阅读说明：本技术 基于电场诱导释放检测技术结合靶标基因片段的转基因检测方法和试剂盒 (Transgene detection method and kit based on electric field induced release detection technology combined with target gene fragment ) 是由 廖玮 莫亚勤 张晨光 于 2018-07-11 设计创作，主要内容包括：本发明“基于电场诱导释放检测技术直接结合靶标基因片段的转基因检测方法和试剂盒”,涉及电化学基因检测技术,所述试剂盒包括检测CP4-EPSPS序列的组合1和检测Lectin序列的组合2,可以快速高效地检测待测大豆材料是否为转基因材料。(The invention discloses a transgene detection method and a kit based on direct combination of an electric field induced release detection technology and a target gene fragment, and relates to an electrochemical gene detection technology, wherein the kit comprises a combination 1 for detecting a CP4-EPSPS sequence and a combination 2 for detecting a Lectin sequence, and can be used for quickly and efficiently detecting whether a soybean material to be detected is a transgene material.)

1. The kit for detecting the transgenic soybean based on the EFIRM technology is characterized by comprising the following probe combinations: detection of CP 4-combination 1 of EPSPS sequences

Capture probe No. 1: 5-AGAGTCAGCTTGTCAGCGTGTCAAAAAAAA-3, and

1, detection probe: 5-GCGCGCGTTAATTTGTGCCATTCTTGAAAGATCTGCT-3, and the components are mixed,

the CP4-EPSPS sequence is: 5-GACACGCTGACAAGCTGACTCTAGCAGATCTTTCAAGAATGGCACAAATTAAC detection of combination 2 of Lectin sequences

Capture probe 2: 5-TTGAAGGAAGCGGCGAAGCTGG-3, and

detection probe 2: 5-GTGTCAGGGGCATAGAAGGTGAAG-3, and the components are mixed,

the Lectin sequence is as follows: CCAGCTTCGCCGCTTCCTTCAACTTCACCTTCTATGCCCCTGACACAAAAAG are provided.

2. The kit of claim 1, wherein the detection probe is labeled at the 3 'end or the 5' end with an affibody for binding a catalytic enzyme; the affinity substance is digoxin, fluorescein isothiocyanate or biotin.

3. The kit of claim 2, further comprising a catalytic enzyme, wherein the catalytic enzyme is horseradish peroxidase or alkaline phosphatase with a label; the marker is digoxin antibody, fluorescein isothiocyanate antibody or streptavidin.

4. The kit of claim 3, further comprising a substrate for said catalytic enzyme;

when the catalytic enzyme is horseradish peroxidase, the substrate is any one of TMB, ABTS and OPD;

when the catalytic enzyme is alkaline phosphatase, the substrate is any one of a combination of BCIP and NBT, p-nitrophenylphosphate, disodium 4-nitrophenylphosphate, naphthol AS-BI phosphate, naphthol-AS-MX-phosphate.

5. The kit of any one of claims 1 to 4, further comprising an anchor for immobilizing the capture probe in a reaction well on a detection well plate, the anchor comprising a conductive polymer and an ionic compound;

the conductive polymer is any one of pyrrole, aniline and thiophene;

the ionic compound is sodium chloride or potassium chloride.

6. The kit of any one of claims 1 to 5, further comprising a wash solution A and a wash solution B, wherein the wash solution A is an SSC buffer containing SDS and the wash solution B is a PBS buffer containing Tween 20.

7. The detection pore plate is characterized in that a working electrode is arranged at the bottom of a reaction pore of the detection pore plate and is configured to apply voltage to form an electric field;

capture probes shown as the following nucleotide sequences are distributed and fixed in reaction holes of the detection pore plate:

capture probe No. 1: 5-AGAGTCAGCTTGTCAGCGTGTCAAAAAAAA

Capture probe 2: 5-TTGAAGGAAGCGGCGAAGCTGG-3;

and only one capture probe is immobilized in one reaction well.

8. The detection well plate of claim 7, wherein: the capture probe is mixed with a conductive polymer and an ionic compound to form a mixed solution, then the mixed solution is added into the reaction hole, and then the mixed solution is fixed on the surface of the inner bottom of the reaction hole after a first square wave electric field is applied through the working electrode;

the parameters of the first electric field are as follows: voltage A: 350mV, 1 s; the voltage B is 950mV for 1 s; 9 cycles were performed.

9. The detection well plate of claim 8, wherein the conductive polymer material is selected from at least one of aniline, thiophene and pyrrole conductive molecular monomers;

the ionic compound is selected from at least one of chloride, nitrate and sulfate;

the chloride salt is one of sodium chloride, potassium chloride, magnesium chloride and ammonium chloride,

the nitrate is one of sodium nitrate, potassium nitrate, magnesium nitrate and ammonium nitrate,

the sulfate is one of sodium sulfate, potassium sulfate, magnesium sulfate and ammonium sulfate.

10. A method for detecting and detecting transgenic soybeans based on an EFIRM technology, which is characterized in that the kit of any one of claims 1 to 6 is adopted, and the steps are as follows:

(1) using a test well plate according to any of claims 7 to 9; or the capture probe is added into a blank detection pore plate, and an electrode is arranged at the bottom in the reaction pore and is used for applying an electric field to the solution in the reaction pore to carry out polymerization reaction after the EFIRM detector is switched on; applying a first electric field to the solution in the reaction hole to perform polymerization reaction after switching on the EFIRM detector; after the electric field treatment is finished, cleaning the detection orifice plate; the parameters of the first electric field treatment are as follows: the voltage is 200 ℃ and 500mV, 1-5 s; the voltage is 800 and 1500mV for 1-5 s; 3-10 cycles;

(2) hybridization of the sample to the capture Probe

Adding a mixed solution of a sample to be detected and the hybridization buffer, and applying a second electric field to the reaction hole: the voltage is 200 ℃ and 500mV, 1-5 s; the voltage is 300 ℃ and 800mV for 1-5 s; 5-150 cycles; cleaning a detection orifice plate;

(3) hybridization of the sample with the detection probe

Adding a detection probe solution corresponding to the capture probe in the reaction well, and then applying a third electric field to the reaction well: the voltage is 200 ℃ and 500mV, 1-5 s; the voltage is 300 ℃ and 800mV for 1-5 s; 3-10 cycles; cleaning the detection pore plate, and immediately carrying out the next operation;

the concentration of the detection probe in the detection probe solution is 0.5-1.5 mu M

(4) Adding the catalytic enzyme solution, incubating and then cleaning;

(5) adding substrate, reading under the electric field treatment of-100 to-300 mV to obtain current value,

(6) the ratio of the value measured by the probe group 1 to the value measured by the probe group 2 is used as a judgment standard, and when the ratio is more than 0.17, the transgenic sample is judged;

when the ratio is less than 0.14, judging the sample as a non-transgenic sample;

when the ratio is between 0.14 and 0.17, the sample is re-measured.

Technical Field

The invention relates to a gene detection technology, in particular to a transgene detection method and a transgene detection kit based on electric field induced release detection technology combined with a target gene fragment

Background

Transgenic crops, also known as biotechnological crops, are plants and their progeny that employ DNA recombination techniques to integrate foreign genes into the genome of the recipient plant, thereby altering its genetic makeup and gene expression. At present, the planting area of transgenic soybean is the largest among dozens of approved transgenic crops, and occupies 56 percent of the global transgenic crop planting area.

The transgenic soybean mainly refers to glyphosate-resistant soybean, is the glyphosate-resistant soybean successfully researched and developed by Monsanto company and has the trade name of Roundup Ready^TM. Glyphosate (also known as Roundup) is a broad-spectrum nonselective organophosphorus herbicide applied to leaves, and the action mechanism of Glyphosate is to specifically inhibit plantsThe activity of shikimate hydroxy ethylene transferase (EPSPS) in the product and bacteria can prevent the amino acid synthesis and kill the product. Since all plants require EPSPS to catalyze the synthesis of amino acids, all plants are killed after use. The main method of glyphosate-resistant soybean gene engineering is to modify the target protein acted by herbicide to make it insensitive to herbicide, i.e. to promote EPSPS over-expression or to use the target protein gene with coding point mutation. Researchers of Monsanto company transfer EPsPs gene of petunia under the control of CaMV35S promoter into soybean genome to obtain transgenic soybean with 20-40 times increased shikimate hydroxyethyltransferase (EPSPS) expression. The soybeans are highly tolerant to the herbicide glyphosate, and the application of glyphosate in the field does not affect the yield of the soybeans.

The currently known exogenous gene segments in transgenic soybeans are Lectin, CP4-EPSPS, PAT, GAT4601, Cry1Ac, FMV35S-P, CaMV35S-P, NOS-3', CaMV 35S-T. In China, soybeans and bean products are traditional foods and the consumption is huge. In recent years, China is changed from the traditional export of soybeans to the import of first-grade soybeans in the world, and in the imported soybeans, the transgenic soybeans account for more than two thirds. The problems of toxicity and potential safety hazard of the transgenic soybean are controversial. The establishment of a corresponding perfect transgenic soybean detection technology is especially important for protecting the consumer's right to know and option of transgenic food.

At present, the transgenic soybean detection technology is developed rapidly. Many corresponding detection methods and means have been derived according to the detection sample and the target. The following methods are commonly used for detecting transgenic soybean, and each method has the characteristics.

Enzyme linked immunosorbent assay (ELISA) and test strip method, the detection accuracy depends on the activity and structure of target protein in the detected matter and is affected by impurity. Because the protein in the bean products can generate denaturation of different degrees along with different processing degrees, especially some bean products subjected to high-temperature high-pressure deep processing, the difficulty in actual detection is high, and the actual detection efficiency and accuracy are poor.

The common PCR technology can quickly and simply amplify the specific fragment CP4-EPSPS of the transgenic soybean, and has high specificity and sensitivity. However, the detection process takes a long time and is not suitable for rapid identification. And PCR amplification products easily pollute an experimental area, and easily cause experimental results such as false positive and the like of later experiments. In addition, the requirements on the skills and laboratories of the workers are high, and the workers need to be trained specially.

Fluorescence PCR detection technology: at present, the detection limit can reach 0.01 percent in literature reports, and compared with the common PCR, the real-time fluorescence PCR technology shows great advantages in the aspects of specificity, sensitivity and accuracy. And makes up for the defects of cross contamination and false positive of the traditional PCR technology. However, there are some problems, and the fluorescent quantitative PCR technique has high requirements for sample preparation and is susceptible to factors such as DNA degradation and complex matrix interference.

Loop-mediated isothermal amplification (LAMP), willow resol and the like perform LAMP amplification by taking CP4-EPSPS exogenous gene as a detection target fragment, and the detection limit of the LAMP is 0.01 percent. LAMP technology has high sensitivity, good specificity, short time consumption and low requirement on an instrument, but primer design is more complex, and improper treatment of an amplification product is easy to cause pollution and influence results.

Direct sequencing method: is easily influenced by factors such as DNA degradation, complex matrix interference and the like, and the low sensitivity can cause a large amount of missed detection. Meanwhile, the sequencing method is complex in detection operation and poor in timeliness, and has obvious limitation on practical application detection requiring high timeliness and high sensitivity.

The gene chip technology comprises the following steps: the specific nucleotide fragments of synthesized reporter gene, promoter and terminator are fixed on the surface of solid phase support medium in preset arrangement mode to form high-density array, the DNA of product to be tested is hybridized with probe, the signal is detected by special device, and the computer is used for analyzing and synthesizing to judge that the sample to be tested is transgenic product. However, the technology for manufacturing the chip is complex, special instruments and equipment are needed, and the cost is too high. In addition, expensive detection instruments and complicated sample preparation limit the gene chip technology as a conventional detection means.

The hybridization detection method comprises the following steps: mainly Southern hybridization and Northern hybridization. Southern hybridization is a technique in which cleaved DNA is transferred to a hybridization membrane and hybridized with a probe, and the position and copy number of foreign DNA can be determined. Northern hybridization is a technique in which RNA of a material is hybridized with a probe, and is used to detect the expression of a gene at the transcriptional level. Both Southern and Northern hybridizations basically involve transferring a nucleic acid fragment to be detected to a solid support of a specific portion, further binding to the solid support, and detecting the nucleic acid to be detected by previously labeling the nucleic acid fragment with a nucleic acid probe. The method is fast, convenient and easy to operate, and can be used for testing a plurality of samples. However, before the sample is tested, the sample cannot be amplified, so that the detection sensitivity is low. In addition, the detection time is too long, and field inspection or primary screening cannot be performed.

In conclusion, various derived systems based on Polymerase Chain Reaction (PCR), such as various variants of PCR reaction, such as real-time fluorescence PCR, LAMP, or sequencing reaction-based NGS, and gene chips based on the principle of molecular hybridization, etc. The methods have advantages and disadvantages, and can meet the detection requirements of food safety transgenosis in some aspects, but because the methods all need to amplify the number of target gene fragments through a chain enzyme amplification reaction, aerosol pollution caused by gene amplification cannot be avoided, so the requirements on experimental environment and experimenters are high, the standard laboratory is high in manufacturing cost, the whole operation cost is high, field detection cannot be carried out, and the clearance speed of soybeans and soybean products is greatly influenced particularly when the methods are used for import and export customs inspection.

Disclosure of Invention

In order to simply and conveniently detect the transgenic conditions of soybeans and bean products in different occasions, the invention provides a simple, easy-to-use, rapid and reliable technology, which is summarized as follows:

the kit for detecting the transgenic soybean based on the EFIRM technology is characterized by comprising the following probe combinations:

detection of CP 4-combination 1 of EPSPS sequences

Capture probe No. 1: 5-AGAGTCAGCTTGTCAGCGTGTCAAAAAAAA-3, and

1, detection probe: 5-GCGCGCGTTAATTTGTGCCATTCTTGAAAGATCTGCT-3, and the CP4-EPSPS sequence is as follows:

5-GACACGCTGACAAGCTGACTCTAGCAGATCTTTCAAGAATGGCACAAATTAAC

detecting combinations 2 of Lectin sequences consisting of

Capture probe 2: 5-TTGAAGGAAGCGGCGAAGCTGG-3, and

detection probe 2: 5-GTGTCAGGGGCATAGAAGGTGAAG-3, and the components are mixed,

the Lectin sequence is as follows:

CCAGCTTCGCCGCTTCCTTCAACTTCACCTTCTATGCCCCTGACACAAAAAG。

preferably, the 3 'end or the 5' end of the detection probe is labeled with an affibody for binding catalytic enzymes; the affinity substance is digoxin, fluorescein isothiocyanate or biotin.

Preferably, the kit further comprises catalytic enzyme, wherein the catalytic enzyme is horseradish peroxidase or alkaline phosphatase with a label; the marker is digoxin antibody, fluorescein isothiocyanate antibody or streptavidin.

Preferably, a substrate corresponding to the catalytic enzyme is also included;

when the catalytic enzyme is horseradish peroxidase, the substrate is any one of TMB, ABTS and OPD;

when the catalytic enzyme is alkaline phosphatase, the substrate is any one of a combination of BCIP and NBT, p-nitrophenylphosphate, disodium 4-nitrophenylphosphate, naphthol AS-BI phosphate, naphthol-AS-MX-phosphate.

Preferably, the kit of any of the above further comprises an anchor for immobilizing the capture probe in a reaction well on a detection well plate, the anchor comprising a conductive polymer and an ionic compound;

the conductive polymer is any one of pyrrole, aniline and thiophene;

the ionic compound is sodium chloride or potassium chloride.

Preferably, any one of the kits further comprises a washing solution, wherein the washing solution comprises a washing solution A and a washing solution B, the washing solution A is an SSC buffer solution containing SDS, and the washing solution B is a PBS buffer solution containing Tween 20.

The detection pore plate is characterized in that a working electrode is arranged at the bottom of a reaction pore of the detection pore plate and is configured to apply voltage to form an electric field;

capture probes shown as the following nucleotide sequences are distributed and fixed in reaction holes of the detection pore plate:

capture probe No. 1: 5-AGAGTCAGCTTGTCAGCGTGTCAAAAAAAA

Capture probe 2: 5-TTGAAGGAAGCGGCGAAGCTGG-3;

and only one capture probe is immobilized in one reaction well.

And only one capture probe is immobilized in one reaction well.

Preferably, the capture probe is mixed with a conductive polymer and an ionic compound to form a mixed solution, then the mixed solution is added into the reaction hole, and then the mixed solution is fixed on the inner bottom surface of the reaction hole after a first square wave electric field is applied through the working electrode;

the parameters of the first electric field are as follows: voltage A: 350mV, 1 s; the voltage B is 950mV for 1 s; the process was carried out for 9 cycles of,

preferably, the conductive polymer material is selected from at least one of aniline, thiophene and pyrrole conductive molecular monomers;

the ionic compound is selected from at least one of chloride, nitrate and sulfate;

the chloride salt is one of sodium chloride, potassium chloride, magnesium chloride and ammonium chloride,

the nitrate is one of sodium nitrate, potassium nitrate, magnesium nitrate and ammonium nitrate,

the sulfate is one of sodium sulfate, potassium sulfate, magnesium sulfate and ammonium sulfate.

A method for detecting and detecting transgenic soybeans based on an EFIRM technology is characterized in that any one of the kits is adopted, and the steps are as follows:

(1) using any of the assay well plates; or the capture probe is added into a blank detection pore plate, and an electrode is arranged at the bottom in the reaction pore and is used for applying an electric field to the solution in the reaction pore to carry out polymerization reaction after the EFIRM detector is switched on; applying a first electric field to the solution in the reaction hole to perform polymerization reaction after switching on the EFIRM detector; after the electric field treatment is finished, cleaning the detection orifice plate; the parameters of the first electric field treatment are as follows: the voltage is 200 ℃ and 500mV, 1-5 s; the voltage is 800 and 1500mV for 1-5 s; 3-10 cycles;

(2) hybridization of the sample to the capture Probe

Adding a mixed solution of a sample to be detected and the hybridization buffer, and applying a second electric field to the reaction hole: the voltage is 200 ℃ and 500mV, 1-5 s; the voltage is 300 ℃ and 800mV for 1-5 s; 5-150 cycles; cleaning a detection orifice plate;

(3) hybridization of the sample with the detection probe

Adding a detection probe solution corresponding to the capture probe in the reaction well, and then applying a third electric field to the reaction well: the voltage is 200 ℃ and 500mV, 1-5 s; the voltage is 300 ℃ and 800mV for 1-5 s; 3-10 cycles; cleaning the detection pore plate, and immediately carrying out the next operation;

the concentration of the detection probe in the detection probe solution is 0.5-1.5 mu M

(4) Adding the catalytic enzyme solution, incubating and then cleaning;

(5) adding substrate, reading under the electric field treatment of-100 to-300 mV to obtain current value,

(6) the ratio of the value measured by the probe group 1 to the value measured by the probe group 2 is used as a judgment standard, and when the ratio is more than 0.17, the transgenic sample is judged;

when the ratio is less than 0.14, judging the sample as a non-transgenic sample;

when the ratio is between 0.14 and 0.17, the sample is diluted or concentrated and then re-assayed.

The transgenic soybean detection method provided by the invention has the following advantages:

one, simple and fast operation

The conventional hybridization method needs long-time incubation, and results such as electrophoresis sequencing and the like need to be carried out after the common PCR amplification, so that the whole process is long in time consumption. The detection of gene chip, real-time fluorescence PCR and direct sequencing method has high requirement for technical personnel.

When the detection method is used for identifying transgenic soybeans based on an EFIRM technology, soybean DNA extraction samples and detection reagents are added into corresponding reaction holes of an e-plate, the e-plate is placed at a corresponding position of an EFIRM detector, a corresponding detection program is started on a computer, detection work can be finished by the instrument after simple ELISA-like plate washing steps, detection data are automatically uploaded to a cloud computing platform, and detection results are directly displayed on a user interface in a graphic or data mode after analysis. The detection process utilizes the effect of an electric field, so that the reaction rate is accelerated, the reaction time is shortened, the whole detection process can be completed within one hour, the detection time can be shortened for workers, and the working efficiency is improved.

Second, high sensitivity and accuracy

The traditional probe fixing method is to fix one end of a probe on a plane support, and the method can reduce the hybridization efficiency of the probe and a target DNA to be detected due to the hydrophobicity of the surface of a chip and other reasons; in the traditional nucleic acid hybridization process, the hybridization efficiency is improved by controlling the hybridization temperature, salt ions, reaction time and the like, and the electric field is added as a fourth control condition, so that the capture efficiency of the capture probe on the target DNA is improved under the action of the electric field; in the method, an electronic signal generated in the oxidation process of the HRP catalytic TMB is measured as a detection result, and the result of the hybridization reaction is indirectly amplified due to high catalytic efficiency of the enzyme, so that the sensitivity of the measurement method is increased. The EFIRM method has ultrahigh sensitivity by three core technologies of instant target molecule capture, ultrahigh activity molecule probe fixation and captured molecule signal specific amplification.

The sensitivity of the ordinary PCR or the fluorescent quantitative PCR is about 0.1 to 1 percent. The sensitivity of the enzyme-linked immunoassay method is 0.1-5%, and the hybridization method has lower sensitivity due to the technical principle limitation. Test data prove that the sensitivity of the detection method can reach 0.1%.

