CN102409094B - Transgenic corn NK603 strain specific quantitative PCR (Polymerase Chain Reaction) accurate detection method - Google Patents
Transgenic corn NK603 strain specific quantitative PCR (Polymerase Chain Reaction) accurate detection method Download PDFInfo
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- CN102409094B CN102409094B CN 201110363639 CN201110363639A CN102409094B CN 102409094 B CN102409094 B CN 102409094B CN 201110363639 CN201110363639 CN 201110363639 CN 201110363639 A CN201110363639 A CN 201110363639A CN 102409094 B CN102409094 B CN 102409094B
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Abstract
The invention belongs to a biotechnology, and relates to a quantitative analysis method for genes, in particular to a transgenic corn NK603 strain specific quantitative PCR (Polymerase Chain Reaction) accurate detection method. In the invention, designed specific upstream primer Event 603-F, downstream primer Event 603-R, a fluorescent probe Event 603-P, DNA (Deoxyribonucleic Acid) diluent of NK603 strain, TaqmanMastermix and water are prepared into a PCR reaction system; and quantitative PCR detection is performed. In the invention, a Taqman quantitative PCR detection technology of high amplification efficiency and high accuracy is mainly established; and the method is suitable for supervision and inspection of domestic agricultural transgenic organisms and products, inspection of entry and exit port transgenic organisms and products and detection of transgenic NK603 strain biological ingredient included 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 strain 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 strain 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 strain 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, Event603-F:5'-TCTCTGGCATTTCCAACCCT-3'
The downstream primer sequence, Event603-R:5'-TCTCAAGCATATGAATGACCTCG-3'
The fluorescent probe sequence, Event603-P:5'-FAM-TGCGTCCCTGGTGGGCTGC-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 strain 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, Event603-F:5'-TCTCTGGCATTTCCAACCCT-3'
The downstream primer sequence, Event603-R:5'-TCTCAAGCATATGAATGACCTCG-3'
The fluorescent probe sequence, Event603-P:5'-FAM-TGCGTCCCTGGTGGGCTGC-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 strain specificity specific site for transgenic corns NK603 and product, be the integration site design of foreign gene and corn gene group DNA, 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 strain 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; Amplification efficiency is 100.2%, in 90%～110% scope.The detection by quantitative result (1.9%) 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 strain
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial
<220>
＜223〉upstream primer (Event603-F)
<400> 1
tctctggcat ttccaaccct 20
<210> 2
<211> 23
<212> DNA
<213> Artificial
<220>
＜223〉downstream primer (Event603-R)
<400> 2
tctcaagcat atgaatgacc tcg 23
<210> 3
<211> 19
<212> DNA
<213> Artificial
<220>
＜223〉fluorescent probe (Event603-P)
<400> 3
tgcgtccctg gtgggctgc 19
Claims (4)
1. the accurate detection method of the special quantitative PCR of transgenic corns NK603 strain, 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, Event603-F:5'-TCTCTGGCATTTCCAACCCT-3'
The downstream primer sequence, Event603-R:5'-TCTCAAGCATATGAATGACCTCG-3'
The fluorescent probe sequence, Event603-P:5'-FAM-TGCGTCCCTGGTGGGCTGC-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 strain 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 strain 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 strain, 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.
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| CN 201110363639 CN102409094B (en) | 2011-11-17 | 2011-11-17 | Transgenic corn NK603 strain specific quantitative PCR (Polymerase Chain Reaction) accurate detection method |
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| CN102409094B true CN102409094B (en) | 2013-06-26 |
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| CN1435489A (en) * | 2002-10-11 | 2003-08-13 | 曹际娟 | Probe sequence and kit for real time fluorescent PCR detection of transgenic corn |
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| 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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| 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 |
| 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 |
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Patent Citations (3)
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|---|---|---|---|---|
| CN1435489A (en) * | 2002-10-11 | 2003-08-13 | 曹际娟 | Probe sequence and kit for real time fluorescent PCR detection of transgenic corn |
| CN101302565A (en) * | 2008-06-30 | 2008-11-12 | 天津市农业科学院中心实验室 | Method for rapid detecting transgenic maize BT176 |
| 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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| Characterisation of the 5" integration site and development of an event-specific real-time PCR assay for NK603 maize from a low starting copy number;Christer R. Nielsen et al.;《Eur Food Res Technol》;20040724;第219卷;421-427 * |
| Christer R. Nielsen et al..Characterisation of the 5" integration site and development of an event-specific real-time PCR assay for NK603 maize from a low starting copy number.《Eur Food Res Technol》.2004,第219卷421-427. |
| Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA;Luke A. Shokere et al.;《Food Control》;20090430;391–401 * |
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| HSIN-YING HUANG et al..Detection of Genetically Modified Maize MON810 and NK603 by Multiplex and Real-Time Polymerase Chain Reaction Methods.《J. Agric. Food Chem.》.2004,第52卷(第11期),3264-3268. |
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