A method for detecting the distribution of residual recombinant lentivirus host cell DNA fragments

Abstract

The application discloses a method for detecting residual fragment distribution of recombinant lentivirus host cell DNA, which uses real-time fluorescent quantitative PCR to accurately detect HEK293T host cell DNA residual in 122bp, 244bp and 562bp three fragment length distribution in a recombinant lentivirus sample. The method of the application adopts a novel prepGEM enzyme to crack the rLVV virus, and the virus is cracked within 5-15 minutes, the enzyme is inactivated within 2-10 minutes, the virus sample amount is small, and the virus cracking solution can be directly applied to a real-time fluorescent quantitative PCR reaction. Compared with the magnetic bead method, the prepGEM enzyme cracking method is more stable and reasonable, and the variance and CV are smaller.

Description

TECHNICAL FIELD

[0001] The present application relates to a detection method, in particular to a method for detecting the distribution of residual viral host cell DNA fragments. BACKGROUND

[0002] According to the report released by the market research agency Fortune Business Insights, the market size of gene therapy was 36.1 billion US dollars in 2019, and is expected to reach 356.7 billion US dollars by 2027, with an annual compound growth rate of 33.6% during the forecast period. At present, the viral carriers of gene drugs mainly include four categories: adeno-associated virus (AAV), lentivirus (LVV), adenovirus (Adenovirus) and retrovirus (Retrovirus).

[0003] Lentivirus (abbreviated as LVV) belongs to the Retroviridae family, has a double-stranded RNA structure, and has a diameter of about 120 nm. Its genome structure is complex and contains genes that can encode three major viral structural proteins: gag, pol and env. The gag gene encodes viral core proteins such as nucleocapsid protein (p7), inner membrane protein (p17) and capsid protein (p24); the pol gene encodes viral replication-related enzymes; the env gene encodes viral envelope glycoprotein. In addition, the viral genome also encodes two regulatory proteins tat and rev. Rev is mainly involved in the expression level of protein regulation, and tat is involved in the control of protein transcription, which promotes the transcription of all genes of the virus after binding with the long terminal repeat (LTR) of the virus. Lentivirus also encodes four auxiliary proteins, namely vif, vpr, vpu and nef. On the genomic structure of HIV-1, there are also other sequence structures required for the life history of the virus, such as signals required for viral replication and packaging, etc.

[0004] Recombinant Lentivirus (abbreviated as rLVV) is a gene therapy vector obtained by modification based on HIV-1 (Human Immunodeficiency Virus-1, abbreviated as HIV-1). By removing the pathogenic gene and reducing the homology between the auxiliary plasmid and the vector plasmid, the lentivirus has the characteristics of higher titer, better biological safety, stronger ability to introduce exogenous fragments, etc. At present, the lentivirus system has been widely used in cell experiments and animal experiments for functional research such as gene overexpression, RNA interference, miRNA research and gene knockout.

[0005] The rLVV viral vector can insert no more than 8 kb of exogenous genes, and is a highly efficient gene delivery vector that can efficiently infect dividing and non-dividing cells. The primary infection of lentivirus is mainly in lymphocytes and macrophages, and in addition, it can also efficiently infect neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells and other types. Lentivirus has low immunogenicity and can stably express transgenes for a long time.

[0006] In cell therapy, rLVV virus is usually used to modify chimeric antigen receptor modified T cells (CAR-T cells) or T cell receptor-gene engineered T cells (TCR-T cells), and rLVV virus is also used for CRISPR / Cas9 library construction.

[0007] As an important intermediate product in the production process of CAR-T cells, the quality of rLVV virus has an important influence on CAR-T cells. Therefore, rLVV virus should also be managed according to the concept of product.

[0008] In the production process of rLVV virus, the residual host cell DNA (HCD) is one of the important parameters for characterizing the quality of rLVV products. Generally, the content of HCD residual should be less than 10 ng / Dose, and the residual DNA fragment should be less than 200 bp. Accurate detection of the residual host cell DNA of rLVV virus and the fragment distribution less than or greater than 200 bp is required by regulations and clinical safety requirements.

[0009] At present, the detection of residual host cell DNA of rLVV virus is generally carried out by using magnetic bead method to extract the DNA wrapped in the viral capsid protein and the DNA outside the capsid, and then detecting the HCD residual. There is also a method of using proteinase K to lyse the virus, and then detecting the residual host cell DNA. These two methods have complicated operation steps, long time-consuming, and large fluctuation of data. The magnetic bead method requires at least 100-200 μL of virus sample, and the elution volume is at least 100-200 μL. Since the magnetic bead method has a large error in recovering DNA itself, the detected HCD data fluctuates greatly, and the variance is large. The method of using proteinase K to lyse the virus requires that the lysis solution be diluted by at least 100 times before being used for qPCR reaction. For the final product of virus with high purity and low HCD residual, the signal may not be detected, or the error of the detection result is large. SUMMARY

