Method for releasing gamma-retroviral vector nucleic acid in PG13 cell culture supernatant and releasing agent thereof

Abstract

The invention provides a method for releasing gamma-retroviral vector nucleic acid in PG13 cell culture supernatant and a releasing agent thereof. The method comprises the following steps: step 1, carrying out enzyme digestion on a cell supernatant sample containing a gamma-retroviral vector produced by packaging cell PG13 culture; 2, a nucleic acid releasing agent is added into the sample subjected to enzyme digestion in the step 1, nucleic acid releasing is conducted, and the nucleic acid releasing agent is composed of guanidine isothiocyanate, absolute ethyl alcohol, isopropyl alcohol and enzyme-free water. The method disclosed by the invention is suitable for a sample nucleic acid release method in which host DNA needs to be removed and virus vector nucleic acid is RNA, and has no interference effect on subsequent quantitative detection. According to the method, the nucleic acid release effect of first enzyme digestion and then release is obviously better than that of an existing method of first nucleic acid release and then enzyme digestion.

Description

Technical Field

[0001] The invention relates to the field of retroviral vectors, and in particular to a method for releasing gamma-retroviral vector nucleic acid in PG13 cell culture supernatant and a releasing agent thereof. Background Art

[0002] PG13 cells are used as packaging cells in the development of cell products such as CAR-T or TCR-T. PG13 cells are used to package retroviral vectors, produce retroviral vectors, and then transduce T cells to make cell preparations. CAR-T cells constructed by γ-retroviral vectors have been widely used in clinical trials for tumor treatment. The production and harvesting of viral vectors is to collect the supernatant of PG13 cell culture. The copy number of the viral vector in the supernatant directly affects the effect of viral vector transduction of T cells, thereby directly affecting the copy number of CAR in CAR-T cells and affecting the quality of CAR-T. Therefore, the detection of its copy number is crucial.

[0003] Since the γ-retroviral vector produced by packaging cell PG13 culture exists in the cell culture supernatant, the cell culture supernatant contains host residual DNA and viral vector nucleic acid RNA, and the host DNA contains the gene sequence of the viral vector. Therefore, the quantitative detection of viral vectors requires the removal of host DNA. In the past, all nucleic acids in the cell culture supernatant were first extracted, and then the host DNA was removed by DNA enzyme digestion, reverse transcription, and finally the viral vector RNA was quantified by quantitative PCR. The nucleic acid extraction process requires multiple steps, the use of multiple reagents and equipment, which is time-consuming, labor-intensive, and costly, and may affect the quality and yield of nucleic acids.

[0004] With the advancement of technology, nucleic acid extraction-free methods have gradually emerged and been applied in the qualitative and quantitative detection of nucleic acids, but this method still has some problems. For example, in Chinese patent CN114990192A, the composition of the nucleic acid releaser is relatively complex, which has an inhibitory interference effect on the subsequent RT-qPCR reagent. If the subsequent RT-qPCR quantitative detection is performed, it will affect the quantitative accuracy and make the result value low; if a qualitative test is performed, the sample contains a low amount of virus, which may cause a false negative in the qualitative result. In order to solve the above problems, the present invention proposes to directly perform enzyme digestion on the sample first, and then add a nucleic acid release component that has no inhibitory interference effect on the subsequent RT-qPCR quantitative detection to obtain nucleic acid. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for releasing γ-retroviral vector nucleic acid in PG13 cell culture supernatant, the method comprising the following steps: step 1, enzymatically digesting a cell supernatant sample containing a γ-retroviral vector produced by culturing packaging cells PG13; and step 2, adding a nucleic acid releaser to the sample after enzymatic digestion in step 1 to release the nucleic acid, the nucleic acid releaser consisting of guanidine isothiocyanate, anhydrous ethanol, isopropanol and enzyme-free water; step 1 and step 2 are reacted in the same tube.

[0006] In one embodiment, the volume content of 50 mmol / L guanidine isothiocyanate in the nucleic acid releasing agent is 25%-40%.

[0007] In one embodiment, the volume content of anhydrous ethanol in the nucleic acid releasing agent is 25%-27%.

[0008] In one embodiment, the volume content of isopropanol in the nucleic acid releasing agent is 15%-30%.

[0009] In one embodiment, the nucleic acid releasing agent is composed of 30% volume of 50 mmol / L guanidine isothiocyanate, 27% volume of anhydrous ethanol, 30% volume of isopropanol and 13% volume of enzyme-free water.

[0010] Nucleic acid releaser: can quickly destroy the protein structure of cells, microorganisms, and viruses that encapsulate nucleic acids, ensuring the effective release of nucleic acids.

[0011] Ribonuclease (RNase) is a nuclease that can hydrolyze RNA into small molecules.

[0012] RNase inhibitors: ribonuclease (RNase) inhibitors, which can bind to RNase in the form of non-covalent bonds to inactivate RNase; protect the released RNA from being degraded by RNase.

[0013] RT-qPCR amplification system: the reagents and volume ratios used for viral nucleic acid RNA amplification reactions. Sample amplification results, Ct values ​​have numerical values. If the Ct value difference of parallel samples is greater than 0.5 for the same batch of samples or different batches of samples amplified at the same time, the data is unreliable and needs to be re-amplified; if the Ct value difference of parallel samples is not greater than 0.5, the data is reliable; Ct values ​​have numerical values, and the smaller the value, the more nucleic acid.

[0014] qPCR (NRT control) amplification system: the reagents and volume ratios used for DNA amplification reactions. The control amplification system set up during RT-qPCR amplification is used to check whether the DNA has been completely removed after enzyme digestion. After the sample is digested, if the Ct value of the amplification result is N / A, that is, there is no sign of amplification, indicating that the DNA fragment has been completely digested; if the Ct value of the amplification result has a value, that is, there is a sign of amplification, indicating that the DNA digestion is not thorough and has not been completely removed.

[0015] The disadvantage of the existing technology is that it is not suitable for quantitative detection of viral vector samples that require the removal of host DNA. After the samples are subjected to extraction-free treatment, they interfere with the subsequent RT-qPCR reaction, inhibiting the quantitative reaction effect, resulting in low quantification and low accuracy.

[0016] PG13 cells are used as packaging cells for viral vectors, and their genome contains the genetic structure of the viral vector. Therefore, when quantitatively detecting the packaged viral vector, it is necessary to remove the DNA of the host cell PG13 in order to accurately quantify the viral vector nucleic acid RNA.

[0017] The nucleic acid of the γ-retroviral vector is two identical single-stranded positive-strand RNAs. The detection of the number of nucleic acid copies is a common method for quantitatively detecting the number of viral vector copies. In the production process of γ-retroviral vectors, the final harvested viral vector is the cell culture supernatant, that is, the viral vector exists in the PG13 cell culture supernatant. To quantitatively detect the nucleic acid of the viral vector, first release the nucleic acid from the shell, and then achieve quantitative detection through reverse transcription, qPCR or RT-qPCR.

[0018] This method is aimed at detecting samples that are cell culture supernatants. In the cell culture supernatants, there are no other sample nucleic acids except for the residual DNA of the host PG13 cells and the viral vector nucleic acid. The viral vector structure is simpler than that of animal and plant tissues, cells, and microorganisms, and the membrane and protein that encapsulate the nucleic acid are more easily damaged. Based on the characteristic that the protein structure that encapsulates the sample nucleic acid is easily damaged, the present invention has developed a nucleic acid releaser that can easily, quickly, and low-costly obtain nucleic acid in the sample. The nucleic acid releaser can promote the rapid release of γ-retroviral vector nucleic acid in the PG13 cell culture supernatant and has no interference with the subsequent RT-qPCR reaction.

[0019] The present invention provides a method for releasing γ-retroviral vector nucleic acid in PG13 cell culture supernatant, which is applicable to a sample nucleic acid release method in which host DNA needs to be removed and the viral vector nucleic acid is RNA, and has no interference with subsequent quantitative detection. In the method of the present invention, the nucleic acid release effect of first enzymatic cleavage and then release is significantly better than the existing method of first nucleic acid release and then enzymatic cleavage.

