Extraction and detection method of host cell residual DNA (Deoxyribose Nucleic Acid)

Abstract

The invention provides a lysis binding solution, an extraction kit and a detection method for extracting residual DNA of host cells, the lysis binding solution comprises guanidine hydrochloride, PEG6000, tRNA, glycogen and isopropanol, the concentration of guanidine hydrochloride is 2.0-2.5 M, the concentration of PEG6000 is 1%-10% (m / v), and the concentration of isopropanol is 50-60%. According to the present invention, the host cell residual DNA extraction is performed by using the lysis binding liquid, such that the compatibility between the reagent and different sample types can be significantly enhanced, the sample pre-dilution is reduced, and the matrix interference is reduced so as to improve the extraction efficiency and the extraction accuracy of the residual DNA, and effectively overcome the limitation of the commercial kit.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid extraction and detection, and particularly relates to a method for extracting and detecting residual DNA in host cells. Background Art

[0002] Residual host cell DNA refers to DNA fragments or longer sequences originating from host tissue cells that may be contaminated during the production of biological products. These fragments pose potential biosafety risks, including immunogenicity and carcinogenicity. Therefore, the detection of residual host cell DNA is a crucial step in the production and quality control of biological products.

[0003] Due to the complexity and high purity requirements of biological products, the accurate quantification of residual host cell DNA has become an extremely challenging task. This requires the detection technology to be able to accurately measure picogram (pg) or even lower concentrations of DNA in milligram (mg) samples to ensure the safety and effectiveness of the product. The appendix of the 2020 edition of the "Chinese Pharmacopoeia" contains three methods for detecting residual host cell DNA, namely DNA probe hybridization, fluorescent staining, and quantitative PCR (qPCR). The European Pharmacopoeia also proposed two efficient analytical methods for quantitative detection of residual host cell DNA: quantitative PCR and immunoenzyme method. qPCR is also the only standard method recommended by the new edition of the "United States Pharmacopoeia" for the detection of residual host cell DNA in biological products. It can be seen that the qPCR method has become the preferred detection method in the field of process development and product quality control of biological products.

[0004] In addition to being used for final product release, residual host cell DNA testing is often used during process control during production process development and validation to assess the effectiveness of the process in removing residual DNA and monitor process robustness. However, the various chemical components contained in intermediate and final product samples at various stages of production, such as proteins, salts, surfactants, and buffers, often cause matrix effects in qPCR detection, interfering with accurate DNA measurement. Therefore, purification and extraction of residual host cell DNA in samples is necessary to reduce or eliminate the impact of matrix effects on residual DNA detection using qPCR.

[0005] In process monitoring applications, the applicability of residual DNA detection methods for intermediates and final products at each production stage should be examined. In particular, it is important to determine whether the test results of the method being used will be interfered with by buffer components in the product and to determine the required dilution ratios for product testing at each stage. Recovery tests are often performed to assess this interference. The Chinese Pharmacopoeia (CP) specifies that for test samples requiring DNA purification prior to testing, a recovery test should be performed starting with the purification step for each test. A recovery test involves adding a predetermined amount of standard DNA (spiked) to the sample, then processing and testing according to the same test procedure. Finally, the ratio of the amount of recovered DNA to the amount of added standard DNA is calculated. Recovery should be within a certain range (e.g., 50% to 150% as specified in the CP and USP), and the relative standard deviation (RSD) should be less than or equal to 30% to demonstrate that the test method is not significantly interfered with by sample matrix components.

[0006] Although both the Chinese Pharmacopoeia and the United States Pharmacopoeia propose the sodium iodide extraction method for extracting residual DNA, this method has high requirements for the technical experience and equipment of the experimenter. The magnetic bead method has gradually become the mainstream sample purification method for residual DNA detection in biological products due to its high efficiency, rapidity, and convenient operation. In actual applications, various biological product companies usually use commercial magnetic bead pretreatment kits to extract and purify nucleic acids (residual DNA) in samples. Given the diversity of intermediates and stock solution sample types in the recombinant protein production process, and the significant differences in residual DNA content and characteristics, these ready-made commercial kits are often difficult to fully cover and perfectly adapt to all types of recombinant protein biological products. Specifically, when the key reagents in the commercial kit (such as magnetic beads, lysis buffer, binding buffer and washing buffer) lack sufficient compatibility with the physical and chemical properties of the specific product (such as isoelectric point, pH value, ionic strength) or complex components (such as high concentrations of salts, sugars, surfactants, proteins, etc.), it may significantly affect the effective binding and capture ability of the magnetic beads with the target DNA, ultimately leading to a decrease in DNA recovery rate and affecting the efficiency and accuracy of extraction. To address this issue, a common strategy is to pre-dilute the sample to reduce matrix effects that interfere with the extraction process. However, this approach also carries a potential risk: the already low DNA content in the sample may be further reduced due to excessive dilution, falling below the detection limit, resulting in undetectable results in the final analysis, making it impossible to accurately assess the effectiveness and stability of the production process.

[0007] In the process of producing recombinant proteins, the applicant uses E. coli to express nanoparticle proteins and CHO cells to express recombinant antigen proteins. For example, patent CN117462666B describes the use of CHO cells to prepare varicella-zoster virus (VZV) recombinant antigens, and the use of E. coli to prepare nanoparticle proteins. After the recombinant antigen is combined with the nanoparticle protein, a nanoparticle vaccine is obtained. Patent CN114395015B describes the preparation and purification methods of nanoparticle proteins. In the purification process of the nanoparticle protein, a large amount of urea, Tris and sucrose are used, and the purified product contains complex components such as high concentrations of urea, Tris and sucrose. It can be seen that the applicant will obtain various types of protein samples in the process of recombinant protein production, and the sample matrix is ​​special and complex. When the applicant uses a commercial magnetic bead pretreatment kit to extract residual DNA from host cells, there are problems such as unstable extraction yield, low recovery rate, and low accuracy.

