CN102618658B - Specific quantitative PCR (Polymerase Chain Reaction) accurate detection method for genetically modified maize TC1507 line - Google Patents
Specific quantitative PCR (Polymerase Chain Reaction) accurate detection method for genetically modified maize TC1507 line Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and relates to a quantitative analysis method of gene, in particular to a specific quantitative PCR (Polymerase Chain Reaction) accurate detection method for a genetically modified maize TC1507 line. A PCR reaction system is prepared from designed specific upstream primer TC1507 event-F, downstream primer TC1507 event-R, fluorescent probe TC1507 event-P, DNA (Deoxyribonucleic Acid) diluent of the TC1507 line, Taqman Master mix and water for performing quantitative PCR detection. According to the invention, a Taqman quantitative PCR detection technology with high amplification efficiency and high accuracy is mainly established and is suitable for supervision inspection of domestic agricultural genetically modified organisms and products,inspection of import and export port genetically modified organisms and products and biological composition detection of genetically modified TC1507 line included in internal import raw materials of enterprises.
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 novel special quantitative PCR of transgenic corns TC1507 strain and necessitate.
Present detection technique about transgenic corns TC1507, 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 TC1507 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 TC1507 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 TC1507 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, TC1507event-F:5'-ACAAAAGCCTCCAAGCGAGTA-3'
The downstream primer sequence, TC1507event-R:5'-ATGGGGGTTACCAGCTGAGA-3'
The fluorescent probe sequence, TC1507event-P:5'-FAM-CCCTAATTATGGTCCCCGACAGTAGCC-TAMARA-3';
(2) the DNA diluent of preparation TC1507 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 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 TC1507 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, TC1507event-F:5'-ACAAAAGCCTCCAAGCGAGTA-3'
The downstream primer sequence, TC1507event-R:5'-ATGGGGGTTACCAGCTGAGA-3'
The fluorescent probe sequence, TC1507event-P:5'-FAM-CCCTAATTATGGTCCCCGACAGTAGCC-TAMARA-3'.
The synthetic concentration of present embodiment primer and fluorescent probe is 10 μ mol/l.
The nucleotide sequence of above primer and fluorescent probe is the strain specificity specific site at transgenic corns TC1507 and product, i.e. goal gene and acceptor corn gene group side site design; Just can precisely detect the transformation event of TC1507 in the transgenic corns by this design.
(2) the DNA diluent of preparation TC1507 strain; Namely adopt conventional DNA extraction means, extracting concentration from transgenic corns TC1507 is the DNA diluent of 50ng/ μ l.
(3) preparation PCR reaction system; Soon get final product 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 b absolute content (ng) | Strain specific fragment Ct value | Strain specific fragment absolute content (ng) | Strain specific fragment relative content (%) |
| 1 | 24.78 | 146.17 | 11.50 | 13.31 | 0.09 |
| 2 | 24.98 | 128.77 | 11.83 | 10.59 | 0.08 |
| 3 | 25.06 | 122.55 | 11.95 | 9.78 | 0.08 |
| 4 | 25.03 | 124.67 | 11.96 | 9.69 | 0.08 |
| 5 | 25.02 | 125.76 | 11.58 | 12.55 | 0.10 |
| 6 | 24.97 | 129.62 | 11.67 | 11.79 | 0.09 |
| 7 | 24.99 | 128.34 | 11.39 | 14.38 | 0.11 |
| 8 | 24.75 | 149.70 | 11.37 | 14.53 | 0.10 |
| 9 | 24.88 | 137.87 | 11.79 | 10.87 | 0.08 |
| 10 | 25.06 | 122.27 | 11.89 | 10.18 | 0.08 |
| 11 | 25.12 | 117.58 | 11.87 | 10.35 | 0.09 |
| 12 | 25.22 | 110.58 | 12.00 | 9.45 | 0.09 |
| 13 | 25.14 | 116.48 | 11.89 | 10.19 | 0.09 |
| 14 | 25.19 | 112.51 | 11.85 | 10.47 | 0.09 |
| 15 | 24.98 | 129.14 | 11.87 | 10.31 | 0.08 |
| 16 | 24.92 | 134.25 | 11.49 | 13.37 | 0.10 |
| Mean value | 25.01 （0.13） | 127.27 (10.95) | 11.74 (0.21) | 11.36 (1.71) | 0.09 (0.01) |
Adopt the present invention can precisely detect TC1507 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, and amplification efficiency is 96.475%, in 90%～110% scope.The detection by quantitative result of sample to be checked (9.0%) is very near actual value (9.2%), 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 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 TC1507 strain
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial
<220>
＜223〉upstream primer (TC1507event-F)
<400> 1
acaaaagcct ccaagcgagt a 21
<210> 2
<211> 20
<212> DNA
<213> Artificial
<220>
＜223〉downstream primer (TC1507event-R)
<400> 2
atgggggtta ccagctgaga 20
<210> 3
<211> 27
<212> DNA
<213> Artificial
<220>
＜223〉fluorescent probe (TC1507event-P)
<400> 3
ccctaattat ggtccccgac agtagcc 27
Claims (4)
1. the accurate method that detects of the special quantitative PCR of transgenic corns TC1507 strain is characterized in that, may further comprise the steps:
(1) synthetic following primer reaches the fluorescent probe that is used with primer,
The upstream primer sequence, TC1507event-F:5'-ACAAAAGCCTCCAAGCGAGTA-3'
The downstream primer sequence, TC1507event-R:5'-ATGGGGGTTACCAGCTGAGA-3'
The fluorescent probe sequence, TC1507event-P:5'-FAM-CCCTAATTATGGTCCCCGACAGTAGCC-TAMARA-3';
(2) the DNA diluent of preparation TC1507 strain;
(3) preparation PCR reaction system;
(4) quantitative PCR detection.
2. the method that precisely detects of the special quantitative PCR of transgenic corns TC1507 strain according to claim 1, it is characterized in that, the concentration of the primer described in the step (1) and fluorescent probe is 10 μ mol/l, and the concentration of the DNA diluent described in the step (2) is 50ng/ μ l.
3. the method that precisely detects of the special quantitative PCR of transgenic corns TC1507 strain according to claim 2, it is characterized in that, PCR reaction system described in the step (3), the DNA diluent that is about to 3 μ l joins in the reaction system of 25 μ l, and the reaction system of described 25 μ l is composed of the following components:
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 the accurate method that detects of the special quantitative PCR of claim 1 or 3 described transgenic corns TC1507 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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| CA2524493C (en) * | 2003-05-02 | 2013-06-25 | Dow Agrosciences Llc | Corn event tc1507 and methods for detection thereof |
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