CN102618658A - 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 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 method of a kind of novel special quantitative PCR of transgenic corns TC1507 strain and necessitate transgenic product.
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
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 transgenic corns TC1507 and product.
The present invention realizes through following technical proposals:
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 concentration of said synthetic primer of step (1) and fluorescent probe 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 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 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 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 to 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 through this design.
(2) the DNA diluent of preparation TC1507 strain; Promptly adopt conventional DNA extraction means, from transgenic corns TC1507, 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.
(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 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 |
| MV | 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 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 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 detection method of the special quantitative PCR of transgenic corns TC1507 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, 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 accurate detection method of the special quantitative PCR of transgenic corns TC1507 strain according to claim 1; It is characterized in that; The concentration of said synthetic primer of step (1) and fluorescent probe 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 accurate detection method of the special quantitative PCR of transgenic corns TC1507 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 accurate detection method of the 3 described special quantitative PCRs of transgenic corns TC1507 strain, 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182585A (en) * | 2018-10-19 | 2019-01-11 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting transgenic corns TC1507 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1485435A (en) * | 2002-09-24 | 2004-03-31 | 深圳市匹基生物工程股份有限公司 | Probe sequence for quantitatively detecting transgenic corn containing CrylA(b) gene using fluorescence PCR and reagent case |
| WO2004099447A2 (en) * | 2003-05-02 | 2004-11-18 | Dow Agrosciences Llc | Corn event tc1507 and methods for detection thereof |
| WO2011075648A1 (en) * | 2009-12-18 | 2011-06-23 | Dow Agrosciences Llc | Endpoint taqman methods for determining zygosity of corn comprising tc1507 events |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1485435A (en) * | 2002-09-24 | 2004-03-31 | 深圳市匹基生物工程股份有限公司 | Probe sequence for quantitatively detecting transgenic corn containing CrylA(b) gene using fluorescence PCR and reagent case |
| WO2004099447A2 (en) * | 2003-05-02 | 2004-11-18 | Dow Agrosciences Llc | Corn event tc1507 and methods for detection thereof |
| WO2011075648A1 (en) * | 2009-12-18 | 2011-06-23 | Dow Agrosciences Llc | Endpoint taqman methods for determining zygosity of corn comprising tc1507 events |
Non-Patent Citations (3)
| Title |
|---|
| REONA TAKABATAKE ET AL: "Evaluation of Quantitative PCR Methods for Genetically Modified Maize (MON863,NK603, TC1507 and T25)", 《FOOD SCI. TECHNOL. RES》, vol. 16, no. 5, 31 December 2010 (2010-12-31), pages 421 - 430 * |
| SEONG-HUN LEE ET AL: "Qualitative PCR Method for Detection of Genetically Modified Maize Lines NK603 and TC1507", 《AGRIC. CHEM. BIOTECHNOL》, vol. 47, no. 4, 31 December 2004 (2004-12-31), pages 185 - 188 * |
| YANAN MENG ET AL: "Applicability of Plasmid Calibrant pTC1507 in Quantification of TC1507 Maize: An Interlaboratory Study", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》, vol. 60, no. 1, 11 January 2012 (2012-01-11), pages 23 - 28 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182585A (en) * | 2018-10-19 | 2019-01-11 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting transgenic corns TC1507 |
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