CN116179762A - Primer group, detection product and detection method for virus titer - Google Patents
Primer group, detection product and detection method for virus titer Download PDFInfo
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Abstract
The invention relates to the field of biological detection, in particular to a primer group, a detection product and a detection method of virus titer. The invention provides a primer set, comprising: the nucleotide sequence shown in any of SEQ ID NO. 1-SEQ ID NO. 20. The invention aims at screening qPCR primers, designing an upstream primer U3-F aiming at a deltaU 3 region remained in a 3' LTR of a self-inactivated SIN carrier, designing a downstream primer SIN-R on a packaging signal, and measuring the functional titer of MMLV-SIN, so that the detection result can truly reflect the actual functional titer of the MMLV-SIN.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a primer group, a detection product and a detection method of virus titer.
Background
Moloney murine leukemia virus (MoloneyMurine LeukemiaVirus, MMLV) is one type of retrovirus whose genome is two identical positive strand RNAs. After infection of the cells by the virus, the RNA genome can be reverse transcribed into DNA for integration into the host genome and stable passaging. The mechanism of integration is that when the virus transduces the target cell, the glycoprotein on the virus surface binds to the receptor on the cell surface, mediating the entry of viral RNA into the host cell, and further reverse transcribing into DNA by means of self-carried reverse transcriptase, followed by random integration of the viral genome located between the two LTRs into the genome of the host cell. However, when MMLV is randomly integrated in the host genome, it can induce up-regulation of gene expression near the site of integration, even activation of protooncogenes, leading to tumorigenesis. To address this potential safety issue, the present company invented MMLV Self-inactivating (SIN) vectors (authorized, CN 112458120B) that lack viral promoter and enhancer sequences in the U3 region of the 3' ltr, which not only reduces the risk of gene upregulation near the insertion site, but also increases autonomy of internal promoter selection.
Viral titer (TU/mL) is an important indicator for measuring MMLV viral dose in MMLV clinical treatment. Because the presence of empty capsids is not therapeutically valuable, measuring viral genome titres during clinical administration, production and analytical testing is the method of choice for measuring titres. The most widely used method for determining the titre of MMLV virus at present is real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR). The qPCR method is to add a fluorescent group into a PCR system to monitor the change of a fluorescent signal in the amplification process in real time, and then to quantify a sample to be detected through a standard curve, and mainly comprises a SYBR Green method and a TaqMan probe method.
At present, the design strategy of the upstream and downstream primers involved in the qPCR method for detecting the conventional MMLV titer mainly aims at simultaneously targeting two primers to a packaging signal region or a target gene sequence, but when plasmid DNA residues are contained in viruses of transduced cells, the copy number detected by the method is the sum of the copy number of the residual plasmid DNA and the integrated copy number of the viruses, and additional repeated rinsing and DNase digestion steps are required to avoid inaccuracy of titer detection, so that the operation duration and uncertainty factors are increased. Unlike conventional MMLV, after the cell is transduced and integrated by the inactivated virus, the residual detaU3 region in the 3'LTR is integrated to the 5' end, and the plasmid DNA itself does not have the structure, so that the primers are designed and developed based on the virus characteristics, and a pair of titer detection primers with good primer specificity, high amplification efficiency and simple and convenient operation and a corresponding detection method system are obtained by screening the primers.
Disclosure of Invention
In view of this, the present invention provides primer sets, detection products and methods for detecting viral titers. The invention aims at screening qPCR primers, designing an upstream primer U3-F aiming at a deltaU 3 region remained in a 3' LTR of a self-inactivated SIN vector, designing a downstream primer SIN-R on a packaging signal, and determining the functional titer of MMLV-SIN, wherein the primers are characterized in that interference of plasmid DNA remained during packaging viruses can be eliminated, so that the detection result can truly reflect the actual functional titer of the MMLV-SIN.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer set, comprising:
(1) Nucleotide sequences shown in any of SEQ ID NO. 1-SEQ ID NO. 20; or (b)
(2) A nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (1) and has the same or similar function as the nucleotide sequence shown in (1); or (b)
(3) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (1) or (2).
The invention also provides a primer group, which comprises:
(9) Nucleotide sequences shown as SEQ ID NO. 11 and SEQ ID NO. 12; or (b)
(10) A nucleotide sequence which encodes the same protein as the nucleotide sequence of (9) but which differs from the nucleotide sequence of (9) due to the degeneracy of the genetic code; or (b)
(11) A nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (9) or (10) and which is functionally identical or similar to the nucleotide sequence shown in (9) or (10); or (b)
(12) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (9), (10) or (11).
In some embodiments of the invention, the primer set comprises one or more of the following combinations:
combination X:
(4) The upstream primer has a nucleotide sequence shown as SEQ ID No (2X-1); and
(5) The downstream primer has a nucleotide sequence shown as SEQ ID No (2X); or (b)
(6) A nucleotide sequence which encodes the same protein as the nucleotide sequence of (4) or (5) but which differs from the nucleotide sequence of (4) or (5) due to the degeneracy of the genetic code; or (b)
(7) A nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (4), (5) or (6) and which is functionally identical or similar to the nucleotide sequence shown in (4), (5) or (6); or (b)
(8) A nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of (4), (5), (6) or (7);
wherein X is selected from any integer from 1 to 10.
In some embodiments of the invention, the upstream primer of the above primer set is located in the U3 region of the self-inactivating SIN vector 3' ltr; the downstream primer is located in the packaging signal region.
In some embodiments of the present invention, the SIN1 sequence in the primer set is as shown in SEQ ID NO. 27: cataactgagaatagagaagttcagatcaaggtcaggaacagatggaacaggagcccacaacccctcact cggggcgccagtcctccgattgactgagtcgcccgggtacccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtgattgactacccgtcagcgggggtctttcatttgggggctcgtccgggatcgggagacccctgcccagggaccaccgacccaccaccgggaggtaagctggccagcaacttatctgtgtctgtccgattgtctagtgtctatgactgattttatgcgcctgcgtcggtactagttagctaactagctctgtatctggcggacccgtggtggaactgacgagtt.
The invention also provides application of the primer group in detecting the titer of the Moroni murine leukemia virus.
The invention also provides a detection product, which comprises the primer group and acceptable auxiliary agents.
In some embodiments of the invention, the detection product described above comprises reagents and/or kits.
The invention also provides a method for detecting the virus titer, which is used for detecting a sample to be detected by using the primer group or the detection product.
In some embodiments of the invention, the detecting in the above detection method comprises the steps of:
s1: taking diluted virus transduction cells, and culturing to obtain the sample to be tested;
s2: extracting nucleic acid of the sample to be detected, and obtaining a melting curve after amplification;
s3: and obtaining the virus titer of the sample to be tested according to the melting curve.
In some embodiments of the invention, in the above detection method, the virus comprises: MMLV-SIN.
In some embodiments of the invention, in the above detection method, the cell comprises: 293T cells.
In some embodiments of the invention, in the above detection method, the method further comprises, before the taking of the diluted virus-transduced cells: step of cell counting, the result of which is denoted as M.
In some embodiments of the invention, in the above detection method, the dilution factor is 100.
In some embodiments of the invention, in the above detection method, the diluted virus has a volume of 5. Mu.L to 50. Mu.L.
In some embodiments of the invention, in the above detection method, the diluted virus has a volume of 50 μl.
In some embodiments of the invention, in the above detection method, the incubation time is 48 hours and the temperature is 37 ℃.
In some embodiments of the present invention, in the above detection method, the detecting further comprises: extracting nucleic acid of the standard substance of the virus, and obtaining a melting curve of the standard substance after amplification.
In some embodiments of the invention, in the above detection method, the amplified reaction system comprises: system 1 and system 2;
wherein the system 1 comprises: amplification reaction solution with a final concentration of 1×and the primer set with a final concentration of 0.1 μm, nucleic acid of the sample to be tested 5 μl, dye solution 0.4 μl, ultrapure water to 20 μl;
the system 2 comprises: the final concentration of 1X amplification reaction solution, the final concentration of 0.1 mu M internal reference primer group, the sample to be tested of nucleic acid 5 mu L, dye solution 0.4 mu L, ultrapure water make up to 20 mu L.
In some embodiments of the invention, in the above detection method, the internal reference primer set has:
(13) Nucleotide sequences shown in any of SEQ ID NO. 21 and SEQ ID NO. 22; or (b)
(14) A nucleotide sequence which encodes the same protein as the nucleotide sequence shown in (13) but which differs from the nucleotide sequence shown in (13) due to the degeneracy of the genetic code; or (b)
(15) A nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (13) or (14) and which is functionally identical or similar to the nucleotide sequence shown in (13) or (14); or (b)
(16) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (13), (14) or (15).
In some embodiments of the invention, in the above detection method, the reaction conditions of the amplification include:
in some embodiments of the present invention, in the above detection method, the virus titer of the obtained sample to be tested uses a titer formula; the titer calculation formula is: titer (TU/mL) =1000×MOI×M×dilution/transduction volume;
the MOI is as follows: MMLV# -quality Mean ≡BMP 2-quality Mean; the transduction volume is the volume of the diluted virus.
The invention provides a primer group, a detection product and application thereof, and also provides a method for detecting virus titer.
The invention aims at screening qPCR primers, designing an upstream primer U3-F aiming at a deltaU 3 region remained in a 3' LTR of a self-inactivated SIN carrier, designing a downstream primer SIN-R on a packaging signal, and measuring the functional titer of MMLV-SIN, so that the detection result can truly reflect the actual functional titer of the MMLV-SIN.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows amplification curves and melting curves for 10 pairs of primer sets; wherein: a shows the melting curve of primer group 1U 3-F1+SIN-R1; b shows an amplification curve of the primer set 1U 3-F1+SIN-R1; c shows the melting curve of the primer group 2U 3-F1+SIN-R2; d shows an amplification curve of primer set 2U 3-F1+SIN-R2; e shows the melting curve of primer set 3U 3-F1+SIN-R3; f shows an amplification curve of the primer set 3U 3-F1+SIN-R3; g shows the melting curve of primer set 4U 3-F1+SIN-R4; h shows the amplification curve of primer set 4U 3-F1+SIN-R4; i shows the melting curve of primer set 5U 3-F1+SIN-R5; j shows an amplification curve of primer set 5U 3-F1+SIN-R5; k represents a melting curve of the primer group 6U 3-F2+SIN-R1; l shows an amplification curve of the primer set 6U 3-F2+SIN-R1; m shows the melting curve of primer set 7U3-F2+SIN-R2; n shows the amplification curve of primer set 7U3-F2+SIN-R2; o shows the melting curve of primer set 8U 3-F2+SIN-R3; p shows the amplification curve of primer set 8U 3-F2+SIN-R3; q shows the melting curve of primer set 9U 3-F2+SIN-R4; r shows the amplification curve of primer set 9U 3-F2+SIN-R4; s shows a melting curve of the primer set 10U 3-F2+SIN-R5; t shows the amplification curve of primer set 10U 3-F2+SIN-R5;
FIG. 2 shows amplification and melting curves for MMLV-SIN virus titer detection primers; wherein: a shows a melting curve of the primer group 6U 3-F2+SIN-R1; b shows an amplification curve of the primer set 6U 3-F2+SIN-R1; c shows the melting curve of BMP2 gene; d shows the amplification curve of BMP2 gene.
Detailed Description
The invention discloses a primer group, a detection product and a detection method for virus titer.
It should be understood that the expression "one or more of … …" individually includes each of the objects recited after the expression and various combinations of two or more of the recited objects unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood as having the same meaning unless otherwise understood from the context.
The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, should generally be construed as open-ended and non-limiting, e.g., not to exclude other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.
It should be understood that the order of steps or order of performing certain actions is not important so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.
The use of any and all examples, or exemplary language, such as "e.g." or "comprising" herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Furthermore, the numerical ranges and parameters setting forth the present invention are approximations that may vary as precisely as possible in the exemplary embodiments. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless explicitly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used in this disclosure are modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.
In examples 1 and 2 of the present invention, the raw materials and reagents used were commercially available.
The invention is further illustrated by the following examples:
EXAMPLE 1 Standard construction
(1) PCR amplification
PCR amplifying the deltaU 3-R-U5 and part of downstream packaging signal sequence (SIN 1 for short) of provirus by using genomic DNA of cells infected with MMLV-SIN virus as a template, running 2% gel electrophoresis on PCR products, cutting gel for recovery, and determining the concentration of the recovered products. The specific information is as follows:
TABLE 1PCR amplification System
| Primer F (10. Mu.M) | 1μL |
| Primer R (10. Mu.M) | 1μL |
| Template | 5μL |
| PremixLATaqVersion2.0 | 20μL |
| ddH 2 O | upto40μL |
TABLE 2PCR amplification primer information
(2) Gibson cloning construction
1) HindIII and XbaI double-digested pMD18-T-MMLV-hBMP2 (https:// en. Vectorkuil der.com/vector/VB 220223-1480ncx.html) plasmid, the digestion system and reaction conditions are as follows:
TABLE 3 cleavage System and reaction conditions
2) The gel recovery kit recovers the skeleton carrier;
3) The linearization skeleton and the PCR product are subjected to Gibson reaction, and the reaction system and the conditions are as follows:
TABLE 4Gibson reaction System
4) Transforming 100 mu L VB UltraStable competent cells with 5 mu L of the ligation product;
5) Colony PCR identified clones on LB plates, with the following primers:
f: gccagggttttcccagtcac (shown as SEQ ID NO: 25)
R: cacagataagttgctggccagctta (shown as SEQ ID NO: 26)
Amplified fragment length: 379bp
6) Enzyme cutting and sequencing to verify the correctness of the sequence;
7) A universal standard was prepared containing two fragments of SIN1 and BMP 2: the standard plasmid was digested with HindIII and BsaAI, run and the corresponding band (663 bp) was recovered, the concentration was measured using a Qubit fluorometer, and the final concentration was averaged 3 times;
8) Standard substance dilution: the number of molecules was calculated based on concentration, fragment size, average base molecular weight per pair (660), diluted to 10 8 And (5) a standard substance. The calculation method comprises the following steps: x=a×6.02×10 23 ×10 -9 /(660×B×2×10 8 ). Wherein X is the multiple of the dilution required, A is the concentration measured for the fragment (in ng/. Mu.L), B is the fragment size (base pair);
9) After dilution, the standard substance is split into 10 mu L of one-tube and stored in a refrigerator at-80 ℃.
(2) Primer screening test
1) Based on the principle that MMLV will replicate the deltaU 3 region in the 3' LTR into the 5' LTR during reverse transcription and integrate into the genome in the form of provirus, in order to be able to obtain an accurate integrated copy number of MMLV-SIN, the upstream primer U3-F is designed for the residual deltaU 3 region in the 3' LTR of the SIN vector, the downstream primer SIN-R is designed on the downstream packaging signal, and the following 10 pairs of primer sets are designed in parallel for amplification detection.
And (3) performing amplification by taking a standard substance as a template, and testing the specificity and the amplification efficiency of the primer.
TABLE 5qPCR test primers
TABLE 6 summary of qPCR test primer results
As can be seen from the amplification curve of FIG. 1 and the results shown in Table 6, the CT values and quality values corresponding to the primer set 1 to primer set 10 have good linear relationship, and R is 2 Are all greater than 0.99; melting curves are unimodal, and Tm values are all greater than 80 ℃, so that the possibility of nonspecific amplification caused by primer dimer is eliminated; however, except for the primer groups 1, 2 and 6, the peaks at the rear ends of the melting curves of the other 7 groups have sudden abnormal, which may be caused by that the target fragment is too long or the corresponding target fragment CG value is too high to cause incomplete unwinding of the corresponding fragment, so that the other 7 groups are excluded; as is clear from the results of the statistics in Table 6, primer sets 1 and 2,6 are 84.193%, 83.193% and 89.986%, respectively, wherein the amplification efficiency of the primer set 6 is highest, and the primer set 6 is determined to be the optimal primer pair.
Example 2 transduction of 293T cells with 3 different MMLV-SIN viruses and determination of functional titres
(1) The day before transduction, 293T cells were plated at 1.5X10 5 Inoculating each well into a 12-well plate, wherein the total liquid volume of each well is 1 mL, and calculating the number of required inoculations according to two compound wells transduced by each virus sample;
(2) Cells from one well were digested prior to transduction and counted, with the count being denoted as M. 10 mu L of MMLV-SIN virus sample is diluted to 1 mL by culture medium, 50 mu L of diluted virus solution is added into 293T cells, and the mixture is put into a 37 ℃ incubator for continuous culture for 48 hours after being gently shaken uniformly. At the same time, one well should be left without any virus as a blank.
(3) Cell genomic DNA extraction
Cells in the 12-well plate were digested separately, washed 2-3 times with PBS, genomic DNA of the cell sample was extracted with a blood tissue cell genomic extraction kit (Tiangen), and finally eluted with 50. Mu.L of 1 XTE buffer.
(4) qPCR detection of viral titres
1) Take 10 8 Is diluted stepwise to 10 with DEPC water 7 、10 6 、10 5 、10 4 、10 3 ；
2) Preparing qPCR reaction systems according to tables 7 and 8, wherein primers used are shown in Table 9, and BMP2 is an internal reference primer and CN108559792B is authorized;
TABLE 7 qPCR reaction System of MMLV sequence
TABLE 8 qPCR reaction System for BMP2 sequences
TABLE 9 amplification primer information
(5) qPCR reactions (qPCR instrument) were performed according to standard protocols;
(6) Calculation of MMLV-SIN virus titer
As can be seen from FIG. 2, the melting curve of the MMLV-SIN amplification primer is unimodal, the Tm value is 87℃and R 2 1, the amplification efficiency is 88.69%, the melting curve of the BMP2 is also unimodal, the Tm value is 81 ℃, and R 2 The amplification efficiency is 0.999 and 90.262%, the error of the amplification efficiency and the amplification efficiency is within 5%, and the data is reliable and has reference value. MMLV# -quality Mean divided by BMP 2-quality Mean for the same sample is the MOI at the time of transduction of the sample. The Titer calculation formula was Titer (TU/mL) =1000×moi×m (cell count value) ×dilution/transduction volume. Taking sample 1 in table 10 as an example, moi= 418.508/6,955.165 =0.060, cell count before transduction is 7.50e+04, 50 μl of 100-fold diluted virus solution is added during transduction, and the amplification efficiencies of BMP2 and MMLV are identical (approaching 100% and differing by less than 5%), so that the titer is 1000×0.06×7.5×10 4 ×100×1/50＝9.03×10 6 TU/mL。
TABLE 10MMLV-SIN virus titer assay-related data
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A primer set, comprising:
(1) Nucleotide sequences shown in any of SEQ ID NO. 1-SEQ ID NO. 20; or (b)
(2) A nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (1) and has the same or similar function as the nucleotide sequence shown in (1); or (b)
(3) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (1) or (2).
2. The primer set of claim 1, wherein the primer set comprises one or more of the following combinations:
combination X:
(4) The upstream primer has a nucleotide sequence shown as SEQ ID No (2X-1); and
(5) The downstream primer has a nucleotide sequence shown as SEQ ID No (2X); or (b)
(6) A nucleotide sequence which encodes the same protein as the nucleotide sequence of (4) or (5) but which differs from the nucleotide sequence of (4) or (5) due to the degeneracy of the genetic code; or (b)
(7) A nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (4), (5) or (6) and which is functionally identical or similar to the nucleotide sequence shown in (4), (5) or (6); or (b)
(8) A nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of (4), (5), (6) or (7);
wherein X is selected from any integer from 1 to 10.
3. The primer set of claim 2, wherein the upstream primer is located in the U3 region of the self-inactivating SIN vector 3' ltr; the downstream primer is located in the packaging signal region.
4. Use of a primer set according to any one of claims 1 to 3 for detecting the titre of the moroni murine leukemia virus.
5. A test product comprising a primer set according to any one of claims 1 to 3 and an acceptable adjuvant.
6. A method for detecting viral titer, characterized in that a sample to be detected is detected with the primer set according to any one of claims 1 to 3 or the detection product according to claim 5.
7. The method of detection according to claim 6, wherein the detecting comprises the steps of:
s1: taking diluted virus transduction cells, and culturing to obtain the sample to be tested;
s2: extracting nucleic acid of the sample to be detected, and obtaining a melting curve after amplification;
s3: and obtaining the virus titer of the sample to be tested according to the melting curve.
8. The method of detection of claim 7, wherein the detecting further comprises: extracting nucleic acid of the standard substance of the virus, and obtaining a melting curve of the standard substance after amplification.
9. The detection method according to claim 7 or 8, wherein the amplified reaction system comprises: system 1 and system 2;
wherein the system 1 comprises: amplification reaction solution with a final concentration of 1×and the primer set with a final concentration of 0.1 μm, nucleic acid of the sample to be tested 5 μl, dye solution 0.4 μl, ultrapure water to 20 μl;
the system 2 comprises: the final concentration of 1X amplification reaction solution, the final concentration of 0.1 mu M internal reference primer group, the sample to be tested of nucleic acid 5 mu L, dye solution 0.4 mu L, ultrapure water make up to 20 mu L.
10. The detection method according to any one of claims 7 to 9, wherein the reaction conditions for the amplification comprise:
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030175698A1 (en) * | 2001-05-10 | 2003-09-18 | Kim Jong-Mook | Retrovirus-specific oligonucleotides and a method for measuring retroviral titer using same |
| CN110607394A (en) * | 2018-06-15 | 2019-12-24 | 云舟生物科技（广州）有限公司 | Moloney murine leukemia virus titer detection kit and titer detection method |
| CN112458120A (en) * | 2020-11-25 | 2021-03-09 | 云舟生物科技（广州）有限公司 | Self-inactivation vector based on Moloney murine leukemia virus and application thereof |
| CN113151587A (en) * | 2021-01-28 | 2021-07-23 | 云舟生物科技（广州）有限公司 | Primer, kit and detection method for detecting titer of lentivirus |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030175698A1 (en) * | 2001-05-10 | 2003-09-18 | Kim Jong-Mook | Retrovirus-specific oligonucleotides and a method for measuring retroviral titer using same |
| CN110607394A (en) * | 2018-06-15 | 2019-12-24 | 云舟生物科技（广州）有限公司 | Moloney murine leukemia virus titer detection kit and titer detection method |
| CN112458120A (en) * | 2020-11-25 | 2021-03-09 | 云舟生物科技（广州）有限公司 | Self-inactivation vector based on Moloney murine leukemia virus and application thereof |
| CN113151587A (en) * | 2021-01-28 | 2021-07-23 | 云舟生物科技（广州）有限公司 | Primer, kit and detection method for detecting titer of lentivirus |
Non-Patent Citations (1)
| Title |
|---|
| "Murine retrovirus shuttle vector pZIPneoSV（TAg），complete sequence", 《GENBANK》, pages 1 - 2 * |
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