CN112359098B - Method for detecting content of herbicide-tolerant transgenic soybean J12 by real-time fluorescence quantitative PCR - Google Patents
Method for detecting content of herbicide-tolerant transgenic soybean J12 by real-time fluorescence quantitative PCR Download PDFInfo
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Abstract
The invention discloses a real-time fluorescence quantitative PCR detection herbicide-tolerant transgeneThe method for controlling the content of soybean J12 comprises the steps of designing and synthesizing specific primers and probes by using a specific sequence of a transformant at the 3' end of herbicide-resistant transgenic soybean J12, and combining soybean internal standard genesLectinA standard curve is made by diluting a standard substance, the percentage content of the transgenic herbicide-tolerant soybean J12 transformant sequence is corrected by utilizing an internal standard gene, and a real-time fluorescent PCR quantitative detection system for an unknown sample is constructed. The real-time fluorescent quantitative PCR detection technology has the advantages of low cost, high sensitivity, strong specificity, relatively simple operation and the like, becomes a main transgenic detection technology recommended by national standards, ministry of agriculture bulletins, entry and exit inspection and quarantine industry standards and the like, and is widely adopted.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a method for detecting a transgenic product, in particular to a method for quantitatively detecting transgenic soybean J12 based on real-time fluorescence PCR (polymerase chain reaction). A standard curve is made through a standard product, and an internal standard gene is used for correcting the percentage content of a transgenic herbicide-resistant soybean J12 transformant sequence to construct a quantitative detection method for transgenic soybean J12.
Background
The transgenic crops develop rapidly in the world, and the planting area of the transgenic soybeans dominates the planting area of the transgenic crops in the world and accounts for more than 50%. The development of the transgenic soybean aims to be matched with the use of glyphosate herbicide, the glyphosate-resistant transgenic crop is the transgenic crop with the largest global sowing area at present, the development of the transgenic soybean can resist the glyphosate herbicide, the glyphosate is a non-selective herbicide, common soybean plants and weeds can be killed together, and the soybean yield is influenced. Consequently, the safety of transgenic food is a focus of current social attention. China stipulates clear policies and regulations for marketing and popularization of transgenic crops, and develops strict biosafety evaluation on the transgenic crops; the related detection standard and supervision and management method are also made for the production and operation of the transgenic food, and the specific regulation is made for the genetic marker.
At present, common methods for detecting transgenic components include a common PCR method, a real-time fluorescence quantitative PCR method and a loop-mediated isothermal amplification technology, wherein the real-time fluorescence quantitative PCR method has the advantages of rapidness, accuracy, simplicity, reliability, strong automation and the like, is widely used in the fields of transgenic detection, crop breeding, medical treatment and the like, and is also a common method for revising national standards of transgenic detection. Compared with a qualitative PCR detection method of the transgenic components, the real-time fluorescent quantitative PCR detection method can detect the transgenic components contained in the product, can relatively quantify the content of the contained transgenic components, and is more beneficial to the management of the transgenic products. At present, various transgenic plants have real-time fluorescence PCR detection methods.
The herbicide-resistant transgenic soybean J12 is developed by the research institute of crop science of Chinese academy of agricultural sciencesG2-EPSPSGenes andGATa new herbicide-resistant soybean strain. The researchers adopt agrobacterium-mediated transformation method to transformG2-EPSPSAndGATthe expression vector DNA of the two genes is introduced into a soybean receptor, and a J12 transformant with resistance to glyphosate is obtained through multi-generation screening, so that a new material with transgenic soybean independent intellectual property rights is created aiming at the common and serious wasteland of soybean production in China, scientific support is provided for improving the competitiveness of the transgenic soybean industry in China, and the method has important industrial application prospect in China. The herbicide-resistant transgenic soybean J12 has no related quantitative detection standard, and the research takes the specific sequence of the transformant at the 3' end of the J12 as a target toLectinA real-time fluorescent quantitative PCR detection method is established for internal standard genes through primer probe screening, specificity testing, standard curve construction and the like, and aims to provide important technical support for safety evaluation, administrative supervision and intellectual property protection of the transformant.
