it impossible to obtain highly specific amplification results through conventional temperature adjustments. The L2 primer pair exhibited a single melting peak at all tested concentrations, and no amplification signal was detected in the template-free control (NTC). Its amplification efficiency and linearity met the requirements for quantitative detection, demonstrating good specificity and reliability. The L3 primer pair produced a single melting peak in the sample at both 60℃ and 62℃, but non-specific amplification of NTC was observed. At 64℃, the melting peak of the NTC amplification product highly overlapped with the melting peak of the target product in the sample, making effective differentiation difficult; therefore, it was unsuitable for establishing a reliable quantitative detection method. The L4 primer pair demonstrated good specificity and amplification efficiency in sample detection. However, at NTC temperatures of 62℃ and 64℃, at least one amplification signal appeared at the same position as the sample melting peak, indicating a risk of generating non-specific background indistinguishable from the target product. Compared to the L3 primer pair, the NTC signal of the L4 primer pair partially showed distinguishable peaks in repeated experiments, suggesting that this phenomenon is somewhat unstable. Based on a systematic comparison of the amplification specificity, efficiency, and background signal of each candidate primer at the preset temperature gradient, the L2 and L4 primer pairs were initially selected for subsequent methodological development.

[0027] Table 3 Amplification results of candidate primer pairs

[0028]

[0029] 1.3 Development and Optimization of TaqMan Probe Method

[0030] 1.3.1 Probe Design: Based on the amplification sequences of the L2 and L4 primer pairs determined in 1.2, and following the design principles of the TaqMan probe method, corresponding specific fluorescent probes were designed and synthesized in the regions between the two primer pairs. The system using the L2 primer pair was designated as combination 1, and the system using the L4 primer pair was designated as combination 2. Combination 3 was designed with reference to the primers and probes of system 2 in patent application CN115851969A as a control system. Specific sequences are shown in Table 4.

[0031] Table 4 Primer and probe sequences

[0032]

[0033] 1.3.2 Validation Experiment: Sf9 DNA residue detection standards were used, and a series of gradient dilutions were prepared as templates for the standard curve to ensure the traceability of the detection system. Three primer-probe combinations (combination 1, combination 2, and combination 3) were used to perform TaqMan qPCR detection on purified actual process samples and low-concentration standards. By comparing the detection concentration and spiked recovery rate of each combination, the quantitative accuracy and stability in complex matrices were evaluated, and primer-probe combinations suitable for subsequent optimization were preliminarily screened. Regarding materials and reagents, the main reagents were commercially available qPCR Mixes from brands A and B, and the Sf9 cell DNA residue detection standard (batch number: 220029-2019). Sample information is shown in Table 5. The method steps are as follows: (1) Preparation of standard solutions: First, dilute the Sf9 cell DNA residue detection standard with TE buffer to 3000 pg / μL, and then dilute it with TE buffer to form a standard curve gradient concentration of 300 pg / μL, 30 pg / μL, 3 pg / μL, 0.3 pg / μL, and 0.03 pg / μL; (2) Samples are extracted and purified according to the method disclosed in patent application CN120665857A (especially the optimal lysis buffer formulation determined in Example 6); (3) PCR detection: Preparation of qPCR Mix (single reaction system): Combination 1 and Combination 2 use qPCR Mix of brand A, and Combination 3 uses qPCR Mix of brand B, as shown in Table 6; (4) Reaction procedure: Perform the procedure recommended in the respective instructions, as shown in Table 7. Based on the screening results of primer-probe combinations (see Table 8), the analysis concludes that combinations 1, 2, and 3 all exhibited acceptable quantitative accuracy in terms of spiked recovery (recovery rates for all samples were within the acceptable range of 50%–150%), indicating their basic applicability in probe-based detection systems. However, combinations 1 and 2 generally showed higher detection values ​​in samples than combination 3, with more stable recovery rates and better repeatability, demonstrating superior reliability and resistance to matrix interference in practical process systems. In contrast, combination 3 showed larger fluctuations in recovery rate in some samples, indicating relatively weaker adaptability to complex matrices. Combinations 1 and 2 outperformed combination 3 in terms of recovery rate control, result stability, and resistance to interference, exhibiting superior detection reliability.

