CN113699278A - Primer, kit and detection method for detecting replication-competent retrovirus based on q-PCR method - Google Patents
Primer, kit and detection method for detecting replication-competent retrovirus based on q-PCR method Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a primer, a kit and a detection method for detecting a replication-competent retrovirus based on a q-PCR method, and relates to the technical field of virus detection. Wherein the primer comprises a primer GALV-F, a primer GALV-R and a probe GALV-probe, and the nucleotide sequence SEQ ID No is shown as follows: GALV-F: GGCACCTTTCCTATGCACAACCC, respectively; GALV-R: GGTCGCCTACTCCACCGACCT, respectively; GALV-probe: CCTTGGGTCCCCCAGCGGCACCGGTC are provided. The kit provided by the invention can rapidly detect whether a cell product has replication-competent retrovirus (RCR) aiming at the GALV (GALV-like retrovirus) vector, so as to reduce the risk of RCR pollution generated in the production stage, has the advantages of high sensitivity and good specificity, can achieve 7 copies/reaction in the limit of quantitation, and can be used for rapid release quality control of the cell product of the retrovirus vector. The detection method provided by the invention has the advantages of good specificity, good repeatability and high sensitivity, and can be used for carrying out quantitative analysis on a sample by utilizing an established standard curve.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to a primer, a kit and a detection method for detecting a replication-competent retrovirus based on a q-PCR method.
Background
The existing cell injection for treating cancer is prepared by packaging retrovirus by using PG13 cells, producing a retrovirus vector, infecting T lymphocytes of a patient and preparing a cell preparation. Wherein the retroviral vector is a replication defective viral vector of a Gibbon Ape Leucovirus (GALV) whose genomic RNA lacks gag, pol and env sequences required for replication of the retrovirus, the risk of producing a replication-competent Virus is in theory substantially reduced. However, the genome of the virus packaging cell PG13 artificially integrates the gag and pol expression sequences of mouse leukemia virus (MoMLV), and the env sequence of GALV, and the packaging elements of these retroviruses are essential for the production of retroviral vectors, but they may be recombined with the PG13 genome, the human genome at the time of cell preparation, and replication-competent retroviruses may be produced. The virus will continuously replicate after integrating its own gene sequence into the host genome (i.e. the T cell to be returned by the patient), and there is a great safety hazard. Meanwhile, the state has issued a directive document to suggest that cell therapy products need to be tested for replication-competent retroviruses (RCR) of viral vectors and cell end products, and therefore, it is necessary to establish a testing method to test replication-competent retroviruses of cell products to reduce the risk of RCR contamination at the production stage.
At present, the detection method of replication-competent retrovirus (RCR) comprises a PCR method or a qPCR method aiming at a specific gene (such as VSVG gene) and a sensitive cell infection test method, and according to the requirements of national food and drug administration, the RCR detection recommends the adoption of a sensitive cell-based amplification culture method, but the method is long in time consumption (4-6 months) and is not suitable for the release of cell end products requiring fresh infusion. The real-time fluorescent quantitative polymerase chain reaction (qPCR) is a nucleic acid detection technology which has high sensitivity and strong specificity and can be quantified by real-time monitoring, the qPCR is the most commonly used two technologies, one is a fluorescent dye method, but the affinity of the qPCR with DNA is strong, and the qPCR generally has an inhibiting effect on the PCR; the other method is a fluorescence probe method, is a TaqMan probe with sequence specificity, has high sensitivity and strong specificity, is applied to some fields of biological pharmacy, and has related literature researches abroad to establish a rapid detection method of replication-competent lentiviruses by utilizing a probe qPCR method; however, different viruses have specificity, and how to utilize a probe qPCR method to establish a rapid detection method for replication-competent retrovirus with the retrovirus vector GALV is to improve rapid detection of cell products and rapid release of quality control.
Disclosure of Invention
The technical problems to be solved by the invention are that the existing method for detecting the replication-competent retrovirus is long in time consumption and low in efficiency.
