CN102409093A - Specific quantitative polymerase chain reaction (PCR) detection method for transgenic corn MON863 strain - Google Patents
Specific quantitative polymerase chain reaction (PCR) detection method for transgenic corn MON863 strain Download PDFInfo
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- CN102409093A CN102409093A CN2011103636246A CN201110363624A CN102409093A CN 102409093 A CN102409093 A CN 102409093A CN 2011103636246 A CN2011103636246 A CN 2011103636246A CN 201110363624 A CN201110363624 A CN 201110363624A CN 102409093 A CN102409093 A CN 102409093A
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Abstract
The invention belongs to a biological technology, and relates to a quantitative analytical method for genes, in particular to a specific quantitative polymerase chain reaction (PCR) detection method for a transgenic corn MON863 strain. The method comprises the following steps of: preparing a PCR reaction system from a specific forward primer Event863-F, a specific reverse primer Event863-R, a fluorescent probe Event863-P, deoxyribonucleic acid (DNA) diluent of the MON863 strain, TaqmanMastermix and water; and performing quantitative PCR detection. In the method, a Taqman quantitative PCR detection technology with high amplification efficiency and accuracy is established, and the method is suitable for the monitoring and inspection of transgenic organisms and transgenic products of domestic agriculture, the inspection of the transgenic organisms and the transgenic products of import and export ports and the detection of ingredients of organisms containing the transgenic MON863 strain in import raw materials of enterprises.
Description
Technical field
The invention belongs to biotechnology, relate to the quantitative analytical procedure of gene.
Background technology
A lot of in the world countries implement limit the quantity of sign and import to transgenic product, 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 are provided with; Remedy and improve China's genetically modified organism and product quantitative measurement technology system simultaneously; Protect right to know and the preference of human consumer better, set up the accurate detection technique of a kind of special quantitative PCR of novel genetically modified corn MON 863 strain and necessitate transgenic product.
And present detection technique about genetically modified corn MON 863; Mainly concentrate on common qualitative PCR method and standard, still have nothing to do in the accurate detection technique of quantitative PCR of the strain specificity specific site (gene order) that detects genetically modified corn MON 863 and product.
Summary of the invention
The object of the invention mainly provides 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 genetically modified corn MON 863 and product.
The present invention realizes through following technical proposals:
The special quantitative PCR detecting method of genetically modified corn MON 863 strain 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, Event863-F:5'-CTACTTGTTCGGATGGGTGTTC-3'
The downstream primer sequence, Event863-R:5'-CCTTTATCGCAATGATGGCA-3'
The fluorescent probe sequence, Event863-P:5'-FAM-CCCCAAAGTGTACCAAGCTTTCCGAT-TAMRA-3';
(2) the DNA diluent of preparation MON863 strain;
(3) preparation PCR reaction system;
(4) quantitative PCR detection.
Further, the concentration of said synthetic primer of step (1) and fluorescent probe all is 10 μ mol/l, and the concentration of the DNA diluent of the said 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 said 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, said PCR reaction conditions is: 95 ℃ of preparatory 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 has broken the transgenic product trade technology barriers that countries and regions such as European Union are provided with.
2, the present invention remedies and perfect China's genetically modified organism and product quantitative measurement technology system.
3, detection technique provided by the invention can be protected right to know and the preference of human consumer to transgenic product better.
4, amplification efficiency of the present invention is high, accuracy is high.
Embodiment
Below in conjunction with embodiment the present invention is done further explanation, but embodiment of the present invention is not limited to this.
Embodiment
The special quantitative PCR detecting method of genetically modified corn MON 863 strain 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, Event863-F:5'-CTACTTGTTCGGATGGGTGTTC-3'
The downstream primer sequence, Event863-R:5'-CCTTTATCGCAATGATGGCA-3'
The fluorescent probe sequence, Event863-P:5'-FAM-CCCCAAAGTGTACCAAGCTTTCCGAT-TAMRA-3'.
Concentration after present embodiment primer and fluorescent probe are synthetic all is 10 μ mol/l.
The nucleotide sequence of above primer and fluorescent probe is the strain specificity specific site to genetically modified corn MON 863 and product; Be the integration site design of foreign gene and corn gene group DNA, just can precisely detect the transformation event of MON863 in the transgenic corns through this design.
(2) the DNA diluent of preparation MON863 strain; Promptly adopt conventional DNA extraction means, from genetically modified corn MON 863, extracting concentration is the DNA diluent of 50ng/ μ l.
(3) preparation PCR reaction system; Get final product in the DNA diluent adding 25ul reaction system that soon 3ul will prepare.
