CN102162012A - Qualitative PCR detection method for transgenic rice kefeng No. 6 - Google Patents
Qualitative PCR detection method for transgenic rice kefeng No. 6 Download PDFInfo
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Abstract
The invention provides a qualitative polymerase chain reaction (PCR) detection method for flanking sequences of transgenic rice kefeng No. 6. The method comprises a step of amplifying deoxyribonucleic acid (DNA) of a sample to be tested by using specific primers, namely KF6-2-F and KF6-2-R, wherein the sample to be tested is the transgenic rice kefeng No. 6 if a target fragment is obtained through amplification; and the sample to be tested is not the transgenic rice kefeng No. 6 if no target fragment is obtained through amplification. The invention provides two other copy flanking sequences of the kefeng No. 6 and an event-specific detection method based on the two copy flanking sequences; and important information is provided for molecular characteristics of the transgenic rice kefeng No. 6.
Description
(1) technical field
The present invention relates to rich No. 6 qualitative PCR detection method of transgenic paddy rice section.
(2) background technology
Paddy rice (Oryza sativa) is a kind of main food crop, also is that early successful applying transgene technique carries out the crop of genetic improvement.Cultivated at present the transgenic paddy rice kind that comprises various trait.China does not ratify the commercialization production of transgenic paddy rice at present as yet, discharges and production experiment but there are some kinds (strain) to get permission environment.Effective supervision to genetically modified crops is prerequisite and the guarantee of safe utilization genetically modified crops.And the transgenic plant external source to insert segmental flanking sequence be one of most important characterization of molecules of transgenic plant strain, therefore, inserting segmental flanking sequence according to external source is the important technology data of setting up transgenic plant strain specificity detection method.
Since Zimmermann in 2000 etc. utilized the inverse PCR technology to decode the flanking sequence of transgenic corns Bt11, set up the strain specificity detection technique system of Bt11, existing at present a plurality of crop transgenosis transformant had all been set up corresponding detecting method.On analysis foundation to existing patent and document, find existing patent (patent No. 200710063780.4) of inserting segmental flanking sequence about rich No. 6 external source of transgenic paddy rice section, because foreign gene is that multiple copied inserts in rich No. 6 of section, only be the flanking sequence of 1 copy wherein and this patent relates to.
(3) summary of the invention
Insert for multiple copied for rich No. 6 at section, the invention provides the flanking sequence of the external source insertion sequence of two other copy, and the transformation event method for detecting specificity of two copies of this strain external source is provided on this basis.
The technical solution used in the present invention is:
Rich No. 6 flanking sequence of a kind of transgenic paddy rice section, described flanking sequence are carrier RB lateral order row.
Rich No. 6 flanking sequence of a kind of transgenic paddy rice section, described flanking sequence are carrier LB lateral order row.
Described RB side flanking sequence can obtain by the HiTail-PCR amplification.The used specificity nested primers of HiTail-PCR is according to the sequences Design of no terminator (accession number DQ005463 in the ncbi database).Wherein N1-1 is positioned at the 11-36 of NOS sequence, and sequence is TCAAACATTTGGCAATAAAGTTTCTT; N
1-2Be positioned at 29-54, sequence is AGTTTCTTAAGATTGAATCCTGTTGC; N
1-3Be positioned at 204-223, sequence is AGCGCGCAAACTAGGATAAA.
Described LB side flanking sequence can obtain by the HiTail-PCR amplification.The used specificity nested primers of HiTail-PCR is according to the sequences Design of hyg resistant gene (accession number AB303069.1 in the ncbi database).Hpt wherein
1-1Be positioned at 427-404, sequence is CGTACACAAATCGCCCGCAGAAGC; Hpt
1-2Be positioned at 398-375, sequence is CCGTCTGGACCGATGGCTGTGTAG; Hpt
1-3Be positioned at 358-338, sequence is GGAAACCGACGCCCCAGCACT.