Disclosure of Invention
The invention overcomes the technical defect that the common PCR detection of the transgenic soybean J12 can only qualitatively and can not quantitatively detect the transgenic soybean J12, and provides a method for quantitatively detecting the transgenic soybean J12. The invention aims to solve the problem of the vacancy of the method for quantitatively detecting the transgenic soybean J12, and has the advantages of simplicity, easiness, high sensitivity, strong specificity and stability.
The technical content of the invention is as follows:
(1) The recombinant strain is characterized in that a 5' end transformant specific sequence including a junction of a J12 exogenous insertion fragment and a flanking sequence is as follows:
forward primer sequence: 5' GGCTTTACTAAAATATAAATCCTAA-3
Reverse primer sequence: 5' GGCGTTAATTCAGTACATTA-3
The probe sequence is as follows: FAM-TGACGCTTAGACAATTACAT-BHQ 1
Endogenous geneLectinThe gene sequence is as follows:
forward primer sequence: 5' GCCCTCTACTCCCCCCCCCCA-3
Reverse primer sequence: 5' GCCCATCTGCAAGTCTTTTT-3
The probe sequence is as follows: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1.
(2) And (3) PCR reaction system: total volume 20. Mu.L, including TaKaRa 2 × Premix ExTaq10 μ L (probe qPCR), 1 μ L each of the upstream and downstream primers (10 μmol/L), 0.5 μ L of the probe (10 μmol/L), 2 μ L of the DNA template (25 ng/μ L), and 20 μ L of the DNA template supplemented with sterile water.
(3) PCR reaction procedure: pre-denaturation at 95 ℃ for 10 min for 1 cycle, denaturation at 95 ℃ for 10 s, annealing at 60 ℃ for 30 s, and annealing at 60 ℃ for 40 cycles, and collecting fluorescence signals at 60 ℃.
(4) Constructing a standard reference substance for quantitative detection of a transgenic herbicide-tolerant soybean J12 transformant sequence, extracting genome DNA of the transgenic herbicide-tolerant soybean J12, performing gradient dilution to respectively obtain 7 concentrations of DNA such as 100%, 20%, 4%, 0.8%, 0.16%, 0.032%, 0.0064% and the like, performing real-time fluorescence PCR amplification by using the 7 concentrations of DNA as a template, and respectively constructing a specific sequence and an endogenous gene of the transgenic herbicide-tolerant soybean J12 transformantLectinStandard curve of gene.
(5) After setting a threshold value, the Ct value and the target concentration of each reaction are automatically calculated by data analysis software of the fluorescence quantitative PCR instrument and recorded. And calculating the percentage content of the transgenic herbicide-tolerant soybean J12 transformant sequence in the sample according to the formula A = B/C × 100%. Wherein A is the percentage content of the transgenic herbicide-tolerant soybean J12 transformant sequence, B is the target concentration of the transgenic herbicide-tolerant soybean J12 transformant sequence, and C isLectinThe target concentration of the endogenous gene.
The invention further discloses an application of the specific primer for quantitatively detecting the transgenic soybean J12 in the aspect of quick, quantitative and efficient screening of the J12, and an experimental result shows that: the method can specifically and quantitatively detect the components of the transgenic herbicide-tolerant soybean J12 transformant, the linearity is more than 0.997, the RSD is 0.12-0.55%, the quantitative limit reaches 0.16%, and the detection limit reaches 0.032%. The method provides a new technical means for accurate quantitative detection of the transgenic herbicide-tolerant soybean J12, and provides technical support for agricultural transgenic supervision.
The invention mainly considers the specificity of a quantitative detection transgenic soybean J12 primer on a transformant, solves the establishment of a molecular characteristic method of a new variety of transgenic herbicide-tolerant soybeans J12, and particularly solves the problem of quantitative detection on the transgenic soybean J12 by screening a proper specific primer.
The test effect of the invention is as follows:
(1) Specificity test
And performing real-time fluorescence PCR amplification by using genome DNAs of the transgenic corn mixed sample, the transgenic soybean mixed sample, the transgenic rape mixed sample, the transgenic rice mixed sample, the transgenic cotton mixed sample and the non-transgenic soybean as templates. The result shows (figure 1), only when the transgenic herbicide-tolerant soybean J12 genome DNA is taken as a template, a typical amplification curve or positive microdroplet is generated, and when other transgenic crops and non-transgenic soybean genome DNA are taken as templates, the typical amplification curve or the positive microdroplet is not generated, which indicates that the specificity of the detection method is good.
