CN102618657B - Specific quantitative PCR (Polymerase Chain Reaction) accurate detection method for transgenic corn 59122 strain - Google Patents
Specific quantitative PCR (Polymerase Chain Reaction) accurate detection method for transgenic corn 59122 strain Download PDFInfo
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- CN102618657B CN102618657B CN 201210114359 CN201210114359A CN102618657B CN 102618657 B CN102618657 B CN 102618657B CN 201210114359 CN201210114359 CN 201210114359 CN 201210114359 A CN201210114359 A CN 201210114359A CN 102618657 B CN102618657 B CN 102618657B
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Abstract
The invention belongs to a biological technology, and relates to the quantitative analyzing method of genes, in particular to a specific quantitative PCR (Polymerase Chain Reaction) accurate detection method for a transgenic corn 59122 strain. In the method, a PCR reaction system is prepared by adopting a designed specific upstream primer 59122event-F, a downstream primer 59122event-R, a fluorescent probe 59122event-P, a DNA diluent of the 59122 strain, TaqmanMastermix and water for performing quantitative PCR detection. In the method, a Taqman quantitative PCR detection technology with 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-exit port transgenic organisms and products, and biological component detection of enterprise internal import raw materials containing transgenic 59122 strains.
Description
Technical field
The invention belongs to biological technical field, relate to the quantitative analytical procedure of gene.
Background technology
A lot of countries implement limit the quantity of sign and import to transgenic product in the world, and China does not still have concrete transgenic product sign threshold value.In order to break the transgenic product trade technology barriers that countries and regions such as European Union arrange; remedy and improve China's genetically modified organism and product quantitative measurement technology system simultaneously; protect the human consumer to right to know and the preference of transgenic product better, set up the accurate detection method of a kind of special quantitative PCR of novel transgenic corns 59122 strains and necessitate.
Present detection technique about transgenic corns 59122, mainly concentrate on common qualitative PCR analytical procedure and standard, still have nothing to do in the accurate detection technique of quantitative PCR of the strain specificity specific site (gene order) of augmentation detection transgenic corns 59122 and product.
Summary of the invention
Purpose of the present invention mainly provides the accurate detection technique of quantitative PCR of the strain specificity specific site of the high detection transgenic corns 59122 of a kind of amplification efficiency height, accuracy and product.
The present invention is achieved through the following technical solutions:
The accurate detection method of the special quantitative PCR of transgenic corns 59122 strains mainly may further comprise the steps:
(1) synthetic primer with following nucleotide sequence reaches the fluorescent probe that is used with primer,
The upstream primer sequence, 59122event-F:5'-AATTCGCCCTATAGTGAGTCGTA-3'
The downstream primer sequence, 59122event-R:5'-GATAAACAAACGGGACCATAGAA-3'
The fluorescent probe sequence, 59122event-P:5'-FAM-CGCAATTCAGTACATTAAAAACGTCCGC-TAMRA-3 ';
(2) the DNA diluent of preparation 59122 strains;
(3) preparation PCR reaction system;
(4) quantitative PCR detection.
Further, the described synthetic primer of step (1) and the concentration of fluorescent probe all are 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 the 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 pre-sex change 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 breaks the transgenic product trade technology barriers of countries and regions settings such as European Union;
2, the present invention remedies and improves China's genetically modified organism and product quantitative measurement technology system;
3, detection technique provided by the invention can protect the human consumer to right to know and the preference of transgenic product better;
4, amplification efficiency height of the present invention, accuracy height.
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 59122 strains mainly may further comprise the steps:
(1) synthetic primer with following nucleotide sequence reaches the fluorescent probe that is used with primer,
The upstream primer sequence, 59122event-F:5'-AATTCGCCCTATAGTGAGTCGTA-3'
The downstream primer sequence, 59122event-R:5'-GATAAACAAACGGGACCATAGAA-3'
The fluorescent probe sequence, 59122event-P:5'-FAM-CGCAATTCAGTACATTAAAAACGTCCGC-TAMRA-3 '.
The synthetic concentration of present embodiment primer and fluorescent probe all is 10 μ mol/l.
The nucleotide sequence of above primer and fluorescent probe is the strain specificity specific site at transgenic corns 59122 and product, be the integration site design of foreign gene and corn gene group DNA, just can precisely detect transformation event in the transgenic corns 59122 by this design.
(2) the DNA diluent of preparation 59122 strains; Namely adopt conventional DNA extraction means, extracting concentration from transgenic corns 59122 is the DNA diluent of 50ng/ μ l.
(3) preparation PCR reaction system; Soon in the DNA diluent adding 25ul reaction system that 3ul prepares.
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.
(4) quantitative PCR detection.
According to above-mentioned PCR reaction system, under following PCR reaction conditions, product is increased and detect, described PCR reaction conditions is: 95 ℃ of pre-sex change 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 present embodiment repeat to do 16 parallel sample continuously, and these 16 samples are detected, and it detects data such as table 1.
