CN102952865B - PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection primer and PCR-DHPLC detection method for transgenic soybean strain RRS - Google Patents
PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection primer and PCR-DHPLC detection method for transgenic soybean strain RRS Download PDFInfo
- Publication number
- CN102952865B CN102952865B CN201110249143.2A CN201110249143A CN102952865B CN 102952865 B CN102952865 B CN 102952865B CN 201110249143 A CN201110249143 A CN 201110249143A CN 102952865 B CN102952865 B CN 102952865B
- Authority
- CN
- China
- Prior art keywords
- dhplc
- primer
- pcr
- rrs
- detection method
- 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
Abstract
The invention discloses a PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection primer and a PCR-DHPLC detection method for a transgenic soybean strain RRS. The primer with high specificity can be used for PCR amplification and DHPLC analysis. The PCR-DHPLC detection method for the transgenic soybean strain RRS is simple and convenient to operate, good in expansibility and high in sensitivity. By using DHPLC for PCR amplification product analysis, fragment size resolution can reach multiple basic groups, and resolution is high. The PCR-DHPLC detection method for the transgenic soybean strain RRS is simple, convenient, effective and reliable and is particularly suitable for use for departments of port inspection and quarantine, agricultural production, plant protection and the like.
Description
Technical field
The present invention relates to a kind of detection of transgenic product, the PCR-DHPLC that particularly relates to a kind of genetically engineered soybean strain detects primer and detection method.
Background technology
At present, the positive rapid growth of global genetically modified crops cultivated area.Genetically engineered soybean is still topmost genetically modified crops, and cultivated area accounts for the over half of the total cultivated area of global genetically modified crops.China in recent years quantity of imported soybean increases year by year.Along with the continuous expansion of genetically modified crops cultivated area, the environment of genetically modified crops and food thereof discharges security and edible safety is also subject to paying close attention to more and more widely.The < < agricultural genetically modified organism safety evaluation management method > > that the Chinese government implemented in 2002, < < agricultural genetically modified organism import security management method > >, < < agricultural genetically modified organism identity management way > > has listed the genetically modified food of first identity management, genetically engineered soybean ranks first.In the face of large batch of imported soybean like this, more need quick, easy, reliable transgenosis detection technique.The most convenient and swift with conventional PCR, real-time fluorescence PCR etc. in current detection method.But for conventional PCR, its Analysis of test results adopts electrophoretic analysis conventionally, modal is gel electrophoresis analysis, this analytical procedure resolving power is not high, and complex operation needs glue, point sample, electrophoresis, photograph etc., and these processes all cannot realize automated operation at present, and workload is large.And real-time fluorescence PCR is due to the restriction of instrument self and fluorescence dye development at present, non-interfering 4 fluorescence channels only can be provided simultaneously, also limit this technology and detected flux expansion.
The analysing and detecting method that it is a kind of high specificity that dhplc analysis detects (Denaturing High-performance Liquid Chromatography, DHPLC), resolving power is high, reproducible, scalability is good.The method can once be analyzed a plurality of samples simultaneously; to meeting the detection level of port quarantine need of work, lifting transgenic product, strengthen China significant to the production safety of the quarantine supervision of transgene component in the plants and plant product that enters the territory, protection China crop.
At present, still there is no the PCR of genetically engineered soybean strain RRS in conjunction with DHPLC detection technique (PCR-DHPLC).
Summary of the invention
The object of this invention is to provide the primer that a kind of PCR-DHPLC for genetically engineered soybean strain RRS detects.
Another object of the present invention is to provide that a kind of scalability based on above-mentioned primer is good, the PCR-DHPLC detection method of sensitivity and the high genetically engineered soybean strain RRS of resolving power.
