CN112798793A - Test strip and test card for detecting PAT/bar protein, and preparation method and application thereof - Google Patents
Test strip and test card for detecting PAT/bar protein, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of immunoassay, and particularly relates to a test strip and a test card for detecting PAT/bar protein, and a preparation method and application thereof. A reaction membrane of the test strip is provided with a detection line and a quality control line, the detection line is coated with a first PAT/bar protein monoclonal antibody, and the quality control line is coated with goat anti-rabbit IgG; the binding pad of the test strip is coated with a second PAT/bar protein monoclonal antibody marked by quantum dots and rabbit IgG marked by quantum dots. The test strip provided by the invention has the advantages of high sensitivity, strong accuracy, good repetition rate, good specificity and simple and convenient operation, can realize qualitative or quantitative detection of the Bar gene in transgenic plants or food, provides technical support for rapidly qualitatively or quantitatively detecting herbicide-resistant transgenic components of plants and food, and has important significance for detecting transgenic components of agricultural products such as plants and food and evaluating the safety of transgenic food.
Description
Technical Field
The invention belongs to the technical field of immunoassay, and particularly relates to a test strip and a test card for detecting PAT/bar protein, and a preparation method and application thereof.
Background
Weeds are produced in association with human productive activities and their presence is a result of long-term acclimatization, competition with cultivated crops for soil, crops, farming systems and social factors. The weed is controlled by the human beings from beginning to engage in agricultural production, and the herbicide spraying is the most efficient mode at present, so that the human beings are liberated from primary labor, and the efficiency is greatly improved. However, because herbicides can cause death of both weeds and crops, commercialization of transgenic crops with herbicide-resistant genes has become one of the most advantageous traits in agricultural production. By 2003, herbicide resistant crops account for over 80% of transgenic crops worldwide. From the development trend, the proportion of transgenic crops is still increasing. As the herbicide-resistant marker is widely applied to commercial transgenic plants, with the rapid development of transgenic food, foreign transgenic crops and food enter China in large quantity, the transgenic food detection technology is an important component for evaluating the safety of the transgenic food, and the technical research has important significance.
Bialaphos is one of many antibiotics produced by streptomyces, and its chemical structure includes the glutamic acid isomer (ppt) and two alanine residues (-alanine). Bialaphos is an inhibitor of glutamine synthetase (glutamine synthetase). The action of glutamine synthetase is inhibited by removing alanine residue through the action of intracellular incision enzyme to generate active glufosinate, and finally, high-concentration ammonia is accumulated in organisms (including plants and bacteria) to generate toxic action on the organisms. Thus, glufosinate is a widely used non-specific selective herbicide. Certain microorganisms are capable of producing an enzyme that detoxifies this effect by acetylating the amino group, this resistance gene being designated Bar. The protein coded by the gene can change phosphinothricin acetylation into nontoxic protein Phosphinothricin Acetyl Transferase (PAT), and the protein expressed by the gene is PAT/bar. Bar gene is widely used in genetic engineering breeding and is also a marker gene in genetic transformation, which makes plants resistant to herbicide with L-phosphinothricin as active component.
The existing method for detecting Bar gene is an immune combination colloidal gold chromatography, and colloidal gold has the characteristics of colloid and is easily influenced by electrolyte, so that the colloidal gold is condensed into large particles. Therefore, colloidal gold has at least one of the following disadvantages: firstly, colloidal gold is a particle visible to naked eyes, and the sensitivity is poor; secondly, the colloidal gold and the protein are combined together by electrostatic attraction, so that nonspecific interference exists, and false positive is easy to appear; and thirdly, the immune combination colloidal gold chromatography can only be used for qualitative detection and can not be used for quantitative detection of the Bar gene.
Therefore, the method which has high sensitivity and can quantitatively detect the Bar gene has important significance for detecting the transgenic components of agricultural products such as plants, foods and the like and evaluating the safety of transgenic foods.
Disclosure of Invention
The test strip is characterized in that a quantum dot labeled antibody is utilized to coat a PAT/Bar protein monoclonal antibody on a detection line, qualitative or quantitative detection of the Bar gene in the plant or food can be realized by carrying out qualitative or quantitative analysis on the PAT/Bar protein in the transgenic plant or food, the sensitivity is high, and the test strip and the test card have important significance for detecting transgenic ingredients of agricultural products such as the plant and the food and evaluating the safety of the transgenic food.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a test strip for detecting PAT/bar protein, wherein a reaction membrane of the test strip is provided with a detection line and a quality control line, the detection line is coated with a first PAT/bar protein monoclonal antibody, and the quality control line is coated with goat anti-rabbit IgG; the binding pad of the test strip is coated with a second PAT/bar protein monoclonal antibody marked by quantum dots and rabbit IgG marked by quantum dots.
In the invention, the test strip detects the PAT/bar protein by using a fluorescence immunochromatography technology, the test strip detects the PAT/bar protein by using a quantum dot labeled antibody, the quantum dot has the advantages of high yield, wide excitation spectrum, narrow emission spectrum, strong fluorescence emission intensity, good photochemical stability, photobleaching resistance, difficult quenching, capability of accepting long-time repeated excitation, capability of realizing time-resolved fluorescence detection and minimizing instantaneous fluorescence interference, so the test strip has high sensitivity, strong accuracy, good repetition rate and simple and convenient operation, can realize qualitative or quantitative detection of Bar gene in transgenic plant or food, provide technical support for rapid qualitative or quantitative detection of herbicide-resistant transgenic components of plant and food, has important significance for detecting transgenic ingredients of agricultural products such as plants, food and the like and evaluating the safety of transgenic food.