The invention still further relates to the rich No. 6 transformation event specificity qualitative PCR detection methods of transgenic paddy rice section, described method is according to carrier RB lateral order row or carrier LB lateral order row.The design Auele Specific Primer, DNA increases to testing sample, if amplification obtains purpose fragment (the purpose clip size is determined by primer), then testing sample is rich No. 6 of transgenic paddy rice section; If amplification does not obtain the purpose fragment, then testing sample is not rich No. 6 of transgenic paddy rice section.Usually testing sample is single rice varieties, can directly reach a conclusion according to the method described above; If testing sample is a mixture, then can judge whether contain transgenic paddy rice section in the mixture rich No. 6 according to the method described above.
Concrete, described Auele Specific Primer is as follows:
KF6-1-F：5’-CGACAAAAGATCAGGATTTGGG-3’
KF6-1-R：5’-GTTTGGAGTTTAAGAGCGA-3’
Primer of above-mentioned primer centering for according in the carrier RB lateral order row 169～189 be designed to forward primer, another primer is designed to reverse primer according to 450～468.Utilize this primer to increase, its purpose fragment is 303bp.
Perhaps, described Auele Specific Primer is as follows:
KF6-2-F：5’-GATCCCGAAGGCCAACACAATAGG-3’，
KF6-2-R：5’-TCGTCCGAGGGCAAAGAAATAGAG-3’
Primer of above-mentioned primer centering for according in the carrier LB lateral order row 134～157 be designed to forward primer, another primer is designed to reverse primer according to 684～707.Utilize this primer to increase, its purpose fragment is 450bp.
Depend on external source and insert the rich No. 6 event-specific qualitative PCR detection method of transgenic paddy rice section of segmental flanking sequence, can detect wherein 1 according to RB side flanking sequence and be inserted in the rice chromosome position No. 6, also can detect another one and insert copy according to LB side flanking sequence.Above-mentioned two pairs of primers can be used for rich No. 6 qualitative PCR of transgenic paddy rice section separately and detect, and rich No. 6 qualitative PCR of transgenic paddy rice section that is used for simultaneously also capable of being combined detects and makes the result more accurate.Further, described method is carried out pcr amplification to testing sample DNA respectively with Auele Specific Primer KF6-1-F and KF6-1-R or KF6-2-F and KF6-2-R simultaneously, obtain the purpose fragment of 303bp and 450bp if same testing sample increases respectively, then testing sample is rich No. 6 of transgenic paddy rice section; If amplification does not obtain the purpose fragment, then testing sample is not rich No. 6 of transgenic paddy rice section.
The T-DNA domain structure of rich No. 6 used carriers of transgenic paddy rice section is seen Fig. 1, is made up of hpt resistant gene expression cassette, CrylAc expression casette and CpTI expression casette.The present invention adopts ordinary method to extract plant genome DNA, utilize the HiTAIL-PCR method, separate to obtain RB side flanking sequence 507bp, find that by analysis wherein the 3926-4092 of 1-167 and binary vector pUB-Hyg DNANCBI accession number AB303069.1 is identical.Filler DNA is 168～189 identical in the repairing sequence body RB lateral order row in the transgenosis process.Oryza sativa genomic dna is positioned at 95684～95962 of No. 6 karyomit(e) NCBI of paddy rice (accession number AP004749.1), mates fully with 190～507 of carrier RB lateral order row.Separate the LB side flanking sequence 607bp obtain, by analyze find its 1～283 with 8984～8701 couplings of paddy rice the 9th karyomit(e) (accession number AP009051.1).284～288 is Filler DNA.289-607 and binary vector pUB-HygDNANCBI accession number AB303069.1's is 41～358 identical.
Beneficial effect of the present invention is mainly reflected in: the invention provides the flanking sequence of rich No. 6 other two copies of section and be based upon these two the transformation event method for detecting specificity on the copy flanking sequence basis, for rich No. 6 characterization of molecules of that paddy rice section of transgenosis provides important data.
