CN101838685A - Primer group for testing Roundup Ready transgenic soy bean EPSPS gene, rapid diagnosis kit and testing method thereof - Google Patents
Primer group for testing Roundup Ready transgenic soy bean EPSPS gene, rapid diagnosis kit and testing method thereof Download PDFInfo
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Abstract
The invention discloses a primer group for testing Roundup Ready transgenic soy bean EPSPS gene based on the loop-mediated isothermal amplification of DNA (LAMP), a rapid diagnosis kit and a testing method thereof. The kit comprises the primer group, Bst DNA polymerase, stabilizing solution, reacting solution, developing solution and positive control solution. The kit applies six sections and four primers, can judge whether the target substance is existent or not according to the amplification, so that the kit has a high specificity. The rapid diagnosis kit of the invention is rapid, efficient and sensitive; the amplification reaction can be realized only needing constant temperature without the special agent or equipment; the testing cost is low and the test process is simple. Pyrophosphate ions separated from the dNTP are combined with Mg<2+> in the reaction solution to generate the byproduct, i.e. magnesium pyrophosphate deposition, which can be observed and appraised by viewing; and the developing differences of the negative and positive results are obviously by adding the developing solution, and the results are more obvious and reliable.
Description
Technical Field
The invention relates to a biological detection reagent, in particular to a primer group for detecting Roundup Ready transgenic soybean EPSPS gene, a rapid diagnosis kit and a detection method.
Background
A glyphosate-resistant variety of soybean developed by Monsanto corporation in 1994 is transformed with a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (CP4EPSPS) from microbial Agrobacterium tumefaciens, and can resist the inhibition of the herbicide glyphosate on the shikimate synthesis pathway, so that the transgenic soybean can grow normally after being sprayed with the herbicide. The receptor of the variety is soybean variety A5403, and exogenous CP4EPSPS gene, E35S promoter and NOS terminator are integrated in transgenic soybean genome. The variety is the transgenic soybean variety with the largest planting area at present and is widely applied to the processing production of edible oil and food.
To enhance the supervision and management of transgenic products, various methods for detecting transgenic components are currently developed, ranging from protein-based ELISA detection techniques to nucleic acid-based Polymerase Chain Reaction (PCR) techniques and rapidly-developed gene chip techniques. The PCR detection technology is most universal due to sensitivity, specificity and high efficiency, and is identified by detecting an exogenous nucleic acid fragment contained in a transgenic product, and with the occurrence of fluorescence real-time quantitative polymerase chain reaction (real time PCR), the method not only can qualitatively identify the transgenic product, but also can quantify the transgenic components.
The detection technology of transgenic products represented by PCR technology also has some problems in practical application, such as the need of special instruments in the common PCR technology, easy cross contamination and complicated operation process. The Real time PCR technology better solves the problem of cross contamination and simplifies the operation process, but needs a more complex quantitative determination instrument, so the Real time PCR technology is not suitable for on-site rapid detection. And the cost of the fluorescent probe in the real-time quantitative polymerase chain reaction PCR technology is higher, thus increasing the difficulty of popularization and application. The gene chip technology can realize rapid and high-flux detection, but is expensive and has high detection cost. Therefore, the latest achievement of timely applying biotechnology development has important significance for continuously improving the detection requirement of the transgenic products. Among them, Isothermal Amplification (Isothermal Amplification) nucleic acid rapid detection technology is a great progress in transgenic product detection technology, loop-mediated Isothermal Amplification (loop-mediated Isothermal Amplification of DNA, abbreviated as LAMP) established at present has many advantages, and no useful kit for rapid diagnosis of transgenic crop EPSPS gene by loop-mediated Isothermal Amplification technology is available at present.
Disclosure of Invention
The invention aims to provide a primer group for detecting Roundup Ready transgenic soybean EPSPS gene by using loop-mediated isothermal amplification technology and having high specificity to the EPSPS gene, aiming at overcoming the defects of the prior art.
The invention also aims to provide a kit for quickly diagnosing the EPSPS gene of the transgenic crop, which has the advantages of low detection cost, convenient use, high detection speed and high specificity and is based on the loop-mediated isothermal amplification technology.
The invention also aims to provide a detection method of the kit for rapidly diagnosing the EPSPS gene of the transgenic crop.
The above purpose of the invention is realized by the following technical scheme:
the primer group for detecting the Roundup Ready transgenic soybean EPSPS gene is based on a loop-mediated isothermal amplification technology, selects a specific sequence of the Roundup Ready transgenic soybean EPSPS gene according to a disclosed Roundup Ready transgenic soybean EPSPS gene sequence, analyzes and designs the specific primer group which can specifically identify the Roundup Ready transgenic soybean EPSPS gene, and identifies the Roundup Ready transgenic soybean EPSPS gene by PCR, wherein the primer group consists of the following four primers:
an outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 1 is shown in the specification;
an outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO: 4, respectively.
The primer group can also consist of the following four primers:
an outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 5 is shown in the specification;
an outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO: 7 is shown in the specification;
the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO: shown in fig. 8.
The kit for quickly diagnosing the EPSPS gene of the transgenic crop consists of two pairs of primers, BstDNA polymerase, reaction liquid, stabilizing liquid, developing liquid and positive control liquid, wherein the six liquids are respectively placed in containers, and the kit comprises the following components in parts by weight:
the two pairs of primers are respectively as follows:
an outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 1 is shown in the specification;
an outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO: 4, respectively.
The two pairs of primers can also be:
an outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 5 is shown in the specification;
an outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO: 7 is shown in the specification;
the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO: shown in fig. 8.
1.6-2 mmol dNTP, 20-25 mmol Tris-HCl, 10-12.5 mmol potassium chloride, 10-12.5 mmol ammonium sulfate, 8-10 mmol magnesium sulfate, 1-1.25 ml TritonX-100, 0.8-1 mol betaine, 1.6-2 mol of each inner primer FIP/BIP and 0.2-0.25 mol of each outer primer F3/B3 are contained in each 1L reaction solution; the preferable ratio is 2mmol dNTP, 25mmol Tris-HCl, 12.5mmol potassium chloride, 12.5mmol ammonium sulfate, 10mmol magnesium sulfate, 1.25ml Triton X-100, 1mol betaine, 2mol each of inner primer FIP/BIP and 0.25mol each of outer primer F3/B3 per 1L reaction solution.
The color developing solution is preferably fluorescent dye SYBR Green I or EvaGreen.
The stabilizing liquid is preferably paraffin oil.
The positive control is EPSPS gene DNA fragment.
Production process of the gene rapid diagnosis kit
1. After the inner primer FIP/BIP and the outer primer F3/B3 are synthesized and purified, quantitative preparation, concentration detection and sampling quality inspection are carried out;
2. aseptically subpackaging the reaction solution, determining the concentration according to the experiment, and sampling and inspecting the quality;
3. aseptically packaging the stable liquid, and sampling for quality inspection;
4. preparing a positive control sample, subpackaging, and sampling for quality inspection;
5. and assembling the kit.
Fourth, the invention gene rapid diagnosis kit detection method
1. Sample processing
The extraction and content measurement of DNA in the sample to be tested are carried out according to the national standard and the regulation in GB/T19495.3-2004. When the DNA is extracted from a sample, the quality and concentration of the DNA are stable, so that the repeatability of the PCR reaction is ensured. It is ensured that the extracted DNA fragment is larger than the amplified fragment.
2. Reaction process of loop-mediated isothermal amplification technology
Adding 38-40% by volume of reaction liquid, 0.9-1.8% by volume of Bst DNA polymerase large fragment, 52-54.5% by volume of stabilizing liquid, 4.5-9% by volume of sample template DNA and reacting at constant temperature of 63-65 ℃ for 45-90 min into a reaction tube. The volume percentage is the volume percentage of the total volume of the four components.
3. Post-reaction treatment
And respectively adding color developing solutions into the reaction tube and the positive control group, and uniformly mixing, wherein the sample group is positive if the color development is the same as that of the control group, and the sample group is negative if the color development is not the same as that of the control group.
The principle of the invention is that Bst DNA polymerase and two pairs of special inner and outer primers (namely inner primer FIP/BIP and outer primer F3/B3) designed according to target gene sequences are utilized to specifically recognize six independent regions on a target sequence, cycle strand displacement reaction is started, complementary strand synthesis is started in the target DNA region, and as a result, a stem-loop DNA mixture with a plurality of loops of cauliflower structures is formed on the same strand in cycles by complementary sequences. In the LAMP reaction process, pyrophosphate ions precipitated from dNTP and Mg in the reaction solution2+Combining to generate a byproduct, namely milky white magnesium pyrophosphate precipitate, and determining the result through visual observation. The LAMP reaction is completed within 45-90 minutes under the condition of constant temperature (63-65 ℃). The relatively mild temperature condition and no temperature cycle simplify the required instrument, and overcome the inherent defects of long detection time, easy pollution, high detection cost and the like of the traditional PCR. In addition, the detection method has low requirement on the technical quality of detection personnel, is extremely simple and convenient to operate practically, does not need special reagents and instruments, and is favorable for establishing a quick screening system with low cost. The LAMP method is a simple, convenient, rapid and highly specific gene amplification method. Comparing the constant temperature gene amplification technology with the PCR technology (including the fluorescent real-time quantitative PCR technology), the technology is equivalent to or superior to the PCR technology in the methodological indexes such as sensitivity, specificity, detection range and the like, can realize on-site high-flux rapid detection without depending on any special instrument and equipment, and has far lower detection cost than the fluorescent quantitative PCR technology. At present, no kit in the aspect is sold at home.
Compared with the prior art, the invention has the following beneficial effects:
1. the gene rapid diagnosis kit can perform amplification reaction only at a constant temperature, does not need special reagents and equipment, and has low detection cost;
2. the gene rapid diagnosis kit of the invention applies six segments and four primers, and can judge whether a target substance exists according to whether amplification exists, thereby having high specificity;
3. the gene rapid diagnosis kit disclosed by the invention is rapid and efficient in amplification, can complete amplification in less than 1 hour, and is high in yield;
4. the gene rapid diagnosis kit has high sensitivity, and the amplification template only needs 10 copies or less;
5. the gene rapid diagnosis kit of the invention has simple and convenient identification, pyrophosphate ions precipitated from dNTP and Mg in reaction solution2+And after the coloring liquid is added, the negative and positive results have obvious color development difference, the verification rate is high, and the method is more obvious and reliable.
Drawings
FIG. 1 is a diagram showing the results of the specific detection in the reagent kit of example 4;
wherein 1 is TE buffer solution, 2 is ddH2O, 3 is soybean non-transgenic sample W794DNA, 4 is rice non-transgenic sample W818DNA, 5 is rape non-transgenic sample W2194DNA, 6 is 250ng soybean transgenic sample W562DNA, 7 is 100ng soybean transgenic sample W562DNA, and 8 is 50ng soybean transgenic sample W562 DNA.
Detailed Description
The present invention will be further described with reference to the following specific examples, which are not intended to limit the invention in any way.
Example 1 preparation of EPSPS Gene Rapid diagnostic kit for transgenic crop
(1) Synthesizing the oligodeoxynucleotide primers by a DNA synthesizer according to the following sequences:
an outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 1 is shown in the specification;
an outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO: 4, respectively.
(2) Purchasing a DNA polymerase: bst DNApolymerase (Large fragment), placed in a container.
