CN102337331A - Primer group and kit for detecting Roundup Ready transgenic soybeans - Google Patents
Primer group and kit for detecting Roundup Ready transgenic soybeans Download PDFInfo
- Publication number
- CN102337331A CN102337331A CN2011102084667A CN201110208466A CN102337331A CN 102337331 A CN102337331 A CN 102337331A CN 2011102084667 A CN2011102084667 A CN 2011102084667A CN 201110208466 A CN201110208466 A CN 201110208466A CN 102337331 A CN102337331 A CN 102337331A
- Authority
- CN
- China
- Prior art keywords
- seq
- nucleotide sequence
- primer
- bip
- inner primer
- 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.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer group and a kit for detecting Roundup Ready transgenic soybeans. The kit consists of the primer group, Bst deoxyribonucleic acid (DNA) polymerase, a stabilizing solution, a reaction solution, a developing solution and a positive control solution. In the kit, six segments and four primers are utilized; and the kit has high specificity according to the condition that whether the existence of target materials can be judged or not through amplification. The quick diagnostic kit provided by the invention has high speed, high efficiency and high sensitivity, can be subjected to an amplification reaction only by a constant temperature without the usage of special reagents and equipment, has low detection cost and is easy and convenient to identify; pyrophosphate ions precipitated from deoxyribonucleoside triphosphate (dNTP) is combined with Mg<2+> in the reaction solution; the produced byproduct, namely magnesium pyrophosphate is precipitated and can be observed and identified through naked eyes; and after the developing solution is added, the kit has obvious development differences of negative and positive results and is more obvious and reliable. A soybean endogenous gene lectin is used as an internal label, so that errors and false negatives caused by the extraction of nucleic acid are prevented; and real-time monitoring is performed, so that detection time is greatly reduced, and human power and material resources are saved.
Description
Technical field
The present invention relates to biological detection reagent, be specifically related to a kind of Roundup Ready genetically engineered soybean and detect with primer sets and diagnostic kit thereof.
Background technology
Monsanto Company has transformed and has come from mikrobe in the resistance glyphosate kind soybean of development in 1994
Agrobacterium tumefaciens5-enol pyruvic acid shikimic acid-3-phosphate synthase (CP4 EPSPS) encoding sox, can resist the inhibition of herbicide glyphosate to the shikimic acid route of synthesis, the render transgenic soybean can normal growth after spraying herbicide.The acceptor of this kind is soybean varieties A5403, has integrated CP4 EPSPS gene, E35S promotor and the NOS terminator of external source in the transgenic soybean gene group.This kind is the maximum genetically engineered soybean kind of present cultivated area, is widely used in the middle of the processing of edible oil and food.
In order to strengthen supervision and management to transgenic product; Developed multiple transgene component detection method at present, from based on proteinic ELISA detection technique to based on the polymerase chain reaction (PCR) of nucleic acid technology and fast-developing biochip technology.Wherein the PCR detection technique is with its susceptibility, specificity, high efficiency and the most general; This technology is identified through detecting the exogenous nucleic acid fragment that contains in the transgenic product; Appearance along with fluorescence real-time quantitative polymerase chain reaction (real time PCR); Not only can carry out the qualitative evaluation of transgenic product, and can carry out quantitatively transgene component.
With the PCR technology is that the transgenic product detection technique of representative also exists some problems in practical application, the instrument special like the regular-PCR Technology Need, and have easy crossed contamination and the loaded down with trivial details shortcoming of operating process.Though and Real time PCR technology has solved the problem of crossed contamination preferably, and has simplified operating process, need more complicated quantitatively determined instrument, therefore be not suitable for field quick detection.And the cost of fluorescent probe is higher in the real-time quantitative polymerase chain reaction PCR technology, has strengthened the difficulty of applying.Biochip technology can realize quick and high-throughout detection, yet costs an arm and a leg, and it is high to detect cost.So in time use the newest fruits of biotech development significant to satisfying improving constantly of transgenic product detection requirement.Wherein isothermal duplication (Isothermal Amplification) nucleic acid Fast Detection Technique is the rapid progress on the transgenic product detection technique; The loop-mediated isothermal amplification technique of at present having set up (loop-mediated isothermal amplication of DNA; Be called for short LAMP) have a lot of meliority, and do not see that at present useful loop-mediated isothermal amplification technique detects the genetically engineered soybean quick diagnosis reagent kit yet.
