CN103484554B - Primer and method for detecting transgenic maize strain MIR162 - Google Patents

Primer and method for detecting transgenic maize strain MIR162 Download PDF

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CN103484554B
CN103484554B CN201310475423.4A CN201310475423A CN103484554B CN 103484554 B CN103484554 B CN 103484554B CN 201310475423 A CN201310475423 A CN 201310475423A CN 103484554 B CN103484554 B CN 103484554B
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primer
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陈双雅
王嘉鹤
许秋贝
张永祥
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Abstract

The invention discloses a primer and a method for detecting a transgenic maize strain MIR162. The primer consists of a forward primer 193F and a reverse primer 751R, wherein the nucleotide sequence of the forward primer 193F is shown as SEQ ID No:1, and the nucleotide sequence of the reverse primer 751R is shown as SEQ ID No:2. The invention also discloses a kit comprising the primer. The detection method has the characteristics of high specificity, rapidness, simplicity, convenience, high accuracy and high sensitivity, has high actual application value in management and inspection and quarantine of transgenic products and is particularly suitable for popularization and application in local laboratories.

Description

Primer and method that a kind of transgenic corns strain MIR162 detects
Technical field
The present invention relates to biological technical field, be specifically related to primer and method that a kind of transgenic corns strain MIR162 detects.
Background technology
Genetically modified crops development in recent years is swift and violent, and all kinds of transgenic plant products of countries in the world approval commercialization plantation are more and more.The service of International Agriculture biotechnology applications organizes (ISAAA) up-to-date research report to claim, within 2012, global genetically modified crops cultivated area reaches 1.703 hundred million hectares, has increased by 6% compared with 2011, has increased by 100 times compared with 1996 (1,700,000 hectares).Wherein, transgenic corns is the second-biggest-in-the-world genetically modified crops that are only second to genetically engineered soybean.In all commercial genetically modified crops, transgenic corns strain is maximum, has 65 transformation events.
But because transgenic technology may produce new toxin and anaphylactogen, cause the reasons such as the genetic drift of resistant gene, international community still has sizable dispute to the security of genetically modified food.World many countries including China has all been put into effect transgenic product laws and regulations on the management, requires transgenic product detect, identify and manage.
Since two thousand nine, China's import corn increases substantially, and 520.74 ten thousand tons of annual corn imports in 2012, increase by 197% on a year-on-year basis, and main body entrance source place is the U.S..But China is permitted importable transgenic corns strain and is only had 13.According to China's laws and regulations, must to immigration corn carry out transgenosis detection with get rid of do not ratify strain enter the territory.
MIR162 is the transgenic corns of the anti-lepidopterous insects developed by Syngenta Co.,Ltd, passed through the plantation license of the U.S., Canada, Argentina, Brazil, Japan and the state such as Russian, but the Ministry of Agriculture does not ratify this transgenic corns strain import.For preventing that illegal genetically modified organism and products thereof from planting at home and selling, very important to the detection of this transgenic corns strain.
The detection of transgene component mainly adopts TaqMan real time fluorescent PCR method and regular-PCR method.TaqMan real-time fluorescence PCR detection method is highly sensitive, and specificity is good, but needs fluorescent PCR instrument, equipment and reagent cost costliness.Regular-PCR method also has higher sensitivity, but cost is lower, easy and simple to handle, is current the most frequently used crop detection of GMOs method in the world, more can meet the generally requirement in basic unit one line laboratory.
According to the amplification object fragment of PCR, the detection method of transgene component is divided into again foreign gene method for detecting specificity, structure method for detecting specificity and event-specific detection method.
At present, external transgenic corns strain MIR162 examination criteria method is European Union's official method " Quantitative PCR method for detection of maize event MIR162 (Charles Delobel et al., 2011) ", domestic transgenic corns strain MIR162 examination criteria method is SN/T 1196-2012.These 2 standards all adopt real time fluorescent PCR method, and the former is event-specific detection method, and the latter is structure method for detecting specificity.
Other researchs comprise: (1) Deng Tingting etc. has delivered " PCR in real time of transgenic corns MIR162 strain and the research of visible die detection method " (Plant Quarantine, 2012,26 (5), 14-18) for 2012, adopt fluorescence PCR method.(2) Jae-Hwan Kim etc. has delivered method (the J. Korean Soc. Appl. Biol. Chem. 2009 that utilizes multiplex PCR to detect four transgenic corns strains (3272, LY038, MIR162 and MON88017) for 2009,52 (1), 105-107).The method is verified can not specific detection go out MIR162 strain (see figure 1) in 14 transgenic strains.(3) road wave making etc. applied for patent " for detection of Auele Specific Primer, probe and the application thereof of transgenic corns MIR162 " (CN201210053592) and " for detection of Auele Specific Primer and fluorescence labeling probe and the application thereof of transgenic corns MIR162 " (CN201210052534).These two Patent Application Publications detect regular-PCR method and the fluorescence PCR method of MIR162.But the amplified production of regular-PCR primer pair only has respectively 94bp and 98bp in two patents, too small target sequence more easily causes false positive results to occur.In addition, the Blast result of primer sequence in patent application CN201210053592 detection method wherein in Genbank shows, its object fragment is not the border sequence of maize plant genome and exogenous dna fragment, and therefore the method is not MIR162 event-specific detection method.The pcr amplification goal gene of patent application CN201210052534 detection method is Vip3Aa20, and this gene is the specificity anti insect gene from the anti-lepidopterid of bacillus thuringiensis, and therefore this detection method is foreign gene method for detecting specificity.Although only have at present MIR162 to proceed to Vip3Aa gene, if there is other plant strain also to proceed to this foreign gene, also can obtain positive findings by the method.
Summary of the invention
The object of the present invention is to provide that a species specificity is good, detection method is fast and convenient, accuracy is high, highly sensitive, the transgenic corns MIR162 event-specific detection method of suitable substrate one line laboratory applications.
For achieving the above object, the invention provides a kind of primer for detection of transgenic corns strain MIR162, formed by forward primer 193F and reverse primer 751R, the base sequence of described forward primer 193F is SEQ ID No:1, and the base sequence of described reverse primer 751R is SEQ ID No:2.
The present invention also protects a kind of test kit for detection of transgenic corns strain MIR162, it is characterized in that: contain above-mentioned primer.
The present invention also protects a kind of detection method for detection of transgenic corns strain MIR162, it is characterized in that: comprise the step that uses described primer or described test kit.
Described method, taking sample gene group DNA as template, utilizes described primer or described test kit to carry out pcr amplification, and reaction finishes rear detected through gel electrophoresis amplified production, and result is judged.
Reaction system in described pcr amplification is 25 μ L, in the PCR of 0.2 mL reaction tubes, adds 12.5 μ L 2 × PCR premixed liquids, the 193F 1 μ L of 10 μ mol/L, the 751R 1 μ L of 10 μ mol/L, 1 μ L genomic dna, 9.5 μ L ultrapure waters; PCR reaction conditions is: 94 DEG C, and 5 min; 94 DEG C of 30 s, 62 DEG C of 40 s, 72 DEG C of 1 min, totally 35 circulations; 72 DEG C are extended 7 min.
Described result judges it is to detect transgenic corns strain MIR162 when the i.e. conduct of specific PCR amplified band of 559 bp appears in electrophoresis.
In the present invention, the object fragment of detection method is the special maize plant genome of MIR162 strain and the border sequence of exogenous dna fragment, contriver for this sequences Design the special Oligonucleolide primers pair of MIR162 strain, set up the event-specific detection method of transgenic corns MIR162 by round pcr, amplified target sequence is 559bp, and PCR reaction finishes the rear location determination laboratory sample according to specific amplification DNA fragmentation and whether contains transgenic corns MIR162 composition.
In order to achieve the above object, technical scheme of the present invention is:
A kind of specific oligonucleotide primer pair detecting for transgenic corns strain MIR162, the present invention is by analyzing transgenic corns strain MIR162 gene order (HI203349) design, be made up of forward primer 193F and reverse primer 751R, the base sequence SEQ ID No:2 of the base sequence SEQ ID No:1 of described forward primer and described reverse primer is as follows.Use primer pair of the present invention, can amplify special, delicately object fragment.
SEQ?ID?No:1:5’-?gagtcccgcaattatacat?-3’
SEQ?ID?No:2:5’-?ggtttgggcaaaatctcaaac?-3’。
The PCR detection kit detecting for transgenic corns strain MIR162, the test kit that contains primer pair described in claim 1.
The PCR detection method detecting for transgenic corns strain MIR162, comprises the steps:
(1) according to nucleotide sequence information synthetic primer 193F and the 751R of above-mentioned primer, using above-mentioned primer be mixed with respectively concentration be the solution of 10 μ mol/L as test kit, for subsequent use;
(2) extract transgenic corns strain MIR162 genomic dna solution for standby;
(3) taking sample gene group DNA as template, utilize described specific oligonucleotide primer 193F and 751R to carry out pcr amplification, reaction finishes rear detected through gel electrophoresis amplified production, according to the specific PCR amplified band result of determination that whether occurs 559 bp.
Described PCR reaction system is 25 μ L, in the PCR of 0.2 mL reaction tubes, adding PCR reaction system is 25 μ L, in the PCR of 0.2 mL reaction tubes, add 12.5 μ L 2 × PCR premixed liquids, 1 μ L 193F(10 μ mol/L), 1 μ L 751R(10 μ mol/L), 1 μ L genomic dna, 9.5 μ L ultrapure waters.
Described PCR reaction conditions is: 94 DEG C, and 5 min; 94 DEG C of 30 s, 62 DEG C of 40 s, 72 DEG C of 1 min, totally 35 circulations; 72 DEG C are extended 7 min.
Described result of determination be there are 559 bp according to electrophoresis specific PCR amplified band as the conclusion that detects transgenic corns strain MIR162.
Detection method in the application is the strain specificity regular-PCR detection method of transgenic corns MIR162, and specificity is good, highly sensitive.Goal gene fragment is 559bp, is difficult for causing false positive to occur when electrophoresis detection.It is a kind of detection method of applicable laboratories application.
Beneficial effect of the present invention is: the present invention, according to transgenic corns strain MIR162 gene order design strain specificity Oligonucleolide primers pair, utilizes PCR method to detect transgenic corns strain MIR162; Whether detection method of the present invention rapidly and accurately judgement sample has transgenic corns strain MIR162, have the advantages that specificity is good, detection method is fast and convenient, accuracy is high, highly sensitive, in transgenic product management and inspection and quarantine real work, there are wide using value and market outlook, be particularly useful for the promotion and application in basic unit one line laboratory.
Brief description of the drawings
Fig. 1 is the test-results figure that (2009) detection methods such as Jae-Hwan Kim detect 14 kinds of transgenic corns strains and non-transgenic corn.
Fig. 2 is the test-results figure that PCR of the present invention detects MIR162 and other 13 kind of plant species.
Fig. 3 is the test-results figure that PCR of the present invention detects 14 kinds of transgenic corns strains and non-transgenic corn.
Fig. 4 is the PCR detection method sensitivity test result figure of transgenic corns strain MIR162 of the present invention.
Embodiment
Describe embodiments of the invention below in detail, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has the element of identical or similar functions from start to finish.Be exemplary below by the embodiment being described with reference to the drawings, be intended to for explaining the present invention, and can not be interpreted as limitation of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Transgenic corns standard substance Bt 176 in the present invention, Bt11, MON810, GA21, NK603, MON863,1507,3272, MIR604,59122,98140 and genetically engineered soybean standard substance GTS40-3-2 purchased from IRMM(European standard material institute); Transgenic corns standard substance MIR162, MON89034, MON88017 and genetically modified rape GT 73 are purchased from AOCS(U.S. oils chemist association); No. 6, transgenic paddy rice BT63 and Ke Feng are from China Inst. of Quarantine Inspection Sciences; Other plant sample is non-transgenic sample, purchased from market.2 × PCR premixed liquid is purchased from Dongsheng bio tech ltd, Guangdong, and its component is 100 mM KCl, 20 mM Tris-Cl, 3 mM MgCl 2, 400 mM dNTP mixtures, 0.1 U/ μ LTaq archaeal dna polymerase and tetrabromophenol sulfonphthalein etc.
embodiment 1: the detection of corn sample
1. design of primers is with synthetic:
According to transgenic corns strain MIR162 gene order (Genbank sequence number HI203349), design primer, primer is made up of forward primer 193F and reverse primer 751R, and wherein the concrete sequence of the sequence SEQ ID No:2 of the sequence SEQ ID No:1 of forward primer 193F and reverse primer 751R is as follows:
SEQ?ID?No:1:5’-?gagtcccgcaattatacat?-3’
SEQ?ID?No:2:5’-?ggtttgggcaaaatctcaaac?-3’。
Above-mentioned primer is by TAKARA company.Be mixed with respectively concentration and be the solution of 10 μ mol/L as test kit, for subsequent use.
the extraction of genomic dna:
DNA extraction method referring to " the molecular cloning experiment guide third edition " (Pehanorm Brooker D.W Russell work. the yellow training hall of the J. of Science Press 2002 [U.S.] waits to be translated).
amplification:
3.1 PCR reaction system and reaction conditionss
Taking the genomic dna of said extracted as template, carry out PCR reaction.
PCR reaction system is 25 μ L, in the PCR of 0.2 mL reaction tubes, add 12.5 μ L 2 × PCR premixed liquids, 1 μ L 193F(10 μ mol/L), 1 μ L 751R(10 μ mol/L), 1 μ L genomic dna (10 ng ~ 50 ng), 9.5 μ L ultrapure waters.
Sample hose is put into PTC-200 PCR instrument (Bio-Rad company of the U.S.), reaction conditions is set is: 94 DEG C, 5 min; 94 DEG C of 30 s, 62 DEG C of 40 s, 72 DEG C of 1 min, totally 35 circulations; 72 DEG C are extended 7 min.
the electrophoresis detection of amplified production
Prepare the sepharose of 1 ~ 1.5 % with 1 × TAE electrophoretic buffer.Get point sample in 10 holes of μ L pcr amplification product on gel slab.With the DNA Marker(TAKARA company of 100 bp) do molecular weight marker.Voltage (3V/cm ~ 5V/cm) is set, and electrophoresis time is 50 min ~ 60 min.With gel imaging instrument (Alpha Imager EP) Taking Pictures recording.
Adopting the transgenic corns strain MIR162 specific gene fragment length of above-mentioned forward primer 193F and reverse primer 751R specific PCR amplification is 559bp, whether produces per sample in the pcr amplification band judgement sample of 559bp whether contain transgenic corns strain MIR162.
embodiment 2: the specificity of the PCR method detecting for transgenic corns strain MIR162 is determined
Get the seed of other plant species or 13 parts of fruits, 14 parts of different transgenic corns strain standard substance, 1 part of non-transgenic corn, extracts respectively genomic dna as stated above, the performing PCR of going forward side by side amplification, detected through gel electrophoresis result.Result shows to only have transgenic corns strain MIR162 specificity to produce the amplified band of 559 bp, and detected result as shown in Figures 2 and 3.
The primer and method are shown in embodiment 1.
Fig. 2 is the experimental result picture that application primer of the present invention and detection method detect MIR162 and other 13 kind of plant species; Swimming lane 1-14 is followed successively by transgenic corns MIR162 (10%), non-transgenic soybean, rice, potato, pea, mung bean, wheat, Carrot Seed, tomato, pawpaw, transgenic paddy rice BT63, rich No. 6 of section, genetically engineered soybean GTS40-3-2 and genetically modified rape GT 73.As can be seen from the figure, only have transgenic corns MIR162 to produce 559bp band (swimming lane 1), other samples are showed no amplified band.
Fig. 3 is the experimental result picture that application primer of the present invention and detection method detect 14 kinds of transgenic corns strains and non-transgenic corn, wherein, swimming lane 1-15 is followed successively by transgenic corns MIR162 (10%), transgenic corns 59122(10%), transgenic corns Bt11(10%), transgenic corns NK603(10%), transgenic corns Bt-176(5%), transgenic corns MON810(5%), transgenic corns MON89034(100%), transgenic corns 1507(10%), transgenic corns MIR604(10%), transgenic corns MON88017(10%), transgenic corns 98140(10%), transgenic corns 3272(100%), transgenic corns GA21(10%), genetically modified corn MON 863 (10%) and non-transgenic corn.As can be seen from the results, only have transgenic corns MIR162 to produce 559bp band (swimming lane 1), other samples are showed no amplified band.
Can find out from the result of Fig. 2 and 3, other plant species, other transgenic corns strains and non-transgenic corn all do not produce the amplified band of 559 bp.Show that the PCR method of setting up has good specificity, can be used for the detection of transgenic corns strain MIR162.
embodiment 3: the Sensitivity determination of the PCR method detecting for transgenic corns strain MIR162
Transgenic corns strain MIR162 standard substance are mixed with non-transgenic corn seed, be mixed with the corn seed sample that the relative percentage composition of transgenic corns strain MIR162 (w/w) is 10 %, 5 %, 1%, 0.5 %, 0.1 %, 0.05%, 0.01 %, extract respectively genomic dna by above-described embodiment method, carry out again pcr amplification, detected through gel electrophoresis result.Detected result as shown in Figure 4.Fig. 4 is the PCR detection method sensitivity test result figure of transgenic corns strain MIR162 of the present invention.Swimming lane 1-8 is followed successively by transgenic corns strain MIR162 content and is respectively 10 %, 5 %, 1 %, 0.5 %, 0.1 %, 0.05%, 0.01% corn seed sample and the PCR detected result of non-transgenic corn seed.
The primer and method are shown in embodiment 1.
Experimental result shows: transgenic corns MIR162 content is that the sample amplification band of 0.1 % is clear, and transgenic corns MIR162 content is that the sample of 0.05 % also has faint band; Illustrate that the PCR method of setting up at least can successfully detect transgenic corns MIR162 from transgenic corns MIR162 content is the sample of 0.1 %, there is higher sensitivity, meet the requirement of detection.
embodiment 4: (2009) detection methods such as Jae-Hwan Kim detect transgenic corns
According to document (Jae-Hwan Kim etc., 2009) disclosed primer and detection method detect 14 kinds of transgenic corns strains and non-transgenic corn, test-results is shown in Fig. 1: swimming lane 1-15 is followed successively by transgenic corns MIR162 (10%), transgenic corns 59122(10%), transgenic corns Bt11(10%), transgenic corns NK603(10%), transgenic corns Bt-176(5%), transgenic corns MON810(5%), transgenic corns MON89034(100%), transgenic corns 1507(10%), transgenic corns MIR604(10%), transgenic corns MON88017(10%), transgenic corns 98140(10%), transgenic corns 3272(100%), transgenic corns GA21(10%), genetically modified corn MON 863 (10%) and non-transgenic corn.As can be seen from the figure,, except the non-transgenic corn of swimming lane 15 has no amplified band, other transgenic corns all produce 140bp amplified band.Illustrate that the disclosed method of document (Jae-Hwan Kim etc., 2009) is not suitable for detecting transgenic corns MIR162.
Although illustrated and described embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention in the situation that not departing from principle of the present invention and aim, amendment, replacement and modification.

Claims (6)

1. for detection of a primer of transgenic corns strain MIR162, be made up of forward primer 193F and reverse primer 751R, the base sequence of described forward primer 193F is SEQ ID No:1, and the base sequence of described reverse primer 751R is SEQ ID No:2.
2. for detection of a test kit of transgenic corns strain MIR162, it is characterized in that, contain primer described in claim 1.
3. for detection of a detection method of transgenic corns strain MIR162, it is characterized in that, comprise that right to use requires the step of test kit described in primer described in 1 or claim 2.
4. described in claim 3, detect the detection method of transgenic corns strain MIR162, it is characterized in that, taking sample gene group DNA as template, utilize described in claim 1 test kit described in primer or claim 2 to carry out pcr amplification, reaction finishes rear detected through gel electrophoresis amplified production, and result is judged.
5. described in claim 4, detect the detection method of transgenic corns strain MIR162, it is characterized in that, reaction system in described pcr amplification is 25 μ L, in the PCR of 0.2 mL reaction tubes, add 12.5 μ L 2 × PCR premixed liquids, the 193F 1 μ L of 10 μ mol/L, the 751R 1 μ L of 10 μ mol/L, 1 μ L genomic dna, 9.5 μ L ultrapure waters; PCR reaction conditions is: 94 DEG C, and 5 min; 94 DEG C of 30 s, 62 DEG C of 40 s, 72 DEG C of 1 min, totally 35 circulations; 72 DEG C are extended 7 min.
6. the detection method that detects transgenic corns strain MIR162 described in claim 3, is characterized in that, described result judges it is to detect transgenic corns strain MIR162 when the i.e. conduct of specific PCR amplified band of 559 bp appears in electrophoresis.
CN201310475423.4A 2013-10-12 2013-10-12 Primer and method for detecting transgenic maize strain MIR162 Expired - Fee Related CN103484554B (en)

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CN101548011A (en) * 2006-06-03 2009-09-30 先正达参股股份有限公司 Corn event MIR162
CN102559921A (en) * 2012-03-02 2012-07-11 山东省农业科学院植物保护研究所 Specific primer and fluorescently-labeled probe for detecting transgenosis corn MIR162 and application thereof

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CN101548011A (en) * 2006-06-03 2009-09-30 先正达参股股份有限公司 Corn event MIR162
CN102559921A (en) * 2012-03-02 2012-07-11 山东省农业科学院植物保护研究所 Specific primer and fluorescently-labeled probe for detecting transgenosis corn MIR162 and application thereof

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