CN104745708A - LAMP (loop-mediated isothermal amplification) detection kit and detection method for transgenic corn NK603 strain - Google Patents
LAMP (loop-mediated isothermal amplification) detection kit and detection method for transgenic corn NK603 strain Download PDFInfo
- Publication number
- CN104745708A CN104745708A CN201510164657.6A CN201510164657A CN104745708A CN 104745708 A CN104745708 A CN 104745708A CN 201510164657 A CN201510164657 A CN 201510164657A CN 104745708 A CN104745708 A CN 104745708A
- Authority
- CN
- China
- Prior art keywords
- strain
- primer
- transgenic corns
- lamp
- detection kit
- 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
Links
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 72
- 240000008042 Zea mays Species 0.000 title claims abstract description 70
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 70
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 70
- 235000005822 corn Nutrition 0.000 title claims abstract description 70
- 238000001514 detection method Methods 0.000 title claims abstract description 51
- 238000007397 LAMP assay Methods 0.000 title abstract description 8
- 230000003321 amplification Effects 0.000 claims abstract description 22
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 22
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 17
- 239000013641 positive control Substances 0.000 claims abstract description 16
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 11
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 11
- 239000013642 negative control Substances 0.000 claims abstract description 8
- 229960003237 betaine Drugs 0.000 claims abstract description 4
- 208000003643 Callosities Diseases 0.000 claims description 54
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 54
- 239000013612 plasmid Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 5
- 108050009160 DNA polymerase 1 Proteins 0.000 claims description 4
- 101150103518 bar gene Proteins 0.000 claims description 3
- 239000000975 dye Substances 0.000 claims description 3
- 239000011541 reaction mixture Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000001712 DNA sequencing Methods 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 238000013492 plasmid preparation Methods 0.000 claims description 2
- 230000009466 transformation Effects 0.000 claims description 2
- 239000013615 primer Substances 0.000 claims 21
- 239000002987 primer (paints) Substances 0.000 claims 21
- 239000000243 solution Substances 0.000 abstract description 23
- 230000035945 sensitivity Effects 0.000 abstract description 7
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 241000209094 Oryza Species 0.000 description 5
- 235000007164 Oryza sativa Nutrition 0.000 description 5
- 235000009566 rice Nutrition 0.000 description 5
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 4
- 108091040857 miR-604 stem-loop Proteins 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 244000037671 genetically modified crops Species 0.000 description 3
- 235000013617 genetically modified food Nutrition 0.000 description 3
- 238000011901 isothermal amplification Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 108010020183 3-phosphoshikimate 1-carboxyvinyltransferase Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000007403 mPCR Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000219146 Gossypium Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- QLULGSLAHXLKSR-UHFFFAOYSA-N azane;phosphane Chemical compound N.P QLULGSLAHXLKSR-UHFFFAOYSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000010421 standard material Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a LAMP (loop-mediated isothermal amplification) detection kit for transgenic corn NK603 strain. The LAMP kit contains amplification primers of SEQ ID NO:1-6 and also comprises betaine, dNTP, a Bst DNA polymerase buffer solution, a fluorescent dye SYTO-9, Bst DNA polymerase, Mg<2+>, a positive control and a negative control. By adopting the kit provided by the invention to perform loop-mediated isothermal amplification, an amplification product is combined with the fluorescent dye SYTO-9 in a reaction solution, and the amplification result is judged by adopting a real-time fluorescent image after reaction. The kit provided by the invention is good in specificity and high in sensitivity, can detect out NK6-3 of which the content is as low as 0.1% in a mixed sample, and is suitable for performing fast and accurate detection in a basic laboratory.
Description
Technical field
The present invention relates to the detection field of Transgenic corn lines, be specifically related to a kind of LAMP kit and detection method of transgenic corns NK603 strain.