In order to solve the above problems, the present invention proposes the following technical solutions:
in a first aspect, the invention provides a primer for detecting a replication-competent retrovirus based on a q-PCR method, which comprises a primer GALV-F, a primer GALV-R and a probe GALV-probe;
the nucleotide sequences of the primer GALV-F, the primer GALV-R and the probe GALV-probe are shown as SEQ ID No, or homofunctional sequences with at least 95% of identity with the sequences;
GALV-F(SEQ ID No 1)：GGCACCTTTCCTATGCACAACCC；
GALV-R(SEQ ID No 2)：GGTCGCCTACTCCACCGACCT；
GALV-probe(SEQ ID No 3)：CCTTGGGTCCCCCAGCGGCACCGGTC。
the technical scheme is that the fluorescence reporter group of the probe GALV-probe is FAM, and the quenching group is NFQ-MGB.
In a second aspect, the present invention provides a replication-competent retrovirus detection kit comprising the primer, wherein the concentration of the primer GALV-F, the primer GALV-R, and the probe GALV-probe is 10. mu.M.
Further, the kit also comprises the following components: buffer solution of a PCR reaction system, fluorescent dye and GALV standard substance.
Further, the reaction system of the kit for carrying out fluorescence quantitative PCR is as follows:
2 XProbe qPCR Premix Ex Taq 10 muL, primer GALV-F, primer GALV-R each 0.4 muL, Probe GALV-Probe 0.8 muL, ROX reference dye II 0.2 muL, sample DNA to be detected or standard substance DNA or quality control point standard substance 5-7 muL, supplement UltraPure water to 20 muL.
In a second aspect, the present invention provides a method for detecting a replication-competent retrovirus based on a q-PCR method, comprising the steps of:
s1, extracting the genomic DNA of the sample to be detected, and determining the concentration of the sample DNA;
s2, preparing a qPCR reaction system which comprises a primer GALV-F, a primer GALV-R and a probe GALV-probe, wherein the nucleotide sequence of the primer GALV-R and the probe GALV-probe is as follows:
primer GALV-F: GGCACCTTTCCTATGCACAACCC, respectively;
primer GALV-R: GGTCGCCTACTCCACCGACCT, respectively;
probe GALV-probe: CCTTGGGTCCCCCAGCGGCACCGGTC, respectively;
s3, preparing a standard substance solution for a standard curve and a standard substance solution for a quality control point;
s4, template-free negative control, negative control of the sample to be detected and sample loading detection of the sample to be detected with the standard substance;
and S5, judging the result.
Further, in S5, the detection result satisfies the following conditions at the same time, and it is determined that the detection result is valid:
(1) correlation coefficient R of standard curve slope2Not less than 0.99, and the amplification efficiency is between 90% and 110%;
(2) ct values for the no template negative control (NTC) should be underdetermined;
(3) the Ct value of the negative control group (NCS) should be underdetermined.
Further, in S5, if the Ct value of the sample to be detected is underdetermined, it is determined that no replication-competent retrovirus is detected from the sample of the batch; and if the Ct value of the sample to be detected is present, judging that the replication-competent retrovirus is detected in the batch of samples.
Further, in S4, qPCR reaction conditions were set as: pre-denaturation at 95 ℃ for 60 s; then, the reaction was carried out for 40 cycles at 95 ℃ for 5 seconds and 62 ℃ for 34 seconds.
Further, in S3, the concentration of each diluted sample in the standard curve of the standard is: 1.4X 105copies/μL，1.4×104copies/μL，1.4×103copies/μL，1.4×102copies/μL，1.4×101copies/μL，1.4copies/μL。
Compared with the prior art, the invention can achieve the following technical effects:
the invention provides a primer for detecting a replication-competent retrovirus based on a q-PCR method, which comprises a primer GALV-F and a primer GALV-R, wherein the nucleotide sequence SEQ ID No is shown as follows: GALV-F: GGCACCTTTCCTATGCACAACCC, respectively; GALV-R: GGTCGCCTACTCCACCGACCT are provided.
The kit provided by the invention can rapidly detect whether a cell product has replication-competent retrovirus (RCR) aiming at the GALV (GALV-like retrovirus) vector, so as to reduce the risk of RCR pollution generated in the production stage, has the advantages of high sensitivity and good specificity, can achieve 7 copies/reaction in the limit of quantitation, and can be used for rapid release quality control of the cell product of the retrovirus vector.
