CN105861733A - Primer, probe and method for detecting CAR-T transduction efficiency - Google Patents
Primer, probe and method for detecting CAR-T transduction efficiency Download PDFInfo
- Publication number
- CN105861733A CN105861733A CN201610424108.2A CN201610424108A CN105861733A CN 105861733 A CN105861733 A CN 105861733A CN 201610424108 A CN201610424108 A CN 201610424108A CN 105861733 A CN105861733 A CN 105861733A
- Authority
- CN
- China
- Prior art keywords
- primer
- car
- probe
- transduction efficiency
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a primer, probe and method for detecting the CAR-T transduction efficiency, and belongs to the technical field of biology. The primer for detecting the CAR-T transduction efficiency includes an upstream primer and a downstream primer. The sequence of the upstream primer is GCTGTAGCTGCCGATTTCC, and the sequence of the downstream primer is TCGTCCTAGATTGAGCTCGTTA. The primer and probe are good in Real-Time PCR detection specificity, the transduction efficiency of CAR-T can be accurately detected, the detection efficiency is improved, and powerful supports are provided for detecting the curative effect of the immune cell treating technology through the primer and probe.
Description
Technical field
The invention belongs to biological technical field, particularly relate to a kind of for detect the primer of CAR-T transduction efficiency, probe and
Method.
Background technology
Chimeric antigen receptor T cell immunotherapy (Chimeric Antigen Receptor T-Cell
Immunotherapy, CAR-T), as the foreword technology of tumor vaccine cells treatment, drench in acute leukemia and non-Hodgkin′s
Significant curative effect is had in the treatment of bar tumor.The ultimate principle of CAR-T is exactly the T cell utilizing patient self, and chimeric antigen is subject to
After body is modified, can specifically identify tumor associated antigen, make the targeting of effector T cell, killing activity and persistency equal
The immunocyte of more conventional application is high, and can overcome tumor by local immunosuppressant microenvironment and break host immune tolerance status.
As the therapy of a kind of tumor disease, CAR-T is before being expelled to realize immunization therapy in the patient, it is desirable to have effect
Quality Control means, it is judged that CAR-T whether reach treat requirement, i.e. need CAR-T transduction efficiency is detected.Existing detection
Method includes Real-Time PCR method, flow cytometry etc..
Chinese patent application 201610079872.0 discloses the detection capture probe of CAR-T cell, this cell content
Detection method；Capture probe includes the part that can be combined in CAR-T cell, and is combined in the fluorescence molecule on this part,
The detection of CAR-T cell is carried out by flow cytometry.
Real-Time PCR method is due to easy and simple to handle, accurately and cost is relatively low, by as detecting CAR-T transduction efficiency
Common method.
Summary of the invention
For solving problems of the prior art, the present invention provide a kind of primer for detecting CAR-T transduction efficiency,
Probe and method, primer and probe are designed for the conserved sequence in CAR-T gene order, the side provided in conjunction with the present invention
Method, it is possible to achieve the detection by quantitative of transduction efficiency, easy to operate, accuracy is good, overcomes prior art special to CAR-T detection
Property is low, accuracy is low and is easily generated false-positive problem.
The present invention provides a kind of primer for detecting CAR-T transduction efficiency, including forward primer and downstream primer；Upstream
The sequence of primer is: GCTGTAGCTGCCGATTTCC, i.e. SEQ ID NO:1, and the sequence of downstream primer is:
TCGTCCTAGATTGAGCTCGTTA, i.e. SEQ ID NO:2.
Correspondingly, the present invention also provide for a kind of with above-mentioned primer with the use of probe, the sequence of described probe is:
ACTCTCAGTTCACATC, i.e. SEQ ID NO:3.
Primer and probe that the present invention provides are designed for the conserved sequence in CAR-T gene order, for Real-
The specificity of Time PCR detection is good, it is possible to accurately detects the transduction efficiency of CAR-T, improves detection efficiency, for it in immunity
The examination of curative effect of cell therapy technology provides strong support.Forward primer and downstream primer are all without primer dimer, and upstream
The annealing temperature gap of primer and downstream primer is less.
Preferably, 5 ' ends of described probe are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
It is highly preferred that described fluorescent reporter group is selected from VIC；Described fluorescent quenching group is MGB.
Additionally, the present invention also provides for a kind of method for detecting CAR-T transduction efficiency, comprise the steps:
The making of S1DNA template: in gnotobasis, takes the CAR-T cell of cultivation, extracts DNA；
The making of S2 serial standards: use plasmid CART-19scFv as standard substance mother solution, extract T lymph before infecting
Cell DNA, is used for diluting CART-19scFv plasmid, makes serial standards；The copy number of serial standards is respectively 1.590E
+08、1.590E+07、1.590E+06、1.590E+05、1.590E+04、1.590E+03、1.590E+02、1.590E+01；
S3Real-Time PCR detects: preparation reaction system, this reaction system include primer described in claim 1 with
And the probe described in any one of claim 2 to 4, DNA profiling is carried out Real-Time pcr amplification reaction, collects fluorescence letter
Number, draw standard curve, draw CAR-T gene copy number and CAR-T transduction efficiency.
Preferably, the condition of described Real-Time pcr amplification reaction is: 95 DEG C of 15min；94 DEG C of 5s, 62 DEG C of 10s, 72
DEG C 20s, totally 40 circulations；72℃300s.
Preferably, the cultivated days in described step S1 is 10～14 days.By existing cultural method, by 10～14 days
Cultivate, obtain the cell of exponential phase, the most final CAR-T product.
The construction method of CART-19scFv plasmid is: synthesized by CAR recombination complete sequence, double by AsisI/NsiI
Enzyme action is connected in slow virus plasmid vector pLent-EF1a, obtains CART-19scFv plasmid.The spectrogram of CART-19scFv plasmid
As shown in Figure 1.
Compared with prior art, the beneficial effect comprise that primer and probe that the present invention provides are for CAR-T base
Because the conserved sequence in sequence is designed, good for the specificity of Real-Time PCR detection, it is possible to accurately to detect CAR-T
Transduction efficiency, improve detection efficiency, for its immune cell therapy technology examination of curative effect provide strong support.This
The Real-Time PCR detection method of bright offer is simple, it is possible to achieve the detection by quantitative of transduction efficiency, easy to operate, accuracy
Good.
Accompanying drawing explanation
The spectrogram of Fig. 1 CART-19scFv plasmid.
The amplification curve of Fig. 2 primer sets of the present invention.
Fig. 3 contrasts the amplification curve of primer sets.
The standard curve of Fig. 4 primer sets of the present invention detection.
Fig. 5 embodiment of the present invention two comprises the amplification curve of sample.
Detailed description of the invention
With embodiment, the present invention is described in further details below in conjunction with the accompanying drawings.
The material related in the present invention can be obtained by commercially available or the ordinary skill in the art.As: DNA extraction kit is purchased
From Axygen company of the U.S., Super Real Real PreMix is purchased from TIANGEN Biotech (Beijing) Co., Ltd., etc..
Embodiment one primer and probe
For detecting the primer of CAR-T transduction efficiency, including forward primer and downstream primer, the sequence of forward primer is:
GCTGTAGCTGCCGATTTCC, i.e. SEQ ID NO:1；The sequence of downstream primer is: TCGTCCTAGATTGAGCTCGTTA, i.e.
SEQ ID NO:2.
With above-mentioned primer with the use of probe, the sequence of described probe is: ACTCTCAGTTCACATC, i.e. SEQ ID
NO:3.5 ' ends of probe are marked with fluorescent reporter group VIC, and 3 ' ends are marked with fluorescent quenching group MGB.The primer of design is transferred to
General biological system (Anhui) company synthesizes, and the synthesis of Life Technology company transferred to by probe.
The present invention devises contrast primer sets simultaneously: the sequence of forward primer is: TGCCGATTTCCAGAAGAAG, i.e. SEQ
ID NO:4, the sequence of downstream primer is: GCGCTCCTGCTGAACTTC, i.e. SEQ ID NO:5.
Embodiment two Real-Time PCR detects
For the method detecting CAR-T transduction efficiency, comprise the steps:
The making of S1 DNA profiling: in gnotobasis, takes the CAR-T cell cultivated 11 days, uses DNA extraction kit to carry
Take DNA；Using the CAR-T cell quantity after cultivating is 4*106Individual；The DNA extracted uses Nanodrop instrument to carry out DNA concentration
And purity analysis, recording sample DNA concentration is 69ng/ μ l.
