CN102409092B - Structure-specific quantitative PCR (polymerase chain reaction) accurate detection method of transgenic maize NK603 - Google Patents
- ️Wed Jun 26 2013
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Publication number
- CN102409092B CN102409092B CN 201110363623 CN201110363623A CN102409092B CN 102409092 B CN102409092 B CN 102409092B CN 201110363623 CN201110363623 CN 201110363623 CN 201110363623 A CN201110363623 A CN 201110363623A CN 102409092 B CN102409092 B CN 102409092B Authority
- CN
- China Prior art keywords
- quantitative pcr
- accurate detection
- primer
- transgenic
- detection method Prior art date
- 2011-11-17 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
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- 230000009261 transgenic effect Effects 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title claims abstract description 25
- 240000008042 Zea mays Species 0.000 title claims abstract description 18
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 18
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 18
- 238000003752 polymerase chain reaction Methods 0.000 title abstract 3
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 title abstract 2
- 235000009973 maize Nutrition 0.000 title abstract 2
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 15
- 239000003085 diluting agent Substances 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 208000003643 Callosities Diseases 0.000 claims description 16
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 16
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 16
- 235000005822 corn Nutrition 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000011144 upstream manufacturing Methods 0.000 claims description 7
- 230000004087 circulation Effects 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 8
- 230000003321 amplification Effects 0.000 abstract description 5
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 238000007689 inspection Methods 0.000 abstract description 3
- 102000053602 DNA Human genes 0.000 abstract 2
- 108020004414 DNA Proteins 0.000 abstract 2
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 239000000047 product Substances 0.000 description 12
- 239000000203 mixture Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 235000003869 genetically modified organism Nutrition 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- VWEWCZSUWOEEFM-WDSKDSINSA-N Ala-Gly-Ala-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(O)=O VWEWCZSUWOEEFM-WDSKDSINSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000007859 qualitative PCR Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to biotechnology, relates to a quantitative analysis method of a gene, and particularly relates to a structure-specific quantitative PCR (polymerase chain reaction) accurate detection method of transgenic maize NK603. The method comprises the steps of: preparing a PCR system by adopting a designed specific forward primer Construct 603-F, a reverse primer Construct 603-R and a fluorescent probe Construct 603-P, an NK603 line DNA (deoxyribonucleic acid) diluent, Taqman Mastermix and water; and carrying out quantitative PCR detection. In the invention, a Taqman quantitative PCR detection technique with high amplification efficiency and high accuracy is mainly built, and the technique is suitable for supervision and inspection of agricultural transgenic organisms and products at home and abroad, inspection of transgenic organisms and products at entry and exit port, and detection of biological components containing transgenic NK603 in imported raw materials in enterprises.
Description
Technical field
The invention belongs to biotechnology, relate to the quantitative analytical procedure of gene.
Background technology
A lot of countries implement to transgenic product limit the quantity of sign and import in the world, and China there is no concrete transgenic product sign threshold value.The transgenic product Trade technique barrier that arranges in order to break the countries and regions such as European Union; make up simultaneously and improve China's genetically modified organism and product quantitative measurement technology system; right to know and the preference of Protection of consumer to transgenic product, set up the accurate detection method of the special quantitative PCR of a kind of novel transgenic corns NK603 structure and necessitate better.
And present detection technique about transgenic corns NK603, mainly concentrate on common qualitative PCR analytical procedure and standard, there is no the accurate detection technique of quantitative PCR about the structure specificity specific site (gene order) of augmentation detection transgenic corns NK603 and product.
Summary of the invention
Purpose of the present invention mainly is to provide the accurate detection technique of quantitative PCR of the strain specificity specific site of a kind of amplification efficiency is high, accuracy is high detection transgenic corns NK603 and product.
The present invention is achieved through the following technical solutions:
The accurate detection method of the special quantitative PCR of transgenic corns NK603 structure mainly comprises the following steps:
Synthetic primer with following nucleotide sequence reaches the fluorescent probe that is used in conjunction with primer,
The upstream primer sequence, Construct603-F:5'-TCCCGACTCTCTTCTCAAGCA-3'
The downstream primer sequence, Construct603-R:5'-ACAGGATCCACTCAAACACTAGAG-3'
The fluorescent probe sequence, Construct603-P:5'-FAM-AGTGTGTCGCGTGGTACCAAGCTTGA-TAMRA-3 ';
(2) the DNA diluent of preparation NK603 strain;
(3) preparation PCR reaction system;
(4) quantitative PCR detection.