The detection method provided by the invention has the advantages of good specificity, good repeatability and high sensitivity, and can be used for carrying out quantitative analysis on a sample by utilizing an established standard curve.
Drawings
FIG. 1 is a fluorescent quantitative PCR standard curve of a standard;
FIG. 2 is a fluorescence amplification chart of the standard.
Detailed Description
The technical solutions in the examples will be clearly and completely described below. It is apparent that the embodiments to be described below are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Replication-competent retrovirus detection kit and method for detecting replication-competent retrovirus by using same
The replication competent retrovirus detection kit provided in this embodiment comprises a primer GALV-F, a primer GALV-R, and a probe GALV-probe.
The specific introduction is as follows:
primer and probe
The reason for selecting the designed target of the primer of the invention is as follows: RCR detection based on the qPCR method can be directed against DNA elements necessary for replication competent retroviruses, such as gag, pol, env (encoding VSV-G, GALV, etc.). There have been studies showing that the env gene has a smaller variation compared to the gag, pol genes. Therefore, we selected the env of GALV, i.e., the DNA sequence fragment of GALVgp3 protein, as the detection target (hereinafter abbreviated as fGALV). We first determined, using the BLASTN program at NCBI, that the fGALV sequence had no similar sequences in the human genome, which had been inserted into the pcrr 2.1 vector.
The nucleotide sequences of the primers and probes are shown in Table 1.
TABLE 1 nucleotide sequences of primers and probes
The standard substance used in the embodiment of the invention is a GALV standard substance, in particular a linearization product of pCR2.1-fGALV plasmid (4430 bp in full length); the preparation process comprises the following steps:
(1) enzyme digestion, wherein the reaction condition is incubation for 60 minutes at 37 ℃;
the reaction system of the enzyme digestion is shown in Table 2 below:
TABLE 2 pCR2.1-fGALV plasmid single enzyme digestion reaction system
(2) The linearized product of the pCR2.1-fGALV plasmid was dephosphorylated using recombinant shrimp alkaline phosphatase (rSAP) to reduce product self-ligation: and (3) directly adding 1.5 mu L of rSAP into the enzyme digestion system after the reaction in the step (1), and uniformly mixing. The reaction was incubated at 37 ℃ for 60 minutes, then warmed to 65 ℃ and stopped for 5 minutes.
(3) And (3) carrying out agarose gel electrophoresis on the product obtained in the step (2) and recovering gel.
(4) The PicoGreen method accurately measures the concentration of gel recovery product (concentration of standards).
(5) Calculating the GALV standard substance copy number concentration according to a calculation formula of the standard substance copy number concentration:
wherein:
c is the concentration of pCR2.1-fGALV gel recovery product accurately quantified by PicoGreen for GALV standards;
·6.02×1023is an Avogastron constant;
660 g/(mol. bp) is the average molecular mass per base pair of double-stranded DNA;
(6) and (4) storing the standard product: the copy number concentration of the GALV standard was diluted to 7X 107copies/. mu.L, designated GALV standard Stock (Stock) -80 ℃ refrigerator storage.
The preparation process of the genome DNA of the healthy person is the same as that of the genome DNA of the detection sample, and the concentration of the genome DNA of the healthy person is 100 ng/mu L and the genome DNA is stored in a refrigerator at the temperature of minus 80 ℃.
1 Material
1.1 sample
A sample to be detected: TCR-T cell injection (TCR-T cells); negative control: patient T cell genome (NC-T cells); and (3) standard substance: GALV standard Stock (Stock) (7X 10)7copies/. mu.L); healthy human genome DNA (100 ng/. mu.L)
1.2 reagents
(1) Blood/tissue/cell genome extraction kit, tiangen;
(2) rnase a, a heaven root organism;
(3)Quant-iTTM PicoGreenTM dsDNA Assay Kit，Invitrogen；
(4)Premix Ex Taq(Probe qPCR)，Takara；
(5) ultrapure DNase/RNase-Free Distilled Water (hereinafter referred to as Ultrapure Water), Invitrogen;
(6) RCR-Taqman-F, RCR-Taqman-R, RCR-Taqman-probe, a biological organism.
2 detection Process
2.1 cell sample treatment
Gently mixing the cells, counting with a blood cell counter under microscope or cell counter, measuring cell density, and taking out the desired cells (less than or equal to 5 × 10)6Tube), centrifuge at 1500rpm for 3min, remove supernatant, add 1mL of 1 XPBS solution, and resuspend cells.
2.2 extraction of genomic DNA (gDNA) of cells
The genomic DNA of TCR-T cells and NC-T cells was extracted according to the instructions of "blood/tissue/cell genome extraction kit" for Tiangen organisms.
2.3 PicoGreen accurately measures the concentration of the genomic DNA of the sample to be detected.
Adopt Quant-iTTM PicoGreenTMThe dsDNA Assay Kit instructions precisely determine the concentration of genomic DNA in a sample.
2.4 qPCR assay
2.4.1 preparation of qPCR reaction System
(1) The reaction system formulation for the standards is shown in table 3:
TABLE 3 qPCR reaction System for standards
(2) The reaction system for the samples to be tested is prepared in Table 4:
TABLE 4 qPCR reaction System for samples to be tested
(3) The reaction system formulation of the quality control points is shown in Table 5:
TABLE 5 qPCR reaction System for quality control points
2.4.2 dilution of the samples
The cell genome DNA was diluted to 20 ng/. mu.L using Ultrapure Water, and the volume of the dilution was not less than 20. mu.L. Centrifuging in a miniature centrifuge for 10 s, and standing at normal temperature for use.
2.4.3 Standard Curve the formulation of the standards is shown in Table 6.
TABLE 6 solutions of standards at different concentrations
2.4.4 preparation of Standard solution for quality control Point
Quality control point: 100ng of NC-T cell genome and 200 copies of standard were added to the reaction wells.
2.4.5 on-machine detection
(1) Setting a target point: the target is named as GALV, the reporter fluorophore is FAM, the quenching fluorophore is NFQ-MGB, and the detection reference fluorescence is ROX.
(2) The reaction program was set up as shown in table 7 below:
TABLE 7 qPCR reaction procedure
The standard curve and amplification result of the standard obtained in this example are shown in FIGS. 1 and 2, and the sample detection result is shown in Table 8.
TABLE 8 test results
2.5 determination of results
2.5.1 the experimental results satisfy the following conditions at the same time, and the experimental results are valid (as shown in fig. 1, fig. 2 and table 8):
(1) correlation coefficient R of standard curve2More than or equal to 0.99, and the amplification efficiency is between 90 and 110 percent;
(2) ct values for the no template negative control (NTC) should be underdetermined;
(3) the Ct value of the negative control group (NCS) should be underdetermined;
(4) the recovery rate of the quality control point is between 80 and 120 percent.
2.5.2 if the Ct value of the sample to be detected is underdetermined, judging that the batch of samples does not detect the replication-competent retrovirus (RCR); if the sample to be detected has a Ct value, determining that the batch sample detects the replication-competent retrovirus (RCR).
In actual production, if the Ct value of the sample to be detected is underdetermined, it is indicated that the batch of samples does not detect the replication retrovirus with the GALV-directed retrovirus vector, the quality inspection is qualified, and the cell products of the batch of samples can be rapidly released.
Specificity (primer specificity) validation test
Possible interfering components in the TCR-T cell injection are: the genomic DNA of T cells, the MSGV viral vector and the TCR of the target gene of the patient or healthy person may be homologous to the primers, and therefore the test sample is set as: patient T cell genome (i.e., NC-T cell genomic DNA), pMSGV-ESO plasmid, healthy human PBMC genomic DNA. Positive control samples were also set: PG13 cell genomic DNA.
The above samples were subjected to fluorescent quantitative PCR detection by the method described in example 1. The results are shown in Table 9.
TABLE 9 results of the test for specificity verification
As can be seen from Table 9, no detection was observed in any of the samples except the positive control sample, indicating that the primers and probes provided by the present invention did not cause any nonspecific reaction in these samples. Therefore, the primer in the replication-competent retrovirus detection kit provided by the invention has good specificity to the replication-competent retrovirus.
And (3) sensitivity test:
GALV standards were diluted with UltraPure Water into a series of concentration gradient samples: 700000 copies/reaction, 70000 copies/reaction, 7000 copies/reaction, 700 copies/reaction, 70 copies/reaction, 7 copies/reaction, respectively labeled: ST1, ST2, ST3, ST4, ST5 and ST6, and the obtained standard curve and the amplification result are shown in FIG. 1 and FIG. 2.
The amplification curves as in figure 2 show: the amplification curves at each concentration had significant exponential growth periods. Standard curve as in fig. 1: linear equation Y ═ 3.175lgX +38.63, R20.999, the result shows that the linear relationship between the CT value and the lg value of the DNA template amount is good in each reaction hole added with the GALV standard, the amplification efficiency is 106.53%, the amplification efficiency is between 90% and 110%, and the detection sensitivity can reach 7 copies/reaction.
As can be seen from the results of the repeated experiments in Table 10, the linearity between the DNA template amount of 7 copies/reaction and 700000 copies/reaction is good for the standard curve of the results of the three additional experiments, R2The amplification efficiency is between 90% and 110%, so the quantitative limit LOQ of the method to the TCR-T cell DNA can reach 7 copies/reaction.
TABLE 10 results of repeated experiments with standard curve of sensitivity test
| Experiment of | Linear equation of equations | Linear decision coefficient R2 | The amplification efficiency% |
| Repetition of 1 | Y＝-3.387lgX+39.141 | 0.999 | 97.338 |
| Repetition 2 | Y＝-3.315lgX+38.984 | 0.999 | 100.273 |
| Repetition of 3 | Y＝-3.452lgX+39.398 | 0.999 | 94.837 |
Accuracy and precision verification test
On the basis of example 1, accuracy and precision analysis were performed by adding a standard (high, medium, and low concentrations) to a sample of TCR-T cell injection, and three different experimenters performed the same test procedure, and the results of accuracy (recovery) are shown in table 11. The results of precision are shown in tables 12-13.
TABLE 11 results of recovery calculation of accuracy test
TABLE 12 results of in-batch precision calculations
TABLE 13 results of inter-batch precision calculations
According to the results in table 11, in the three experiments, the high, medium and low concentration standard recovery rates of the three batches of samples are all between 70% and 130%, and the accuracy verification of the kit and the detection method provided by the invention meets the standard requirements.
According to the results in tables 12-13, the intra-batch variation coefficient and the inter-batch variation coefficient of the measured values of the high, medium and low spiking contents of the three batches of samples are not more than 10% and not more than 20% in the three experiments. Therefore, the precision verification of the kit and the detection method provided by the invention meets the standard requirement.
In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.
While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence listing
<110> Shenzhen jinuo immune Limited, Shenzhen city jinuo transformation medical research institute
<120> primer, kit and detection method for detecting replication-competent retrovirus based on q-PCR method
<130> 1
<141> 2021-09-17
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ggcacctttc ctatgcacaa ccc 23
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 2
ggtcgcctac tccaccgacc t 21
<210> 3
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 3
ccttgggtcc cccagcggca ccggtc 26
Claims (10)
1. A primer for detecting replication-competent retrovirus based on q-PCR method is characterized by comprising a primer GALV-F, a primer GALV-R and a probe GALV-probe;
the nucleotide sequences of the primer GALV-F, the primer GALV-R and the probe GALV-probe are shown as SEQ ID No, or homofunctional sequences with at least 95% of identity with the sequences;
GALV-F：GGCACCTTTCCTATGCACAACCC；
GALV-R：GGTCGCCTACTCCACCGACCT；
GALV-probe：CCTTGGGTCCCCCAGCGGCACCGGTC。
2. the q-PCR-based primer for detecting a replication-competent retrovirus according to claim 1, wherein the fluorescent reporter of the probe GALV-probe is FAM and the quencher is NFQ-MGB.
3. A kit for detecting a replication competent retrovirus by q-PCR, comprising the primers for detecting a replication competent retrovirus by q-PCR according to claims 1 to 2.
4. A replication competent retrovirus detection kit according to claim 3, further comprising the following components: buffer solution of a PCR reaction system, fluorescent dye and GALV standard substance.
5. A replication competent retrovirus detection kit according to claim 4, wherein the reaction system for performing fluorescent quantitative PCR is:
2 XProbe qPCR Premix Ex Taq 10 muL, primer GALV-F, primer GALV-R each 0.4 muL, Probe GALV-Probe 0.8 muL, ROX reference dye II 0.2 muL, sample DNA to be detected or standard DNA5-7 muL, supplement UltraPure water to 20 muL;
wherein the concentration of the primer GALV-F, the primer GALV-R and the probe GALV-probe is 10. mu.M.
6. A method for detecting a replication-competent retrovirus based on a q-PCR method, comprising the steps of:
s1, extracting the genomic DNA of the sample to be detected, and determining the concentration of the sample DNA;
s2, preparing a qPCR reaction system which comprises a primer GALV-F, a primer GALV-R and a probe GALV-probe, wherein the nucleotide sequence of the primer GALV-R and the probe GALV-probe is as follows:
primer GALV-F: GGCACCTTTCCTATGCACAACCC, respectively;
primer GALV-R: GGTCGCCTACTCCACCGACCT, respectively;
probe GALV-probe: CCTTGGGTCCCCCAGCGGCACCGGTC, respectively;
s3, preparing a standard substance solution for a standard curve and a standard substance solution for a quality control point;
s4, template-free negative control, negative control of the sample to be detected and sample loading detection of the sample to be detected with the standard substance;
and S5, judging the result.
7. The method for detecting a replication-competent retrovirus according to q-PCR of claim 6, wherein the detection result in S5 satisfies the following conditions at the same time, and the detection result is judged to be valid:
(1) correlation coefficient R of standard curve slope2Not less than 0.99, and the amplification efficiency is between 90% and 110%;
(2) the Ct value of the no-template negative control group should be undermined;
(3) the Ct value of the negative control group should be underdetermined.
8. The method for detecting a replication-competent retrovirus according to q-PCR of claim 7, wherein in S5, if the Ct value of the sample to be tested is underdetermined, it is determined that no replication-competent retrovirus is detected from the sample of the batch; and if the Ct value of the sample to be detected is present, judging that the replication-competent retrovirus is detected in the batch of samples.
9. The method for detecting a replication-competent retrovirus according to q-PCR of claim 6, wherein in S4, the qPCR reaction conditions are set as follows: pre-denaturation at 95 ℃ for 60 s; then, the reaction was carried out for 40 cycles at 95 ℃ for 5 seconds and 62 ℃ for 34 seconds.
10. The method for detecting a replication competent retrovirus according to q-PCR of claim 6, wherein the concentration of each diluted sample in the standard curve of the standard substance in S3 is: 1.4X 105copies/μL，1.4×104copies/μL，1.4×103copies/μL，1.4×102copies/μL，1.4×101copies/μL、1.4copies/μL。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109804089A (en) * | 2016-07-29 | 2019-05-24 | 朱诺治疗学股份有限公司 | For assessing the present or absent method of duplicating virus |
| CN111918972A (en) * | 2018-01-31 | 2020-11-10 | 朱诺治疗学股份有限公司 | Methods and reagents for assessing the presence or absence of replication competent viruses |
| CN111910015A (en) * | 2019-05-08 | 2020-11-10 | 深圳宾德生物技术有限公司 | Probe, primer pair, fluorescent quantitative PCR kit and method for detecting replication type lentivirus |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109804089A (en) * | 2016-07-29 | 2019-05-24 | 朱诺治疗学股份有限公司 | For assessing the present or absent method of duplicating virus |
| CN111918972A (en) * | 2018-01-31 | 2020-11-10 | 朱诺治疗学股份有限公司 | Methods and reagents for assessing the presence or absence of replication competent viruses |
| CN111910015A (en) * | 2019-05-08 | 2020-11-10 | 深圳宾德生物技术有限公司 | Probe, primer pair, fluorescent quantitative PCR kit and method for detecting replication type lentivirus |
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