[0034] Table 5 Sample Information

[0035]

[0036] Table 6. Preparation of qPCR Mix (single reaction system)

[0037]

[0038] Table 7 qPCR reaction procedure.

[0039]

[0040] Table 8. Screening and detection results of primer-probe combinations

[0041]

[0042] 1.4 Confirmation of Primer and Probe Combinations: Primer pairs and probes from combinations 1 and 2 were used to detect DNA from appropriately diluted cell supernatant samples expressing HA antigens of B / victoria, H3N2, and H1N1 (referred to as BV, AH3, and AH1), purified using the method described in patent application (CN120665857A). A standard procedure was employed, as shown in Table 7 (Brand A), to screen for the optimal primer pairs and probes. The results are shown in Table 9. Combination 1 showed significantly higher detection values ​​than Combination 2 in all samples, and its recovery rate remained within a reasonable range (79.8%-113%). Combination 2, however, showed a significant deviation in recovery rate in some samples (e.g., 162% in AH3 sample), exceeding the acceptable range of 50%-150%. In summary, Combination 1 demonstrated superior sensitivity and accuracy, and was therefore selected as the final primer pair and probe.

[0043] Table 9. Screening and detection results of primer-probe combinations

[0044]

[0045] 1.5 Determination of Standard Curve Linearity Range: Using insect cell Sf9 DNA residue detection standards as templates, six graded standard solutions with concentrations ranging from 300 pg / μL to 0.003 pg / μL were prepared by serially diluting the standards 10-fold with TE buffer. The primer pairs from combination 1 were used with the probes and reaction system for the reaction. Results showed that within the concentration range of 300 pg / μL to 0.003 pg / μL, the standard curve exhibited good linearity, with a coefficient of determination R² = 1, an amplification efficiency of 98.8%, a slope of -3.35, and relative standard deviations (RSDs) between replicates less than 20%. The 300 pg / μL to 0.003 pg / μL range met the criteria for linearity in quantitative detection; therefore, the standard curve range was determined to be 300 pg / μL to 0.003 pg / μL. (See standard curve example 1). Figure 1 .

[0046] In summary, the TaqMan probe method was confirmed for the detection of residual DNA in Sf9 host cells. The primer and probe combination used was as follows: upstream primer was 5'-AACCGACTCACACTTGGC-3'; downstream primer was 5'-CCTAGACCACTTCCAATAGTCG-3'; and probe was 5'FAM-ACCTTGACCTAAAGACCTACCTCCATGC-TAMRA3'.

[0047] Example 2 Methodological Validation

[0048] 2.1 Linearity and Range: The insect cell SF9 residue detection standard was serially diluted 10-fold using TE buffer to obtain six concentration gradients from 300 pg / μL to 0.003 pg / μL. The method of this invention was used to detect each concentration of the standard, with each concentration repeated three times. A standard curve was plotted with Ct value on the ordinate and the logarithm of the standard concentration (Log value) on the abscissa. The results showed that within the concentration range of 300 pg / μL to 0.003 pg / μL, the standard curve exhibited good linearity, with a coefficient of determination R² = 1 (acceptable standard: R² > 0.99), an amplification efficiency of 99.7% (acceptable standard: 90%-110%), a slope of -3.33 (acceptable standard: -3.1 to -3.8), and relative standard deviations (RSD) between replicates and relative deviations (CV) between measured and theoretical values ​​were both less than 20% (acceptable standard: ≤20%). The linearity and range of the quantitative detection method of the present invention are from 300 pg / μL to 0.003 pg / μL.

[0049] Table 10 Results of Linearity and Range Measurements

[0050]

[0051] 2.2 Specificity: Using the method of this invention, genomic DNA from 300 pg / μL CHO cells, 300 pg / μL E. coli cells, 300 pg / μL Vero cells, negative control (H2O), and positive control 300 pg / μL Sf9 cells (genomic DNA was extracted from expanded cultured Sf9 cells using an Omega HP Tissue DNA Midi Kit, and the concentration and purity were determined using a NanoDrop2000, the same below) were amplified, and the amplification curves were compared. Figure 2 The results showed that only Sf9 cell genomic DNA showed specific amplification, while CHO, E. coli, Vero cell genomic DNA and H2O showed no amplification signal, indicating that the method of the present invention has good specificity.