[0008] In response to the problems and shortcomings of current commercial kits in processing residual DNA in biological products, which are often caused by insufficient compatibility of reagents (including magnetic beads, lysis buffer, binding buffer, washing buffer, etc.) with complex sample components, resulting in low DNA extraction efficiency and affected accuracy, the technical problem solved by the present invention is to provide a more universal, efficient and stable method for pre-treatment of residual DNA in recombinant protein biological products. This method can significantly enhance the compatibility between reagents and different sample types, reduce sample pre-dilution and reduce matrix interference, thereby improving the extraction efficiency and accuracy of residual DNA, and effectively overcoming the limitations of commercial kits. Summary of the Invention

[0009] In order to solve the above technical problems, the present application provides a lysis and binding solution, a kit, an extraction method and a detection method for extracting residual DNA from host cells.

[0010] The technical solutions adopted by the present invention include the following aspects:

[0011] In a first aspect, the present invention provides a lysis and binding solution comprising guanidine hydrochloride and PEG6000.

[0012] In some embodiments, the concentration of guanidine hydrochloride in the lysis and binding solution of the present invention is 2.0 to 2.5 M. Preferably, the concentration of guanidine hydrochloride in the lysis and binding solution of the present invention is 2.5 M.

[0013] In some embodiments, the concentration of PEG6000 in the lysis and binding solution of the present invention is 1% to 10% (m / v). Preferably, the concentration of PEG6000 in the lysis and binding solution of the present invention is 10% (m / v).

[0014] Preferably, the lysis and binding solution of the present invention comprises 2.5 M guanidine hydrochloride and 10% (m / v) PEG6000.

[0015] In some embodiments, the lysis and binding solution of the present invention further comprises 50% to 60% isopropanol. Preferably, the lysis and binding solution of the present invention comprises 50% isopropanol.

[0016] In some embodiments, the lysis and binding solution of the present invention further comprises a nucleic acid precipitation aid.

[0017] In some embodiments, the nucleic acid precipitation aid of the present invention comprises tRNA and glycogen. Preferably, the concentration of tRNA is 5 μg to 20 μg / mL, and the concentration of glycogen is 50 μg to 125 μg / mL.

[0018] In some embodiments, the lysis and binding solution of the present invention comprises 50 μg / mL glycogen and 5 μg / mL tRNA.

[0019] In some embodiments, the lysis and binding solution of the present invention comprises 2 M guanidine hydrochloride, 1% (m / v) PEG6000, 60% isopropanol, 50 μg / mL glycogen and 5 μg / mL tRNA.

[0020] In a preferred embodiment, the lysis and binding solution of the present invention comprises 2.5 M guanidine hydrochloride, 10% (m / v) PEG6000, 50% isopropanol, 50 μg / mL glycogen and 5 μg / mL tRNA.

[0021] In some embodiments, the preparation method of the lysis and binding solution of the present invention comprises the step of dissolving PEG6000 powder in 5M guanidine hydrochloride solution.

[0022] In some embodiments, the lysis and binding solution of the present invention can be used to extract nucleic acids from samples.

[0023] In a second aspect, the present invention provides a kit for extracting residual DNA from host cells, wherein the kit comprises the lysis and binding solution of the present invention.

[0024] In some embodiments, the kit of the present invention further comprises a washing solution A and a washing solution B, wherein the washing solution A comprises guanidine hydrochloride and anhydrous ethanol, and the washing solution B comprises anhydrous ethanol.

[0025] Preferably, the washing solution A of the present invention comprises 24 mM guanidine hydrochloride and 60% ethanol.

[0026] Preferably, the washing solution B of the present invention comprises 70% anhydrous ethanol.

[0027] In some embodiments, the kit of the present invention further comprises a DNA elution buffer.

[0028] Preferably, the DNA elution buffer of the present invention is TE buffer (EDTA, Tris-HCl, pH 7.8).

[0029] In some embodiments, the kit of the present invention further comprises magnetic beads.

[0030] In some embodiments, the magnetic beads used in the present invention are carboxyl magnetic beads.

[0031] In some embodiments, the kit of the present invention further comprises proteinase K.

[0032] The present invention provides a kit for extracting residual DNA from host cells, comprising the following components:

[0033] 1) lysis and binding solution, comprising 2.5 M guanidine hydrochloride, 10% (m / v) PEG6000, 50% isopropanol, 50 μg / mL glycogen, and 5 μg / mL tRNA;

[0034] 2) Washing solution A, which contains 24 mM guanidine hydrochloride and 60% ethanol;

[0035] 3) washing solution B, which contains 70% anhydrous ethanol;

[0036] 4) DNA elution buffer, which is TE buffer;

[0037] 5) Carboxyl magnetic beads.

[0038] In some embodiments, the kit of the present invention can be used to extract nucleic acid from a sample.

[0039] In a third aspect, the present invention provides a method for extracting residual DNA from host cells, comprising the steps of sample digestion, DNA binding, DNA cleaning, and DNA elution.

[0040] In some embodiments, the method for extracting residual DNA from host cells of the present invention includes a step of purifying the DNA using magnetic beads.

[0041] In some embodiments, the method for extracting residual host cell DNA of the present invention can be used in conjunction with an automated nucleic acid extraction instrument.

[0042] Furthermore, the method for extracting residual DNA from host cells of the present invention comprises the following steps:

[0043] 1) Use proteinase K to digest sample proteins and release residual host DNA;

[0044] 2) Mixing the sample digestion solution with the lysis and binding solution to lyse the sample and release DNA, which is then bound to the magnetic beads, enriching the magnetic beads and separating the magnetic bead-DNA complex from the liquid;

[0045] 3) Adding washing solution A to the magnetic bead-DNA complex to wash away impurities such as proteins, lipids, and salt ions on the surface of the complex, and obtaining a washed complex under the action of a magnetic field;

[0046] 4) adding washing solution B to the magnetic bead-DNA complex to wash away impurities such as guanidine salts and ions on the surface of the complex, and obtaining a washed complex under the action of a magnetic field;

[0047] 5) Add DNA elution solution to the washed magnetic bead-DNA complex to elute the nucleic acid from the magnetic beads, and separate the magnetic beads and nucleic acid.

[0048] Purified host DNA was obtained.

[0049] In some embodiments, the samples of the present invention include all sample types in the production process of recombinant protein biological products.

[0050] In some embodiments, the samples of the present invention include all sample types in the processes of recombinant protein fermentation, purification, concentration, preparation, and canning, such as fermentation filtration stock solution, centrifugation supernatant stock solution, chromatography fluid, cleaning fluid, and eluate during purification.

[0051] Furthermore, the present invention provides a method for digesting the above sample, comprising using proteinase K for digestion.

[0052] In some embodiments, the proteinase K concentration in the digestion method of the present invention is not less than 20%, the digestion temperature is 55-60° C., and the digestion time is not less than 1 hour.

[0053] Preferably, the proteinase K digestion system used in the present invention comprises 10 μL of 5 M NaCl, 10 μL of proteinase K, 100 μL of proteinase K buffer and the test sample, and the digestion condition is 55° C. for 1 h.

[0054] In a fourth aspect, the present invention provides a method for detecting residual DNA in host cells, the method comprising pre-treating a sample using the method described in the present invention to obtain a DNA extract, and performing qPCR fluorescence quantitative detection on the DNA extract.

[0055] In some embodiments, the method for detecting residual host cell DNA of the present invention comprises the following steps:

[0056] 1) Preparation of DNA standard solution;

[0057] 2) Sample processing, including sample dilution;

[0058] 3) DNA purification: extracting and purifying DNA using the method for extracting residual DNA from host cells provided by the present invention;

[0059] 4) qPCR detection.

[0060] In some embodiments, the present invention uses the national standard for CHO cell / E. coli DNA content determination provided by the China Food and Drug Inspection Institute as a standard.

[0061] In some embodiments, the present invention uses an automated nucleic acid extractor to extract and purify DNA.

[0062] Preferably, the present invention uses a pre-treatment system of an automated nucleic acid extractor to extract and purify DNA.

[0063] In some embodiments, the qPCR of the present invention uses the primers, probe sequences and reaction systems provided in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0064] In some embodiments, the qPCR reaction procedure of the present invention is performed in accordance with Part III of the 2020 version of the Pharmacopoeia of the People's Republic of China.

[0065] All materials and reagents used in the present invention can be purchased commercially.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] (1) The lysis and binding solution used in the present invention has simple components, which prevents unnecessary components from interfering with subsequent separation, purification, or detection steps. The relatively small number of components makes it easier to control the quality stability of the lysate and reduce performance variations caused by interactions between the components. The simple composition also effectively reduces the cost of use and eliminates the need for a complex mixing and preparation process, reducing the possibility of operational errors and improving experimental efficiency.

[0068] (2) The method of the present invention has a strong ability to resist matrix interference. By optimizing the reagents and formula, the compatibility between the reagents and various recombinant protein sample types (including but not limited to samples with different pH values, ionic strengths, high concentrations of salts, sugars, proteins and other complex components) is significantly improved, reducing the decrease in binding efficiency due to mismatch, ensuring rapid and efficient DNA extraction in a wider range of samples while maintaining high accuracy and repeatability.

[0069] (3) The method of the present invention requires less pre-dilution, reducing the reliance on sample pre-dilution, thereby avoiding the risk of DNA loss due to over-dilution, while reducing matrix interference and improving DNA recovery rate.

[0070] (4) Using the method of the present invention, the extraction recovery rate of residual DNA in the intermediate products and stock solutions of recombinant protein biological products containing complex matrices at each production stage is between 75% and 120%. The recovery effect is stable and meets the requirements of the Chinese Pharmacopoeia and the United States Pharmacopoeia (50% to 150%), and is better than commercially available kits (70% to 130%). DETAILED DESCRIPTION

[0071] The principles and features of the present invention are described below with reference to examples. The examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific specific embodiments and are not intended to limit the scope of protection of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions or according to the conditions recommended by the respective manufacturers. When numerical ranges are given in the examples, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints may be used. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the examples of the present invention may also be used to implement the present invention based on the prior art knowledge of those skilled in the art and the description of the present invention. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

[0072] Example 1: Extraction of residual host cell DNA using commercially available kits

[0073] The inventors analyzed the composition of the sample matrix buffer at each step of the recombinant protein production process. They also used a commercially available host cell residual DNA sample pretreatment kit (qPCR) to extract DNA from samples using different matrix buffers and measured spike recovery. Specific experimental procedures were performed in accordance with the Pharmacopoeia of the People's Republic of China (2020 edition, Part III) and the instructions for the commercially available kit.

[0074] The specific experimental methods are as follows:

[0075] 1. Preparation of CHO DNA Standard Solution

[0076] Take seven 1.5 mL nuclease-free EP tubes and label them: STD0, STD1, STD2, STD3, STD4, STD5, and STD6. First, dilute the national standard solution for CHO cell DNA content determination to 3000 pg / μL with TE buffer, which is STD0. Then, dilute the solution with TE buffer according to Table 1 to a standard curve gradient of 300 pg / μL, 30 pg / μL, 3 pg / μL, 0.3 pg / μL, 0.03 pg / μL, and 0.003 pg / μL.

[0077] Table 1 CHO DNA standard dilution reference table

[0078] DNA standard number Volume of DNA standard added Volume of TE buffer added Final concentration STD1 10 μL of STD0 90μL 300 pg / μL STD2 10 μL of STD1 90μL 30 pg / μL STD3 10 μL of STD2 90μL 3 pg / μL STD4 10 μL of STD3 90μL 0.3 pg / μL STD5 10 μL of STD4 90μL 0.03 pg / μL STD6 10 μL of STD5 90μL 0.003 pg / μL

[0079] 2. Preparation of E. coli DNA standard solution

[0080] Prepare six 1.5 mL nuclease-free EP tubes and label them: STD0, STD1, STD2, STD3, STD4, and STD5. First, dilute the E. coli DNA national standard solution with TE buffer to 3000 pg / μL, designated STD0. Then, dilute the solution with TE buffer according to Table 2 to create a standard curve gradient of 300 pg / μL, 30 pg / μL, 3 pg / μL, 0.3 pg / μL, and 0.03 pg / μL.

[0081] Table 2 E. coli DNA standard dilution reference table

[0082] DNA standard number Volume of DNA standard added Volume of TE buffer added Final concentration STD1 10 μL of STD0 90μL 300 pg / μL STD2 10 μL of STD1 90μL 30 pg / μL STD3 10 μL of STD2 90μL 3 pg / μL STD4 10 μL of STD3 90μL 0.3 pg / μL STD5 10 μL of STD4 90μL 0.03 pg / μL

[0083] 3. Preparation of spiked and recovered samples

[0084] Take an appropriate amount of test sample and dilute it with 1× PBS (without magnesium ions and calcium ions) to obtain the test solution. Take 100 μL of the test solution into a 1.5 mL nucleic acid-free EP tube for DNA purification and PCR detection.

[0085] 4. DNA Purification

[0086] (1) Sample digestion

[0087] According to the instructions of the commercially available brand A kit, add a certain volume of 5M NaCl solution and a certain volume of proteinase K digestion solution to each tube of test solution, vortex and mix for at least 1 minute, then centrifuge and incubate at a suitable temperature.

[0088] (2) Extraction, purification and sample loading

[0089] After removing the sample from the dry block, quickly centrifuge it and arrange it in a 96-deep-well plate with the appropriate solutions (see Table 3 for details). Add the digested sample after all other reagents have been added. Finally, the sample purification solution will be in columns 5 and 11 of the row containing the added sample.

[0090] Table 396 Deep Well Plate Sample Loading Table

[0091]

[0092] (3) Start the DNA extraction process

[0093] Use a commercially available automated nucleic acid extraction instrument (brand A). Place the loaded 96-deep-well plate into a fixed position in the instrument and insert the plastic sleeve into the corresponding position of the magnetic head. Select the program corresponding to the pretreatment of residual host cell DNA sample to start DNA extraction. See Table 4 for details.

[0094] Table 4 Pre-treatment system extraction procedures

[0095]

[0096] 5. qPCR detection: qPCR detection was performed using the reagents and methods of the host cell residual DNA detection kit from Brand A.

[0097] 6. Result processing and calculation:

[0098] The external standard curve was used for quantification, i.e. the Logarithm of the DNA concentration (pg / μL) of each standard STD1 to STD5 / STD6 was used as the abscissa and the corresponding Ct average value was used as the ordinate. The linear regression equation y=Ax+B (A: slope, B: intercept, y: Ct average value; x: Logarithm of DNA concentration (pg / μL)) was fitted. The Ct value of each sample solution was substituted into the linear regression equation to calculate the Logarithm of its DNA concentration (L). Then the antilogarithm was calculated. L (i.e., 10 to the power of L), the DNA concentration in the sample (pg / μL) is obtained. The above data calculation can be directly output in the software. The spike recovery results are shown in Tables 5 and 6:

[0099] Table 5 Summary of CHO DNA extraction recovery rates using different matrix buffers

[0100]

[0101] Table 6 Summary of E. coli DNA extraction recovery rates using different matrix buffers

[0102]

[0103] 7. Experimental conclusion:

[0104] It can be seen that the sample matrix buffers at each stage of the recombinant protein production process are diverse and complex, and the content and characteristics of residual DNA also vary. Commercially available brand test kits lack compatibility, resulting in low DNA recovery rates, affecting the efficiency and accuracy of extraction. In addition, commercial test kits also have problems such as long delivery time and high detection costs. Based on this, the inventors have optimized and innovated from the following aspects to develop a host cell residual DNA sample pretreatment method and related reagents suitable for their company.

[0105] Example 2: Optimization of lysis and binding solution components

[0106] 1. Experimental purpose: By adjusting the types and ratios of the components of the lysis and binding buffer, including the addition of a DNA nucleic acid precipitation aid, the precipitation and purification process of nucleic acids was optimized, thereby preliminarily determining the optimal binding buffer composition that can significantly improve DNA extraction efficiency and recovery rate.

[0107] 2. Experimental Design: Based on an in-depth study of relevant literature and patents, the inventors preliminarily screened and determined the basic components of the lysis and binding solution. On this basis, they further introduced different DNA nucleic acid precipitation aids, such as polyethylene glycol (PEG) and polysaccharide compounds. These substances can promote the precipitation of DNA molecules or enhance their binding to magnetic beads through charge interactions or hydrophobic interactions, thereby improving the DNA detection effect and recovery rate of the test sample and its spiked solution. At the same time, a commercial pretreatment kit was set up as a control group, and CHO cell DNA was used as the experimental sample to compare the detection effect and recovery rate of DNA under different conditions.

[0108] 3. Main reagents and their preparation:

[0109] (1) Proteinase K buffer: Measure 4 mL of 1 M Tris-HCl solution, 8 mL of 0.5 M EDTA-2Na solution, 10 mL of 20% SDS solution, and 14 mL of ultrapure water and mix well.

[0110] (2) 2% Proteinase K solution: Dissolve 100 mg of Proteinase K powder in 5 mL of sterile water. Aliquot the solution according to experimental requirements and store at -20 ± 5°C to avoid repeated freezing and thawing.

[0111] (3) 5 M guanidine hydrochloride solution: Weigh 238.8 g of guanidine hydrochloride powder, add 250 mL of ultrapure water and dissolve in a 50°C water bath, then dilute to 500 mL and filter using a 0.22 μm microporous filter membrane.

[0112] (4) Washing solution A: Add 16 mL of 60 mM guanidine hydrochloride solution to 24 mL of anhydrous ethanol and mix well.

[0113] (5) Washing solution B (80% ethanol): Add sterile water to 40 mL of anhydrous ethanol to make up to 50 mL and mix well.

[0114] (6) Lysis and Binding Solution 1: Take 360 ​​μL of isopropanol and 240 μL of 5 M guanidine hydrochloride solution, mix well, and prepare the solution according to the actual amount.

[0115] (7) Lysis and binding solution 2: Take 360 ​​μL of isopropanol, 240 μL of 5 M guanidine hydrochloride solution, weigh the actual amount of PEG6000 powder according to the volume fraction of 1% (m / v), mix well, and prepare the actual amount for use.

[0116] (8) Lysis and binding solution 3: Take 360 ​​μL of isopropanol, 240 μL of 5 M guanidine hydrochloride solution, and 1 μL of glycogen (50 μg / mL), mix well, and prepare according to the actual amount before use.

[0117] (9) Lysis and binding solution 4: Take 360 ​​μL of isopropanol, 240 μL of 5 M guanidine hydrochloride solution, 1 μL of glycogen (50 μg / mL), weigh the actual amount of PEG6000 powder according to a volume fraction of 1% (m / v), mix well, and prepare the actual amount for use.

[0118] (10) Lysis and binding solution 5: Take 360 ​​μL of isopropanol, 240 μL of 5 M guanidine hydrochloride solution, 0.2 μL of tRNA (5 μg / mL) and 1 μL of glycogen (50 μg / mL), weigh the actual amount of PEG6000 powder according to the volume fraction of 1% (m / v), mix well, and prepare the actual amount for use.

[0119] (11) Commercial carboxyl magnetic beads.

[0120] 4. Test sample: CHO cell culture medium expressing SARS-CoV-2 RBD antigen, diluted at a factor of 100,000.

[0121] 5. The optimization scheme for adjusting the binding fluid composition is as follows:

[0122] Table 7 Experimental plan for adjusting the composition of the binding solution

[0123]

[0124]

[0125] 6.DNA extraction:

[0126] The experimental group using carboxyl magnetic beads for extraction had the corresponding solutions pre-added according to the 96-deep-well plate layout shown in Table 8. The control group and other experimental groups were treated according to the machine-assembled protocol of the Brand A pretreatment kit (magnetic bead method). Extraction and purification were performed using a Brand A automated nucleic acid extraction instrument for the control group and all experimental groups, following the extraction steps in Example 1. Samples were pretreated using the above method to obtain purified DNA.

[0127] Table 896 Deep Well Plate Sample Loading Table

[0128]

[0129] 7. Perform qPCR fluorescence quantitative detection on the DNA purification solution according to the qPCR system and method provided in the Chinese Pharmacopoeia.

[0130] 8. Result analysis:

[0131] Table 9 Summary of results of experimental group 1

[0132]

[0133]

[0134] PCS: Positive Control

[0135] As shown in the table above, the DNA concentration of the test samples extracted using the basic lysis and binding solution (60% isopropanol + 2M guanidine hydrochloride) was lower than that of the control group. This indicates that the extraction efficiency of the basic lysis and binding solution is relatively low under the current experimental conditions. Adding 1% (m / v) PEG6000 and 1 μL glycogen to the basic lysis and binding solution increased the recovery rate and the DNA concentration of the test samples, but it was still lower than the control group and did not reach the ideal level.

[0136] The results of this set of experiments show that the basic lysis and binding solution has certain limitations in DNA extraction efficiency and requires further optimization. Although the addition of glycogen and PEG6000 each has a positive effect on improving DNA extraction efficiency, with glycogen being more effective than PEG6000, it still does not reach the level of Brand A kit. Therefore, further optimization options will be explored based on the basic lysis and binding solution + glycogen formulation.

[0137] Table 10 Summary of results of experimental group 2

[0138]

[0139] As shown in the table above, after switching to the Proteinase K digestion method, the detection concentration of the sample obtained from Experimental Group 2-1, which used the basic lysis and binding solution plus glycogen, increased significantly, reaching a level comparable to that of the control group. This suggests that the new Proteinase K digestion method may have improved DNA release or increased its affinity for the binding solution, resulting in higher DNA extraction efficiency using the basic lysis and binding solution plus glycogen. Experimental Group 2-2, which added both glycogen and PEG6000 to the basic lysis and binding solution, showed the highest detection concentration of the sample (8050.0 pg / μL) among all experimental and control groups. This further confirms the positive impact of PEG6000 on DNA extraction efficiency, particularly when combined with glycogen, where the two exhibited a significant synergistic effect, jointly promoting improved DNA extraction. However, the recovery rates of both experimental groups exhibited some volatility, lacking the stability of the recovery rate of the control group. Therefore, further refinement of the ratio of the lysis and binding solution components is required to ensure stable and efficient recovery rates.

[0140] The results of Experimental Group 3 are as follows:

[0141] Table 11 Summary of results of experimental group 3

[0142]

[0143] Lysis and Binding Solution 4 (containing 60% isopropanol, 2M guanidine hydrochloride, 1μL glycogen, and 1% (m / v) PEG6000) demonstrated good DNA extraction efficiency when treating PCS, the test sample, and the spiked test sample. The detected concentration of the test sample did not differ significantly from that of the control group, demonstrating the feasibility of Lysis and Binding Solution 4 for DNA extraction. Experimental Group 3-2 added tRNA to the binding solution based on Experimental Group 3-1, forming Binding Solution 5. This adjustment significantly increased the detected concentration of the test sample, reaching the highest value among the experimental groups (5758.6pg / μL), demonstrating that the addition of tRNA has a positive effect on DNA extraction efficiency.

[0144] 9. Experimental conclusion:

[0145] The final concentration of the lysis and binding solution for a single sample was preliminarily determined to be 60% isopropanol, 2 M guanidine hydrochloride, 50 μg / mL glycogen, 1% (m / v) PEG6000, and 5 μg / mL tRNA. The formula needs to be further optimized and verified to achieve higher extraction efficiency.

[0146] Example 3: Optimization of the ratio of lysate components

[0147] 1. Experimental purpose: To adjust the ratio of isopropyl alcohol to guanidine hydrochloride in the binding solution of Example 2 and determine the optimal binding solution composition by comparing the extraction effects under different binding solution ratios.

[0148] 2. Experimental design: Under the premise of keeping all other experimental conditions unchanged (the final concentration of the lysis and binding solution formula of a single sample is fixed at a certain amount of isopropanol and guanidine hydrochloride, 1μL glycogen (50μg / mL), 1% (m / v) PEG6000 and 0.2μL tRNA (5μg / mL); the final concentration of the washing solution A is 24mM guanidine hydrochloride and 60% ethanol; the washing solution B is 80% ethanol), the isopropanol and guanidine hydrochloride content in the lysis and binding solution was adjusted, and Escherichia coli DNA was used as the experimental sample. By comparing the DNA concentration and recovery rate of the test solution extracted using binding solutions with different component ratios, the optimal amount of isopropanol and guanidine hydrochloride was screened out.

[0149] 3. The sample used in this example is the SARS-CoV-2 RBD-NPM binding stock solution, and the dilution factor is 1. The following table shows the binding solution ratio adjustment scheme.

[0150] Table 12 Adjustment scheme of binding liquid ratio

[0151]

[0152]

[0153] The extraction and purification were started according to the extraction procedure of Example 1 and the qPCR system and detection method provided by the pharmacopoeia were used to perform the determination steps.

[0154] 3. Result analysis:

[0155] Table 13 Binding solution usage ratio adjustment results

[0156]

[0157] Because the detection concentration of the low-concentration test sample was extremely low (close to or below the detection limit), the results of this experiment could not be used to directly evaluate the specific impact of the lysis and binding solution with different volume ratios of isopropanol and 5M guanidine hydrochloride on the results based solely on the mean detection concentration (pg / μL). Therefore, it is necessary to compare the recovery data. The recovery of PCS and the spiked test sample in the lysis and binding solution with a volume ratio of isopropanol to 5M guanidine hydrochloride of 3:2 was higher than that in the lysis and binding solution with a volume ratio of isopropanol to 5M guanidine hydrochloride of 1:1. The recovery of the spiked test sample exceeded the acceptance standard (150%). The recovery of PCS and the spiked test sample in the lysis and binding solution with a volume ratio of isopropanol to 5M guanidine hydrochloride of 1:1 was approximately 130%.

[0158] 4. Experimental conclusions:

[0159] Based on the above analysis, the extraction accuracy was higher when the ratio of isopropanol to 5M guanidine hydrochloride in the lysis and binding solution was 1:1 (i.e., the final concentration of the binding solution components for a single sample was 50% isopropanol, 2.5M guanidine hydrochloride, 50 μg / mL glycogen, 1% (m / v) PEG6000, and 5 μg / mL tRNA). Therefore, we chose to continue to optimize the extraction system on this basis.

[0160] Example 4: Optimization of the formulation of washing liquid A

[0161] 1. Experimental purpose: To optimize the nucleic acid extraction process by adjusting the formula of Wash Buffer A to improve the purity and efficiency of nucleic acids extracted from CHO cells and Escherichia coli.

[0162] 2. Experimental design: With other conditions unchanged (the final concentrations of the lysis and binding buffer components of a single sample were 50% isopropanol, 2.5 M guanidine hydrochloride, 50 μg / mL glycogen, 1% (m / v) PEG6000, and 5 μg / mL tRNA, and the formula of wash buffer B was 70% ethanol), three different wash buffer A formulas were set, each containing different concentrations of guanidine hydrochloride, ethanol, and other components that may help improve the extraction effect (such as EDTA and Tris-HCl). CHO cells and Escherichia coli were used as experimental samples. By comparing the DNA concentrations and recoveries of the test sample solutions extracted using wash buffer A with different component ratios, the effects of different wash buffer A formulas on the nucleic acid extraction of these two cell types were evaluated, and the optimal wash buffer A formula was screened out.

[0163] 3. The sample information of this embodiment is as follows:

[0164] Table 14

[0165]

[0166]

[0167] Digestion was performed according to the proteinase K digestion method of Example 2, and the composition of wash solution A was adjusted according to the table below.

[0168] Table 15 Detergent A formula adjustment table

[0169]

[0170] The extraction and purification were started according to the extraction procedure of Example 1 and the qPCR system and detection method provided by the pharmacopoeia were used to perform the determination steps.

[0171] 4. Result analysis:

[0172] Table 16 Washing solution A formula adjustment results table (CHO cells)

[0173]

[0174] As shown in the table above, wash solution A1 (24 mM guanidine hydrochloride + 60% ethanol) achieved the highest detection concentration and a spike recovery rate of nearly 100%, indicating that this formulation is effective for nucleic acid extraction from CHO cells. Wash solutions A2 and A3 achieved slightly lower detection concentrations, and A3 achieved a higher spike recovery rate, potentially indicating that this formulation failed to completely remove impurities during the extraction process.

[0175] Table 17: Cleaning solution A formula adjustment results table (Escherichia coli)

[0176]

[0177]

[0178] As shown in the table above, Washing Solution A1 also exhibited the highest detected concentration, but the spiked recovery was lower (84.9%). This may be due to experimental error or incomplete specific extraction of E. coli nucleic acid by Washing Solution A1. Washing Solution A2 exhibited an unusually high spiked recovery (133%), possibly due to interference from impurities promoted by certain ingredients in this formulation. Washing Solution A3 exhibited a more normal recovery (90.0%), but the detected concentration was lower, indicating that this formulation may not be as effective as Washing Solution A1 in extracting E. coli nucleic acid.

[0179] 5. Experimental conclusion:

[0180] The experimental results show that the final concentration of Wash Buffer A, 24 mM guanidine hydrochloride and 60% ethanol, achieved the highest detection concentrations for both CHO cells and E. coli samples. However, the positive quality control recoveries for E. coli were high, likely due to the influence of certain components in the binding buffer on nucleic acid extraction from E. coli. Therefore, further optimization of the binding buffer formulation is necessary to further improve the accuracy and reliability of nucleic acid extraction.

[0181] Example 5: Optimization of the formulation of washing liquid B

[0182] 1. Experimental purpose: By adjusting the ethanol concentration in Washing Buffer B, compare the effects of different formulations on DNA extraction concentration and recovery rate, and select the optimal Washing Buffer B formulation to optimize DNA extraction results.

[0183] 2. Experimental design: Ethanol within a certain concentration range can effectively remove salt ions from DNA. Different concentrations of ethanol have different effects on the removal of salt ions from DNA. Therefore, under the condition that other conditions remain unchanged (the final concentrations of the lysis and binding solution components of a single sample are 50% isopropanol, 2.5M guanidine hydrochloride, 1μL glycogen, 1% (m / v) PEG6000, 0.2μL tRNA, and the final concentrations of the wash solution A are 24mM guanidine hydrochloride and 60% ethanol), different wash solution B formulas were set, and CHO cell DNA was used as the experimental sample. The DNA concentration and recovery rate measured by the test solution extracted using different wash solutions B were compared.

[0184] 3. The specific sample used was a culture medium harvested from CHO cells expressing SARS-CoV-2 RBD, with a dilution factor of 100,000. Digestion was performed according to the proteinase K digestion method of Example 2, and the following method parameters were adjusted for extraction and determination.

[0185] Table 18 Detergent B formula adjustment table

[0186]

[0187] The corresponding solutions were pre-added according to the following 96-deep-well plate arrangement, and the extraction and purification were started according to the extraction procedure of Example 1, and the qPCR system and detection method provided by the pharmacopoeia were used to perform the determination steps.

[0188] Table 19 96 deep well plate sample loading table

[0189]

[0190]

[0191] 4. Result analysis:

[0192] Table 20 Washing solution B formula adjustment results table (CHO cells)

[0193]

[0194] As can be seen from the above table, when using washing solution B1 (70% ethanol), the detection concentration of the test sample (2933.7 pg / μL) is significantly higher than the detection concentration (974.0 pg / μL) when using washing solution B2 (80% ethanol); although the recovery rate of washing solution B1 (whether PCS or spiked test sample) fluctuates greatly (58.6% and 49.5%), it is generally higher than that of washing solution B2 (50.5% and 48.5%).

[0195] 5. Experimental conclusion:

[0196] Wash Buffer B, formulated with 70% ethanol, demonstrated superior DNA extraction, with higher detection concentrations and relatively high recovery rates. Therefore, we continued to use 70% ethanol as Wash Buffer B in subsequent experiments, and further optimized the binding buffer formulation based on this.

[0197] Example 6: Optimization of lysis and binding solution components

[0198] 1. Experimental purpose: To evaluate the effect of adjusting the PEG6000 concentration in the lysis and binding buffer on the nucleic acid extraction of CHO cells and Escherichia coli, and to screen out the optimal PEG6000 concentration.

[0199] 2. Experimental design: With other conditions unchanged (the final concentrations of the lysis and binding solution components of a single sample are 50% isopropanol, 2.5M guanidine hydrochloride, 50μg / mL glycogen, a certain concentration of PEG6000, and 5μg / mL tRNA, the final concentrations of the wash solution A formula are 24mM guanidine hydrochloride and 60% ethanol, and the wash solution B formula is 70% ethanol), different PEG6000 concentrations are set in the lysis and binding solution, and CHO cells and Escherichia coli are used as experimental samples. The DNA concentrations and recovery rates of the two cell types' nucleic acid test solutions extracted using lysis and binding solutions with different PEG6000 concentrations are compared.

[0200] 3. Sample information:

[0201] Table 21

[0202] Test sample Sample name Dilution multiple 1 60℃ treatment of E. coli (expressing NPM) precipitate and re-dissolved clarified liquid 600 2 CHO cell (expressing SARS-CoV-2 RBD antigen) culture harvest 100000

[0203] 4. Experimental process:

[0204] Digestion was performed according to the proteinase K digestion method determined in Example 2, and the following method parameters were adjusted to perform extraction and determination.

[0205] Table 22 Lysis and Binding Solution Composition Parameter Adjustment Table

[0206]

[0207] The corresponding solutions were pre-added according to the following 96-deep-well plate arrangement, and the extraction and purification were started according to the extraction procedure of Example 1, and the qPCR system and detection method provided by the pharmacopoeia were used to perform the determination steps.

[0208] Table 23 96 deep well plate loading table

[0209]

[0210] 5. Result analysis:

[0211] Table 24 PEG6000 volume fraction adjustment results (CHO cells)

[0212]

[0213] As can be seen from the above table, compared with 1% (m / v) PEG6000, the lysis and binding solution with 10% (m / v) PEG6000 significantly improved the detection concentration and recovery rate of DNA from CHO cells.

[0214] Table 25 PEG6000 volume fraction adjustment results (Escherichia coli)

[0215]

[0216]

[0217] As can be seen from the above table, similar to CHO cells, the lysis and binding solution containing 10% (m / v) PEG6000 also significantly increased the detection concentration and recovery rate of Escherichia coli DNA.

[0218] 6. Experimental conclusion:

[0219] When the volume fraction of PEG6000 in the lysis and binding buffer was 10% (m / v), the detection concentration and recovery rate of DNA from both cell types were significantly improved. Therefore, the optimal lysis and binding buffer composition and final concentration were: 50% isopropanol + 2.5M guanidine hydrochloride + 10% (m / v) PEG6000 + 50 μg / mL glycogen + 5 μg / mL tRNA.

[0220] Example 7: Precision Test

[0221] 1. Use the following scheme to conduct precision test:

[0222] 1) The proteinase K digestion system consists of 10 μL 5 M NaCl, 10 μL proteinase K, 100 μL proteinase K buffer, and the test sample. The digestion condition is set at 55°C for 1 h.

[0223] 2) The composition of the lysis and binding solution for a single sample is: 50% isopropanol + 2.5 M guanidine hydrochloride + 10% (m / v) PEG6000 + 50 μg / mL glycogen + 50 μg / mL tRNA.

[0224] 3) The formula of washing solution A is 24 mM guanidine hydrochloride and 60% ethanol.

[0225] 4) The formula of washing solution B is 70% ethanol.

[0226] 5) The amount of magnetic beads used is 10 μL / well (stock solution concentration 50 mg / mL).

[0227] 2. The qPCR detection method for residual exogenous DNA was determined as follows: the extraction and purification were started according to the extraction procedure of Example 1, and the qPCR system and detection method provided in the pharmacopoeia were used to perform the determination steps.

[0228] 3. Experimental plan:

[0229] (1) 15,000 pg, 300 pg, and 3 pg CHO DNA standards were added to the SARS-CoV-2 RBD-NPM binding stock solution, respectively. The nucleic acid of the samples with and without the addition of the standard was extracted using the method of the present invention. The sample recovery rate and the intermediate precision of different personnel on different days were analyzed. The results showed that the spiked recovery rate was 78.2%-107%, which was consistent with the spiked recovery rate specified in the pharmacopoeia (50%-150%). Different analysts repeated the measurement of each concentration level 24 times on different days. The RSD of the recovery rate was 4.4%-9.8%, all <10%, which was far lower than the RSD ≤ 30% specified in the pharmacopoeia.

[0230] Table 26 Precision and accuracy of the method of the present invention

[0231]

[0232]

[0233] (2) The SARS-CoV-2RBD-NPM binding stock solution was added with or without 0.03 pg CHO DNA standard, and the sample nucleic acid was extracted using the method of the present invention. The sample recovery rate was analyzed. The results showed that even when an extremely low concentration of DNA standard (0.003 pg / μL, the lowest concentration point of the qPCR standard curve) was added to the stock solution with complex components, the average spiked recovery rate of the sample extracted by the method of the present invention was still 87%, with an RSD of <10%.

[0234] Table 27 Accuracy of the method of the present invention

[0235]

[0236] Example 8: Comparative Experiment

[0237] 1. Main reagents:

[0238] Host cell residual DNA sample preparation kits of brands A, B and C, CHO residual DNA detection kit of brand A, and E. coli residual DNA detection kit of brand A.

[0239] 2. Sample information

[0240] Table 28

[0241]

[0242]

[0243] 3. Experimental Plan

[0244] Table 29 Experimental group settings

[0245]

[0246]

[0247] The pretreatment methods of the test samples of Brand A, Brand B and Brand C were all carried out according to the instructions of their respective kits, and the qPCR assay of Brand A was also carried out according to the instructions of its corresponding kit.

[0248] 4. Result analysis:

[0249] Table 30 Comparative experimental results (CHO)

[0250]

[0251] Table 31 Comparative experimental results (Escherichia coli)

[0252]

[0253] From the experimental results in Table 30, it can be seen that the detection concentration of the method of the present invention in most samples is similar to or slightly higher than that of the brand A kit, and the spiked recovery rate is generally high, ranging from 86.0% to 96.5%, which meets the acceptance standard (50% to 150%); while the spiked recovery rate of the brand A kit is low in some samples, especially the SARS-CoV-2RBD-NPM combined stock solution, with a spiked recovery rate of only 48.0%, which does not meet the acceptance standard. The results obtained using the brand B and brand C kits are lower than those of the method of the present invention and the brand A kit method, and the spiked recovery rate is generally low. Comprehensive evaluation shows that the method of the present invention shows good stability in both detection concentration and spiked recovery rate. The spiked recovery rate of the brand A kit fluctuates, and the overall performance of the brand B and brand C kits does not meet the acceptance standard.

[0254] From the experimental results in Table 31, it can be seen that the detection concentration of the method of the present invention in most Escherichia coli samples is similar to or slightly higher than that of the brand A kit, and the spiked recovery rate is generally high, ranging from 84.5% to 115%, which meets the acceptance standard (50% to 150%); the spiked recovery rate of the brand A kit is too high or too low in some samples (between 48.8% and 131%), and there are cases where it does not meet the acceptance standard. The results obtained using the brand B and brand C kits are lower than those of the method of the present invention and the brand A kit method, especially in high-concentration samples, and the spiked recovery rate is also generally low, and there are cases where it does not meet the acceptance standard. Comprehensively evaluated, the method of the present invention shows good stability in both detection concentration and spiked recovery rate. The performance of the brand A kit in some samples is acceptable, but the recovery rate fluctuates. The overall performance of the brand B and brand C kits does not meet the acceptance standard.

[0255] In summary, the above embodiments 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 in the scope of protection of the present invention.

Claims

1. A lysis and binding solution for DNA extraction, characterized in that: The invention comprises guanidine hydrochloride, PEG6000, tRNA, glycogen and isopropanol, wherein the concentration of the guanidine hydrochloride is 2.0-2.5M, the concentration of the PEG6000 is 1%-10% (m / v), and the concentration of the isopropanol is 50-60%.

2. The lysis and binding solution according to claim 1, wherein the concentration of guanidine hydrochloride is 2.5 M, the concentration of PEG6000 is 10% (m / v), The concentration of the isopropanol is 50%, the concentration of the glycogen is 50-125 μg / mL, and the concentration of the tRNA is 5-20 μg / mL; Preferably, the concentration of glycogen is 50 μg / mL, and the concentration of tRNA is 5 μg / mL.

3. A kit for DNA extraction, characterized in that: The method comprises the lysis and binding solution according to claim 1 or 2, washing solution A, washing solution B, DNA elution solution and magnetic beads. 4 . The kit according to claim 3 , wherein the washing solution A comprises 24 mM guanidine hydrochloride and 60% ethanol, and / or the washing solution B comprises 70% ethanol.

5. The kit according to claim 3 or 4, wherein the DNA elution solution is TE buffer.

6. The kit according to any one of claims 3 to 5, wherein the magnetic beads are carboxyl magnetic beads.

7. A method for pre-treating residual DNA samples of host cells, characterized in that: The residual DNA of the host cells is extracted using the lysis and binding solution according to claim 1 or 2, or the kit according to any one of claims 3 to 6.

8. The method according to claim 7, wherein the host cells comprise Chinese hamster ovary cells (CHO cells) and Escherichia coli.

9. The method according to claim 7 or 8, further comprising detecting the amount of residual host cell DNA using qPCR.

10. Use of the lysis and binding solution according to claim 1 or 2 or the kit according to any one of claims 3 to 6 in extracting nucleic acids from a sample.

Images