Summary of the invention
The objective of the invention is to deficiency, provide a kind of and detect Roundup Ready transgenic soy bean EPSPS-NOS gene, and primer sets is used in the detection that this EPSPS-NOS gene has high specific can be used for loop-mediated isothermal amplification technique to prior art.
Another object of the present invention is to provide a kind of genetically engineered soybean quick diagnosis reagent kit based on loop-mediated isothermal amplification technique that cost is low, easy to use, detection speed is fast, specificity is high that detects.
Above-mentioned purpose of the present invention is achieved through following technical scheme:
One, Roundup Ready transgenic of the present invention detects and uses primer sets; Be based on loop-mediated isothermal amplification technique; According to disclosed Roundup Ready transgenic soy bean EPSPS-NOS gene order; Choose the specific sequence of Roundup Ready transgenic soy bean EPSPS-NOS gene, analysis is designed then, and the ability specificity is differentiated the Auele Specific Primer group of Roundup Ready transgenic soy bean EPSPS-NOS gene; Identify Roundup Ready transgenic soy bean EPSPS-NOS gene through the LAMP method, this EPSPS-NOS gene primer group is made up of following four primers:
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:1;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:2;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:3;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:4;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:5;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:6;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:7;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:8;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:9;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:2;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:10;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:4;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:11;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:12;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:13;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:14;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:15;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:16;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:17;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:18.
Two, primer sets is used in soybean endogenous gene lectin of the present invention gene test; Be based on loop-mediated isothermal amplification technique; According to disclosed lectin gene order, choose the specific sequence of lectin gene, analysis is designed then; Identify the lectin gene through the LAMP method, this endogenous gene lectin primer sets is made up of following four primers:
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:19;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:20;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:21;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:22;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:23;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:24;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:25;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:26.
Three, Roundup Ready genetically engineered soybean quick diagnosis reagent kit of the present invention, be by EPSPS-NOS gene primer, lectin gene primer,
BstDNA polysaccharase, reaction solution, stable liquid, colour developing liquid and positive control solution are formed, more than seven kinds of liquid place container respectively, wherein:
Contain 1.6～2mmol dNTP, 20～25mmol Tris-HCl, 10～12.5mmol Repone K, 10～12.5mmol ammonium sulfate, 8～10mmol sal epsom, 1～1.25ml TritonX-100,0.8～1mol trimethyl-glycine, each 1.6～2mol of inner primer FIP/BIP and each 0.2～0.25mol of outer primer F3/B3 in above-mentioned every 1L reaction solution; Preferred ratio is to contain 2mmol dNTP, 25mmol Tris-HCl, 12.5mmol Repone K, 12.5mmol ammonium sulfate, 10mmol sal epsom, 1.25ml TritonX-100,1mol trimethyl-glycine, each 2mol of inner primer FIP/BIP and each 0.25mol of outer primer F3/B3 in every 1L reaction solution.
Above-mentioned colour developing liquid is preferably optical dye SYBR Green I or EvaGreen.
Aforementioned stable liquid is preferably Yellow Protopet 2A.
Above-mentioned positive control is an EPSPS-NOS gene DNA fragment.
Four, the production technique of gene quick diagnosis kit of the present invention
1, respectively with behind EPSPS-NOS gene and lectin gene inner primer FIP/BIP and the outer primer F3/B3 synthesizing and purifying, quantitatively preparation, concentration detects, the sampling quality inspection;
2, reaction solution is aseptic subpackaged, and confirm the quality inspection of sampling with carrying out concentration according to experiment;
3, stable liquid is aseptic subpackaged, the sampling quality inspection;
4, with the positive control sample preparations, packing, sampling quality inspection;
5, assembling test kit.
Five, the detection method of gene quick diagnosis kit of the present invention
1, sample preparation
The extraction of DNA and assay are carried out according to the regulation among the GB/T 19495.3-2004 by GB in the testing sample.When being carried out the DNA extraction, sample to guarantee quality and the steady concentration of DNA, to guarantee the repeatability of PCR reaction.The DNA fragment of guaranteeing to extract is greater than amplified fragments.
2, loop-mediated isothermal amplification technique reaction process
In reaction tubes, add reaction solution 38～40 volume %, big fragment 0.9～1.8 volume % of Bst archaeal dna polymerase, stable liquid 52～54.5 volume %, sample template DNA 4.5～9 volume %, 63～65 ℃ of isothermal reaction 45～90min.Said volume percent is meant the volume percent that accounts for four component TVs.
