CN102586442A - Specific primer and probe for assaying genetically modified corn MIR162 and application thereof - Google Patents
Specific primer and probe for assaying genetically modified corn MIR162 and application thereof Download PDFInfo
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Abstract
The invention discloses a specific primer for assaying genetically modified corn MIR162, the sequence of which is shown as SEQ ID NO:1 and SEQ ID NO:2. The invention also discloses a fluorescently labeled probe for assaying genetically modified corn MIR162, the sequence of which is shown as SEQ ID NO:3, the 5' end of the probe is connected with a fluorescent reporter FAM, and the 3' end of the probe is connected with a fluorescent quencher Eclipse. The invention also discloses an assay method which utilizes the primer and the probe to carry out gene-specific qualitative PCR (Polymerase Chain Reaction), SYBR Green I real-time fluorogenic quantitative PCR and TaqMan probe real-time fluorogenic quantitative PCR to assay whether corn and related products thereof contain MIR162 transformation events, and an experiment proves that the assay method is sensitive, accurate, simple and reliable, and has a high application value and a wide market prospect.
Description
Technical field
The present invention relates to be used to detect Auele Specific Primer and the probe of transgenic corns MIR162; And whether contain the application in the MIR162 transformation event at gene specific qualitative PCR, SYBR Green I real-time fluorescence quantitative PCR and TaqMan probe for real-time fluorescence quantitative PCR detection corn and related prods thereof, belong to biology field.
Background technology
At the beginning of the nineties, first genetically modified foodGMF appears at the U.S. on the market, is a kind of fresh-keeping tomato.More than ten years subsequently, transgenic is produced product and is born successively, especially at food, obtain very great achievement aspect medical.Because the ecological security and the edible safety of transgenic product are controversial always, there are more than 40 countries and regions to set up and implemented transgenic sign system in succession.Along with the foundation of these systems and constantly perfect, people have proposed strict requirement to the accuracy and the sensitivity of transgenic detection technique, and the detection technique of various transgenic product also becomes the research focus.
Testing goal according to different transgenic product is different, identifies that the main mode of transgenic product has examination method, gene specific detection method, specificity of transformant detection method and makes up method for detecting specificity.Wherein the specificity of transformant detection is that specificity is the strongest, uses maximum detection methods.Say that from technological layer what application was maximum in above-mentioned detection is round pcr, comprises qualitative PCR, nest-type PRC, composite PCR, competitive quantitative PCR and quantitative fluorescent PCR etc.
Transgenic corns MIR162 is the novel anti lepidopterous insects kind that U.S. Syngenta Co.,Ltd develops.The applicant does not find any report about transgenic corns MIR162 specificity of transformant qualitative PCR, SYBR Green I real-time fluorescence quantitative PCR and TaqMan probe for real-time fluorescence quantitative PCR detection as yet through the retrieval to existing patent and other documents.
Summary of the invention
To above-mentioned prior art; The invention provides the Auele Specific Primer and the probe that are used to detect transgenic corns MIR162; And whether contain the application in the MIR162 transformation event at gene specific qualitative PCR, SYBR Green I real-time fluorescence quantitative PCR and TaqMan probe for real-time fluorescence quantitative PCR detection corn and related prods thereof, belong to biology field.
The present invention realizes through following technical scheme:
A kind of Auele Specific Primer that is used to detect transgenic corns MIR162, said primer is made up of upstream primer and downstream primer, and its sequence is:
MIR162-F：5′-CTGTCTAATAGTTTGAGTGA-3′；
MIR162-R：5′-GTGACTCCCTTAATTCTC-3′；
Shown in SEQ ID NO.1 and SEQ ID NO.2.
