Human DNA residue detection primer probe composition, detection kit and detection method

Abstract

The invention discloses a Human DNA residue detection primer probe composition, a detection kit and a detection method. The primer probe composition comprises a Human forward primer, a Human reverse primer, a Human Taqman probe, an IPC forward primer, an IPC reverse primer and an IPC probe. The sequence of the Human forward primer is shown as Seq ID No: 1, and the sequence of the Human forward primer is 5 '-TAAMAATYAGCGSGCGTAGKGGC-3'; the sequence of the Human forward primer is shown as Seq ID No: 1; the sequence of the Human reverse primer is as shown in Seq ID No: 2, and the sequence of the Human reverse primer is 5 '-TCGKGTTCAYGCCAT-3'; the sequence of the Human Taqman probe is shown as Seq ID No: 3 and is 5 '-CTGCCTSAGCCTCCGGGTA-3', the sequence of the IPC forward primer is shown as Seq ID No: 4 and is 5 '-CCGTGCTTCCCTATGCAAG-3', the sequence of the IPC reverse primer is shown as Seq ID No: 5 and is 5 '-TCCGTGAGATCCCGACCA-3', and the sequence of the IPC probe is shown as Seq ID No: 6. The detection kit and the detection method provided by the invention can provide flexible and accurate Human DNA residue detection for any different biological agents produced by human cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer-probe composition, a detection kit and a detection method for detecting human DNA residues. Background Art

[0002] Biological products refer to preparations for the prevention, treatment and diagnosis of human diseases that are prepared through biological technology using microorganisms, cells, animal or human tissues and body fluids as starting materials. In the production process of biological products, human cell residues are an issue that needs to be strictly controlled because these residues may carry potential carcinogenic and infectious risks.

[0003] In the production process of biological products, residual DNA from human cells may exist as impurities in semi-finished products, intermediates and finished products. Due to the stability of DNA, it is easy to produce residues during the production process, and these biological products will bring uncontrollable risk factors when used to treat human diseases.

[0004] Residual nucleic acids in human cells, especially DNA, may transmit tumor or virus-related genes, which poses potential carcinogenic and infectious risks. Therefore, drug regulatory agencies in various countries are cautious and clear about the attitude of residual nucleic acids in host cells in biological products, requiring pharmaceutical companies to establish detailed, feasible and highly sensitive detection methods to ensure that the purification process of drugs can remove residual DNA and strictly control the release of final products.

[0005] Residual host DNA in biological products is not only an impurity brought about during production, but also poses certain safety risks. Residual DNA has potential tumorigenic and infectious risks, so the drug regulatory authorities of various countries have very strict limits on DNA impurities. The WHO and drug registration regulatory agencies of various countries generally only allow residual DNA below 100pg / dose in biological products, and encourage minimizing the amount and fragment length of residual DNA (less than the length of a functional gene, which is approximately 200bp based on current evidence). Depending on the source of impurities and the process, a maximum of 10ng / dose is allowed under special circumstances.

[0006] The third part of the 2020 edition of the Pharmacopoeia of the People's Republic of China stipulates that the detection of exogenous DNA residues adopts DNA probe hybridization method, fluorescent dye colorimetry and quantitative PCR method. At present, the detection of residual DNA in host cells on the market mainly adopts the fluorescent probe qPCR method. The qPCR method has extremely high sensitivity, sequence specificity and accuracy, and can provide a reliable detection method for the biopharmaceutical industry in process research and finished product quality control. Based on the various detection methods in the General Chapters 509 and 1130 of the United States Pharmacopoeia and the General Chapter 3407 of the Chinese Pharmacopoeia, the more accurate, reliable and scientific fluorescent probe was finally selected. qPCR was used as a method to detect residual cellular DNA. A fluorescent probe is an oligonucleotide probe that carries a fluorescent group (reporter group), such as FAM, TET, VIC, etc., at its 5' end, and a quencher group, such as TAMRA, BHQ1, etc., at its 3' end. During PCR amplification, a specific fluorescent probe is added at the same time as a pair of primers. When the probe is intact, the fluorescent signal emitted by the fluorescent group is absorbed by the quencher group. During PCR amplification, the 5'-3' exonuclease activity of the Taq enzyme cleaves and degrades the probe, separating the fluorescent group and the quencher fluorescent group, so that the fluorescent monitoring system (fluorescence quantitative PCR instrument) can receive the fluorescent signal, that is, for each DNA chain amplified, a fluorescent molecule is formed, achieving complete synchronization between the accumulation of the fluorescent signal and the formation of the PCR product. This method has the characteristics of strong specificity and high sensitivity, and is currently widely used in the detection of residual nucleic acids in biopharmaceutical cells. Summary of the invention

[0007] The purpose of the present invention is to provide a primer probe composition, a detection kit and a detection method for detecting human DNA residues, which can provide flexible and accurate human DNA residue detection for any different biological preparations produced by human cells.