Preferably, the reaction membrane is a nitrocellulose membrane (NC membrane).
In any of the above schemes, preferably, the distance between the detection line and the quality control line is 3-5 mm.
In any of the above schemes, preferably, the working concentration of the first PAT/bar protein monoclonal antibody is 0.1 mg/mL-1 mg/mL.
In any of the above embodiments, preferably, the working concentration of the goat anti-rabbit IgG is 0.1mg/mL to 1 mg/mL.
In any of the above schemes, preferably, the solution used for diluting the first PAT/bar protein monoclonal antibody and the goat anti-rabbit IgG is a coating diluent, and the coating diluent comprises 0.005g/mL to 0.015g/mL Bovine Serum Albumin (BSA) and 0.005g/mL to 0.015g/mL NaCl.
In any of the above embodiments, preferably, the coating diluent is a Phosphate Buffer (PB) containing 0.005g/mL to 0.015g/mL Bovine Serum Albumin (BSA) and 0.005g/mL to 0.015g/mL NaCl, the concentration of the phosphate buffer is 50mM to 200mM, and the pH of the phosphate buffer is 6.0 to 8.0.
In any of the above schemes, preferably, the test strip comprises a sample pad, a binding pad, a reaction membrane and an absorption pad which are sequentially overlapped; the quality control line is positioned at one end of the reaction membrane close to the absorption pad, and the distance between the quality control line and the absorption pad is 7-10 mm; the detection line is positioned at one end of the reaction membrane close to the combination pad, and the distance between the detection line and the combination pad is 7-10 mm.
In any of the above embodiments, the sample pad treatment solution used in the preparation of the sample pad preferably includes 0.005g/mL to 0.015g/mL Bovine Serum Albumin (BSA) and 0.005g/mL to 0.015g/mL naci.
In any of the above embodiments, the sample pad treatment solution used in the preparation of the sample pad is a Tris-HCl buffer solution containing 0.005 g/mL-0.015 g/mL Bovine Serum Albumin (BSA) and 0.005 g/mL-0.015 g/mL NaCl.
In any of the above embodiments, the concentration of the Tris-HCl buffer is preferably 40 mM-80 mM.
In any of the above schemes, preferably, the pH value of the Tris-HCl buffer solution is 7.0-9.0.
In any of the above embodiments, the binding pad treatment solution used for preparing the binding pad is Tris-HCl (Tris-HCl) buffer solution containing 0.005 g/mL-0.015 g/mL Bovine Serum Albumin (BSA) and 0.005 g/mL-0.015 g/mL NaCl; the concentration of the Tris-HCl buffer solution is 40 mM-80 mM; the pH value of the Tris-HCl buffer solution is 7.0-9.0.
In any of the above schemes, the mass ratio of the second PAT/bar protein monoclonal antibody marked by the quantum dots to the rabbit IgG marked by the quantum dots on the binding pad is preferably 1 (1-3).
In any of the above schemes, preferably, the preparation method of the second PAT/bar protein monoclonal antibody labeled by the quantum dot comprises:
and mixing the activated quantum dot microspheres with the second PAT/bar protein monoclonal antibody for reaction, and covalently coupling the quantum dot microspheres with the antibody to obtain the quantum dot labeled second PAT/bar protein monoclonal antibody.
In any of the above schemes, preferably, the mass ratio of the second PAT/bar protein monoclonal antibody to the quantum dot microspheres is 0.03-0.08; the particle size of the quantum dot microspheres is 80 nm-400 nm; the temperature of the mixing reaction is 20-30 ℃; the activators used for the activation include 1-ethyl- (3-dimethylaminopropyl) -carbodiimides hydrochloride (EDC) and N-hydroxysuccinimide (NHS).
In any of the above embodiments, preferably, the final concentration of 1-ethyl- (3-dimethylaminopropyl) -carbonyldiimine hydrochloride (EDC) is 40mM to 80 mM; the final concentration of the N-hydroxysuccinimide (NHS) is 40 mM-80 mM.
In any of the above embodiments, preferably, the quantum dot is a single compound quantum dot or a composite quantum dot assembled by at least two compounds.
In any of the above embodiments, preferably, the compound is at least one selected from CdSe, CdTe, ZnS, InP, InAs, InGaAs, and InGaP.
In a second aspect, the present invention provides a method for preparing the test strip of the first aspect, the method comprising the following steps:
(1) preparing a second PAT/bar protein monoclonal antibody marked by quantum dots and rabbit IgG marked by quantum dots, mixing the marked antibodies, and spraying a film to obtain a binding pad;
(2) diluting goat anti-rabbit IgG, then scribing on a reaction membrane to form a quality control line, diluting the first PAT/bar protein monoclonal antibody, and then scribing on the reaction membrane to form a detection line;
(3) and sequentially fixing the sample pad, the combination pad, the reaction membrane and the absorption pad on the bottom plate to obtain the test strip.
Preferably, in the step (1), the spraying amount of the sprayed film is 3-6 muL/cm; in the step (2), the concentration of the scribing line is 0.5-1.5 muL/cm.
In any of the above schemes, preferably, the preparation of the bonding pad and the reaction membrane further comprises a drying operation, wherein the drying operation is to dry the bonding pad or the reaction membrane for 12 to 36 hours in an oven with a temperature of 20 to 50 ℃ and a humidity of less than 30%.
In any of the above schemes, preferably, the preparation method of the test strip comprises the following steps:
(1) mixing and reacting the activated quantum dot microspheres with the second PAT/bar protein monoclonal antibody, wherein the mass ratio of the second PAT/bar protein monoclonal antibody to the quantum dot microspheres is 0.03-0.08; the quantum dot microspheres are covalently coupled with antibodies, and the particle size of the quantum dot microspheres is 80-400 nm; obtaining a second PAT/bar protein monoclonal antibody marked by quantum dots; diluting and mixing the marked antibodies, loading the diluted and mixed antibodies into a film-scribing gold spraying machine, spraying a film according to the spraying amount of 3-6 mu L/cm to obtain a bonding pad, wherein the mass ratio of the second PAT/bar protein monoclonal antibody marked by the quantum dots to the rabbit IgG marked by the quantum dots on the bonding pad is 1 (1-3);
(2) diluting goat anti-rabbit IgG, and then marking the diluted goat anti-rabbit IgG on a reaction membrane to form a quality control line, wherein the working concentration of the goat anti-rabbit IgG is 0.1-1 mg/mL; diluting a first PAT/bar protein monoclonal antibody, and then scribing the diluted first PAT/bar protein monoclonal antibody onto the reaction membrane to form a detection line, wherein the working concentration of the first PAT/bar protein monoclonal antibody is 0.1-1 mg/mL; the concentration of the scribing is 0.5-1.5 muL/cm;
(3) and sequentially fixing the sample pad, the combination pad, the reaction membrane and the absorption pad on the bottom plate to obtain the test strip.
In a third aspect, the present invention provides a test card for detecting PAT/bar protein, comprising the test strip of the first aspect and a plastic casing for packaging the test strip.
Preferably, the upper cover of the clamping shell of the plastic shell is provided with a sample adding hole and an observation window, and the sample adding hole is arranged above the sample pad and used for observing the detection line and the quality control line through the observation window.
In a fourth aspect, the present invention provides the use of the test strip of the first aspect for detecting a herbicide-resistant transgenic plant.
Preferably, the herbicide-resistant transgenic plant is a transgenic bar plant.
In a fifth aspect, the present invention provides a use of the test strip of the first aspect for detecting PAT/bar protein.
Drawings
Fig. 1 is a schematic structural diagram of the test strip for detecting PAT/bar protein provided by the present invention, wherein 1 is a sample pad, 2 is a binding pad, 3 is a reaction membrane, 4 is a detection line, 5 is a quality control line, 6 is an absorption pad, 7 is a bottom plate, 8 is a dropwise added sample, and 9 is a sample flow direction;
FIG. 2 is a linear fitting graph of a sample of corn material for quantitatively detecting Bar gene by using the test strip provided by the invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the raw materials, instruments, equipment and the like used in the following examples are either commercially available or available by existing methods; the dosage of the reagent is the dosage of the reagent in the conventional experiment operation if no special description exists; the experimental methods are conventional methods unless otherwise specified.
In a first aspect, the embodiment of the invention provides a test strip for detecting PAT/bar protein, wherein a reaction membrane of the test strip is provided with a detection line and a quality control line, the detection line is coated with a first PAT/bar protein monoclonal antibody, and the quality control line is coated with goat anti-rabbit IgG; the binding pad of the test strip is coated with a second PAT/bar protein monoclonal antibody marked by quantum dots and rabbit IgG marked by quantum dots.
The goat anti-rabbit IgG on the quality control line can be combined with the rabbit IgG marked by the quantum dots on the combination pad to determine the validity of the detection result, and under the action of exciting light, if no optical signal with a certain wavelength is emitted at the quality control line, the test strip is invalid; rabbit IgG marked by quantum dots is coated on the bonding pad, so that cross reaction between the rabbit IgG and a mouse antibody on a detection line can be avoided; when immunoreaction occurs on the detection line and the quality control line, if one quantum dot labeled antibody is shared, and the PAT/bar protein content in the sample is changed, the fluorescence intensity value of the quality control line is correspondingly changed, so that the stability of the C value and the T/C value is influenced, and the quantitative accuracy is seriously influenced.
In the embodiment of the invention, the test strip detects the PAT/bar protein by using a fluorescence immunochromatography technology. The test strip adopts the quantum dot labeled antibody to detect the PAT/bar protein, and as the quantum dots have the advantages of high yield, wide excitation spectrum, narrow emission spectrum width, strong fluorescence emission intensity, good photochemical stability, light bleaching resistance, difficult quenching, capability of receiving long-time repeated excitation, capability of realizing time-resolved fluorescence detection and minimum instantaneous fluorescence interference, therefore, the test strip has high sensitivity and high accuracy, and has the advantages of stable detection result, good repetition rate, good specificity, simple operation, low cost and wide application, can realize qualitative or quantitative detection of Bar gene in transgenic plant or food, provide technical support for rapid qualitative or quantitative detection of herbicide-resistant transgenic components of plant and food, has important significance for detecting transgenic ingredients of agricultural products such as plants, food and the like and evaluating the safety of transgenic food.
During detection, a sample to be detected is dripped on a sample pad of the test strip, under the capillary effect, PAT/bar protein in the sample is combined with a second PAT/bar protein monoclonal antibody marked by quantum dots on a combination pad, the combined PAT/bar protein monoclonal antibody is diffused to a reaction membrane and captured by the coated first PAT/bar protein monoclonal antibody, and a complex is formed and gathered on a detection line; under the action of exciting light, the fluorescent substance emits light signals with certain wavelength, and the light signals are identified by the fluorescence immunoassay analyzer and converted into certain numerical values; and the fluorescence immunoassay analyzer judges the concentration of the PAT/bar protein in the sample according to a built-in reference range, so as to judge whether the detection result of the sample to be detected is negative or positive.
Further, the reaction membrane is a nitrocellulose membrane (NC membrane).
Further, the distance between the detection line and the quality control line is 3-5 mm, for example, the distance between the detection line and the quality control line can be 3mm, 4mm or 5 mm. The distance is too close, the fluorescence signal intensity of the detection line and the quality control line are easy to interfere with each other, so that the repetition rate or accuracy of the test result of the test strip is reduced; the distance is too far, the fluorescence detection signals of the detection line and the quality control line are unstable, the error is large, the detection result is unstable, and the repetition rate of the test result of the test strip is low.
Further, the working concentration of the first PAT/bar protein monoclonal antibody is 0.1mg/mL to 1mg/mL, for example, the working concentration of the first PAT/bar protein monoclonal antibody may be 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, or 1mg/mL, etc., preferably, the working concentration of the first PAT/bar protein monoclonal antibody is 0.5 mg/mL.
Further, the working concentration of the goat anti-rabbit IgG is 0.1mg/mL to 1mg/mL, for example, the working concentration of the goat anti-rabbit IgG is 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, or 1mg/mL, and preferably the working concentration of the goat anti-rabbit IgG is 0.5 mg/mL.
Further, the solution used in diluting the first PAT/bar protein monoclonal antibody and goat anti-rabbit IgG is a coating diluent comprising 0.005g/mL to 0.015g/mL (e.g., 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, or 0.015g/mL, etc., preferably 0.01g/mL) Bovine Serum Albumin (BSA), and 0.005g/mL to 0.015g/mL (e.g., 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.006g/mL, 0.013g/mL, or the like, preferably 0.0085g/mL) NaCl.
Further, the coating diluent is a phosphate buffer (preferably a phosphate buffer) containing 0.005g/mL to 0.015g/mL (for example, 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, or 0.015g/mL, and preferably 0.01g/mL) of Bovine Serum Albumin (BSA), and 0.005g/mL to 0.015g/mL (for example, 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, or 0.015g/mL of NaCl, and preferably 0.85 g/mL).
Further, the phosphate buffer may have a concentration of 50mM to 200mM, for example, a concentration of 50mM, 100mM, 150mM, or 200mM, and preferably a concentration of 100 mM.
Further, the pH value of the phosphate buffer is 6.0 to 8.0, for example, the pH value may be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0, and preferably, the pH value is 7.4.
Further, the test strip comprises a sample pad, a combination pad, a reaction membrane and an absorption pad which are sequentially overlapped; the quality control line is positioned at one end of the reaction membrane close to the absorption pad, and the distance between the quality control line and the absorption pad is 7 mm-10 mm, for example, the distance can be 7mm, 8mm, 9mm or 10 mm; the detection line is positioned at one end of the reaction membrane close to the binding pad, and is 7 mm-10 mm away from the binding pad, for example, the distance may be 7mm, 8mm, 9mm, 10mm, or the like.
Further, the sample pad treatment solution used for preparing the sample pad includes 0.005g/mL to 0.015g/mL (for example, 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, or 0.015g/mL, and the like, and preferably 0.01g/mL) of Bovine Serum Albumin (BSA), and 0.005g/mL to 0.015g/mL (for example, 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, or 0.015g/mL, and the like, and preferably 0.85 g/mL of NaCl).
Further, the sample pad treatment solution used for preparing the sample pad is a buffer solution containing 0.005g/mL to 0.015g/mL (for example, 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, or 0.015g/mL, and preferably 0.01g/mL) Bovine Serum Albumin (BSA), and 0.005g/mL to 0.015g/mL (for example, 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, or 0.015g/mL, and preferably 0.0085g/mL) of NaCl. The sample pad treatment solution must be capable of adjusting the pH value of an added sample, has a wide buffering capacity range, and cannot be precipitated with heavy metal ions in transgenic plants.
Further, the concentration of the Tris-HCl buffer is 40 mM-80 mM, for example, the concentration may be 40mM, 50mM, 60mM, 70mM, or 80mM, and preferably, the concentration is 50 mM.
Further, the pH value of the Tris-HCl buffer is 7.0-9.0, for example, the pH value can be 7.0, 7.5, 8.0, 8.5 or 9.0, and the like, and preferably, the pH value of the Tris-HCl buffer is 8.0.
Further, the binding pad treatment solution used for preparing the binding pad is a buffer solution containing 0.005g/mL to 0.015g/mL (e.g., 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, or 0.015g/mL, and preferably 0.01g/mL) Bovine Serum Albumin (BSA), and 0.005g/mL to 0.015g/mL (e.g., 0.005g/mL, 0.006g/mL, 0.007g/mL, 0.008g/mL, 0.009g/mL, 0.01g/mL, 0.011g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, or 0.015g/mL, and preferably 0.0085g/mL) of NaCl; the concentration of the Tris-HCl buffer is 40 mM-80 mM, for example, the concentration can be 40mM, 50mM, 60mM, 70mM or 80mM, and the like, and preferably, the concentration is 50 mM; the pH value of the Tris-HCl buffer solution is 7.0-9.0, for example, the pH value can be 7.0, 7.5, 8.0, 8.5 or 9.0, and the like, and preferably, the pH value of the Tris-HCl buffer solution is 8.0.
Further, the mass ratio of the second PAT/bar protein monoclonal antibody labeled by the quantum dots on the binding pad to the rabbit IgG labeled by the quantum dots is 1: (1-3), for example, the mass ratio of the quantum dot labeled second PAT/bar protein monoclonal antibody to the quantum dot labeled rabbit IgG may be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, etc., preferably, the mass ratio is 1: 1.5; the mass does not contain the mass of the quantum dot covalently coupled to the antibody, e.g., the mass of the antibody is a, the mass of the quantum dot covalently coupled to the antibody is b, and then the mass of the quantum dot-labeled antibody is a.
Further, the preparation method of the second PAT/bar protein monoclonal antibody marked by the quantum dots comprises the following steps: and mixing the activated quantum dot microspheres with the second PAT/bar protein monoclonal antibody for reaction, and covalently coupling the quantum dot microspheres with the antibody to obtain the quantum dot labeled second PAT/bar protein monoclonal antibody. In the embodiment of the invention, the rabbit IgG is labeled by the same method as the second PAT/bar protein monoclonal antibody.
Further, the mass ratio of the second PAT/bar protein monoclonal antibody to the quantum dot microspheres is 0.03-0.08, for example, the mass ratio may be 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, or the like.
Further, the mass ratio of the rabbit IgG to the quantum dot microspheres is 0.05-0.1, for example, the mass ratio can be 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1.
Further, the particle size of the quantum dot microsphere is 80nm to 400nm, for example, the particle size may be 80nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm or 400nm, and preferably, the particle size may be 100nm, 200nm or 300nm, and the quantum dot microsphere has a carboxyl functional group, which is beneficial for coupling an antibody. The coupling efficiency and coupling strength of the quantum dot microspheres and the antibody can be better improved by limiting the particle size of the quantum dot microspheres within the range of 80 nm-400 nm.
Further, in order to improve the distinction degree between the signal and the background, in the embodiment of the present invention, the emission wavelength range of the quantum dot microsphere is 200 to 1500nm, for example, the emission wavelength is 200nm, 400nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1200nm, 1400nm, or 1500nm, and the like, and preferably, the emission wavelength range is 400 to 900 nm. The fluorescence intensity of the reaction film and the base plate is very weak in a near infrared region (600-800 nm), so that the emission wavelength range is more preferably 600-800 nm, and the emission wavelength of the quantum dot microspheres is more preferably 610 nm.
Further, the temperature of the mixing reaction is 20 to 30 ℃, for example, the temperature may be 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 28 ℃ or 30 ℃, and preferably, the temperature is 25 ℃.
Further, the activating agents used for the activation include 1-ethyl- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).
Further, the final concentration of the 1-ethyl- (3-dimethylaminopropyl) -carbonyldiimine hydrochloride (EDC) is 40mM to 80mM, and for example, the final concentration may be 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, or 80mM, and preferably, the final concentration is 50 mM.
Further, the final concentration of the N-hydroxysuccinimide (NHS) is 40 mM-80 mM, for example, the final concentration may be 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, or 80mM, and preferably, the final concentration is 50 mM.
Further, the quantum dot is a single compound quantum dot or a composite quantum dot assembled by at least two compounds.
Further, the compound is selected from at least one of CdSe, CdTe, ZnS, InP, InAs, InGaAs, and InGaP.
Further, the quantum dots are CdSe quantum dots, CdTe quantum dots, CdSe/ZnS quantum dots, CdTe/CdSe quantum dots, InP quantum dots, InAs quantum dots, InGaAs quantum dots, InGaP/ZnS quantum dots, or the like.
Further, in the method for preparing the second PAT/bar protein monoclonal antibody labeled by the quantum dot, the pH of the labeling buffer used for labeling may be 5.5 to 6.7, for example, 5.5, 5.6, 5.8, 6, 6.2, 6.4, 6.5, or 6.7, and preferably, the pH is 6.0 or 6.5.
Further, the labeling buffer was 2- (N-morpholino) ethanesulfonic acid (MES) buffer.
The pH of the labeling preservative solution used after the labeling is completed is 7 to 7.5, for example, the pH may be 7, 7.1, 7.2, 7.3, 7.4, 7.5, or the like, and preferably the pH is 7.4.
Further, the marker preservation solution is a phosphate buffer solution containing 3 to 8mg/mL (for example, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL or the like, preferably 5mg/mL) of Bovine Serum Albumin (BSA).
Illustratively, the method for labeling rabbit IgG comprises the following steps:
and (3) activation: taking a marked 100 mu L quantum dot microsphere (with the concentration of 10mg/mL) as an example, adding 100 mu L quantum dot microsphere into a marked buffer solution, shaking and mixing uniformly, adding an activating agent, and carrying out incubation reaction at room temperature; centrifuging, removing supernatant, collecting precipitate, resuspending with labeled buffer solution, diluting to 500 μ L, and ultrasonically dispersing;
antibody labeling: adding rabbit IgG 0.075mg into the activated 500 μ L microsphere buffer (about 100 μ L corresponding to the original concentration of the microsphere), and performing concussion coupling reaction;
blocking the antibody: after the reaction was completed, 400. mu.L of 0.1g/mL BSA solution was added and mixed well, and blocked at 25 ℃ for 0.5 h.
And (3) purification: centrifuging, removing supernatant, collecting precipitate, redissolving with 100 μ L labeled preservative solution, and shaking for dispersion to obtain quantum dot labeled rabbit IgG stock solution; storing at 2-8 deg.C (such as 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C or 8 deg.C), and using within 72 h.
In a second aspect, the embodiment of the present invention provides a method for preparing the test strip of the first aspect, where the method for preparing the test strip includes the following steps:
(1) preparing a second PAT/bar protein monoclonal antibody marked by quantum dots and rabbit IgG marked by quantum dots, mixing the marked antibodies, and spraying a film to obtain a binding pad;
(2) diluting goat anti-rabbit IgG, then scribing on a reaction membrane to form a quality control line, diluting the first PAT/bar protein monoclonal antibody, and then scribing on the reaction membrane to form a detection line;
(3) and sequentially fixing the sample pad, the combination pad, the reaction membrane and the absorption pad on the bottom plate to obtain the test strip.
In the step (1), the spraying amount of the sprayed film is 3-6 μ L/cm, for example, the spraying amount can be 3 μ L/cm, 4 μ L/cm, 5 μ L/cm or 6 μ L/cm, and preferably the spraying amount is 4 μ L/cm.
Further, in the step (2), the concentration of the scribe line is 0.5 to 1.5. mu.L/cm, for example, the concentration may be 0.5. mu.L/cm, 0.6. mu.L/cm, 0.8. mu.L/cm, 1. mu.L/cm, 1.2. mu.L/cm, 1.4. mu.L/cm or 1.5. mu.L/cm, and the like, and preferably, the concentration is 1. mu.L/cm.
Further, the preparation of the conjugate pad and the reaction membrane further includes a drying operation, wherein the drying operation is to dry the conjugate pad or the reaction membrane for 12h to 36h (for example, the drying time may be 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, etc., preferably, the drying time is 24h) in an oven with a temperature of 20 ℃ to 50 ℃ (for example, the temperature may be 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, etc., preferably, the temperature is 45 ℃), and a humidity of less than 30% (for example, the humidity may be 0%, 5%, 10%, 15%, 20%, 25%, etc.).
Further, the preparation method of the test strip comprises the following steps:
(1) mixing and reacting the activated quantum dot microspheres with the second PAT/bar protein monoclonal antibody, wherein the mass ratio of the second PAT/bar protein monoclonal antibody to the quantum dot microspheres is 0.03-0.08; the quantum dot microspheres are covalently coupled with an antibody, and the particle size of the quantum dot microspheres is 80-400 nm, so that a second PAT/bar protein monoclonal antibody marked by quantum dots is obtained; the preparation method of the quantum dot labeled rabbit IgG is the same as that of the quantum dot labeled second PAT/bar protein monoclonal antibody; diluting and mixing the labeled antibodies, loading the diluted antibodies into a film-scribing gold spraying machine, spraying a film according to the spraying amount of 3-6 mu L/cm to obtain a bonding pad, wherein the mass ratio of the quantum dot labeled second PAT/bar protein monoclonal antibody to the quantum dot labeled rabbit IgG on the bonding pad is 1: (1-3);
(2) diluting goat anti-rabbit IgG, and then marking the diluted goat anti-rabbit IgG on a reaction membrane to form a quality control line, wherein the working concentration of the goat anti-rabbit IgG is 0.1-1 mg/mL; diluting a first PAT/bar protein monoclonal antibody, and then scribing the diluted first PAT/bar protein monoclonal antibody onto the reaction membrane to form a detection line, wherein the working concentration of the first PAT/bar protein monoclonal antibody is 0.1-1 mg/mL; the concentration of the scribing is 0.5-1.5 muL/cm;
(3) and sequentially fixing the sample pad, the combination pad, the reaction membrane and the absorption pad on the bottom plate to obtain the test strip.
Illustratively, the embodiment of the invention provides a test strip for detecting PAT/bar protein, the test strip includes a bottom plate, a sample pad, a binding pad, a reaction membrane and an absorption pad are sequentially overlapped on the bottom plate, the material of the bottom plate is PVC or a hard non-absorbent material, the material of the sample pad is glass fiber, cotton fiber, filter paper fiber or polyester fiber, the material of the binding pad is glass fiber or polyester fiber, the material of the absorption pad is absorbent filter paper, and the specific preparation steps are as follows:
(1) preparation of a reaction film: scribing a quality control line, diluting the goat anti-rabbit IgG to 0.5mg/mL by using a coating diluent for coating, wherein the scribing concentration is 1 mu L/cm; carrying out detection line scribing, namely diluting the first PAT/bar protein monoclonal antibody to 0.5mg/mL, wherein the scribing concentration is 1 muL/cm; and after the reaction is finished, placing the mixture in an oven with the humidity of less than 30% and the temperature of 45 ℃, drying the mixture for 24 hours, taking out the dried mixture, and sealing and storing the dried mixture.
(2) Preparation of the bonding pad: soaking the bonding pad for 1.5h by using the bonding pad treatment solution, and then drying the bonding pad in an oven with the humidity of less than 30% and the temperature of 45 ℃ for 18 h; wherein the treatment solution of the combination pad is Tris-HCl buffer solution containing 0.01g/mL bovine serum albumin, 8.5mg/mL NaCl and having a pH value of 8.0 and 50 mM.
Diluting the rabbit IgG marked by the quantum dots and the second PAT/bar protein monoclonal antibody by using a marking treatment solution, loading the diluted solution into a film-scratching gold spraying machine, spraying the diluted solution onto a treated combination pad according to the spraying amount of 4 mu L/cm, placing the treated combination pad in an oven with the humidity of less than 20% and the temperature of 45 ℃, drying for 24h, and sealing and storing at 4-8 ℃.
(3) Preparation of sample pad: soaking the sample pad in Tris-HCl buffer solution containing 0.01g/mL bovine serum albumin, 8.5mg/mL NaCl, pH value of 8.0 and 50mM for 2h, then placing the sample pad in an oven with humidity of less than 30% and temperature of 45 ℃, drying for 18h, and sealing and storing at 4-8 ℃.
(4) Assembling the test strip: the sample pad, the combination pad, the reaction film and the absorption pad are sequentially overlapped and stuck and fixed on the bottom plate from left to right, the overlap is 2mm, the tail end of the sample pad is connected with the initial end of the combination pad, the tail end of the combination pad is connected with the initial end of the reaction film, the tail end of the reaction film is connected with the initial end of the absorption pad, the initial end of the sample pad is aligned with the initial end of the bottom plate, the tail end of the absorption pad is aligned with the tail end of the bottom plate, and then a machine is used for cutting the sample pad, the combination pad, the reaction film and the absorption pad into a plurality of test strips with the.
In a third aspect, the embodiment of the present invention provides a test card for detecting PAT/bar protein, including the test strip of the first aspect and a plastic housing for packaging the test strip; the upper cover of the clamping shell of the plastic shell is provided with a sample adding hole and an observation window, wherein the sample adding hole is arranged above the sample pad and passes through the observation window to observe the detection line and the quality control line.
In a fourth aspect, the embodiments of the present invention provide a use of the test strip of the first aspect, for detecting a herbicide-resistant transgenic plant.
Further, the herbicide-resistant transgenic plant is a bar transgenic plant.
In a fifth aspect, the embodiments of the present invention provide a use of the test strip of the first aspect, wherein the test strip is used for detecting PAT/bar protein.
In the present invention, the sources of the first PAT/bar protein monoclonal antibody, the goat anti-rabbit IgG, the second PAT/bar protein monoclonal antibody and the rabbit IgG are not particularly limited, and commercially available products meeting the above requirements, which are well known to those skilled in the art, may be used, or antibodies meeting the above requirements may be prepared according to methods commonly used by those skilled in the art.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
The invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.
The test strip for detecting the PAT/bar protein is used for detecting based on a fluorescence immunochromatography method and an immunocapture method, and the working principle of the test strip is briefly described with reference to FIG. 1 as follows:
the reaction membrane of the test strip comprises a detection line (T line) and a quality control line (C line), wherein the detection line is coated with a first PAT/bar protein monoclonal antibody, and the quality control line is coated with goat anti-rabbit IgG;
a second PAT/bar protein monoclonal antibody and rabbit IgG marked by quantum dot fluorescent microspheres are fixed on the binding pad of the test strip;
during detection, a sample to be detected is dripped on a sample pad of the test strip and is diffused under the capillary effect, if the sample to be detected contains PAT/bar protein, the PAT/bar protein is combined with a second PAT/bar protein monoclonal antibody marked by a quantum dot and is diffused to a detection area, and the PAT/bar protein is captured by the coated first PAT/bar protein monoclonal antibody to form a compound to be gathered on the detection line of the reaction membrane;
the rabbit IgG on the binding pad continuously diffuses, is combined with the goat anti-rabbit IgG and is gathered on the quality control line of the reaction membrane;
under the action of exciting light, the quantum dots emit optical signals with certain wavelength, are identified by a fluorescence immunoassay analyzer and are converted into certain numerical values; and the fluorescence immunoassay analyzer judges the concentration of the PAT/bar protein in the sample according to a built-in reference range.