(4) description of drawings
Fig. 1 is the T-DNA domain structure figure of rich No. 6 used carriers of transgenic paddy rice section;
Fig. 2 is primer KF6-1-F and KF6-1-R amplification electrophorogram; Wherein, Y6, Y7, Y8, Y9, Y10 represent transgenic paddy rice strain section successively rich No. 6, rich No. 6 of the excellent section of II, extensive No. 1 of China, Bt63, Kemingdao; YS6, YS7, YS8, YS9, YS10 represent excellent No. 10 of excellent 63, bright extensive 63, the Xian of excellent bright 86, the Xian of bright extensive 86, the II of non-transgenic check variety respectively; BLK: extract blank; The contrast of water:PCR amplifing reagent; M:100bp DNAladder;
Fig. 3 is primer KF6-2-F and KF6-2-R amplification electrophorogram; Wherein, 1～10 represents genetically engineered soybean GTS40-3-2, genetically modified corn MON 863, BT11, BT176, MON810, genetically modified rape GT 73, transgene cotton MON531, extensive No. 1 of transgenic paddy rice China, Bt63, Kemingdao respectively; 11～20 are respectively bright extensive 86 (11,12) of non-transgenic paddy rice check variety, excellent bright 86 (13,14) of II, excellent 63 (15,16) of Xian, bright extensive 63 (17,18), Xian excellent No. 10 (19,20) repeats 2 times separately; 21: the extract blank; The contrast of 22:PCR amplifing reagent; 23: rich No. 6 of transgenic paddy rice section; M:100bp DNAladder.
Fig. 4 primer KF6-1-F and KF6-1-R are to the amplification electrophorogram of the biased sample of rich No. 6 different contents of section; Wherein, Y6: rich No. 6 of section; YS6: bright extensive 86; BLK: extract blank; The blank water contrast of water:PCR; 10%～0.05% shows rich No. 6 biased samples of the section that contains different content; M:100bp DNA ladder.
Fig. 5 primer KF6-2-F and KF6-2-R are to the amplification electrophorogram of the biased sample of rich No. 6 different contents of section; Wherein, 100%～0.01% shows rich No. 6 biased samples of the section that contains different content; Ck-is that non-transgenic is to throwing light on extensive 86; BLK: extract blank; Y7 is rich No. 6 of the rich No. 6 excellent sections of derived varieties II of section, and YS7 is II excellent bright 86.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the flanking sequence clone of rich No. 6 exogenous insertion vectors of transgenic paddy rice strain section
One. experiment material
1, vegetable material: rich No. 6 and acceptor check variety of transgenic paddy rice section is bright extensive 86, and the academy of agricultural sciences provides by Fujian;
2, reagent: Taq archaeal dna polymerase, 10 * PCR Buffer (100mM Tris-HCl pH8.3,500mMKCl, 15mM MgCl
2), dNTP is available from precious biotechnology Dalian company limited; 100bp ladder DNA Marker is available from the Beijing Quanshijin Biotechnology Co., Ltd.PCR product purification test kit (Cat.NO.SK1141) and PCR product cloning test kit (Cat.NO.SK2213) are given birth to worker's biotechnology company limited available from Shanghai.Primer is synthetic to be finished by Invitrogen company with cloning and sequencing.Other biochemical reagents are import packing or homemade analytical pure.
3, laboratory apparatus: pcr amplification instrument: PTC-200 (Bio-Rad production)
Biotype spectrophotometer 6131 (Eppendorf production)
Nucleic acid electrophoresis apparatus: DYY-III type nucleic acid electrophoresis apparatus (Liuyi Instruments Plant, Beijing)
Gel imaging instrument: Gel Doc XR (Bio-Rad production)
Other instruments comprise: whizzer, electronic balance, incubator, Bechtop, metal thermostatic bath etc.
Two. experimental technique and process
1, the extraction of oryza sativa genomic dna and detection
1.1.1, extract preparation (100mL): in 60mL water, add 6.93g glucose, 2g polyvinylpyrrolidone, 100mg DIECA, fully dissolving adds 1mol/L Tris-HCl (pH7.5) 10mL, 0.5mol/L EDTA (pH8.0) 1mL then, add water and be settled to 100mL, autoclaving.The beta-mercaptoethanol that adds 0.2% (V/V) (200 μ L) before using.
1.1.2, lysate preparation (100ml): in 60mL water, add 8.17g sodium-chlor, 2g cetyl trimethylammonium bromide (CTAB), 2g polyvinylpyrrolidone (K30) (PVP), 100mg DIECA, fully dissolving adds 1mol/L Tris-HCl (pH7.5) 10mL, 0.5mol/L EDTA (pH8.0) 0.4mL then, add water and be settled to 100mL, behind the autoclaving 4 ℃.The beta-mercaptoethanol that adds 0.2% (V/V) during use.
1.1.3, extracting method: water intaking rice blade or the about 200mg of rice powder clay into power in liquid nitrogen.Add 1mL and be chilled to 4 ℃ paddy DNA extract in advance, put upside down mixing after, leave standstill 5min on ice, the centrifugal 15min of 10000g abandons supernatant liquor under 4 ℃ of conditions.Add 600 μ L and be preheating to 65 ℃ lysate, abundant resuspended precipitation.Keep 60min at 65 ℃ of constant temperature.After adding A37 ℃ of insulation of 2 μ LRNase 30min, use isopyknic phenol: chloroform: primary isoamyl alcohol mixing solutions (25: 24: 1) and chloroform: twice of primary isoamyl alcohol (24: 1) solution difference extracting.Under the room temperature condition, the centrifugal 10min of 10000g.Supernatant adds 2/3 volume Virahol, 1/10 volume 3mol/L sodium acetate solution (pH 5.6).Under 4 ℃ of conditions, the centrifugal 15min of 10000g with 70% washing with alcohol precipitation once, pours out ethanol, dries precipitation.Add 50 μ L TE (pH8.0) dissolution precipitations.
1.1.4, the detection of DNA: gets 2.0 μ L DNA and detect its integrity, and with biotype spectrophotometric determination DNA concentration, and it is diluted to concentration with TE is 100ng/ μ L with 0.8% agarose gel electrophoresis.
2, the acquisition of flanking sequence
2.1 the amplification of flanking sequence: utilize HiTail-PCR to carry out with reference to methods such as Liu, first and second takes turns reaction volume 25uL, third round reaction volume 50uL.Reaction system is 1 * PCR buffer, 2.0mmol/L MgCl
2, 200umol/L dNTP, first round amplification: degenerated primer 1.0 μ mol/L such as LAD1-1, LAD1-2, LAD1-3, LAD1-4 or LAD1-5 in the 25 μ L reaction systems, nested special primer N
1-1Or Hpt
1-10.3 μ mol/L, 0.5U Taq enzyme, template DNA 1.0 μ L; The 2nd takes turns amplification: primer AC in the 25 μ L reaction systems
10.3 μ mol/L, N
1-2Or Hpt
1-20.3 μ mol/L, 0.625U Taq enzyme, first round amplified production is got 1.0 μ L as template after diluting 50 times; The 3rd takes turns amplification: primer AC in the 50 μ L reaction systems
10.3 μ mol/L, N
1-3Or Hpt
1-30.3 μ mol/L, 1.0U Taq enzyme, the 2nd gets 1.0 μ L as template after taking turns 10 times of amplified production dilutions.Amplified production is with 2.0% agarose gel electrophoresis and observe imaging.The primer sequence sees Table 1, and amplification program sees Table 2.
Table 1:HiTAIL-PCR the primer
Table 2:HiTAIL-PCR amplification program
2.2 the clone of flanking sequence and order-checking: the PCR product purification test kit (Cat.NO.SK1141) and the PCR product cloning test kit (Cat.NO.SK2213) that utilize Shanghai to give birth to worker's biotechnology company limited carry out PCR product purification and clone.To the pUCm-T carrier, give birth to the limited order-checking of worker's biotechnology by Shanghai.The blast software of sequence comparing analysis utilization NCBI website carries out.
3, result
3.1 according to 3 nested special primers of no terminator sequences Design, in the 2nd and 3 take turns, all only in rich No. 6 of transgenic paddy rice section, amplify a band when having only, it the 3rd taken turns amplified production and reclaim, clone and check order with degenerated primer LAD1-4.Sequential analysis finds that the T-DNA on position is No. 6 karyomit(e) in the rice genome, the T-DNA fracture position has more the sequence of 22bp at inboard the 4th the base place of RB sequence (tgacaggatatattggcgggtaaa) between T-DNA sequence and rice genome sequence.
3.2 according to 3 nested special primers of hpt gene design, in the 2nd and 3 take turns, all amplify a band when having only, it the 3rd taken turns amplified production and reclaim, clone and check order with degenerated primer LAD1-4.Sequential analysis finds that the T-DNA on position is No. 9 karyomit(e) in the rice genome, the T-DNA fracture position has more the sequence of 5bp at inboard the 14th the base place of LB sequence (tggcaggata tattgtggtg taaaca) between T-DNA sequence and rice genome sequence.
Embodiment 2: the strain specificity qualitative PCR that inserts flanking sequence based on rich No. 6 two external sources of transgenic paddy rice strain section detects
One, experiment material: rich No. 6 of transgenic paddy rice strain section, and rich No. 6 of the excellent section of derived varieties II, the academy of agricultural sciences provides by Fujian; Other transgenic paddy rices China extensive No. 1 and Bt63 by Hua Zhong Agriculture University provide, Kemingdao provides by Zhejiang University; The non-transgenic paddy rice to throw light on extensive 86 and II excellent bright 86 provide by the academy of agricultural sciences, Fujian, Xian excellent 63 is provided by Hua Zhong Agriculture University, Xian is provided by Zhejiang University for excellent No. 10.Test used enzyme and reagent and be equal to embodiment 1.
Two, experimentation and method:
1, the extraction of DNA and detection: with embodiment 1.
2, based on two different specific detection of inserting the site: according to two sequences measuring among the embodiment 1, at rice genome part and two pairs of PCR primers of carrier part design, primer sequence sees Table 4 respectively.
Table 4: rich No. 6 two different specific detection primers that insert the site of section
The PCR reaction system is: 1 * PCR buffer, 200umol/L dNTP, 0.2umol/L special primer, 0.025U/uLTaq enzyme.Response procedures is 95 ℃, 5min; 94 ℃ of 30s, 59 ℃ of 45s, 35 circulations of 72 ℃ of 45s; 72 ℃ are extended 3min.Utilize 2.0% agarose gel electrophoresis to carry out product analysis.
3, result
Primer KF6-1-F and KF6-1-R only obtain the 303bp amplified production in rich No. 6 of section rich No. 6 and the excellent section of derived varieties II, and all do not obtain amplification in other transgenic paddy rice strains and check variety, see Fig. 2.Primer KF6-2-F and KF6-2-R obtain the amplified band of 450bp in rich No. 6 of transgenic paddy rice section, and all do not obtain amplification (Fig. 3) in its contrast non-transgenic rice varieties and other transgenic plant (comprising soybean, corn).
Embodiment 3: the susceptibility that strain specificity detects detects
One, experiment material: transgenic paddy rice strain section rich No. 6 and contrast acceptor kind bright extensive 86.Test used enzyme and reagent and be equal to embodiment 1.
Two, experimentation and method:
1, the extraction of DNA and detection, after section's bright seed of extensive 86 of rich No. 6 and non-transgenic contrast acceptor worn into powder, prepare serial biased sample, the contained section of each sample is followed successively by 10%, 5.0%, 2.5%, 1.25%, 0.5%, 0.1%, 0.05% and 0.01% (W/W) rich No. 6, extracts the DNA method behind the mixing with embodiment 1.
2, detect based on two different susceptibilitys of inserting the site: the primer sees Table 4, and the PCR reaction system is 1 * PCR buffer, 200umol/L dNTP, 0.2umol/L special primer, 0.025U/uLTaq enzyme.Response procedures is 95 ℃, 5min; 94 ℃ of 30s, 59 ℃ of 45s, 35 circulations of 72 ℃ of 45s; 72 ℃ are extended 3min.Utilize 2.0% agarose gel electrophoresis to carry out product analysis.
3, result
Can in rich No. 6 content of transgenic paddy rice section are 0.1～0.05% biased sample, the increase target product (seeing Fig. 4,5) of expection size of primer KF6-1-F, KF6-1-R and KF6-2-F, KF6-2-R.
SEQUENCE?LISTING
＜110〉China Paddy Rice Inst
＜120〉rich No. 6 qualitative PCR detection method of transgenic paddy rice section
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<170> PatentIn?version?3.4
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<212> DNA
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＜223〉artificial sequence
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<213> Unknown
<220>
＜223〉artificial sequence
<400> 2
gtttggagtt?taagagcga 19
<210> 3
<211> 24
<212> DNA
<213> Unknown
<220>
＜223〉artificial sequence
<400> 3
gatcccgaag?gccaacacaa?tagg 24
<210> 4
<211> 24
<212> DNA
<213> Unknown
<220>
＜223〉artificial sequence
<400> 4
tcgtccgagg?gcaaagaaat?agag 24
<210> 5
<211> 33
<212> DNA
<213> Unknown
<220>
＜223〉artificial sequence
<220>
<221> misc_feature
<222> (25)..(25)
<223> n?is?a,?c,?g,?or?t
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<213> Unknown
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＜223〉artificial sequence
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＜223〉artificial sequence
<220>
<221> misc_feature
<222> (25)..(25)
<223> n?is?a,?c,?g,?or?t
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<221> misc_feature
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<213> Unknown
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＜223〉artificial sequence
<220>
<221> misc_feature
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<213> Unknown
<220>
＜223〉artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n?is?a,?c,?g,?or?t
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<213> Unknown
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<220>
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agtttcttaa?gattgaatcc?tgttgc 26
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<211> 20
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Claims (2)
1. rich No. 6 qualitative PCR detection method of transgenic paddy rice section, described method is with Auele Specific Primer KF6-2-F and KF6-2-R testing sample DNA to be increased, if amplification obtains the purpose fragment, then testing sample is rich No. 6 of transgenic paddy rice section; If amplification does not obtain the purpose fragment, then testing sample is not rich No. 6 of transgenic paddy rice section;
Described Auele Specific Primer is as follows:
KF6-2-F：5’-GATCCCGAAGGCCAACACAATAGG-3’，
KF6-2-R：5’-TCGTCCGAGGGCAAAGAAATAGAG-3’
The purpose fragment is 450bp.
2. the method for claim 1, it is characterized in that described method is respectively testing sample DNA to be carried out pcr amplification with Auele Specific Primer KF6-1-F and KF6-1-R or KF6-2-F and KF6-2-R, obtain the purpose fragment of 303bp and 450bp if same testing sample increases respectively, then testing sample is rich No. 6 of transgenic paddy rice section; If amplification does not obtain the purpose fragment, then testing sample is not rich No. 6 of transgenic paddy rice section;
Described Auele Specific Primer is as follows:
KF6-1-F：5’-CGACAAAAGATCAGGATTTGGG-3’
KF6-1-R：5’-GTTTGGAGTTTAAGAGCGA-3’
The purpose fragment is 303bp;
KF6-2-F：5’-GATCCCGAAGGCCAACACAATAGG-3’，
KF6-2-R：5’-TCGTCCGAGGGCAAAGAAATAGAG-3’
The purpose fragment is 450bp.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102344965A (en) * | 2011-10-20 | 2012-02-08 | 曹际娟 | Detection method for genetically modified wheat B73-6-1 |
| CN102864245A (en) * | 2012-10-16 | 2013-01-09 | 中国水稻研究所 | Specific PCR (polymerase chain reaction) molecular markers for detecting high-silicon content allele qHUS6 of rice variety Miyang 46 |
| CN103160533A (en) * | 2013-03-19 | 2013-06-19 | 中国检验检疫科学研究院 | Standard molecule for specifically detecting transgenic rice strain Kefeng No.6 and application thereof |
| CN103525936A (en) * | 2013-10-24 | 2014-01-22 | 中国农业科学院生物技术研究所 | Specific identification for transgenic rice kefeng No.6 strain via adoption of RPA (Recombinase Ploymerase Amplification) technology |
| CN105039526A (en) * | 2015-07-02 | 2015-11-11 | 吉林省农业科学院 | Method for identifying transgene paddy rice Jishengjing No.2 based on exogenous gene flanking sequences |
| CN107312819A (en) * | 2016-04-25 | 2017-11-03 | 中国检验检疫科学研究院 | Primed probe, kit and the method precisely quantitatively detected for the rich No. 6 strain-specific gene compositions of genetically modified rice section |
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| CN102344965A (en) * | 2011-10-20 | 2012-02-08 | 曹际娟 | Detection method for genetically modified wheat B73-6-1 |
| CN102344965B (en) * | 2011-10-20 | 2013-06-26 | 曹际娟 | Detection method for genetically modified wheat B73-6-1 |
| CN102864245A (en) * | 2012-10-16 | 2013-01-09 | 中国水稻研究所 | Specific PCR (polymerase chain reaction) molecular markers for detecting high-silicon content allele qHUS6 of rice variety Miyang 46 |
| CN102864245B (en) * | 2012-10-16 | 2014-11-26 | 中国水稻研究所 | Specific PCR (polymerase chain reaction) molecular markers for detecting high-silicon content allele qHUS6 of rice variety Miyang 46 |
| CN103160533A (en) * | 2013-03-19 | 2013-06-19 | 中国检验检疫科学研究院 | Standard molecule for specifically detecting transgenic rice strain Kefeng No.6 and application thereof |
| CN103160533B (en) * | 2013-03-19 | 2015-01-07 | 中国检验检疫科学研究院 | Standard molecule for specifically detecting transgenic rice strain Kefeng No.6 and application thereof |
| CN103525936A (en) * | 2013-10-24 | 2014-01-22 | 中国农业科学院生物技术研究所 | Specific identification for transgenic rice kefeng No.6 strain via adoption of RPA (Recombinase Ploymerase Amplification) technology |
| CN103525936B (en) * | 2013-10-24 | 2015-10-28 | 中国农业科学院生物技术研究所 | Application RPA technology is to the rich No. 6 strain specificities qualification of transgenic paddy rice section |
| CN105039526A (en) * | 2015-07-02 | 2015-11-11 | 吉林省农业科学院 | Method for identifying transgene paddy rice Jishengjing No.2 based on exogenous gene flanking sequences |
| CN105039526B (en) * | 2015-07-02 | 2018-04-10 | 吉林省农业科学院 | Method based on the lucky raw round-grained rice 2 of foreign gene flanking sequence identification transgenic paddy rice |
| CN107312819A (en) * | 2016-04-25 | 2017-11-03 | 中国检验检疫科学研究院 | Primed probe, kit and the method precisely quantitatively detected for the rich No. 6 strain-specific gene compositions of genetically modified rice section |
| CN107312819B (en) * | 2016-04-25 | 2021-03-02 | 中国检验检疫科学研究院 | Primer probe, kit and method for accurate quantitative detection of specific gene components of transgenic rice Kefeng No. 6 strain |
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