CN102952861B - Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize - Google Patents
Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize Download PDFInfo
- Publication number
- CN102952861B CN102952861B CN201110248327.7A CN201110248327A CN102952861B CN 102952861 B CN102952861 B CN 102952861B CN 201110248327 A CN201110248327 A CN 201110248327A CN 102952861 B CN102952861 B CN 102952861B
- Authority
- CN
- China
- Prior art keywords
- primer
- seq
- sequence shown
- primer pair
- dhplc
- 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 multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and a detection method for genetically modified maize. The primer has strong specificity and can be used for PCR amplification and DHPLC analysis. The detection method is simple and convenient to operate, good in expansion performance and high in sensitivity, and multi-target detection of the genetically modified maize is realized. The DHPLC is used to analyze PCR amplified products, fragment size resolution can reach multiple bases, and resolution ratio is high. The multiplex PCR-DHPLC detection primer and the detection method for genetically modified maize have the advantages that the detection method is simple, convenient, effective, reliable, high in throughput and especially suitable for departments of port inspection and quarantine and the like.
Description
Technical field
The present invention relates to a kind of detection of transgenic product, particularly relate to a kind of transgenic corns multiplex PCR-DHPLC and detect primer and detection method.
Background technology
At present mainly multiplex PCR is adopted to the detection method of transgenic corns, the detection methods such as multiple real time fluorescence PCR, PCR-gene chip.Traditional multiple Standard PCR detection method has certain limitation in platform extension method, and along with the increase of target to be checked, need to re-start optimization to the consumption often overlapping primer in system and ratio, and take into account the factors such as amplification efficiency, workload is larger; On the other hand, the method for the gel electrophoresis analysis amplified production usually adopted, distinguish efficiency not high, detected result is undesirable.Although multiple real time fluorescence PCR more multiple Standard PCR detection method in detection sensitivity etc. has superiority, but due to the restriction that current instrument self and fluorescence dye are developed, non-interfering 4 fluorescence channels only can be provided simultaneously, also limit this technology in the expansion of detection flux.Conventional many cover PCR-gene chip detection methods, need many cover primers to increase, complex operation step, and are not suitable for large-scale high throughput testing.Denaturing high performance liquid chromatography (Denaturing High-performance Liquid Chromatography, DHPLC) be a kind of method for nucleic acid analysis of simple, quick, non-gel, have that extendability is strong, good resolution, sensitivity advantages of higher.The method is analytic sample under 50 DEG C of conditions, and the wash-out at sample peak only determines elution order by the quantity of base pair, and the acetonitrile concentration having served as post improves, and nucleic acid fragment can according to molecular weight order from small to large by wash-out out.To be compared with marker by the elution peak that obtains and determine molecular size range, determine whether containing target detect gene.At present, the PCR of transgenic corns is not still had in conjunction with the detection technique (PCR-DHPLC) of DHPLC.
Summary of the invention
The object of this invention is to provide a kind of primer detected for transgenic corns multiplex PCR-DHPLC.
Another object of the present invention is to provide that a kind of scalability based on above-mentioned primer is good, sensitivity and the high transgenic corns multiplex PCR-DHPLC detection method of resolving power.
For achieving the above object, present invention employs following technical scheme:
The invention discloses the primer detected for transgenic corns multiplex PCR-DHPLC, described primer comprises at least one pair of primer in primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6 and primer pair 7; 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; 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; The upstream primer of primer pair 3 contains sequence shown in Seq ID No.5, and downstream primer contains sequence shown in Seq ID No.6; The upstream primer of primer pair 4 contains sequence shown in Seq ID No.7, and downstream primer contains sequence shown in Seq ID No.8; The upstream primer of primer pair 5 contains sequence shown in Seq ID No.9, and downstream primer contains sequence shown in Seq ID No.10; The upstream primer of primer pair 6 contains sequence shown in Seq ID No.11, and downstream primer contains sequence shown in Seq ID No.12; The upstream primer of primer pair 7 contains sequence shown in Seq ID No.13, and downstream primer contains sequence shown in Seq ID No.14.
It is pointed out that above-mentioned Seq ID No.1 to Seq ID No.14 is the distinguished sequence with the detection target sequence complementary pairing of Transgenic corn lines, there is very strong specific recognition.
Further, described primer pair 3, primer pair 4, primer pair 5 and primer pair 6,5 ' end of its upstream primer also comprises sequence shown in Seq ID No.15, and 5 ' end of its downstream primer also comprises sequence shown in Seq ID No.16;
Seq?ID?No.15：5’-CGTGGCCTCGCGATCTGACT-3’
Seq?ID?No.16：5’-CTCAGCGGCGGAGCTACAGA-3’。
It is to be noted, above-mentioned Seq ID No.15 and Seq ID No.16 holds the one section sequence irrelevant with detecting target sequence of adding at 5 ' of upstream primer and downstream primer respectively, this sequence can be the sequence of other species far with detecting target sequence homology, also can be the sequence of one section of stochastic generation.This sequence may be used for the size of pcr amplification product, so that the further analysis of pcr amplification product.
In the present invention, preferably, the upstream primer of described primer pair 3 has sequence shown in Seq ID No.17, and downstream primer has sequence shown in Seq ID No.18; The upstream primer of primer pair 4 has sequence shown in Seq ID No.19, and downstream primer has sequence shown in Seq ID No.20; The upstream primer of primer pair 5 has sequence shown in Seq ID No.21, and downstream primer has sequence shown in Seq ID No.22; The upstream primer of primer pair 6 has sequence shown in Seq ID No.23, and downstream primer has sequence shown in Seq ID No.24.
It should be noted that, described Seq ID No.17 to Seq ID No.24 sequence adds regulating and controlling sequence at its 5 ' end respectively by above-mentioned Seq ID No.5 to Seq ID No.12 sequence to form, illustrate that although above-mentioned regulating and controlling sequence shown in Seq ID No.15 and Seq ID No.16 can be the sequence of stochastic generation, but, be not that arbitrary sequence can be added, specific in the present invention, the unified of the physico-chemical property considering sequence shown in regulating and controlling sequence and Seq ID No.5 to Seq ID No.12 is needed to coordinate, and need to consider that shown in Seq ID No.17 to the Seq ID No.24 after adding regulating and controlling sequence, sequence is as the overall performance detecting primer.
In the present invention, preferred, described primer pair 1 is the primer detecting Transgenic corn lines NK603, primer pair 2 is the primer detecting Transgenic corn lines MON863, primer pair 3 is the primer detecting Transgenic corn lines MON810, primer pair 4 is the primer detecting Transgenic corn lines T25, primer pair 5 is the primer detecting Transgenic corn lines MON88017, primer pair 6 is the primer detecting Transgenic corn lines MIR604, and primer pair 7 is the primer detecting Transgenic corn lines 59122.
The invention also discloses a kind of for transgenic corns multiplex PCR-DHPLC detection method, described detection method comprises the above-mentioned primer of employing, be that template carries out pcr amplification with maize dna, and dhplc analysis (Denaturing High-performance Liquid Chromatography, HPLC) is carried out to pcr amplification product.
Further, it is that reference standard carries out dhplc analysis that described detection method also comprises with marker, the dhplc analysis result of pcr amplification product and the dhplc analysis result of marker is compared.
Preferred above-mentioned detection method comprises the steps:
(A) adopt above-mentioned primer, take maize dna as template, carry out pcr amplification;
(B) be that sample carries out DHPLC analysis with the pcr amplification product of step (A), be that reference standard carries out DHPLC analysis with marker 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, thus judge whether the target sequence containing detecting.
Owing to adopting above technical scheme, beneficial effect of the present invention is:
Transgenic corns Multiple detection primer of the present invention has stronger specificity, can be used in follow-up multiplexed PCR amplification and DHPLC analysis.The present invention is directed to the problem that traditional electrophoretic analysis pcr amplification result is undesirable, PCR and DHPLC is combined, provide a kind of easy and simple to handle, scalability good, sensitivity and the high transgenic corns multiple detection method of resolving power, achieves the multiplex detection of transgenic corns.Utilize DHPLC to analyze pcr amplification product, several base can be reached to the PCR primer fragment differentiation rate of different size, even can distinguish the fragment of 1 base difference in size.Primer of the present invention and detection method are that Transgenic corn lines BT11 detection provides a kind of simple, convenient, effective, reliable high-flux detection method, are particularly suitable for the departments such as Check and Examination of Port quarantine and use.
Accompanying drawing explanation
Figure is the partial results that in the embodiment of the present invention, DHPLC analyzes, wherein the elution peak of the elution peak, 6 of the elution peak, 4 of the elution peak, 2 of 1 Transgenic corn lines MON863 to be the elution peak, 3 of Transgenic corn lines NK603 be Transgenic corn lines MON810 to be the elution peak, 5 of Transgenic corn lines T25 be Transgenic corn lines MON88017 to be the elution peak, 7 of Transgenic corn lines MIR604 be Transgenic corn lines 59122; The Auele Specific Primer that curve A is the DHPLC elution curve of the 7 heavy pcr amplification products that the Auele Specific Primer of 7 strains carries out with the DNA hybrid template of 7 strains, curve B is 7 strains take non-transgenic corn as the DHPLC elution curve of the 7 heavy pcr amplification products that template is carried out, curve M is that each peak value of marker, marker is followed successively by 80bp, 102bp, 174bp, 257bp, 267bp, 296bp, 434bp, 458bp, 587bp from left to right.
Embodiment
The present invention discloses the primer detected for transgenic corns multiplex PCR-DHPLC, and this primer designs for the distinguished sequence of Transgenic corn lines.Comprise at least one in primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6 and primer pair 7.Further, also the upstream and downstream of primer pair 3, primer pair 4 primer pair 5 and primer pair 6 with the addition of respectively one section with detect target sequence and have nothing to do or regulating and controlling sequence that homology is far, for realizing the regulation and control of the clip size to amplified production.What it is pointed out that regulating and controlling sequence adds just in order to obtain more excellent Detection results, and therefore, the present invention is preferred, adds regulating and controlling sequence, have sequence shown in Seq ID No.17 to Seq ID No.24 respectively to primer pair 3-6.
The invention also discloses a kind of for transgenic corns multiplex PCR-DHPLC detection method, described detection method comprises the described primer of employing, is that template carries out pcr amplification, carries out dhplc analysis to pcr amplification product with maize dna.Further, it is that reference standard carries out dhplc analysis that described detection method also comprises with marker, the dhplc analysis result of pcr amplification product and the dhplc analysis result of marker are compared, judge the size of pcr amplification product according to comparison result, thus judge whether the target sequence containing detecting.In the present invention, the DHPLC elution peak of primer pair 1 amplified production is 141bp, the DHPLC elution peak of primer pair 2 amplified production is 164bp, the DHPLC elution peak of primer pair 3 amplified production is 220bp, the DHPLC elution peak of primer pair 4 amplified production is 241bp, the DHPLC elution peak of primer pair 5 amplified production is 267bp, and the DHPLC elution peak of primer pair 6 amplified production is 283bp, and the DHPLC elution peak of primer pair 7 amplified production is 196bp.
Also by reference to the accompanying drawings the present invention is described in further detail below by specific experiment example.Following experimental example is only further detailed the present invention, should not be construed as limitation of the present invention.
Embodiment 1DNA extracts
CTAB method is adopted to extract sample DNA, specific as follows:
A) take sample 5g, in mortar, add the powder that liquid nitrogen grinding to sample is about 0.5mm 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 DEG C of preheatings, mixing, puts into 65 DEG C of water-bath water-bath 30min;
C) 5 μ L RNase (10mg/mL) are added, 37 DEG C 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 the mixing of isopyknic chloroform/primary isoamyl alcohol (24: 1), the centrifugal 15min of 12000r/min;
F) get supernatant, add the mixing of isopyknic chloroform/primary isoamyl alcohol (24: 1), the centrifugal 15min of 12000r/min;
G) add the Virahol of equal-volume precooling, jiggle, be placed in-20 DEG C of refrigerators and leave standstill 30min, 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, carry out drying, add 50 μ L ~ 100 μ L TE or aseptic deionized waters with freeze drier, after fully dissolving, the purity of measurement DNA and concentration are placed in-20 DEG C of refrigerators preserves.
Embodiment 2DNA concentration determination
Concentration and purity testing are carried out to the sample DNA extracted; Adopt ultraviolet spectrophotometer to measure 260nm and 280nm place absorption value, calculate purity and the concentration of nucleic acid respectively, 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 increases
According to Transgenic corn lines MON863, NK603, MON810, T25, MON88017, MIR604, the binding site of 59122 design specific detection primers, and at MON810, T25, MON88017, 5 ' end of the specific detection primer binding site of MIR604 strain adds regulating and controlling sequence, the detection primer (table 1) of synthesis containing regulating and controlling sequence, synthetic primer is to 1 up/down trip primer specificity binding site, primer pair 2 up/down trip primer specificity binding site, the primer pair 3 up/down trip primer of primer pair 7 up/down trip primer specificity binding site and interpolation regulating and controlling sequence, add the primer pair 4 up/down trip primer of regulating and controlling sequence, add the primer pair 5 up/down trip primer of regulating and controlling sequence, that adds the primer pair 6 up/down trip primer of regulating and controlling sequence carries out pcr amplification as primer, PCR sample arranges and comprises, the above-mentioned biased sample of 7 Transgenic corn lines DNA and the DNA of non-transgenic corn.
Table 1 transgenic corns 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, 10 μm of ol/L primer each 1 μ L, DNA 2 μ L, 5U/ μ L Taq enzyme 0.25 μ L, supplying with sterilizing distilled water is 50 μ L.
PCR reaction conditions: 94 DEG C of sex change 1min; Then 35 circulations are entered, 94 DEG C of 30s, 57 DEG C of 1min, 72 DEG C of 1min; After loop ends, 72 DEG C extend 10min.
Embodiment 4DHPLC detects
PCR primer is placed on the automatic sampling platform of DHPLC; Open DHPLC control software design Navigator, in detection method, select Multiplex, be i.e. multiple clips analysis, set upper limit of detection is 600bp simultaneously, and lower limit is 70bp; Run two blank, i.e. empty needles, balance chromatographic column; Run a marker, for reference to contrast, detect the nucleic acid fragment size of sample; Analyze sample to be tested successively, partial results is shown in Fig. 1.
Result judges: observe the elution peak that DHPLC obtains, by the DNA fragmentation size with marker comparison and WAVE 4500 automatic analysis determination elution peak position, judge accordingly.
Elution peak 141bp is the amplified production of primer pair 1, elution peak 164bp is the amplified production of primer pair 2, elution peak 220bp is the amplified production of primer pair 3, elution peak 241bp is the amplified production of primer pair 4, elution peak 267bp is the amplified production of primer pair 5, elution peak 283bp is the amplified production of primer pair 6, and elution peak 196bp is the amplified production of primer pair 7.To the sample of mixing Transgenic corn lines DNA, the display of its detected result is containing above-mentioned 7 elution peaks; And non-transgenic corn sample is without elution peak.
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, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
Claims (6)
1., for the primer that transgenic corns multiplex PCR-DHPLC detects, it is characterized in that: described primer comprises primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6 and primer pair 7;
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;
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;
The upstream primer of primer pair 3 contains sequence shown in Seq ID No.5, and downstream primer contains sequence shown in Seq ID No.6;
The upstream primer of primer pair 4 contains sequence shown in Seq ID No.7, and downstream primer contains sequence shown in Seq ID No.8;
The upstream primer of primer pair 5 contains sequence shown in Seq ID No.9, and downstream primer contains sequence shown in Seq ID No.10;
The upstream primer of primer pair 6 contains sequence shown in Seq ID No.11, and downstream primer contains sequence shown in Seq ID No.12;
The upstream primer of primer pair 7 contains sequence shown in SeqIDNo.13, and downstream primer contains sequence shown in Seq ID No.14;
Seq?ID?No.1：5’-TTATTTTGGACTATCCCGACTCTC-3’
Seq?ID?No.2：5’-CTCAGCGGCGGAGCTACAGAGAGATAACAGGATCCACTCAAACAC-3’
Seq?ID?No.3：5’-AACTATTGACCCTACTTGTTCGGAT-3’
Seq?ID?No.4：5’-TCGGCAGAGGCATCTTGAAT-3’
Seq?ID?No.5：5’-CGTCAACGTGCCCGGTACT-3’
Seq?ID?No.6：5’-AAAGGACCTGACTGCTCGCA-3’
Seq?ID?No.7：5’-GGCAGCTACGACATGATACTCCT-3’
Seq?ID?No.8：5’-CCGAGGAGGTTTCCGGATATTA-3’
Seq?ID?No.9：5’-TTTTCTGTACTTGTGTAATCGGCTAA-3’
Seq?ID?No.10：5’-AGTTGACCATCCAAACCCGA-3’
Seq?ID?No.11：5’-TCTGCGCACGCAATTCAAC-3’
Seq?ID?No.12：5’-CGTTGTGGTCTCCATCCTCTTAC-3’
Seq?ID?No.13：5’-AAGCGAACGATTCAGATGGC-3’
Seq?ID?No.14：5’-CGTTTCCCGCCTTCAGTTTA-3’。
2. primer according to claim 1, it is characterized in that: described primer pair 3, primer pair 4, primer pair 5 and primer pair 6,5 ' end of its upstream primer also comprises sequence shown in SeqIDNo.15, and 5 ' end of its downstream primer also comprises sequence shown in Seq ID No.16;
Seq?ID?No.15：5’-CGTGGCCTCGCGATCTGACT-3’
Seq?ID?No.16：5’-CTCAGCGGCGGAGCTACAGA-3’。
3. primer according to claim 2, is characterized in that:
The upstream primer of described primer pair 3 has sequence shown in Seq ID No.17, and downstream primer has sequence shown in Seq ID No.18;
The upstream primer of primer pair 4 has sequence shown in Seq ID No.19, and downstream primer has sequence shown in Seq ID No.20;
The upstream primer of primer pair 5 has sequence shown in Seq ID No.21, and downstream primer has sequence shown in Seq ID No.22;
The upstream primer of primer pair 6 has sequence shown in Seq ID No.23, and downstream primer has sequence shown in Seq ID No.24;
Seq?ID?No.17：5’-CGTGGCCTCGCGATCTGACTCGTCAACGTGCCCGGTACT-3’
Seq?ID?No.18：5’-CTCAGCGGCGGAGCTACAGAAAAGGACCTGACTGCTCGCA-3’
Seq?ID?No.19：5’-CGTGGCCTCGCGATCTGACTGGCAGCTACGACATGATACTCCT-3’
Seq?ID?No.20：5’-CTCAGCGGCGGAGCTACAGACCGAGGAGGTTTCCGGATATTA-3’
Seq?ID?No.21：5’-CGTGGCCTCGCGATCTGACTTTTTCTGTACTTGTGTAATCGGCTAA-3’
Seq?ID?No.22：5’-CTCAGCGGCGGAGCTACAGAAGTTGACCATCCAAACCCGA-3’
Seq?ID?No.23：5’-CGTGGCCTCGCGATCTGACTTCTGCGCACGCAATTCAAC-3’
Seq?ID?No.24：5’-CTCAGCGGCGGAGCTACAGACGTTGTGGTCTCCATCCTCTTAC-3’。
4. the primer according to any one of claim 1-3, is characterized in that:
Described primer pair 1 is the primer detecting Transgenic corn lines NK603,
Primer pair 2 is the primer detecting Transgenic corn lines MON863,
Primer pair 3 is the primer detecting Transgenic corn lines MON810,
Primer pair 4 is the primer detecting Transgenic corn lines T25,
Primer pair 5 is the primer detecting Transgenic corn lines MON88017,
Primer pair 6 is the primer detecting Transgenic corn lines MIR604,
Primer pair 7 is the primer detecting Transgenic corn lines 59122.
5. one kind for transgenic corns multiplex PCR-DHPLC detection method, it is characterized in that: described detection method comprises the primer adopted described in any one of claim 1-4, be that template carries out pcr amplification with maize dna, and dhplc analysis is carried out to pcr amplification product.
6. detection method according to claim 5, it is characterized in that: it is that reference standard carries out dhplc analysis that described detection method also comprises with marker, the dhplc analysis result of pcr amplification product and the dhplc analysis result of marker is compared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110248327.7A CN102952861B (en) | 2011-08-26 | 2011-08-26 | Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110248327.7A CN102952861B (en) | 2011-08-26 | 2011-08-26 | Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102952861A CN102952861A (en) | 2013-03-06 |
| CN102952861B true CN102952861B (en) | 2014-12-31 |
Family
ID=47762242
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110248327.7A Expired - Fee Related CN102952861B (en) | 2011-08-26 | 2011-08-26 | Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102952861B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343162A (en) * | 2013-06-06 | 2013-10-09 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PCR-DHPLC detection primers and detection method for maize ubiquitin protein promoter gene |
| CN104195260B (en) * | 2014-09-22 | 2015-12-30 | 天津出入境检验检疫局动植物与食品检测中心 | A kind of new Transgenic corn lines MIR604 detection kit and detection method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1332246A (en) * | 2000-06-22 | 2002-01-23 | 孟山都技术有限公司 | Corn individual PV-IMGT 32 (NK 603) and composition and method for detecting it |
| CN101646782A (en) * | 2007-01-29 | 2010-02-10 | 科学公共卫生研究所（Iph） | The transgenic plant event detection |
| CN101812517A (en) * | 2010-01-18 | 2010-08-25 | 华中农业大学 | Wheat scab ear rot and seedling resistance molecular marker |
-
2011
- 2011-08-26 CN CN201110248327.7A patent/CN102952861B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1332246A (en) * | 2000-06-22 | 2002-01-23 | 孟山都技术有限公司 | Corn individual PV-IMGT 32 (NK 603) and composition and method for detecting it |
| CN101646782A (en) * | 2007-01-29 | 2010-02-10 | 科学公共卫生研究所（Iph） | The transgenic plant event detection |
| CN101812517A (en) * | 2010-01-18 | 2010-08-25 | 华中农业大学 | Wheat scab ear rot and seedling resistance molecular marker |
Non-Patent Citations (4)
| Title |
|---|
| Detection of genetically modified maize MON810 and NK603 by multiplex and real-time polymerase chain reaction methods;HY Huang et al;《J.Agric.Food Chem.》;20041231;第53卷;3264-3268 * |
| 利用变性高效液相色谱(dHPLC)进行小麦等位基因差异表达分析;韩宗福等;《自然科学进展》;20081130;第18卷(第11期);1256-1263 * |
| 多重PCR结合变性高效液相色谱技术转基因小麦检测方法的建立;白月等;《麦类作物学报》;20110715;第31卷(第4期);577-581 * |
| 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》;20110630;第25卷(第3期);129-134 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102952861A (en) | 2013-03-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Onishi et al. | Development of a multiplex polymerase chain reaction method for simultaneous detection of eight events of genetically modified maize | |
| CN102952856B (en) | PCR-DHPLC detection primers for transgenic rice line Kefeng 6 and detection method | |
| US20200017900A1 (en) | Genetic markers for distinguishing the phenotype of a cannabis sativa sample | |
| CN101824411B (en) | Flanking sequence of transgenic rice Kefeng No. 6 and qualitative PCR detection method | |
| CN102952861B (en) | Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize | |
| CN102952858B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified rice strain KMD | |
| CN102952863B (en) | Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified cotton | |
| CN102952862B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain MON89034 | |
| CN102952864B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT176 | |
| CN102952860B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT11 | |
| CN101343666A (en) | Pest-resistant transgenic rice multiple PCR detection reagent kit and detection method thereof | |
| CN102952859A (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain CBH351 | |
| CN102952857B (en) | Primer and method for PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection for endogenous genes of wheat | |
| CN103667475A (en) | Detection method for transgenic Cry1Ab insect-resistant gene rice variety mfb-MH86 | |
| CN103173548A (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) assay primer and assay method for transgenic rapeseed RF2 strain | |
| CN103509875B (en) | Detection of CaMV 35S promoter and nos terminator by adopting RPA (Recombinase Ploymerase Amplification) technology | |
| CN103173550A (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) assay primer and assay method for transgenic rapeseed RT73 strain | |
| CN103820566B (en) | Primer, probe and method for specific quantitative polymerase chain reaction (PCR) accurate detection of transgenic maize DAS-40278-9 strain | |
| CN102952865B (en) | PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection primer and PCR-DHPLC detection method for transgenic soybean strain RRS | |
| CN102776268A (en) | Kit and method for detection of transgenic maize Mon89034 and derivative varieties thereof through gene amplification at constant temperature | |
| CN101792814A (en) | PCR method for detecting specificity of transformant of transgenic pest-resistant cotton GK12 | |
| CN103173551A (en) | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) assay primer and assay method for transgenic rapeseed MS1 strain | |
| CN102367489B (en) | Primer for detecting freesia mosaic virus (FreMV) and detection method of freesia mosaic virus | |
| CN102618658B (en) | Specific quantitative PCR (Polymerase Chain Reaction) accurate detection method for genetically modified maize TC1507 line | |
| CN103031376A (en) | Genetic diagnosis method for detecting different cattle groups at home and abroad |
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: 20141231 Termination date: 20180826 |