Digital PCR (polymerase chain reaction)-based method for detecting residual DNA (deoxyribonucleic acid) of multiple host cells

Abstract

The invention belongs to the technical field of gene detection of cross fusion of biology and a new generation of information technology, and particularly relates to a method for detecting residual DNA of multiple host cells based on digital PCR (Polymerase Chain Reaction). The invention discloses a multiplex digital PCR quantitative detection primer pair and a probe group for detecting residual DNA of CHO cells, Vero cells, pichia pastoris, escherichia coli and NS0 cells for the first time. By utilizing the method, not only can the residual DNA of five host cells be simultaneously subjected to single or multiple detection in a single reaction, but also non-specific amplification, signal interference and detection limitation of the residual DNA of the host cells in the qPCR detection process can be solved friendly, and meanwhile, the original concentration of a sample is absolutely quantified by the method; the method does not depend on the establishment of a standard curve, reduces the influence caused by a standard product, and is superior to the prior art.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection technology that crosses biology and new generation information technology, and particularly relates to a method for detecting multiple host cell residual DNA based on digital PCR. Background Art

[0002] In the field of biopharmaceuticals, CHO cells, Vero cells, Pichia pastoris (P. pastoris), Escherichia coli (E. coli) and NS0 cells are widely used as host cells for the production of various vaccines, therapeutic proteins, cell products, gene drugs, etc. because of their rapid growth, easy operation and culture, high adaptability to the production environment and compatibility with biomanufacturing processes.

[0003] However, during the production and processing of host cells, insufficient process optimization may result in residual host cell DNA in intermediates or final products. Residual host cell DNA not only reduces the purity and quality of the product, but also poses potential biosafety risks such as immunogenicity or carcinogenicity. Therefore, from a safety perspective, domestic and international regulatory agencies have established limit standards for residual DNA. For example, the World Health Organization (WHO) and the European Union stipulate that the acceptable amount of residual host cell DNA is no more than 10 ng / dose. The limit standard of the 2025 edition (Part III) of the Chinese Pharmacopoeia is also mostly less than 10 ng / dose in each monograph. The US Food and Drug Administration (FDA) stipulates that the residual host cell DNA content of each biological product shall not exceed 100 pg. Therefore, strictly controlling the residual host cell DNA content in biological products is an important indicator for ensuring the quality and safety of biological products.

[0004] Regarding the detection methods for residual exogenous DNA, the 2025 edition of the Chinese Pharmacopoeia includes DNA probe hybridization method, fluorescent staining method and quantitative PCR method (qPCR). Each method has its own characteristics. The most widely used method at present is qPCR method, which is mainly because this method is a nucleic acid detection technology with strong specificity, wide quantitative range and real-time quantification. However, although qPCR technology has obvious advantages, it still has certain limitations in dealing with complex samples, detection accuracy and detection sensitivity, and is easily affected by standard products (including but not limited to degradation failure caused by improper transportation or storage of standard products). In addition, when dealing with samples with multiple biological components or biological product samples containing interfering substances, qPCR technology may encounter problems such as nonspecific amplification, signal interference and detection limitations.

[0005] Digital PCR (dPCR) is a new generation of absolute quantitative PCR technology for nucleic acid molecules that has been developed in recent years. This technology evenly distributes the reaction solution into tens of thousands of independent nanoscale droplets using an oil-in-water method. PCR amplification reactions are performed in single-molecule units, and the "1" or "0" endpoint signal of each unit is counted. The target concentration is then calculated using a Poisson distribution. This technology achieves absolute quantification of nucleic acid molecules without relying on the construction of a standard curve. dPCR technology surpasses qPCR in terms of interference resistance, detection rate, sensitivity, accuracy, and efficiency.