(3) Preparing a reaction solution: the reaction solution was prepared so that 1L of the reaction solution contained 2mmol of dNTP, 25mmol of Tris-Cl, 12.5mmol of potassium chloride, 12.5mmol of ammonium sulfate, 10mmol of magnesium sulfate, 1.25ml of Triton X-100, 1mol of betaine, 2mol each of inner primers FIP/BIP and 0.25mol each of outer primers F3/B3, and the mixture was placed in a container.
(4) Purchasing a stabilizing solution: paraffin oil, put into container.
(5) Purchasing a color developing solution: SYBR Green I, placed in a container.
(6) Extracting a positive control: preparation of EPSPS gene DNA fragments, placing in containers respectively.
(8) Packaging the 5 containers into a kit, and packaging.
The preparation process is briefly described as follows:
1. after the inner primer FIP/BIP and the outer primer F3/B3 are synthesized and purified, quantitative preparation, concentration detection and sampling quality inspection are carried out;
2. aseptically subpackaging the reaction solution, determining the concentration according to the experiment, and sampling and inspecting the quality;
3. subpackaging the stable liquid, and sampling for quality inspection;
4. preparing a positive control sample, subpackaging, and sampling for quality inspection;
5. and assembling the kit.
Example 2 preparation of EPSPS Gene Rapid diagnostic kit for transgenic crop
The two pairs of primers are:
an outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 5 is shown in the specification;
an outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO: 7 is shown in the specification;
the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO: shown in fig. 8.
The formula of the reaction solution is as follows: 1.6mmol dNTP, 20mmol Tris-HCl, 10mmol potassium chloride, 10mmol ammonium sulfate, 8mmol magnesium sulfate, 1ml Triton X-100, 0.8mol betaine, 1.6mol of each inner primer FIP/BIP and 0.2mol of each outer primer F3/B3 are contained in each 1L reaction solution;
the color developing solution is EvaGreen.
The rest is the same as example 1.
Example 3 application of EPSPS gene rapid diagnosis kit for transgenic crop
In this example, the kit for rapidly diagnosing EPSPS gene of transgenic crop prepared in example 1 was used to rapidly diagnose EPSPS gene in a sample to be tested.
1. Sample processing (template DNA extraction)
1) Grinding 100mg of the pretreated sample in liquid nitrogen to powder, adding into 700 μ L CTAB extraction buffer I (directly adding the sample without grinding), shaking, mixing, keeping at 65 deg.C for 30min, and mixing by gradually reversing for 2-3 times.
2) Adding 700. mu.L chloroform-isoamyl alcohol, and mixing the solution by gentle inversion for 2-3 times. Centrifuge at 12000g for 5min to phase separate.
3) Transferring the supernatant to a clean centrifuge tube, adding 0.6 times volume of 4 deg.C pre-cooled isopropanol, standing at-20 deg.C for 5min, and centrifuging at 12000g for 5 min.
4) Discarding the supernatant, adding 1000 μ L70% ethanol, gently rotating the centrifuge tube, centrifuging at 8000g at 4 deg.C for 1min, discarding the supernatant, adding 20 μ L RNase A enzyme (10 μ g/μ L), and warm bathing at 37 deg.C for 30 min.
5) Adding 600 μ L sodium chloride solution, and warm bathing at 65 deg.C for 10 min. Adding 600 mu L of chloroform-Tris saturated phenol, reversing, mixing uniformly, centrifuging for 5min at 12000g, and transferring the supernatant into a 1.5mL centrifuge tube.
6) Adding 0.6 times volume of 4 deg.C pre-cooled isopropanol, standing at 4 deg.C for 30min, centrifuging at 12000g for 10min, and discarding supernatant.
7) Adding 1000 μ L of pre-cooled 70% ethanol at 4 deg.C, gently rotating the centrifuge tube, centrifuging at 12000g at 4 deg.C for 10min, and discarding the supernatant. The operation was repeated once. The liquid was evaporated at room temperature.
8) After the precipitate was dried, 50. mu.L of TE buffer was added to dissolve the DNA sufficiently, and the DNA was stored at-20 ℃ for further use.