Table 1
| Experiment number | ZSS II b Ct value | ZSS II absolute content (ng) | Strain specific fragment Ct value | The strain specific fragment contains extremely amount (ng) | Strain specific fragment relative content (%) |
| 1 | 23.410 | 141.323 | 30.245 | 1.274 | 0.9 |
| 2 | 23.450 | 137.747 | 30.368 | 1.177 | 0.9 |
| 3 | 23.460 | 136.800 | 30.384 | 1.165 | 0.9 |
| 4 | 23.476 | 135.457 | 29.941 | 1.550 | 1.1 |
| 5 | 23.446 | 138.115 | 30.531 | 1.059 | 0.8 |
| 6 | 23.485 | 134.630 | 30.369 | 1.176 | 0.9 |
| 7 | 23.450 | 137.743 | 30.349 | 1.192 | 0.9 |
| 8 | 23.316 | 150.133 | 30.391 | 1.160 | 0.8 |
| 9 | 23.397 | 142.517 | 30.234 | 1.283 | 0.9 |
| 10 | 23.454 | 137.374 | 30.628 | 0.996 | 0.7 |
| 11 | 23.504 | 132.999 | 30.338 | 1.200 | 0.9 |
| 12 | 23.576 | 126.954 | 30.505 | 1.078 | 0.8 |
| 13 | 23.564 | 127.983 | 30.568 | 1.035 | 0.8 |
| 14 | 23.492 | 134.086 | 30.628 | 0.995 | 0.7 |
| 15 | 23.506 | 132.815 | 30.321 | 1.214 | 0.9 |
| 16 | 23.355 | 146.359 | 30.286 | 1.241 | 0.9 |
| Mean value | 30.926(0.097) | 1.430(0.134) | 25.271(0.151) | 61.765(6.679) | 1.240 |
Adopt the present invention can precisely detect 59122 strain specific fragment and content thereof in the 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 of sample to be checked (0.9%) is very near actual value (1%), 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 that by above detected result each index of present method all satisfies the scope of the accurate gene quantification method of inspection of international endorsement, amplification efficiency height of the present invention, accuracy height.
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 59122 strains
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> Artificial
<220>
＜223〉upstream primer (59122event-F)
<400> 1
aattcgccct atagtgagtc gta 23
<210> 2
<211> 23
<212> DNA
<213> Artificial
<220>
＜223〉downstream primer (59122event-R)
<400> 2
gataaacaaa cgggaccata gaa 23
<210> 3
<211> 28
<212> DNA
<213> Artificial
<220>
＜223〉probe sequence (59122event-P)
<400> 3
cgcaattcag tacattaaaa acgtccgc 28
Claims (4)
1. the accurate detection method of the special quantitative PCR of transgenic corns 59122 strains is characterized in that, mainly may further comprise the steps:
(1) synthetic primer with following nucleotide sequence reaches the fluorescent probe that is used with primer,
The upstream primer sequence, 59122event-F:5'-AATTCGCCCTATAGTGAGTCGTA-3'
The downstream primer sequence, 59122event-R:5'-GATAAACAAACGGGACCATAGAA-3'
The fluorescent probe sequence, 59122event-P:5'-FAM-CGCAATTCAGTACATTAAAAACGTCCGC-TAMRA-3 ';
(2) the DNA diluent of preparation 59122 strains;
(3) preparation PCR reaction system;
(4) quantitative PCR detection.
2. the accurate detection method of the special quantitative PCR of transgenic corns 59122 strains according to claim 1, it is characterized in that, the described synthetic primer of step (1) and the concentration of fluorescent probe are 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 59122 strains 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 the 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. according to claim 1 or the accurate detection method of the 3 described special quantitative PCRs of transgenic corns 59122 strains, it is characterized in that described PCR reaction conditions is: 95 ℃ of pre-sex change 10min, 1 circulation; 95 ℃ of sex change 15s, 59 ℃ of annealing 60s, 45 circulations.
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| CN 201210114359 CN102618657B (en) | 2012-04-18 | 2012-04-18 | Specific quantitative PCR (Polymerase Chain Reaction) accurate detection method for transgenic corn 59122 strain |
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Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AR050891A1 (en) * | 2004-09-29 | 2006-11-29 | Du Pont | EVENT DAS-59122-7 OF CORN AND METHODS FOR DETECTION |
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2012
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Non-Patent Citations (6)
| Title |
|---|
| Event-specific analytical methods for biotech maize MIR 604 and DAS-59122-7;Seong-Hun Lee, et. al;《J. Sci. Food. Agric.》;20091007;第89卷(第15期);第2616-2624页 * |
| Seong-Hun Lee, et. al.Event-specific analytical methods for biotech maize MIR 604 and DAS-59122-7.《J. Sci. Food. Agric.》.2009,第89卷(第15期),第2616-2624页. |
| 标准分子构建及其在转基因玉米59122定量检测中的应用;陆姣等;《食品科学》;20111231;第32卷(第02期);第136-140页 * |
| 许文涛等.转基因玉米59122品系的特异性检测.《食品科学》.2011,第32卷(第04期),第139-142页. |
| 转基因玉米59122品系的特异性检测;许文涛等;《食品科学》;20111231;第32卷(第04期);第139-142页 * |
| 陆姣等.标准分子构建及其在转基因玉米59122定量检测中的应用.《食品科学》.2011,第32卷(第02期),第136-140页. |
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