Further, the described synthetic primer of step (1) and the concentration of fluorescent probe are all 10 μ mol/l, and the concentration of the DNA diluent of the described preparation of step (2) is 50ng/ μ l.
In order to reach best detection effect, the described preparation of step (3) PCR reaction system, the DNA diluent that is about to 3 μ l joins in 25 μ l reaction systems, and the reaction system of described 25 μ l comprises following component:
Taqman Master mix 12.5μl
Upstream primer 1 μ l
Downstream primer 1 μ l
Fluorescent probe 0.5 μ l
Water 10ul.
As the reaction conditions of optimum, described PCR reaction conditions is: 95 ℃ of denaturation 10min, 1 circulation; 95 ℃ of sex change 15s, 59 ℃ of annealing 60s, 45 circulations.
The present invention has the following advantages and beneficial effect:
1, the present invention has broken the transgenic product Trade technique barrier that the countries and regions such as European Union arrange.
2, the present invention makes up and perfect China's genetically modified organism and product quantitative measurement technology system.
3, detection technique provided by the invention right to know and the preference of Protection of consumer to transgenic product better.
4, amplification efficiency of the present invention is high, accuracy is high.
Embodiment
The present invention is described further below in conjunction with embodiment, but embodiments of the present invention are not limited to this.
Embodiment
The accurate detection method of the special quantitative PCR of transgenic corns NK603 structure mainly comprises the following steps:
(1) synthetic primer with following nucleotide sequence reaches the fluorescent probe that is used in conjunction with primer,
The upstream primer sequence, Construct603-F:5'-TCCCGACTCTCTTCTCAAGCA-3'
The downstream primer sequence, Construct603-R:5'-ACAGGATCCACTCAAACACTAGAG-3'
The fluorescent probe sequence, Construct603-P:5'-FAM-AGTGTGTCGCGTGGTACCAAGCTTGA-TAMRA-3 '.
The synthetic concentration of the present embodiment primer and fluorescent probe is all 10 μ mol/l.
The nucleotide sequence of above primer and fluorescent probe is the structure specificity specific site for transgenic corns NK603 and product, i.e. goal gene and controlling element specific site design; Just can precisely detect the transformation event of NK603 in transgenic corns by this design.
(2) the DNA diluent of preparation NK603 strain; Namely adopt conventional DNA extraction means, extracting concentration from transgenic corns NK603 is the DNA diluent of 50ng/ μ l.
(3) preparation PCR reaction system; Being about to DNA diluent that 3ul prepares adds in the 25ul reaction system and gets final product.
Above 25ul reaction system comprises following component:
Taqman Master mix 12.5μl
Upstream primer 1 μ l
Downstream primer 1 μ l
Fluorescent probe 0.5 μ l
Water 10 μ l.
Taqman Master mix adopts is the Taqman Master mix that ABI company produces.
(4) quantitative PCR detection.
According to above-mentioned PCR reaction system, under following PCR reaction conditions, product is increased and detects, described PCR reaction conditions is: 95 ℃ of denaturation 10min, 1 circulation; 95 ℃ of sex change 15s, 59 ℃ of annealing 60s, 45 circulations.What adopt in the present embodiment is the 7500 type quantitative fluorescent PCR instruments that ABI company produces.
Method data to the present embodiment repeat to do 13 parallel sample continuously, and these 13 samples are detected, and it detects data such as table 1.
Table 1
Adopt the present invention can precisely detect NK603 structure specific fragment and content thereof in transgenic corns, obtain slope of standard curve, between-3.6～-3.1, relation conefficient is greater than 0.99, and amplification efficiency is 98.1%, in 90%～110% scope.The detection by quantitative result (1.6%) of sample to be checked is very near actual value (2%), and the deviation ratio of detected result is less than 25% of international endorsement, and the uncertainty of detected result is less than 10%.
Can be learnt by above detected result, each index of present method all satisfies the scope of the accurate gene quantification method of inspection of international endorsement, and amplification efficiency of the present invention is high, accuracy is high.
