CN102134603A - Primer, probe, test kit and method for testing genetically modified rice or products thereof - Google Patents
Primer, probe, test kit and method for testing genetically modified rice or products thereof Download PDFInfo
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Abstract
The invention relates to an oligonucleotides primer and probe for testing genetically modified rice containing an ubiquitin promoter or products thereof. The invention relates to a real-time fluorescent PCR (Polymerase Chain Reaction) testing method of the genetically modified rice containing the ubiquitin promoter or products thereof, in which the specific oligonucleotides primer and the probe are used. The invention also relates to a test kit for testing the genetically modified rice containing the ubiquitin promoter or products thereof, which comprises the specific oligonucleotides primer and the probe. The invention also relates to the application of the specific oligonucleotides primer and the probe or the test kit to testing the genetically modified rice containing the ubiquitin promoter or products thereof. By using the specific oligonucleotides prime, the probe, the test kit and the PCR testing method, the genetically modified rice containing the ubiquitin promoter or products thereof can be tested simply, quickly, specifically and sensitively.
Description
Technical field
The invention belongs to biological technical field, particularly, the present invention relates to be used to contain the transgenic paddy rice of corn ubiquitin promoter or Oligonucleolide primers that its goods detect and probe, test kit, be used to measure the transgenic paddy rice that contains the corn ubiquitin promoter or the real-time fluorescence PCR detection method of its goods, and specific oligonucleotide primer of the present invention and probe or test kit contain the transgenic paddy rice of corn ubiquitin promoter or the application in its goods in detection.
Background technology
Genetically modified organism is meant and utilizes biotechnology, foreign gene transferred in his species transforming its hereditary property, thereby obtained the proterties, nutritional quality of necessary for human and the biological new variety that produce.With genetically modified organism or with its food that is raw material processing comes is exactly genetically modified food.From 1994 first routine genetically modified food (transgenic Fructus Lycopersici esculenti) be born so far in the U.S., genetically modified organism has been widely used in field of food.
The Status of development of genetically modified organism
The research and development of international and domestic genetically modified organism, produce flourishly, cultivate the developing direction that new crop varieties is present crop breeding, improving crop yield, improve quality, improving aspects such as resistance and have great potential by the genetically engineered mode.Paddy rice (Oryza sativa L.) is the food crop of China's maximum, and sown area is about 3,100 ten thousand hectares throughout the year, and total yield accounts for 40% of China's total output of grain about 200,000,000 tons.Rice Production is occupied critical role in the agriculture production of China.
1991, usefulness particle gun transformation technologies such as Christou obtained transgenic rice plant, and coming years, gene gun technology are ripe day by day, and the report that all kinds of goal gene is imported paddy rice acquisition transfer-gen plant occurs in a large number.1994, Hiei etc. have set up agriculture bacillus mediated efficient japonica rice genetic conversion system, the agriculture bacillus mediated transformation system of java rice and long-grained nonglutinous rice is also successively set up, progressively reach the practical stage, become the mainstream technology of rice conversion, successively with the antiweed that do not have in the paddy gene storehouse, pest-resistant, disease-resistant, antiviral, salt tolerant, improve genes such as rice quality and introduce in the commercial rice varieties.China has also obtained important achievement in transgenic paddy rice cultivation aspect, import in multiple hybrid rice restorer line, the maintenance line with the cowpea trypsin inhibitor gene modified, Bt toxoprotein gene, Snowdrop lectin gene, antibacterial peptide gene with from the anti insect genes such as Xa21 gene of wild-rice, through the approval of the national Ministry of Agriculture, more than ten kind of transgenic paddy rice strain carried out environment release, interim test, wherein has 2 pest-resistant transgenic rices to obtain the genetically modified organism safety certificate.At present China is carrying out that environment discharges, the transgenosis of a plurality of pest-resistant transgenic rices of interim test contains the corn ubiquitin promoter in making up, so adopt ubiquitin promoter structure specificity method can specially detect pest-resistant transgenic rice delicately.