[0006] CHO cells, Vero cells, Pichia pastoris (P. pastoris), Escherichia coli (E. coli), and NS0 cells are all widely used host cells in the biopharmaceutical field. Currently, there are no reports on dPCR detection methods for simultaneously detecting residual DNA in these host cells. Summary of the Invention

[0007] To overcome the problems of the prior art, the present invention proposes a method for detecting multiple residual host cell DNA based on digital PCR. This method enables simultaneous single or multiplex detection of multiple common residual host cell DNA in a single reaction, accommodating complex samples, saving manpower, material resources, and production costs, and improving the efficiency of residual host cell DNA detection.

[0008] The object of the present invention is achieved like this:

[0009] In a first aspect, the present invention provides a multiplex digital PCR detection product comprising primer pairs for detecting residual DNA in multiple host cells, including CHO cells, Vero cells, Pichia pastoris, Escherichia coli, and NSO cells, wherein the primer pairs are composed of the following:

[0010] A primer pair for detecting residual DNA in CHO cells, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0011] A primer pair for detecting residual Vero cell DNA, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0012] A primer pair for detecting residual Pichia pastoris DNA, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0013] A primer pair for detecting residual Escherichia coli DNA, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO: 7 and SEQ ID NO: 8;

[0014] The sequences of the upstream and downstream primers of the primer pair used to detect residual DNA in NS0 cells are shown in SEQ ID NO: 9 and SEQ ID NO: 10.

[0015] Furthermore, the detection product also includes the following probe group:

[0016] The sequence of the probe for detecting residual DNA in CHO cells is shown in SEQ ID NO: 11;

[0017] The sequence of the probe for detecting residual DNA in Vero cells is shown in SEQ ID NO: 12;

[0018] The sequence of the probe for detecting residual Pichia pastoris DNA is shown in SEQ ID NO: 13;

[0019] The sequence of the probe for detecting residual DNA of Escherichia coli is shown in SEQ ID NO: 14;

[0020] The sequence of the probe for detecting residual DNA in NS0 cells is shown in SEQ ID NO:15.

[0021] Furthermore, each probe sequence has different fluorescent labels at both ends;

[0022] The fluorescent markers include one or more of FAM, VIC, CY5, CY5.5, HEX, and JOE. Preferably, the fluorescent reporter group connected to the 5' end can be selected from any one or a combination of FAM, VIC, CY5, CY5.5, HEX, and JOE, preferably a fluorescent reporter group in the fluorescent signal channel configured for digital PCR; the fluorescent quencher group connected to the 3' end is preferably MGB.

[0023] More preferably, the two ends of the CHO host cell residual DNA detection probe sequence are modified with 5'6-FAM / 5'CY5, 3'MGB respectively, the two ends of the Vero host cell residual DNA detection probe sequence are modified with 5'6-FAM / 5'VIC, 3'MGB respectively, the two ends of the Pichia pastoris host cell residual DNA detection probe sequence are modified with 5'VIC, 3'MGB respectively, the two ends of the Escherichia coli host cell residual DNA detection probe sequence are modified with 5'6-FAM, 3'MGB respectively, and the two ends of the NS0 host cell residual DNA detection probe sequence are modified with 5'CY5, 3'MGB respectively.

[0024] The second aspect of the present invention provides a multiplex host cell residual DNA detection kit, which comprises the multiplex digital PCR detection product as described in the first aspect.

[0025] The third aspect of the present invention provides a method for detecting multiple host cell residual DNA based on digital PCR, wherein the multiple digital PCR detection product described in the first aspect or the detection kit described in the second aspect is used to detect the sample to be detected, and the detection method comprises the following steps:

[0026] 1) Collect samples to be tested and extract DNA;

[0027] 2) using the DNA sample obtained in step 1) as a template and the primer pair and probe set described in the first aspect to prepare a PCR reaction mixture;

[0028] 3) adding the reaction mixture prepared in step 2) to a droplet generation chip, adding droplet generation oil to each well, and generating a droplet emulsion in each well via a droplet generator. After the droplet emulsion is formed, all of the prepared droplet emulsion is further transferred to corresponding wells of a 96-well PCR plate, and then heat-sealing the plate with a puncturable heat-sealing film using a PCR plate sealer;

[0029] 4) placing the 96 PCR plate sealed with the heat-sealing film obtained in step 3) into a PCR instrument for PCR amplification;

[0030] 5) The 96-well PCR plate that completed PCR amplification in step 4) is placed in a digital PCR droplet reader for data reading and result analysis. The absolute concentration of each sample is automatically reported by calculating the ratio of positive droplets to total droplets and combining it with a Poisson distribution.

[0031] Furthermore, in step 2), the PCR reaction system mixture is as follows:

[0032]

[0033] In the PCR reaction system mixture, the final concentration of the upstream primer or the downstream primer is 200nM to 2000nM, and the final concentration of the probe is 100nM to 1000nM.

[0034] Furthermore, in step 4), the reaction conditions of the PCR are:

[0035] ① Pre-denaturation at 95°C for 10 min, one cycle;

[0036] ② Denaturation at 95°C for 30 seconds, annealing and extension at 50-65°C, preferably at 60°C for 1 minute, for 30-50 cycles;

[0037] ③Inactivate the enzyme at 98°C for 10 min, one cycle;

[0038] The heating and cooling rate is 1-3℃ / sec.

[0039] The fourth aspect of the present invention provides a detection kit comprising the primer pair and probe group as described in the first aspect, for use in preparing a multiplex digital PCR detection product for detecting the residual amount of host cell DNA, characterized in that the host cells include CHO cells, Vero cells, Pichia pastoris, Escherichia coli, and NS0 cells.

[0040] Furthermore, the test samples include intermediates or products expressed by CHO cells, Vero cells, Pichia pastoris, Escherichia coli and NS0 cells.

[0041] Furthermore, the products include cell products, protein products, gene drugs, vaccines or antibodies.

[0042] The advantages and beneficial effects of the present invention are:

[0043] 1. Compared with the existing qPCR technology for detecting residual host cell DNA, the detection method of the present invention is based on digital PCR. The reaction solution is evenly distributed into tens of thousands of independent nanoscale droplets through oil-in-water, and the PCR amplification reaction is carried out in single-molecule units. The PCR amplification reactions in the single-molecule units do not interfere with each other. Therefore, it can handle complex component samples and samples with multiple detection requirements that are difficult for qPCR technology to handle. In addition, the detection method of the present invention does not rely on the construction of a standard curve to achieve absolute quantification of nucleic acid molecules. The method of the present invention is significantly superior to traditional detection technologies in terms of anti-interference, detection rate, sensitivity, accuracy and detection efficiency.

[0044] 2. The present invention can simultaneously perform single or multiplex detection of residual DNA from multiple host cells, including CHO cells, Vero cells, Pichia pastoris (P. pastoris), Escherichia coli (E. coli), and NS0 cells, in a single reaction. It can also solve the nonspecific amplification, signal interference, and detection limitations encountered by traditional detection methods during the detection process, saving manpower, material resources, and production costs, and improving the detection efficiency of residual host cell DNA.

[0045] 3. The detection method described in this invention offers strong specificity, high sensitivity, good reproducibility, high detection efficiency, and accurate experimental results. It is highly suitable for detecting residual host cell DNA in intermediates, semi-finished products, and finished products of biopharmaceutical products or biological products. This method provides a more objective assessment of the risk of residual host cell DNA and offers a new alternative and supplement to existing quality control tests for residual impurities in biological products. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be further described below with reference to the accompanying drawings and examples.

[0047] Figure 1 Figure 2 is the annealing temperature optimization result. A is the annealing temperature optimization result for CHO-FAM, B is the annealing temperature optimization result for CHO-CY5, C is the annealing temperature optimization result for Vero-FAM, D is the annealing temperature optimization result for Vero-VIC, E is the annealing temperature optimization result for P. pastoris-VIC, F is the annealing temperature optimization result for E. coli-FAM, and G is the annealing temperature optimization result for NS0-CY5. The annealing temperatures from left to right in each figure are 52°C, 54°C, 56°C, 58°C, 60°C, and 62°C.