The said Auele Specific Primer that is used to detect transgenic corns MIR162 can be used for the qualitative detection corn and whether related prods contains the MIR162 transformation event, and when specifically using, step is:
(1) extracts the genomic dna of detected sample, and, utilize MIR162-F and MIR162-R combination of primers to carry out pcr amplification as template;
(2) pcr amplification product separates with agarose gel electrophoresis, and EB dyeing back identifies whether there is amplified production; If there is amplified production, then contain the MIR162 transformation event in the detected sample; If there is not amplified production, then do not contain the MIR162 transformation event in the detected sample.
In the said step (1), the PCR program is: 95 ℃ of preparatory sex change of 5min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ are extended 7min.
The said Auele Specific Primer that is used to detect transgenic corns MIR162 can be used for that SYBR Green I real-time fluorescence quantitative PCR detects corn and whether related prods contains the MIR162 transformation event, and when specifically using, step is:
(1) set up the SYBR Green I real-time fluorescence quantitative PCR typical curve of MIR162 specificity of transformant primer and the SYBR Green I real-time fluorescence quantitative PCR typical curve of zSSIIb gene:
1. extract transgenic corns MIR162 standard substance genomic dna and carry out gradient dilution, in each diluent, add the Auele Specific Primer of MIR162 transformation event then, and the national standard primer of zSSIIb gene, increase;
2. after the amplification, measure the Ct value of fluorescence associated affinity tag in each diluent, the natural logarithm of this Ct value and copy number is linear, draws the quantitative PCR typical curve of MIR162 transformation event and the quantitative PCR typical curve of zSSIIb gene in view of the above;
(2) extract the detected sample genomic dna, get extracting genome DNA liquid, in extracting genome DNA liquid, add the Auele Specific Primer of MIR162 gene then, and the national standard primer of zSSIIb gene, increase; After the amplification; Measure the Ct value of fluorescence associated affinity tag in the extracting genome DNA liquid; Then according to the quantitative PCR typical curve of the MIR162 gene of above-mentioned drafting and the quantitative PCR typical curve of zSSIIb gene; Calculate corresponding copy number, the ratio of the copy number of transgenic corns MIR162 gene and the copy number of corn internal standard gene zSSIIb gene then is the percentage composition of transgenic corns MIR162 in the detected sample.
Said step (1) 1. with (2) in, zSSIIb primer sequence (national standard) as follows:
zSSIIb-F：5′-CGGTGGATGCTAAGGCTGATG-3′；
zSSIIb-R：5′-AAAGGGCCAGGTTCATTATCCTC-3′。
Said step (1) 1. with (2) in, the parameter of amplification is following: the amplified reaction volume is 20uL, 2 * Premix Ex Taq (Rox) 10uL; Concentration is the Forward primer 0.4uL of 10umol/L; Concentration is the Reverse primer0.4uL of 10umol/L, ddH2O 8.2uL, dna profiling 1uL;
Amplification reaction condition: 95 ℃ of preparatory sex change of 10min; 95 ℃ of 15s, 50 ℃ of 30s, 72 ℃ of 30s collect fluorescent signal at 72 ℃, amount to 40 circulations.
A kind of fluorescence labeling probe that is used to detect transgenic corns MIR162; Sequence is: MIR162-P:FAM-5 '-CAGATTGTCGTTTCCCGCCTTC-3 '-Eclipse; Shown in SEQ ID NO.3; 5 of probe ' end connects the FAM of fluorescence report group, and 3 ' end is connected with an Eclipse of fluorescent quenching group.