The management expectancy of genetically modified food
Genetically modified food is used widely, but transgenosis as a kind of new biotechnology, it is to the influence of human health, the eubiosis, the security of potential security genetically modified food potential more and more becomes human consumer's focus.
United Nations had passed through " Biosafety Protocol " in 2000, this protocol to except medicine all pass in and out active genetically modified organism about pass by, examine, detection, risk assessment and management, liability for damage etc. have made detailed regulation.Major country's legislation manages transgenic product in the world, the U.S., Canada, European Union, Japan, Korea S, Australia, Zelanian law are clearly stipulated, genetically modified organism needs with approval of authority, and through the Biosafety of strictness, environmental safety test just can field planting, environment discharges and as food, feed, European Union, Japan, Korea S, Australia, New Zealand etc. require genetically modified food to identify, and have stipulated the respective threshold level.China is in promulgation on May 23 calendar year 2001 " agriculture genetically modified organism security control regulations ", and regulation is carried out inspection and quarantine to agriculture genetically modified organism." the agriculture genetically modified organism identity management way " that came into effect on March 20th, 2002 also stipulated the sign system of genetically modified food.For the rules measure of carrying out above-mentioned United Nations and various countries, the detection of transgenic product is one of key measure, need determine genetically modified kind by qualitative detection, differentiate its whether be approved or got permission to be used for food and feed, any diffusion with the unknown transgenic product that prevents to have risk produces harm to society; Also need to determine by detection by quantitative the content of transgenic product, the clear and definite threshold level that whether has reached the country one belongs to's regulation is because the threshold level of each national transgenosis sign is all inequality.
Detection Method for Transgenic Food
Genetically modified food detects and is broadly divided into two classes, one class is a detection of nucleic acids, when foreign gene is inserted into recipient cell karyomit(e), generally to make up promotor, terminator, selectable marker gene and reporter gene, as cauliflower mosaic virus (CaMV) 35s promotor, nopaline synthase NOS terminator etc., the target of genetically modified food detection of nucleic acids is the foreign gene that inserts, the integration site that comprises foreign gene, promotor, terminator, the nucleotide sequence of selectable marker gene and reporter gene, present transgene component detect to have in the database and surpass 400 pairs of PCR detection primers and and the endogenous reference gene of kind more than 40.Another kind of is Protein Detection, promptly detects by protein or its function of inserting exogenous gene expression, and existing about 20 kinds of transgene protein detection methods have dropped into use in the transgenosis detection range.
Detection of nucleic acids is mainly used PCR method and biochip technology.PCR method has very high sensitivity, uses the most extensive in the transgenosis field.Have tissue specificity with protein and compare, round pcr is not subjected to the restriction of material.In addition, nucleic acid is than protein stabilization, and easy renaturation still can detect in processed food after the sex change.The key of PCR method is a design of primers, the general long 17-30nt that is of primer, the pairing of strict restriction upstream and downstream primer and primer self pairing.
Transgene protein detects euzymelinked immunosorbent assay (ELISA) (ELISA), immunochromatography (test strip method) and Western blotting etc.Although use the transgene protein detection method that some advantages are arranged, but many materials that are present in the food substrate, as tensio-active agent (Saponin/TSM), phenolic compound, lipid acid, endogenous phosphoric acid (ester) enzyme, all can suppress the special interaction of antigen one antibody, in addition, 3 grades or 4 level structures that immunodetection needs transgene protein to be kept perfectly, but the albumen of exogenous gene expression can be degraded in the course of processing, the detectivity of immunization method descends, thereby only is fit to unprocessed raw-material detection.In addition, some exogenous protein expression is specificity in a organized way, and for example some BT albumen major parts are expressed in the leaf in transgenic corns, and not in grain.These drawbacks limit the application of transgene protein detection method.
Genetically modified sign demand and some rules require transgene component in the food is carried out detection by quantitative to the restriction of gm content.
At present, rare both at home and abroad report can be quick, simple, special and be detected the method for the transgenic paddy rice and the goods thereof that contain the corn ubiquitin promoter delicately.
Therefore, the transgenic paddy rice that contains the corn ubiquitin promoter or its goods detection method that this area needs are a kind of fast, specificity is good, highly sensitive contain the detection of transgenic paddy rice or its goods of corn ubiquitin promoter.
Summary of the invention
One object of the present invention is, is provided for rapid detection and contains the transgenic paddy rice of corn ubiquitin promoter or the specific oligonucleotide primer and the probe of its goods.
Another object of the present invention is, provides rapid determination to contain the transgenic paddy rice of corn ubiquitin promoter or the real-time fluorescence PCR detection method of its goods.
A further object of the present invention is, is provided for rapid detection and contains the transgenic paddy rice of corn ubiquitin promoter or the test kit of its goods.
A further object of the present invention is, provides specific oligonucleotide primer of the present invention and probe or test kit to contain the transgenic paddy rice of corn ubiquitin promoter or the application in its goods in detection.
At the foregoing invention purpose, the invention provides following technical scheme:
According to one embodiment of the invention, the invention provides and be used for real time fluorescent PCR method and detect and to contain the transgenic paddy rice of corn ubiquitin promoter or the specific oligonucleotide primer and the probe of its goods.Oligonucleolide primers of the present invention is to designing according to corn ubiquitin promoter gene order with probe.In one embodiment, described primer is to being made up of upstream primer and downstream primer, described upstream primer is Ubiquitin-F2:TAGCCCTGCCTTCATACGCTA (SEQ ID No.1), and described downstream primer is Ubiquitin-R2:TGATCCTCTAGAGTCGACCTGC (SEQ ID No.2); Described probe is that Ubiquitin-P2:TGTCGATGCTCACCCTGTTGTTTGGTGT (SEQ IDNo.3) is connected with a fluorescent quenching group B HQ1 at 3 ' end of probe, and 5 ' end is connected with a fluorescence report group FAM.
According to another embodiment of the invention, the invention provides the transgenic paddy rice that contains the corn ubiquitin promoter or the real-time fluorescence PCR detection method of its goods, described method comprise use at the specific oligonucleotide primer of corn ubiquitin promoter gene order to and probe.In one embodiment, in the real-time fluorescence PCR detection method of the transgenic paddy rice that contains the corn ubiquitin promoter of the present invention or its goods, employed specific oligonucleotide primer is to being made up of upstream primer and downstream primer, the base sequence of described upstream primer is SEQ ID No.1, and the base sequence of described downstream primer is SEQ ID No.2; The base sequence of employed probe is SEQ ID No.3, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.
In one embodiment, of the present inventionly contain the transgenic paddy rice of corn ubiquitin promoter or the real-time fluorescence PCR detection method of its goods comprises the steps:
(a) from product to be measured, extract the DNA sample;
(b) provide the condition of nucleic acid amplification reaction;
(c) use specific oligonucleotide primer of the present invention to carry out nucleic acid amplification reaction and detect amplified production by real time fluorescent PCR method to reaching probe.
According to another embodiment of the invention, the invention provides and be used to detect the transgenic paddy rice that contains the corn ubiquitin promoter or the test kit of its goods, described test kit comprise specific oligonucleotide primer of the present invention to and probe.In the preferred embodiment of test kit of the present invention, the specific oligonucleotide primer that comprises in the described test kit is to being made up of upstream primer and downstream primer, the base sequence of described upstream primer is SEQ ID No.1, and the base sequence of described downstream primer is SEQ ID No.2; The base sequence of the probe that comprises in the described test kit is SEQ ID No.3, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.In preferred embodiments, described test kit also comprises reagent that is used for the sample DNA extraction and reagent and the working instructions that are used for the PCR reaction.In a preferred embodiment, the working instructions in the described test kit comprise being used for the description that rapid detection contains the pcr amplification condition of the transgenic paddy rice of corn ubiquitin promoter or its goods.In a preferred embodiment, the pcr amplification condition that provides in the specification sheets of described test kit is: 95 ℃, and 10min; 95 ℃ of 15s; 60 ℃, 1min, 45 circulations.