Background technology
Genetically modified crops or genetic modification crop are by importing foreign gene be incorporated in genome, make a certain or some proterties obtain the crop of improveing.Genetically modified crops play important effect in raising resistance and improvement quality.But because transgenic technology may produce the reason such as new toxin and anaphylactogen, the genetic drift causing resistant gene, the security of international community to genetically modified food still has sizable dispute.European Union, the U.S., Japan and other countries have promulgated relevant decree all in succession, carry out identity management, and made relevant regulations to threshold value to genetically modified crops and derivative food thereof.The Ministry of Health of China has also put into effect " genetically modified food hygiene control way " in 2002.Should " way " specify: must identify " food being raw material production with genetically modified animals and plants, microorganism or its direct processed goods and foodstuff additive ".Therefore setting up a set of technology of GMO detection is easily and efficiently implement the important prerequisite of mark system.
Transgenic corns NK603 strain is developed by About Monsanto Chemicals (Monsanto Company), and imported in corn by Bar gene CP4 EPSPS, the CP4 EPSPS albumen of this genes encoding, can make the anti-careless ammonium phosphine of corn.Though this transgenic corns European Union's official approval is at present used for food and feed, its security still receives much concern.
At present the detection of transgenic product is mainly detected based on the PCR of nucleic acid level, comprise multiplex PCR (Multiplex PCR), real-time quantitative PCR (Real-time PCR) etc., but these methods need the heat circulating equipment with real-time fluorescence measuring ability, there is complex operation step, detection time is longer, is not suitable for on-the-spot detection in real time and tracing detection.2000, Japanese scholars Notomi etc. develops a kind of constant temperature nucleic acid amplification method---loop-mediated isothermal amplification technique (loop-mediatedisothermal amplification of novelty, LAMP), the method is for 6 zone design 4 species-specific primers of target gene, improve the specificity of detection, comparatively regular-PCR is high for its amplification efficiency simultaneously, improve sensitivity largely, LAMP technology has simply, fast, the advantage such as high specificity, in detecting for pathogenic bacterium, virus, parasite, anaphylactogen, species identification etc.
Along with increasing transgenic product large-scale commercial is produced, the rapid detection of transgenosis component is subject to extensive concern, and LAMP amplification technique is constant-temperature amplification, can not rely on thermal cycler, and common thermostatical instrument both can detect.Therefore fast and convenient LAMP has been successfully applied to the detection of the crops such as genetically engineered soybean, cotton, corn, paddy rice.
Summary of the invention
The object of the present invention is to provide a kind of LAMP detection kit and detection method of transgenic corns NK603 strain.
A LAMP detection kit for transgenic corns NK603 strain, its reaction system comprises: trimethyl-glycine, dNTP, Bst DNA polymerase buffer liquid, fluorescence dye SYTO-9, Bst archaeal dna polymerase, Mg2+, positive control, negative control; It is characterized in that, also comprise following primer, the base sequence of described primer is as follows:
Outer primer F3:5 '-GACCAGGTAATCTTACCTTTGT-3 '
Outer primer B3:5 '-TGAAACCGCTTTCAAGAGAA-3 '
Inner primer FIP:5 '-GGCCGCGTTAACAAGCTTACTTTTTGGACTATCCCGACTCTCT-3 '
Inner primer BIP:5 '-CTTGGTACCACGCGACACATTTTCTGTTATGGTTCGAGAAGAGAT-3 '
Ring primer LF:5 '-TCGAGGTCATTCATATGCTTGA-3 '
Ring primer LB:5 '-AGTGTTTGAGTGGATCCTGTT-3 '.
Preferably, in described reaction system, trimethyl-glycine concentration is 0.3-1.5mM, dNTP concentration be 0.2-3.5mM, Mg2+ concentration is 2-18mM.
Preferably, the ratio of the concentration of the inner primer described in described reaction system and outer primer is 1-8: 1; The ratio of ring primer and outer primer concentration is 1-2: 1.
Preferably, described reaction system contains 0.2 μM of outer primer, 1.6 μMs of inner primers, 0.4 μM of ring primer.
Preferably, Bst archaeal dna polymerase content is 4-10U, and more preferably, Bst archaeal dna polymerase content is 8U.
Preferably, it is characterized in that described positive control is the genomic dna of transgenic corns NK603 strain.
Preferably, described positive control is the recombinant plasmid containing NK603 strain Bar gene CP4EPSPS gene.