The above-mentioned fluorescence labeling probe that is used to detect the Auele Specific Primer of transgenic corns MIR162 and is used to detect transgenic corns MIR162; Can be used for TaqMan probe for real-time fluorescence quantitative PCR detection corn and related prods thereof and whether contain the MIR162 transformation event; During concrete the application, step is:
(1) set up the TaqMan probe for real-time fluorescence quantitative PCR typical curve of MIR162 specificity of transformant primer and the TaqMan probe for real-time fluorescence quantitative PCR typical curve of zSSIIb gene:
1. extract transgenic corns MIR162 standard substance genomic dna and carry out gradient dilution; The Auele Specific Primer and the fluorescence labeling probe that in each diluent, add the MIR162 transformation event then; And the national standard primer and the fluorescence labeling probe of zSSIIb gene, increase;
2. after the amplification, measure the Ct value of fluorescence associated affinity tag in each diluent, the natural logarithm of this Ct value and copy number is linear, draws the quantitative PCR typical curve of MIR162 transformation event and the quantitative PCR typical curve of zSSIIb gene in view of the above;
(2) extract the detected sample genomic dna; Get extracting genome DNA liquid; The Auele Specific Primer and the fluorescence labeling probe that in extracting genome DNA liquid, add the MIR162 gene then, and the national standard primer and the fluorescence labeling probe of zSSIIb gene increase; After the amplification; Measure the Ct value of fluorescence associated affinity tag in the extracting genome DNA liquid; Then according to the quantitative PCR typical curve of the MIR162 gene of above-mentioned drafting and the quantitative PCR typical curve of zSSIIb gene; Calculate corresponding copy number, the ratio of the copy number of transgenic corns MIR162 gene and the copy number of corn internal standard gene zSSIIb gene then is the percentage composition of transgenic corns MIR162 in the detected sample.
Said step (1) 1. with (2) in, zSSIIb primer and probe sequence (national standard) as follows:
zSSIIb-F：5′-CGGTGGATGCTAAGGCTGATG-3′；
zSSIIb-R：5′-AAAGGGCCAGGTTCATTATCCTC-3′；
zSSIIb-P：FAM-5′-TAAGGAGCACTCGCCGCCGCATCTG-3′-TAMRA。
Said step (1) 1. with (2) in; The parameter of amplification is following: the amplified reaction volume is 20uL, 2 * Premix Ex Taq (Rox) 12.5uL, and concentration is the Forward primer 1uL of 10umol/L; Concentration is the Reverse primer 1uL of 10umol/L; Concentration is the TaqMan probe0.5uL of 10umol/L, ddH2O 4uL, dna profiling 1uL;
Amplification reaction condition: 95 ℃ of preparatory sex change of 10min; 95 ℃ of 15s, 50 ℃ of 30s, 72 ℃ of 30s collect fluorescent signal at 72 ℃, amount to 45 circulations.
The present invention has designed primer and the probe sequence that inserts carrier side gene to transgenic corns MIR162 transformation event foreign; Transgenic corns MIR162 specificity of transformant qualitative PCR detection method, SYBR Green I real-time fluorescence quantitative PCR detection method and TaqMan probe for real-time fluorescence quantitative PCR detecting method have been set up; This detection method is through experimental verification; Sensitive, accurate, simple, reliable, have wide application value and market outlook.
Description of drawings
Fig. 1 is a transgenic corns MIR162 specificity of transformant qualitative PCR amplification electrophoresis synoptic diagram among the embodiment 1.
Fig. 2 is the SYBR Green I real-time fluorescence quantitative PCR typical curve of internal standard gene zSSIIb among the embodiment 2.
Fig. 3 is a transgenic corns MIR162 transformant SYBR Green I real-time fluorescence quantitative PCR typical curve among the embodiment 2.
Fig. 4 is the TaqMan probe for real-time fluorescence quantitative PCR typical curve of internal standard gene zSSIIb among the embodiment 3.
Fig. 5 is a transgenic corns MIR162 transformant TaqMan probe for real-time fluorescence quantitative PCR typical curve among the embodiment 3.
Embodiment
Below in conjunction with accompanying drawing the present invention is further described.