[0008] In order to achieve the above purpose, this application adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a primer-probe composition for detecting human DNA residues, the primer-probe composition comprising a human forward primer, a human reverse primer, a human Taqman probe, an IPC forward primer, an IPC reverse primer and an IPC probe;

[0010] The sequence of the Human forward primer is shown in Seq ID No: 1, which is 5'-TAAMAATYAGCCGSGCGTAGKGGC-3';

[0011] The sequence of the Human reverse primer is shown in Seq ID No: 2, which is 5'-TCGKGTTCAYG CCAT-3';

[0012] The sequence of the Human Taqman probe is shown in Seq ID No: 3, which is 5'-CTGCCTSAGCCTCCKGGGTA-3';

[0013] The sequence of the IPC forward primer is shown in Seq ID No: 4, which is 5'-CCGTGCTTTCCCTC TATGCAAG-3';

[0014] The sequence of the IPC reverse primer is shown in Seq ID No: 5, which is 5'-TCCGTGAGATCCC GACCA-3';

[0015] The sequence of the IPC probe is shown in Seq ID No: 6, which is 5'-ATACGCGCTTCCCCAT GCCAT-3'.

[0016] In the above technical solution, the 5' end of the Human Taqman probe is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a quencher group BHQ1;

[0017] The 5' end of the IPC probe is labeled with a fluorescent reporter group VIC, and the 3' end is labeled with a quencher group BHQ1.

[0018] In a second aspect, the present invention provides a Human DNA residue detection kit, wherein the kit contains the primer-probe combination according to claim 1.

[0019] In the above technical solution, the kit includes a PCR amplification reaction solution, a primer-probe mixture containing the primer-probe combination, a DNA standard and a DNA diluent;

[0020] The PCR amplification reaction solution comprises a heat-resistant DNA polymerase, deoxyribonucleoside triphosphates and a buffer containing magnesium ions;

[0021] In the primer-probe mixture, the forward primer concentration is 0.2-0.5 μmol / L, the reverse primer concentration is 0.2-0.5 μmol / L, and the oligonucleotide probe concentration is 0.1-0.3 μmol / L.

[0022] In the above technical solution, the kit includes 1 1.5mL PCR amplification reaction solution, 1 50uL 30ng / uL DNA standard, 1 0.5mL primer probe mixture containing the primer probe combination, and 3 1.5mL DNA diluents. The PCR amplification reaction solution includes Taq enzyme, dNTPs, Mg 2+ and BSA, the reaction system is 30uL, and the DNA standard is 30ng / uL of 293T cell genomic DNA.

[0023] In a third aspect, the present invention provides the use of the primer-probe combination described above for detecting the amount of residual DNA in products produced by human cells.

[0024] In the above technical solutions, the products include cell products, recombinant vaccines or monoclonal antibodies.

[0025] In a fourth aspect, the present invention provides a method for detecting residual human DNA for non-diagnostic or therapeutic purposes, comprising the following steps:

[0026] S1. Sample DNA extraction;

[0027] S2. Using the primer-probe combination of claim 1 to establish a multiplex real-time fluorescence quantitative qPCR reaction system, and perform PCR amplification;

[0028] S3. Analysis of test sample results: Calculate the amount of residual human DNA in the test sample based on the obtained standard curve.

[0029] In the above technical solution, the multiplex real-time fluorescence quantitative qPCR reaction system is 30 μL, which is composed of 15 μL PCR amplification reaction solution, 5 μL primer-probe mixture containing the primer-probe combination, 10 μL DNA standard and sample nucleic acid.

[0030] In the above technical solution, the reaction conditions of the multiplex real-time fluorescence quantitative qPCR reaction system are: pre-denaturation at 95°C for 2 min, followed by 95°C for 10 s, 60°C for 34 s, and 40 cycles.

[0031] The beneficial effects of the present invention are:

[0032] 1. The present invention utilizes the taqman probe technology of real-time fluorescence quantitative PCR to perform quantitative detection of human DNA residues, and the minimum detection limit is as low as 30fg / uL, indicating that the kit has good sensitivity and detection limit.

[0033] 2. The present invention overcomes the technical defect that it is difficult for the DNA probe hybridization method and the fluorescent dye method to achieve the sensitivity of impurity limit detection. When enterprises adopt this kit in production and research and development, they can greatly reduce time-consuming, labor-intensive and costly methodological investigations. Only a small number of method adaptability experiments are needed to play a practical role in optimizing the production process and quality control system, while meeting the requirements of relevant standards of the US FDA.

[0034] 3. The kit prepared by the present invention pre-mixes the required raw material components, which reduces the tediousness and complexity of the operation process, improves work efficiency and accuracy, and reduces intra-batch and inter-batch differences.

[0035] 4. The present invention adds an internal standard IPC, i.e., an internal positive control (Internal Positive Control), which is a key component in the kit for ensuring the reliability and accuracy of the experimental results. IPC is usually included in the qPCR kit to monitor the entire qPCR process, including sample preparation, amplification, and detection steps, to ensure that there are no experimental operating errors or the presence of inhibitors that affect the experimental results. IPC can monitor the entire experimental process to ensure that there is no non-specific amplification or inhibitor effect caused by operating errors, and whether there are inhibitors that affect the qPCR reaction. It not only monitors the reliability of the experimental process, but also provides quality control, verifies the accuracy of the experimental results, and helps standardize experimental conditions.

[0036] 5. The kit of the present invention can provide flexible and accurate human gene residue detection for any different biological preparations produced by human cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the fluorescence quantitative PCR amplification curve of the present invention, wherein Figure 1 a is the Human amplification curve, 1b is the IPC 10-10 5 Amplification curve, Figure 1 c is IPC10 3 Amplification curve of plasmid added to Human system;

[0038] Figure 2 is the Human fluorescent quantitative PCR standard curve of the present invention;

[0039] Figure 3 The specificity analysis of the fluorescent quantitative PCR method of the present invention is as follows: Figure 3 a is Huamn, Human+CHO, Figure 3 b stands for Huamn, Human+E.coli. DETAILED DESCRIPTION

[0040] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0041] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0042] Unless otherwise specified, the reagents used in the following examples can be purchased from conventional biochemical reagent manufacturers.

[0043] PCR amplification reaction solution was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; ABI7500 fluorescence quantitative PCR instrument was purchased from ABI (Applied Biosystems, USA); DNA dilution solution was prepared using analytical grade reagents, and genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0044] Example 1 Primer design and reference material preparation and detection for detecting residual human DNA by fluorescence quantitative PCR

[0045] 1. Design and synthesis of primers and probes

[0046] By comparing the existing Human nucleic acid sequences in the Genbank database and the nucleic acid sequences reported in published literature at home and abroad, the main genes related to Human were used as amplification target sites, and highly conserved segments without secondary structures were selected. According to the basic principles of primer and probe design, multiple pairs of primers and probes were designed using Oligo7 software and artificial methods. At the same time, the internal reference genes of plant genes with low homology and distant genetic relationship with human cells were selected as internal positive controls (Internal Positive Control), i.e., IPCs. Also according to the basic principles of primer and probe design, primers and probes were designed using Oligo7 software and artificial methods.

[0047] Through preliminary experiments, select the best combination:

[0048] Human forward primer sequence: 5'-TAAMAATYAGCCGSGCGTAGKGGC-3', Seq ID No: 1; Human reverse primer sequence: 5'-TCGKGTTCAYGCCAT-3', Seq ID No: 2;

[0049] Human Taqman probe: 5'-CTGCCTSAGCCTCCKGGGTA-3', Seq ID No: 3;

[0050] IPC forward primer sequence: 5'-CCGTGCTTTCCCTCTATGCAAG-3', Seq ID No: 4 IPC reverse primer sequence: 5'-TCCGTGAGATCCCGACCA-3', Seq ID No: 5;

[0051] IPC probe sequence: 5'-ATACGCGCTTCCCCATGCCAT-3', Seq ID No: 6.

[0052] The 5' end of the Human Taqman probe is labeled with a fluorescent reporter group, which is FAM, and the 3' end is labeled with a quencher group, which is BHQ1. The 5' end of the IPC probe is labeled with a fluorescent reporter group, which is VIC, and the 3' end is labeled with a quencher group, which is BHQ1.

