CN103849688A - Primer and probe for specific quantitative polymerase chain reaction (PCR) accurate detection of transgenic maize 98140 strain and method thereof - Google Patents
Primer and probe for specific quantitative polymerase chain reaction (PCR) accurate detection of transgenic maize 98140 strain and method thereof Download PDFInfo
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- CN103849688A CN103849688A CN201410092491.7A CN201410092491A CN103849688A CN 103849688 A CN103849688 A CN 103849688A CN 201410092491 A CN201410092491 A CN 201410092491A CN 103849688 A CN103849688 A CN 103849688A
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- 98140event
- primer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention belongs to the technical field of an organism, relates to a quantitative analysis method of a gene, and particularly relates to a method for specific quantitative polymerase chain reaction (PCR) accurate detection of a transgenic maize 98140 strain. A PCR reaction system is prepared from a specific upstream primer sequence 98140event-F, a downstream primer sequence 98140event-R, a fluorescence probe sequence 98140event-P, a deoxyribonucleic acid (DNA) diluent of a 98140 strain, and TaqmanMastermix (2x) and water, and quantitative PCR detection is carried out. A Taqman quantitative PCR detection technique with high amplification efficiency and high accuracy is mainly built. The primer and the probe are applicable to supervision and inspection of domestic agricultural genetically modified organisms and products, inspection of genetically modified organisms and products of import and export ports, and biological ingredients detection of a transgenic gene 98140 strain in the internal imported raw materials.
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 limitation mark and import to transgenic product in the world, and China there is no concrete transgenic product mark threshold value.The transgenic product Trade technique barrier arranging 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 better, sets up the accurate detection method of a kind of novel transgenic corns special quantitative PCR of 98140 strain and necessitates.
The current detection technique about transgenic corns 98140, 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 98140 and product.
Summary of the invention
Object of the present invention is mainly 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 98140 and product.
The present invention is achieved through the following technical solutions:
Primer and probe that the special quantitative PCR of transgenic corns 98140 strain precisely detects,
Wherein, upstream primer sequence, 98140event-F:5'-GCGTTTTTTTGTGTGTGTATGTCTCT-3';
Downstream primer sequence, 98140event-R:5'-CGTTTCCCGCCTTCAGTTTA-3';
Fluorescent probe sequence, 98140event-P:5'-FAM-TGCTTGGTCTTTCTCTATCGATCCCCCTC-TAMRA-3'.
The accurate detection method of the transgenic corns special quantitative PCR of 98140 strain, comprises the following steps:
(1) fluorescent probe that synthesizes following primer and be used in conjunction with primer,
Upstream primer sequence, 98140event-F:5'-GCGTTTTTTTGTGTGTGTATGTCTCT-3'
Downstream primer sequence, 98140event-R:5'-CGTTTCCCGCCTTCAGTTTA-3'
Fluorescent probe sequence, 98140event-P:5'-FAM-TGCTTGGTCTTTCTCTATCGATCCCCCTC-TAMRA-3';
(2) prepare the DNA diluent of 98140 strains;
(3) preparation PCR reaction system;
(4) quantitative PCR detection.
Further, the concentration of the described synthetic primer of step (1) and fluorescent probe is 10 μ mol/l, and the concentration of the DNA diluent of the described preparation of step (2) is 50ng/ μ l.
Again further, the preparation PCR reaction system that step (3) is described, the DNA diluent that is about to 3 μ l joins in 20 μ l reaction systems, and the reaction system of described 20 μ l comprises following component:
Taqman Master mix（2×） 10μl
Upstream primer 1 μ l
Downstream primer 1 μ l
Fluorescent probe 0.5 μ l
50×ROX 0.4μl
DNA diluent 3.0 μ l
Water 4.1 μ l.
In addition, described PCR reaction conditions is: 95 ℃ of denaturation 10min, 1 circulation; 95 ℃ of sex change 15s, 60 ℃ of annealing 60s, 45 circulations.
The present invention has the following advantages and beneficial effect:
(1) the present invention breaks the transgenic product Trade technique barrier that the countries and regions such as European Union arrange;
(2) the present invention makes up and improves 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
Below in conjunction with embodiment, the present invention is described further, but embodiments of the present invention are not limited to this.
Embodiment
The accurate detection method of the transgenic corns special quantitative PCR of 98140 strain, mainly comprises the following steps:
(1) fluorescent probe that synthesizes following primer and be used in conjunction with primer,
Upstream primer sequence, 98140event-F:5'-GCGTTTTTTTGTGTGTGTATGTCTCT-3'
Downstream primer sequence, 98140event-R:5'-CGTTTCCCGCCTTCAGTTTA-3'
Fluorescent probe sequence, 98140event-P:5'-FAM-TGCTTGGTCTTTCTCTATCGATCCCCCTC-TAMRA-3'.
The synthetic concentration of the 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 for transgenic corns 98140 and product, i.e. goal gene and the design of acceptor Maize genome side site; Just can precisely detect in transgenic corns 98140 transformation event by this design.
(2) prepare the DNA diluent of 98140 strains; Adopt conventional DNA extraction means, from transgenic corns 98140, extracting concentration is the DNA diluent of 50ng/ μ l.
(3) preparation PCR reaction system; The DNA diluent preparing by 3 μ l adds in 20 μ l reaction systems.
Above 20 μ l reaction systems comprise following component:
Taqman Master mix（2×） 10μl
Upstream primer 1 μ l
Downstream primer 1 μ l
Fluorescent probe 0.5 μ l
50×ROX 0.4μl
DNA diluent 3.0 μ l
Water 4.1 μ l.
(4) quantitative PCR detection.
According to above-mentioned PCR reaction system, under following PCR reaction conditions, product is increased and detected, described PCR reaction conditions is: 95 ℃ of denaturation 10min, 1 circulation; 95 ℃ of sex change 15s, 60 ℃ of annealing 60s, 45 circulations.What in the present embodiment, adopt is the 7500 type quantitative fluorescent PCR instruments that ABI company produces.
The method data of the present embodiment are repeated to do 15 parallel sample continuously, these 15 samples are detected, it detects data as table 1.
Table 1
| Experiment number | ZSS II bCt value | ZSS II b absolute content (ng) | Strain specific fragment Ct value | Strain specific fragment absolute content (ng) | Strain specific fragment relative content (%) |
| 1 | 22.904 | 148.204 | 31.536 | 0.919 | 0.620 |
| 2 | 22.912 | 147.532 | 31.297 | 0.88 | 0.596 |
| 3 | 22.742 | 165.364 | 31.554 | 1.076 | 0.651 |
| 4 | 22.886 | 150.085 | 31.672 | 0.839 | 0.559 |
| 5 | 22.778 | 158.422 | 31.352 | 0.864 | 0.545 |
| 6 | 22.884 | 161.754 | 31.550 | 0.857 | 0.530 |
| 7 | 22.910 | 159.872 | 31.471 | 0.866 | 0.542 |
| 8 | 22.792 | 155.524 | 31.422 | 0.891 | 0.573 |
| 9 | 22.863 | 160.231 | 31.557 | 0.89 | 0.555 |
| 10 | 22.880 | 164.287 | 31.601 | 0.907 | 0.552 |
| 11 | 22.903 | 156.033 | 31.495 | 0.918 | 0.588 |
| 12 | 22.877 | 158.594 | 31.689 | 0.947 | 0.597 |
| 13 | 22.895 | 157.998 | 31.411 | 0.965 | 0.611 |
| 14 | 22.908 | 160.371 | 31.327 | 0.902 | 0.562 |
| 15 | 22.798 | 162.669 | 31.516 | 1.004 | 0.577 |
| Mean value | 22.862 | 157.796 | 31.497 | 0.915 | 0.580 |
Adopt the present invention can precisely detect 98140 transformation events and content thereof in transgenic corns, obtain slope of standard curve, between-3.52～-3.20, relation conefficient is greater than 0.99, and amplification efficiency is 98.62%, in 90%～110% scope.The detection by quantitative result (0.58%) of sample to be checked approaches actual value (0.5%) very much, and the relative deviation of detected result is less than 25% of international endorsement, and the uncertainty of detected result is less than 10%.
In addition, contriver has collected transgenic corns, paddy rice, soybean, cotton and non-transgenic corn, paddy rice, soybean, cotton, rape totally 36 samples, detect with the present invention, result does not detect 98140 signal, proves that the present invention has higher specificity.
Can be learnt by above detected result, each index of present method all meets 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
Institute of Analysis of <110> Sichuan Academy of Agricultural Sciences
Primer and probe and method that the special quantitative PCR of <120> transgenic corns 98140 strain precisely detects
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> DNA
<213> Artificial
<220>
<223> upstream primer (98140event-F)
<400> 1
gcgttttttt gtgtgtgtat gtctct 26
<210> 2
<211> 20
<212> DNA
<213> Artificial
<220>
<223> downstream primer (98140event-R)
<400> 2
cgtttcccgc cttcagttta 20
<210> 3
<211> 29
<212> DNA
<213> Artificial
<220>
<223> fluorescent probe (98140event-P)
<400> 3
tgcttggtct ttctctatcg atccccctc 29
Claims (5)
1. the special quantitative PCR of transgenic corns 98140 strain precisely detects primer and probe, is characterized in that,
Upstream primer sequence, 98140event-F:5'-GCGTTTTTTTGTGTGTGTATGTCTCT-3';
Downstream primer sequence, 98140event-R:5'-CGTTTCCCGCCTTCAGTTTA-3';
Fluorescent probe sequence, 98140event-P:5'-FAM-TGCTTGGTCTTTCTCTATCGATCCCCCTC-TAMRA-3'.
2. the accurate detection method of the transgenic corns special quantitative PCR of 98140 strain, is characterized in that, comprises the following steps:
(1) fluorescent probe that synthesizes following primer and be used in conjunction with primer,
Upstream primer sequence, 98140event-F:5'-GCGTTTTTTTGTGTGTGTATGTCTCT-3'
Downstream primer sequence, 98140event-R:5'-CGTTTCCCGCCTTCAGTTTA-3'
Fluorescent probe sequence, 98140event-P:5'-FAM-TGCTTGGTCTTTCTCTATCGATCCCCCTC-TAMRA-3';
(2) prepare the DNA diluent of 98140 strains;
(3) preparation PCR reaction system;
(4) quantitative PCR detection.
3. the accurate detection method of the transgenic corns special quantitative PCR of 98140 strain according to claim 2, it is characterized in that, the concentration of the described synthetic primer of step (1) and fluorescent probe is 10 μ mol/l, and the concentration of the DNA diluent of the described preparation of step (2) is 50ng/ μ l.
4. the accurate detection method of the transgenic corns special quantitative PCR of 98140 strain according to claim 3, it is characterized in that, the preparation PCR reaction system that step (3) is described, the DNA diluent that is about to 3 μ l joins in 20 μ l reaction systems, and the reaction system of described 20 μ l comprises following component:
Taqman Master mix（2×） 10μl
Upstream primer 1 μ l
Downstream primer 1 μ l
Fluorescent probe 0.5 μ l
50×ROX 0.4μl
DNA diluent 3.0 μ l
Water 4.1 μ l.
5. according to the accurate detection method of the transgenic corns special quantitative PCR of 98140 strain described in claim 2 or 4, it is characterized in that, described PCR reaction conditions is: 95 ℃ of denaturation 10min, 1 circulation; 95 ℃ of sex change 15s, 60 ℃ of annealing 60s, 45 circulations.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101641443A (en) * | 2006-10-30 | 2010-02-03 | 先锋高级育种国际公司 | Maize event DP-098140-6 and compositions and methods for the identification and/or detection thereof |
| CN102634593A (en) * | 2012-01-16 | 2012-08-15 | 广州迪澳生物科技有限公司 | LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic maize EVENT98140 and derived varieties thereof |
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- 2014-03-13 CN CN201410092491.7A patent/CN103849688B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101641443A (en) * | 2006-10-30 | 2010-02-03 | 先锋高级育种国际公司 | Maize event DP-098140-6 and compositions and methods for the identification and/or detection thereof |
| CN102634593A (en) * | 2012-01-16 | 2012-08-15 | 广州迪澳生物科技有限公司 | LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic maize EVENT98140 and derived varieties thereof |
Non-Patent Citations (1)
| Title |
|---|
| C.SAVINI: "Event-specific Method for the Quantification of Maize 98140 Using Real-time PCR", 《EURL FOR GM FOOD&FEED》 * |
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Granted publication date: 20160106 Termination date: 20190313 |