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.
That 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 detect, said PCR reaction conditions is: 95 ℃ of preparatory 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 15 parallel sample continuously, and said 15 samples are detected, and it detects data such as table 1.
Table 1
Adopt present method can precisely detect MON863 transformation event 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 102.2%, in 90%～110% scope.The detection by quantitative result of sample to be checked (1.1%) 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 of the present invention is high, accuracy is high.
SEQUENCE?LISTING
< 110>Institute of Analysis of Sichuan Academy of Agricultural Sciences
< 120>the special quantitative PCR detecting method of genetically modified corn MON 863 strain
<130>
<160> 3
<170> PatentIn?version?3.3
<210> 1
<211> 22
<212> DNA
<213> Artificial
<220>
< 223>upstream primer (Event863-F)
<400> 1
ctacttgttc?ggatgggtgt?tc 22
<210> 2
<211> 20
<212> DNA
<213> Artificial
<220>
< 223>downstream primer (Event863-R)
<400> 2
cctttatcgc?aatgatggca 20
<210> 3
<211> 26
<212> DNA
<213> Artificial
<220>
< 223>fluorescent probe (Event863-P)
<400> 3
ccccaaagtg?taccaagctt?tccgat 26
Claims (4)
1. the special quantitative PCR detecting method of genetically modified corn MON 863 strain 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, Event863-F:5'-CTACTTGTTCGGATGGGTGTTC-3'
The downstream primer sequence, Event863-R:5'-CCTTTATCGCAATGATGGCA-3'
The fluorescent probe sequence, Event863-P:5'-FAM-CCCCAAAGTGTACCAAGCTTTCCGAT-TAMRA-3';
(2) the DNA diluent of preparation MON863 strain;
(3) preparation PCR reaction system;
(4) quantitative PCR detection.
2. the special quantitative PCR detecting method of genetically modified corn MON 863 strain according to claim 1; It is characterized in that; The concentration of said synthetic primer of step (1) and fluorescent probe all is 10 μ mol/l, and the concentration of the DNA diluent of the said preparation of step (2) is 50ng/ μ l.
3. the special quantitative PCR detecting method of genetically modified corn MON 863 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 the 25 μ l reaction systems, and the reaction system of said 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 special quantitative PCR detecting method of 3 described genetically modified corn MON 863 strains, it is characterized in that said PCR reaction conditions is: 95 ℃ of preparatory sex change 10min, 1 circulation; 95 ℃ of sex change 15s, 59 ℃ of annealing 60s, 45 circulations.
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| CN2011103636246A CN102409093A (en) | 2011-11-17 | 2011-11-17 | Specific quantitative polymerase chain reaction (PCR) detection method for transgenic corn MON863 strain |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1724689A (en) * | 2005-07-21 | 2006-01-25 | 上海交通大学 | Strain specificity PCR detection method of heredity modified corn strain MON863 |
| CN1740322A (en) * | 2005-07-21 | 2006-03-01 | 上海交通大学 | Para-gene sequence for exogenous insertion vector of corn strain MON863 |
| EP1724360A1 (en) * | 2005-05-17 | 2006-11-22 | Eppendorf Array Technologies S.A. | Identification and/or quantification method of nucleotide sequence(s) elements specific of genetically modified plants on arrays |
| CN101519693A (en) * | 2009-04-02 | 2009-09-02 | 天津市农业科学院中心实验室 | Method for rapidly detecting genetically modified corn MON863 |
-
2011
- 2011-11-17 CN CN2011103636246A patent/CN102409093A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1724360A1 (en) * | 2005-05-17 | 2006-11-22 | Eppendorf Array Technologies S.A. | Identification and/or quantification method of nucleotide sequence(s) elements specific of genetically modified plants on arrays |
| CN1724689A (en) * | 2005-07-21 | 2006-01-25 | 上海交通大学 | Strain specificity PCR detection method of heredity modified corn strain MON863 |
| CN1740322A (en) * | 2005-07-21 | 2006-03-01 | 上海交通大学 | Para-gene sequence for exogenous insertion vector of corn strain MON863 |
| CN101519693A (en) * | 2009-04-02 | 2009-09-02 | 天津市农业科学院中心实验室 | Method for rapidly detecting genetically modified corn MON863 |
Non-Patent Citations (1)
| Title |
|---|
| HONG ZHU等: "A specific qualitative and real-time PCR detection of MON863 maize based on the 5"-transgene integration sequence", 《JOURNAL OF CEREAL SCIENCE》 * |
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Application publication date: 20120411 |