[0053] The primers were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0054] 2. Preparation and testing of test reference materials

[0055] The detection reference is cell gene DNA, and the human DNA quantitative reference is 293T cell genomic DNA. 293T cells were cultured, collected by centrifugation, and washed twice with PBS aseptically. 293T genomic DNA was extracted according to the instructions of the genomic DNA extraction kit. The extraction steps are as follows:

[0056] (1) Take a cell suspension sample of 1E7 cells and add it to an EP tube. If the volume is less than 200 μL, make it up to 200 μL with water.

[0057] (2) Add 20 μL Proteinase K and 350 μL Lysis Buffer GHL. Lyse in a constant temperature shaking metal bath at 75°C, 1,500 rpm for 15-30 min until there are no clumps in the sample.

[0058] (3) Add 350 μL of isopropanol and oscillate for 10 seconds. Add 15 μL of magnetic bead suspension GH and oscillate for 1 minute. Let stand for a total of 9 minutes, oscillating and mixing for 1 minute every 3 minutes.

[0059] (4) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid.

[0060] (5) Add 900 μL of buffer GDZ (please check whether anhydrous ethanol has been added before use) and vortex to mix for 2 min.

[0061] (6) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid;

[0062] (7) Add 500 μL of buffer GDZ and vortex to mix for 2 min;

[0063] (8) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid;

[0064] (9) Remove the centrifuge tube from the magnetic rack, add 900 μL of PWD (please check whether anhydrous ethanol has been added before use), and oscillate to mix for 2 min.

[0065] (10) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid.

[0066] (11) Remove the centrifuge tube from the magnetic rack, add 300 μL of PWD, and mix by shaking for 2 min.

[0067] (12) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid.

[0068] (13) Place the centrifuge tube on a magnetic rack and dry it at room temperature for 10-15 minutes.

[0069] Note: Ethanol residue will inhibit subsequent enzyme reactions, so make sure that the ethanol evaporates completely when drying. But do not dry for too long, otherwise it will be difficult to elute the DNA.

[0070] (14) Remove the centrifuge tube from the magnetic rack, add 100 μL of elution buffer TB, vortex to mix, and incubate at 56°C for 10 min. Invert and mix three times, 3-5 times each time.

[0071] (15) Place the centrifuge tube on the magnetic rack and let it stand for 2 min. After the magnetic beads are completely adsorbed, carefully transfer the DNA solution to a new centrifuge tube and store it under appropriate conditions.

[0072] Then, Nanodrop was used to perform preliminary concentration and purity tests: the concentration and purity of the extracted 293T cell genomic DNA were measured using an ultra-micro UV spectrophotometer, with an OD260 / OD280 ratio between 1.8 and 2.0, and OD260 / OD230> 2. Qubit was used to examine the quality of the genomic DNA and calibrate the concentration of the genomic DNA to 30 ng / uL, and stored in aliquots at -20°C for use as a standard.

[0073] The standard is Human genomic DNA with a concentration of 30 ng / uL; when used, it is diluted with DNA diluent into 6 concentration gradients, namely 3000 pg / uL, 300 pg / uL, 30 pg / uL, 3 pg / uL, 0.3 pg / uL, and 0.03 pg / uL.

[0074] The reaction system of fluorescence quantitative qPCR detection is 30 μL, and the specific components are: 15 μL PCR amplification reaction solution, 1 μL of Human and IPC upstream primers, 1 μL of Human and IPC downstream primers, 0.4 μL Humanh and IPC probes, 0.2 μL10 3 copy / μL of IPC plasmid fragments, 10μL of Human genomic DNA.

[0075] After the sample addition, PCR amplification was performed on an ABI7500 real-time fluorescence quantitative PCR instrument. The reaction conditions were: 95°C pre-denaturation for 2 min; 95°C for 10 s, 60°C for 34 s, and 40 cycles.

[0076] Analyzing the standard curve, we can obtain from the experimental results of fluorescence quantitative PCR that the amplification curve shows that the amplification curve of each concentration has an obvious exponential growth period ( Figure 1 ).like Figure 2 Standard curve: The distance between the Cq values ​​of each gradient of the amplification curve is the same, and the linear correlation coefficient R of the standard curve is 2 =1, the amplification efficiency E (E = 10-1 / slope-1) is 97.1%, and IPC has an amplification curve. From the standard curve, it can be seen that the primer pairs and PCR conditions meet the experimental requirements of fluorescent quantitative PCR.