[0010] The application aims to provide a method for accurately detecting the length distribution of HEK293T host cell DNA residues at 122bp, 244bp and 562bp in a recombinant lentivirus (rLVV) sample by using real-time fluorescence quantitative polymerase chain reaction (PCR). The application has the advantages that the method is simple to operate, the enzyme lysis and enzyme inactivation are performed in the same microcentrifuge tube, no reagent is added in the middle, and the labor cost is low. From the treatment of the virus to the acquisition of the DNA residue data, the whole process only needs 2.5 hours; while the magnetic bead method has 33 steps in the sample preparation stage, the labor cost is high, and from the extraction of the virus DNA to the acquisition of the DNA residue data, the whole process needs at least 5.5 hours. The HEK293T host cell DNA residue data obtained by the detection method is stable, and the variance and coefficients of variation (CV) at the three fragment lengths of 122bp, 244bp and 562bp are smaller than or equivalent to those of the magnetic bead method.

[0011] To achieve the above object, the application adopts the following technical solutions:

[0012] A method for detecting the length distribution of host cell DNA residues in a recombinant lentivirus, comprising the following steps:

[0013] (1) 5-10ul of 10X buffer and 1-5ul of prepGEM enzyme are added to 5-50ul of rLVV virus sample, then nuclease-free water is added to a total volume of 50-100ul, and the mixture is mixed uniformly by blowing, so that the prepGEM enzyme can lyse the virus, and then the mixture is incubated to inactivate the enzyme;

[0014] (2) Detecting the residual fragment distribution of HEK293T host cell DNA (Host Cell DNA, abbreviated as HCD) in the rLVV virus sample: take 5-10 μL of the virus lysate sample, according to the content of the residual host cell DNA, do not dilute or dilute by an appropriate multiple, add 15-17 μL of 2X PCR Supermix and 2-5 μL of primer probe mixture, and then add nuclease-free water to make the volume of the polymerase chain reaction (PCR) system 30 μL; transfer the PCR reaction system into a 96-well plate, seal the 96-well plate, centrifuge for 30 seconds, and let the droplets settle at the bottom of the 96-well plate hole, and then place the 96-well plate into a real-time fluorescent quantitative PCR instrument for PCR amplification. After the real-time fluorescent quantitative PCR reaction is completed, analyze the residual fragment distribution of host cell DNA (Host Cell DNA, abbreviated as HCD) in the rLVV sample.

[0015] Preferably, in step (2), the PCR amplification conditions are as follows: a: 95℃, 10 minutes; b: 95℃, 15 seconds; c: 58-62℃, 30 seconds; d: 72℃, 90 seconds; e: repeat b to d for 40 cycles; f: 4℃, hold.

[0016] Preferably, in step (2), the residual fragment distribution of HEK293T host cell DNA in the rLVV virus sample is detected by using a commercial kit (Huzhou Shenke, Cat. No. SK030306S-H).

[0017] The present application has the following innovations and advantages:

[0018] 1. The present application uses a new type of enzyme prepGEM to directly lyse the rLVV virus sample, only two steps of enzyme lysis and enzyme inactivation, 5-15 minutes for lysis of the virus, and 2-10 minutes for enzyme inactivation, simple and convenient operation. The two steps are completed in the same microcentrifuge tube, without the need to open the microcentrifuge tube to add reagents in between, and the labor cost is low. Compared with the commonly used proteinase K lysis of the virus, the proteinase K needs to lyse the virus at 55℃ for 1 hour, and then incubate at 95℃ for 30 minutes to inactivate the proteinase K. Similarly, compared with the method of using magnetic bead method to extract DNA, the operation of using magnetic bead method to extract DNA is complicated, with a total of 33 operation steps, and the labor cost is high, and at least 3.5 hours are needed to process one sample; if multiple samples are processed, the labor cost and time are longer.

[0019] 2. Using prepGEM enzyme to lyse the rLVV virus sample only needs 5-50 μL of sample. The virus sample required by the magnetic bead method is at least 2.5 times or more than the enzyme lysis method.

[0020] 3. Viral solutions lysed with prepGEM enzyme can be directly used in real-time fluorescence quantitative PCR (RT-PCR), while viral solutions lysed with proteinase K require at least a 100-fold dilution before PCR. Extracting total DNA using magnetic beads involves numerous steps, and DNA loss is easily caused during these steps, leading to significant errors. 100-200 μL of elution buffer is needed to elute DNA, effectively diluting the hepatocellular carcinoma (HCD). For viral samples with high purity and low HCD residue, prepGEM enzyme lysis provides more accurate signal detection. With proteinase K lysis or magnetic beads, HCD may not detect the signal or may result in significant detection errors.

[0021] 4. The distribution of residual HCD fragments of rLVV virus in HEK293T cells was detected using a commercially available kit (Huzhou Shenke, Cat. No. SK030306S-H). Comparing the prepGEM enzyme lysis method and the magnetic bead method, the prepGEM enzyme lysis method yielded more stable and smaller variance data for DNA standard recovery and lentiviral sample HCD data, while the magnetic bead method showed greater fluctuations and larger variance. Figures 1 to 5 The different fragment sizes are all displayed. This difference is in Figure 1 The recovery rate of DNA standards was particularly high for fragments of size 562. The recovery rates of DNA standards obtained by the prepGEM enzyme lysis method were all within the acceptable range (50-150%), and the coefficients of variation (CV) were also within the acceptable range (≤25%). The recovery rates and CVs of DNA standards obtained by the magnetic bead method sometimes fell outside the acceptable range.

[0022] 5. The data from the enzyme lysis method are more stable and reasonable for the following two reasons.

[0023] (1) In HCD remnants, theoretically, the proportion of small fragments is higher than that of large fragments, so the distribution percentage of large fragments relative to small fragments is theoretically no higher than 100%. Figure 2In the rLVV virus sample, the distribution percentages of the 562 bp fragments relative to the 122 bp fragments were 93.76%, 119.16%, 140.98%, 107.32%, and 104.38%, respectively, with an average value ± standard deviation of 113.12% ± 18.01% and a CV of 15.92%; in comparison, the distribution percentages of the 562 bp fragments relative to the 122 bp fragments were 77.15%, 87.28%, 61.50%, 79.25%, and 79.38%, respectively, with an average value ± standard deviation of 76.31% ± 9.34% and a CV of 12.24%. The two methods had a significant difference, with a P value of 0.004 (in the present application, P value≤0.05 is defined as a significant difference, and P value>0.05 is defined as no significant difference, which is the same below). The data of the magnetic bead method was higher than 100%, and the data of the enzyme cleavage method was more reasonable and had smaller fluctuations. Similarly, Figure 4 In the rLVV virus sample, the distribution percentages of the 562 bp fragments relative to the 122 bp fragments were 93.76%, 119.16%, 140.98%, 107.32%, and 104.38%, respectively, with an average value ± standard deviation of 113.12% ± 18.01% and a CV of 15.92%; in comparison, the distribution percentages of the 562 bp fragments relative to the 122 bp fragments were 77.15%, 87.28%, 61.50%, 79.25%, and 79.38%, respectively, with an average value ± standard deviation of 76.31% ± 9.34% and a CV of 12.24%. The two methods had a significant difference, with a P value of 0.004 (in the present application, P value≤0.05 is defined as a significant difference, and P value>0.05 is defined as no significant difference, which is the same below). The data of the magnetic bead method was higher than 100%, and the data of the enzyme cleavage method was more reasonable and had smaller fluctuations. Similarly,

[0024] (2). In the HCD residue, the small fragments are theoretically more, and the recovery rate of the small fragments is theoretically 100%. In the rLVV virus sample, the distribution percentages of the 562 bp fragments relative to the 122 bp fragments were 93.76%, 119.16%, 140.98%, 107.32%, and 104.38%, respectively, with an average value ± standard deviation of 113.12% ± 18.01% and a CV of 15.92%; in comparison, the distribution percentages of the 562 bp fragments relative to the 122 bp fragments were 77.15%, 87.28%, 61.50%, 79.25%, and 79.38%, respectively, with an average value ± standard deviation of 76.31% ± 9.34% and a CV of 12.24%. The two methods had a significant difference, with a P value of 0.004 (in the present application, P value≤0.05 is defined as a significant difference, and P value>0.05 is defined as no significant difference, which is the same below). The data of the magnetic bead method was higher than 100%, and the data of the enzyme cleavage method was more reasonable and had smaller fluctuations. Similarly, Figure 3 In the rLVV virus sample, the distribution percentages of the 562 bp fragments relative to the 122 bp fragments were 93.76%, 119.16%, 140.98%, 107.32%, and 104.38%, respectively, with an average value ± standard deviation of 113.12% ± 18.01% and a CV of 15.92%; in comparison, the distribution percentages of the 562 bp fragments relative to the 122 bp fragments were 77.15%, 87.28%, 61.50%, 79.25%, and 79.38%, respectively, with an average value ± standard deviation of 76.31% ± 9.34% and a CV of 12.24%. The two methods had a significant difference, with a P value of 0.004 (in the present application, P value≤0.05 is defined as a significant difference, and P value>0.05 is defined as no significant difference, which is the same below). The data of the magnetic bead method was higher than 100%, and the data of the enzyme cleavage method was more reasonable and had smaller fluctuations. Similarly,

[0025] 6. The prepGEM enzyme lysis method has good tolerance. Two batches of prepGEM enzyme were used to lyse rLVV virus samples, and the percentage of HCD residual and large fragment distribution in HEK293T cells was stable, and there was no significant difference, as shown in Figure 7 and Figure 8 .

[0026] 7. The present application has low cost. The cost of the method of prepGEM enzyme lysis is only 1 / 6 of that of the magnetic bead method.

[0027] 8. The present application applies real-time fluorescent quantitative PCR technology to quickly, accurately and specifically detect the distribution of host cell DNA residual fragments in recombinant lentivirus (rLVV) samples, which is suitable for recombinant lentivirus intermediates in the production process, and recombinant lentivirus final products. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Comparison of DNA standard recovery rate at different fragment sizes by enzyme lysis method and magnetic bead method. The mean values of the two methods have no significant difference, P value is: 122bp, P=0.199; 244bp, P=0.235; 562bp, P=0.413.

[0029] The variances of the two methods have no significant difference at 122bp and 244bp fragment sizes (P value≤0.05 is defined as significant difference, P value>0.05 is defined as no significant difference, the same below), P value is: 122bp, P=0.330; 244bp, P=0.425. But there is significant difference at 562bp fragment size, the variance of magnetic bead method is larger than that of enzyme lysis method, P=0.024. The recovery rate of enzyme lysis method at 562bp is 65.76%, 107.09%, 83.54%, 93.65%, 99.17% respectively, the average value±standard deviation is 89.85%±15.96%, CV is 17.76%; while the recovery rate of magnetic bead method at 562bp is 70.17%, 94.39%, 192.68%, 93.73%, 75.91% respectively, the average value±standard deviation is 104.98%±50.09%, CV is 47.72%. It can be seen that the recovery rate of magnetic bead method for recovering DNA standard is out of the acceptable standard range (acceptable standard is that the recovery rate is within 50-150%), the data fluctuation is large, and the CV is also out of the acceptable standard range (acceptable standard is that CV≤25%). In comparison, the recovery rate and CV of enzyme lysis method are within the acceptable standard range, the data is more stable and has less fluctuation than that of magnetic bead method.

[0030] Figure 2 The effect of enzyme cleavage method and magnetic bead method on the percentage of large fragments relative to small fragments of DNA standard was compared. There was no significant difference in the mean percentage of 244 bp relative to 122 bp fragments between the two methods, with a P value of 0.789; but there was a significant difference in the mean percentage of 562 bp relative to 122 bp fragments, with a P value of 0.004. The percentage of 562 bp relative to 122 bp fragments by enzyme cleavage method was 77.15%, 87.28%, 61.50%, 79.25% and 79.38%, respectively, with a mean value ± standard deviation of 76.31% ± 9.34% and a CV of 12.24%; the percentage of 562 bp relative to 122 bp fragments by magnetic bead method was 93.76%, 119.16%, 140.98%, 107.32% and 104.38%, respectively, with a mean value ± standard deviation of 113.12% ± 18.01% and a CV of 15.92%. The percentage of 562 bp relative to 122 bp fragments by magnetic bead method was significantly higher than that by enzyme cleavage method, with a value greater than 100% and a large fluctuation between data. In HCD residues, the larger the fragment, the smaller the theoretical proportion, so the percentage of 562 bp relative to 122 bp fragments should not be higher than 100%. Compared with magnetic bead method, the data of enzyme cleavage method were more reasonable and stable.

[0031] There was no significant difference in the variance between the two methods, with P values of 244 bp / 122 bp, P = 0.196; and 562 bp / 122 bp, P = 0.116. The variance of magnetic bead method was slightly larger than that of enzyme cleavage method.

[0032] Figure 3In rLVV virus samples, 2-fold, 5-fold and 10-fold DNA standard was added to rLVV virus sample host cell DNA (Host Cell DNA, abbreviated as HCD) residue respectively, and the recovery rate of DNA standard obtained by enzyme lysis method and magnetic bead method was compared at different fragment lengths. The average of the recovery rates of the two methods at 244bp and 562bp had no significant difference, and the P value was: 244bp, P=0.777; 562bp, P=0.962. But the average of the recovery rates of the two methods at 122bp had significant difference, and the P value was 0.0187. When the added amount of DNA standard was 2-fold, 5-fold and 10-fold of rLVV virus sample HCD respectively, the recovery rate of enzyme lysis method at 122bp was 110.28%, 105.33%, 100.47% respectively, and the average value ± standard deviation was 105.36% ± 4.90%, and the CV was 4.66%; compared with this, the recovery rate of magnetic bead method at 122bp was 69.64%, 83.60%, 89.03% respectively, and the average value ± standard deviation was 80.76% ± 10.00%, and the CV was 12.39%. The recovery rate of magnetic bead method at 122bp was significantly lower than that of enzyme lysis method. In HCD residue, the proportion of small fragments was high in theory, and the theoretical value of the recovery rate of DNA standard at 122bp was 100%. Compared with the two methods, the recovery rate of magnetic bead method at 122bp fragment size was significantly lower than 100%, while the data of enzyme lysis method was very close to 100%. It is shown that the data of enzyme lysis method is more stable and reasonable.

[0033] The variance of the two methods had no significant difference, and the P value was: 122bp, P=0.194; 244bp, P=0.122; 562bp, P=0.394. The variance of magnetic bead method was slightly larger than that of enzyme lysis method.

[0034] Figure 4In the rLVV virus sample, the DNA standard was added to the rLVV virus sample at 2 times, 5 times and 10 times the HCD, respectively, and the distribution percentages of large fragments relative to small fragments of the DNA standard recovered by enzyme lysis and magnetic bead methods were compared. The two methods had no significant difference in the mean of 244bp relative to 122bp, with a P value of 0.068, but had a significant difference in the distribution percentage of 562bp relative to 122bp fragments, with a P value of 0.030. When the spiked amount of the DNA standard was 2 times, 5 times and 10 times the HCD of the rLVV virus sample, respectively, the distribution percentages of 562bp relative to 122bp fragments by enzyme lysis were 85.47%, 74.38% and 70.80%, respectively, with a mean ± standard deviation of 76.89% ± 7.65% and a CV of 9.95%. In comparison, the distribution percentages of 562bp relative to 122bp fragments by magnetic bead method were 150.14%, 104.90% and 116.61%, respectively, with a mean ± standard deviation of 123.88% ± 23.48% and a CV of 18.95%. The distribution percentage of 562bp relative to 122bp fragments by magnetic bead method was significantly higher than that by enzyme lysis, and was higher than 100%, with large fluctuations. In the HCD residue, the proportion of small fragments was theoretically higher than that of large fragments, so the theoretical value of the distribution percentage of large fragments relative to small fragments was not higher than 100%. The distribution percentage of 562bp relative to 122bp fragments by magnetic bead method was higher than 100%, and the data by enzyme lysis was more stable and reasonable.

[0035] The variances of the two methods had no significant difference, with P values of 244bp / 122bp, P=0.236; 562bp / 122bp, P=0.096, respectively. The variance of the magnetic bead method was slightly larger than that of the enzyme lysis method.

[0036] Figure 5 The amounts of 122bp, 244bp and 562bp fragments in the HCD of the rLVV virus sample obtained by enzyme lysis and magnetic bead methods were compared. The means of the HCD of the rLVV virus sample at the three fragment sizes obtained by the two methods had no significant difference, with P values of 122bp, P=0.922; 244bp, P=0.385; 562bp, P=0.202, respectively.

[0037] The variances of the two methods also had no significant difference, with P values of 122bp, P=0.243; 244bp, P=0.116; 562bp, P=0.069, respectively. The variance of the magnetic bead method was slightly larger than that of the enzyme lysis method.

[0038] Figure 6HCD in rLVV virus samples was detected using both enzymatic lysis and magnetic bead methods, and the distribution percentages of large fragments relative to small fragments obtained by the two methods were compared. There was no significant difference in the means between the two methods, with P values ​​of 244bp / 122bp (P = 0.236) and 562bp / 122bp (P = 0.090), respectively.

[0039] There was no significant difference in variance between the two methods, with P values ​​of 0.235 for 244bp / 122bp and 0.258 for 562bp / 122bp.

[0040] Figure 7 The effects of two batches of enzyme on the detection of hematocrit (HCD) in rLVV virus samples were compared. There were no significant differences in the mean HCD values ​​of the two batches of enzyme at 122 bp, 244 bp, and 562 bp, with P values ​​of: 122 bp, P = 0.265; 244 bp, P = 0.220; 562 bp, P = 0.151, respectively.

[0041] The variances of the rLVV virus samples HCD obtained from the two batches of enzymes were not significantly different at 122bp, 244bp, and 562bp, with P values ​​of 0.441, 0.435, and 0.063, respectively.

[0042] from Figure 7 It can be seen that different batches of enzyme have no significant effect on the HCD of rLVV virus samples, indicating that the enzyme lysis method has good tolerance.

[0043] Figure 8 The effects of two batches of enzyme on the distribution of large fragments relative to small fragments in HCD of rLVV virus samples were compared. There were no significant differences in the mean percentages of 244bp to 122bp and 562bp to 122bp fragments obtained from the two batches of enzyme, with P values ​​of 0.770 for 244bp / 122bp and 0.427 for 562bp / 122bp.

[0044] There were no significant differences in the variance of the percentage distribution of HCD fragments at 244bp relative to 122bp and 562bp relative to 122bp between the two batches of enzymes for rLVV virus samples, with P values ​​of 0.162 for 244bp / 122bp and 0.303 for 562bp / 122bp.

[0045] from Figure 8It can be seen that different batches of enzymes have no significant effect on the distribution of HCD large fragments relative to small fragments of rLVV virus samples, indicating that the enzyme cleavage method has good tolerance. DETAILED DESCRIPTION

[0046] Example 1:

[0047] 1.1 prepGEM enzyme cleavage method to recover DNA standard.

[0048] Take 3 ng / μL DNA standard and dilute to 300 pg / μL. Take 20-50 μL of DNA standard with a concentration of 300 pg / μL, add 5-10 μL of 10X buffer, 1-5 μL of prepGEM enzyme, and add nuclease-free water to 50-100 μL. Gently blow and mix evenly. Incubate the mixture at 75°C for 5-15 minutes to cleave the virus with prepGEM enzyme. Then incubate the mixture at 95°C for 2-10 minutes to inactivate the enzyme.

[0049] 1.2 Magnetic bead method to recover DNA standard.

[0050] Commercial kits are used for DNA extraction / recovery. Add 100-200 μL of DNA standard with a concentration of 300 pg / μL to a 1.5 mL centrifuge tube. Follow the kit instructions for specific steps for DNA recovery.

[0051] 1.3 Real-time fluorescent quantitative PCR amplification reaction

[0052] Commercial host cell DNA residual fragment analysis detection kit is used for real-time fluorescent quantitative PCR amplification (Huzhou Shenke, Cat. No. SK030306S-H). Take 5-10 μL of virus lysate or DNA sample obtained in 1.1 and 1.2, add 15-17 μL of 2X PCR Supermix, 2-5 μL of primer probe mixture, and add nuclease-free water to make the PCR reaction system volume 30 μL. Transfer the PCR reaction system into a 96-well plate, seal the 96-well plate, and centrifuge gently for 30 seconds to make the droplets settle at the bottom of the 96-well plate. Place the 96-well plate into a real-time fluorescent quantitative PCR instrument for PCR amplification, and the amplification conditions are as follows:

[0053] a: 95°C, 10 minutes;

[0054] b: 95°C, 15 seconds;

[0055] c: 58-62°C, 30 seconds;

[0056] d: 72°C, 90 seconds;

[0057] e: Repeat b to d for 40 cycles;

[0058] f: 4°C, hold.

[0059] After the real-time fluorescent quantitative PCR amplification, the content of HCD at 122 bp, 244 bp and 562 bp in the HEK293T cells was analyzed.

[0060] Since the volume of standard DNA sample required by the enzyme cleavage method is 2.5 times or more lower than that of the magnetic bead method, for the convenience of comparison, the data is uniformly converted to the HCD obtained by loading 1000 μL of sample.

[0061] As shown in Figure 1 , the experimental results show that there is no significant difference between the average values of the two methods for recovering DNA standard by prepGEM enzyme cleavage method and magnetic bead method (Pvalue≤0.05 is defined as significant difference, Pvalue>0.05 is defined as no significant difference, the same below), at 122 bp, 244 bp and 562 bp, the Pvalue of the average value is 122 bp, P=0.199; 244 bp, P=0.235; 562 bp, P=0.413. The variance of the two methods has no significant difference at 122 bp and 244 bp, the Pvalue is 122 bp, P=0.330; 244 bp, P=0.425. But for the 562 bp fragment, the variance of the two methods has significant difference, the variance of the magnetic bead method is larger than that of the enzyme cleavage method, P=0.024. The recovery rate of the enzyme cleavage method at 562 bp is 65.76%, 107.09%, 83.54%, 93.65%, 99.17%, respectively, and the average value ± standard deviation is 89.85% ± 15.96%, and the CV is 17.76%; while the recovery rate of the magnetic bead method at 562 bp is 70.17%, 94.39%, 192.68%, 93.73%, 75.91%, respectively, and the average value ± standard deviation is 104.98% ± 50.09%, and the CV is 47.72%. It can be seen that the recovery rate of the magnetic bead method for recovering DNA standard is out of the acceptable standard range (the acceptable standard is that the recovery rate is within 50%-150%), the data fluctuation is large, and the CV is also out of the acceptable standard range (the acceptable standard is that the CV is ≤25%). In comparison, the recovery rate and CV obtained by the enzyme cleavage method are within the acceptable standard range, and the data is more stable and less fluctuant than the magnetic bead method.

[0062] The amount of large fragment DNA recovered is divided by the amount of small fragment DNA to obtain the percentage distribution of large fragments relative to small fragments. As shown in Figure 2As shown, the experimental results show that the mean of the percentage of distribution of the two methods at 244bp relative to 122bp fragments has no significant difference, P value is 0.789; but the mean of the percentage of distribution at 562bp relative to 122bp fragments has significant difference, P value is 0.004. Among them, the percentage of distribution of 562bp relative to 122bp fragments of the enzyme cleavage method is 77.15%, 87.28%, 61.50%, 79.25% and 79.38%, the average value ± standard deviation is 76.31% ± 9.34%, and the CV is 12.24%; the percentage of distribution of 562bp relative to 122bp fragments of the magnetic bead method is 93.76%, 119.16%, 140.98%, 107.32% and 104.38%, the average value ± standard deviation is 113.12% ± 18.01%, and the CV is 15.92%. It can be concluded that the percentage of distribution of 562bp fragments relative to 122bp fragments of the magnetic bead method is significantly higher than that of the enzyme cleavage method, the value is greater than 100%, and the data fluctuates greatly. In the HCD residue, the larger the fragment, the smaller the theoretical proportion, so the percentage of distribution of 562bp fragments relative to 122bp fragments is not higher than 100%. It can be seen from the results that the data of the enzyme cleavage method is more reasonable.

[0063] The variance of the two methods has no significant difference, P value is: 244bp / 122bp, P=0.196; 562bp / 122bp, P=0.116. The variance of the magnetic bead method is slightly larger than that of the enzyme cleavage method.

[0064] Example 2:

[0065] In 20-100 μL rLVV virus sample, add DNA standard, the amount of DNA standard is 2 times, 5 times and 10 times of the HCD of rLVV virus sample, respectively. The total HCD obtained is subtracted from the HCD of the lentivirus sample to obtain the recovered DNA standard. The amount of the recovered DNA standard is divided by the amount of the original added DNA standard to obtain the recovery rate of the added DNA standard.

[0066] The same as example 1, use enzyme cleavage method and magnetic bead method respectively to detect the recovered DNA standard. At 122bp, 244bp and 562bp fragment length, calculate the recovery rate of the DNA standard respectively.

[0067] From Figure 3As can be seen, the DNA standard recovery rates obtained by the two methods had no significant difference at 244 bp and 562 bp, with P values of 244 bp, P = 0.777; 562 bp, P = 0.962; but the mean values of the recovery rates at 122 bp by the two methods had significant difference, with P value of 0.0187. When the spiked amounts of the DNA standard were 2 times, 5 times and 10 times of the rLVV viral sample HCD, respectively, the recovery rates of the enzyme cleavage method at 122 bp were 110.28%, 105.33%, 100.47%, respectively, with the mean value ± standard deviation of 105.36% ± 4.90% and CV of 4.66%; in comparison, the recovery rates of the magnetic bead method at 122 bp were 69.64%, 83.60%, 89.03%, respectively, with the mean value ± standard deviation of 80.76% ± 10.00% and CV of 12.39%; the recovery rate of the magnetic bead method at 122 bp was significantly lower than that of the enzyme cleavage method. In the HCD residue, the proportion of small fragments was high in theory, and the proportion of large fragments was low. The theoretical recovery rate of the DNA standard at 122 bp fragment size was 100%. Compared with the two methods, the recovery rate of the magnetic bead method at 122 bp was significantly lower than 100%, while the data of the enzyme cleavage method was very close to 100%. Therefore, the data of the enzyme cleavage method was more stable and reasonable.

[0068] The variances of the two methods had no significant difference, with P values of 122 bp, P = 0.194; 244 bp, P = 0.122; 562 bp, P = 0.394. The variance of the magnetic bead method was slightly larger than that of the enzyme cleavage method.

[0069] Figure 4 For comparison of the large fragment distribution percentages relative to the small fragment distribution percentages of the enzyme cleavage method and the magnetic bead method. From the above data, it can be seen that the large fragment distribution percentages of the two methods were very close to each other, with P values of 244 bp, P = 0.999; 562 bp, P = 0.999; 122 bp, P = 0.999. Figure 4As can be seen, there was no significant difference between the two methods in the mean of 244bp versus 122bp (P value: 0.068); however, there was a significant difference in the percentage distribution of the 562bp versus 122bp fragment (P value: 0.030). When the DNA standard was spiked at 2, 5, and 10 times the HCD of the rLVV virus sample, the percentage distribution of the 562bp versus 122bp fragment using the enzyme lysis method was 85.47%, 74.38%, and 70.80%, respectively, with a mean ± standard deviation of 76.89% ± 7.65% and a CV of 9.95%. In contrast, the percentage distribution of the 562bp versus 122bp fragment using the magnetic bead method was 150.14%, 104.90%, and 116.61%, respectively, with a mean ± standard deviation of 123.88% ± 23.48% and a CV of 18.95%. The magnetic bead method showed a significantly higher percentage of 562bp fragments relative to 122bp fragments compared to the enzyme lysis method, exceeding 100%, but with large data fluctuations. Theoretically, smaller fragments should account for a higher proportion of larger fragments than larger fragments in HCD residues, so the theoretical percentage of larger fragments relative to smaller fragments should not exceed 100%. While the magnetic bead method showed a higher percentage of 562bp fragments relative to 122bp fragments, the enzyme lysis method provided more stable and reasonable data.

[0070] There was no significant difference in variance between the two methods, with P-values ​​of 0.236 for 244bp / 122bp and 0.096 for 562bp / 122bp. The variance of the magnetic bead method was slightly larger than that of the enzyme lysis method.

[0071] Example 3:

[0072] Take 20-100 μL of rLVV virus samples and use enzyme lysis method and magnetic bead method to detect the distribution of HCD fragment lengths at 122bp, 244bp and 562bp in the rLVV virus samples.

[0073] Experimental results are as follows Figure 5 As shown. From Figure 5 It can be seen that the mean values ​​of the rLVV virus samples HCD obtained by the two methods were not significantly different at the three fragment sizes of 122bp, 244bp and 562bp, respectively, with P values ​​of: 122bp, P=0.922; 244bp, P=0.385; 562bp, P=0.202.

[0074] There was no significant difference in variance between the two methods, with P values ​​of: 122bp, P = 0.243; 244bp, P = 0.116; 562bp, P = 0.069. However, the variance of the magnetic bead method was slightly larger than that of the enzyme lysis method.

[0075] Figure 6This compares the percentage distribution of large fragments relative to small fragments using enzyme lysis and magnetic bead methods. From... Figure 6 As can be seen, there was no significant difference in the mean distribution percentage of 244bp to 122bp fragments and the distribution percentage of 562bp to 122bp fragments between the two methods, with P values ​​of 244bp / 122bp, P = 0.236 and 562bp / 122bp, respectively, P = 0.090.

[0076] There was no significant difference in variance between the two methods, with P values ​​of 0.235 for 244bp / 122bp and 0.258 for 562bp / 122bp.

[0077] Example 4:

[0078] Take 5-50 μL of rLVV virus samples and lyse the rLVV virus with two batches of prepGEM enzyme respectively. Detect the effect of different batches of enzyme on the HCD fragment length distribution in the rLVV virus samples at 122bp, 244bp and 562bp.

[0079] Experimental results are as follows Figure 7 As shown, the two batches of enzyme had no significant effect on rLVV viral HCD. The mean sizes of the rLVV viral HCD obtained from the two batches of enzyme were not significantly different at 122bp, 244bp, and 562bp, respectively, with P values ​​of: 122bp, P = 0.265; 244bp, P = 0.220; 562bp, P = 0.151.

[0080] The variances of the rLVV viral HCD fragment sizes obtained from the two batches of enzymes at 122bp, 244bp, and 562bp were not significantly different, with P values ​​of 0.441 for 122bp, 0.435 for 244bp, and 0.063 for 562bp.

[0081] like Figure 8 As shown, neither batch of enzymes had a significant effect on the distribution of large fragments relative to small fragments of rLVV viral HCD. From Figure 8 It can be seen that there was no significant difference in the mean distribution percentage of the rLVV virus HCD at 244bp relative to 122bp and at 562bp relative to 122bp between the two batches of enzymes, with P values ​​of 244bp / 122bp, P = 0.770 and 562bp / 122bp, respectively, P = 0.427.

[0082] The variance of the percentage distribution of the 244bp versus 122bp and 562bp versus 122bp fragments for the two batches of enzymes also did not show significant difference, P value were: 244bp / 122bp, P=0.162; 562bp / 122bp, P=0.303, respectively.

[0083] From Figure 7 and Figure 8 It can be seen that the enzyme lysis method has good tolerance. The HCD of the rLVV virus samples obtained by different batches of enzymes at three fragment sizes of 122bp, 244bp and 562bp, and the percentage distribution of large fragments relative to small fragments, all have no significant difference.

Claims

1. A method for detecting the distribution of residual fragments of DNA of a recombinant lentivirus host cell, characterized in that, The host cell is a HEK293T cell, and the method comprises the following steps: (1) 5-10 μL of 10X buffer and 1-5 μL of prepGEM enzyme are added to 5-50 μL of an rLVV virus sample containing HEK293T host cells, then nuclease-free water is added to a total volume of 50-100 μL, gently blown and mixed, the mixture is incubated at 75°C for 5-15 minutes to allow the prepGEM enzyme to lyse the virus, and then the mixture is incubated at 95°C for 2-10 minutes to inactivate the enzyme; (2) Detecting the HEK293T host cell DNA residual fragment distribution of the rLVV virus sample: 5-10 μL of the virus lysate sample is taken, according to the content of the host cell DNA residue, without dilution or dilution by an appropriate multiple, 15-17 μL of 2X PCR Supermix and 2-5 μL of primer probe mixture are added, the primer probe mixture is component in the commercial kit Huzhou Shenke Cat. No. SK030306S-H, and then nuclease-free water is added to make the PCR reaction system volume 30 μL; the PCR reaction system is transferred into a 96-well plate, the 96-well plate is sealed, centrifuged for 30 seconds to make the droplets settle at the bottom of the 96-well plate, and the 96-well plate is placed into a real-time fluorescent quantitative PCR instrument for PCR amplification; after the real-time fluorescent quantitative PCR reaction is completed, the fragment distribution of the HEK293T host cell DNA residue of the rLVV virus sample is analyzed.

2. The method for detecting the distribution of residual DNA fragments in recombinant lentiviral host cells according to claim 1, characterized in that, In step (2), the PCR amplification conditions are as follows: a: 95°C, 10 minutes; b: 95°C, 15 seconds; c: 58-62°C, 30 seconds; d: 72°C, 90 seconds; e: repeat b to d for 40 cycles; f: 4°C, hold.

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