CN106048049A - LAMP primer group for detecting transgenic soybean GTS 40-3-2 and detection method - Google Patents
LAMP primer group for detecting transgenic soybean GTS 40-3-2 and detection method Download PDFInfo
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- CN106048049A CN106048049A CN201610566417.3A CN201610566417A CN106048049A CN 106048049 A CN106048049 A CN 106048049A CN 201610566417 A CN201610566417 A CN 201610566417A CN 106048049 A CN106048049 A CN 106048049A
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- 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
Abstract
The invention relates to an LAMP primer group for detecting transgenic soybean GTS 40-3-2 and a detection method. The method comprises the steps of extracting DNA of a soybean variety to be detected, performing amplification on a template DNA through four designed specific LAMP primers, performing 2 percent agarose gel electrophoresis detection, and determining whether the soybean to be detected is transgenic soybean GTS 40-3-2 by judging whether a strap electrophoresis strip appears or not. According to the method, the DNA amount of each system template is limited to 0.001ng. The primer group has the advantages of high simplicity, high speediness, high specificity and high sensitivity, and a new LAMP detection primer group is provided for the detection of the transgenic soybean GTS 40-3-2.
Description
Technical field
The present invention relates to agricultural and technical field of plant quarantine, be specifically related to genetically engineered soybean Roundup Ready (GTS
LAMP detection primer group 40-3-2) and detection method.
Background technology
Semen sojae atricolor is one of the most important oil crop, 2015, and soybean oil accounts for the 44% of Chinese vegetable oils raw material,
The annual demand of China Semen sojae atricolor is about 85,000,000 tons.But, soybean in China was difficult to meet domestic demand, from 1996
Since, China starts a large amount of imported soybean, and within 2014, soybean import reaches 73,000,000 tons, accounts for world soybean yield
20%.At present, the Semen sojae atricolor of China's import is essentially from U.S., Argentinian and Brazilian, and wherein, 2014 from imported from America Semen sojae atricolor amount
Account for the 42% of import total amount.And the U.S. be genetically engineered soybean plant big country, it is the most public that the genetically engineered soybean of the U.S. is mainly Meng Shan
The resistance glyphosate genetically engineered soybean (Roundup Ready) that department produces.Wherein, GTS 40-3-2 is by Agrobacterium
The 5-enolpyruvyl shikimic acid-3-phosphate synthase of isolated resistance glyphosate in tumefaciens strain CP4
(EPSPS) it is bred as during gene proceeds to commercial soy kind " A5403 ".
The development of genetically modified crops causes the whole world to human health and the deep worry of Environmental security, therefore, science
Genetically modified crops are carried out effective monitoring, sets up effective detection method, worried for eliminating the common people, promote normal business between country
Industry trade has great importance.At present, many countries have launched respectively strict laws and regulations on the management, to genetically modified food in the world
Carry out strict control.Such as, during European Union specifies food, the content of genetic modification material is necessary for more than 1% indicating.The Chinese government
Regulation, every list in indicate administrative directory and for sell agriculture genetically modified crops all should indicate.
At present, the method for detection GMOs is mainly based upon the detection of target protein and detection based on purpose nucleic acid.Its
In, it is mainly ELISA detection and colloidal gold fast detecting test paper strip method, both approaches valency with albumen for mesh object detection method
Lattice are expensive, are difficult to popularize in routine testing.Detection method based on nucleic acid is mainly hybridization and PCR skill
Art.At present, qualitative PCR and real-time quantitative PCR detection method it are widely used that both at home and abroad.But, PCR method reaction system and
Operating process is complicated, needs professional person.It addition, PCR proliferation time is 1.5-2 hour, required PCR instrument price is 5-10 ten thousand yuan
Left and right.Therefore, find a kind of quickly, easy, easily operation and accurately genetically modified crops detection method imperative.
Loop-mediated isothermal amplification technique (Loop-Mediated Isothermal Amplification, LAMP) is Japan
The gene amplification that Rong Yan chemistry Zhu Shi commercial firm developed before and after 2000, have fast and convenient, need not large-scale instrument, behaviour
Make the advantages such as convenient.At present, utilized GTS 40-3-2 insertion sequence and Semen sojae atricolor endogenous gene border sequence, CaMV35S sequence,
GTS40-3-2 exogenous gene insertion sequence etc. develops the serial LAMP detection method of genetically engineered soybean GTS 40-3-2.
The present invention utilizes 5 '-junction insertion sequence (the GenBank accession of genetically engineered soybean GTS 40-3-2
Number:AJ308514.1) devise new LAMP primer, can be used for the quick detection of genetically engineered soybean GTS 40-3-2.This
Invention has important potential using value for detecting genetically engineered soybean GTS 40-3-2 quickly and accurately.
Summary of the invention
There is provided new solution for genetically engineered soybean GTS 40-3-2 detection, the invention provides a kind of genetically engineered soybean
The LAMP method for quick of GTS40-3-2, it is therefore intended that provide the LAMP of a kind of quick, accurate, sensitive GTS40-3-2 fast
Speed detection method.
To achieve these goals, the present invention is by the following technical solutions:
The LAMP method for quick of a kind of genetically engineered soybean GTS40-3-2, comprises the steps:
A) design primer, primer sequence is:
B) extraction of soybean gene group: utilize Cwbiotech DNA extraction kit to extract soybean gene group.Ultraviolet spectrometry
Photometer Nanodrop 2000 detects DNA extraction quality and concentration, chooses the DNA that OD260/OD280 detected value is 1.7-1.9
Sample expands for LAMP, and-20 DEG C save backup.
C) LAMP amplification: the genome obtaining step b) carries out LAMP amplification, and LAMP system arranges reaction temperature gradient
It is 61 DEG C, 63 DEG C and 65 DEG C；Total system 25 μ L: wherein primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ
ID NO:1), B3 (SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM or 0.6 μM: 0.6 μM: 0.1 μM: 0.1 μ
M；4mM MgSO4,1.6mM dNTPs,0.8M betaine(Sigma-Aldrich,Tokyo,Japan),1μL Bst DNA
polymerase(New England Biolabs,Beijing,China)；The amplified reaction time is 40min or 60min.
D) analyze: the amplified production obtaining step c) carries out 2% agarose gel electrophoresis.2% agarose gel electrophoresis
Go out belt-like strip for sample containing genetically engineered soybean GTS 40-3-2, blank for not containing genetically engineered soybean GTS 40-
The sample of 3-2.
As preferably, primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4) in step c), F3 (SEQ ID NO:
1), B3 (SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, and the amplified reaction time is 40min or 60min,
LAMP amplified reaction temperature is 61 DEG C or 63 DEG C.
As preferably, primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4) in step c), F3 (SEQ ID NO:
1), B3 (SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, and the amplified reaction time is 40min, LAMP amplification
Reaction temperature is 61 DEG C.
The invention has the beneficial effects as follows:
The present invention utilizes the 5 '-junction insertion sequences of the genetically engineered soybean GTS 40-3-2 that GenBank announces
(GenBank accession number:AJ308514.1) gene order design LAMP (loop-mediated
Isothermal amplification) primer, establish the LAMP detection system of genetically engineered soybean GTS 40-3-2, and to instead
Condition is answered to be optimized.LAMP at primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1),
B3 (SEQ ID NO:2) ratio is under the 25 μ L systems of 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, and 61 DEG C of reaction 40min are optimal
Reaction condition.Result shows, the LAMP detection system of foundation is simple, quick, sensitivity is good, specificity is high, at genetically engineered soybean
The LAMP context of detection of GTS 40-3-2 has a high potential.
Accompanying drawing explanation
The 2% sepharose electrophoresis figure of Fig. 1 LAMP primer amplification GTS 40-3-2；
In figure: M is marker, 1,2 swimming band is GTS 40-3-2 for template DNA；3,4 swimming bands add water comparison for template.
Fig. 1 illustrates: belt-like strip occurs in 1,2 swimming band, shows that the LAMP primer of design and amplification system can amplify GTS
The specific band of 40-3-2；There is not band in 3,4 swimming bands, show that reaction system is not affected by DNA pollution.
Fig. 2 is specific detection figure；
In Fig. 2: M, marker；1-12 swimming band represent respectively template DNA from genetically engineered soybean (GTS 40-3-2,
MON87701, CV127,356043, MON89788,305423) and Semen Tritici aestivi, Semen sojae atricolor, Semen Maydis, Brassica campestris L, Oryza sativa L. and blank (water) are right
According to；
Fig. 2 illustrates: belt-like strip occurs in 1 swimming band, shows genetically engineered soybean GTS 40-3-2 to be detected；2-11 swimming band occurs
Blank, shows to be not detected by genetically engineered soybean GTS 40-3-2, illustrates that genetically engineered soybean GTS 40-3-2 is had by the primer of design
There is specificity；There is blank in 12 swimming bands, show that reaction system is not affected by DNA pollution.
Fig. 3 is susceptiveness detection figure；
In Fig. 3, M is that marker, 1-9 swimming band represents every individual system (25 μ L) interior DNA profiling genome amount respectively respectively
60,40,10,1,0.1,0.01,0.005,0.001 and 0.0001ng；
Fig. 3 illustrates: banding swimming band all occurs in 1-8 swimming band, shows that when template DNA is 0.001-60ng, this system can detect that
Genetically engineered soybean GTS 40-3-2；9 swimming bands are blank, show can not detect when template DNA is 0.0001ng GTS 40-3-2.Cause
This, this system Monitoring lower-cut is 0.001ng/25 μ L.
Detailed description of the invention
Below by being embodied as case, technical scheme is further illustrated.Should be appreciated that this
Inventive embodiment is not limited to the following examples, and any pro forma accommodation being made the present invention and/or change are all
Scope will be fallen into.
Method in following embodiment, if no special instructions, is the conventional method of this area.
LAMP method Bst DNA polymerase is provided by New England Biolabs company, and Soybean genomic DNA makes
Extracting by Cwbiotech DNA extraction kit, agarose Agarose H (the raw work in Shanghai), Betaine is by Sigma-
Aldrich provides.
Utilize the 5 '-junction insertion sequence (GenBank of the genetically engineered soybean GTS 40-3-2 that GenBank announces
Accession number:AJ308514.1) gene order design LAMP (loop-mediated isothermal
Amplification) primer.
Embodiment 1
The LAMP method for quick of genetically engineered soybean GTS40-3-2, comprises the steps:
A) design primer, primer sequence is:
B) extraction of soybean gene group: weigh 2-3g soybean seed, grinds, utilizes Cwbiotech DNA extraction kit
Extract soybean gene group.Ultraviolet spectrophotometer Nanodrop 2000 detects DNA extraction quality and concentration, chooses OD260/
OD280 detected value is that the DNA sample of 1.7-1.9 expands for LAMP, and-20 DEG C save backup.
C) LAMP amplification: the genome obtaining step b) carries out LAMP amplification, and LAMP system arranges reaction temperature gradient
It it is 61 DEG C；Total system 25 μ L: wherein primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1),
B3 (SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, 4mM MgSO4,1.6mM dNTPs,0.8M
betaine(Sigma-Aldrich,Tokyo,Japan),1μL Bst DNA polymerase(New England
Biolabs,Beijing,China)；Arranging the LAMP amplified reaction time is 40min.
D) analyze: the amplified production obtaining step c) carries out 2% agarose gel electrophoresis.2% agarose gel electrophoresis
Go out belt-like strip for sample containing genetically engineered soybean GTS 40-3-2, blank for not containing genetically engineered soybean GTS 40-
The sample of 3-2.
Embodiment 2
The LAMP method for quick of genetically engineered soybean GTS40-3-2, comprises the steps:
A) design primer, primer sequence is:
B) extraction of soybean gene group: weigh 2-3g soybean seed, grinds, utilizes Cwbiotech DNA extraction kit
Extract soybean gene group.Ultraviolet spectrophotometer Nanodrop 2000 detects DNA extraction quality and concentration, chooses OD260/
OD280 detected value is that the DNA sample of 1.7-1.9 expands for LAMP, and-20 DEG C save backup.
C) LAMP amplification: the genome obtaining step b) carries out LAMP amplification, and LAMP system arranges reaction temperature gradient
It it is 63 DEG C；Total system 25 μ L: wherein primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1),
B3 (SEQ ID NO:2) ratio is 0.6 μM: 0.6 μM: 0.1 μM: 0.1 μM, 4mM MgSO4,1.6mM dNTPs,0.8M
betaine(Sigma-Aldrich,Tokyo,Japan),1μL Bst DNA polymerase(New England
Biolabs,Beijing,China)；Arranging the LAMP amplified reaction time is 40min.
D) analyze: the amplified production obtaining step c) carries out 2% agarose gel electrophoresis.2% agarose gel electrophoresis
Go out belt-like strip for sample containing genetically engineered soybean GTS 40-3-2, blank for not containing genetically engineered soybean GTS 40-
The sample of 3-2.
Embodiment 3
The LAMP method for quick of genetically engineered soybean GTS40-3-2, comprises the steps:
A) design primer, primer sequence is:
B) extraction of soybean gene group: weigh 2-3g soybean seed, grinds, utilizes Cwbiotech DNA extraction kit
Extract soybean gene group.Ultraviolet spectrophotometer Nanodrop 2000 detects DNA extraction quality and concentration, chooses OD260/
OD280 detected value is that the DNA sample of 1.7-1.9 expands for LAMP, and-20 DEG C save backup.
C) LAMP amplification: the genome obtaining step b) carries out LAMP amplification, and LAMP system arranges reaction temperature gradient
It it is 65 DEG C；Total system 25 μ L: wherein primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1),
B3 (SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, 4mM MgSO4,1.6mM dNTPs,0.8M
betaine(Sigma-Aldrich,Tokyo,Japan),1μL Bst DNA polymerase(New England
Biolabs,Beijing,China)；Arranging the LAMP amplified reaction time is 60min.
D) analyze: the amplified production obtaining step c) carries out 2% agarose gel electrophoresis.2% agarose gel electrophoresis
Go out belt-like strip for sample containing genetically engineered soybean GTS 40-3-2, blank for not containing genetically engineered soybean GTS 40-
The sample of 3-2.
Susceptiveness detects: will utilize the genetically engineered soybean GTS 40-3-2 genomic DNA dilution that step b) of the present invention obtains,
With ensure every individual system (25 μ L) interior DNA profiling genome amount be respectively 60,40,10,1,0.1,0.01,0.005,0.001 and
0.0001ng, checking LAMP optimization system is for the Detection results of genetically engineered soybean GTS 40-3-2.LAMP result such as Fig. 3 institute
Show: LAMP detection system sensitivity can reach 0.001ng/ system to genetically engineered soybean GTS 40-3-2.
Specific detection: utilize DNA extraction kit (Cwbiotech) to extract soybean gene group.Ultraviolet spectrophotometer
Nanodrop 2000 detects DNA extraction quality and concentration, carries out LAMP and PCR amplification according to DNA profiling concentration 20ng/ system,
The specificity of checking LAMP optimization system.From electrophoretogram 1 it can be seen that contain the amplification sample liquid of genetically engineered soybean GTS 40-3-2
Gradient LAMP band clearly can be run out of, and, can substantially distinguish genetically engineered soybean GTS 40-3-2 (Fig. 2) according to band
With Non-transgenic soybean and other crops.Result shows, the LAMP for genetically engineered soybean GTS 40-3-2 that the present invention sets up
Detection method high specificity.
Herein invention in use genetically engineered soybean (GTS 40-3-2, MON87701, CV127,356043, MON89788,
305423) and the material such as Semen Tritici aestivi, Semen sojae atricolor, Semen Maydis, Brassica campestris L, Oryza sativa L. is China Agricultural University Huang Kunlun professor's laboratory and provides.
Claims (3)
1. the LAMP method for quick of a genetically engineered soybean GTS40-3-2, it is characterised in that step is as follows:
A) design primer, primer sequence is:
B) extraction of soybean gene group: utilize Cwbiotech DNA extraction kit to extract soybean gene group；Uv-spectrophotometric
Meter Nanodrop 2000 detects DNA extraction quality and concentration, chooses the DNA sample that OD260/OD280 detected value is 1.7-1.9
Expanding for LAMP ,-20 DEG C save backup；
C) LAMP amplification: the genome obtaining step b) carries out LAMP amplification, and it is 61 that LAMP system arranges reaction temperature gradient
DEG C, 63 DEG C and 65 DEG C；Total system 25 μ L: wherein primers F IP, the ratio of BIP, F3, B3 is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM
Or 0.6 μM: 0.6 μM: 0.1 μM: 0.1 μM；4mM MgSO4, 1.6mM dNTPs, 0.8M betaine, 1 μ L Bst DNA
polymerase；The amplified reaction time is 40min or 60min；
D) analyze: the amplified production obtaining step c) carries out 2% agarose gel electrophoresis；2% agarose gel electrophoresis goes out band
Shape band is the sample containing genetically engineered soybean GTS 40-3-2, blank for not contain genetically engineered soybean GTS 40-3-2's
Sample.
The LAMP method for quick of a kind of genetically engineered soybean GTS40-3-2 the most as claimed in claim 1, it is characterised in that institute
Stating primers F IP in step c), the ratio of BIP, F3, B3 is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, and the amplified reaction time is
40min or 60min, LAMP amplified reaction temperature is 61 DEG C or 63 DEG C.
The LAMP method for quick of a kind of genetically engineered soybean GTS40-3-2 the most as claimed in claim 1, it is characterised in that institute
Stating primers F IP in step c), the ratio of BIP, F3, B3 is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, and the amplified reaction time is
40min, LAMP amplified reaction temperature is 61 DEG C.
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Cited By (1)
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| CN109055501A (en) * | 2018-10-19 | 2018-12-21 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting genetically engineered soybean GST40-3-2 |
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| CN109055501A (en) * | 2018-10-19 | 2018-12-21 | 浙江省农业科学院 | A kind of primer, probe and kit and method detecting genetically engineered soybean GST40-3-2 |
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Application publication date: 20161026 |