2. Reaction process of loop-mediated isothermal amplification technology
1) Prepare the reaction system in a 200. mu.l reaction tube: mu.l of the reaction solution, 0.5. mu.l (4U) of Bst DNA polymerase and 2.5. mu.l of template DNA.
2) The prepared reaction tube is reacted for 1h at the constant temperature of 64 ℃.
3. Post-reaction treatment
Adding 2 mu l of SYBR Green I into the PCR reaction product, uniformly mixing, simultaneously adding SYBR Green I into a positive control tube, uniformly mixing, wherein the reaction tube is positive if the reaction tube and the control tube are Green, and the reaction tube is negative if the reaction tube is orange.
Example 4 specificity verification of EPSPS Gene Rapid diagnostic kit for transgenic crop
In this example, the kit for rapidly diagnosing the EPSPS gene of the transgenic crop prepared in example 2 was used to perform specific detection of the kit.
The kit for rapidly diagnosing the EPSPS gene of the transgenic crop prepared in the example 2 is adopted to respectively perform the detection on a soybean transgenic sample W562, a soybean non-transgenic sample W794, a rice non-transgenic sample W818, a rape non-transgenic sample W2194 and ddH2And amplifying in O and TE buffer solutions, wherein 250ng of non-transgenic sample DNA is added, 250ng, 100ng and 50ng of soybean transgenic sample DNA are added, and 2 repeats are set for each sample.
The reaction system is as follows: 22. mu.l of reaction solution, 0.5. mu.l of Bst DNA polymerase, 30. mu.l of stabilizing solution and 2.5. mu.l of DNA template.
Reaction procedure: the temperature in the metal bath is 65 ℃ and 1 hour.
After the reaction, 2.0. mu.l of a color developing solution was added, and the results were observed as shown in Table 1 and FIG. 1.
TABLE 1 kit specificity test results
In Table 1 above, P represents positive amplification; n represents negative amplification.
As can be seen from the results in Table 1, the target gene products were amplified positively in the reactions containing 50ng, 100ng and 250ng of DNA from the soybean transgenic samples, while the amplification was positive in the reactions containing DNA from the soybean non-transgenic samples, DNA from the rice non-transgenic samples and DNA from the canola non-transgenic samples, and ddH2Products can not be amplified in O and TE, and amplification negativity is presented, so that the kit for quickly diagnosing the EPSPS gene of the transgenic crop has high specificity and can correctly identify a transgenic soybean sample.
Primer group for detecting Roundup Ready transgenic soybean EPSPS gene, rapid diagnosis kit and detection method sequence table
SEQUENCE LISTING
<110> Guangzhou Huafeng Biotechnology Ltd
Primer group for detecting Roundup Ready transgenic soybean EPSPS gene, rapid diagnostic kit and detection method
<130>
<160>8
<170>PatentIn version 3.5
<210>1
<211>18
<212>DNA
<213> Artificial sequence
<400>1
gcgtgcaggt gaaatcgg 18
<210>2
<211>18
<212>DNA
<213> Artificial sequence
<400>2
cgcacgtcat gatcggct 18
<210>3
<211>40
<212>DNA
<213> Artificial sequence
<400>3
gcggtaggtg atcggcgtct tttggtgacc gtcttcccgt 40
<210>4
<211>41
<212>DNA
<213> Artificial sequence
<400>4
cgatggcctc cgcacaggtt tttcgatgac cgtcgtgatg c 41
<210>5
<211>16
<212>DNA
<213> Artificial sequence
<400>5
cctaccgcgt gccgat 16
<210>6
<211>16
<212>DNA
<213> Artificial sequence
<400>6
acttggccgg tgagct 16
<210>7
<211>42
<212>DNA
<213> Artificial sequence
<400>7
gctcgatgac cgtcgtgatg cttttgtgaa gtccgccgtg ct 42
<210>8
<211>44
<212>DNA
<213> Artificial sequence
<400>8
cggaaaagat gctgcagggc ttttttcctt ccaggcggat ggtg 44
Claims (7)
1. A primer group for detecting Roundup Ready transgenic soybean EPSPS gene is characterized by consisting of an outer primer F3, an outer primer B3, an inner primer FIP and an inner primer BIP, wherein:
the nucleotide sequence of the outer primer F3 is shown as SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of the outer primer B3 is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO: 4 is shown in the specification;
or,
the nucleotide sequence of the outer primer F3 is shown as SEQ ID NO: 5 is shown in the specification;
the nucleotide sequence of the outer primer B3 is shown as SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO: 7 is shown in the specification;
the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO: shown in fig. 8.