Primer is synthetic by Dalian Takara company, and it is subsequent use to be diluted to 10umol/L.The synthetic primer sequence is following:
MIR162-F：5′-CTGTCTAATAGTTTGAGTGA-3′；
MIR162-R：5′-GTGACTCCCTTAATTCTC-3′。
Extract transgenic corns MIR162, MON810, NK603, Bt176, MON88017, GA21, TC1507, MIR604, Bt11 respectively; Genetically engineered soybean GTS40-3-2, MON87701; Non-transgenic soybean 1138-2, transgene cotton MON88913, LLcotton25, MON531, MON1445, rich No. 6 of transgenic paddy rice TT51, section; Non-transgenic corn Zheng Dan 958 gene DNAs are template, carry out pcr amplification with MIR162-F and MIR162-R combination of primers respectively.The PCR program is: 95 ℃ of preparatory sex change of 5min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ are extended 7min.Pcr amplification product separates with agarose gel electrophoresis, and EB dyeing back identifies whether there is amplified production.
The result: utilizing institute's designed primer is that template is carried out pcr amplification with the genome that extracts; The result is as shown in Figure 1; Visible by figure; Have only transgenic corns MIR162 genome successfully to amplify 94bp specificity product, and other transgenics and non-transgenic sample template all do not have observable amplified production.Prove that thus this primer has good specificity, is suitable for the qualitative detection of transgenic corns MIR162 specificity of transformant.
Embodiment 2 transgenic corns MIR162 specificity of transformant SYBR Green I real-time fluorescence quantitative PCR detection methods
Extract transgenic corns MIR162 standard substance genomic dna, be diluted to 5 respectively by 5 times of multiple proportions
0, 5
-1, 5
-2, 5
-3, 5
-4Corresponding copy number is made as 100000,20000,4000,800,160, and each concentration is established 3 repetitions, detects the repeatability of amplification.
Primer is synthetic by Dalian Takara company, and it is subsequent use to be diluted to 10umol/L.Synthetic MIR162 primer sequence is following:
MIR162-F：5′-CTGTCTAATAGTTTGAGTGA-3′；
MIR162-R：5′-GTGACTCCCTTAATTCTC-3′。
Corn interior label primer (zSSIIb) adopts the national standard primer, and its sequence is following:
zSSIIb-F：5′-CGGTGGATGCTAAGGCTGATG-3′；
zSSIIb-R：5′-AAAGGGCCAGGTTCATTATCCTC-3′。
The amplified reaction volume is 20uL, 2 * Premix Ex Taq (Rox) 10uL, and concentration is the Forward primer0.4uL of 10umol/L, concentration is the Reverse primer 0.4uL of 10umol/L, ddH2O 8.2uL, dna profiling 1uL.Amplification reaction condition: 95 ℃ of preparatory sex change of 10min; 95 ℃ of 15s, 50 ℃ of 30s, 72 ℃ of 30s collect fluorescent signal at 72 ℃, amount to 45 circulations.Each sample repeats 3 times, sets up aseptic double-distilled water (ddH simultaneously
2O, blank) and non-transgenic sample (Zheng Dan 958, feminine gender) contrast.
Result: through being 100000,20000,4000 to different MIR162 standard substance copy numbers; 800,160 DNA carries out the amplification of SYBR Green I real-time fluorescence quantitative PCR, and computer software generates typical curve automatically; Ordinate zou is Ct, and X-coordinate is the natural logarithm of initial copy number.Linear calculation formula according to the typical curve gained; Through transgenic corns MIR162 standard substance (1%, 0.5%, 0.1% to different transgenic content; 0.05%; 0.01%), the mensuration of positive, negative sample and blank, with the Ct value substitution formula of sample, can obtain the percentage composition of testing sample transgene component.
The specificity of transformant primer of corn internal standard gene zSSIIb and transgenic corns MIR162 is carried out the amplification of SYBR Green I real-time fluorescence quantitative PCR to the transgenic corns DNA of same group of standard substance DNA and different percentage compositions; Obtain the pcr amplification curve respectively, and generate typical curve automatically by system software.The typical curve of corn internal standard gene zSSIIb is as shown in Figure 2, and this slope of a curve is-3.25, coefficient R
2Be 0.999.The typical curve of the specificity of transformant primer of transgenic corns MIR162 is as shown in Figure 3; This slope of a curve is-3.332; Coefficient R 2 is that therefore 0.999. needs only the Ct value that obtains unknown sample, can calculate the initial copy number of this sample from typical curve.According to formula:
Transgenic corns MIR162 content in the sample=(foreign gene MIR162 copy number/internal standard gene zSSIIb copy number) * 100%
Can calculate the percentage composition of transgene component MIR162 in the testing sample.
Embodiment 3 transgenic corns MIR162 specificity of transformant TaqMan probe for real-time fluorescence quantitative PCR detecting methods
Extract transgenic corns MIR162 standard substance genomic dna, be diluted to 5 respectively by 5 times of multiple proportions
0, 5
-1, 5
-2, 5
-3, 5
-4Corresponding copy number is made as 100000,20000,4000,800,160, and each concentration is established 3 repetitions, detects the repeatability of amplification.
Primer and probe are synthetic by Dalian Takara company, and it is subsequent use to be diluted to 10umol/L.Synthetic MIR162 primer and probe sequence are following:
MIR162-F：5′-CTGTCTAATAGTTTGAGTGA-3′；
MIR162-R：5′-GTGACTCCCTTAATTCTC-3′；
MIR162-P：FAM-5′-CAGATTGTCGTTTCCCGCCTTC-3′-Eclipse。
Corn interior label primer and probe (zSSIIb) adopt national standard primer and probe, and its sequence is following:
zSSIIb-F：5′-CGGTGGATGCTAAGGCTGATG-3′；
zSSIIb-R：5′-AAAGGGCCAGGTTCATTATCCTC-3′；
zSSIIb-P：FAM-5′-TAAGGAGCACTCGCCGCCGCATCTG-3′-TAMRA。
The amplified reaction volume is 20uL, 2 * Premix Ex Taq (Rox) 12.5uL, and concentration is the Forwardprimer 1uL of 10umol/L; Concentration is the Reverse primer 1uL of 10umol/L; Concentration is the TaqMan probe0.5uL of 10umol/L, ddH2O 4uL, dna profiling 1uL; Amplification reaction condition: 95 ℃ of preparatory sex change of 10min; 95 ℃ of 15s, 50 ℃ of 30s, 72 ℃ of 30s collect fluorescent signal at 72 ℃, amount to 45 circulations, and each sample repeats 3 times, sets up aseptic double-distilled water (ddH simultaneously
2O, blank) and non-transgenic sample (Zheng Dan 958, feminine gender) contrast.
Result: through being 100000,20000,4000 to different MIR162 standard substance copy numbers; 800,160 DNA carries out the amplification of TaqMan real-time fluorescence quantitative PCR, and computer software generates typical curve automatically; Ordinate zou is Ct, and X-coordinate is the natural logarithm of initial copy number.Linear calculation formula according to the typical curve gained; Through transgenic corns MIR162 standard substance (1%, 0.5%, 0.1% to different transgenic content; 0.05%; 0.01%), the mensuration of positive, negative sample and blank, with the Ct value substitution formula of sample, can obtain the percentage composition of testing sample transgene component.
The specificity of transformant primer of corn internal standard gene zSSIIb and transgenic corns MIR162 is carried out the amplification of TaqMan real-time fluorescence quantitative PCR to the transgenic corns DNA of same group of standard substance DNA and different percentage compositions; Obtain the pcr amplification curve respectively, and generate typical curve automatically by system software.The typical curve of corn internal standard gene zSSIIb is as shown in Figure 4, and this slope of a curve is-3.557, coefficient R
2Be 0.999.The typical curve of the specificity of transformant primer of transgenic corns MIR162 is as shown in Figure 5; This slope of a curve is-3.556; Coefficient R 2 is that therefore 0.997. needs only the Ct value that obtains unknown sample, can calculate the initial copy number of this sample from typical curve.According to formula:
Transgenic corns MIR162 content in the sample=(foreign gene MIR162 copy number/internal standard gene zSSIIb copy number) * 100%
Can calculate the percentage composition of transgene component MIR162 in the testing sample.
Above result can prove; The present invention can be used for the detection of transgenic corns MIR162 for qualitative PCR, SYBR Green I real-time fluorescence quantitative PCR and the TaqMan probe for real-time fluorescence quantitative PCR detection of transgenic corns MIR162 provide simply, reliable measuring method.The present invention provides a kind of strong technical support for transgenic sign, for the control of transgenic product provides necessary means.
Claims (10)
1. Auele Specific Primer that is used to detect transgenic corns MIR162, it is characterized in that: said primer is made up of upstream primer and downstream primer, and its sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.
2. whether the described Auele Specific Primer that is used for detecting transgenic corns MIR162 of claim 1 contains the application of MIR162 transformation event at qualitative detection corn and related prods thereof.
3. the described Auele Specific Primer that is used for detecting transgenic corns MIR162 of claim 1 detects the application whether corn and related prods thereof contain the MIR162 transformation event at SYBR Green I real-time fluorescence quantitative PCR.
4. fluorescence labeling probe that is used to detect transgenic corns MIR162; It is characterized in that: the sequence of said probe is shown in SEQ ID NO.3; 5 of probe ' end connects the FAM of fluorescence report group, and 3 ' end is connected with an Eclipse of fluorescent quenching group.
5. whether the described fluorescence labeling probe that is used to detect transgenic corns MIR162 of described Auele Specific Primer that is used for detecting transgenic corns MIR162 of claim 1 and claim 4 contains the application of MIR162 transformation event at TaqMan probe for real-time fluorescence quantitative PCR detection corn and related prods thereof.
6. whether qualitative detection corn and related prods thereof contain the method for MIR162 transformation event, and it is characterized in that: step is:
(1) extracts the genomic dna of detected sample, and, utilize MIR162-F and MIR162-R combination of primers to carry out pcr amplification as template;
(2) pcr amplification product separates with agarose gel electrophoresis, and EB dyeing back identifies whether there is amplified production; If there is amplified production, then contain the MIR162 transformation event in the detected sample; If there is not amplified production, then do not contain the MIR162 transformation event in the detected sample.
7. a SYBR Green I real-time fluorescence quantitative PCR detects the method whether corn and related prods thereof contain the MIR162 transformation event, and it is characterized in that: step is:
(1) set up the SYBR Green I real-time fluorescence quantitative PCR typical curve of MIR162 specificity of transformant primer and the SYBR Green I real-time fluorescence quantitative PCR typical curve of zSSIIb gene:
1. extract transgenic corns MIR162 standard substance genomic dna and carry out gradient dilution, in each diluent, add the Auele Specific Primer of MIR162 transformation event then, and the national standard primer of zSSIIb gene, increase;
2. after the amplification, measure the Ct value of fluorescence associated affinity tag in each diluent, the natural logarithm of this Ct value and copy number is linear, draws the quantitative PCR typical curve of MIR162 transformation event and the quantitative PCR typical curve of zSSIIb gene in view of the above;
(2) extract the detected sample genomic dna, get extracting genome DNA liquid, in extracting genome DNA liquid, add the Auele Specific Primer of MIR162 gene then, and the national standard primer of zSSIIb gene, increase; After the amplification; Measure the Ct value of fluorescence associated affinity tag in the extracting genome DNA liquid; Then according to the quantitative PCR typical curve of the MIR162 gene of above-mentioned drafting and the quantitative PCR typical curve of zSSIIb gene; Calculate corresponding copy number, the ratio of the copy number of transgenic corns MIR162 gene and the copy number of corn internal standard gene zSSIIb gene then is the percentage composition of transgenic corns MIR162 in the detected sample.
8. method according to claim 7; It is characterized in that: said step (1) 1. with (2) in, the parameter of amplification is following: the amplified reaction volume is 20uL, 2 * Premix Ex Taq (Rox) 10uL; Concentration is the Forward primer 0.4uL of 10umol/L; Concentration is the Reverse primer 0.4uL of 10umol/L, ddH2O 8.2uL, dna profiling 1uL; Amplification reaction condition: 95 ℃ of preparatory sex change of 10min; 95 ℃ of 15s, 50 ℃ of 30s, 72 ℃ of 30s collect fluorescent signal at 72 ℃, amount to 40 circulations.
9. whether TaqMan probe for real-time fluorescence quantitative PCR detection corn and related prods thereof contain the method for MIR162 transformation event, and it is characterized in that: step is:
(1) set up the TaqMan probe for real-time fluorescence quantitative PCR typical curve of MIR162 specificity of transformant primer and the TaqMan probe for real-time fluorescence quantitative PCR typical curve of zSSIIb gene:
1. extract transgenic corns MIR162 standard substance genomic dna and carry out gradient dilution; The Auele Specific Primer and the fluorescence labeling probe that in each diluent, add the MIR162 transformation event then; And the national standard primer and the fluorescence labeling probe of zSSIIb gene, increase;
2. after the amplification, measure the Ct value of fluorescence associated affinity tag in each diluent, the natural logarithm of this Ct value and copy number is linear, draws the quantitative PCR typical curve of MIR162 transformation event and the quantitative PCR typical curve of zSSIIb gene in view of the above;
(2) extract the detected sample genomic dna; Get extracting genome DNA liquid; The Auele Specific Primer and the fluorescence labeling probe that in extracting genome DNA liquid, add the MIR162 gene then, and the national standard primer and the fluorescence labeling probe of zSSIIb gene increase; After the amplification; Measure the Ct value of fluorescence associated affinity tag in the extracting genome DNA liquid; Then according to the quantitative PCR typical curve of the MIR162 gene of above-mentioned drafting and the quantitative PCR typical curve of zSSIIb gene; Calculate corresponding copy number, the ratio of the copy number of transgenic corns MIR162 gene and the copy number of corn internal standard gene zSSIIb gene then is the percentage composition of transgenic corns MIR162 in the detected sample.
10. method according to claim 9 is characterized in that: said step (1) 1. with (2) in, the parameter of amplification is following: the amplified reaction volume is 20uL; 2 * Premix Ex Taq (Rox) 12.5uL; Concentration is the Forward primer 1uL of 10umol/L, and concentration is the Reverse primer 1uL of 10umol/L, and concentration is the TaqMan probe0.5uL of 10umol/L; DdH2O 4uL, dna profiling 1uL;
Amplification reaction condition: 95 ℃ of preparatory sex change of 10min; 95 ℃ of 15s, 50 ℃ of 30s, 72 ℃ of 30s collect fluorescent signal at 72 ℃, amount to 45 circulations.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205498A (en) * | 2013-04-09 | 2013-07-17 | 山东省农业科学院植物保护研究所 | Plasmid molecule for gene specific detection of insect-resistant transgenic maize, preparation and application method of plasmid molecule |
| CN103215261A (en) * | 2013-04-09 | 2013-07-24 | 山东省农业科学院植物保护研究所 | Gene specific primer of phytase (phyA2) transgenic corns, probe and applications of gene specific primer and probe in PCR (Polymerase Chain Reaction) detection |
| CN103352084A (en) * | 2013-07-26 | 2013-10-16 | 山东省农业科学院植物保护研究所 | Transgenic maize MIR162 transformant composite PCR detection primer, probe and application of primer |
| CN105112538A (en) * | 2015-09-15 | 2015-12-02 | 中国检验检疫科学研究院 | Double digital PCR fluorescent quantitative detection method for transgenic maize MIR162 |
| CN105177172A (en) * | 2015-10-30 | 2015-12-23 | 中国农业科学院生物技术研究所 | Transgenic maize T4-1-1 strain specific detection method and reagent kit |
| CN105274229A (en) * | 2015-10-30 | 2016-01-27 | 中国农业科学院生物技术研究所 | Method and kit for detecting homozygous or heterozygous state of exogenous gene of genetically-modified corn T4-1-1 |
| CN110551842A (en) * | 2019-09-04 | 2019-12-10 | 中国农业科学院油料作物研究所 | method for identifying MIR604 genotype of corn transformant based on insertion site genome sequence establishment |
| CN112980930A (en) * | 2021-04-30 | 2021-06-18 | 四川省农业科学院分析测试中心 | Primer group for specific PCR (polymerase chain reaction) accurate quantitative detection of transgenic corn 2A-7 strain and detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548011A (en) * | 2006-06-03 | 2009-09-30 | 先正达参股股份有限公司 | Corn event MIR162 |
-
2012
- 2012-03-02 CN CN2012100535924A patent/CN102586442A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548011A (en) * | 2006-06-03 | 2009-09-30 | 先正达参股股份有限公司 | Corn event MIR162 |
Non-Patent Citations (2)
| Title |
|---|
| 翟志芳 等: "转基因玉米LY038转化事件的特异性检测", 《农业生物技术学报》 * |
| 袁亚男 等: "实时荧光定量PCR技术得类型、特点与应用", 《中国畜牧兽医》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205498A (en) * | 2013-04-09 | 2013-07-17 | 山东省农业科学院植物保护研究所 | Plasmid molecule for gene specific detection of insect-resistant transgenic maize, preparation and application method of plasmid molecule |
| CN103215261A (en) * | 2013-04-09 | 2013-07-24 | 山东省农业科学院植物保护研究所 | Gene specific primer of phytase (phyA2) transgenic corns, probe and applications of gene specific primer and probe in PCR (Polymerase Chain Reaction) detection |
| CN103352084A (en) * | 2013-07-26 | 2013-10-16 | 山东省农业科学院植物保护研究所 | Transgenic maize MIR162 transformant composite PCR detection primer, probe and application of primer |
| CN105112538A (en) * | 2015-09-15 | 2015-12-02 | 中国检验检疫科学研究院 | Double digital PCR fluorescent quantitative detection method for transgenic maize MIR162 |
| CN105177172A (en) * | 2015-10-30 | 2015-12-23 | 中国农业科学院生物技术研究所 | Transgenic maize T4-1-1 strain specific detection method and reagent kit |
| CN105274229A (en) * | 2015-10-30 | 2016-01-27 | 中国农业科学院生物技术研究所 | Method and kit for detecting homozygous or heterozygous state of exogenous gene of genetically-modified corn T4-1-1 |
| CN105177172B (en) * | 2015-10-30 | 2018-04-13 | 中国农业科学院生物技术研究所 | 11 event-specific detection methods of transgenic corns T4 and kit |
| CN105274229B (en) * | 2015-10-30 | 2018-04-13 | 中国农业科学院生物技术研究所 | Detect the method and kit of 11 foreign gene homozygosis of transgenic corns T4/heterozygous state |
| CN110551842A (en) * | 2019-09-04 | 2019-12-10 | 中国农业科学院油料作物研究所 | method for identifying MIR604 genotype of corn transformant based on insertion site genome sequence establishment |
| CN110551842B (en) * | 2019-09-04 | 2022-10-11 | 中国农业科学院油料作物研究所 | Method for identifying genotype of corn transformant MIR604 based on insertion site genome sequence establishment |
| CN112980930A (en) * | 2021-04-30 | 2021-06-18 | 四川省农业科学院分析测试中心 | Primer group for specific PCR (polymerase chain reaction) accurate quantitative detection of transgenic corn 2A-7 strain and detection method |
| CN112980930B (en) * | 2021-04-30 | 2022-01-18 | 四川省农业科学院分析测试中心 | Primer group for specific PCR (polymerase chain reaction) accurate quantitative detection of transgenic corn 2A-7 strain and detection method |
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