In the aspect of accuracy, false positive is easy to appear due to the self limitation of the PCR detection technology, the ELISA detection method has higher requirement on a detection sample, and the protein denaturation of the processed soybean can seriously influence the detection result. The method is based on the EFIRM detection platform technology, and the sample does not need high-temperature treatment; the design of the specific capture probe can specifically capture the required gene fragment, avoid false positive experimental results, and finally, the accuracy rate is more than 97 percent and the detection repeatability is more than 96 percent through capturing the specific amplification of molecular signals.

Thirdly, establishment of a methodology for detecting transgenes based on EFIRM technology

Through a large amount of experimental data statistics, the ratio of the detected current values of the target gene probe pair and the reference gene probe pair is used as the judgment standard of transgene detection:

when the transgenic sample is detected, the ratio of the detection value of the target gene probe pair to the detection value of the reference gene probe pair is more than 0.17;

when non-transgenic samples are detected, the ratio is less than 0.14.

When the ratio is between 0.14 and 0.17, the sample is re-measured,

fourthly, the cost is low

First, in terms of detection equipment, whether direct sequencing, gene chip or fluorescence quantification, the detection equipment is very expensive, and the price of the current commercially available fluorescence quantification instrument and gene chip hybridization instrument is tens of thousands yuan. The high price severely restricts the popularization and the application of the method in the practical detection. Moreover, the instruments need strict training on staff, and the popularization and application of the instruments on a common detection platform are greatly limited by high price and high requirements on detection staff. Compared with the EFIRM platform, the EFIRM platform adopts the original electric field guided release and measurement technology, the detection process utilizes the electric field effect, the reaction is fast, and the final result is detected in the form of an electric signal, so that the detection equipment is not provided with an expensive fluorescence detection system like a fluorescence quantitative PCR, the cost of the equipment is greatly reduced, and the detection equipment is only about half of that of a fluorescence quantitative PCR instrument.

Secondly, the EFIRM technology in terms of detection reagents is based on the principle of nucleic acid hybridization, and adopts a uniquely designed electrochemical technology. The nucleic acid probe used in the invention adopts an artificially synthesized oligonucleotide probe, a common Biotin modification method is adopted in the detection probe, the capture probe does not need to be modified, the preparation of the probe is finished by a commercialized DNA chemical synthesis company, the technical difficulty is low, the stability is good, and the cost is low.

Therefore, from the cost perspective, the EFIRM detection, whether the slave device or the reagent, is lower in cost than the PCR-based technology, and the development of portable EFIRM instrument devices in the future will further reduce the detection cost.

In a word, compared with the prior art, the developed product has the obvious advantages of accuracy, reliability, rapidness, convenience and economy, is favorable for repeated sampling detection, has wide application range, can cover the detection of various transgenic soybeans and products thereof, and is an ideal transgenic soybean identification technology.

Drawings

FIG. 1 is a schematic diagram of the operation of the detection kit of the present invention.

Detailed Description

The present invention is described below by way of exemplary examples, in which the specific conditions are not specified, and the procedures are performed according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Reagent and instrument

The EFIRM detector adopted by the invention is produced by Guangzhou Living organisms, and is recorded in Electrochemical Sensor for multiple biomarkers Detection, Clin Cancer Res.2009Jul 1, which is published in 2009 by Fang Wei and the like on client Cancer Research; 4446-4452, namely an electrochemical detector used therein.

Those skilled in the art can apply square wave (csw E-field) to the reaction wells using a general square wave generating apparatus, or using EFIRM apparatus and associated software developed by readily available Biotechnology Inc., according to the description of the present invention and the prior art mentioned above.

Or an EFIRM instrument as described in application No. 201610658321.X, entitled holding Structure and Detector comprising a holding Structure.

The blank detection pore plate with the electrode arranged at the bottom adopted by the invention is a product of Guangzhou living biotechnology limited company, and the structure and the working principle of the blank detection pore plate are disclosed in the utility model patent 201620769829.2.

The sequences to which this patent application relates are shown in table 1.

TABLE 1 Probe sequences

The Lectin sequence detected by the YHTS-2 probe pair is an internal reference sequence, and the CaMV35S promoter sequence and the CP4-EPSPS sequence detected by the YHTS-1 probe pair and the YHTS-3 probe pair are target gene internal sequences.