[0048] Figure 2 Figure 2 is a graph showing primer / probe concentration optimization results; A is the CHO-FAM primer / probe concentration optimization result, B is the CHO-CY5 primer / probe concentration optimization result, C is the Vero-FAM primer / probe concentration optimization result, D is the Vero-VIC primer / probe concentration optimization result, E is the P. pastoris-VIC primer / probe concentration optimization result, F is the E. coli-FAM primer / probe concentration optimization result, and G is the NS0-CY5 primer / probe concentration optimization result; the primer / probe concentrations from left to right in each figure are 500nM / 125nM, 500nM / 250nM, 500nM / 400nM, 900nM / 125nM, 900nM / 250nM, and 900nM / 400nM;

[0049] Figure 3Figures 2 and 3 are the specificity verification results of different samples; among them, A is the specificity verification result of CHO-FAM, B is the specificity verification result of CHO-CY5, C is the specificity verification result of Vero-FAM, D is the specificity verification result of Vero-VIC, E is the specificity verification result of P. pastoris-VIC, F is the specificity verification result of E. coli-FAM, and G is the specificity verification result of NS0-CY5; the templates input from left to right in each figure are CHO cell DNA, Vero cell DNA, Pichia pastoris (P. pastoris) DNA, Escherichia coli (E. coli) DNA, NS0 cell DNA and NTC (no template control);

[0050] Figure 4 Figure 1 shows the results of digital PCR-based detection of multiple residual host cell DNA. The fluorescence of single-positive and double-positive droplets is clustered and classified in a two-dimensional plot, with the x-axis and y-axis representing the fluorescence amplitude of different channels, respectively. A is a two-dimensional cluster plot of FAM and VIC, showing single-positive targets in the FAM (blue) or VIC (green) channel, and double-positive targets (red) in both channels. B is a two-dimensional cluster plot of FAM and CY5, showing single-positive targets in the FAM (blue) or CY5 (pink) channel, and double-positive targets (red) in both channels. C is a two-dimensional cluster plot of VIC and CY5, showing single-positive targets in the VIC (green) or CY5 (pink) channel, and double-positive targets (red) in both channels. Negative droplets are displayed in gray. DETAILED DESCRIPTION

[0051] The present invention has no special restrictions on the synthesis method of the primers and probes, and can be commissioned to a gene synthesis company well known in the art. The sequences of primers, probes, etc. involved in the following embodiments of the present invention were all commissioned to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. When performing digital PCR amplification using the method described in the present invention, a droplet digital PCR reaction premix reagent is required. The present invention has no special restrictions on the source of the droplet digital PCR reaction premix reagent, and conventional commercially available products in this field can be used. The droplet digital PCR reaction premix reagent described in the embodiments of the present invention was purchased from Guangzhou Yongnuo Company. In the present invention, the reagents and consumables also include droplet generation oil, droplet detection oil, droplet generation chip, 96PCR plate and pierceable heat-sealed film. These reagents and consumables can be used according to the model of the digital PCR instrument. These reagents and consumables described in the embodiments of the present invention were purchased from Guangzhou Yongnuo Company.

[0052] The primers and probes described in the following examples of this application are shown in Table 1.

[0053] Table 1

[0054]

[0055]

[0056] A digital PCR-based method for detecting multiple residual host cell DNA. This method utilizes a digital PCR instrument to simultaneously detect the residual host cell DNA in intermediates or products expressed in CHO cells, Vero cells, Pichia pastoris, Escherichia coli, and NS0 cells. Specifically, the method includes the following steps:

[0057] 1) Collect samples to be tested and extract DNA.

[0058] 2) Prepare the PCR reaction system. The reference reaction system is as follows:

[0059]

[0060] In the digital PCR system, the final concentration of the upstream primer or the downstream primer is 200 nM-2000 nM, and the final concentration of the probe is 100 nM-1000 nM.

[0061] 3) Chip loading: 20 μl of the reaction mixture prepared in step 2) was added to the droplet generation chip, 50 μl of droplet generation oil was added to each well, and a droplet emulsion was generated in each well by a droplet generator. After the droplet emulsion was formed, all the prepared droplet emulsions were further transferred to the corresponding wells of a 96-well PCR plate, and then the plate was heat-sealed with punctureable heat-sealing film using a PCR plate sealer.

[0062] 4) The 96 PCR plate sealed with the heat-sealing film obtained in step 3) is placed in a PCR instrument for PCR amplification.

[0063] 5) Data reading and result analysis: PCR plates were read using a digital PCR droplet reader to measure fluorescence signals, and analysis was performed using the droplet reader software. The software automatically reports the absolute concentration of each sample by calculating the proportion of positive droplets to total droplets and applying a Poisson distribution.

[0064] Example 1: Optimization of Multiplex Digital PCR Annealing Temperature

[0065] To optimize the annealing temperature in this example, the final concentrations of each primer and probe set were fixed at 900 nM and 250 nM, respectively. The optimized annealing temperatures for each primer / probe set were set at 52°C, 54°C, 56°C, 58°C, 60°C, and 62°C. Digital PCR amplification was performed to detect the changes in the results under different annealing temperature reaction conditions to determine the appropriate annealing temperature range for each primer / probe set.

[0066] This example is to optimize the annealing temperature. The specific implementation method is as follows: each group of positive templates is diluted 10 times in series, and the final dilution is 1.0×10 4 copies·μL -1 As a detection template. The reaction system was prepared according to Table 2, with the final concentration of primers used being 900 nM and the final concentration of probes used being 250 nM. Amplification was performed using a droplet digital PCR reaction premix solution. The reaction procedure is shown in Table 3. Amplification was performed using Applied Biosystems TM PCR amplification was performed using a PCR instrument. After completion, the 96-well PCR plate was placed in a digital PCR droplet reader for data reading and analysis. QuantDrop-Pro4 software was used to automatically report the absolute concentration of each sample by calculating the proportion of positive droplets to the total droplets and applying a Poisson distribution. One-dimensional plots of the test results were also generated using QuantDrop-Pro4 software.

[0067] Table 2 dPCR amplification system

[0068] Components Volume (μL) Reaction premix 10 Primer F (10 μM) 1.8 Primer R (10 μM) 1.8 Probe P (10 μM) 0.5 <![CDATA[Template 1*10 4 (copies / μL)]]> 1 Deionized water 4.9 total 20

[0069] Table 3 dPCR reaction procedure

[0070]

[0071] The nucleotide sequences of the primers and probes are shown in Table 1.

[0072] The results show that Figure 1 As shown, each primer / probe set can tolerate a wide temperature range, with no significant difference in the separation between positive and negative droplets across the six annealing temperatures. However, the highest accuracy of copy number concentration was achieved at 58°C and 60°C for each set. Therefore, the final optimized annealing temperature for the droplet digital PCR protocol was 58°C or 60°C.

[0073] Example 2: Optimization of primer / probe concentrations for multiplex digital PCR

[0074] Based on the results of Example 1, the annealing temperature for each set was fixed at 58°C. In this example, to optimize primer / probe concentrations, the final concentrations of each primer and probe set were set to 500nM / 125nM, 500nM / 250nM, 500nM / 400nM, 900nM / 125nM, 900nM / 250nM, and 900nM / 400nM, respectively. Digital PCR amplification was performed to detect the results of each primer / probe set under different reaction conditions to determine the appropriate detection concentration for each primer / probe set.