[0020] In the present invention, the host DNA removal and release process is completed in one reaction tube, which simplifies the operation process, avoids degradation caused by the transfer of sample nucleic acid RNA in different containers, and ensures that there is no loss of nucleic acid after release; and because of the simple formula, after being treated with two reagents, an enzyme cutting reagent and a nucleic acid releaser, especially the nucleic acid releaser of the present invention has a simple composition, the treated sample and reagent mixture has no inhibitory effect on the subsequent RT-qPCR reaction. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0022] Example 1

[0023] 1. Open the biosafety cabinet, sterilize with UV for 30 minutes, and then run for 15 minutes. Take out the cell supernatant sample A containing the CD19-CAR retroviral vector (γ-retroviral vector) produced by packaging cell PG13 culture and stored in a -80℃ refrigerator. The copy number of the viral vector is 1.0×10 8 copies / mL, put it into the biosafety cabinet, thaw it at room temperature, and use it for the next step. Take out the reagents used for enzyme digestion, nucleic acid release, RT-qPCR amplification system, and qPCR (NRT control) amplification system, put them into the biosafety cabinet, and wait for use.

[0024] 2. Enzyme Digestion Process

[0025] 2.1.DNase I and 10×Reaction Buffer were purchased from Novizan Biotechnology Co., Ltd.; RNase inhibitor was purchased from Beyotime Biotechnology Co., Ltd.; the enzyme digestion premix was prepared according to the following ratio: 10×Reaction Buffer: DNase I: ultrapure water: RNase inhibitor (2U / μL) = 8:8:23.6:0.4: volume ratio, take 80μL 10×Reaction Buffer, 80μL DNase I, 236μL ultrapure water, 4μL RNase inhibitor, add to a 1.5mL centrifuge tube, vortex mix for 15 seconds, which is the enzyme digestion premix;

[0026] 2.2. Take the melted sample and vortex mix for 15 seconds;

[0027] 2.3. Take four 1.5 ml centrifuge tubes, label them 1, 2, 3, and 4, add 40 μL of enzyme digestion premix and 5 μL of sample to each tube, and vortex mix for 15 seconds;

[0028] 2.4. Keep the mixture in 2.3 in a 37℃ water bath for 15 minutes and in a metal bath at 90℃ for 5 minutes.

[0029] 2.5. Return to room temperature for subsequent operations.

[0030] 3. Viral vector nucleic acid release

[0031] 3.1. Prepare the following formula of nucleic acid release agent according to volume ratio:

[0032] Formula 1 (see the formula of "Development of Trace Nucleic Acid Releaser Based on RT-RPA", see "China Frontier Health and Quarantine Journal", Volume 45, Issue 3, June 2022, pages 172-174. Guanidine isothiocyanate in the formula is a powerful protein denaturant, and its main function is to lyse cells to release proteins and nucleic acids in cells. Tween-20 is a non-ionic surfactant that can be used for cell lysis. Triton X-100 can promote the dissolution of lipids and proteins and is used for cell lysis, protein extraction, etc. Isopropanol can protect the hydrophilic groups in the RNA chain through the hydrophobic effect of hydroxyl groups, and dissolve some organic impurities and proteins through the washing effect of ethanol): 40% 50mmol / L guanidine isothiocyanate + 0.05% Tween20 + 0.05% TritonX100 + 25% anhydrous ethanol + 20% isopropanol + 14.9% enzyme-free water;

[0033] Formula 2: 40% 50mmol / L guanidine thiocyanate + 0.05% Tween20 + 25% anhydrous ethanol + 20% isopropanol + 14.95% enzyme-free water;

[0034] Formula 3: 40% 50mmol / L guanidine thiocyanate + 0.05% TritonX100 + 25% anhydrous ethanol + 20% isopropanol + 14.95% enzyme-free water;

[0035] Formula 4: 40% 50mmol / L guanidine thiocyanate + 25% anhydrous ethanol + 20% isopropanol + 15% enzyme-free water.