[0052] 2.3 Accuracy. Sf9 genomic DNA samples at concentrations of 150 pg / μL, 15 pg / μL, and 0.15 pg / μL were selected and three replicate tests were performed to evaluate the accuracy of the method. The results showed (Table 11) that the relative deviations of the detected values ​​from the theoretical values ​​at each concentration level were all <20%, and the RSDs of the replicate tests were also <20%, indicating that the method has good accuracy and repeatability.

[0053] Table 11 Accuracy Measurement Results

[0054]

[0055] 2.4 Reproducibility: Sf9 genomic DNA samples at concentrations of 100 pg / μL, 10 pg / μL, and 1 pg / μL were subjected to six replicate assays to evaluate the reproducibility of the method. The results (Table 12) showed that the RSD of the six replicate assays at each concentration level was <20%, indicating that the method has good reproducibility.

[0056] Table 12 Repeatability Test Results

[0057]

[0058] 2.5 Intermediate Precision: Sf9 genomic DNA samples at concentrations of 100 pg / μL, 10 pg / μL, and 1 pg / μL were selected and measured six times by different personnel at different time points to evaluate the intermediate precision of the method. The results (Table 13) showed that the coefficient of variation (CV) of the detection results for each concentration sample under different personnel and time conditions was less than 20%, indicating that the method has good intermediate precision.

[0059] Table 13 Results of intermediate precision determination

[0060]

[0061] 2.6 Limit of Quantification: Multiple analyses were performed on 3 fg / μL and 6 fg / μL Sf9 genomic DNA samples. The CV value of 15 replicate wells for concentrations of 3 fg / μL and above was <20%, and the recoveries were all in the range of 50% to 150%, indicating that the limit of quantification of the method of this invention can reach 3 fg / μL.

[0062] Table 14 Results of Limit of Quantitation Determination

[0063]

[0064] 2.7 Limit of Detection: Multiple tests were performed on 0.3 fg / μL and 0.6 fg / μL Sf9 genomic DNA samples. One out of 18 results for the 0.3 fg / μL sample was not detected, resulting in a detection rate of 94%; the detection rate for the 0.6 fg / μL sample was 100%.

[0065] Table 15 Results of Detection Limit Determination

[0066]

[0067] 2.8 Effect of DNA Fragmentation: After ultrasonic disruption for 20 min, the Sf9 genomic DNA sample was diluted to obtain five concentration gradients (300 pg / μL-0.03 pg / μL). The recovery rate was then analyzed using the method of this invention, and the recovery rate was ≥70% for all samples. This indicates that the present invention can effectively detect fragmented Sf9 DNA, and the degree of fragmentation of Sf9 DNA will not affect the detection results.

[0068] Table 16 Results of Sf9 genomic DNA assay after ultrasound treatment

[0069]

[0070] Example 3: Comparative Experiment

[0071] 3.1 Comparison with the method of patent CN114075594B: The comparison scheme is shown in Table 17. Experimental group 1-1 performed qPCR detection on the test samples according to the method determined in Example 1; Experimental group 1-2 performed qPCR detection on the test samples according to its patented method. The comparison results are shown in Table 18. Based on the same pretreated samples, the detection values ​​of the qPCR method of this invention (1-1) in both samples were significantly higher than those of the patented method (1-2) of CN114075594B, and the spiked recovery rate was close to 100%, also superior to the method of patent CN114075594B. This indicates that the sensitivity of the method of this invention is superior to that of the patented method of CN114075594B.

[0072] Table 17 Comparison Schemes

[0073]

[0074] Table 18 Comparison Results

[0075]

[0076] 3.2 Comparison with Commercial Kits: Information on commercial kits is shown in Table 19. The experimental objectives and procedures were: 1) to evaluate the extraction recovery rate of the pretreatment method of this invention and to compare the extraction efficiency of different commercial pretreatment kits (Comparative Experiment 1 is shown in Table 20); 2) to determine the background value and directly compare the anti-interference, accuracy, and specificity of different detection methods (Comparative Experiment 2 is shown in Table 21); 3) to verify the performance of commercial kits in their own system (Comparative Experiment 3 is shown in Table 22). Extraction and purification were performed according to the instructions of each kit; qPCR detection was performed on the purified samples according to the instructions of each kit; the method of this invention was used to perform qPCR detection on the samples according to the above method. The comparison results are shown in Tables 23, 24, and 25.