[0075] This example is to carry out primer / probe optimization, and the specific implementation method is as follows:

[0076] Each group of positive templates was serially diluted 10-fold, and the final dilution was 1.0×10 4 copies·μL -1 As a detection template. The reaction system was prepared according to Table 4. The primer / probe concentrations in Table 4 dPCR amplification system were added as needed based on the optimized concentrations (500nM / 125nM, 500nM / 250nM, 500nM / 400nM, 900nM / 125nM, 900nM / 250nM and 900nM / 400nM). Amplification was performed using a droplet digital PCR reaction premix. The reaction procedure is shown in Table 5. Amplification was performed using Applied Biosystems. TM PCR amplification was performed using a PCR instrument. After the PCR reaction was completed, the 96-well plate was placed in a digital PCR droplet reader for data reading and analysis. QuantDrop-Pro4 software was used to automatically report the absolute concentration of each sample by calculating the proportion of positive droplets to the total droplets and applying a Poisson distribution. One-dimensional plots of the test results were also generated using QuantDrop-Pro4 software.

[0077] Table 4 dPCR amplification system

[0078] Components Volume (μL) Reaction premix 10 Primer F (10 μM) Add as needed Primer R (10 μM) Add as needed Probe P (10 μM) Add as needed <![CDATA[Template 1*10 4 (copies / μL)]]> 1 Deionized water Make up to 20 total 20

[0079] Table 5 dPCR reaction procedure

[0080]

[0081] The nucleotide sequences of the primers and probes are shown in Table 1.

[0082] The results show that Figure 2 As shown, for each primer / probe set, when the same primer concentration is used, as the probe concentration increases, the fluorescence intensity of both positive and negative droplets increases, and the dispersion and separation of positive droplets also increase. When the probe concentration is constant, increasing the primer concentration from 500nM to 900nM, the fluorescence intensity of negative droplets remains essentially unchanged, while the fluorescence intensity of positive droplets increases relatively, improving the separation of negative and positive droplets. According to the experimental results of each test group, each primer and probe set can achieve effective separation of negative and positive droplets. When the primer concentration is 500nM or 900nM and the probe concentration is 250nM, the fluorescence intensity of negative droplets is relatively moderate, the separation between negative and positive is relatively good, and the total number of droplets is relatively stable.

[0083] Example 3: Specificity Verification

[0084] The present invention relates to multiple reactions. In order to avoid interference or cross-reaction between each set of primers / probes, the specificity of the designed and synthesized primers / probes needs to be verified. According to the results of the condition optimization of Example 1 and Example 2, the annealing temperature was fixed at 58°C and the primer / probe concentration was fixed at 500nM / 250nM. This example verified the specificity of quantitative digital PCR detection by detecting five types of host cell DNA. The digital PCR method was adopted to fix the primer probes, replace different templates, and comprehensively evaluate the cross-reactions of different host cell DNA samples to verify the specificity of quantitative digital PCR detection. Deionized water was used instead of the sample as a no template control (NTC).

[0085] This example is to carry out specificity verification, and the specific implementation method is as follows:

[0086] Each group of positive templates was serially diluted 10-fold, and the final dilution was 1.0×10 4 copies·μL -1 As a detection template. The reaction system was prepared according to Table 6, with the final concentration of primers used being 500nM and the final concentration of probes used being 250nM. Amplification was performed using a droplet digital PCR reaction premix solution. The reaction procedure is shown in Table 5. Amplification was performed using Applied Biosystems. TM PCR amplification was performed using a PCR instrument. After the PCR reaction was completed, the 96-well plate was placed in a digital PCR droplet reader for data reading and analysis. QuantDrop-Pro4 software was used to automatically report the absolute concentration of each sample by calculating the proportion of positive droplets to the total droplets and applying a Poisson distribution. One-dimensional plots of the test results were also generated using QuantDrop-Pro4 software.

[0087] Table 6 dPCR amplification system

[0088] Components Volume (μL) Reaction premix 10 Primer F (10 μM) 1 Primer R (10 μM) 1 Probe P (10 μM) 0.5 <![CDATA[Template 1*10 4 (copies / μL)]]> 1 Deionized water Make up to 20 total 20

[0089] The nucleotide sequences of the primers and probes are shown in Table 1.

[0090] The results show that Figure 3 As shown in the figure, the specificity of quantitative digital PCR was verified by detecting five types of host cell DNA. The specificity results showed that, except for the positive template corresponding to the primer / probe set, other mismatched templates and the no-template control (NTC) did not produce positive fluorescence signals, indicating that digital PCR detection has high specificity for multiple host cell DNA and no cross-reaction.

[0091] Example 4: Establishment of a Multiplex Digital PCR Detection Method for CHO Cells, Vero Cells, Pichia Pastoris, Escherichia coli, and NS0 Cells

[0092] To establish a multiplex digital PCR detection method for CHO cells, Vero cells, Pichia pastoris (P. pastoris), Escherichia coli (E. coli), and NS0 cells, this example optimized and evaluated multiplex digital PCR technology for detecting residual DNA from multiple host cells in test samples in a single-step procedure. Specifically, digital PCR was performed using a mixture of primers / probes and templates in the same system to verify the multiplex detection performance of digital PCR.

[0093] This example establishes and verifies a multiplex digital PCR detection method for CHO cells, Vero cells, Pichia pastoris, Escherichia coli, and NS0 cells. The specific implementation method is as follows:

[0094] Each group of positive templates was serially diluted 10-fold, and the final dilution was 1.0×10 4 copies·μL -1 As a detection template. The reaction system was prepared according to Table 7. The final concentration of the mixed primer was 1000nM and the final concentration of the mixed probe was 600nM. Amplification was performed using a droplet digital PCR reaction premix reaction solution. The reaction procedure is shown in Table 5. Amplification was performed using Applied Biosystems. TM PCR amplification was performed using a PCR instrument. After the PCR reaction was completed, the 96-well plate was placed in a digital PCR droplet reader for data reading and analysis. QuantDrop-Pro4 software was used to automatically report the absolute concentration of each sample by calculating the proportion of positive droplets to the total droplets and applying a Poisson distribution. The test results were analyzed using 2D plots using QuantDrop-Pro4 software.

[0095] Table 7 dPCR amplification system

[0096] Components Volume (μL) Reaction premix 10 Primer F mix 2 Primer R Mix 2 Probe P Mix 1.2 <![CDATA[Template 1*10 4 (copies / μL)]]> 1 Deionized water Make up to 20 total 20

[0097] The nucleotide sequences of the primers and probes are shown in Table 1.

[0098] The results show that Figure 4As shown, for the 2D plots of the FAM and VIC channels, the single-positive targets CHO and E. coli are displayed only in the FAM channel, the single-positive target P. pastoris is displayed only in the VIC channel, and the double-positive target Vero is displayed in both channels. For the 2D plots of the FAM and CY5 channels, the single-positive targets Vero and E. coli are displayed only in the FAM channel, the single-positive target NS0 is displayed only in the CY5 channel, and the double-positive target CHO is displayed in both channels. For the 2D plots of the VIC and CY5 channels, the single-positive targets P. pastoris and Vero are displayed only in the VIC channel, and the single-positive targets CHO and NS0 are displayed only in the CY5 channel.

[0099] In summary, the present invention establishes a method for detecting multiple host cell residual DNA based on digital PCR, which can realize single or multiple detection of five types of host cell residual DNA, namely CHO cells, Vero cells, Pichia pastoris, Escherichia coli and NS0 cells, in a single reaction, and can also solve the non-specific amplification, signal interference and detection limitations encountered by traditional detection methods during the detection process, saving manpower, material resources and production costs, and improving the detection efficiency of host cell residual DNA. It is very suitable for the detection of host cell DNA residues in intermediates, semi-finished products and finished products in biopharmaceutical products or biological products. The present invention is more objective in the assessment of the risk of host cell residual DNA and provides a new option and supplement to the current quality control detection of residual impurities in biological products.

[0100] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and is not limiting. Although the present invention is described in detail with reference to the preferred arrangement scheme, ordinary technicians in this field should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A multiplex digital PCR detection product, characterized in that: The multiplex digital PCR detection product comprises primer pairs for detecting residual DNA of multiple host cells including CHO cells, Vero cells, Pichia pastoris, Escherichia coli and NS0 cells, and the composition of the primer pairs is as follows: A primer pair for detecting residual DNA in CHO cells, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2; A primer pair for detecting residual Vero cell DNA, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO: 3 and SEQ ID NO: 4; A primer pair for detecting residual Pichia pastoris DNA, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO: 5 and SEQ ID NO: 6; A primer pair for detecting residual Escherichia coli DNA, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO: 7 and SEQ ID NO: 8; The sequences of the upstream and downstream primers of the primer pair used to detect residual DNA in NS0 cells are shown in SEQ ID NO: 9 and SEQ ID NO:

10.

2. The multiplex digital PCR detection product according to claim 1, characterized in that: The detection product also includes the following probe group: The sequence of the probe for detecting residual DNA in CHO cells is shown in SEQ ID NO: 11; The sequence of the probe for detecting residual DNA in Vero cells is shown in SEQ ID NO: 12; The sequence of the probe for detecting residual Pichia pastoris DNA is shown in SEQ ID NO: 13; The sequence of the probe for detecting residual DNA in Escherichia coli cells is shown in SEQ ID NO: 14; The sequence of the probe for detecting residual DNA in NS0 cells is shown in SEQ ID NO:

15.

3. The multiplex digital PCR detection product according to claim 2, characterized in that: Each probe sequence has different fluorescent labels at both ends; The fluorescent markers include one or more of FAM, VIC, CY5, CY5.5, HEX, and JOE.

4. A multiple host cell residual DNA detection kit, characterized in that: The kit comprises the multiplex digital PCR detection product according to any one of claims 1 to 3.

5. A method for detecting multiple host cell residual DNA based on digital PCR, characterized in that: The multiplex digital PCR detection product according to any one of claims 1 to 3, or the detection kit according to claim 4 is used to detect the sample to be detected, and the detection method comprises the following steps: 1) Collect samples to be tested and extract DNA; 2) using the DNA sample obtained in step 1) as a template and the primer pair and probe set according to any one of claims 1 to 3 to prepare a PCR reaction mixture; 3) adding the reaction mixture prepared in step 2) to a droplet generation chip, adding droplet generation oil to each well, and generating a droplet emulsion in each well via a droplet generator. After the droplet emulsion is formed, all of the prepared droplet emulsion is further transferred to corresponding wells of a 96-well PCR plate, and then heat-sealing the plate with a puncturable heat-sealing film using a PCR plate sealer; 4) placing the 96 PCR plate sealed with the heat-sealing film obtained in step 3) into a PCR instrument for PCR amplification; 5) The 96-well PCR plate that completed PCR amplification in step 4) is placed in a digital PCR droplet reader for data reading and result analysis. The absolute concentration of each sample is automatically reported by calculating the ratio of positive droplets to total droplets and combining it with a Poisson distribution.

6. The detection method according to claim 5, characterized in that In step 2), the PCR reaction system mixture is as follows: In the PCR reaction system mixture, the final concentration of the upstream primer or the downstream primer is 200nM to 2000nM, and the final concentration of the probe is 100nM to 1000nM.

7. The detection method according to claim 5, characterized in that In step 4), the reaction conditions of the PCR are: ① Pre-denaturation at 95°C for 10 min, one cycle; ② Denaturation at 95°C for 30 seconds, annealing and extension at 50-65°C, preferably at 60°C for 1 minute, for 30-50 cycles; ③Inactivate the enzyme at 98°C for 10 min, one cycle; The heating and cooling rate is 1-3℃ / sec.

8. Use of the primer pair and probe set according to any one of claims 1 to 3, or the detection kit according to claim 4, in the preparation of a multiplex digital PCR detection product for detecting residual host cell DNA, characterized in that: The host cells include CHO cells, Vero cells, Pichia pastoris, Escherichia coli and NS0 cells.

9. The use according to claim 8, characterized in that The test samples include intermediates or products expressed by CHO cells, Vero cells, Pichia pastoris, Escherichia coli and NS0 cells.

10. The use according to claim 9, characterized in that The products include cell products, protein products, gene drugs, vaccines or antibodies.

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