2. A Roundup Ready transgenic soybean EPSPS gene rapid diagnosis kit is characterized by comprising the primer group in claim 1, Bst DNA polymerase, reaction liquid, stabilizing liquid, developing liquid and positive control liquid, wherein the six liquids are respectively placed in containers, and the formula of the reaction liquid is as follows: 1.6-2 mmol dNTP, 20-25 mmol Tris-HCl, 10-12.5 mmol potassium chloride, 10-12.5 mmol ammonium sulfate, 8-10 mmol magnesium sulfate, 1-1.25 ml TritonX-100, 0.8-1 mol betaine, 1.6-2 mol of each inner primer FIP/BIP and 0.2-0.25 mol of each outer primer F3/B3 are contained in each 1L reaction solution; the positive control is EPSPS gene DNA fragment.
3. The rapid diagnostic kit according to claim 2, wherein the color developing solution is SYBR Green I or EvaGreen I.
4. The rapid diagnostic kit according to claim 2, wherein each 1L of the reaction solution contains 2mmol dNTP, 25mmol Tris-HCl, 12.5mmol potassium chloride, 12.5mmol ammonium sulfate, 10mmol magnesium sulfate, 1.25ml Triton X-100, 1mol betaine, 2mol each of inner primer FIP/BIP and 0.25mol each of outer primer F3/B3.
5. The rapid diagnostic kit according to claim 2, wherein the stabilizing solution is paraffin oil.
6. A method for detecting the Roundup Ready transgenic soybean EPSPS gene using the rapid diagnostic kit of claim 2, comprising the steps of:
(1) preparing DNA of a sample to be detected;
(2) adding 38-40% by volume of reaction liquid, 0.9-1.8% by volume of Bst DNA polymerase, 52-54.5% by volume of stabilizing liquid and 4.5-9% by volume of sample template DNA into a reaction tube, and reacting at constant temperature;
(3) and respectively adding color developing solutions into the reaction tube and the positive control group, and uniformly mixing, wherein the sample group is positive if the color development is the same as that of the control group, and the sample group is negative if the color development is not the same as that of the control group.
7. The detection method according to claim 6, wherein in the step (2), the reaction conditions of the isothermal reaction are 63-65 ℃ and the reaction time is 45-90 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559897A (en) * | 2012-01-13 | 2012-07-11 | 广州华峰生物科技有限公司 | Primer group for NPT(Noctumal Penile Tumescence)II gene detection, corresponding reagent kit for and use method thereof |
| WO2022226870A1 (en) * | 2021-04-29 | 2022-11-03 | 中国科学院大学宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature conditions, kit, and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1470646A (en) * | 2002-07-26 | 2004-01-28 | 深圳市匹基生物工程股份有限公司 | Primer sequence for EPSPS gene containing transgenic crop nucleic acid amplification |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559897A (en) * | 2012-01-13 | 2012-07-11 | 广州华峰生物科技有限公司 | Primer group for NPT(Noctumal Penile Tumescence)II gene detection, corresponding reagent kit for and use method thereof |
| WO2022226870A1 (en) * | 2021-04-29 | 2022-11-03 | 中国科学院大学宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature conditions, kit, and application |
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| CN101838685B (en) | 2012-10-03 |
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