(2) Drawing of standard curve
The extracted transgenic herbicide-tolerant soybean genome DNA (25 ng/. Mu.L) is diluted to 20%, 4%, 0.8%, 0.16%, 0.032% and 0.0064% for PCR amplification. And (3) performing real-time fluorescent PCR amplification by using DNA (containing 100%) with 7 concentration gradients as a template to construct a transgenic herbicide-tolerant soybean J12 transformant specific sequence standard curve so as to realize relative quantitative analysis on the transgenic herbicide-tolerant soybean J12. The results show (figure 2; figure 3), the linearity of the method is more than 0.997 within the range of 0.006% to 100% of template content, the amplification efficiency is 107.9%, the detection limit LOD is 0.032%, and the quantification limit LOQ is 0.16%, so that the method is suitable for further quantitative analysis of transgenic herbicide-tolerant soybeans J12.
(3) Calculating the sequence content of transgenic herbicide-resistant soybean J12 transformant in a sample
Target concentration and endogenous gene of transgenic herbicide-tolerant soybean J12 transformant sequenceLectinThe target concentration of the transgenic herbicide-tolerant soybean J12 transformant is substituted into a formula, and the percentage content of the transgenic herbicide-tolerant soybean J12 transformant sequence in the sample is calculated. Calculating the percentage content of the transgenic herbicide-tolerant soybean J12 transformant sequence in the sample according to the formula:
a-percentage (%) of transgenic herbicide tolerant soybean J12 transformant sequences in the sample;
b-target concentration of transgenic herbicide-tolerant soybean J12 transformant sequence;
C—Lectintarget concentration of endogenous gene;
conclusion of the experiment
The research establishes a method for relatively quantitatively detecting the transgenic soybean J12 by real-time fluorescent PCR. The method for relatively quantitatively detecting the transgenic soybean J12 by real-time fluorescence PCR takes a transformant specific sequence thereof as a target, and establishes a real-time fluorescence quantitative PCR detection method based on a Taqman hydrolysis probe. The method can specifically and quantitatively detect the components of the transgenic herbicide-tolerant soybean J12 transformant, the linearity is more than 0.997, the RSD is 0.12-0.55%, the quantitative limit reaches 0.16%, and the detection limit reaches 0.032%. The method provides a new technical means for accurate quantitative detection of the transgenic herbicide-tolerant soybean J12, and provides technical support for agricultural transgenic supervision.
Drawings
FIG. 1: detecting the specificity of real-time fluorescent quantitative PCR; as shown in fig. 1: blank control; 2: transgenic herbicide tolerant soybean J12;3: mixing transgenic corn samples; 4: mixing transgenic soybean; 5: mixing the transgenic rape; 6: mixing transgenic rice samples; 7: mixing transgenic cotton; 8: a non-transgenic soybean sample;
FIG. 2 is a schematic diagram: amplifying a specific sequence of the real-time fluorescent quantitative PCR transformant;
FIG. 3: real-time fluorescence quantitative PCR standard curve.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The herbicide-resistant transgenic soybean J12 is researched and developed by the crop science research institute of Chinese academy of agricultural sciences, and can be freely provided for research and use in scientific research.
Example 1
(1) Sample 1: the transgenic herbicide-tolerant soybean J12 component is a self-made simulation mixed sample with 5 percent, and the mixed mode is that 500 ng of transgenic herbicide-tolerant soybean J12 genome DNA and 9500 ng of non-transgenic soybean WT genome DNA are fully and evenly mixed.
(2) Reagent: premix Premix Ex Taq (Probe qPCR) manufactured by TaKaRa; soybean endogenous Lectin gene synthesized by Shanghai and transformant specific sequence amplification primers.
Transformant specific sequence primers:
forward primer sequence: 5' GGCTTTACTAAAATATAAATCCTAA-3
Reverse primer sequence: 5' GGCGTTAATTCAGTACATTA-3
The probe sequence is as follows: FAM-TGACGCTTAGACACTTAATAACAAT-BHQ 1
Endogenous geneLectinThe gene sequence is as follows:
forward primer sequence: 5' GCCCTCTACTCCCCCCCCCCA-3
Reverse primer sequence: 5' GCCCATCTGCAAGTCTTTTT-3
The probe sequence is as follows: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1;
(3) And (3) PCR reaction system: total volume 20. Mu.L, including TaKaRa 2 × Premix ExTaq10 μ L (probe qPCR), 1 μ L each of the upstream and downstream primers (10 μmol/L), 0.5 μ L of the probe (10 μmol/L), 2 μ L of the DNA template (25 ng/μ L), and 20 μ L of the DNA template supplemented with sterile water.
(4) PCR reaction procedure: pre-denaturation at 95 ℃ for 10 min for 1 cycle, denaturation at 95 ℃ for 10 s, annealing at 60 ℃ for 30 s, and annealing at 60 ℃ for 40 cycles, and collecting fluorescence signals at 60 ℃.
(5) A standard curve was constructed by PCR amplification of 100% of transgenic soybean J12 genomic DNA diluted to 20%, 4%, 0.8%, 0.16%. 5 DNA (containing 100 percent) with concentration gradient is used as a template for real-time fluorescence PCR amplification to construct a specific sequence and an endogenous gene of a transgenic herbicide-tolerant soybean J12 transformantLectinStandard curve of gene.
(6) After setting a threshold value, the Ct value and the target concentration of each reaction are automatically calculated by data analysis software of the fluorescence quantitative PCR instrument and recorded. The target concentration of the specific sequence of the transgenic herbicide-tolerant soybean J12 transformant and the target concentration of the Lectin endogenous gene are substituted into a formula, and the percentage content of the specific sequence of the transgenic herbicide-tolerant soybean J12 transformant in a sample is calculated to be 5.57%, and the result is shown in table 1.
Calculating the percentage content of the transgenic herbicide-tolerant soybean J12 transformant sequence in the sample according to the formula:
in the formula:
a-percentage (%) of transgenic herbicide tolerant soybean J12 transformant sequences in the sample;
b-target concentration of transgenic herbicide-tolerant soybean J12 transformant sequence;
C—Lectintarget concentration of endogenous gene;
table 1 blind sample 1 test results
Conclusion of the experiment
The method for relatively quantitatively detecting the J12 content of the transgenic soybean by using the real-time fluorescent PCR established in the research is used for measuring 5 percent of self-made simulated mixed samples. The target concentration of the specific sequence of the transgenic herbicide-tolerant soybean J12 transformant and the target concentration of the Lectin endogenous gene are obtained by constructing a standard curve, the content of the self-made blind sample 1 is calculated by substituting a formula to be 5.57%, the deviation from a true value is 11.4%, and the requirement that the quantitative detection standard specification is not more than 25% is met. The method provides a new technical means for accurate quantitative detection of the transgenic herbicide-tolerant soybean J12, and provides technical support for agricultural transgenic supervision.
Example 2
(1) Sample 2: a self-made simulation mixed sample with 3% of transgenic herbicide-tolerant soybean J12 components is prepared by fully and uniformly mixing 300 ng of transgenic herbicide-tolerant soybean J12 genome DNA and 9700 ng of non-transgenic soybean WT genome DNA.
(2) Reagent: premix Premix Ex Taq (Probe qPCR) manufactured by TaKaRa; soybean endogenous Lectin gene synthesized by Shanghai and transformant specific sequence amplification primers.
Transformant specific sequence primers:
forward primer sequence: 5' GGCTTTACTAAAATATAAATCCTAA-3
Reverse primer sequence: 5' GGCGTTAATTCAGTACATTA-3
The probe sequence is as follows: FAM-TGACGCTTAGACAATTACAT-BHQ 1
Endogenous geneLectinThe gene sequence is as follows:
forward primer sequence: 5' GCCCTCTACTCCCCCCCCA + 3
Reverse primer sequence: 5' GCCCATCTGCAAGTCTTTTT-3
The probe sequence is as follows: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1;
(3) And (3) PCR reaction system: total volume 20. Mu.L, including TaKaRa 2 × Premix ExTaq10 μ L (probe qPCR), 1 μ L each of the upstream and downstream primers (10 μmol/L), 0.5 μ L of the probe (10 μmol/L), 2 μ L of the DNA template (25 ng/μ L), and 20 μ L of the DNA template supplemented with sterile water.
(4) PCR reaction procedure: pre-denaturation at 95 ℃ for 10 min for 1 cycle, denaturation at 95 ℃ for 10 s, annealing at 60 ℃ for 30 s for 40 cycles, and collecting fluorescence signals at 60 ℃.
(5) A standard curve was constructed by PCR amplification of 100% of transgenic soybean J12 genomic DNA diluted to 20%, 4%, 0.8%, 0.16%. 5 DNA (containing 100%) with concentration gradient is used as a template for real-time fluorescence PCR amplification to construct a specific sequence and an endogenous source of a transgenic herbicide-tolerant soybean J12 transformantLectinStandard curve of gene.
(6) After setting a threshold value, the Ct value and the target concentration of each reaction are automatically calculated by data analysis software of the fluorescence quantitative PCR instrument and recorded. The target concentration of the specific sequence of the transgenic herbicide-tolerant soybean J12 transformant and the target concentration of the Lectin endogenous gene are substituted into a formula, and the percentage content of the specific sequence of the transgenic herbicide-tolerant soybean J12 transformant in a sample is calculated to be 3.03%, and the result is shown in a table 2.
Calculating the percentage content of the transgenic herbicide-tolerant soybean J12 transformant sequence in the sample by the following formula:
a-percentage (%) of transgenic herbicide tolerant soybean J12 transformant sequences in the sample;
b-target concentration of transgenic herbicide-tolerant soybean J12 transformant sequence;
C—Lectintarget concentration of endogenous gene;
table 2 blind sample 2 test results
Conclusion of the experiment
The method for relatively quantitatively detecting the content of the transgenic soybean J12 by using the real-time fluorescent PCR established in the research is used for measuring 3 percent of self-made simulated mixed samples. The target concentration of the specific sequence of the transgenic herbicide-tolerant soybean J12 transformant and the target concentration of the Lectin endogenous gene are obtained by constructing a standard curve, the content of the self-made blind sample 2 is calculated to be 3.03% by substituting a formula, the deviation from a true value is 1%, and the requirement that the quantitative detection standard is not more than 25% is met. The method provides a new technical means for accurate quantitative detection of the transgenic herbicide-tolerant soybean J12, and provides technical support for agricultural transgenic supervision.
It will be apparent to those skilled in the art that various changes and modifications can be made in the above embodiments without departing from the scope and spirit of the invention, and it is intended that all such changes and modifications as fall within the true spirit and scope of the invention be interpreted in accordance with the principles of the invention. And the invention is not limited to the example embodiments set forth in the description.
SEQUENCE LISTING
<110> Tianjin City academy of agricultural sciences
<120> method for detecting content of herbicide-tolerant transgenic soybean J12 by real-time fluorescence quantitative PCR
<160> 6
<170> PatentIn version 3.5
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<212> DNA
<213> Artificial sequence
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ggctttacta aaatataaat cctaa 25
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<213> Artificial sequence
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<212> DNA
<213> Artificial sequence
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tgacgcttag acaacttaat aacacat 27
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<213> Artificial sequence
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agcttcgccg cttccttcaa cttcac 26
Claims (1)
1. The application of the transformant specific primer and probe for quantitatively detecting the transgenic soybean J12 in the aspect of identifying the soybean J12 is characterized by comprising the primer and probe for detecting the transformant specific sequence at the 5' end of the soybean J12:
forward primer sequence: 5' GGCTTTACTAAAATATAAATCCTAA-3
Reverse primer sequence: 5' GGCGTTAATTCAGTACATTA-3
The probe sequence is as follows: FAM-TGACGCTTAGACACTTAATAACAAT-BHQ 1
Detection of endogenous genesLectinPrimers and probes for gene sequences:
forward primer sequence: 5' GCCCTCTACTCCCCCCCCCCA-3
Reverse primer sequence: 5' GCCCATCTGCAAGTCTTTTT-3
The probe sequence is as follows: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1.
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