[0077] Example 2 Application of Fluorescence Quantitative PCR to Detect Human Residual DNA and Its Specificity Analysis

[0078] 1. Detection steps

[0079] 1) Sample processing and DNA extraction

[0080] The samples measured in this example include 293T cell genomic DNA (30pg / μl), E. coli cell genomic DNA (30pg / μl), and CHO genomic DNA (30pg / μl). The DNA was extracted using the magnetic bead enrichment method. When extracting the sample, the positive quality control and negative quality control were extracted at the same time. The negative quality control was used to determine whether there was contamination during the extraction process, and the positive quality control was used to determine the extraction yield.

[0081] All samples to be tested must be extracted and dissolved in the DNA diluent or pure water provided in the kit.

[0082] 2) Establishing a real-time fluorescence quantitative qPCR reaction system using the Human detection primer probe combination

[0083] The reaction system for fluorescence quantitative qPCR detection is 30 μL, and its composition is:

[0084] 15 μL PCR amplification reaction solution, 1 μL of Human and IPC upstream primers, 1 μL of Human and IPC downstream primers, 0.4 μL of Human and IPC probes, 0.2 μL of 10 3 copy / μL of IPC plasmid fragment, 10μL Human genomic DNA.

[0085] The negative control substance is pure water. The quantitative standard substance is human genomic DNA, with a concentration of 30 ng / uL.

[0086] 3) PCR amplification and result analysis

[0087] Standards, negative quality controls and positive quality controls should be set up for each test. When using standards, dilute the standards with DNA, add the standards and PCR amplification reaction solution to a 96-well PCR reaction plate, with a total volume of 30μl, including 15μl of PCR amplification reaction solution and 10μl of the DNA solution of the sample to be tested.

[0088] PCR amplification was performed on an ABI 7500Fast fluorescent quantitative PCR instrument under the following conditions: 95°C pre-denaturation for 2 min, 95°C for 10 s, 60°C for 34 s, 40 cycles. Standard samples were added for detection to make a standard curve.

[0089] After the reaction is completed, the test data file is saved. According to the curve graph obtained from the PCR amplification results, the analysis parameters are adjusted to optimize the standard curve under the standard curve window (i.e., the correlation coefficient is greater than 0.97, and the amplification efficiency is between 95% and 105%). The test results are processed by the instrument to calculate the residual DNA in the test sample according to the standard curve, and the interference of the commonly used engineered cell genomic DNA on the Human detection reagent is evaluated. The interference group overlaps with the control group, and no interference is observed (see Figure 3 ).

[0090] Example 3: Human residual DNA detection kit detection sample and accuracy analysis

[0091] 1. The composition of the kit is shown in Table 1:

[0092] Table 1 Components of Human Residual DNA Detection Kit

[0093]

[0094] Take 6 new 1.5ml low-adsorption centrifuge tubes, label them as SD0, SD1, SD2, SD3, SD4, and SD5, and add 90μl of DNA dilution buffer to each tube. Add 10μl of Human DNA Control to the SD0 tube, vortex the mixer to mix thoroughly, and then add 10μl of the previous concentration to each tube in turn.

[0095] 2. Detection steps

[0096] a) Sample processing and DNA extraction

[0097] Different amounts of human DNA (300pg, 30pg, 3pg) were added to three sample matrices (basic samples) as recovery samples and purified by a pretreatment kit. When extracting the samples, positive quality control products and negative quality control products were extracted at the same time. The negative quality control products were used to determine whether there was contamination during the extraction process, and the positive quality control products were used to determine the extraction yield.

[0098] All samples to be tested must be extracted and dissolved in the DNA diluent or pure water provided in the kit.

[0099] b) PCR amplification and result analysis

[0100] Standards, negative quality controls and positive quality controls should be set up for each test. When using standards, dilute the standards with DNA, add the standards and PCR amplification reaction solution to a 96-well PCR reaction plate, with a total volume of 30μl, including 15μl PCR amplification reaction solution, 5μl primer and probe mixture (including Human and IPC primer probes and IPC positive plasmid), and 10μl DNA solution of the sample to be tested. PCR amplification was performed on an ABI 7500Fast fluorescent quantitative PCR instrument under the conditions of 95℃ pre-denaturation for 2min; 95℃10s, 60℃34s, 40 cycles. At the same time, add standards to test and make a standard curve.