Described positive control recombinant plasmid preparation method is as described below: extract transgenic corns NK603 strain genomic dna, primers F 3/B3 is utilized to carry out PCR, increase and reclaim CP4EPSPS gene and be cloned in vector plasmid, transformation of E. coli competent cell, extract plasmid, DNA sequencing confirms the plasmid that sequence is correct.
Present invention also offers a kind of detection method applying LAMP detection kit detection transgenic corns NK603 strain, comprise following key step:
(1) detected sample genomic dna is extracted, with sample gene group DNA for template;
(2) according to detection sample number, LAMP amplification reaction solution is prepared: get 2 × LAMP reaction mixture 12.5 μ L according to every sample 25 microlitre reaction system, comprising 40mmol/L Tris-HClpH 8.8,20mmol/L KCl, 16mmol/L MgSO
4, 20mmol/L (NH
4)
2sO
4, 0.2%Tween-20,0.8mol/L Betaine, 2.8mmol/L of dNTPs; Add the outer primer B3 solution 0.5 μ L of the outer primer F3 solution 0.5 μ L of 10 μm of ol/L, 10 μm of ol/L; Add the inner primer BIP solution 2 μ L of the inner primer FIP solution 2 μ L of 20 μm of ol/L, 20 μm of ol/L; Add the ring primer LB solution 2 μ L of the ring primer LF solution 2 μ L of 5 μm of ol/L, 5 μm of ol/L; Add fluorescence dye SYTO-9 0.5 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, mixing;
(3) sample DNA template 2 μ L to be detected is added, mixing reaction system;
(4) isothermal duplication: add 20 μ L sealing liquids at PCR pipe inwall; 63 DEG C of isothermal reaction 45min, after reaction terminates, compared with showing fluorescence with positive control;
(5) result interpretation: reaction terminates rear employing real-time fluorescence figure to judge amplification, and the dyestuff used in reaction is fluorescence dye SYTO-9.Described result of determination be according to fluorescence dye SYTO-9 formed amplification curve as the conclusion detecting Transgenic corn lines NK603, otherwise result is not for detect Transgenic corn lines NK603.
The invention provides a kind of stopped pipe fluorescent detection system, the present invention is according to LAMP technology principle, the method is combined with real-time fluorescence technology, in LAMP reaction system, adds fluorescence dye, after amplification terminates, the DNA obtained that increases combines with fluorescence dye, does not need electrophoresis, naked eyes or fluorescence detector conveniently can observe amplification, this real-time fluorescence LAMP method for quick, is adapted at basic unit and detects promoting the use of in unit and on-the-spot fast inspection work.
Beneficial effect of the present invention is: the LAMP detection kit of the transgenic corns NK603 strain of the present invention's development and detection method, be applicable to the detection of transgenic corns NK603 strain, specificity is good, detection sensitivity high (0.1% sample can detect), method quick and precisely, is particularly useful for the promotion and application of basic unit one line laboratory and Site Detection.
Accompanying drawing explanation
The LAMP detection method specificity experiments result figure of Fig. 1: transgenic corns NK603 strain (1-6 be respectively transgenic corns NK603, MON863, MON810, transgenic paddy rice Bt63, transgenic corns biased sample (containing MIR604, Bt11, GA21,59122, Bt176), negative control).
The LAMP detection method sensitivity experiment result figure (1-7 is the transgenic corns of 5%, 1%, 0.5%, 0.1%, 0.05%, 0% respectively, negative control) of Fig. 2: transgenic corns NK603 strain.
Embodiment
In the present invention, transgenic corns standard substance NK603, MON863, MON810 are purchased from national standard material information center, transgenic paddy rice Bt63 and transgenic corns biased sample (containing MIR604, Bt11, GA21,59122, Bt176) presented by Entry-Exit Inspection and Quarantine Bureau of Hunan Province, non-transgenic corn sample is purchased from market.
The design of embodiment 1:LAMP primer sets
According to transgenic corns NK603 strain-specific gene sequence (GenBank sequence number AX342369), design LAMP primer group, LAMP primer group is made up of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer LF, ring primer LB, and the concrete sequence of each primer is in table 1.
Table 1 LAMP primer sequence
Above-mentioned primer is synthesized by Shanghai Sheng Gong biotechnology limited-liability company.Be mixed with respectively concentration be the outer primer F3 solution of 10 μm of ol/L, the outer primer B3 solution of 10 μm of ol/L, the inner primer FIP solution of 20 μm of ol/L, the inner primer BIP solution of 20 μm of ol/L, the ring primer LF solution of 5 μm of ol/L, 5 μm of ol/L ring primer LB solution as test kit, for subsequent use.
Embodiment 2: the preparation of positive control recombinant plasmid T-NK
Extract transgenic corns NK603 strain genomic dna according to CTAB method, utilize forward outer primer F3, oppositely outer primer B3 to carry out PCR, amplification object band, reaction conditions is as follows:
PCR reaction system (50 μ L): primer NK603-F3/NK603-B3 1 μ L, ddH respectively of 10 × PCR buffer 5 μ L, 5U/ μ L Taq archaeal dna polymerase 1 μ L, 10 μm of ol/L
2o 37 μ L, genomic dna 5 μ L; Be placed in PCR amplification instrument to react, response procedures: 94 DEG C of 4min denaturations; 95 DEG C of sex change 30sec, 58 DEG C of annealing 30sec, 72 DEG C of extensions 30sec, totally 30 circulations; 72 DEG C extend 5min.
Reclaim object band, be connected with carrier pMD18-T, transform DH5 α competent cell, extract plasmid, PCR sends to the order-checking of Shanghai Sheng Gong biotechnology company limited after identifying the positive.The plasmid markers that sequence is correct is the positive control of T-NK as transgenic corns NK603 strain.
The preparation of recombinant plasmid, is not limited only to the pMD18-T carrier in the present embodiment, and any common molecular clone common carrier, intestinal bacteria all can be used for preparing positive control plasmid.
Transgenic corns NK603 strain genomic dna also can use as the positive control in the present invention.
Embodiment 3: the reaction system of isothermal duplication and reaction condition optimization
In the present invention, sample gene group DNA is extracted by conventional CTAB method, for carrying out isothermal amplification.In an experiment, we are optimized the primer proportioning of isothermal duplication, be 0.3-1.5mM, dNTP concentration be 0.2-3.5mM, Mg2+ concentration are 2-18mM in trimethyl-glycine concentration.The ratio of described inner primer and the concentration of outer primer is 1-8: 1; The ratio of ring primer and outer primer concentration is 1-2: 1, all effectively augmentation detection can go out NK603 strain sample.Most preferred embodiment is as follows:
Isothermal amplification system is 25 μ L, namely adds in the PCR reaction tubes of 0.2mL: 2 × LAMPreaction mixture (40mmol/L Tris-HCl pH 8.8,20mmol/L KCl, 16mmol/LMgSO
4, 20mmol/L (NH
4)
2sO
40.2%Tween-20,0.8mol/L Betaine, 2.8mmol/Lof dNTPs) 12.5 μ L, the outer primer F3 solution 0.5 μ L of 10 μm of ol/L, the outer primer B3 solution 0.5 μ L of 10 μm of ol/L, the inner primer FIP solution 2 μ L of 20 μm of ol/L, the inner primer BIP solution 2 μ L of 20 μm of ol/L, the ring primer LF solution 2 μ L of 5 μm of ol/L, ring primer LB solution 2 μ L, the fluorescence dye SYTO-90.5 μ L of 5 μm of ol/L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, recombinant plasmid T-NK DNA 2 μ L, add 20 μ L sealing liquids at PCR pipe inwall after mixing.Isothermal amplification condition is: 63 DEG C of isothermal reaction 45-60min.
Reaction terminates rear employing real-time fluorescence figure to judge amplification, and the dyestuff used in reaction is fluorescence dye SYTO-9.Described result of determination be according to fluorescence dye SYTO-9 formed amplification curve as the conclusion detecting transgenic corns NK603 strain, otherwise for not detect transgenic corns NK603 strain.
Embodiment 4: the LAMP detection kit preparing transgenic corns NK603 strain
For convenience of using, according to embodiment 1,2,3, assembling detection kit (50 times), with easy to use.
A test solution, is LAMP reaction system, contains: 2 × LAMP reaction mixture, fluorescence dye SYTO-9, Bst archaeal dna polymerase, LAMP detection primer group; Every agent A test solution contains 2 × LAMPreaction mixture 625 μ L, fluorescence dye SYTO-9 25 μ L, 8U/ μ L Bst archaeal dna polymerase 50 μ L, LAMP primer group 450 μ L, wherein outer primer F3 and each 25 μ L of B3 solution; Inner primer FIP and each 100 μ L of BIP solution; Ring primer LF and each 100 μ L of LB solution; Primers F 3, B3, FIP, BIP, LF, LB have the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.6 respectively.
B test solution is recombinant plasmid T-NK603 100 μ L, and concentration is 0.1ng/ μ L, as positive control;
C test solution is deionized water 100 μ L, as negative control.
Embodiment 5: the specificity experiments of the LAMP kit that transgenic corns NK603 strain detects
Get transgenic corns NK603, MON863, MON810, transgenic paddy rice Bt63 and transgenic corns biased sample (containing MIR604, Bt11, GA21,59122, Bt176) 1 part respectively, genomic dna is extracted respectively by CTAB method, and carry out LAMP isothermal duplication, for determining the specificity of test kit.
Fig. 1 is the experimental result picture that application primer sets of the present invention and detection method detect transgenic corns NK603 strain and other 5 kinds of samples, 1-6 be respectively transgenic corns NK603, MON863, MON810, transgenic paddy rice Bt63, transgenic corns biased sample (containing MIR604, Bt11, GA21,59122, Bt176), negative control, judge amplification according to real-time fluorescence figure, only transgenic corns NK603 genome has amplification curve.
As can be seen from the result of Fig. 1, all not there is specific amplification in other Transgenic corn lines, other plant and non-transgenic corn.Experimental result shows that the LAMP method set up has good specificity, can be used for the detection of transgenic corns NK603 strain.
Embodiment 6: the sensitivity experiment of the LAMP kit that transgenic corns NK603 strain detects
Transgenic corns NK603 strain standard substance are mixed with non-transgenic corn sample, be mixed with the corn seed sample that transgenic corns NK603 strain percentage contents (w/w) is 5%, 1%, 0.5%, 0.1%, 0.05%, 0%, CTAB method extracts genomic dna respectively, carries out isothermal duplication according to embodiment 3 most preferred embodiment.
Fig. 2 is the LAMP detection method sensitivity experiment result figure of transgenic corns NK603 strain of the present invention, 1-7 is the transgenic corns of 5%, 1%, 0.5%, 0.1%, 0.05%, 0%, the LAMP detected result of negative control corn sample respectively.
Experimental result show: transgenic corns NK603 content be 5%, 1%, 0.5%, 0.1% sample all have amplification curve to occur, illustrate that the LAMP method set up can be successfully detect transgenic corns NK603 the sample of 0.1% from transgenic corns NK603 strain content, there is higher sensitivity, can be used for the detection containing transgenic corns NK603 strain sample.
Above embodiment is only the preferred embodiment of this creation, and not in order to limit this creation, any amendment made within all spirit in this creation and principle, equivalent replacement, improvement etc., within the protection domain that all should be included in this creation.
Claims (10)
1. the LAMP detection kit of a transgenic corns NK603 strain, it is characterized in that, its reaction system comprises trimethyl-glycine, dNTP, Bst DNA polymerase buffer liquid, fluorescence dye SYTO-9, BstDNA polysaccharase, Mg2+, positive control, negative control and primer sets, and its base sequence is as follows:
Outer primer F3:5 '-GACCAGGTAATCTTACCTTTGT-3 '
Outer primer B3:5 '-TGAAACCGCTTTCAAGAGAA-3 '
Inner primer FIP:5 '-GGCCGCGTTAACAAGCTTACTTTTTGGACTATCCCGACTCTCT-3 '
Inner primer BIP:5 '-CTTGGTACCACGCGACACATTTTCTGTTATGGTTCGAGAAGAGAT-3 '
Ring primer LF:5 '-TCGAGGTCATTCATATGCTTGA-3 '
Ring primer LB:5 '-AGTGTTTGAGTGGATCCTGTT-3 '.
2. the LAMP detection kit of transgenic corns NK603 strain as claimed in claim 1, is characterized in that: in described reaction system, trimethyl-glycine concentration is 0.3-1.5mM, dNTP concentration be 0.2-3.5mM, Mg2+ concentration is 2-18mM.
3. the LAMP detection kit of transgenic corns NK603 strain as claimed in claim 1, is characterized in that: the ratio of the concentration of the inner primer described in described reaction system and outer primer is 1-8: 1; The ratio of ring primer and outer primer concentration is 1-2: 1.
4. the LAMP detection kit of transgenic corns NK603 strain as claimed in claim 3, is characterized in that: described reaction system contains 0.2 μM of outer primer, 1.6 μMs of inner primers, 0.4 μM of ring primer.
5. the LAMP detection kit of the transgenic corns NK603 strain as described in as arbitrary in Claims 1-4, is characterized in that: Bst archaeal dna polymerase content is 4-10U.
6. the LAMP detection kit of transgenic corns NK603 strain as claimed in claim 5, is characterized in that: Bst archaeal dna polymerase content is 8U.
7. the LAMP detection kit of transgenic corns NK603 strain as claimed in claim 1, is characterized in that: described positive control is the genomic dna of transgenic corns NK603 strain.
8. the LAMP detection kit of transgenic corns NK603 strain as claimed in claim 1, is characterized in that: described positive control is the recombinant plasmid containing NK603 strain Bar gene CP4EPSPS gene.
9. the LAMP detection kit of transgenic corns NK603 strain as claimed in claim 8, described positive control recombinant plasmid preparation method is as described below: extract transgenic corns NK603 strain genomic dna, primers F 3/B3 is utilized to carry out PCR, increase and reclaim CP4EPSPS gene and be cloned in vector plasmid, transformation of E. coli competent cell, extract plasmid, DNA sequencing confirms the plasmid that sequence is correct.
10. application rights requires that described in 1, LAMP detection kit detects a detection method for transgenic corns NK603 strain, comprises following key step:
(1) detected sample genomic dna is extracted, with sample gene group DNA for template;
(2) according to detection sample number, LAMP amplification reaction solution is prepared: get 2 × LAMP reaction mixture 12.5 μ L according to every sample 25 microlitre reaction system, comprising 40mmol/L Tris-HClpH 8.8,20mmol/L KCl, 16mmol/L MgSO
4, 20mmol/L (NH
4)
2sO
4, 0.2%Tween-20,0.8mol/L Betaine, 2.8mmol/L of dNTPs; Add the outer primer B3 solution 0.5 μ L of the outer primer F3 solution 0.5 μ L of 10 μm of ol/L, 10 μm of ol/L; Add the inner primer BIP solution 2 μ L of the inner primer FIP solution 2 μ L of 20 μm of ol/L, 20 μm of ol/L; Add the ring primer LB solution 2 μ L of the ring primer LF solution 2 μ L of 5 μm of ol/L, 5 μm of ol/L; Add fluorescence dye SYTO-9 0.5 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, mixing;
(3) sample DNA template 2 μ L to be detected is added, mixing reaction system;
(4) isothermal duplication: add 20 μ L sealing liquids at PCR pipe inwall, 63 DEG C of isothermal reaction 45min, after reaction terminates, compared with showing fluorescence with positive control;
(5) result interpretation: reaction terminates rear employing real-time fluorescence figure to judge amplification, and the dyestuff used in reaction is fluorescence dye SYTO-9.Described result of determination be according to fluorescence dye SYTO-9 formed amplification curve as the conclusion detecting Transgenic corn lines NK603, otherwise result is not for detect Transgenic corn lines NK603.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510164657.6A CN104745708A (en) | 2015-04-02 | 2015-04-02 | LAMP (loop-mediated isothermal amplification) detection kit and detection method for transgenic corn NK603 strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510164657.6A CN104745708A (en) | 2015-04-02 | 2015-04-02 | LAMP (loop-mediated isothermal amplification) detection kit and detection method for transgenic corn NK603 strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104745708A true CN104745708A (en) | 2015-07-01 |
Family
ID=53585955
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510164657.6A Pending CN104745708A (en) | 2015-04-02 | 2015-04-02 | LAMP (loop-mediated isothermal amplification) detection kit and detection method for transgenic corn NK603 strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104745708A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1332246A (en) * | 2000-06-22 | 2002-01-23 | 孟山都技术有限公司 | Maize Individual PV-IMGT32(NK603) and Compositions and Methods for its Detection |
-
2015
- 2015-04-02 CN CN201510164657.6A patent/CN104745708A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1332246A (en) * | 2000-06-22 | 2002-01-23 | 孟山都技术有限公司 | Maize Individual PV-IMGT32(NK603) and Compositions and Methods for its Detection |
Non-Patent Citations (2)
| Title |
|---|
| RAJESH K. BHOGE ET AL.: "Event-specific analytical methods for six genetically modified maize events using visual and real-time loop-mediated isothermal amplification", 《FOOD CONTROL》 * |
| 兰青阔等: "环状等温扩增技术快速检测转基因玉米MON863的研究", 《玉米科学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN116064462A (en) | Taq DNA polymerase mutant and preparation method thereof | |
| CN103924000B (en) | LAMP detecting primer group, kit and detecting method for cry3A gene in transgenic plant | |
| CN107868849A (en) | Detect primer sets and its application of prawn IHHNV viruses | |
| KR20130055040A (en) | Pcr primer for detecting salmonella | |
| CN105063207B (en) | Genetically engineered soybean MON87705 LAMP detection primer group, kit and detection method | |
| KR20130007815A (en) | Primer and probe set for detection and quantification of listeria monocytogenes | |
| CN104388578B (en) | Utilize cross primer and the method for double probe constant-temperature amplification detection NOS terminator | |
| CN104745708A (en) | LAMP (loop-mediated isothermal amplification) detection kit and detection method for transgenic corn NK603 strain | |
| KR102293251B1 (en) | Nucleic Acid Molecule for Detecting Antagonistic Microorganism against Pathogen Causing Root Rot Disease of Ginseng and Detecting Kit Comprising the Same | |
| KR20200059761A (en) | Development of a Singleplex Real-Time PCR Kit for Rapid Detection of Listeria monocytogenes ssrA target gene | |
| CN110157780A (en) | A primer set, kit and detection method for bacterial drug resistance detection | |
| CN103409535A (en) | Primer pair, probe and method for detecting Cronobacter sakazakii | |
| KR101670934B1 (en) | Primer set for detection of Clostridium botulinum, composition, and kit comprising the same | |
| CN101942521A (en) | Molecular Marker Detection Method and Primers of Avr-Pit Avirulent Gene of Magnaporthe grisea | |
| Wancham et al. | Sex identification of sun conure (Aratinga solstitialis) using loop-mediated isothermal amplification of W and Z spindlin chromosomes | |
| KR101906217B1 (en) | Porcine gelatin detection kit and porcine gelatin detection process using the kit | |
| KR101999815B1 (en) | Sets of primers of Ultra-Fast PCR-based assay for discriminating red horsehead from yellow horsehead, or method of discriminating species using thereof | |
| KR101855984B1 (en) | composition comprising SNP markers for a differentiation of Cudrania tricuspidata Bureau lines, and method for a differentiation of Cudrania tricuspidata Bureau lines and hybrid using the same | |
| KR20200063566A (en) | Primer set, composition and kit for detecting genetically modified crops introduced EPSPS gene, and methods using the same | |
| TWI607090B (en) | Materials and methods for detecting the aryloxyalkanoate dioxygenase gene (aad-12) in plants | |
| CN101519693B (en) | Method for rapidly detecting genetically modified corn MON863 | |
| CN105039556B (en) | Genetically engineered soybean MON87769 LAMP detection primer group, kit and detection method | |
| KR101631114B1 (en) | Primer set for loop-mediated isothermal amplification reaction for meat species identification, and method for meat species identification using the same | |
| CN105506071B (en) | LAMP detection primer group, LAMP detection kit and the detection method of transgenic pest-resistant soybean MON87701 and its derived varieties | |
| CN103525910B (en) | Quantitative PCR (Polymerase Chain Reaction) detection method for specificity of phytase transgenic corn BVLA430101 strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150701 |
|
| WD01 | Invention patent application deemed withdrawn after publication |