CN103484528A - Detection method for detecting Te9 gene in transgenic product, and kit thereof - Google Patents

Detection method for detecting Te9 gene in transgenic product, and kit thereof Download PDF

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CN103484528A
CN103484528A CN201210202227.5A CN201210202227A CN103484528A CN 103484528 A CN103484528 A CN 103484528A CN 201210202227 A CN201210202227 A CN 201210202227A CN 103484528 A CN103484528 A CN 103484528A
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王述柏
高宏伟
周茜
孙敏
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Qingdao Agricultural University
Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
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Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
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    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q2561/113—Real time assay

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Abstract

The invention belongs to a transgenic plant product detection technology, and concretely relates to a detection method for detecting Te9 gene in a transgenic product through a real-time fluorescent PCR technology. Reagents used in the method comprise a PCR amplification reaction solution, an upstream primer Te9-F: 5'-TTTGAGAATGAACAAAAGGACCATAT-3', a downstream primer Te9-R: 5'-GGTTTTCGCTATCGAACTGTGA-3' and a Taqman-MGB probe Te9-MGBP: FAM-ATTCATTAACTCTTCTCCATCC-MGB. The method for detecting the Te9 gene in the transgenic product comprises the following steps: extracting DNA from the transgenic plant product, carrying out real-time fluorescent PCR amplification of the Te9 gene, and analyzing amplification results through fluorescent quantitative PCR instrument random software to carry out result determination. The method has the advantages of fastness, strong specificity, high sensitivity and simple operation.

Description

The detection method that in transgenic product, the Te9 gene test is used and test kit
Technical field
The invention belongs to the detection technique of transgenic plant product, specifically use detection method and the test kit for detection of Te9 gene in real-time fluorescence PCR technology for detection transgenic product.
Background technology
In developing country, population growth and the contradiction be short of food are becoming increasingly acute, reduce in the simultaneous arable land, biology is planted and the deterioration of living environment, transgenic technology is the effective way addressed these problems, transgenic technology has not only greatly improved the efficiency of crop breeding, be bred as some and there are the new variety of outstanding feature, and genetically modified crops are, at aspects such as output, quality or anti-adversity abilities, significant advantage is all arranged, more outstanding is the cost that transgenic technology can greatly reduce agriculture production, alleviates the agroecological environment constantly worsened.Since nineteen eighty-three, the first case transfer-gen plant came out, the world has obtained transfer-gen plant in more than 120 of 35 sections plant, and mainly concentrates on soybean, corn, rape, cotton, potato, watermelon, summer squash and pawpaw.
When enjoying the huge material benefit that transgenic technology brings, potential ecological safety, the food-safety problem of transgenic product also causes people's concern day by day, and various countries formulate one after another corresponding laws and regulations transgenic product is supervised.Except the U.S., Canada, Argentina and hk Hong Kong are carried out, sign is outside-the-system voluntarily, and most countries has all been formulated mandatory sign system as EU countries, Hungary, Switzerland, Poland, Japan, Korea S etc. in the world.China, in calendar year 2001,2002 issuing and implementation " agriculture genetically modified organism security control regulations " and " agriculture genetically modified organism identity management way ", has started the supervision oversight mechanism of agriculture genetically modified organism.Chinese government regulation, everyly list identity management catalogue the agriculture genetically modified crops for selling in, should label, and sign and not sign in accordance with regulations, must not import and sale, therefore, must effectively detect transgenic product.
The at present detection of transgenic product, mainly adopt the method for Protein Detection and DNA detection.Two class methods, for detecting and detection by quantitative of transgene component in variant production, respectively have excellent, shortcoming.The method that the DNA of take is tested object with take the method that albumen is tested object and compare, under the product composition complicated situation, target dna can effectively extract, and is subject to the impact of product mechanism less; In addition, DNA is more stable, is subject to the impact of complete processing unlike protein forms.The Te9 gene is the terminator that derives from pea diphosphoribulose carboxylase gene, can be used as the screening-gene of transgenic plant product, utilizes the Fluorescence PCR assay detection to have advantages of sensitive, special.
Summary of the invention
The invention belongs to the detection technique of transgenic plant product; specifically use the detection method of Te9 gene in real-time fluorescence PCR technology for detection transgenic product; to overcome the limitation of detection method in some product detects based on albumen; for detecting, transgenic product provides effective tool; thereby strengthen the supervision supervision of transgenic product; guarantee the consistence of Product labelling, the right to know of Protection of consumer and preference.
Cardinal principle of the present invention is: for conserved sequence, design one group of primer (upstream primer: Te9-F:5 '-tttgagaatgaacaaaaggaccatat-3; Downstream primer Te9-R:5 '-ggttttcgctatcgaactgtga-3 '; Taqman-MGB probe Te9-MGBP:FAM-attcattaactcttctccatcc-MGB.)。Adjust while detecting, template DNA is after being heated to 94-95 ℃ of certain hour, and the DNA double chain dissociates, primer and Taqman-MGB probe and template specificity set.5 ends of MGB probe are marked with report group, and 3 ends, by non-fluorescent quenching group, also are connected with MGB simultaneously and modify group (MinorGroove Binder).When probe is complete, the fluorescence that report group is launched can be quenched group and absorb, and instrument can't detect signal.Along with the carrying out of PCR, the Taq enzyme is in the chain extension process, and while running into the probe of being combined with template, its 3 ' → 5 ' exonuclease activity will cut off probe, report group principle cancellation group, and its energy can not be absorbed, and produces fluorescent signal.Along with the increase of PCR circulation, purpose fragment exponentially increases, and fluorescent signal also synchronously strengthens, and the power of fluorescent signal directly reflects template number.
The detection method of Te9 gene in the transgenic product the present invention relates to, reagent wherein comprises as follows:
(1) pcr amplification reaction liquid A
Comprise 10 * PCR reaction buffer, 0.1-0.4mmol/L dNTP, 2-4mmol/L sal epsom, 1-2U Taq enzyme, 0.2-0.6 μ mol/L upstream primer, 0.2-0.6 μ mol/L downstream primer, 0.2-0.6 μ mol/L Taqman-MGB probe;
Trihydroxy methyl aminomethane-hydrochloric acid, 500mmol/L Repone K and 1% triton x-100 that wherein 10 * PCR reaction buffer contains 100mmol/L pH8.8;
Upstream primer Te9-F:5 '-tttgagaatgaacaaaaggaccatat-3 '; Downstream primer Te9-R:5 '-ggttttcgctatcgaactgtga-3 '; Taqman-MGB probe Te9-MGBP:FAM-attcattaactcttctccatcc-MGB.
Wherein the volume ratio of upstream primer, downstream primer, Taqman-MGB probe is: 1: 1: 1;
Wherein the mass ratio of the mixture of four kinds of thymus nucleic acids in dNTP is dTTP: dATP: dGTP: dCTP=1: 1: 1: 1.
(2) positive control dna
Above described pcr amplification reaction liquid A, the best group of every pipe 24 μ L becomes: 2.5 μ L 10 * PCR reaction buffers, 2 μ L 2.5mmol/LdNTP (mixtures of four kinds of thymus nucleic acids), 2 μ L 25mmol/L sal epsom, 1 μ L 10 μ mol/L upstream primers, 1 μ L 10 μ mol/L downstream primers, 1 μ L 10 μ mol/L Taqman-MGB probes, 0.3 μ L Taq enzyme, 14.2 μ L ddH 2o.
Use the mentioned reagent box to detect the method for Te9 gene in the transgenic plant product, comprise the following steps successively (1)-(3):
(1) extraction of sample DNA to be checked
DNA extraction can adopt the DNA extraction test kit.
(2) real-time fluorescence PCR of Te9 gene amplification in the transgenic plant product
A. add 1 μ L sample DNA to be checked in the reaction tubes that 24 μ L pcr amplification reaction liquid A are housed, mix.
B. the PCR reaction tubes is put into to the fluorescent PCR instrument, by following reaction conditions, completes pcr amplification:
95 ℃ of 3min, 1 circulation;
95 ℃ of 30s, 60 ℃ of 30s, totally 40 circulations.
(3) application quantitative real time PCR Instrument accompanying software, analysing amplified result.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1
Detect as follows genetically modified rape GT 73, use MS1xRF1, MS1xRF2, MS8xRF3 transgene rape strain to do negative control:
Comprise 10 * PCR reaction buffer, 0.2mmol/L dNTP, 2mmol/L sal epsom, 1.5U Taq enzyme, 0.4 μ mol/L upstream primer Te9-F:5 '-tttgagaatgaacaaaaggaccatat-3 '; Downstream primer Te9-R:5 '-ggttttcgctatcgaactgtga-3 '; Taqman-MGB probe Te9-MGBP:FAM-attcattaactcttctccatcc-MGB.Trihydroxy methyl aminomethane-hydrochloric acid, 500mmol/L Repone K and 1% triton x-100 that wherein 10 * PCR reaction buffer contains 100mmol/L pH8.8;
According to following program, detected:
(1) extraction of transgenic plant sample DNA to be measured
Adopt An Biao test kit (GenoDNA Plant Mini Kit)
A. fully grind
B. add 400 μ g Buffer AP1 in the bright material of maximum every 75mg or 15mg dry substance sample, 0.5 μ l RNaseA, fully mix;
C. cultivate 10min at 65 ℃, during turn upside down centrifuge tube 2-3 time;
D. add 130 μ l Buffer AP2, mix, place 5min on ice, the centrifugal 5min of 14000rpm;
E. supernatant is proceeded in new 1.5ml centrifuge tube, add the Buffer AP3/E of 1.5 times of volumes, put upside down 3-4 time, mix;
F. solution is carefully proceeded in the adsorption column of 2ml collection tube, the centrifugal 1min of 10000rpm, if once from not complete, repeatedly application of sample, until the mistake post completes, is outwelled the liquid in collection tube;
G. add 500 μ l Buffer AW1, the centrifugal 1min of 10000rpm, outwell the liquid in collection tube;
H. add 500 μ l Buffer AW2, the centrifugal 1min of 10000rpm, outwell the liquid in collection tube;
I. repeat 8 steps once;
J.14000rpm centrifugal 3min, be transferred to centrifugal column in one clean 1.5ml centrifuge tube;
K. carefully add 50-100 μ l Buffer AE to the adsorption column filter membrane, the standing 1-5min of room temperature, the centrifugal 1min of 10000rpm; 4 ℃ of preservations.But also adopting by equation CTAB method.
(2) real-time fluorescence PCR of transgenic plant sample DNA to be measured amplification
A. add 24 μ L PCR reaction solution A and 1 μ L sample DNA in the PCR reaction tubes, mix.
B. the PCR reaction tubes is put into to quantitative real time PCR Instrument, by following reaction conditions, completes pcr amplification:
95 ℃ of 3min, 1 circulation;
95 ℃ of 30s, 60 ℃ of 30s, totally 40 circulations.
(3) application quantitative real time PCR Instrument accompanying software, analysing amplified result.
Embodiment 2
Detect as follows genetically engineered soybean MON89788, use MS1xRF1, MS1xRF2, MS8xRF3 transgene rape strain to do negative control:
Comprise 10 * PCR reaction buffer, 0.2mmol/L dNTP, 2mmol/L sal epsom, 1.5U Taq enzyme, 0.4 μ mol/L upstream primer Te9-F:5 '-tttgagaatgaacaaaaggaccatat-3 '; Downstream primer Te9-R:5 '-ggttttcgctatcgaactgtga-3 '; Taqman-MGB probe Te9-MGBP:FAM-attcattaactcttctccatcc-MGB.Trihydroxy methyl aminomethane-hydrochloric acid, 500mmol/L Repone K and 1% triton x-100 that wherein 10 * PCR reaction buffer contains 100mmol/L pH8.8;
According to following program, detected:
(1) extraction of transgenic plant sample DNA to be measured
Adopt An Biao test kit (GenoDNA Plant Mini Kit)
A. fully grind
B. add 400 μ g Buffer AP1 in the bright material of maximum every 75mg or 15mg dry substance sample, 0.5 μ l RNaseA, fully mix;
C. cultivate 10min at 65 ℃, during turn upside down centrifuge tube 2-3 time;
D. add 130 μ l Buffer AP2, mix, place 5min on ice, the centrifugal 5min of 14000rpm;
E. supernatant is proceeded in new 1.5ml centrifuge tube, add the Buffer AP3/E of 1.5 times of volumes, put upside down 3-4 time, mix;
F. solution is carefully proceeded in the adsorption column of 2ml collection tube, the centrifugal 1min of 10000rpm, if once from not complete, repeatedly application of sample, until the mistake post completes, is outwelled the liquid in collection tube;
G. add 500 μ l Buffer AW1, the centrifugal 1min of 10000rpm, outwell the liquid in collection tube;
H. add 500 μ l Buffer AW2, the centrifugal 1min of 10000rpm, outwell the liquid in collection tube;
I. repeat 8 steps once;
J.14000rpm centrifugal 3min, be transferred to centrifugal column in one clean 1.5ml centrifuge tube;
K. carefully add 50-100 μ l Buffer AE to the adsorption column filter membrane, the standing 1-5min of room temperature, the centrifugal 1min of 10000rpm; 4 ℃ of preservations.But also adopting by equation CTAB method.
(2) real-time fluorescence PCR of transgenic plant sample DNA to be measured amplification
A. add 24 μ L PCR reaction solution A and 1 μ L sample DNA in the PCR reaction tubes, mix.
B. the PCR reaction tubes is put into to quantitative real time PCR Instrument, by following reaction conditions, completes pcr amplification:
95 ℃ of 3min, 1 circulation;
95 ℃ of 30s, 60 ℃ of 30s, totally 40 circulations.
(3) application quantitative real time PCR Instrument accompanying software, analysing amplified result.
The accompanying drawing explanation
The graphic representation of Fig. 1 modified rape GT 73 fluorescence PCR products.The above positive sample of real horizontal line beyond coordinate axis in figure, below negative sample or blank.
The graphic representation of Fig. 2 soybean MON89788 fluorescence PCR products.The above positive sample of real horizontal line beyond coordinate axis in figure, below negative sample or blank.
Figure ISA00000736933200011

Claims (2)

1. the detection method of Te9 gene in a transgenic product is characterized in that primer and probe wherein:
Upstream primer Te9-F 5 '-TTTGAGAATGAACAAAAGGACCATAT-3 ';
Downstream primer Te9-R:5 '-TTACTGTGTTTTTTATTCGGTTTTCG-3 '.
Probe Te9-MGBP:F AM-ATTCATTAACTCTTCTCCATCC-MGB
2. a right to use requires the method for Te9 gene in a described rapid detection transgenic product, it is characterized in that comprising the following steps: successively
(1) DNA extraction of sample to be checked;
(2) real-time fluorescence PCR of Te9 gene amplification in transgenic product:
Add the sample DNA to be checked of 1 μ L in the reaction tubes of the pcr amplification reaction liquid that 24 μ L are housed, mix.Complete pcr amplification according to following step.
95 ℃ of 3min, 1 circulation;
95 ℃ of 30s, 60 ℃ of 30s, totally 40 circulations.
(3) use amplification curve to analyze the PCR product.
CN201210202227.5A 2012-06-13 2012-06-13 Detection method for detecting Te9 gene in transgenic product, and kit thereof Pending CN103484528A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008030614A2 (en) * 2006-09-08 2008-03-13 Ambrx, Inc. Suppressor trna transcription in vertebrate cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008030614A2 (en) * 2006-09-08 2008-03-13 Ambrx, Inc. Suppressor trna transcription in vertebrate cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
张丽等: "转基因油菜筛查检测策略研究", 《中国油料作物学报》 *
潘良文等: "转基因抗草甘膦油菜内源特异参照基因BnACCg8和外源E9 3’终止子的检测研究", 《中国油料作物学报》 *

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