[0101] After the reaction is completed, save the test data file. Adjust the analysis parameters according to the curve graph obtained from the PCR amplification results to make the standard curve in the standard curve window reach the best (that is, the correlation coefficient is greater than 0.97, and the amplification efficiency is between 95% and 105%). See the attached Figure 3 Finally, the automatic analysis device of the instrument calculates the determination value of the unknown sample and the recovery rate. The results are shown in Table 2. The recovery rate is greater than 70%, and the accuracy of the kit is high.

[0102] Table 2. Recovery analysis

[0103]

[0104] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A primer-probe composition for detecting human DNA residues, characterized in that: The primer-probe composition includes a Human forward primer, a Human reverse primer, a Human Taqman probe, an IPC forward primer, an IPC reverse primer and an IPC probe; The sequence of the Human forward primer is shown in Seq ID No: 1, which is 5'-TAAMAATYAGC CGSGCGTAGKGGC-3'; The sequence of the Human reverse primer is shown in Seq ID No: 2, which is 5'-TCGKGTTCAYG CCAT-3'; The sequence of the Human Taqman probe is shown in Seq ID No: 3, which is 5'-CTGCCTSAG CCTCCKGGGTA-3'; The sequence of the IPC forward primer is shown in Seq ID No: 4, which is 5'-CCGTGCTTTCCCTC TATGCAAG-3'; The sequence of the IPC reverse primer is shown in Seq ID No: 5, which is 5'-TCCGTGAGATCCC GACCA-3'; The sequence of the IPC probe is shown in Seq ID No: 6, which is 5'-ATACGCGCTTCCCCAT GCCAT-3'.

2. The primer-probe composition according to claim 1, characterized in that: The 5' end of the Human Taqman probe is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a quencher group BHQ1; The 5' end of the IPC probe is labeled with a fluorescent reporter group VIC, and the 3' end is labeled with a quencher group BHQ1.

3. A Human DNA residue detection kit, characterized in that: The kit contains the primer-probe combination according to claim 1.

4. The kit according to claim 3, characterized in that: The kit comprises a PCR amplification reaction solution, a primer-probe mixture containing the primer-probe combination, a DNA standard and a DNA diluent; The PCR amplification reaction solution comprises a heat-resistant DNA polymerase, deoxyribonucleoside triphosphates and a buffer containing magnesium ions; In the primer-probe mixture, the forward primer concentration is 0.2-0.5 μmol / L, the reverse primer concentration is 0.2-0.5 μmol / L, and the oligonucleotide probe concentration is 0.1-0.3 μmol / L.

5. The kit according to claim 3, characterized in that: The kit includes 1 1.5mL PCR amplification reaction solution, 1 50uL 30ng / uL DNA standard, 1 0.5mL primer probe mixture containing the primer probe combination, and 3 1.5mL DNA diluents. The PCR amplification reaction solution includes Taq enzyme, dNTPs, Mg 2+ and BSA, the reaction system is 30uL, and the DNA standard is 30ng / uL of 293T cell genomic DNA.

6. Use of the primer-probe combination according to claim 1 in detecting the amount of residual DNA in products produced by human cells.

7. The use according to claim 6, characterized in that: The products include cell products, recombinant vaccines or monoclonal antibodies.

8. A method for detecting residual human DNA for non-diagnostic or therapeutic purposes, characterized in that: The following steps are involved: S1. Sample DNA extraction; S2. Using the primer-probe combination of claim 1 to establish a multiplex real-time fluorescence quantitative qPCR reaction system, and perform PCR amplification; S3. Analysis of test sample results: Calculate the amount of residual human DNA in the test sample based on the obtained standard curve.

9. The detection method according to claim 8, characterized in that: The multiplex real-time fluorescence quantitative qPCR reaction system is 30 μL, and is composed of 15 μL PCR amplification reaction solution, 5 μL primer-probe mixed solution containing the primer-probe combination, and 10 μL DNA standard and sample nucleic acid.

10. The detection method according to claim 8, characterized in that: The reaction conditions of the multiplex real-time fluorescence quantitative qPCR reaction system are: pre-denaturation at 95° C. for 2 min, followed by 95° C. for 10 s, 60° C. for 34 s, and 40 cycles.

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