CN106591340B - Standard plasmid molecule for qualitative detection of transgenic organism and product gene thereof - Google Patents
Standard plasmid molecule for qualitative detection of transgenic organism and product gene thereof Download PDFInfo
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Abstract
The invention discloses a standard plasmid molecule for qualitative detection of transgenic organisms and genes of products thereof, belonging to the technical field of detection of transgenic organisms. The invention splices 17 genes according to a specific sequence to obtain the core sequence of the plasmid standard molecule. The core sequence of the plasmid standard molecule is connected with the skeleton vector to obtain the plasmid standard molecule, and when the obtained plasmid standard molecule is used as a positive control for detecting transgenic crops with different parameters, the plasmid standard molecule can correctly detect the exogenous gene and the internal standard gene corresponding to each transformation event.
Description
Technical Field
The invention relates to a standard plasmid molecule for qualitative detection of transgenic organisms and genes of products thereof, in particular to a standard plasmid molecule simultaneously suitable for qualitative detection of 17 genes of 5 transgenic crops and construction thereof, belonging to the technical field of detection of transgenic organisms.
Background
At present, more and more transgenic crops are commercially grown and applied in a large scale. The biological safety supervision of transgenic products is perfected in all countries in the world, transgenic biological detection is an important component of the safety supervision, and nucleic acid detection is the most effective and conventional method for detecting transgenic components. In order to prevent the occurrence of false positive, the current detection standard requires that a positive standard substance control (referred to as a positive control for short) must be set in the nucleic acid detection process to monitor the detection process and the result.
The currently commonly used positive standard is a matrix standard prepared from raw materials, however, it is complicated to prepare, expensive, and not conducive to long-term storage. Therefore, the matrix standard substance has become a bottleneck limiting the detection of transgenic products, and the development of a substitute for the matrix standard substance is urgently needed.
The standard molecule is a linearized recombinant plasmid molecule containing a specific segment of a transgenic test foreign insert gene and/or an internal standard gene, so that a plurality of target genes can be detected in one standard molecule. Plasmid standard molecules have been internationally recognized and widely studied and used as a standard substance substitute in the detection of transgenic components. Some plasmid standard molecules for transgene detection are reported at present, but the plasmid standard molecules only aim at one or a few exogenous genes or universal element sequences for detection, so that plasmid standard molecules containing most detection parameters need to be constructed.
Disclosure of Invention
The invention aims to provide a standard plasmid molecule which is simultaneously suitable for qualitative detection of 17 genes of 5 transgenic crops, and meets the detection requirements of the transgenic crops. When the transgenic screening is detected, the transgenic screening is used as a positive control to detect various transgenic crops, so that the aim of detecting a plurality of exogenous genes by using a single positive control is fulfilled.
The core sequence of the plasmid standard molecule is shown as SEQ ID NO. 1.
The construction method of the plasmid standard molecule is formed by splicing 17 sequences of P-CaMV35S, P-FMV35S, P-NOS, T-CaMV35S, NPTII, HPT, PMI, bar, pat, CP4-epsps, Bt, Lectin, zSSIIb, SPS and HMG I/Y, Sad1 in sequence.
The invention screens 12 detection parameters (general elements P-CaMV35S, P-FMV35S, P-NOS, T-CaMV35S, marker genes NPTII, HPT, PMI, bar and pat fragments, herbicide-resistant genes CP4-epsps, insect-resistant genes Bt, 5 internal standard genes Lectin, zSSIIb, SPS and HMG I/Y, Sad1) with the detection coverage rate of 95 percent by analyzing exogenous genes, general elements and marker genes in the transformation events of the current international commercialized transgenic crops, and splices the fragments according to a specific sequence to obtain the core sequence of the standard plasmid molecules. Connecting a core sequence of the plasmid standard molecule with a skeleton vector to obtain a plasmid standard molecule, and amplifying specific PCR bands by using 17-parameter detection primers in the existing transgenic component detection standard when the obtained plasmid standard molecule is subjected to PCR amplification; the PCR amplification sensitivity is 50 copies, and has good reproducibility and stability, and can be completely used as a positive control for qualitative PCR analysis of actual samples. When the obtained plasmid standard molecules are used as positive controls to detect transgenic crops (A2704-12, GTS40-3-2, MON89788, BT11, BT176, MIR162, MON863, MON89034, NK603, T25, TT51-1, Kefeng 6, rice borer, GT73, MS1, TOPAS19/2, LL25, MON1445 and MON15985) with different parameters, the plasmid standard molecules can correctly detect the foreign gene and the internal standard gene corresponding to each transformation event.
Drawings
FIG. 1 is a schematic diagram of the splicing sequence of candidate sequences of standard molecules.
FIG. 2 shows PCR amplification results of 17 pairs of primer pair candidate sequences 1, 2 and 3 in the detection standard.
FIG. 3 shows the structure of the PSM17 plasmid standard molecule gene.
FIG. 4 detection sensitivity analysis of PSM 17; 1, DL2000DNA marker; 2-8, PSM17 copy number of 1000, 500, 100, 50, 10, 5, 1 copies of template PCR results.
FIG. 5 detection reproducibility analysis of PSM17, 1, DL2000DNA marker, 2-4, 1.32 × 106Copy template amplification of 3 replicates 5-7, 1.32 × 1083 replicates of copy template amplification 8-10, 1.32 × 1010Copy template amplification of 3 parallel.
FIG. 6 analysis of the suitability of PSM17 for practical detection; 1, DL2000DNA marker; 2-21 are respectively PSM17, A2704-12, GTS40-3-2, MON89788, BT11, BT176, MIR162, MON863, MON89034, NK603, T25, TT51-1, Kefeng 6, rice stem borer, GT73, MS1, TOPAS19/2, LL25, MON1445 and MON 15985.
Detailed Description
Example 1 plasmid Standard molecule construction
P-CaMV35S, P-FMV35S, P-NOS, T-CaMV35S, NPTII, HPT, PMI, bar, pat, CP4-epsps, Bt, Lectin, zSSIIb, SPS, HMG I/Y, Sad1 fragments are searched and obtained by combining with a GenBank database by using published national standards for related detection of transgenic plants. As the new sequence formed by splicing the 17 sequences is likely to generate a high GC content sequence or a hairpin structure and the like which influence PCR amplification, the 17 target fragments are spliced into 3 standard molecule candidate sequences according to different sequences, the candidate sequences are respectively named as candidate sequences 1, 2 and 3, the splicing sequence is shown in figure 1, the length of the obtained combined sequence is 4208bp, the 3 sequences are obtained in a gene synthesis mode, the 3 sequences are connected with a cloning vector pMD18T (2692bp), 3 candidate plasmid standard molecules with the length of 6900bp are obtained, after the sequence correctness is verified by sequencing, the candidate plasmid standard molecules are transformed into escherichia coli to be subjected to competent proliferation to extract corresponding plasmids, the 3 plasmid standard molecules are used as templates for PCR amplification, and template plasmids suitable for amplification of all 17 detection parameters are selected.
Example 2 verification of plasmid Standard molecular PCR amplification
As the plasmid standard molecule is finally used as a positive control to be applied to the qualitative PCR detection of the transgenic components, the current transgenic component detection standard in China should be selected to carry out effect verification. Therefore, PCR amplification was performed using 17-parameter detection primers (Table 1) derived from the current transgenic component detection standard, using 3 standard plasmid molecules obtained in example 1 as templates, respectively, in the following reaction scheme: the total volume was 25. mu.L, wherein 5. mu.L of 10-fold Taq buffer, 2.5. mu.L of dNTPs (2.5mM), 0.5. mu.L of each of the upstream and downstream primers (10. mu.M), 50ng of template, and the total volume was made up to 50. mu.L with ddH 2O; the reaction procedure is as follows: 95 ℃ for 5min, 35 cycles (95 ℃ for 30s, 56-63 ℃ for 30s, 72 ℃ for 30s), 72 ℃ for 5min, the specific annealing temperatures for each primer are shown in Table 1. The results are shown in FIG. 2: when the candidate sequence 1 is amplified by 17 pairs of primers respectively, all the primers can amplify specific PCR bands; when the candidate sequence 2 is amplified by 17 pairs of primers respectively, non-specific amplification bands appear on the amplification products of the primers P-CaMV35S-F/R and NPTII-F/R; when the candidate sequence 3 is amplified by 17 pairs of primers respectively, non-specific amplification bands appear on the amplification products of the primers P-CaMV35S-F/R and NPTII-F/R, and diffusion bands appear on the amplification products of the primer Bt-F/R.
Therefore, the candidate sequence 1 is determined as a 'plasmid standard molecule core sequence', the specific sequence is shown in SEQ ID NO.1, the candidate sequence is named as 'PSM 17' after being connected with a pMD18T vector, the complete vector structure is shown in figure 3, and the corresponding strain is preserved at-70 ℃.
TABLE 1 primers used for PCR amplification validation of plasmid Standard molecules
Example 3 determination of the detection sensitivity of plasmid Standard molecule PSM17
To verify the sensitivity of PSM17 in the qualitative PCR detection process, 7 DNA template samples (1000, 500, 100, 50, 10, 5, 1 copies) were prepared at different concentrations and PCR amplified separately for "PSM 17" using 17 pairs of primers. The results show that the sensitivity of PSM17 to different primers can reach 50 or 10 copies (FIG. 4), and the combined results determine that the sensitivity is 50 copies.
Example 4 determination of reproducibility of detection of plasmid Standard molecule PSM17
To verify the reproducibility of the detection system, different copy numbers (1.32 × 10) were used respectively6Copy, 1.32 × 108Copy, 1.32 × 1010Copies) of the standard plasmid molecule DNA as templates, 3 replicates per template, and 3PCR reproducibility tests were performed (PCR system see example 2). The target bands can be detected by PCR among 3 parallel reaction systems and 3 times of repetition (figure 5), and the PCR detection of PSM17 has good reproducibility and high stability, and can be completely used as a positive controlThe method is used for qualitative PCR analysis of actual samples.
Example 5 application of plasmid Standard molecules in practical assays
The applicability of the standard substance is examined by using the developed standard substance as a positive control to detect an actual sample with a known result so as to verify the feasibility and the effectiveness of the standard substance. According to the current transgenic component detection standard in China, the PSM16 plasmid standard molecule obtained by the method is used as a positive control sample to verify the applicability of the PSM16 plasmid in the PCR detection process of the transgenic component.
In the experiment, transgenic crops (A2704-12, GTS40-3-2, MON89788, BT11, BT176, MIR162, MON863, MON89034, NK603, T25, TT51-1, Kefeng 6, Ke dao, GT73, MS1, TOPAS19/2, LL25, MON1445 and MON15985) with different parameters are selected as templates, qualitative PCR detection is carried out by taking PSM17 as a positive control and utilizing 17 pairs of primers in a detection standard (a PCR system is shown in example 2), the used detection samples and detection results are summarized in Table 2, the electrophoresis result of the PCR detection is shown in FIG. 6, and the result shows that: the PSM17 plasmid standard molecule can correctly detect the corresponding foreign gene and internal standard gene of each transformation event.
TABLE 2
Note: in Table 2, "+" indicates that the PCR result was positive; "-" indicates that the PCR result was negative.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> northeast university of agriculture
<120> a standard plasmid molecule for qualitative detection of transgenic organism and product gene thereof
<160>35
<170>PatentIn version 3.3
<210>1
<211>4208
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<213> Artificial sequence
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gctcctacaa atgccatcat tgcgataaag gaaaggctat cattcaagat gcctctgccg 60
acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc 120
caaccacgtc ttcaaagcaa gtggattgat gtgatacttc cactgacgta agggatgacg 180
cacaatccca ctatcaagac atccaccgaa gacttaaagt tagtgggcat ctttgaaata 240
atctttgtca acatcgagca gctggcttgt ggggaccaga caaaaaagga atggtgcaga 300
attgttaggc gcacctacca aaagcatctt tgcatttatt gcaaagataa agcagattcc 360
tctagtacaa gtggggaaca aaataacgtg gaaaagagct gtcctgccgt tttacgtttg 420
gaactgacag aaccgcaacg ttgaaggagc cactcagccg cgggtttctg gagtttaatg 480
agctaagcac atacgtcaga aaccattatt gcgcgttcaa aagtcgccta aggtcactat 540
cagctagcaa atatttcttg tcaaaaatgc tccactgacg ttccataaga atcctgttgc 600
cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 660
catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 720
catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataagt ttcgctcatg 780
tgttgagcgt ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata 840
aaatttctaa ttcctaaaac caaaatccag tactaaaatc cagatcccca ctgggcacaa 900
cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 960
ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg 1020
ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 1080
gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 1140
cttgctcctg ccgagaaagt atccatcatg gctgatgcga agtgcttgac attggggagt 1200
ttagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc 1260
tgcctgaaac cgaactgccc gctgttctac aaccggtcgc ggaggctatg gatgcgatcg 1320
ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc 1380
aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc 1440
aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc 1500
tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca 1560
atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg 1620
gggattccca atacgaggtc gccaacatct agcaaaacgg cgttgactga actttatggt 1680
atggaaaatc cgtccagcca gccgatggcc gagctgtgga tgggcgcaca tccgaaaagc 1740
agttcacgag tgcagaatgc cgccggagat atcgtttcac tgcgtgatgt gattgagagt 1800
gataaatcga ctctgctcgg agaggccgtt gccaaacgct ttggcgaact gcctttcctg 1860
ttcaaagtat tatgcgcagc acagccactc tccattcagg ttcatccaaa cgaaggcacg 1920
caacgcctac gactggacgg ccgagtcgac cgtgtacgtc tccccccgcc accagcggac 1980
gggactgggc tccacgctct acacccacct gctgaagtcc ctggaggcac agggcttcaa 2040
gagcgtggtc gctgtcatcg ggctgcccaa cgacccgagc gtgcgcatgc acgaggcgct 2100
cggatatgcc ccccgcggca tgctgcgggc ggccggcttc aagcacggga actggcatga 2160
cgtgggtttc tgggaaggca cgcaacgcct acgactggac ggccgagtcg accgtgtacg 2220
tctccccccg ccaccagcgg acgggactgg gctccacgct ctacacccac ctgctgaagt 2280
ccctggaggc acagggcttc aagagcgtgg tcgctgtcat cgggctgccc aacgacccga 2340
gcgtgcgcat gcacgaggcg ctcggatatg ccccccgcgg catgctgcgg gcggccggct 2400
tcaagcacgg gaactggcat gacgtgggtt tctggacggt gaccgtcttc ccgttacctt 2460
gcgcgggccg aagacgccga cgccgatcac ctaccgcgtg ccgatggcct ccgcacaggt 2520
gaagtccgcc gtgctgctcg ccggcctcaa cacgcccggc atcacgacgg tcatcgagcc 2580
gatcatgacg cgcgatcata cggaaaagat gctgcagggc tttggcgcca accttaccgt 2640
cgagacggat gcggacggcg tgcgcaccat ccgcctggaa ggccgcggca agctcaccgg 2700
ccaagtcatc gacgtgccgg gcgacccgtc ctcgacggcc ttcccgctgg ttgcggccct 2760
gcttgttcga aggattgagc aatctctacc aaatctatgc agagagcttc agagagtggg 2820
aagccgatcc tactaaccca gctctccgcg aggaaatgcg tattcaattc aacgacatga 2880
acagcgcctt gaccacagct atcccattgt tcgcagtcca gaactaccaa gttcctctct 2940
tgtccgtgta cgttcaagca gctaatcttc acctcagcgt gcttcgagac gttagcgtgt 3000
ttgggcaaag gtggggattc gatgctgcaa ccatcaatag ccgttacaac gaccttacta 3060
ggctgattgg cgatcgagta gtgagagtcg tcttattaca ctttcttcct tcgatctgtc 3120
acatttagat ggcctcatgc aacacaaagc ttgtaagatc caaaggatca atgttactgc 3180
tagcgtgtgg caaattggaa gcaaaagacc aagaaagcac gtcatgcgat tccccaggta 3240
tgtcgagtcc cgtggcagca gagaacccta tcctcacccc tcccaatcct ttgacatctg 3300
ctccgaagca aagtcagagc gctgcaatgc aaaacggaac gagtgggggc agcagcgcga 3360
gcaccgccgc gccggtgtcc ggacccaaag ctgatcatcc atcagctcct gtcaccaaga 3420
gagaaatcga atctgtttac tcgtcaagtg tcatctcctg aagtggactg gagctatggg 3480
gagcctactg aaatgttaac ctccggttcc actgacggag agggaagcgg tgagagtgct 3540
ggtgcgtaca ttgtgcgcat tccgtgcggt ccaagggaca agtacctccg taaagaggcc 3600
ctgtggcctt acctccaaga gtttgtcgac ggagctctcg cgcatatcct gaacatgtcc 3660
aaggctctgg gggaacaggt tagcaatggg aagctggtct tgccatatgt aatccatggc 3720
tccttccgtt tcctcgccga ggcctagagg tcgtcctcct aaggcgaaag gaccttcctc 3780
ggaggtggag acgaaagttg cggcaccgag tggctccggg aggccacgtg gacgaccgcc 3840
gaagaagcag aagacggaat ccgaggcggt taaagccgat gttgaacctg cggaggctcc 3900
ggctggggag cggagagggc gtggaatggc ctctaatcat tgttatgatg agatttgaac 3960
ttttacaaat tatgatattg ttgtctttga atccttggta ataatgatga tgtatgagta 4020
tgagaatgaa agtaatccca aagttggttt atttttactg aacacagaga ggttgggaat 4080
ctgaaaaagc ctttcacgcc tccaaaggag gtgcctgttc agatcaccca ctccatgccg 4140
cctcacaaga ttgagatctt taaatctttg gagggctggg ctgagaacaa cattctgact 4200
cacctcaa 4208
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gctcctacaa atgccatcat tgc 23
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gatagtggga ttgtgcgtca tccc 24
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aagacatcca ccgaagactt a 21
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aggacagctc ttttccacgt t 21
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gccgttttac gtttggaact g 21
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ggggatctgg attttagtac tg 22
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actgggcaca acagacaatc g 21
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gcatcagcca tgatggatac ttt 23
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<211>22
<212>DNA
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gaagtgcttg acattgggga gt 22
<210>15
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<213> Artificial sequence
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agatgttggc gacctcgtat t 21
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gtttggatga acctgaatgg aga 23
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gaaggcacgc aacgcctacg a 21
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ccagaaaccc acgtcatgcc a 21
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<400>20
gaaggctagg aacgcttacg a 21
<210>21
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<400>21
ccaaaaacca acatcatgcc a 21
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acggtgaccg tcttcccgtt ac 22
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<211>21
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gaacaagcag ggccgcaacc a 21
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gaaggtttga gcaatctcta c 21
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cgatcagcct agtaaggtcg t 21
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<211>21
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<400>26
gggtgaggat agggttctct g 21
<210>27
<211>21
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gcgatcgagt agtgagagtc g 21
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<211>22
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ctcccaatcc tttgacatct gc 22
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tcgatttctc tcttggtgac agg 23
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atctgtttac tcgtcaagtg tcatctc 27
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gccatggatt acatatggca aga 23
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<211>18
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tggcctctaa tcattgttat gatg 24
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ttgaggtgag tcagaatgtt gttc 24
Claims (6)
1. A plasmid standard molecule is characterized by consisting of a skeleton carrier and a core sequence, wherein the nucleotide sequence of the core sequence is shown as SEQ ID NO. 1.
2. The plasmid standard molecule of claim 1 wherein the backbone vector is pMD 18T.
3. A method for constructing the plasmid standard molecule of claim 1 or 2, which is characterized in that the plasmid standard molecule is formed by splicing 17 sequences of P-CaMV35S, P-FMV35S, P-NOS, T-CaMV35S, NPTII, HPT, PMI, bar, pat, CP4-epsps, Bt, Lectin, zSSIIb, SPS and HMG I/Y, Sad1 segments in sequence.
4. Use of the plasmid standard molecule of claim 1 or 2 for the detection of a transgene component.
5. A kit comprising the plasmid standard molecule of claim 1 or 2.
6. Use of the kit of claim 5 for the detection of transgenic components.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN201587947U (en) * | 2009-11-18 | 2010-09-22 | 东北农业大学 | Standard molecule kit for detecting foreign genes of transgenic soya beans, corns, and paddy rice |
| CN104651511A (en) * | 2015-02-14 | 2015-05-27 | 中国农业科学院油料作物研究所 | Positive plasmid molecule pBI121-Screening and application thereof |
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| CN201587947U (en) * | 2009-11-18 | 2010-09-22 | 东北农业大学 | Standard molecule kit for detecting foreign genes of transgenic soya beans, corns, and paddy rice |
| CN104651511A (en) * | 2015-02-14 | 2015-05-27 | 中国农业科学院油料作物研究所 | Positive plasmid molecule pBI121-Screening and application thereof |
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| Title |
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| 新型转基因标准物质与标记基因检测方法研究;岳运锋;《中国优秀硕士学位论文全文数据库基础科学辑》;20120715(第7期);摘要及第55-56页 * |
| 用于四种主要作物转基因筛查检测的标准质粒分子的构建及应用;徐俊锋等;《农业生物技术学报》;20150925;第23卷(第9期);1167-1177页 * |
| 转基因产品检测标准物质研究;张丽;《中国博士学位论文全文数据库基础科学辑》;20121015;7-8页1.2.2及93-100页4.2 * |
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