3, post-reaction treatment
In above-mentioned reaction tubes and positive controls, add colour developing liquid respectively, mixing, the sample sets colour developing is identical with control group then positive, otherwise negative.
Principle of the present invention is to utilize
BstArchaeal dna polymerase and the special inside and outside primer (being inner primer FIP/BIP and outer primer F3/B3) of two couples that designs according to target-gene sequence; Six isolated areas on the specific recognition target sequence; Start the endless chain replacement(metathesis)reaction; Start complementary strand in target DNA district synthetic, and result's complementary sequence on same chain goes round and begins again and is formed with the very stem of polycyclic Cauliflower structure-circular DNA mixture.In the LAMP reaction process, pyrophosphate ion of separating out from dNTP and the Mg the reaction soln
2+In conjunction with, produce by product---magnesium pyrophosphate milky white precipitate, can be through the visual inspection result of determination.The LAMP reaction is under constant temperature (63～65 ℃) condition, to accomplish in 45～90 minutes.This comparatively gentle temperature condition and do not have temperature cycle that required instrument is oversimplified, overcome conventional P CR inherent detection time long, pollute easily and detect shortcoming such as cost height.In addition, this detection method is lower to testing staff's technical quality requirement, and actually operating is very easy, does not need special reagent and plant and instrument, helps setting up rapid screening system with low cost.The LAMP method is a kind of gene amplification method of easy, quick, high degree of specificity.Constant temperature gene amplification technology and round pcr (comprising the fluorescence real-time quantitative PCR technology) are compared; Can find and technology on methodology indexs such as sensitivity, specificity and sensing range, to be equivalent to or to be superior to round pcr; And do not rely on any special plant and instrument and can realize on-the-spot high-throughput rapid detection, and detect cost far below fluorescent quantitative PCR technique.Domestic do not have the test kit of this respect to sell at present as yet.
Compared with prior art, the present invention has following beneficial effect:
1. gene quick diagnosis kit of the present invention only needs just ability amplified reaction of a steady temperature, does not need special reagent and equipment, and it is low to detect cost;
2. gene quick diagnosis kit of the present invention is used six sections, four primers, and whether the existence that just can judge target substance according to whether increasing to be, so has high specific;
3. gene quick diagnosis kit of the present invention amplification fast and efficient can accomplish amplification less than 1 hour, and productive rate is high;
4. gene quick diagnosis kit of the present invention is highly sensitive, and amplification template only needs 10 copies or still less;
5. gene quick diagnosis kit of the present invention is identified easy, pyrophosphate ion of separating out from dNTP and the Mg the reaction soln
2+In conjunction with, produce by product-magnesium pyrophosphate deposition, can identify through visual inspection, and after adding colour developing liquid, yin and yang attribute colour development difference as a result is remarkable, the checking rate is high, and is more obviously reliable.
Description of drawings
Fig. 1 is test kit specific detection figure (goal gene EPSPS-NOS) as a result;
Fig. 2 is test kit specific detection figure (goal gene lectin) as a result;
Wherein, 1 is the TE damping fluid, and 2 is ddH
2O, 3 is rape non-transgenic sample DNA, 4 is corn non-transgenic sample DNA; 5 is rice non-transgenic sample DNA, and 6 is soybean non-transgenic sample DNA, and 7 is 50ng soybean transgene sample DNA; 8 is 100ng soybean transgene sample DNA, and 9 is 250ng soybean transgene sample DNA.
Embodiment
Below in conjunction with specific embodiment the present invention is done to set forth further, but specific embodiment is not done any qualification to the present invention.
The preparation of embodiment 1 Roundup Ready genetically engineered soybean quick diagnosis reagent kit
(1) synthesize the EPSPS-NOS gene primer by following sequence through dna synthesizer:
Outer primer F3, its nucleotide sequence is shown in SEQ ID NO:1;
Outer primer B3, its nucleotide sequence is shown in SEQ ID NO:2;
Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:3;
Inner primer BIP, its nucleotide sequence is shown in SEQ ID NO:4.
(2) synthesize the lectin gene primer by following sequence through dna synthesizer:
Outer primer F3, its nucleotide sequence is shown in SEQ ID NO:19;
Outer primer B3, its nucleotide sequence is shown in SEQ ID NO:20;
Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:21;
Inner primer BIP, its nucleotide sequence is shown in SEQ ID NO:22.
(3) purchase archaeal dna polymerase:
BstDNA polymerase (big fragment) places container.