The making of S2 serial standards: use plasmid CART-19scFv as standard substance mother solution, plasmid CART-19scFv
Concentration be 1.47 μ g/mL, extract infect prolymphocyte DNA, be used for dilute CART-19scFv plasmid, make series standard
Product；The copy number of serial standards be respectively 1.590E+08,1.590E+07,1.590E+06,1.590E+05,1.590E+04,
1.590E+03、1.590E+02、1.59E+01；
S3 Real-Time PCR detects: preparation reaction system is as shown in table 1, and the condition of amplified reaction is as shown in table 2, right
DNA profiling carries out Real-Time pcr amplification reaction, collects fluorescence signal, uses primer sets (the SEQ ID that the present invention provides
NO:1 and SEQ ID NO:2) amplification curve as in figure 2 it is shown, use contrast primer sets (SEQ ID NO:4 and SEQ ID NO:
5) amplification curve is as shown in Figure 3.By comparison diagram 2 and Fig. 3, the amplification curve effect of the primer sets that the present invention provides is more
Good, primer is good with probe matching.With Cq value as vertical coordinate, with the logarithm of Starting Quantity as abscissa, draw this
The examination criteria curve of invention primer sets is as shown in Figure 4.
Table 1 reaction system
Table 2 amplification reaction condition
The Real-Time PCR detection method that the present invention provides achieves the detection by quantitative of CAR-T transduction efficiency, detection
Unit is Copies/100ng genomic DNA (the CAR-T gene copy number contained in every 100ng genome).Bent according to standard
Line, carries out quantitatively, conversing the CAR-T gene copy number contained in every 100ng genome, with this to the CAR-T copy number of sample
Draw transduction efficiency.This sample is for be drawn by 90ng genomic DNA amplification, and sample DNA is from 4.0 × 106Individual cell obtains
6.90ug genomic DNA take out, the transduction efficiency calculating sample (Cq value is 26.34) is a 0.55 copy number/cell, the moon
Property comparison Cq value be 39.80.The criterion of CAR-T product transduction efficiency is >=0.2 copy number/cell, as judgement
Whether CAR-T product reaches the criterion of the requirement of immunization therapy.
Above content is to combine concrete preferred implementation further description made for the present invention, it is impossible to assert
Being embodied as of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of present inventive concept, it is also possible to make some simple deduction or replace, all should be considered as belonging to the present invention's
Protection domain.
SEQUENCE LISTING
<110>suitable clear-cells bio tech ltd
<120>a kind of for detecting the primer of CAR-T transduction efficiency, probe and method
<130>
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
gctgtagctg ccgatttcc 19
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
tcgtcctaga ttgagctcgt ta 22
<210> 3
<211> 19
<212> DNA
<213>artificial sequence
<400> 3
actctcagtt cacatcctc 19
<210> 4
<211> 19
<212> DNA
<213>artificial sequence
<400> 4
tgccgatttc cagaagaag 19
<210> 5
<211> 18
<212> DNA
<213>artificial sequence
<400> 5
gcgctcctgc tgaacttc 18
Claims (7)
1. the primer being used for detecting CAR-T transduction efficiency, it is characterised in that: include forward primer and downstream primer；Described
The sequence of forward primer is: GCTGTAGCTGCCGATTTCC, i.e. SEQ ID NO:1；The sequence of described downstream primer is:
TCGTCCTAGATTGAGCTCGTTA, i.e. SEQ ID NO:2.
2. one kind with the primer described in claim 1 with the use of probe, it is characterised in that: the sequence of described probe is:
ACTCTCAGTTCACATCCTC, i.e. SEQ ID NO:3.
Probe the most according to claim 2, it is characterised in that: the 5 ' of described probe are held and are marked with fluorescent reporter group, and 3 '
End is marked with fluorescent quenching group.
Probe the most according to claim 3, it is characterised in that: described fluorescent reporter group is selected from VIC；Described fluorescent quenching
Group is MGB.
5. the method being used for detecting CAR-T transduction efficiency, it is characterised in that: comprise the steps:
The making of S1 DNA profiling: in gnotobasis, takes the CAR-T cell after cultivation, extracts DNA；
The making of S2 serial standards: use plasmid CART-19scFv as standard substance mother solution, extract and infect pre-T lymphocyte
DNA, is used for diluting CART-19scFv plasmid, makes serial standards；
S3 Real-Time PCR detects: preparation reaction system, this reaction system includes the primer described in claim 1 and power
Profit requires the probe described in 2 to 4 any one, and DNA profiling carries out Real-Time pcr amplification reaction, collects fluorescence signal, paints
Standard curve processed, draws CAR-T gene copy number and CAR-T transduction efficiency.
Method for detecting CAR-T transduction efficiency the most according to claim 5, it is characterised in that: described Real-Time
The condition of pcr amplification reaction is: 95 DEG C of 15min；94 DEG C of 5s, 62 DEG C of 10s, 72 DEG C of 20s, totally 40 circulations；72℃300s.
Method for detecting CAR-T transduction efficiency the most according to claim 5, it is characterised in that: in described step S1
Cultivated days be 10～14 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610424108.2A CN105861733A (en) | 2016-06-14 | 2016-06-14 | Primer, probe and method for detecting CAR-T transduction efficiency |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610424108.2A CN105861733A (en) | 2016-06-14 | 2016-06-14 | Primer, probe and method for detecting CAR-T transduction efficiency |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105861733A true CN105861733A (en) | 2016-08-17 |
Family
ID=56650499
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610424108.2A Pending CN105861733A (en) | 2016-06-14 | 2016-06-14 | Primer, probe and method for detecting CAR-T transduction efficiency |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105861733A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017133222A1 (en) * | 2016-02-04 | 2017-08-10 | 北京艺妙神州医疗科技有限公司 | Capture probe for detecting car-t cell, method for detecting cell content, and application |
| CN108285920A (en) * | 2017-01-09 | 2018-07-17 | 上海恒润达生生物科技有限公司 | A kind of technology and application thereof of vivo detection CART cells expression |
| CN109722468A (en) * | 2019-02-28 | 2019-05-07 | 上海邦耀生物科技有限公司 | Detect the PCR primer combination and application of BCMA Chimeric antigen receptor gene |
| WO2019129048A1 (en) * | 2017-12-28 | 2019-07-04 | 上海细胞治疗研究院 | Method and kit for determining car copy number by using dual fluorescence quantitative pcr |
| WO2019129055A1 (en) * | 2017-12-28 | 2019-07-04 | 上海细胞治疗研究院 | Locked nucleic acid-modified probe and method for determining car copy number |
| CN109971837A (en) * | 2017-12-28 | 2019-07-05 | 上海细胞治疗研究院 | A kind of detection method and kit of exogenous antibodies gene copy number |
| CN111378651A (en) * | 2018-12-28 | 2020-07-07 | 上海细胞治疗集团有限公司 | Method for detecting intracellular PB gene residue and detection kit |
| CN111500690A (en) * | 2020-04-15 | 2020-08-07 | 深圳科诺医学检验实验室 | Method for detecting copy number of virus vector in CAR-T cell genome |
| CN111635899A (en) * | 2019-03-01 | 2020-09-08 | 上海细胞治疗集团有限公司 | Detection kit for foreign plasmid residue based on resistance gene and use method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928355A (en) * | 2014-03-19 | 2015-09-23 | 李跃 | Method and kit thereof for detecting BRAF gene mutation |
| CN105368961A (en) * | 2015-12-15 | 2016-03-02 | 天津脉络生物科技有限公司 | Shewanella oneidensis quantitative detection fluorescent PCR reagent kit and application thereof |
| CN105504042A (en) * | 2016-02-04 | 2016-04-20 | 北京艺妙神州医疗科技有限公司 | Capture probe used for detecting CAR-T (Chimeric Antigen Receptor-T) cells, method for detecting content of cells, and application |
-
2016
- 2016-06-14 CN CN201610424108.2A patent/CN105861733A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928355A (en) * | 2014-03-19 | 2015-09-23 | 李跃 | Method and kit thereof for detecting BRAF gene mutation |
| CN105368961A (en) * | 2015-12-15 | 2016-03-02 | 天津脉络生物科技有限公司 | Shewanella oneidensis quantitative detection fluorescent PCR reagent kit and application thereof |
| CN105504042A (en) * | 2016-02-04 | 2016-04-20 | 北京艺妙神州医疗科技有限公司 | Capture probe used for detecting CAR-T (Chimeric Antigen Receptor-T) cells, method for detecting content of cells, and application |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017133222A1 (en) * | 2016-02-04 | 2017-08-10 | 北京艺妙神州医疗科技有限公司 | Capture probe for detecting car-t cell, method for detecting cell content, and application |
| CN108285920A (en) * | 2017-01-09 | 2018-07-17 | 上海恒润达生生物科技有限公司 | A kind of technology and application thereof of vivo detection CART cells expression |
| WO2019129048A1 (en) * | 2017-12-28 | 2019-07-04 | 上海细胞治疗研究院 | Method and kit for determining car copy number by using dual fluorescence quantitative pcr |
| WO2019129055A1 (en) * | 2017-12-28 | 2019-07-04 | 上海细胞治疗研究院 | Locked nucleic acid-modified probe and method for determining car copy number |
| CN109971836A (en) * | 2017-12-28 | 2019-07-05 | 上海细胞治疗研究院 | The method and kit of double fluorescent quantitative PCR measurement CAR copy number |