According to another embodiment of the present invention, the invention provides specific oligonucleotide primer of the present invention to containing the transgenic paddy rice of corn ubiquitin promoter or the application in its goods in detection with probe.In the preferred embodiment according to application of the present invention, described specific oligonucleotide primer is to being made up of upstream primer and downstream primer, and the base sequence of described upstream primer is SEQ ID No.1, and the base sequence of described downstream primer is SEQID No.2; The base sequence of described probe is SEQ ID No.3, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.In another embodiment, the present invention also provides test kit of the present invention to contain the transgenic paddy rice of corn ubiquitin promoter or the application in its goods in detection.Preferably, in above-mentioned application of the present invention, described test kit comprise specific oligonucleotide primer of the present invention to and probe.
The present invention detects the basis with the DNA of transgenic paddy rice or its goods, according to corn ubiquitin promoter gene order design primer and probe, utilizes the real-time fluorescence PCR method to detect transgenic paddy rice or its goods that contain the corn ubiquitin promoter.The about 2kb of corn ubiquitin protein promotor total length (containing first intron), transcriptional capability is strong, expression is stable because of starting, in recent years in monocotyledonous transgenic researches such as paddy rice, corn, wheat, obtain to be extensive use of, in the present invention, 3 ' zone at corn ubiquitin protein promotor, 16 primer/probe combinations have been designed, through the screening of specificity test, sensitivity test, obtained high specificity, sensitivity reach relative content 100,000/, can detect primer/probe combinations that all contain corn ubiquitin protein promotor.
In the methods of the invention, the product that is detected is transgenic paddy rice and the goods thereof that contain the corn ubiquitin promoter.
Real-time fluorescence quantitative PCR is to set up on the basis of conventional PCR method.In this PCR system, except two common primers, also have one 5 ' and 3 ' end respectively mark report fluorescence dye group (R), quench fluorescence dye groups (Q) and with the oligonucleotide probe of PCR product specific combination.When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group; During pcr amplification, 5 ' 5 prime excision enzyme activity of Tag enzyme is cut degraded with the probe enzyme, the report fluorophor is separated with quenching group, thereby the fluorescence monitoring system can receive fluorescent signal, it is DNA chain of every amplification, just have fluorescence molecule to form, it is synchronous fully that the accumulation that has realized fluorescent signal and PCR product form.By the initial copy number of analysis pcr template, and, judge the transgenosis content in the product to be checked with standard substance preparation standard curve.
Real-time fluorescence PCR detection method of the present invention is owing to use fluorescence dye to show the dynamic accumulation of PCR product in real time, and the stopped pipe operation does not have the PCR last handling process in whole testing process, has solved PCR after stain problem effectively.Use real-time fluorescence PCR detection method of the present invention, can detect the transgenic paddy rice or its goods that contain the corn ubiquitin promoter simple, quick, special and delicately.
Description of drawings
Fig. 1 shows rice conversion event-specific real-time fluorescence PCR detected result, wherein use the specific oligonucleotide primer that SEQ ID No.1 and SEQ ID No.2 and probe SEQ ID No.3 are detected, the fluorescence curve numbering is corresponding with sample as follows: 1: rich No. 6 of section; 2: rich No. 8 of section; 3: Kemingdao (KMD1); 4: change the Cry2A paddy rice; 5: change the Cry1C paddy rice; 6: change the Cry1Ah paddy rice; 7: anti-excellent 97; 8: crust Annexation rice; 9:K105; 10: bright extensive 63; 11: training assorted 35; 12: Tianjin rice 9618; 13: Anhui rice 181; 14: the spring excellent 59; 15: blank, each sample carry out repetition in triplicate.The above fluorescence curve of baseline is rich No. 6 of section, rich No. 8 of section, Kemingdao (KMD1), changes the Cry2A paddy rice, changes the Cry1C paddy rice, changes Cry1Ah paddy rice sample, and baseline position is assorted 35 for anti-excellent 97, crust Annexation rice, K105, bright extensive 63, training, Tianjin rice 9618, Anhui rice 181, spring excellent 59 and blank.These rice varieties are named by national agricultural sector, for well known in the art.