(4) prepare EPSPS-NOS gene and lectin gene reaction solution respectively: the prescription of reaction solution contains each 0.25mol preparation of 2mmol dNTP, 25mmol Tris-Cl, 12.5mmol Repone K, 12.5mmol ammonium sulfate, 10mmol sal epsom, 1.25ml TritonX-100,1mol trimethyl-glycine, each 2mol of inner primer FIP/BIP and outer primer F3/B3 by every 1L solution, places container.
(5) purchase stable liquid: Yellow Protopet 2A places container.
(6) purchase colour developing liquid: SYBR Green I places container.
(7) extract positive control: preparation EPSPS gene DNA fragment places container respectively.
(8) above-mentioned 6 containers are dressed up test kit, encapsulation.
Preparation technology is summarized as follows:
1, with behind inner primer FIP/BIP and the outer primer F3/B3 synthesizing and purifying, quantitatively preparation, concentration detects, the sampling quality inspection;
2, reaction solution is aseptic subpackaged, and confirm the quality inspection of sampling with carrying out concentration according to experiment;
3, with the stable liquid packing, the sampling quality inspection;
4, with the positive control sample preparations, packing, sampling quality inspection;
5, assembling test kit.
The preparation of embodiment 2 Roundup ready genetically engineered soybean quick diagnosis reagent kits
Two pairs of primers of EPSPS-NOS gene are:
Outer primer F3, its nucleotide sequence is shown in SEQ ID NO:5;
Outer primer B3, its nucleotide sequence is shown in SEQ ID NO:6;
Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:7;
Inner primer BIP, its nucleotide sequence is shown in SEQ ID NO:8.
Two pairs of primers of Lectin gene are:
Outer primer F3, its nucleotide sequence is shown in SEQ ID NO:23;
Outer primer B3, its nucleotide sequence is shown in SEQ ID NO:24;
Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:25;
Inner primer BIP, its nucleotide sequence is shown in SEQ ID NO:26.
The prescription of reaction solution is: contain 1.6mmol dNTP, 20mmol Tris-HCl, 10mmol Repone K, 10mmol ammonium sulfate, 8mmol sal epsom, 1ml TritonX-100,0.8mol trimethyl-glycine, each 1.6mol of inner primer FIP/BIP and each 0.2mol of outer primer F3/B3 in every 1L reaction solution;
Colour developing liquid is EvaGreen.
Other are with embodiment 1.
The preparation of embodiment 3 Roundup ready genetically engineered soybean quick diagnosis reagent kits
Two pairs of primers of EPSPS-NOS gene are:
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:9;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:2;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:10;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:4;
Two pairs of primers of Lectin gene are:
Outer primer F3, its nucleotide sequence is shown in SEQ ID NO:23;
Outer primer B3, its nucleotide sequence is shown in SEQ ID NO:24;
Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:25;
Inner primer BIP, its nucleotide sequence is shown in SEQ ID NO:26.
The prescription of reaction solution is: contain 2mmol dNTP, 25mmol Tris-HCl, 11mmol Repone K, 12mmol ammonium sulfate, 9mmol sal epsom, 1.25 ml TritonX-100,1 mol trimethyl-glycine, each 2mol of inner primer FIP/BIP and each 0.25mol of outer primer F3/B3 in every 1L reaction solution;
Colour developing liquid is EvaGreen.
Other are with embodiment 1.
The preparation of embodiment 4 Roundup ready genetically engineered soybean quick diagnosis reagent kits
Two pairs of primers of EPSPS-NOS gene are:
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:11;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:12;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:13;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:14;
Two pairs of primers of Lectin gene are:
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:19;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:20;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:21;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:22.
The prescription of reaction solution is: contain 1.6mmol dNTP, 22mmol Tris-HCl, 10mmol Repone K, 10mmol ammonium sulfate, 8mmol sal epsom, 1ml TritonX-100,0.8mol trimethyl-glycine, each 1.6mol of inner primer FIP/BIP and each 0.2mol of outer primer F3/B3 in every 1L reaction solution;
Colour developing liquid is EvaGreen.
Other are with embodiment 1.
The preparation of embodiment 5 Roundup ready genetically engineered soybean quick diagnosis reagent kits
Two pairs of primers of EPSPS-NOS gene are:
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:15;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:16;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:17;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:18.
Two pairs of primers of Lectin gene are:
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:23;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:24;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:25;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:26.