[0036] 3.2. Take 45 μL of each of the nucleic acid release agents of formula 1, 2, 3, and 4, add them to the enzyme-cut sample tubes, vortex and mix for 60 seconds, let stand at room temperature for 5 minutes, and then add 410 μL of PBS. Use for subsequent qPCR amplification. The components of the RT-qPCR amplification system and the qPCR (NRT control) amplification system are as follows:

[0037]

[0038] The RT-qPCR amplification system program is as follows

[0039]

[0040] 3.3. The amplification data are shown in the following table. The results show that after the nucleic acid obtained by formula 4 was amplified by RT-qPCR, the Ct value was the smallest. The smaller the Ct value, the more nucleic acid. After the samples were released by nucleic acid in each group of formulas, the RT-qPCR amplification results are shown in the following table.

[0041]

[0042] 3.4 Conclusion: In the method of the present invention, the nucleic acid release amount of formula 4 without Tween20 and TritonX100 is higher, and this formula has a good nucleic acid release effect. There is no sign of amplification in the NRT control, indicating that the DNA has been completely removed after enzyme digestion.

[0043] Example 2

[0044] 1. Open the biosafety cabinet, sterilize with UV for 30 minutes, and then run for 15 minutes. Take out the cell supernatant sample B containing the CD19-CAR retroviral vector (γ-retroviral vector) produced by packaging cell PG13 culture and stored in a -80℃ refrigerator. The copy number of the viral vector is 1.0×10 10 copies / mL, put it into the biosafety cabinet, thaw it at room temperature, and use it in the next step. Take out the reagents used for enzyme digestion, nucleic acid release, RT-qPCR amplification system, and qPCR (NRT) amplification system, put them into the biosafety cabinet, and wait for use.

[0045] 2. Enzyme Digestion Process

[0046] 2.1. DNase I and 10×Reaction Buffer were purchased from Novizan Biotechnology Co., Ltd.; RNase inhibitor was purchased from Beyotime Biotechnology Co., Ltd.; the enzyme digestion premix was prepared according to the following ratio: 10×Reaction Buffer: DNase I: ultrapure water: RNase inhibitor (2U / μL) = 8:8:23.6:0.4: volume ratio, take 80μL 10×Reaction Buffer, 80μL DNase I, 236μL ultrapure water, 4μL RNase inhibitor, add to a 1.5mL centrifuge tube, vortex mix for 15 seconds, which is the enzyme digestion premix;

[0047] 2.2. Take the melted sample and vortex mix for 15 seconds;

[0048] 2.3. Take four 1.5 ml centrifuge tubes, label them 4, 5, 6, and 7, add 40 μL of enzyme digestion premix and 5 μL of sample to each tube, and vortex mix for 15 seconds;

[0049] 2.4. Keep the mixture in 2.3 in a 37℃ water bath for 15 minutes and in a metal bath at 90℃ for 5 minutes.

[0050] 2.5. Restore to room temperature for subsequent operations.

[0051] 3. Viral vector nucleic acid release

[0052] 3.1. In the preferred embodiment 1, the proportion of guanidine isothiocyanate is adjusted to prepare the following formula by volume ratio for nucleic acid release:

[0053] Formula 4: 40% 50mmol / L guanidine thiocyanate + 25% anhydrous ethanol + 20% isopropanol + 15% enzyme-free water

[0054] Formula 5: 30% 50mmol / L guanidine isothiocyanate + 25% anhydrous ethanol + 20% isopropanol + 25% enzyme-free water

[0055] Formula 6: 35% 50mmol / L guanidine thiocyanate + 25% anhydrous ethanol + 20% isopropanol + 20% enzyme-free water

[0056] Formula 7: 45% 50mmol / L guanidine isothiocyanate + 25% anhydrous ethanol + 20% isopropanol + 10% enzyme-free water

[0057] 3.2. Take 45 μL of each of the nucleic acid release agents of formula 4, 5, 6, and 7, add them to the enzyme-cut sample tubes, vortex mix for 60 seconds, let stand at room temperature for 5 minutes, and then add 410 μL of PBS. Use for subsequent qPCR amplification. The components of the RT-qPCR amplification system and the qPCR (NRT control) amplification system are as follows:

[0058]

[0059] The RT-qPCR amplification system program is as follows

[0060]

[0061] 3.3 The amplification data are shown in the following table. The results show that after the nucleic acid obtained from formula 5 is amplified by RT-qPCR, the Ct value is the smallest. The smaller the Ct value, the more nucleic acid there is.