SEQUENCE LISTING
＜110〉Institute of Analysis of Sichuan Academy of Agricultural Sciences
＜120〉the accurate detection method of the special quantitative PCR of transgenic corns NK603 structure
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial
<220>
＜223〉upstream primer (Construct603-F)
<400> 1
tcccgactct cttctcaagc a 21
<210> 2
<211> 24
<212> DNA
<213> Artificial
<220>
＜223〉downstream primer (Construct603-R)
<400> 2
acaggatcca ctcaaacact agag 24
<210> 3
<211> 26
<212> DNA
<213> Artificial
<220>
＜223〉fluorescent probe (Construct603-P)
<400> 3
agtgtgtcgc gtggtaccaa gcttga 26
Claims (4)
1. the accurate detection method of the special quantitative PCR of transgenic corns NK603 structure, is characterized in that, mainly comprises the following steps:
(1) primer of synthetic following nucleotide sequence reaches the fluorescent probe that is used in conjunction with primer,
The upstream primer sequence, Construct603-F:5'-TCCCGACTCTCTTCTCAAGCA-3'
The downstream primer sequence, Construct603-R:5'-ACAGGATCCACTCAAACACTAGAG-3'
The fluorescent probe sequence, Construct603-P:5'-FAM-AGTGTGTCGCGTGGTACCAAGCTTGA-TAMRA-3 ';
(2) the DNA diluent of preparation NK603 strain;
(3) preparation PCR reaction system;
(4) quantitative PCR detection.
2. the accurate detection method of the special quantitative PCR of transgenic corns NK603 structure according to claim 1, it is characterized in that: the described synthetic primer of step (1) and the concentration of fluorescent probe are all 10 μ mol/l, and the concentration of the DNA diluent of the described preparation of step (2) is 50ng/ μ l.
3. the accurate detection method of the special quantitative PCR of transgenic corns NK603 structure according to claim 2, it is characterized in that, the described preparation of step (3) PCR reaction system, the DNA diluent that is about to 3 μ l joins in 25 μ l reaction systems, and the reaction system of described 25 μ l comprises following component:
Taqman Master mix 12.5μl
Upstream primer 1 μ l
Downstream primer 1 μ l
Fluorescent probe 0.5 μ l
Water 10ul.
4. the according to claim 1 or 3 accurate detection methods of the special quantitative PCR of described transgenic corns NK603 structure, is characterized in that, described PCR reaction conditions is: 95 ℃ of denaturation 10min, 1 circulation; 95 ℃ of sex change 15s, 59 ℃ of annealing 60s, 45 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 201110363623 CN102409092B (en) | 2011-11-17 | 2011-11-17 | Structure-specific quantitative PCR (polymerase chain reaction) accurate detection method of transgenic maize NK603 |
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| CN 201110363623 CN102409092B (en) | 2011-11-17 | 2011-11-17 | Structure-specific quantitative PCR (polymerase chain reaction) accurate detection method of transgenic maize NK603 |
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| CN102409092A CN102409092A (en) | 2012-04-11 |
| CN102409092B true CN102409092B (en) | 2013-06-26 |
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* Cited by examiner, † Cited by third party| Publication number | Priority date | Publication date | Assignee | Title |
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| BRPI0100752B1 (en) * | 2000-06-22 | 2015-10-13 | Monsanto Co | DNA Molecules and Pairs of Molecules, Processes for Detecting DNA Molecules and for Creating a Glyphosate Tolerant Trait in Corn Plants, as well as DNA Detection Kit |
| CN1203187C (en) * | 2002-10-11 | 2005-05-25 | 曹际娟 | Probe sequence and kit for real time fluorescent PCR detection of transgenic corn |
| CN101302565B (en) * | 2008-06-30 | 2012-09-05 | 天津市农业科学院中心实验室 | Method for rapid detecting transgenic maize BT176 |
| CN101812527B (en) * | 2010-04-20 | 2014-09-17 | 北京农业职业学院 | Method for quickly detecting six kinds of genetically modified corns |
| CN102206632A (en) * | 2011-03-11 | 2011-10-05 | 山东省农业科学院植物保护研究所 | Para-gene for exogenous insertion vector of transgenic maize transformation event MON88017 and application thereof |
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