| CN109971835A (en) * | 2017-12-28 | 2019-07-05 | 上海细胞治疗研究院 | A kind of probe that lock nucleic acid is modified and a kind of method for measuring CAR copy number |
| CN109971837A (en) * | 2017-12-28 | 2019-07-05 | 上海细胞治疗研究院 | A kind of detection method and kit of exogenous antibodies gene copy number |
| CN111378651A (en) * | 2018-12-28 | 2020-07-07 | 上海细胞治疗集团有限公司 | Method for detecting intracellular PB gene residue and detection kit |
| CN109722468A (en) * | 2019-02-28 | 2019-05-07 | 上海邦耀生物科技有限公司 | Detect the PCR primer combination and application of BCMA Chimeric antigen receptor gene |
| CN111635899A (en) * | 2019-03-01 | 2020-09-08 | 上海细胞治疗集团有限公司 | Detection kit for foreign plasmid residue based on resistance gene and use method thereof |
| CN111500690A (en) * | 2020-04-15 | 2020-08-07 | 深圳科诺医学检验实验室 | Method for detecting copy number of virus vector in CAR-T cell genome |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105861733A (en) | Primer, probe and method for detecting CAR-T transduction efficiency | |
| Chacko et al. | The lncRNA RZE1 controls cryptococcal morphological transition | |
| CN105524924B (en) | Cyclic RNA circ-ZKSCAN1 use | |
| CN103028119B (en) | Application of miR-132 in preparation of drug for treating parkinsonism | |
| CN111206109B (en) | Multiple RPA detection primer group and kit for Brucella melitensis of cattle, sheep and pig species | |
| CN108624720A (en) | The primed probe group and kit of RAA Fluorometric assay Rift Valley fever virus | |
| CN103374573B (en) | Liver cancer cell nucleic acid aptamer sequence and uses thereof | |
| CN107190005B (en) | Applications of the lncRNA as biomarker in adenocarcinoma of lung diagnosis and treatment | |
| CN108411014A (en) | Differentiate the primer and probe and detection method of A types and the dual real-time fluorescence quantitative PCR of Type B ox pasteurella multocida | |
| CN111518954A (en) | Primer group, kit and method for double nano PCR detection of H5 and N8 subtype avian influenza virus | |
| CN106103714B (en) | In vivo infection microorganism, parasitic microbe, the noncoding RNA of symbiotic microorganism and its identification and application | |
| CN107723348B (en) | NASBA detection method for identifying Listeria monocytogenes 1/2c serotype | |
| CN115779084A (en) | Application of preparation for activating pig TUSC1 gene expression in preparation of pig pseudorabies virus infection resisting medicine | |
| CN112322711A (en) | Primer group, kit and method for detecting copy number of human CAR-T cells in mouse model | |
| CN112226460B (en) | Screening and breeding method of cell line for stably expressing porcine host restriction factor A3Z2 molecule and application thereof | |
| CN108611442A (en) | The fluorescence quantitative RT-PCR primer and probe and method of a kind of detection pig atypia pestivirus and application | |
| CN110951918B (en) | Kit for combined detection of influenza A and B viruses based on RNA isothermal amplification-gold probe chromatography technology and application thereof | |
| CN106521038A (en) | High-sensitivity BHV-2 (bovine herpes virus 2) quantitative real-time PCR (polymerase chain reaction) detection method and kit | |
| CN102212611B (en) | Standard molecule for detecting tilletia walkeri castebury&carris and construction method thereof | |
| CN104178575B (en) | Method and the kit of a kind of quick discriminating campylobacter fetus and venereal disease subspecies | |
| CN107236023B (en) | Antigen composition for detecting tuberculosis infection and application thereof | |
| CN104531904A (en) | Apple chlorotic leaf spot virus real-time fluorescent quantitative PCR (polymerase chain reaction) detection method | |
| CN110699471B (en) | Primer pair and kit for rapidly detecting mycoplasma and application of primer pair and kit | |
| Tomasek et al. | Comparison of three template preparation methods for routine detection of beak and feather disease virus and avian polyomavirus with single and nested polymerase chain reaction in clinical specimens | |
| CN116426668B (en) | Primer group for detecting mucor based on nucleic acid mass spectrometry technology and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160817 |