[0062]

[0063] 3.4. Conclusion: When the content of 50mmol / L guanidine isothiocyanate in the formula is 25%-40%, the effect is relatively high in nucleic acid release. Among them, formula 5: 30% 50mmol / L guanidine isothiocyanate + 25% anhydrous ethanol + 20% isopropanol + 25% enzyme-free water has a relatively high nucleic acid release. This formula has a good nucleic acid release effect. There is no sign of amplification in the NRT control, indicating that the DNA has been completely removed after enzyme digestion.

[0064] Example 3

[0065] 1. Open the biosafety cabinet, sterilize with UV for 30 minutes, and run for 15 minutes. Take out the cell supernatant sample C containing the CD19-CAR retroviral vector (γ-retroviral vector) produced by packaging cell PG13 culture and stored in a -80℃ refrigerator. The copy number of the viral vector is 9.2×10 9 copies / mL, put it into the biosafety cabinet, thaw it at room temperature, and use it for the next step. Take out the reagents used for enzyme digestion, nucleic acid release, RT-qPCR amplification system, and qPCR (NRT control) amplification system, put them into the biosafety cabinet, and wait for use.

[0066] 2. Enzyme Digestion Process

[0067] 2.1.DNase I and 10×Reaction Buffer were purchased from Novizan Biotechnology Co., Ltd.; RNase inhibitor was purchased from Beyotime Biotechnology Co., Ltd.; the enzyme digestion premix was prepared according to the following ratio: 10×Reaction Buffer: DNase I: ultrapure water: RNase inhibitor (2U / μL) = 8:8:23.6:0.4: volume ratio, take 80μL 10×Reaction Buffer, 80μL DNase I, 236μL ultrapure water, 4μL RNase inhibitor, add to a 1.5mL centrifuge tube, vortex mix for 15 seconds, which is the enzyme digestion premix;

[0068] 2.2. Take the melted sample and vortex mix for 15 seconds;

[0069] 2.3. Take four 1.5 ml centrifuge tubes, label them 5, 8, 9, and 10, add 40 μL of enzyme digestion premix and 5 μL of sample to each tube, and vortex mix for 15 seconds;

[0070] 2.4. Keep the mixture in 2.3 in a 37℃ water bath for 15 minutes and in a metal bath at 90℃ for 5 minutes.

[0071] 2.5. Return to room temperature for subsequent operations.

[0072] 3. Viral vector nucleic acid release

[0073] 3.1 In the preferred embodiment 2, the proportion of anhydrous ethanol was adjusted to prepare the following formula by volume ratio for nucleic acid release and RT-qPCR amplification test:

[0074] Formula 5: 30% 50mmol / L guanidine isothiocyanate + 25% anhydrous ethanol + 20% isopropanol + 25% enzyme-free water

[0075] Formula 8: 30% 50mmol / L guanidine isothiocyanate + 20% anhydrous ethanol + 20% isopropanol + 30% enzyme-free water

[0076] Formula 9: 30% 50mmol / L guanidine isothiocyanate + 30% anhydrous ethanol + 20% isopropanol + 20% enzyme-free water

[0077] Formula 10: 30% 50mmol / L guanidine isothiocyanate + 35% anhydrous ethanol + 20% isopropanol + 15% enzyme-free water

[0078] 3.2 Take 45 μL of each of the nucleic acid release agents of formula 5, 8, 10, and 10, add them to the enzyme-cut sample tubes, vortex mix for 60 seconds, let stand at room temperature for 5 minutes, and then add 410 μL of PBS. Use for subsequent qPCR amplification. The components of the RT-qPCR amplification system and the qPCR (NRT control) amplification system are as follows:

[0079]

[0080] The RT-qPCR amplification system program is as follows

[0081]

[0082] 3.3 The amplification data are shown in the following table. The results show that after the nucleic acid obtained from formula 9 is amplified by RT-qPCR, the Ct value is the smallest. The smaller the Ct value, the more nucleic acid there is.

[0083]

[0084]

[0085] 3.4 Conclusion: The release effect is better when the content of anhydrous ethanol is 25%-27%. Formula 9: 30% 50mmol / L guanidine isothiocyanate + 27% anhydrous ethanol + 20% isopropanol + 23% enzyme-free water, the nucleic acid release amount is higher, and this formula has a good nucleic acid release effect. There is no sign of amplification in the NRT control, indicating that the DNA has been completely removed after enzyme digestion.

[0086] Example 4

[0087] 1. Open the biosafety cabinet, sterilize with UV for 30 minutes, and run for 15 minutes. Take out the cell supernatant sample D containing the CD19-CAR retroviral vector (γ-retroviral vector) produced by packaging cell PG13 culture and stored in a -80℃ refrigerator. The copy number of the viral vector is 9.6×10 9 copies / mL, put it into the biosafety cabinet, thaw it at room temperature, and use it for the next step. Take out the reagents used for enzyme digestion, nucleic acid release, RT-qPCR amplification system, and qPCR (NRT control) amplification system, put them into the biosafety cabinet, and wait for use.

[0088] 2. Enzyme Digestion Process

[0089] 2.1.DNase I and 10×Reaction Buffer were purchased from Novizan Biotechnology Co., Ltd.; RNase inhibitor was purchased from Beyotime Biotechnology Co., Ltd.; the enzyme digestion premix was prepared according to the following ratio: 10×Reaction Buffer: DNase I: ultrapure water: RNase inhibitor (2U / μL) = 8:8:23.6:0.4: volume ratio, take 80μL 10×Reaction Buffer, 80μL DNase I, 236μL ultrapure water, 4μL RNase inhibitor, add to a 1.5mL centrifuge tube, vortex mix for 15 seconds, which is the enzyme digestion premix;

[0090] 2.2. Take the melted sample and vortex mix for 15 seconds;

[0091] 2.3. Take four 1.5 ml centrifuge tubes, label them 9, 11, 12, and 13, add 40 μL of enzyme digestion premix and 5 μL of sample to each tube, and vortex mix for 15 seconds;

[0092] 2.4. Keep the mixture in 2.3 in a 37℃ water bath for 15 minutes and in a metal bath at 90℃ for 5 minutes.

[0093] 3. Viral vector nucleic acid release

[0094] 3.1. In the preferred embodiment 3, the proportion of isopropanol in formula 9 is adjusted, and the following formula is prepared by volume ratio for nucleic acid release and RT-qPCR amplification test.

[0095] Formula 9: 30% 50mmol / L guanidine isothiocyanate + 27% anhydrous ethanol + 20% isopropanol + 23% enzyme-free water

[0096] Formula 11: 30% 50mmol / L guanidine isothiocyanate + 27% anhydrous ethanol + 15% isopropanol + 28% enzyme-free water

[0097] Formula 12: 30% 50mmol / L guanidine isothiocyanate + 27% anhydrous ethanol + 25% isopropanol + 18% enzyme-free water

[0098] Formula 13: 30% 50mmol / L guanidine isothiocyanate + 27% anhydrous ethanol + 30% isopropanol + 13% enzyme-free water

[0099] 3.2. Take 45 μL of each of the nucleic acid release agents of formula 9, 11, 12, and 13, add them to the enzyme-cut sample tubes, vortex and mix for 60 seconds, let stand at room temperature for 5 minutes, and then add 410 μL of PBS. Use for subsequent qPCR amplification. The components of the RT-qPCR amplification system and the qPCR (NRT control) amplification system are as follows:

[0100]

[0101] The RT-qPCR amplification system program is as follows

[0102]

[0103] 3.3. The amplification data are shown in the following table. The results show that the Ct value is the smallest after RT-qPCR amplification of the nucleic acid obtained from formula 13.

[0104] Note: The smaller the Ct value, the more nucleic acid there is.

[0105]

[0106] 3.4 Conclusion: The release effect is better when the content of isopropanol is between 15% and 30%. Formula 13: 30% 50mmol / L guanidine isothiocyanate + 27% anhydrous ethanol + 30% isopropanol + 13% enzyme-free water, the nucleic acid release amount is higher, and this formula has a good nucleic acid release effect. There is no sign of amplification in the NRT control, indicating that the DNA has been completely removed after enzyme digestion.

[0107] Example 5

[0108] Refer to the specific process of Example 4, except that the order of the enzyme cleavage process and the nucleic acid release process is reversed, and the experiment is carried out using Formula 13.

[0109] Solution 1: The sample is first digested by enzymes, and then nucleic acid is released. The final dilution factor of the sample is 100 times. It will be used for subsequent RT-qPCR amplification.

[0110] Solution 2: The sample is first subjected to nucleic acid release, then enzyme digestion, and the final sample dilution factor is 100 times. It will be used for subsequent RT-qPCR amplification.

[0111]

[0112] From the analysis of the experimental results, the effect of scheme 1 in which the sample is first digested by enzyme and then the nucleic acid is released is higher than that of scheme 2 in which the sample is first released by nucleic acid and then digested by enzyme.

[0113] Example 6

[0114] 1. Open the biosafety cabinet, sterilize with UV for 30 minutes, and run for 15 minutes. Take out the cell supernatant sample E containing the CD19-CAR retroviral vector (γ-retroviral vector) produced by packaging cell PG13 culture and stored in a -80℃ refrigerator. The copy number of the viral vector is 6.0×10 9 copies / mL, put it into the biosafety cabinet, thaw it at room temperature, and use it for the next step. Take out the reagents used for enzyme digestion, nucleic acid release, RT-qPCR amplification system, and qPCR (NRT control) amplification system, put them into the biosafety cabinet, and wait for use.

[0115] 2. Enzyme Digestion Process

[0116] 2.1.DNase I and 10×Reaction Buffer were purchased from Novizan Biotechnology Co., Ltd.; RNase inhibitor was purchased from Beyotime Biotechnology Co., Ltd.; the enzyme digestion premix was prepared according to the following ratio: 10×Reaction Buffer: DNase I: ultrapure water: RNase inhibitor (2U / μL) = 8:8:23.6:0.4: volume ratio, take 80μL 10×Reaction Buffer, 80μL DNase I, 236μL ultrapure water, 4μL RNase inhibitor, add to a 1.5mL centrifuge tube, vortex mix for 15 seconds, which is the enzyme digestion premix;

[0117] 2.2. Take the melted sample and vortex mix for 15 seconds;

[0118] 2.3. Take 6 1.5mL centrifuge tubes, label 1, 2, 3, 4, 5, 6, add 40μL of enzyme digestion premix and 5μL of sample to each tube, vortex mix for 15 seconds; vortex mix for 15 seconds;

[0119] 2.4. Keep the mixture in 2.3 in a 37℃ water bath for 15 minutes and in a metal bath at 90℃ for 5 minutes.

[0120] 3. Viral vector nucleic acid release

[0121] 3.1. Add 40 μL, 80 μL, 160 μL, 200 μL, 225 μL, and 250 μL of the nucleic acid releaser of Formula 13 in Example 3 in sequence, vortex mix for 15 seconds, and let stand at room temperature for 5 minutes.

