CN103451292B - Identification of specificity of transgenic rice Huahui 1 by applying recombinase polymerase amplification (RPA) technology - Google Patents
Identification of specificity of transgenic rice Huahui 1 by applying recombinase polymerase amplification (RPA) technology Download PDFInfo
- Publication number
- CN103451292B CN103451292B CN201310391525.8A CN201310391525A CN103451292B CN 103451292 B CN103451292 B CN 103451292B CN 201310391525 A CN201310391525 A CN 201310391525A CN 103451292 B CN103451292 B CN 103451292B
- Authority
- CN
- China
- Prior art keywords
- rpa
- primer
- amplification
- seq
- dna
- 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.)
- Expired - Fee Related
Links
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 45
- 235000009566 rice Nutrition 0.000 title claims abstract description 43
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 40
- 230000003321 amplification Effects 0.000 title claims abstract description 25
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 23
- 102000018120 Recombinases Human genes 0.000 title claims abstract description 11
- 108010091086 Recombinases Proteins 0.000 title claims abstract description 11
- 238000005516 engineering process Methods 0.000 title abstract description 12
- 240000007594 Oryza sativa Species 0.000 title description 3
- 241000209094 Oryza Species 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000000523 sample Substances 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 241000607479 Yersinia pestis Species 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000011901 isothermal amplification Methods 0.000 claims description 6
- 230000003416 augmentation Effects 0.000 claims description 4
- 238000011529 RT qPCR Methods 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 2
- 238000011156 evaluation Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 2
- 229940069446 magnesium acetate Drugs 0.000 claims description 2
- 235000011285 magnesium acetate Nutrition 0.000 claims description 2
- 239000011654 magnesium acetate Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 abstract description 27
- 241000238631 Hexapoda Species 0.000 abstract 4
- 102000053602 DNA Human genes 0.000 abstract 2
- 238000001917 fluorescence detection Methods 0.000 abstract 1
- 239000004615 ingredient Substances 0.000 abstract 1
- 238000013461 design Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 208000035657 Abasia Diseases 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 1
- 101710116602 DNA-Binding protein G5P Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 102000002151 Microfilament Proteins Human genes 0.000 description 1
- 108010040897 Microfilament Proteins Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 101710162453 Replication factor A Proteins 0.000 description 1
- 101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 description 1
- 101710176276 SSB protein Proteins 0.000 description 1
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 238000000498 ball milling Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000003632 microfilament Anatomy 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer and probe combination which is suitable for identifying transgenic insect-resistant rice Huahui 1 (TT51-1) by using a recombinase polymerase amplification (RPA) technology. A forward primer sequence of the primer and probe combination is shown by SEQ ID No.1, a reverse primer sequence is shown by SEQ ID No.2, and a probe sequence is shown by SEQ ID No.3. Meanwhile, the invention also discloses a method for identifying the transgenic insect-resistant rice TT51-1. The method comprises the following steps: extracting DNA (Deoxyribonucleic Acid) of a sample to be tested as a template; performing RPA rapid amplification and real-time fluorescence detection by using the primer; if an obvious amplification curve is obtained, proving that the tested sample contains the ingredients of the transgenic insect-resistant rice TT51-1. The invention provides an RPA detection method for the specificity of the transgenic insect-resistant rice TT51-1 for the first time.
Description
Technical field
The invention belongs to biological technical field, relate to the strain specificity method of application recombinase polysaccharase isothermal amplification technique (Recombinase Ploymerase Amplification, RPA) technical evaluation transgenic pest-resistant rice China extensive No. 1 (TT51-1).
Background technology
DNA cloning is the main method of detection of nucleic acids at present, and conventional PCR detects needs accurate instrument and loaded down with trivial details testing sequence, is difficult to meet the requirement of Site Detection under non-lab environment.In recent years, nucleic acid constant-temperature amplification technology has obtained significant progress, and comparing nucleic acid constant-temperature amplification technology with normal PCR does not need expensive PCR instrument, and rapid amplifying goes out object fragment at short notice, has the advantages such as easy, quick, sensitive.RPA technology is DNA replication dna in simulation organism, based on the polymerase-mediated amplification principle of recombinase, develops, while utilizing recombinase and primer to form microfilament to search the sequence of complete complementary with it on template DNA, under the help of single-stranded DNA binding protein, template DNA is unwind, primer and template DNA start pairing formation and copy required 3 ' C-terminal freely, under the effect of archaeal dna polymerase, copy extension, forming new DNA complementary strand reaction product is also to increase with exponential.Different from conventional PCR reaction, RPA reacts required primer length and is generally 30-35nt.During design of primers for fear of form primer inner and between secondary structure, the increase of its length also makes design of primers and selects difficulty to increase, the design of primer sequence with select most important to the result of RPA.In RPA amplification system, add a fluorescently-labeled probe just can realize the Real-Time Monitoring of template amplification, each mark Yi Ge fluorescence group (FAM and BHQ1) in these two T bases in probe middle part, between Liang Ge group, there is an abasic site (dSpacer), this site can be identified from colibacillary exonuclease by a kind of, this enzyme has 3 '-5 ' 5 prime excision enzyme activity, can Shi Liangge fluorescence group separated, thus make the accumulation synchronised of fluorescent signal and amplified production.In conjunction with a portable amplified fluorescence detector, just can in 10-20 minute, fluorescence curve be detected.RPA technology has greatly shortened detection time, has simplified response procedures, combines make field detection become possibility with DNA rapid extraction technology, is with a wide range of applications.
The method that round pcr detects transgenic product has screening to detect, and gene specific detects, and builds specific detection and specificity of transformant and detects.Due to the uniqueness characteristic of foreign gene in acceptor gene group insertion point, therefore according to the connecting zone design RPA primer of external source insertion DNA sequence dna and rice genome, detect and there is very high specificity, can detect specifically this transformant and derivative strain thereof.
In August, 2009, the Ministry of Agriculture provided the safety certificate of transgenic pest-resistant rice TT51-1 in Hubei Province, caused the extensive concern of the public to its security.At present China not yet ratifies the commercialization plantation of TT51-1, but its spread condition happens occasionally, in the urgent need to setting up fast and convenient detection method, for market surpervision and routine monitor.
At present, in the transgenic plant detection method of having reported, be mainly to utilize PCR instrument in laboratory, to carry out conventional detection, the method can't further meet the rapid detection of transgenic product.Also not utilizing at present RPA technology to do strain specificity to transgenic paddy rice China extensive No. 1 (TT51-1) both at home and abroad identifies.
Summary of the invention
For the blank in above-mentioned field, the invention provides the RPA detection method of extensive No. 1 (TT51-1) strain specificity of accurate, quick, easy detection transgenic paddy rice China.
Technical scheme provided by the invention is: a kind of for identify the primer that contains transgenic pest-resistant rice China extensive No. 1 (TT51-1) by recombinase polysaccharase isothermal amplification technique, its forward primer sequence is as shown in SEQ ID No.1, reverse primer sequence is as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.
The present invention also provides a kind of test kit contain extensive No. 1 of transgenic pest-resistant rice China of identifying by recombinase polysaccharase isothermal amplification technique, and this test kit comprises above-mentioned primer and probe.
The present invention also provides a kind of method that contains transgenic pest-resistant rice TT51-1 of identifying by recombinase polysaccharase isothermal amplification technique: extract the DNA of testing sample as template, utilize the primer described in claim 1 to carry out fluorescence rapid detection, if obtain obvious amplification curve, prove that institute's sample product are extensive No. 1 of transgenic pest-resistant rice China.Implementation step is: in the 50mL amplification system of RPA amplification kit recommendation response, add each 2mL(10mmol/L of primer), probe 0.5mL(10mmol/L), template DNA 50ng.39 degrees Celsius of reactions of RPA augmentation detection instrument (or quantitative real time PCR Instrument) 15 minutes.
The inventive method is to design a large amount of RPA Auele Specific Primers according to the connecting zone of external source insertion DNA sequence dna and rice genome, therefrom filters out a set of primer and probe combinations that can effectively detect fast extensive No. 1 (TT51-1) composition of transgenic paddy rice China.Utilize this to primer, to carry out fluorescence rapid detection, extensive No. 1 (TT51-1) genomic dna of transgenic paddy rice China of take can obtain obvious amplification curve as template, but not the genomic dna of transgenic paddy rice bright extensive 63 and 3 kinds of rice materials of No. 6 grades of other strain Kefengs is that template amplification does not all have amplification curve.Template is diluted with water to 2000,400,200,100,20 copies, result has amplification curve, and the method has higher sensitivity, and the present invention provides the RPA detection method of extensive No. 1 (TT51-1) strain specificity of transgenic pest-resistant rice China first.The method is improved the ability of biological technology products aspect accurate, rapid detection.
Accompanying drawing explanation
Fig. 1 is TT51-1 specific detection figure, wherein, and 1: transgenic paddy rice TT51-1; 2: rich No. 6 of transgenic paddy rice section; 3: transgenic paddy rice Kemingdao 1; 4: non-transgenic paddy rice bright extensive 63; 5: water
Fig. 2 is sensitivity test figure, and from 1 to 6 template copy number is followed successively by 2000; 400; 200; 100; 20; 0
Embodiment
Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does example explanation.
The experimental technique of unreceipted actual conditions in embodiment below, conventionally according to normal condition, < < molecular cloning such as Sambrook etc.: laboratory manual > > (New York:Cold Spring Harbor Laboratory Press, 2001) condition described in, or the condition of advising according to instrument or reagent manufacturer.
First, design primer: according to TT51-1 transformant distinguished sequence (GenBank No. EU880444) design primer, and probe.RPA requires as 30-35nt primer length, just thereby easily causing producing a large amount of primer dimers under constant temperature affects experiment effect for this, RPA experiment need to design multipair primer and be optimized from target sequence two ends, screening, the replacement of indivedual bases or increase and decrease all can produce material impact to experimental result.In this experiment, designed a large amount of primers, and therefrom filtered out a pair of highly sensitive and primer that specificity is good for RPA fluoroscopic examination, primer and probe sequence are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3(table 1).
Note: FAM: luminophore; DSpacer: abasic site; BHQ1: quenching group; Phosphate: phosphate group
1. experiment material
(1) vegetable material
Transgenic paddy rice TT51-1, rich No. 6 of transgenic paddy rice section, transgenic paddy rice Kemingdao 1, non-transgenic paddy rice bright extensive 63.
(2) enzyme and reagent
Molecular biology reagent, TwistAmp DNA Amplification Exo Kits is purchased from TwistDX company, and other biochemical reagents are import packing or domestic analytical pure.Primer is synthetic by Beijing Sheng Gong Bioisystech Co., Ltd.
(3) laboratory apparatus
DNA process instrumentation: low-temperature mixed ball milling instrument MM400(Retsch)
Fluorescence detector: RPA augmentation detection instrument (Twista) or quantitative real time PCR Instrument.
Other Instruments comprises: thermostat water bath, electronic balance, whizzer, vortex instrument, pure water instrument, constant incubator etc.
2. experimental technique and process
(1) extraction of oryza sativa genomic dna
Single-seed rice plantation, gets young leaflet tablet as DNA extraction material, according to TianGen Plant Genomic DNA Kit(Cat#DP-305) operational manual of test kit, carries out the extraction of rice total dna.
(2) DNA concentration and purity testing
Use NanoDrop 1000 spectrophotometers (Thermo Scientific) to measure purity and the concentration of DNA, and regulate DNA concentration to 50ng/mL with deionization distilled water.
(3) primer amplification
The transformant distinguished sequence inserting between DNA and plant genome DNA for PRA method amplification external source in the present embodiment is identified transgenic paddy rice TT51-1, and template concentrations is 50ng/mL.
RPA amplification system is: total system 50mL, in the 0.2mL TwistAmp Exo reaction tubes that contains lyophozyme powder, add rehydration damping fluid 29.5mL, magnesium acetate solution 2.5mL(280mmol/L), each 2mL(10mmol/L of primer), probe 0.5mL(10mmol/L), template DNA 50ng, residue water is supplied;
Primer amplification program: 39 degrees Celsius of reactions of RPA augmentation detection instrument 15 minutes.
3. experimental result
According to forward primer and the reverse primer of the design of transformant distinguished sequence, to bright extensive 63, rich No. 6 of section, Kemingdao 1 and TT51-1 oryza sativa genomic dna carry out RPA fluoroscopic examination, can identify rapidly and accurately transgenic paddy rice TT51-1, wherein at transgenic paddy rice TT51-1, have obvious fluorescence curve, but not rich the grade in material for No. 6 of transgenic paddy rice Ming Hui63He transgenic paddy rice section all do not have amplification curve (Fig. 1).Template is diluted with water to 2000,400,200,100,20 copies, result has amplification curve (Fig. 2).Explanation establishes primer with the present invention and method identifies that transgenic paddy rice TT51-1 has higher sensitivity and accuracy, and easy and simple to handle.
<110> Biological Technology institute, Chinese Academy of Agricultural Sciences
<120> application RPA technology is identified extensive No. 1 strain specificity of transgenic paddy rice China
<160>?3
<210>?1
<211>?35
<212>?DNA
<400>?1
CCCGGCGTCA?ATACGGGATA?ATACCGCGCC?ACATA
<210>?2
<211>?35
<212>?DNA
<400>?2
GGAACATATG?AGTGGTAGCG?TCCAGAAGGA?AAAGG
<210>?3
<211>?46
<212>?DNA
<400>?3
CTTTAACCCCCGAACATCGCCTCGCTCCAGCAGACC?GCTGTTATGC
Claims (4)
1. primer pair and the probe combinations for identifying to contain transgenic pest-resistant rice by recombinase polysaccharase isothermal amplification technique, it is characterized in that: its forward primer sequence is as shown in SEQ ID No.1, reverse primer sequence is as shown in SEQ ID No.2, probe sequence is as shown in SEQ ID No.3, and wherein said paddy rice is extensive No. 1 of China.
2. the test kit that evaluation contains extensive No. 1 of transgenic pest-resistant rice China, is characterized in that: this test kit comprises primer pair claimed in claim 1 and probe combinations.
3. a method of identifying to contain extensive No. 1 of transgenic pest-resistant rice China by recombinase polysaccharase isothermal amplification technique, it is characterized in that: extract the DNA of testing sample as template, utilize the primer pair described in claim 1 and probe combinations is carried out rapid amplifying and real-time fluorescence detects, if obtain obvious amplification curve, prove that institute's sample product contain extensive No. 1 composition of transgenic pest-resistant rice China.
4. method as claimed in claim 3, is characterized in that:
RPA amplification system is: total system 50mL adds rehydration damping fluid 29.5mL, 280mmol/L magnesium acetate solution 2.5mL in the 0.2mL TwistAmp Exo reaction tubes that contains lyophozyme powder, each 2mL of 10mmol/L primer, 10mmol/L probe 0.5mL, template DNA 50ng, residue water is supplied;
Amplification program is: RPA augmentation detection instrument or quantitative real time PCR Instrument were in 39 degrees Celsius of reactions 15 minutes.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310391525.8A CN103451292B (en) | 2013-09-02 | 2013-09-02 | Identification of specificity of transgenic rice Huahui 1 by applying recombinase polymerase amplification (RPA) technology |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310391525.8A CN103451292B (en) | 2013-09-02 | 2013-09-02 | Identification of specificity of transgenic rice Huahui 1 by applying recombinase polymerase amplification (RPA) technology |
Publications (2)
Publication Number | Publication Date |
---|---|
CN103451292A CN103451292A (en) | 2013-12-18 |
CN103451292B true CN103451292B (en) | 2014-11-26 |
Family
ID=49734125
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201310391525.8A Expired - Fee Related CN103451292B (en) | 2013-09-02 | 2013-09-02 | Identification of specificity of transgenic rice Huahui 1 by applying recombinase polymerase amplification (RPA) technology |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103451292B (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107016258B (en) * | 2016-01-27 | 2020-06-05 | 应清界 | Method for fluorescence quantitative calculation based on recombinase-mediated isothermal nucleic acid amplification (RAA) method |
CN106755488A (en) * | 2017-01-22 | 2017-05-31 | 中国农业科学院生物技术研究所 | Transgenic corn BT 11 strain specificity is identified using RPA technologies |
CN106868137B (en) * | 2017-03-01 | 2021-01-26 | 浙江省农业科学院 | Multiple digital PCR quantitative detection method for transgenic rice |
CN109234434A (en) * | 2018-10-19 | 2019-01-18 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting transgenic paddy rice G6H1 |
CN109136339A (en) * | 2018-10-19 | 2019-01-04 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting transgenic paddy rice TT51-1 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101240277A (en) * | 2007-02-09 | 2008-08-13 | 中国农业科学院植物保护研究所 | Side sequence of exogenous insert of transgene paddy strain Bt Shanyou 63 |
CN101302520A (en) * | 2008-07-01 | 2008-11-12 | 中国农业科学院油料作物研究所 | Transgenic rice TT51-1 transformation event foreign vector integration site complete sequence and use thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7399590B2 (en) * | 2002-02-21 | 2008-07-15 | Asm Scientific, Inc. | Recombinase polymerase amplification |
-
2013
- 2013-09-02 CN CN201310391525.8A patent/CN103451292B/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101240277A (en) * | 2007-02-09 | 2008-08-13 | 中国农业科学院植物保护研究所 | Side sequence of exogenous insert of transgene paddy strain Bt Shanyou 63 |
CN101302520A (en) * | 2008-07-01 | 2008-11-12 | 中国农业科学院油料作物研究所 | Transgenic rice TT51-1 transformation event foreign vector integration site complete sequence and use thereof |
Also Published As
Publication number | Publication date |
---|---|
CN103451292A (en) | 2013-12-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104099425B (en) | A kind of test kit for detecting B-raf transgenation | |
CN106755488A (en) | Transgenic corn BT 11 strain specificity is identified using RPA technologies | |
CN103451292B (en) | Identification of specificity of transgenic rice Huahui 1 by applying recombinase polymerase amplification (RPA) technology | |
CN102719526A (en) | MicroRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize fluorescent nano silver cluster probe | |
CN101487051B (en) | Detecting probe and liquid phase chip for BRAF gene mutation and detecting method thereof | |
CN103525936B (en) | Application RPA technology is to the rich No. 6 strain specificities qualification of transgenic paddy rice section | |
CN106801092A (en) | Transgenic corns Bt176 strain specificities are identified using RPA technologies | |
CN103451291B (en) | RPA (Recombinase Polymerase Amplification) detection method for CrylAb/CrylAc insect-resistant gene | |
CN103509875B (en) | Detection of CaMV 35S promoter and nos terminator by adopting RPA (Recombinase Ploymerase Amplification) technology | |
CN102094079B (en) | Fast detection method of loop-mediated isothermal nucleic acid amplification of in-situ QCM (Quartz Crystal Microbalance) | |
CN109706226A (en) | A method of miRNA is carried out based on asymmetric PCR and LAMP cyclic amplification reaction and is quickly detected | |
CN104593504A (en) | Composite PCR (polymerase chain reaction) amplification fluorescence detection kit for 27 plant transgenic loci | |
Chaouachi et al. | A strategy for designing multi-taxa specific reference gene systems. Example of application––ppi Phosphofructokinase (ppi-PPF) used for the detection and quantification of three taxa: maize (Zea mays), cotton (Gossypium hirsutum) and rice (Oryza sativa) | |
CN103698516A (en) | Novel isothermal loop-mediated yersinia pestis nucleic acid mark detection reagent | |
CN103397108A (en) | HCLV (hog cholera lapinized virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection kit | |
Li et al. | Development of a novel reference plasmid for accurate quantification of genetically modified Kefeng6 rice DNA in food and feed samples | |
CN1932037B (en) | Method of screening transgenic wheat | |
CN101948926A (en) | Method for analyzing droplet polymerase chain reaction based on capillary gel electrophoresis detection | |
Xu et al. | Event-specific real-time RPA detection of transgenic rice kefeng 6 | |
CN104830857B (en) | The primer and probe and method that the special quantitative PCR of transgenic corns MON88017 strains precisely detects | |
CN101792814B (en) | PCR method for detecting specificity of transformant of transgenic pest-resistant cotton GK12 | |
CN110564822B (en) | LAMP technology-based transgenic corn Bt 176-related gene detection method and kit | |
CN106399302A (en) | Duplex fluorescent PCR method for efficiently detecting specific genes of transgenic soybean MON87701 strain and transgenic soybean MON87708 strain simultaneously | |
CN103276096A (en) | Nicking endonuclease constant-temperature amplification technology based ATP or NAD system and detection method thereof | |
CN105331734A (en) | Food pathogenic bacterium detection kit |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141126 |