[0077] Horizontal Comparative Analysis: Table 23 analyzes (evaluating pretreatment efficiency, with the qPCR method fixed as the method of this invention). The qPCR method of this invention can effectively detect Sf9 DNA in samples extracted by different pretreatment methods. In high-concentration baculovirus-free samples, the pretreatment method disclosed in patent CN120665857A shows the highest detection value and the best recovery rate. The detection values ​​of brands B and D are on the same order of magnitude, but the recovery rate of brand B is relatively low. The detection value obtained by pretreatment of brand C is the lowest. In samples containing trace amounts of baculovirus (BV ultrafiltration concentrate), the recovery rates of all pretreatment methods are close to 100%, indicating that baculovirus DNA does not significantly interfere with Sf9 quantification; the difference mainly stems from DNA extraction efficiency. In summary, the pretreatment method disclosed in patent CN120665857A shows stable and high DNA extraction efficiency in various samples, while the qPCR method of this invention has good detection sensitivity and anti-interference ability in different sample matrices. Table 24 analyzes (evaluating qPCR detection performance, with the pretreatment method as disclosed in CN120665857A). Based on the same pretreatment samples, the qPCR method of this invention (2-1) exhibits the highest detection value and stable recovery rate in both types of samples. In contrast, the three commercial qPCR kits show significant deviations: Brand B (2-5) shows significantly lower detection values ​​and insufficient sensitivity; Brand C (2-6) has an abnormally high recovery rate, indicating a bias in its quantification system; Brand D (2-7) has a recovery rate of 63.0% in baculovirus-containing samples, indicating that its detection process is susceptible to interference from the baculovirus genome. Table 25 analyzes (performance of the commercial kits' own systems), showing inconsistent performance of each commercial kit in its supporting system (pretreatment + detection + premixed standards). The detection values ​​of Brand B system (2-8) were relatively low, but the recovery rate was relatively controllable. Brand C (2-9) showed a recovery rate of only 10.2% in samples containing baculoviruses, indicating significant limitations in complex samples. Brand D system (2-10) had relatively good detection values ​​and recovery rates, but the recovery rate in samples containing baculoviruses (80.4%) was still below the ideal level. The detection values ​​of all commercial kits were significantly lower than those of the method of this invention (2-1).

[0078] Longitudinal comparative analysis: Brand B (experimental groups 2-2, 2-5, 2-8) showed systematically low detection values ​​under different configurations. Its pretreatment efficiency was acceptable (2-2), but its qPCR detection component lacked sensitivity; the detection values ​​were significantly lower than those of the method described in this invention, both in the pretreatment of this invention (2-5) and in its own system (2-8). Brand C (experimental groups 2-3, 2-6, 2-9) had the lowest pretreatment efficiency among all methods (2-3). Its qPCR detection component showed deviations in the pretreated samples of this invention (2-6), and the recovery rate of baculovirus-containing samples in its own system was only 10.2% (2-9). The detection values ​​of background samples were significantly lower than those of the method described in this invention, indicating that its detection system has significant limitations in complex samples, possibly due to inhibition by baculovirus DNA, and that the pretreatment step is inefficient when extracting complex samples. This kit did not demonstrate reliable quantitative ability in either simple or complex matrices, and its detection sensitivity or quantitative benchmark deviated significantly from the true values. Brand D (experimental groups 2-4, 2-7, 2-10) showed good processing efficiency (2-4). Its qPCR detection performed reasonably well in simple samples, but the recovery rate in samples containing baculovirus was only 63.0% (2-7), indicating weak anti-interference ability. In its own system (2-10), the recovery rate improved, presumably because its premixed standards contained AcNPV DNA background, which calibrated the detection system and partially masked the interference. The kit achieved acceptable recovery in samples containing only Sf9 DNA, but the background sample detection value (3763.8 pg / μl) was significantly lower than that of the method of this invention (12766.2 pg / μl), suggesting low sensitivity for real samples. In samples containing baculovirus, detection was significantly interfered with, indicating insufficient ability to distinguish homologous sequences and insufficient accuracy in complex matrices.

[0079] Based on the results of the above comparative experiments, it can be seen that the qPCR method of the present invention is superior to the compared commercial kits in terms of specificity, accuracy and anti-interference ability.

[0080] Table 19 Information on Commercial Reagent Kits

[0081]

[0082] Table 20 Comparative Experiment 1

[0083]

[0084] Table 21 Comparative Experiment 2

[0085]

[0086] Table 22 Comparative Experiment 3

[0087]

[0088]

[0089] Example 4: Practical Testing Application

[0090] 4.1 Detection of Samples Containing Different Recombinant Baculoviruses: DNA extraction and purification were performed on the supernatant of recombinant influenza vaccine cells containing different exogenous gene sequences from the Sf9 insect cell-baculovirus expression system using the method disclosed in patent CN120665857A. The residual Sf9 DNA was then determined using the method of this invention. This invention's method was tested on three types of recombinant influenza vaccine cell harvest supernatant samples. Experimental results showed that all samples exhibited good linearity at different dilutions, with spiked recoveries ranging from 86.6% to 105.6%, meeting the validation requirements (50%-150%) for residual DNA detection in biological products. These results demonstrate that the method of this invention maintains good specificity and accuracy in different recombinant baculovirus application scenarios. The high spiked recoveries indicate that the method is not interfered with by baculovirus DNA; the detection stability at different dilutions further demonstrates its anti-interference ability and reliability. In summary, the Sf9 cell-specific target gene regions screened in this invention have no homology with the baculovirus genome and its common exogenous insertion sequences, which can effectively avoid false positive results.

[0091] Table 26 Results of Sf9 Residual DNA Content Determination in Test Samples

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[0093] 4.2. Detection of intermediate purification samples of recombinant influenza vaccine: The purification DNA of intermediate purification samples of recombinant BV influenza vaccine expressed by the Sf9 insect cell-baculovirus expression system was extracted using the method disclosed in patent CN120665857A. The residual Sf9 DNA was then determined using the method of this invention. Experimental results show that the method of this invention can clearly reflect the gradual decreasing trend of residual DNA during purification and accurately assess the DNA removal efficiency of different purification steps. The method of this invention exhibits good accuracy, precision, and high sensitivity for test samples of different types and purification stages, and is suitable for the full-process monitoring of residual Sf9 host cell DNA.

[0094] Table 27 Results of Sf9 Residual DNA Content Determination in Test Samples

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[0096] In summary, the above embodiments and accompanying drawings are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A reagent for detecting residual DNA in Sf9 host cells, said reagent comprising the following primer pairs: Upstream primer: 5'-AACCGACTCACACTTGGC-3' (SEQ ID NO: 4); Downstream primer: 5'-CCTAGACCACTTCCAATAGTCG-3' (SEQ ID NO: 5).

2. The reagent according to claim 1, characterized in that, The reagent further comprises a probe, the sequence of which is: 5'-ACCTTGACCTAAAGACCTACCTCCATGC-3' (SEQ ID NO: 10).

3. The reagent according to claim 2, characterized in that, The probe is labeled with a fluorescent reporter group at its 5' end and with a fluorescent quencher group at its 3' end.

4. The reagent according to claim 3, characterized in that, The fluorescent reporter group is FAM, and the fluorescent quencher group is TAMRA.

5. A kit for detecting residual DNA in Sf9 host cells, characterized in that, The kit comprises the reagent according to any one of claims 1-4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes reagents for real-time quantitative PCR.

7. A method for detecting residual DNA in Sf9 host cells, characterized in that, The method is real-time quantitative PCR, which includes performing the reaction using the reagents of any one of claims 1-4 or the kits of any one of claims 5-6.

8. The method according to claim 7, characterized in that, The reaction procedure for the real-time quantitative PCR is as follows: Step 1: 95℃ for 30 seconds to 1 hour; Step 2: 95℃ for 5-15s, 60℃ for 30-60s, 40 cycles.

9. The use of the reagent according to any one of claims 1-4 or the kit according to any one of claims 5-6, characterized in that, The stated application is for the detection of residual Sf9 host cell DNA in biological products.

10. The use according to claim 9, characterized in that, The biological products mentioned include recombinant proteins, vaccines, antibodies, and gene therapy vectors.

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