For achieving the above object, the present invention has adopted following technical scheme:
The invention discloses the primer detecting for genetically engineered soybean strain RRS PCR-DHPLC, described primer comprises primer pair 2, and the upstream primer of primer pair 2 contains sequence shown in Seq ID No.3, and downstream primer contains sequence shown in Seq ID No.4;
Seq?ID?No.3：5’-CGTGGCCTCGCGATCTGACTTCAATTTAACCGATGCTAATGAGTT-3’
Seq?ID?No.4：5’-CTCAGCGGCGGAGCTACAGATGCGAAGGATAGTGGGATTGT-3’。
Further, described primer also comprises primer pair 1, and the upstream primer of primer pair 1 contains sequence shown in Seq ID No.1, and downstream primer contains sequence shown in Seq ID No.2;
Seq?ID?No.1：5’-TCCTCCAAGGAGACGCTATTG-3’
Seq?ID?No.2：5’-GGTTTTGGGGTGCCGTTT-3’。
Further, the upstream primer 5 ' end of described primer pair 1 also comprises sequence shown in Seq ID No.5, and downstream primer 5 ' end also comprises sequence shown in Seq ID No.6;
Seq?ID?No.5：5’-CGTGGCCTCGCGATCTGACT-3’
Seq?ID?No.6：5’-CTCAGCGGCGGAGCTACAGA-3’。
It is to be noted, above-mentioned Seq ID No.5 and Seq ID No.6 are irrelevant two sections of sequences with detecting target sequence of adding at 5 ' end of upstream primer and downstream primer respectively, these two sections of sequences can be the sequences of other species far with detecting target sequence homology, can be also the random sequences generating.These two sections of sequences, can be for controlling the size of pcr amplification product, so that the further analysis of pcr amplification product as regulating and controlling sequence.
In the present invention, preferred, the upstream primer of described primer pair 1 has sequence shown in Seq ID No.7, and downstream primer has sequence shown in Seq ID No.8; The upstream primer of primer pair 2 has sequence shown in Seq ID No.3, and downstream primer has sequence shown in Seq ID No.4;
Seq?ID?No.7：5’-CGTGGCCTCGCGATCTGACTTCCTCCAAGGAGACGCTATTG-3’
Seq?ID?No.8：5’-CTCAGCGGCGGAGCTACAGAGGTTTTGGGGTGCCGTTT-3’。
It should be noted that, described Seq ID No.7 and Seq ID No.8 sequence are by above-mentioned Seq ID No.1 and Seq ID No.2 sequence, to add regulating and controlling sequence respectively at its 5 ' end to form, and it is also regulating and controlling sequence that 5 ' of described Seq ID No.3 and Seq ID No.4 sequence held front 20 bases.Although above-mentioned, illustrated that regulating and controlling sequence shown in Seq ID No.5 and Seq ID No.6 can be the random sequence generating, but, be not that arbitrary sequence can be added, in the present invention, need to consider that the unified of specificity site sequence physico-chemical property of regulating and controlling sequence and design coordinate, and need to consider to add primer after regulating and controlling sequence as the overall performance that detects primer.
In the present invention, preferred, described primer pair 1 is for detecting the primer of soybean native gene Lectin gene, and primer pair 2 is for detecting the primer of genetically engineered soybean strain RRS.
The invention also discloses a kind of PCR-DHPLC detection method for genetically engineered soybean strain RRS, described detection method comprises at least one pair of primer adopting in above-mentioned primer pair 1 and 2, the soy bean DNA of take carries out pcr amplification as template, and pcr amplification product is carried out to dhplc analysis.
Further, described detection method also comprises that take marker carries out dhplc analysis as reference standard, compares the dhplc analysis result of the dhplc analysis result of pcr amplification product and marker.
Preferred above-mentioned detection method comprises the steps:
(A) adopt described primer, take soy bean DNA as template, carry out pcr amplification;
(B) take the pcr amplification product of step (A) carries out DHPLC analysis as sample, and the marker of take carries out DHPLC analysis as reference standard simultaneously;
(C) the DHPLC analytical results of sample in step (B) and the DHPLC analytical results of marker are compared, determine the molecular size range of sample, thereby judge whether the target sequence that contains detection.
Owing to adopting above technical scheme, beneficial effect of the present invention is:
Genetically engineered soybean RRS strain of the present invention detects primer and has stronger specificity, can be used in follow-up pcr amplification and DHPLC and analyzes.The present invention combines PCR with DHPLC, provide a kind of easy and simple to handle, scalability good, highly sensitive genetically engineered soybean strain RRS detection method, can realize the multiplex detection of genetically engineered soybean strain RRS.Utilize DHPLC to analyze pcr amplification product, to the PCR product fragment differentiation rate of different sizes, can reach several bases, resolving power is high.Primer of the present invention and detection method provide a kind of simple, convenient, effective, reliable detection method for genetically engineered soybean strain RRS detects, the departments such as Check and Examination of Port quarantine, agriculture production, plant protection that are particularly suitable for are used.
Accompanying drawing explanation
Fig. 1 to Fig. 3 is the partial results that in the embodiment of the present invention, DHPLC analyzes, wherein 1 is that non-transgenic soybean, 2 is that genetically engineered soybean 256043,3 is that genetically engineered soybean A2704-12,4 is that genetically engineered soybean 305423,5 is that genetically engineered soybean MON89788,6 is that genetically engineered soybean A5547-127,7 is genetically engineered soybean RRS strain GTS-4-3-2, the negative contrast of 9-12, is respectively the DNA of corn, rape, paddy rice, cotton; Marker-puc is puc18marker, and each peak value of marker is followed successively by 80bp, 102bp, 174bp, 257bp, 267bp, 296bp, 434bp, 458bp, 587bp from left to right.
Embodiment
The present invention has announced the primer for the PCR-DHPLC detection of genetically engineered soybean strain RRS, and this primer designs for the native gene of genetically engineered soybean and the distinguished sequence of genetically engineered soybean strain RRS respectively.Comprise at least one pair of in primer pair 1 and primer pair 2.Further, also the upstream and downstream in primer pair 1 and 2 has added respectively one section of irrelevant with detection target sequence or homology is far regulating and controlling sequence, for realizing the regulation and control to the clip size of amplified production.It is to be noted, at above-mentioned detection primer, the specificity and the stability that detect have been guaranteed, can realize the Multiple detection analysis to genetically engineered soybean, adding just in order to obtain more excellent detection effect of regulating and controlling sequence, therefore, the present invention is preferred, for the PCR of genetically engineered soybean, in conjunction with the primer of DHPLC technology for detection, is the primer that has added regulating and controlling sequence.Also it is pointed out that primer pair 1 of the present invention is the primer for soybean native gene Lectin gene design, for detection of whether containing soybean components in sample; Primer pair 2 is the primers for soybean line RRS design, for detection of whether containing genetically engineered soybean RRS strain.
The invention also discloses a kind of PCR-DHPLC detection method for genetically engineered soybean strain RRS, described detection method comprises the above-mentioned primer of employing, and the soy bean DNA of take carries out pcr amplification as template, and pcr amplification product is carried out to dhplc analysis.Further, described detection method also comprises that take marker carries out dhplc analysis as reference standard, the dhplc analysis result of the dhplc analysis result of pcr amplification product and marker is compared, according to the size of comparison result judgement pcr amplification product, thereby judge whether the target sequence that contains detection.In the present invention, the DHPLC elution peak of primer pair 1 amplified production is 119bp, and the DHPLC elution peak of primer pair 2 amplified productions is 404bp.Concrete, elution peak is 119bp, judges in sample and contains soybean components; Elution peak is 404bp, judges in sample and contains genetically engineered soybean RRS strain.
Below by specific experiment example, also by reference to the accompanying drawings the present invention is described in further detail.Following experimental example is only further detailed the present invention, should not be construed as limitation of the present invention.
Embodiment 1DNA extracts
Adopt CTAB method to extract sample DNA, specific as follows:
A) take sample 5g, in mortar, add liquid nitrogen grinding to sample to be the powder of 0.5mm left and right size;
B) take the sample that 300mg grinds, proceed to rapidly in 2mL centrifuge tube, add the CTAB extracting solution 700 μ L of 65 ℃ of preheatings, mix, put into 65 ℃ of water-bath water-bath 30min;
C) add 5 μ L RNase (10mg/mL), 37 ℃ of water-bath 30min;
D) add the saturated phenol of equal-volume Tris, fully mix, the centrifugal 15min of 12000r/min;
E) get supernatant, add isopyknic chloroform/primary isoamyl alcohol (24: 1) to mix, the centrifugal 15min of 12000r/min;
F) get supernatant, add isopyknic chloroform/primary isoamyl alcohol (24: 1) to mix, the centrifugal 15min of 12000r/min;
G) add the Virahol of equal-volume precooling, jiggle, be placed in-20 ℃ of standing 30min of refrigerator, the centrifugal 15min of 12000r/min;
H) abandon supernatant, add 70% ethanol 500 μ L, the centrifugal 3min of 12000r/min, removes supernatant, repeats 2 times;
I) obtain DNA precipitation, with freeze drier, be dried, add 50 μ L～100 μ L TE or aseptic deionized waters, after fully dissolving, purity and the concentration of measuring DNA are placed in-20 ℃ of refrigerators preserves.
Embodiment 2DNA concentration determination
The sample DNA extracting is carried out to concentration and purity testing; Adopt ultraviolet spectrophotometer to measure 260nm and 280nm place absorption value, calculate respectively purity and the concentration of nucleic acid, calculation formula is as follows:
DNA purity=OD260/OD280
DNA concentration=50 * OD260mg/mL
The purity ratio of DNA is between 1.7～1.9, and concentration is greater than 10ng/ μ L.
Embodiment 3PCR amplification
According to the binding site of Lectin gene and RRS strain design specific detection primer, and add regulating and controlling sequence at 5 ' end of specific detection primer binding site, the synthetic detection primer pair (table 1) that contains regulating and controlling sequence, adopt the primer that adds regulating and controlling sequence to carry out pcr amplification, sample setting comprises: non-transgenic soybean, genetically engineered soybean 256043, genetically engineered soybean A2704-12, genetically engineered soybean 305423, genetically engineered soybean MON89788, genetically engineered soybean A5547-127, genetically engineered soybean RRS strain GTS-4-3-2, corn, rape, paddy rice, the DNA of cotton etc.
Table 1 genetically engineered soybean strain RRS detects primer binding site and primer
PCR reaction system cumulative volume is 50 μ L, each composition is respectively: multi-PRC reaction mixed solution Multiplex PCR Mix (TaKaRa) 25 μ L, each 1 μ L of 10 μ mol/L upstream primers, each 1 μ L of 10 μ mol/L downstream primers, DNA2 μ L, 5U/ μ L Taq enzyme 0.25 μ L, with sterilizing distilled water, supplying is 50 μ L.
PCR reaction conditions: 94 ℃ of sex change 1min; Then enter 35 circulations, 94 ℃ of 30s, 57 ℃ of 1min, 72 ℃ of 1min; After loop ends, 72 ℃ are extended 10min.
Embodiment 4DHPLC detects
PCR product is placed on the automatic sampling platform of DHPLC; Open DHPLC and control software Navigator, in detection method, select Multiplex, i.e. multi-disc piecewise analysis, set upper limit of detection is 600bp simultaneously, under be limited to 70bp; Move two blank, i.e. empty needle, balance chromatographic column; Move a marker, for reference to contrast, detect the nucleic acid fragment size of sample; Successively sample to be tested is analyzed, partial results is shown in Fig. 1-3.
Result judgement: observe the elution peak that DHPLC obtains, by comparing with marker and WAVE 4500 automatic analysis are determined and judged the DNA fragmentation size of elution peak position accordingly.
Elution peak 119bp, is the amplified production of primer pair 1, i.e. the lectin gene for judging that sample contains soybean components; Elution peak 404bp, is the amplified production of primer pair 2, for judging that sample contains genetically engineered soybean RRS strain.
The analysis of the soybean sample that contains transgenic strain RRS is shown, this sample has two elution peaks, is respectively 119bp (containing soybean components), 404bp (containing RRS strain) (Fig. 2, sample 7); To non-transgenic soybean or be not that the analysis of the genetically engineered soybean sample of RRS strain shows, this sample only has an elution peak, i.e. 119bp elution peak (Fig. 1 and 2, sample 1 to 6); Blank and other non-transgenic sample do not have the elution peak (Fig. 3, sample 9 to 12) of above-mentioned 119bp or 404bp.
Above content is in conjunction with concrete embodiment further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (4)
1. the primer detecting for genetically engineered soybean strain RRSPCR-DHPLC, is characterized in that: described primer comprises primer pair 1 and primer pair 2, the upstream primer of primer pair 2 is sequence shown in SeqIDNo.3, and downstream primer is sequence shown in SeqIDNo.4;
SeqIDNo.3：5’-CGTGGCCTCGCGATCTGACTTCAATTTAACCGATGCTAATGAGTT-3’
SeqIDNo.4：5’-CTCAGCGGCGGAGCTACAGATGCGAAGGATAGTGGGATTGT-3’；
The upstream primer of described primer pair 1 is sequence shown in SeqIDNo.7, and downstream primer is sequence shown in SeqIDNo.8;
SeqIDNo.7：5’-CGTGGCCTCGCGATCTGACTTCCTCCAAGGAGACGCTATTG-3’
SeqIDNo.8：5’-CTCAGCGGCGGAGCTACAGAGGTTTTGGGGTGCCGTTT-3’。
2. primer according to claim 1, is characterized in that: described primer pair 1 is for detecting the primer of soybean native gene Lectin gene, and primer pair 2 is for detecting the primer of genetically engineered soybean strain RRS.
3. the PCR-DHPLC detection method for genetically engineered soybean strain RRS, it is characterized in that: described detection method comprises the primer described in employing claim 1 or 2, the soy bean DNA of take carries out pcr amplification as template, and pcr amplification product is carried out to dhplc analysis.
4. detection method according to claim 3, it is characterized in that: described detection method also comprises that take marker carries out dhplc analysis as reference standard, compares the dhplc analysis result of the dhplc analysis result of pcr amplification product and marker.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110249143.2A CN102952865B (en) | 2011-08-26 | 2011-08-26 | PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection primer and PCR-DHPLC detection method for transgenic soybean strain RRS |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110249143.2A CN102952865B (en) | 2011-08-26 | 2011-08-26 | PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection primer and PCR-DHPLC detection method for transgenic soybean strain RRS |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102952865A CN102952865A (en) | 2013-03-06 |
| CN102952865B true CN102952865B (en) | 2014-09-17 |
Family
ID=47762246
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110249143.2A Expired - Fee Related CN102952865B (en) | 2011-08-26 | 2011-08-26 | PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection primer and PCR-DHPLC detection method for transgenic soybean strain RRS |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102952865B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1584049A (en) * | 2003-08-22 | 2005-02-23 | 国家质量监督检验检疫总局动植物检疫实验所 | Preparing method for tr-gene products detecting oligonucleotides chip and use thereof |
| CN102154454A (en) * | 2010-12-30 | 2011-08-17 | 暨南大学 | Method for detecting transgenosis constituents in plant by GeXP multi-PCR (polymerase chain reaction) technology and application thereof |
-
2011
- 2011-08-26 CN CN201110249143.2A patent/CN102952865B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1584049A (en) * | 2003-08-22 | 2005-02-23 | 国家质量监督检验检疫总局动植物检疫实验所 | Preparing method for tr-gene products detecting oligonucleotides chip and use thereof |
| CN102154454A (en) * | 2010-12-30 | 2011-08-17 | 暨南大学 | Method for detecting transgenosis constituents in plant by GeXP multi-PCR (polymerase chain reaction) technology and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| 应用多重PCR-DHPLC方法快速检测转基因马铃薯及EH92-527-1品系鉴定;白月等;《中国马铃薯》;20110625;第25卷(第3期);第131页第1.4.5、2.2节、第134页左栏第2段 * |
| 张海波.适合转基因大豆GTS40-3-2复合PCR检测标准分子研制及番茄内标准基因国际协同验证.《中国优秀硕士学位论文全文数据库 农业科技辑》.2008,(第07期), |
| 白月等.应用多重PCR-DHPLC方法快速检测转基因马铃薯及EH92-527-1品系鉴定.《中国马铃薯》.2011,第25卷(第3期), |
| 适合转基因大豆GTS40-3-2复合PCR检测标准分子研制及番茄内标准基因国际协同验证;张海波;《中国优秀硕士学位论文全文数据库 农业科技辑》;20080715(第07期);摘要、附录以及第28页最后一段 * |
| 适合转基因大豆GTS40-3-2复合PCR检测标准分子研制及番茄内标准基因国际协同验证;张海波;《中国优秀硕士学位论文全文数据库 农业科技辑》;20080715(第07期);摘要及附录 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102952865A (en) | 2013-03-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103740815B (en) | Primer for multi-PCR (Polymerase Chain Reaction) detection aiming at fusaria and application of primer | |
| CN102634593B (en) | LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic maize EVENT98140 and derived varieties thereof | |
| CN102634588B (en) | LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic maize MIR604 and derived varieties thereof | |
| CN102634591B (en) | Genetically modified rice BT63 and LAMP (loop mediated isothermal amplification) detection primer group, detection kit and detection method of derived variety thereof | |
| CN102154454B (en) | Method for detecting transgenosis constituents in plant by GeXP multi-PCR (polymerase chain reaction) technology and application thereof | |
| CN102952856B (en) | PCR-DHPLC detection primers for transgenic rice line Kefeng 6 and detection method | |
| CN103451291B (en) | RPA (Recombinase Polymerase Amplification) detection method for CrylAb/CrylAc insect-resistant gene | |
| CN102649976B (en) | LAMP (Loop-mediated isothermal amplification) detection primer set of transgenic soybeans GTS 40-3-2 as well as derived varieties thereof, kit and detection method | |
| CN104726582A (en) | Probe method based constant temperature detection primer group, detection kit and detection method of genetically modified soybeans MON89788 | |
| CN102952865B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection primer and PCR-DHPLC detection method for transgenic soybean strain RRS | |
| CN104404161B (en) | The multiplex PCR examination detection primer of transgenic corns and detection method | |
| CN102952858B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified rice strain KMD | |
| CN103509875B (en) | Detection of CaMV 35S promoter and nos terminator by adopting RPA (Recombinase Ploymerase Amplification) technology | |
| CN102634592B (en) | LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic maize BT176 and derived varieties thereof | |
| CN102952863B (en) | Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified cotton | |
| CN105063187A (en) | Method and primer composition for rapid detection of resistance of botrytis cinerea to SDH1 fungicides | |
| CN102776268A (en) | Kit and method for detection of transgenic maize Mon89034 and derivative varieties thereof through gene amplification at constant temperature | |
| CN102952861A (en) | Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize | |
| CN102586303B (en) | Standard plasmid of transgenic Bt rice and application of standard plasmid | |
| CN102952864B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT176 | |
| CN102952862B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain MON89034 | |
| CN102952860B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT11 | |
| CN102586302B (en) | Standard plasmid of transgenic Bt cotton and application of standard plasmid | |
| CN102168103A (en) | Reference molecule for specifically detecting genetically modified rapeseed RT73 and application of reference molecule | |
| CN102952857B (en) | Primer and method for PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection for endogenous genes of wheat |
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 |
Granted publication date: 20140917 Termination date: 20180826 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |