CN104031982A - Real-time fluorogenic quantitative PCR detection primers, detection method and kit for transgenic rice Kefeng No. 2 line specificity - Google Patents
Real-time fluorogenic quantitative PCR detection primers, detection method and kit for transgenic rice Kefeng No. 2 line specificity Download PDFInfo
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Abstract
The invention discloses real-time fluorogenic quantitative PCR detection primers, a detection method and a kit for a transgenic rice Kefeng No. 2 line specificity. Nucleotide sequences of the PCR detection primers are shown as SEQ ID No.2 and SEQ ID No.3. The nucleotide sequence of a probe is shown as SEQ ID No.4. The real-time fluorogenic quantitative PCR detection method for the transgenic rice Kefeng No. 2 line specificity is also disclosed. The method includes steps of: (1) extracting DNA of a rice sample to be detected; (2) establishing a PCR reaction system by adopting the extracted DNA as a template and by adopting the detection primers, and performing amplification; and (3) determining the detection result according to a collected fluorescence curve and a Ct value. Results of specificity experiments, sensitivity experiments, reliability experiments and detection limit experiments show that the detection method has characteristics of high specificity, high sensitivity and high reliability.
Description
Technical field
The PCR that the present invention relates to transgenic paddy rice strain specificity detects primer and detection kit, the real-time fluorescence quantitative PCR that relates in particular to rich No. 2 strain specificities of transgenic paddy rice section detects primer, detection method and detection kit, belongs to transgenic paddy rice event-specific detection field.
Background technology
Along with the extensive plantation of transgenic plant, the safety issue of genetically modified food also causes that people pay close attention to greatly, and existing more than 30 country implements transgenic product mark system in succession, comprises China.The enforcement of transgenic labeling system depends on the composition detection to transgenic plant and converted products thereof.Round pcr is the most widely used method in transgenic product that detects both at home and abroad at present.In the time that this technology of application is carried out genetically modified crops qualitative detection, according to its specificity, be divided into screening detection method, gene specific method, built specificity method and strain specificity method.Event-specific detection is that the joining region sequence by detecting insertion vector and Plant Genome realizes, due to each transgenic plant strain, all there is the joining region sequence of special exogenous insertion vector and Plant Genome, with first three methods comparison, strain specificity has higher specificity and accuracy, and the most applicable transgenic product of doing detects.
At present, existing partial monopoly and bibliographical information the event-specific detection method of transgenic plant.For example: the event-specific detection method of magnifying the people such as soldier and set up in 2006 transgenosis MON863 corn; The people such as Xie Jiajian have set up a series of event-specific detection methods such as rich No. 8 of transgenic paddy rice Kemingdao, Bt Shanyou 63, section rich No. 6 and section; The people such as Lu Changming have set up the event-specific detection method of a series of transgene rapes in 2007.But, up to now, lack the real-time fluorescence quantitative PCR detection method of the strain specificity of rich No. 2 of a kind of transgenic paddy rice section.
Summary of the invention
One of object of the present invention is to provide for detection of the strain specificity real-time fluorescence quantitative PCR of rich No. 2 of transgenic paddy rice section and detects primer and probe;
Two of object of the present invention is to set up the strain specificity real-time fluorescence quantitative PCR detection method of rich No. 2 of a kind of transgenic paddy rice section;
Three of object of the present invention is to provide the strain specificity real-time fluorescence quantitative PCR detection kit of rich No. 2 of a kind of transgenic paddy rice section.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
First the present invention provides a pair of strain specificity real-time fluorescence quantitative PCR for detection of rich No. 2 of transgenic paddy rice section to detect primer and probe, and the nucleotide sequence of described primer pair is respectively shown in SEQ ID No.2 and SEQ ID No.3; The nucleotides sequence of described probe is classified as shown in SEQ ID No.4.
The present invention is that SCK gene (SEQ ID No.5) obtains external source insertion gene 5 ' end paddy rice flanking sequence and 3 ' end paddy rice flanking sequence by Hi-TAIL PCR according to the goal gene inserting in rich No. 2 strains of transgenic paddy rice section, and the joining region nucleotides sequence of the exogenous insertion vector that contains SCK gene obtaining and both sides 5 ' end and the 3 ' distolateral wing paddy rice sequence is classified as shown in SEQ ID No.1; The joining region nucleotide sequence forming according to rich No. 2 exogenous insertion vectors of section and 3 ' end paddy rice flanking sequence has filtered out a pair of high specificity, highly sensitive specificity of transformant primer and probe sequence, and its nucleotides sequence is classified as shown in SEQ ID No.2, SEQ ID No.3; The nucleotides sequence of described probe is classified as shown in SEQ ID No.4.
Two of object of the present invention is to provide the real-time fluorescence quantitative PCR detection method of the strain specificity of rich No. 2 of a kind of transgenic paddy rice section, and the method comprises: (1) extracts the DNA of paddy rice sample to be detected; (2) taking the DNA that extracts as template, taking Nucleotide shown in SEQ ID No.2 and SEQ ID No.3 as amplification upstream and downstream primer, classify probe as with the nucleotides sequence shown in SEQ ID No.4, set up the performing PCR amplification of going forward side by side of pcr amplification system; (3) after PCR reaction finishes, according to fluorescence curve and the Ct value result of determination of collecting;
Wherein, in amplification sample, carry out the gene amplification of SPS internal standard, in the time of the gene amplification of SPS internal standard, blank is without typical amplification curve, and typical amplification curve appears in negative control and positive control; In the time of specificity of transformant sequence amplification, blank and negative control are without typical amplification curve, and typical amplification curve appears in positive control, show that reaction system is working properly; In the normal situation of reaction system, there is typical amplification curve in test sample, and Ct value is less than or equal to 38, shows the detection rich No. 2 transformant compositions of graduating from old-type opera school.
Wherein, the described method of extracting DNA from paddy rice sample to be detected, can be the various common methods of extracting DNA from vegetable material, for example, can be the whole bag of tricks such as CTAB method, guanidine isothiocyanate method or guanidine hydrochloride method.
Pcr amplification system described in the present invention can be set up with reference to following methods: the cumulative volume of reaction system is 20.0 μ L, wherein, 2 × Mix10.0 μ L, upstream primer 0.8 μ L shown in 10.0 μ mol/L SEQ ID No.2, downstream primer 0.8 μ L shown in 10.0 μ mol/LSEQ ID No.3, probe 0.4 μ L shown in 10.0 μ mol/LSEQ ID No.4,25ng/ μ L DNA profiling 2.0 μ L, surplus is distilled water.
Described pcr amplification condition optimization is: first stage 95 DEG C/10min; 95 DEG C/15s of subordinate phase, 95 DEG C/60s, cycle number 40;
Further aim of the present invention is to provide the real-time fluorescence quantitative PCR detection kit of the strain specificity of rich No. 2 of a kind of transgenic paddy rice section, comprising: 2 × Mix damping fluid, amplimer pair, probe and distilled water.
In described PCR detection kit, also contain SPS internal standard gene amplification system.
Specificity, sensitivity, reliability, detectability test result show, strain specificity real-time fluorescence Fluorescent Quantitative PCR detection method high specificity, highly sensitive (sensitivity can reach 20 copies), good reliability that transgenic paddy rice section that the present invention sets up is rich No. 2.
Brief description of the drawings
Fig. 1 is Hi-TAIL pcr amplification result; M:1Kb plus Marker1: PCR product 2 is taken turns in downstream second: downstream third round PCR product; 3: PCR product 4 is taken turns in upstream second: upstream third round product.
Fig. 2 is upstream and downstream order-checking splicing result (2614bp).
Fig. 3 is the nested primers from the two ends design Hi-TAIL PCR of known array.
Fig. 4 is Hi-TAIL pcr amplification result; PCR product is taken turns in M:1Kb plus Marker1, upstream second; 2, upstream third round product; Note: downstream primer is without band.
Fig. 5 is upstream and downstream order-checking splicing result.
In Fig. 6, M:1Kb plus Marker1: blank; 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Fig. 7 is the integrated structure that long segment checking obtains; 1., 2., 3., 4., 5. representative respectively: increase from illustrated Position Design long segment primer.
Fig. 8 is No. 1 primer amplification result: M:1Kb plus Marker; 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Fig. 9 is No. 2 primer amplification results: M:100bp Marker; 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Figure 10 is No. 3 primer amplification results: M:1Kb plus Marker, 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Figure 11 is No. 4 primer amplification results: M:1Kb plus Marker, 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Figure 12 is No. 5 primer amplification results: M:1Kb plus Marker, 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Figure 13 is the specificity test result of quantivative approach of the present invention.
Figure 14 is the failtests result of quantivative approach of the present invention.
Figure 15 is sensitivity test result; When copy number is 5copies and 1copies, real-time fluorescence amplification curve and be positioned at the position of typical curve.
Figure 16 is the 1st test-results of limit of detection; A:SPS real-time fluorescence figure and canonical plotting; B:KF2 real-time fluorescence figure and canonical plotting.
Figure 17 is the 2nd test-results of limit of detection; A:SPS real-time fluorescence figure and canonical plotting; B:KF2 real-time fluorescence figure and canonical plotting.
Figure 18 is the 3rd test-results of limit of detection; A:SPS real-time fluorescence figure and canonical plotting; B:KF2 real-time fluorescence figure and canonical plotting.
Figure 19 is limit of detection the 4th test-results; A:SPS real-time fluorescence figure and canonical plotting; B:KF2 real-time fluorescence figure and canonical plotting.
Figure 20 is limit of detection the 5th test-results; A:SPS real-time fluorescence figure and canonical plotting; B:KF2 real-time fluorescence figure and canonical plotting.
Embodiment
Further describe the present invention below in conjunction with specific embodiment; these embodiment are only exemplary; what those skilled in the art should understand that is; can the details of technical solution of the present invention and form be modified or be replaced lower without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The acquisition of the flanking sequence of the SCK gene that in rich No. 2 strains of experimental example 1 transgenic paddy rice section, external source is inserted
It is SCK gene that the rich No. 2 strain external sources of transgenic paddy rice section are inserted goal gene, 415bp altogether, and its base sequence is shown in SEQ ID No.5.Obtain integrated structure (external source is inserted gene flanking sequence) by Hi-TAIL PCR.
1, the first step: the nested primers that designs Hi-TAIL PCR on goal gene SCK
1. downstream primer:
Sck-sp1 CTTTCTCATCATCTTCATCCCTGGACTTG
Sck-sp2 ACGATGGACTCCAGTCCGGCCGATTTGCAAGCCGAGTGACACGAATT
Sck-sp3 TTGATTTAGTGCAGATGCATGAATCGC
Upstream primer:
Sck-Ap1 GCACCATCTTCTTTGCTCTCTTTCTCTGT
Sck-Ap2 ACGATGGACTCCAGTCCGGCCTTTCGTTTTAAAGGTGTGTGTGCTGGTAC
Sck-Ap3 TGATGACTCAAGCGATGAACCTTCTGAG
Random primer:
AD1-1 ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
AD1-2 ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
AD1-3 ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
AD1-4 ACGATGGACTCCAGAGCGGCCGCBDNBNNNAGGT
AD-C ACGATGGACTCCAGAG
2. PCR the results are shown in Figure 1.
3. upstream and downstream order-checking splicing result (2614bp) is shown in Fig. 2.
2, second step: the nested primers of establishing Hi-TAIL PCR from the two ends of known array
The schematic diagram that Fig. 3 designs for nested primers.
1. downstream primer:
OSP-SP1 AGATTAAAATAGCTTTCCCCCGTTGTAGC
OSP-SP2 ACGATGGACTCCAGTCCGGCCCAAAGTGCTATCCACGATCCATAGCAAG
OSP-SP3 CAATAGTCTCCACACCCCCCCACTATC
Upstream primer:
hptII-AP1 GCGACGTCTGTCGAGAAGTTTCTGATC
hptII-AP2 ACGATGGACTCCAGTCCGGCCAGGGCGAAGAATCTCGTGCTTTCAG
hptII-AP3 CGTTATGTTTATCGGCACTTTGCATCG
Random primer: the same.
2. PCR the results are shown in Figure 4.
3. upstream and downstream order-checking splicing result (3840bp) is shown in Fig. 5.
3. the 3rd step: the paddy rice sequence obtaining according to 5 ' end, obtains 3 ' end paddy rice sequence.Then, on rice starter, establish upstream primer, on paddy rice 3 ' terminal sequence, establish downstream primer, increase and obtain 3 ' end flanking sequence by long segment.
1. downstream primer:
OSJ-3'-AP：CGCTTTGACCTAAGTTTGCACGGAC
Upstream primer:
OSP-SP：CAATAGTCTCCACACCCCCCCACTATC
2. PCR the results are shown in Figure 6.
3. order-checking splicing result (5423bp) is shown in Fig. 7.
4, whole sequencing results: whole joining region sequences (joining region of the paddy rice flanking sequence of exogenous insertion vector (containing SCK gene) and both sides) is for shown in SEQ ID No.1.
5, the integrated structure that long segment checking obtains
Increase according to the Position Design long segment primer that in Fig. 7,1., 2., 3., 4., 5. representative is illustrated respectively, expection size is respectively: 1.4Kb, 1176bp, 817bp, 2.3kb, 4443bp; Amplification is shown in respectively Fig. 8, Fig. 9, Figure 10, Figure 11, Figure 12.
The foundation of the experimental example 2 rich No. 2 strain specificity real-time fluorescence quantitative PCR detection methods of transgenic paddy rice section
1, quantitative primer, probe and amplified fragments
1 TAGAGCAGCTTGAGCTTGGATCAGATTGTTTGCTCTAGTTGCGAATCGCGCATATGAAAT
61 CACACCATG
TAGTGTATTGACCGATTCCTTGCGGTCCGAATGGGC
121
CGGCCGCAGCGATCGCATCCTTAGGTCAAAGCGGTTTGTTTGCTCTA
ACAT
181
CACAACTTGTATAGTCCGTGC
Kefeng2-F-dl：TAGTGTATTGACCGATTCCTTGC（SEQ ID No.2）
Kefeng2-R-dl：GCACGGACTATACAAGTTGTGATGT（SEQ ID No.3）
Kefeng2-P：CGAACCCGCTCGTCTGGCTAAGAT（SEQ ID No.4）
Expection amplified fragments size: 132bp.
2, DNA extraction
Adopt a day root Plant Genome to extract test kit and extract the DNA of vegetable material to be detected, and DNA is diluted to 25ng/ μ L.
3, PCR reaction system is set up
Table 1 real-time fluorescence PCR detecting reaction system
| Reagent | Final concentration | Volume (μ L) |
| Water | 6 | |
| 2×Mix | 1× | 10 |
| F(10μM) | 400nM | 0.8 |
| R(10μM) | 400nM | 0.8 |
| Probe(10μM) | 200nM | 0.4 |
| DNA profiling (25ng/ μ L) | 2.5ng/μL | 2 |
| Cumulative volume | 20 |
PCR reaction moves by following program: first stage 95 DEG C/10min; 95 DEG C/15s of subordinate phase, 95 DEG C/60s, cycle number 40; Extend the period in the annealing of subordinate phase and collect fluorescent signal, after PCR reaction finishes, according to fluorescence curve and the Ct value result of determination of collecting.In the time of the gene amplification of SPS internal standard, blank to without typical amplification curve, there is typical amplification curve in negative control and positive control; In the time of specificity of transformant sequence amplification, blank and negative control without typical amplification curve, there is typical amplification curve in positive control, shows that reaction system is working properly.In the normal situation of reaction system, test sample occurs that typical amplification curve and Ct value are less than or equal to 38, shows to detect the rich No. 2 transformant compositions of graduating from old-type opera school.
The experiment of specificity, reliability, sensitivity and the detectability of the real-time fluorescence quantitative PCR detection method that experimental example 3 the present invention set up
One, experiment material
1, positive control sample: the rich No. 2 paddy rice content of section are 1%;
2, positive control sample: the rich No. 2 paddy rice content of section are 0.1%
3, negative control sample: the rich No. 2 paddy rice acceptor-Ming extensive 86 of section;
4, non-transgenic soybean;
5, genetically engineered soybean 356043,305423, CV127, MON89788, A5547-127, A2704-12, GTS40-3-2; Be mixed and made into 1 sample, content each 5%;
6, non-transgenic corn;
7, transgenic corns Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034,59122, MIR604, MON88017, is mixed and made into 1 sample, content each 5%;
8, non-transgenic rape;
9, transgene rape MS1, MS8, RF1, RF2, RF3, T45, Oxy235, Topas19/2, GT73(CTP1),, be mixed and made into 1 sample, content each 5%;
10, non-transgenic cotton;
11, transgene cotton MON1445, MON531, MON15985, LLCOTTON25, MON88913, sGK321 (CPTI), sGK9708 (CPTI) are mixed and made into 1 sample, content each 5%;
12, rich No. 6 of transgenic paddy rice section, rich No. 8 of section, Kemingdao, M12, TT51, extensive No. 1 of China, Shan are excellent 10, Shanyou 63, Anhui 80-4B,, Anhui 21B, anti-excellent 97, eastern agriculture, bar68-1 be mixed and made into a sample, content each 5%;
13, the lucky round-grained rice 88 of non-transgenic paddy rice, Japan fine, raise rice No. 6, bright extensive 86, balanced mix is made a sample.
Two, experimental technique
1, specificity test
13 samples in experiment material are carried out to real-time fluorescence PCR test according to the method for experimental example 2, each sample do 3 parallel, do and repeat experiment for 3 times, statistics CT value.
2, the reliability testing of quantivative approach
By the homozygote of rich section No. 2 homozygote DNA(empirical tests) be diluted to 50ng/ μ L, gradient dilution is to 5ng/ μ L, 0.5ng/ μ L, 0.05ng/ μ L, 0.005ng/ μ L, the corresponding copy number of each gradient is respectively 106382copies/ μ L, 10638copies/ μ L, 1063copies/ μ L, 106copies/ μ L, 10copies/ μ L, get 2 μ L and do template, each point do 3 parallel, Criterion curve, statistics R2, amplification efficiency E.Amplification program cycle number becomes 50 circulations.This step in triplicate, is added up three times experimental result, and calculates SD and RSD value.
3, the test of detection sensitivity
Continue to do gradient dilution to 5copies/ μ L, 1copies/ μ L according to the result of the reliability testing of above-mentioned quantivative approach, the sensitivity of test quantivative approach.Get 1 μ L and do template, each point do 10 parallel, carry out pcr amplification with the reliability testing of quantivative approach simultaneously.
4, the test of detectability
Rich No. 2 genomic dnas of homozygote section and acceptor gene group DNA are diluted to 25ng/ μ L, being mixed with transgenosis content is respectively: the sample of 5% (Y1), 1% (Y2), 0.1% (Y3), 0.04% (Y4), measure its transgenosis content, 3 parallel 3 repetitions, statistics three point value.Internal standard gene adopts SPS, and reaction system and program are the same.
Three, experimental result
1, specificity test data Classified statistics
Table 2 specificity test result
2, the reliability result data Classified statistics of quantivative approach
The reliability result of table 3 quantivative approach
3, detection sensitivity result data Classified statistics
Repeat experimental result the 1st time of table 4 detection sensitivity
Repeat experimental result the 2nd time of table 5 detection sensitivity
Repeat experimental result the 3rd time of table 6 detection sensitivity
4, limit of detection result data Classified statistics
Table 7 limit of detection repeats experimental result the 1st time
Table 8 limit of detection repeats experimental result the 2nd time
Table 9 limit of detection repeats experimental result the 3rd time
Table 10 limit of detection the 4th repeats experimental result
Table 11 limit of detection the 5th repeats experimental result
Specificity, sensitivity, reliability, detectability test result show, strain specificity PCR quantitative detecting method high specificity, highly sensitive (sensitivity can reach 20 copies), good reliability that transgenic paddy rice section that the present invention sets up is rich No. 2.
Claims (7)
1. detect primer and probe for detection of the real-time fluorescence quantitative PCR of rich No. 2 strain specificities of transgenic paddy rice section, it is characterized in that: the nucleotide sequence of described primer is respectively shown in SEQ ID No.2 and SEQ ID No.3; The nucleotides sequence of described probe is classified as shown in SEQ ID No.4.
2. a real-time fluorescence quantitative PCR detection method for rich No. 2 strain specificities of transgenic paddy rice section, is characterized in that, comprising: (1) extracts the DNA of paddy rice sample to be detected; (2), taking the DNA that extracts as template, set up the performing PCR amplification of going forward side by side of PCR reaction system taking Nucleotide shown in SEQ ID No.2 and SEQ ID No.3 as amplification upstream and downstream primer; (3) according to fluorescence curve and the Ct value result of determination of collecting.
3. according to real-time fluorescence quantitative PCR detection method claimed in claim 2, it is characterized in that, comprise: in the normal situation of reaction system, if there is typical amplification curve in detected sample, and Ct value is less than or equal to 38, show to contain in detected sample the rich No. 2 transformant compositions of transgenic paddy rice section.
4. according to real-time fluorescence quantitative PCR detection method claimed in claim 2, it is characterized in that: the cumulative volume of described PCR reaction system is 20.0 μ L, wherein, 2 × Mix10.0 μ L, upstream primer 0.8 μ L shown in 10.0 μ mol/L SEQ ID No.2, the downstream primer 0.8 μ L shown in 10.0 μ mol/L SEQ ID No.3, probe 0.4 μ L shown in 10.0 μ mol/L SEQ ID No.3,25ng/ μ L DNA profiling 2.0 μ L, surplus is distilled water.
5. according to real-time fluorescence quantitative PCR detection method claimed in claim 2, it is characterized in that: described pcr amplification condition is: 95 DEG C of first stage, 10min; 95 DEG C of subordinate phase, 15s, 95 DEG C, 60s, cycle number 40.
6. a real-time fluorescence quantitative PCR detection kit for the strain specificity of rich No. 2 of transgenic paddy rice section, comprising: 2 × Mix, amplimer pair, probe and distilled water; It is characterized in that: the right nucleotide sequence of described amplimer is respectively shown in SEQ ID No.2 and SEQ ID No.3; The nucleotides sequence of described probe is classified as shown in SEQ ID No.4.
7. according to real-time fluorescence quantitative PCR detection kit claimed in claim 6, it is characterized in that: contain SPS internal standard gene amplification system.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868137A (en) * | 2017-03-01 | 2017-06-20 | 浙江省农业科学院 | Transgenic rice multiple digital pcr quantitative detecting method |
| CN107164514A (en) * | 2017-06-22 | 2017-09-15 | 中华人民共和国黄埔出入境检验检疫局 | Transgenic beet GTSB77 strain specificities real-time fluorescent PCR testing primer, probe, method and kit |
| CN110724704A (en) * | 2019-10-16 | 2020-01-24 | 安徽省农业科学院水稻研究所 | Positive plasmid for identifying transgenic rice transformant and construction method and application thereof |
| CN114657276A (en) * | 2022-03-04 | 2022-06-24 | 江汉大学 | Primer pair combination, kit and detection method for detecting rice transgenic line |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2014
- 2014-03-06 CN CN201410081751.0A patent/CN104031982A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 农业部2031号公告-7-2013: "转基因植物及其产品成分检测抗虫水稻科丰2号及其衍生品种定性PCR方法", 《中华人民共和国国家标准》, 4 December 2013 (2013-12-04) * |
| 王渭霞等: "实时定量PCR检测转基因水稻科丰6号***拷贝数和转基因含量", 《农业生物技术学报》, vol. 20, no. 1, 25 January 2012 (2012-01-25) * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868137A (en) * | 2017-03-01 | 2017-06-20 | 浙江省农业科学院 | Transgenic rice multiple digital pcr quantitative detecting method |
| CN107164514A (en) * | 2017-06-22 | 2017-09-15 | 中华人民共和国黄埔出入境检验检疫局 | Transgenic beet GTSB77 strain specificities real-time fluorescent PCR testing primer, probe, method and kit |
| CN110724704A (en) * | 2019-10-16 | 2020-01-24 | 安徽省农业科学院水稻研究所 | Positive plasmid for identifying transgenic rice transformant and construction method and application thereof |
| CN114657276A (en) * | 2022-03-04 | 2022-06-24 | 江汉大学 | Primer pair combination, kit and detection method for detecting rice transgenic line |
| CN114657276B (en) * | 2022-03-04 | 2023-07-07 | 江汉大学 | Primer pair combination, kit and detection method for detecting rice transgenic line |
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Application publication date: 20140910 |