Fig. 2 carries out the sensitivity evaluation at event-specific primer probe.With the relative mass mark of 100ng is the rich No. 64 times of dilutions of 5 gradients of genomic dna of 5% (W/W) transgenic paddy rice section, the result shows, when the transgenic paddy rice section of containing the corn ubiquitin promoter can obtain reliable result at relative content greater than 0.001% the time rich No. 6.
Embodiment
The present invention is further illustrated for mode by embodiment, but the present invention is not limited only to following examples.
Present embodiment be the application of the invention primer to probe test transformation event specificity.
By detecting corn ubiquitin promoter sequence, test transformation event specificity.
Use primer of the present invention to the combination of probe, under the few situation of sample size, with respect to other primers to still amplifying the purpose fragment special, delicately.
In the present embodiment employed be used to detect the specific primer of transformation event to and probe sequence be:
Ubiquitin-F2：TAGCCCTGCCTTCATACGCTA(SEQ?ID?No.1)
Ubiquitin-R2：TGATCCTCTAGAGTCGACCTGC?(SEQ?ID?No.2)
Ubiquitin-P2:TGTCGATGCTCACCCTGTTGTTTGGTGT (SEQ ID No.3) is connected with a fluorescent quenching group B HQ1 at 3 ' end of probe, and 5 ' end is connected with a fluorescence report group FAM.
In the present embodiment, detected 14 increments this: rich No. 6 of section, rich No. 8 of section, Kemingdao (KMD1), change the Cry2A paddy rice, change the Cry1C paddy rice, change the Cry1Ah paddy rice, anti-excellent 97, crust Annexation rice, K105, bright extensive 63, training are assorted 35, Tianjin rice 9618, Anhui rice 181, spring excellent 59.
Employed detection key instrument:
Micropipet (10 μ L, 100 μ L, 1000 μ L Eppendorf), quantitative real time PCR Instrument, high speed tabletop centrifuge (Pico17 Thermo), high speed disintegrator (IKA-WEARKE GERMANY), nucleic acid-protein analyser (DYY-6C Liuyi Instruments Plant, Beijing) etc.
Detect main agents:
Taq enzyme, dNTPs, 10 * PCR Buffer, ethidium bromide, DNA Ladder Marker (Takara); TaqMan Universal PCR Master Mix (ABI); Primer and probe (by sequence generate a reagent box), pipettor Tips: must use the model of band filter core, otherwise when mixing and branch sample, be very easy to pollute; The pipettor of 10uL and 2.5uL must use long Tips simultaneously, and common short Tips is when work, and pipettor bar portion may contact with the centrifuge tube inwall, pollutes.
Detect key step:
1 DNA extraction
Testing sample is: rich No. 6 of section, rich No. 8 of section, Kemingdao (KMD1), change the Cry2A paddy rice, change the Cry1C paddy rice, change the Cry1Ah paddy rice, anti-excellent 97, crust Annexation rice, K105, bright extensive 63, training are assorted 35, Tianjin rice 9618, Anhui rice 181, spring excellent 59.
Take by weighing the 50.0mg sample powder, add 1.0ml CTAB and extract damping fluid and 4.0 μ L Proteinase Ks (10mg/ml), 65 ℃ of temperature are bathed hatching 1h; The centrifugal 15min of 12000rpm gets and is close to limpid supernatant 700 μ l; Add 500 μ L chloroforms, high speed vortex mixed 30 seconds; The centrifugal 10min of 12000rpm collects 500 μ L supernatants, transfers in the new 1.5ml reaction tubes; Add the CTAB precipitation buffering liquid of two volumes, room temperature is hatched 1h; 12000rpm is centrifugal, and 5min abandons supernatant, precipitation is dissolved in the sodium chloride solution of 1.2mol/L of 350 μ L, fully dissolving; Add 350 μ L trichloromethanes, careful high speed vortex mixed 30 seconds; With the centrifugal 10min of 12000rpm, supernatant is transferred in the new reaction tubes; The Virahol that adds 0.8 times, incubated at room is 20min at least; The centrifugal 10min of 12000rpm abandons supernatant; Add 500 μ L, 70% ethanol in precipitation, high speed vortex 30 seconds is with the centrifugal 10min of 12000rpm; Abandon supernatant, 60 ℃ of dry 15-25min, and be dissolved in 50 μ LTE (pH 8.0) ,-20 ℃ of preservations are standby.Corresponding blank (replacing sample with distilled water) is all set up in each extraction.
2 real-time fluorescence PCRs detect used transformation event Auele Specific Primer and probe
Primer sequence is SEQ ID No.1 and SEQ ID No.2;
Probe sequence is SEQ ID No.3, and 3 ' end is connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.
3 real-time fluorescence PCR reaction systems:
TaqMan?Universal?PCR?Master?Mix?12.5μL
Probe (10 μ M) 0.5 μ L
Upstream primer (10 μ M) 0.5 μ L
Downstream primer (10 μ M) 0.5 μ L
Add ddH
2O to cumulative volume be 25 μ L
Annotate: each PCR detects and all sets up corresponding blank (ultrapure water with the preparation reaction system replaces dna profiling, and whether detection reagent is polluted);
4 real-time fluorescence PCR reaction parameters:
95℃?10min
95℃?15s
60℃?1min
45 circulations.
Annotate: different instruments should be done suitable adjustment with each reagent of PCR and reaction parameter.
As shown in Figure 1, use the DNA of primer amplification testing sample, test transformation event specificity, rich No. 6 of section, rich No. 8 of section, Kemingdao (KMD1), change the Cry2A paddy rice, change the Cry1C paddy rice, change the Cry1Ah paddy rice amplified fluorescence curve can occur more than baseline, anti-excellent 97, crust Annexation rice, K105, bright extensive 63, training are assorted 35, the fluorescence curve of Tianjin rice 9618, Anhui rice 181, spring excellent 59 and blank is at baseline position.
Above result shows that rich No. 6 of section, rich No. 8 of section, Kemingdao (KMD1), commentaries on classics Cry2A paddy rice, commentaries on classics Cry1C paddy rice, commentaries on classics Cry1Ah paddy rice sample are the transgenic paddy rice that contains the corn ubiquitin promoter.This shows that primer that the present invention is designed and probe can detect transgenic paddy rice or its goods that contain ubiquitin promoter specifically.
Present embodiment for by following test to primer of the present invention to carrying out sensitivity test with probe.
In the present embodiment the employed primer that is used to detect transformation event sensitivity to and probe sequence be:
Primer is SEQ ID No.1 and SEQ ID No.2;
Probe is SEQ ID No.3, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.
DNA extraction step and PCR reaction system are described with embodiment 1.
For determining the detectability of Auele Specific Primer and probe combinations, rich No. 6 is example with section, with the relative mass mark of 100ng is that 5 gradients of rich No. 6 DNA of 5% (W/W) transgenic paddy rice section are diluted in the sterilized water for 4 times, carry out the real-time fluorescence PCR amplification by the condition described in the embodiment 1 respectively, the result as shown in Figure 2.
The result shows, when rich No. 6 dna contents of the genetically modified section of containing the corn ubiquitin promoter can obtain detected result reliably greater than 0.001% the time.
Use primer of the present invention to the combination of probe, with respect to other primers for and probe, can amplify the purpose fragment special, delicately, thus high special, identify transgenic paddy rice or its goods that contain corn ubiquitin promoter gene with sensitivity.
Though specific embodiments of the present invention is described, those skilled in the art will appreciate that under the prerequisite that does not depart from scope of the present invention or spirit and can carry out multiple change and modification to the present invention.Thereby, this invention is intended to contain all these changes and modification of dropping in claims and the coordinator scope thereof.
Claims (7)
1. be used for specific oligonucleotide primer that real time fluorescent PCR method detects the transgenic paddy rice contain the corn ubiquitin promoter or its goods to and probe, wherein said primer is to being made up of upstream primer and downstream primer, the base sequence of described upstream primer is SEQ ID No.1, the base sequence of described downstream primer is SEQ ID No.2, the base sequence of described probe is SEQ ID No.3,3 ' end at probe is connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.
2. be used for real time fluorescent PCR method and detect and to contain the transgenic paddy rice of corn ubiquitin promoter or the test kit of its goods, it comprise the described Oligonucleolide primers of claim 1 to and probe.
3. test kit according to claim 2, it also comprises the reagent that is used to extract DNA, the reagent that is used for real-time fluorescence PCR, blank and working instructions.
4. contain the transgenic paddy rice of corn ubiquitin promoter or the real-time fluorescence PCR detection method of its goods, described method comprises uses the described Oligonucleolide primers of claim 1 to reaching probe or claim 2 or 3 described test kits.
5. real-time fluorescence PCR detection method according to claim 4, described method comprises the steps:
(a) from product to be measured, extract the DNA sample;
(b) provide the condition of nucleic acid amplification reaction;
(c) use the described Oligonucleolide primers of claim 1 to reaching the test kit of probe or claim 2 or 3, carry out nucleic acid amplification reaction and detect amplified production by real time fluorescent PCR method.
6. the described Oligonucleolide primers of claim 1 contains the transgenic paddy rice of corn ubiquitin promoter or the application in its goods to reaching probe in detection.
7. claim 2 or 3 described test kits contain the transgenic paddy rice of corn ubiquitin promoter or the application in its goods in detection.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103060458A (en) * | 2013-01-17 | 2013-04-24 | 中国检验检疫科学研究院 | Primer, probe, kit and method for detecting transgenic rice strain T1c-19 |
| CN103834646A (en) * | 2014-03-06 | 2014-06-04 | 中国农业科学院生物技术研究所 | PCR (Polymerase Chain Reaction) detection primer and qualitative detection method and kit for specificity of transgenic rice PA110-15 strain |
| CN105543238A (en) * | 2016-01-07 | 2016-05-04 | 中国检验检疫科学研究院 | Transgenic maize IE 034 exogenous insertion element 3'-end flanking sequence and detection method |
| CN106701909A (en) * | 2015-11-18 | 2017-05-24 | 中国检验检疫科学研究院 | Primer probe for detecting sweet potato-derived components as well as method and kit |
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2010
- 2010-12-21 CN CN 201010615237 patent/CN102134603B/en not_active Expired - Fee Related
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| ERTAO WANG ET AL.: "Control of rice grain-filling and yield by a gene with a potential signature of domestication", 《NATURE GENETICS》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060458A (en) * | 2013-01-17 | 2013-04-24 | 中国检验检疫科学研究院 | Primer, probe, kit and method for detecting transgenic rice strain T1c-19 |
| CN103834646A (en) * | 2014-03-06 | 2014-06-04 | 中国农业科学院生物技术研究所 | PCR (Polymerase Chain Reaction) detection primer and qualitative detection method and kit for specificity of transgenic rice PA110-15 strain |
| CN106701909A (en) * | 2015-11-18 | 2017-05-24 | 中国检验检疫科学研究院 | Primer probe for detecting sweet potato-derived components as well as method and kit |
| CN105543238A (en) * | 2016-01-07 | 2016-05-04 | 中国检验检疫科学研究院 | Transgenic maize IE 034 exogenous insertion element 3'-end flanking sequence and detection method |
| CN105543238B (en) * | 2016-01-07 | 2020-04-07 | 中国检验检疫科学研究院 | 3' end flanking sequence of exogenous insertion segment of transgenic maize IE034 and detection method |
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| Publication number | Publication date |
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| CN102134603B (en) | 2013-02-13 |
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