The prescription of reaction solution is: contain 1.6mmol dNTP, 21mmol Tris-HCl, 10mmol Repone K, 10mmol ammonium sulfate, 8mmol sal epsom, 1ml TritonX-100,0.8mol trimethyl-glycine, each 1.6mol of inner primer FIP/BIP and each 0.2mol of outer primer F3/B3 in every 1L reaction solution;
Colour developing liquid is EvaGreen.
Other are with embodiment 1.
The application of embodiment 6 Roundup ready genetically engineered soybean quick diagnosis reagent kits
Present embodiment adopts the Roundup ready genetically engineered soybean quick diagnosis reagent kit of embodiment 1 preparation to carry out the quick diagnosis in the testing sample.
1, sample preparation (template DNA extraction)
1) with 100mg through pretreated sample; The last people of the adding 700 μ L CTAB that in liquid nitrogen, fully pulverize extract in the damping fluid
(sample that need not grind directly adds); Vibration back mixing; 65 ℃ of insulation 30 min light and slow frequently put upside down mixing 2-3 time therebetween.
2) add people's 700 μ L trichloromethane-primary isoamyl alcohol, light and slowly put upside down mixing solution 2-3 time.Centrifugal 5 min of 12000g are to phase-splitting.
3) supernatant is transferred in the clean centrifuge tube, adds the Virahol of 4 ℃ of precoolings of 0.6 times of volume of people, leave standstill 5min, centrifugal 5 min of 12 000 g in-20 ℃.
4) abandon supernatant, add 1000 μ L, 70% ethanol, rotate centrifuge tube gently, 4 ℃ of centrifugal 1 min of following 8000g, abandon supernatant after, add 20 μ L Rnase A enzymes (10 μ g/μ L) again, 37 ℃ of temperature are bathed 30min.
5) add 600 μ L sodium chloride solutions, 65 ℃ of temperature are bathed 10min.Add the saturated phenol of 600 μ L trichloromethane-Tris, put upside down mixing after, centrifugal 5 min of 12000g shift supernatant to the 1.5mL centrifuge tube.
6) add the Virahol of 4 ℃ of precoolings of 0.6 times of volume of people, leave standstill 30min, centrifugal 10 min of 12 000 g, abandoning supernatant in 4 ℃.
7) add 4 ℃ of precooling 70% ethanol of 1000 μ L, rotate centrifuge tube gently, 4 ℃ of following centrifugal 10 min of 12 000 g abandon supernatant.Repeat single job.Volatilised liq under the room temperature.
8) after the deposition drying, add the abundant dissolving DNA of 50 μ L TE damping fluids ,-20 ℃ of preservations are subsequent use.
2, the reaction process of loop-mediated isothermal amplification technique
1) in 200 μ L reaction tubess preparation reaction system: reaction solution 22 μ L,
BstArchaeal dna polymerase 0.5 μ L (4U), template DNA 2.5 μ L.
2) with the reaction tubes for preparing in 64 ℃ of isothermal reaction 1h.
3, post-reaction treatment
In above-mentioned PCR reaction product, add 2 μ L SYBR Green I, mixing also adds SYBR Green I mixing in the heliotropism control tube, simultaneously if the same shows green with control tube of reaction tubes is then positive, if reaction tubes manifests orange then negative.
The specificity checking of embodiment 7 Roundup Ready genetically engineered soybean quick diagnosis reagent kits
Present embodiment adopts the Roundup Ready genetically engineered soybean quick diagnosis reagent kit of embodiment 2 preparations to carry out the specific detection of test kit.
Adopt the Roundup Ready genetically engineered soybean quick diagnosis reagent kit of embodiment 2 preparations, respectively to soybean transgene sample, soybean non-transgenic sample, rape non-transgenic sample, corn non-transgenic sample, rice non-transgenic sample, ddH
2O and TE damping fluid increase, and the non-transgenic sample DNA adds 250ng respectively, and the soybean transgene sample DNA adds 250ng, 100ng and 50ng respectively, and each sample is provided with 2 repetitions.
Reaction system is: reaction solution 22 μ l,
BstDNA polysaccharase 0.5 μ l, stable liquid 30 μ l and dna profiling 2.5 μ l.
Response procedures: in the metal bath 65 ℃, 1 hour.
Add colour developing liquid 2.0 μ l after reaction finishes, observations is like table 1, table 2 and Fig. 1, shown in Figure 2.
Table 1 test kit specific detection result (goal gene EPSPS-NOS)
In the above-mentioned table 1, P representative amplification is positive; N representative amplification is negative.
Table 2 test kit specific detection result (goal gene lectin)
In the above-mentioned table 2, P representative amplification is positive; N representative amplification is negative.
Can find out from the result of table 1; In the reaction that contains 50ng, 100ng and 250ng soybean transgene sample DNA, can amplify the product of EPSPS-NOS gene; It is positive to present amplification, and is containing soybean non-transgenic sample DNA, rice non-transgenic sample DNA and rape non-transgenic DNA and ddH
2All can not amplify product among O and the TE, it is negative to present amplification, explanation thus, genetically modified crops EPSPS-NOS gene quick diagnosis kit of the present invention has high specific, can be correct identify the genetically engineered soybean sample.Can find out from the result of table two, in the reaction of soybean transgene sample DNA and soybean non-transgenic sample DNA, all can amplify the product of lectin gene, present the amplification positive, no false-negative existence is described, guarantee result's safety.
SEQUENCE?LISTING
< 110>Guangzhou Huafeng Biotech Co., Ltd.
< 120>Roundup Ready genetically engineered soybean detects with primer sets and detection kit thereof
<130>
<160> 26
<170> PatentIn?version?3.5
<210> 1
<211> 20
<212> DNA
< 213>artificial sequence
<400> 1
catgagcttc?ctcgtcatgg 20
<210> 2
<211> 19
<212> DNA
< 213>artificial sequence
<400> 2
cgggaattgg?atccggtac 19
<210> 3
<211> 40
<212> DNA
< 213>artificial sequence
<400> 3
atgaactccg?ggaagctcgt?gctcgtgtcg?gaaaaccctg 40
<210> 4
<211> 40
<212> DNA
< 213>artificial sequence
<400> 4
gctgggcgcg?aagatcgaac?gctcgaattc?gagctcatca 40
<210> 5
<211> 18
<212> DNA
< 213>artificial sequence
<400> 5
gccacccatc?tcgatcac 18
<210> 6
<211> 20
<212> DNA
< 213>artificial sequence
<400> 6
gctcgaattc?gagctcatca 20
<210> 7
<211> 39
<212> DNA
< 213>artificial sequence
<400> 7
tcgtccaccg?tgacagggtt?catcgccatg?agcttcctc 39
<210> 8
<211> 41
<212> DNA
< 213>artificial sequence
<400> 8
acgagcttcc?cggagttcat?gagccttcgt?atcggagagt?t 41
<210> 9
<211> 19
<212> DNA
< 213>artificial sequence
<400> 9
catcgccatg?agcttcctc 19
<210> 10
<211> 40
<212> DNA
< 213>artificial sequence
<400> 10
gtccatgaac?tccgggaagc?tcaaaaccct?gtcacggtgg 40
<210> 11
<211> 19
<212> DNA
< 213>artificial sequence
<400> 11
ggagttcatg?gacctgatg 19
<210> 12
<211> 23
<212> DNA
< 213>artificial sequence
<400> 12
cccatctcat?aaataacgtc?atg 23
<210> 13
<211> 39
<212> DNA
< 213>artificial sequence
<400> 13
tccggtaccg?agctcgaatt?gaagatcgaa?ctctccgat 39
<210> 14
<211> 49
<212> DNA
< 213>artificial sequence
<400> 14
ttcttaagat?tgaatcctgt?tgccgcatgc?ttaacgtaat?tcaacagaa 49
<210> 15
<211> 18
<212> DNA
< 213>artificial sequence
<400> 15
gttcatggac?ctgatggc 18
<210> 16
<211> 22
<212> DNA
< 213>artificial sequence
<400> 16
atctcataaa?taacgtcatg?ca 22
<210> 17
<211> 39
<212> DNA
< 213>artificial sequence
<400> 17
attggatccg?gtaccgagct?gaactctccg?atacgaagg 39
<210> 18
<211> 47
<212> DNA
< 213>artificial sequence
<400> 18
ttcttaagat?tgaatcctgt?tgccgtgctt?aacgtaattc?aacagaa 47
<210> 19
<211> 19
<212> DNA
< 213>artificial sequence
<400> 19
accagcaagg?caaactcag 19
<210> 20
<211> 18
<212> DNA
< 213>artificial sequence
<400> 20
ttgaaggaag?cggcgaag 18
<210> 21
<211> 46
<212> DNA
< 213>artificial sequence
<400> 21
ccgaggaggt?cacaatagcg?tctttttcag?ctggaacaag?ttcgtg 46
<210> 22
<211> 44
<212> DNA
< 213>artificial sequence
<400> 22
aacggcaccc?caaaaccctc?ttttctaccg?gtttctttgt?ccca 44
<210> 23
<211> 19
<212> DNA
< 213>artificial sequence
<400> 23
gccgaagcaa?ccaaacatg 19
<210> 24
<211> 20
<212> DNA
< 213>artificial sequence
<400> 24
ggggcataga?aggtgaagtt 20
<210> 25
<211> 45
<212> DNA
< 213>artificial sequence
<400> 25
tggggtgccg?ttttcgtcaa?cttttgagac?gctattgtga?cctcc 45
<210> 26
<211> 42
<212> DNA
< 213>artificial sequence
<400> 26
accctcgtct?cttggtcgcg?ttttggcaac?gctaccggtt?tc 42
Claims (6)
1. the EPSPS-NOS gene primer group of a Roundup Ready genetically engineered soybean detection usefulness is characterized in that, form by outer primer F3, outer primer B3, inner primer FIP and inner primer BIP, wherein:
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:1;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:2;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:3;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:4;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:5;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:6;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:7;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:8;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:9;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:2;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:10;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:4;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:11;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:12;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:13;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:14;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:15;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:16;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:17;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:18.
2. the endogenous gene lectin primer sets of a Roundup Ready genetically engineered soybean detection usefulness is characterized in that, form by outer primer F3, outer primer B3, inner primer FIP and inner primer BIP, wherein:
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:19;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:20;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:21;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:22;
Perhaps,
The nucleotide sequence of outer primer F3 is shown in SEQ ID NO:23;
The nucleotide sequence of outer primer B3 is shown in SEQ ID NO:24;
The nucleotide sequence of inner primer FIP is shown in SEQ ID NO:25;
The nucleotide sequence of inner primer BIP is shown in SEQ ID NO:26.
3. Roundup Ready genetically engineered soybean quick detection kit, it is characterized in that this test kit by the said EPSPS-NOS gene primer of claim 1 group, the said endogenous gene lectin of claim 2 primer sets,
BstDNA polysaccharase, reaction solution, stable liquid, colour developing liquid and positive control solution are formed; More than seven kinds of liquid place container respectively; Wherein, the prescription of said reaction solution is: contain 1.6～2mmol dNTP, 20～25mmol Tris-HCl, 10～12.5mmol Repone K, 10～12.5mmol ammonium sulfate, 8～10mmol sal epsom, 1～1.25ml TritonX-100,0.8～1mol trimethyl-glycine, each 1.6～2mol of inner primer FIP/BIP and each 0.2～0.25mol of outer primer F3/B3 in every 1L reaction solution; Said positive control is the EPSPS-NOS gene DNA fragment.
4. quick detection kit according to claim 3 is characterized in that described colour developing liquid is SYBR Green I or EvaGreen.
5. quick detection kit according to claim 3 is characterized in that containing in said every 1L reaction solution 2mmol dNTP, 25mmol Tris-HCl, 12.5mmol Repone K, 12.5mmol ammonium sulfate, 10mmol sal epsom, 1.25ml TritonX-100,1mol trimethyl-glycine, each 2mol of inner primer FIP/BIP and each 0.25mol of outer primer F3/B3.
6. quick detection kit according to claim 3 is characterized in that said stable liquid is Yellow Protopet 2A.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110208466 CN102337331B (en) | 2011-07-25 | 2011-07-25 | Primer group and kit for detecting Roundup Ready transgenic soybeans |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110208466 CN102337331B (en) | 2011-07-25 | 2011-07-25 | Primer group and kit for detecting Roundup Ready transgenic soybeans |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102337331A true CN102337331A (en) | 2012-02-01 |
| CN102337331B CN102337331B (en) | 2013-07-10 |
Family
ID=45513280
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110208466 Active CN102337331B (en) | 2011-07-25 | 2011-07-25 | Primer group and kit for detecting Roundup Ready transgenic soybeans |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102337331B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104881701A (en) * | 2015-06-11 | 2015-09-02 | 飞天诚信科技股份有限公司 | Intelligent card and manufacturing method thereof |
| CN105420359A (en) * | 2015-12-08 | 2016-03-23 | 许昌学院 | LAMP primer group for Lectin detection and gene isothermal amplification method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1786195A (en) * | 2005-09-08 | 2006-06-14 | 中国疾病预防控制中心营养与食品安全所 | Transgene agricultural product DNA detection chip, its preparation method and application |
| CN101906473A (en) * | 2010-07-22 | 2010-12-08 | 中华人民共和国北京出入境检验检疫局 | Nucleic acid isothermal amplification kit and method for detecting EPSPS transgenic crops |
-
2011
- 2011-07-25 CN CN 201110208466 patent/CN102337331B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1786195A (en) * | 2005-09-08 | 2006-06-14 | 中国疾病预防控制中心营养与食品安全所 | Transgene agricultural product DNA detection chip, its preparation method and application |
| CN101906473A (en) * | 2010-07-22 | 2010-12-08 | 中华人民共和国北京出入境检验检疫局 | Nucleic acid isothermal amplification kit and method for detecting EPSPS transgenic crops |
Non-Patent Citations (4)
| Title |
|---|
| 兰青阔等: "LAMP在检测转基因抗草甘膦大豆cp4-epsps基因上的应用", 《安徽农业科学》 * |
| 刘彩霞等: "抗草甘膦转基因大豆及加工品LAMP检测研究", 《大豆科学》 * |
| 柳毅等: "改良环介导等温扩增技术快速检测转基因大豆", 《大豆科学》 * |
| 田芳等: "抗草甘膦转基因大豆及其加工产品的环介导等温扩增检测技术", 《动物营养学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104881701A (en) * | 2015-06-11 | 2015-09-02 | 飞天诚信科技股份有限公司 | Intelligent card and manufacturing method thereof |
| CN105420359A (en) * | 2015-12-08 | 2016-03-23 | 许昌学院 | LAMP primer group for Lectin detection and gene isothermal amplification method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102337331B (en) | 2013-07-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106544443A (en) | The dry powdered LAMP quick detection kit of Pseudomonas aeruginosa and its using method | |
| CN101654706B (en) | Rapid diagnostic reagent kit of legionella pneumophilia genes based on loop-mediated isothermal amplification technique and detecting method thereof | |
| CN101660005B (en) | Rapid diagnostic kit based on loop-mediated isothermal amplification technique for hepatitis A virus genes and detection method thereof | |
| CN110982922B (en) | Primer composition and method for rapidly detecting rice bakanae disease pathogenic bacteria fusarium granatum based on LAMP | |
| CN106544444A (en) | The dry powdered LAMP quick detection kits of streptococcus fecalis and its using method | |
| CN113999921B (en) | Method and kit for rapidly and visually detecting shigella flexneri | |
| CN101942508B (en) | Escherichia coli detection kit and use method thereof | |
| CN101403001B (en) | Rapid diagnosis reagent kit and detection method for pseudomonas aeruginosa gene | |
| CN102094090A (en) | Cholera toxin virulence gene detection kit and detection method thereof | |
| CN102337331B (en) | Primer group and kit for detecting Roundup Ready transgenic soybeans | |
| CN104593504B (en) | Composite PCR (polymerase chain reaction) amplification fluorescence detection kit for 27 plant transgenic loci | |
| CN101307356A (en) | Rapid diagnosis kit for staphylococcus aureus gene based on loop-mediated isothermal amplification technology and detecting method thereof | |
| CN102140516B (en) | Primer for detecting CAMV35S genes, relevant kit and detecting method | |
| CN101838685B (en) | Primer group for testing Roundup Ready transgenic soy bean EPSPS gene, rapid diagnosis kit and testing method thereof | |
| CN108277290A (en) | The dry powdered LAMP quick detection kits of staphylococcus aureus | |
| CN101307351A (en) | Rapid diagnosis kit for listeria monocytogenes gene based on loop-mediated isothermal amplification technology and detecting method thereof | |
| CN101979660B (en) | Brucella detection kit and using method thereof | |
| CN102719548B (en) | Kit for detecting brucella and use method thereof | |
| CN102251044A (en) | Enterorrhagia colibacillus stx1 gene detection kit and application method thereof | |
| CN101824482A (en) | Detection kit for vibrio cholerae O1 group and detection method thereof | |
| CN107312858A (en) | A kind of LAMP primer composition thing and its kit for being used to detect Peanut Allergen Ara h6 genes | |
| CN102559897B (en) | Primer group for NPT(Noctumal Penile Tumescence)II gene detection, corresponding reagent kit for and use method thereof | |
| CN101403005B (en) | Rapid diagnosis reagent kit and detection method for cholera vibrio gene | |
| CN103484456B (en) | Loop-mediated isothermal amplification primer and kit for detecting apium graveolens ingredients and application of loop-mediated isothermal amplification primer | |
| CN103602725B (en) | Loop-mediated isothermal amplification primer for sesame component, kit and applications of the primer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |