CN104195256A - Qualitative PCR (Polymerase Chain Reaction) detection primer, qualitative PCR detection method and qualitative PCR detection kit for transgenic maize - Google Patents

Qualitative PCR (Polymerase Chain Reaction) detection primer, qualitative PCR detection method and qualitative PCR detection kit for transgenic maize Download PDF

Info

Publication number
CN104195256A
CN104195256A CN201410457456.0A CN201410457456A CN104195256A CN 104195256 A CN104195256 A CN 104195256A CN 201410457456 A CN201410457456 A CN 201410457456A CN 104195256 A CN104195256 A CN 104195256A
Authority
CN
China
Prior art keywords
qualitative pcr
transformant
specificity
transgenic corns
detection method
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201410457456.0A
Other languages
Chinese (zh)
Inventor
李飞武
龙丽坤
李葱葱
徐俊锋
刘允军
闫伟
董立明
武奉慈
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jilin Academy of Agricultural Sciences
Original Assignee
Jilin Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jilin Academy of Agricultural Sciences filed Critical Jilin Academy of Agricultural Sciences
Priority to CN201410457456.0A priority Critical patent/CN104195256A/en
Publication of CN104195256A publication Critical patent/CN104195256A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Abstract

The invention belongs to the field of biotechnology, and relates to a transgenic plant and a detection method of a transgenic plant product, and particularly relates to a qualitative PCR (Polymerase Chain Reaction) detection primer, a qualitative PCR detection method and a qualitative PCR detection kit for the transgenic maize. According to the method, a specific primer pair for precisely detecting the transgenic maize IE034 is disclosed. On the basis, the invention establishes a qualitative PCR detection method for the transgenic maize IE034, and the qualitative PCR detection method is strong in specificity and high in sensitivity. The invention further discloses a qualitative PCR detection kit for the transgenic maize IE034. Experiments prove that the qualitative PCR detection primer, method and kit for the transgenic maize have the advantages of being strong in specificity and high in sensitivity, and are applicable to accurate detection on specificity of the transgenic maize IE034 and derivative strains thereof.

Description

The qualitative PCR of transgenic corns IE034 detects primer, method and test kit
Technical field
The invention belongs to biological technical field, relate to the detection method of transgenic plant and products thereof, the qualitative PCR that is specifically related to transgenic corns IE034 detects primer, method and test kit.
Background technology
Transgenic corns IE034 is the killing gene through optimizing by codon cry1Ieby agrobacterium-mediated transformation, proceed in Maize genome an insect-resistant transgenic new corn strain of acquisition.At present, IE034 corn has entered the industrial experimentation stage, has great commercial application prospect.Setting up the accurate detection method of specificity of transformant of IE034 corn, is this transgenic corns to be carried out to the important means of safety evaluation and security control.
Round pcr is the method being most widely used during the outer transgenic product of Present Domestic detects.Up to now, lack a kind of for the high specificity of transgenic insect-resistant corn IE034 and derived varieties thereof, highly sensitive qualitative PCR detection method.
Summary of the invention
The qualitative PCR that the object of the invention is to open transgenic corns IE034 detects primer.
Another object of the present invention is to disclose the qualitative PCR detection method of a kind of transgenic corns IE034.
Another object of the present invention is to disclose the qualitative PCR detection kit of a kind of transgenic corns IE034.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
According to the specificity of transformant sequence of transgenic corns IE034, the specificity qualitative PCR of the synthetic IE034 corn of design detects primer, and its nucleotide sequence is as SEQ ID NO:1 ~ 2; Determine reaction system and the response procedures of qualitative PCR; Qualitative PCR detection method is carried out to the tests such as specificity, sensitivity.
Transgenic corns IE034 specificity of transformant qualitative PCR detects primer, and its nucleotides sequence is classified as:
Upstream primer sequence, IE034-F1:5 '-TGCAGGAGAAGTTTGATGGA-3 ' (SEQ ID NO:1);
Downstream primer sequence, IE034-R1:5 '-GGGTTTCGCTCATGTGTTG-3 ' (SEQ ID NO:2).
Transgenic corns IE034 specificity of transformant qualitative PCR detection method, comprises the following steps:
(1) synthetic upstream primer (SEQ ID NO:1) and downstream primer (SEQ ID NO:2);
(2) prepare the genomic dna solution of testing sample;
(3) preparation qualitative PCR reaction system;
(4) operation qualitative PCR response procedures;
(5), if amplify the specific band of 258bp in testing sample, in this sample, contain transgenic corns IE034 transformant composition; If do not amplify the band of 258bp in testing sample, in this sample, do not contain transgenic corns IE034 transformant composition.
Further, the concentration of the described synthetic upstream primer of step (1) and downstream primer is 10 μ mol/L, and the concentration of the DNA solution of the described preparation of step (2) is 50ng/ μ L.
Further, the cumulative volume of the preparation qualitative PCR reaction system that step (3) is described is 25 μ L, comprises following component: 10 * PCR damping fluid, 2.5 μ L, dNTPs mixing solutions 2 μ L, upstream primer 0.5 μ L, downstream primer 0.5 μ L, Taq archaeal dna polymerase 0.125 μ L, DNA solution 2 μ L, water 17.375 μ L.
Further, the described qualitative PCR response procedures of step (4) is: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out altogether 35 circulations; 72 ℃ are extended 7min.
In addition, transgenic corns IE034 specificity of transformant qualitative PCR detection kit disclosed by the invention, comprises following component: 10 * PCR damping fluid, dNTPs mixing solutions, upstream primer IE034-F1 and downstream primer IE034-R1, Taq archaeal dna polymerase and water.
Prove by experiment, the qualitative PCR of transgenic corns IE034 provided by the invention detects primer, method and test kit and has the advantages such as high specificity, sensitivity height, and the specificity that is applicable to transgenic corns IE034 and derived varieties thereof precisely detects.
Accompanying drawing explanation
Fig. 1 is the selection result of the response procedures of IE034 corn qualitative PCR detection method, and a is 56 ℃ of annealing, and b is 58 ℃ of annealing, and c is 60 ℃ of annealing, and d is 62 ℃ of annealing, and e is 64 ℃ of annealing; 1 is blank, 2 negative contrasts, and 3 is the massfraction IE034 corn that is 1%, 4 is the massfraction IE034 corn that is 0.1%;
Fig. 2 is the specificity test result of IE034 corn qualitative PCR detection method, 1 is blank, 2 negative contrasts, 3 is the massfraction IE034 corn that is 1%, 4 is transgenic corns MON89034, MON810, Bt11, Bt176, MON863, MON88017, MIR604, TC1507, 59122 and the biased sample of NK603, 5 is genetically engineered soybean MON89788, GTS40-3-2, A2704-12, A5547-127, 305423 and 356043 biased sample, 6 is transgenic paddy rice KF-6, the biased sample of TT51-1 and KMD-1, 7 is transgene cotton MON531, MON15985, the biased sample of MON1445 and LLCOTTON25, 8 is transgene rape RF1, MS1, the biased sample of Topas19/2 and T45, M is DNA molecular amount Marker,
Fig. 3 is the sensitivity test result of IE034 corn qualitative PCR detection method, 1 is blank, 2 negative contrasts, 3 ~ 7 are followed successively by IE034 corn massfraction is respectively 1%, 0.5%, 0.1%, 0.05%, 0.01% test sample, and M is DNA molecular amount Marker.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
The response procedures optimization of the qualitative PCR detection method of embodiment 1 transgenic corns IE034
It is tested object that the massfraction of take is respectively 1% and 0.1% IE034 corn, with non-transgenic corn, combine 31 negative contrasts, take sterilizing ultrapure water as blank, 5 annealing temperatures such as 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃ are set, the primer final concentration that adopts 0.2 μ mol/L, carries out qualitative PCR amplification.
Result shows (Fig. 1), while using 5 kinds of different annealing temperatures to carry out pcr amplification, at IE034 corn massfraction, it is the specific band of acquisition 258bp of all increasing in 1% and 0.1% sample, show that detection method of the present invention has good permission to annealing temperature, final definite annealing temperature is preferably 58 ℃, and primer final concentration is 0.2 μ mol/L.
The specificity test of the qualitative PCR detection method of embodiment 2 transgenic corns IE034
Specificity to the qualitative PCR detection method of the transgenic corns IE034 described in embodiment 1 is tested, and test sample comprises: the IE034 corn that massfraction is 1%, by transgenic corns MON89034, MON810, Bt11, Bt176, MON863, MON88017, MIR604, TC1507, 59122 and the biased sample (massfraction of every kind of transformant is 1%) made of NK603, by genetically engineered soybean MON89788, GTS40-3-2, A2704-12, A5547-127, 305423 and 356043 biased samples of making (massfraction of every kind of transformant is 1%), by transgenic paddy rice KF-6, the biased sample that TT51-1 and KMD-1 make (massfraction of every kind of transformant is 1%), by transgene cotton MON531, MON15985, the biased sample that MON1445 and LLCOTTON25 make (massfraction of every kind of transformant is 1%), by transgene rape RF1, MS1, the biased sample that Topas19/2 and T45 make (massfraction of every kind of transformant is 1%), non-transgenic corn combines 31.
Result shows (Fig. 2), in the IE034 corn sample that only massfraction is 1%, amplify the specific band of 258bp, and in other genetically modified crops and non-transgenic corn, all do not amplify the band of 258bp, show that detection method of the present invention has good specificity to transgenic corns IE034.
The sensitivity test of the qualitative PCR detection method of embodiment 3 transgenic corns IE034
Transgenic corns IE034 is combined to 31 in mass ratio with corresponding non-transgenic corn, be mixed and made into IE034 corn massfraction and be respectively 1%, 0.5%, 0.1%, 0.05%, 0.01% sample, the sensitivity of the qualitative PCR detection method of the transgenic corns IE034 described in embodiment 1 is tested.
Result shows (Fig. 3), at IE034 corn massfraction, is all can stablize the specific band that amplifies 258bp in the sample that more than 0.05% (contains 0.05%), shows that the sensitivity of detection method of the present invention can reach 0.05%.
Should be noted that; above-described embodiment is specific embodiments of the invention; but embodiments of the present invention are not restricted to the described embodiments; all distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.

Claims (6)

1. transgenic corns IE034 specificity of transformant qualitative PCR detects primer, it is characterized in that:
Upstream primer sequence, IE034-F1:5 '-TGCAGGAGAAGTTTGATGGA-3 ' (SEQ ID NO:1);
Downstream primer sequence, IE034-R1:5 '-GGGTTTCGCTCATGTGTTG-3 ' (SEQ ID NO:2).
2. transgenic corns IE034 specificity of transformant qualitative PCR detection method, is characterized in that, comprises the following steps:
(1) synthetic upstream primer claimed in claim 1 and downstream primer;
(2) prepare the genomic dna solution of testing sample;
(3) preparation qualitative PCR reaction system;
(4) operation qualitative PCR response procedures;
(5), if amplify the specific band of 258bp in testing sample, in this sample, contain transgenic corns IE034 transformant composition; If do not amplify the band of 258bp in testing sample, in this sample, do not contain transgenic corns IE034 transformant composition.
3. transgenic corns IE034 specificity of transformant qualitative PCR detection method according to claim 2, it is characterized in that, the concentration of the described synthetic upstream primer of step (1) and downstream primer is 10 μ mol/L, and the concentration of the DNA solution of the described preparation of step (2) is 50ng/ μ L.
4. according to the transgenic corns IE034 specificity of transformant qualitative PCR detection method described in claim 2 and 3, it is characterized in that, the cumulative volume of the preparation qualitative PCR reaction system that step (3) is described is 25 μ L, comprises following component: 10 * PCR damping fluid, 2.5 μ L, dNTPs mixing solutions 2 μ L, upstream primer 0.5 μ L, downstream primer 0.5 μ L, Taq archaeal dna polymerase 0.125 μ L, DNA solution 2 μ L, water 17.375 μ L.
5. according to the transgenic corns IE034 specificity of transformant qualitative PCR detection method described in claim 2～4, it is characterized in that, the described qualitative PCR response procedures of step (4) is: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out altogether 35 circulations; 72 ℃ are extended 7min.
6. transgenic corns IE034 specificity of transformant qualitative PCR detection kit, comprises following component: 10 * PCR damping fluid, dNTPs mixing solutions, upstream primer claimed in claim 1 and downstream primer, Taq DNA polysaccharase and water.
CN201410457456.0A 2014-09-10 2014-09-10 Qualitative PCR (Polymerase Chain Reaction) detection primer, qualitative PCR detection method and qualitative PCR detection kit for transgenic maize Pending CN104195256A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410457456.0A CN104195256A (en) 2014-09-10 2014-09-10 Qualitative PCR (Polymerase Chain Reaction) detection primer, qualitative PCR detection method and qualitative PCR detection kit for transgenic maize

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410457456.0A CN104195256A (en) 2014-09-10 2014-09-10 Qualitative PCR (Polymerase Chain Reaction) detection primer, qualitative PCR detection method and qualitative PCR detection kit for transgenic maize

Publications (1)

Publication Number Publication Date
CN104195256A true CN104195256A (en) 2014-12-10

Family

ID=52080618

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410457456.0A Pending CN104195256A (en) 2014-09-10 2014-09-10 Qualitative PCR (Polymerase Chain Reaction) detection primer, qualitative PCR detection method and qualitative PCR detection kit for transgenic maize

Country Status (1)

Country Link
CN (1) CN104195256A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105483241A (en) * 2015-12-26 2016-04-13 吉林省农业科学院 LAMP (Loop-mediated isothermal amplification) detection primer group and kit and detection method for gene cry1Ie of transgenic plants
CN105543238A (en) * 2016-01-07 2016-05-04 中国检验检疫科学研究院 Transgenic maize IE 034 exogenous insertion element 3'-end flanking sequence and detection method
CN114292842A (en) * 2021-12-29 2022-04-08 四川省农业科学院农业质量标准与检测技术研究所 Amplification primer and detection method for realizing specific qualitative PCR detection of transgenic corn 2A-7 transformation event

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102604940A (en) * 2012-03-13 2012-07-25 中国农业科学院作物科学研究所 Flanking sequence of foreign insert segment in maize genetic modification event IE034 and application of flanking sequence

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102604940A (en) * 2012-03-13 2012-07-25 中国农业科学院作物科学研究所 Flanking sequence of foreign insert segment in maize genetic modification event IE034 and application of flanking sequence

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105483241A (en) * 2015-12-26 2016-04-13 吉林省农业科学院 LAMP (Loop-mediated isothermal amplification) detection primer group and kit and detection method for gene cry1Ie of transgenic plants
CN105543238A (en) * 2016-01-07 2016-05-04 中国检验检疫科学研究院 Transgenic maize IE 034 exogenous insertion element 3'-end flanking sequence and detection method
CN105543238B (en) * 2016-01-07 2020-04-07 中国检验检疫科学研究院 3' end flanking sequence of exogenous insertion segment of transgenic maize IE034 and detection method
CN114292842A (en) * 2021-12-29 2022-04-08 四川省农业科学院农业质量标准与检测技术研究所 Amplification primer and detection method for realizing specific qualitative PCR detection of transgenic corn 2A-7 transformation event

Similar Documents

Publication Publication Date Title
CA2673748C (en) Transgenic plant event detection
CN103923999B (en) The qualitative PCR detection method of transgenic pest-resistant herbicide-resistant paddy rice G6H1 and derived varieties thereof
Shrestha et al. Advances in detection of genetically engineered crops by multiplex polymerase chain reaction methods
Fraiture et al. An innovative and integrated approach based on DNA walking to identify unauthorised GMOs
CN102206632A (en) Para-gene for exogenous insertion vector of transgenic maize transformation event MON88017 and application thereof
CN103924000B (en) LAMP detecting primer group, kit and detecting method for cry3A gene in transgenic plant
EP3228187A1 (en) Novel means and methods for cereal pathogen resistance
CN104195256A (en) Qualitative PCR (Polymerase Chain Reaction) detection primer, qualitative PCR detection method and qualitative PCR detection kit for transgenic maize
CN104195257A (en) Primers, probe and method for specific quantitative PCR detection of transformant of transgenic maize IE034
Su et al. Integrated structure and event-specific real-time detection of transgenic cry1Ac/SCK rice Kefeng 6
CN103966208B (en) Transgenic paddy rice PA110-15 external source Insert Fragment flanking sequence and application
CN103773867B (en) The LAMP detection primer group of cry2Ab gene, test kit and detection method in genetically modified crops
Wang et al. Multiplex event-specific qualitative polymerase chain reaction for detecting three transgenic rice lines and application of a standard plasmid as a quantitative reference molecule
CN102649976A (en) LAMP (Loop-mediated isothermal amplification) detection primer set of transgenic soybeans GTS 40-3-2 as well as derived varieties thereof, kit and detection method
CN102134603A (en) Primer, probe, test kit and method for testing genetically modified rice or products thereof
La Paz et al. Interlaboratory transfer of a real-time polymerase chain reaction assay for quantitative detection of genetically modified maize event TC-1507
Pan et al. Interlaboratory trial validation of an event-specific qualitative polymerase chain reaction-based detection method for genetically modified RT73 rapeseed
Dong-Myung Quantitative analysis of two genetically modified maize lines by real-time PCR
CN105063207B (en) Genetically engineered soybean MON87705 LAMP detection primer group, kit and detection method
KR100723069B1 (en) Qualitative and quantitative detection methods using specific primers probes and standard plasmid for GM corns
CN103509875A (en) Detection of CaMV 35S promoter and nos terminator by adopting RPA (Recombinase Ploymerase Amplification) technology
Li et al. Proficiency testing results and analysis of qualitative detection of genetically modified component in maize.
CN102634592A (en) LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic maize BT176 and derived varieties thereof
Su et al. A construct-specific qualitative and quantitative PCR detection method of transgenic maize BVLA430101
CN104388579A (en) Arm-PCR detection method of transgenic soybeans and derived varieties thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C12 Rejection of a patent application after its publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20141210