[0122] 3.2.5 minutes later, add 415μL, 375μL, 295μL, 255μL, 230μL, and 205μL to tubes 1, 2, 3, 4, 5, and 6 respectively to make up the total volume to 500μL, and vortex to mix. Use for subsequent qPCR amplification. The components of the RT-qPCR amplification system and the qPCR (NRT control) amplification system are as follows:

[0123]

[0124] The RT-qPCR amplification system program is as follows

[0125]

[0126] The results are shown in the following table. The nucleic acid amplification Ct of samples with tube numbers 5 and 6 is relatively small, indicating that when the volume of nucleic acid releaser was 225 and 250, more nucleic acid was obtained in this sample. The smaller the Ct value, the more nucleic acid is obtained.

[0127]

[0128]

[0129] 7. Conclusion: The volume of the viral vector is 5 μL, and the copy number is less than 1.0×10 10 The volume of the nucleic acid releaser is within the range of 200-250 μL, and the viral vector nucleic acid can be fully released. There is no sign of amplification in the NRT control, indicating that the DNA has been completely removed after enzyme digestion.

[0130] Example 7

[0131] A cell supernatant sample containing the CD19-CAR retroviral vector produced by culturing the packaging cell PG13 was taken, and the final dilution multiple of the sample to be tested was 100 times. The nucleic acid obtained by the method of the present invention (the nucleic acid releaser is the nucleic acid releaser of Formula 13 in Example 3) was compared with the nucleic acid obtained after extraction and enzyme digestion with a domestic superior nucleic acid extraction kit, as shown in the following table. The results of RT-qPCR amplification of nucleic acids obtained by the two schemes showed that the Ct value of the sample nucleic acid amplification result obtained by the method of the present invention was smaller than the Ct value of the sample nucleic acid amplification result after extraction and enzyme digestion with the extraction kit, which means that the amount of nucleic acid obtained by the method of the present invention is higher than the amount of nucleic acid obtained by the kit first extracted and then enzyme digested. NRT is a control to verify whether there is residual DNA for amplification, and N / A is DNA no amplification data, which means that the DNA is completely removed.

[0132]

[0133] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A method for releasing γ-retroviral vector nucleic acid from PG13 cell culture supernatant, characterized in that: The method comprises the following steps: step 1, enzymatically digesting a cell supernatant sample containing a gamma-retroviral vector produced by culturing packaging cells PG13; and step 2, adding a nucleic acid releaser to the sample digested in step 1 to release nucleic acid, wherein the nucleic acid releaser is composed of guanidine isothiocyanate, anhydrous ethanol, isopropanol and enzyme-free water; and steps 1 and 2 are reacted in the same tube.

2. The method for releasing nucleic acid according to claim 1, characterized in that: The volume content of 50mmol / L guanidine isothiocyanate in the nucleic acid releaser is 25%-40%.

3. The method for releasing nucleic acid according to claim 1, characterized in that: The volume content of anhydrous ethanol in the nucleic acid releaser is 25%-27%.

4. The method for releasing nucleic acid according to claim 1, characterized in that: The volume content of isopropanol in the nucleic acid releaser is 15%-30%.

5. The method for releasing nucleic acid according to claim 1, characterized in that: The nucleic acid releaser is composed of 30% by volume of 50mmol / L guanidine isothiocyanate, 27% by volume of anhydrous ethanol, 30% by volume of isopropanol and 13% by volume of enzyme-free water.

6. The nucleic acid releasing agent used in any one of the methods of claims 1 to 5, wherein the nucleic acid releasing agent consists of guanidine isothiocyanate, anhydrous ethanol, isopropanol and enzyme-free water.

7. The nucleic acid releasing agent according to claim 6, characterized in that The volume content of 50mmol / L guanidine isothiocyanate in the nucleic acid releaser is 25%-40%.

8. The nucleic acid releasing agent according to claim 6, characterized in that The volume content of anhydrous ethanol in the nucleic acid releaser is 25%-27%.

9. The nucleic acid releasing agent according to claim 6, characterized in that The volume content of isopropanol in the nucleic acid releaser is 15%-30%.

10. The nucleic acid releasing agent according to claim 6, characterized in that: The nucleic acid releaser is composed of 30% by volume of 50mmol / L guanidine isothiocyanate, 27% by volume of anhydrous ethanol, 30% by volume of isopropanol and 13% by volume of enzyme-free water.

Images