And (3) judging standard: the fluorescent test strip is matched with a corresponding fluorescence immunoassay analyzer for detection, and the fluorescence immunoassay analyzer reads the fluorescence signal intensity of a detection line (T line) and a quality control line (C line) on the fluorescent test strip and calculates the T/C value. Critical values (Cutoff values) of different sample types are preset in the instrument, and if the T/C value of a certain sample is larger than or equal to the Cutoff value, the sample is judged to be positive; otherwise, it is negative. Note that even the blank control test results have lower levels of T and C values, which can be understood as background values. The inside of the instrument sets a value slightly higher than the background value as the cutoff value according to a large amount of data of the early test.
In the following examples, T/C > 0.03 determined the test result to be positive, and T/C < 0.03 determined the test result to be negative.
In the following examples, the quantum dot microspheres used were purchased from Shanghai Kunmao Biotechnology Co., Ltd, and have a product name of QBC620, a particle size of 100nm, an emission wavelength of 610nm, and carboxyl functional groups on the surface;
the excitation wavelength is 365 nm;
the first PAT/bar protein monoclonal antibody, the goat anti-rabbit IgG, the second PAT/bar protein monoclonal antibody and the rabbit IgG are prepared by conventional means;
the bottom plate is made of a PVC material, and the PVC bottom plate has the advantages of stable chromatography, no fluorescent substance and high bonding strength; the sample pad and the combination pad are made of glass fibers; the reaction membrane is a nitrocellulose membrane (NC membrane); the material of the absorption pad is absorbent filter paper.
Example 1
The embodiment provides a test strip for rapidly detecting PAT/bar protein, which comprises a PVC base plate, wherein a sample pad, a combination pad, a nitrocellulose membrane (NC membrane) and an absorption pad are connected on the PVC base plate.
In this example, the composition of each solution used is shown in table 1 below:
TABLE 1
The preparation method of the test strip comprises the following steps:
(1) preparation of sample pad: and soaking the sample pad in the sample pad treatment solution for 2h, then placing the sample pad in an oven with the humidity of less than 30% and the temperature of 45 ℃, drying the sample pad for 18h, and then sealing and storing the sample pad at 6 ℃.
(2) Preparation of the bonding pad: soaking the bonding pad in the bonding pad treating solution for 2h, taking out the soaked bonding pad, drying in an oven with humidity less than 30% and temperature of 45 deg.C for 18h, and sealing at 5 deg.C for storage.
Mixing a quantum dot-labeled rabbit IgG stock solution, a quantum dot-labeled second PAT/bar protein monoclonal antibody stock solution and a labeling treatment solution in a volume ratio of 1:1:108, loading the mixture into a film-scribing gold spraying machine, spraying the mixture onto treated glass fibers according to a spraying amount of 4 mu L/cm, placing the treated glass fibers in an oven with the humidity of less than 20% and the temperature of 45 ℃, drying for 24 hours, and sealing and storing at 4 ℃.
Wherein, the labeling method of the second PAT/bar protein monoclonal antibody comprises the following steps:
1. and (3) activation: taking a marked 100 mu L (with the concentration of 10mg/mL) quantum dot microsphere as an example, adding 100 mu L of quantum dot microsphere into a first marking buffer solution, shaking and uniformly mixing, adjusting the concentration of the quantum dot microsphere to be 2mg/mL, adding an activating agent 1 and an activating agent 2 to enable the final concentration of the activating agent 1 to be 50mM and the final concentration of the activating agent 2 to be 50mM, carrying out incubation reaction at room temperature for 20min, then centrifuging at 14000rpm for 20min, removing supernatant, collecting precipitate, carrying out resuspension by using a second marking buffer solution, fixing the volume to 500 mu L, and then carrying out ultrasonic dispersion to obtain a quantum dot microsphere resuspension solution;
2. antibody labeling: dispersing 0.05mg of a second PAT/bar protein monoclonal antibody in the quantum dot microsphere resuspension (about 100 mu L of the original concentration of the microspheres) to perform oscillation coupling reaction, and reacting at room temperature for 1 h;
3. blocking the antibody: after completion of the reaction, 400. mu.L of a 0.1g/mL BSA solution was added thereto and mixed well, followed by reaction at room temperature for 0.5 h.
4. And (3) purification: centrifuging at room temperature of 14000rpm for 10min, removing supernatant, separating to obtain precipitate, redissolving with 100 μ L of labeled preservation solution, and dispersing by shaking to obtain quantum dot labeled second PAT/bar protein monoclonal antibody storage solution; stored at 4 ℃ and used within 72 h. The labeling method for rabbit IgG was essentially the same as for the second PAT/bar protein monoclonal antibody, except that the mass of rabbit IgG in step 2 was 0.075 mg.
(3) Preparation of NC film: drawing a quality control line: the distance between the line C and the upper edge of the nitrocellulose membrane is 10 mm; diluting goat anti-rabbit IgG to 0.5mg/mL by using a coating diluent for coating, wherein the streak concentration is 1 mu L/cm; line marking of a detection line: the distance between the T line and the lower edge of the nitrocellulose membrane is 11 mm; diluting the first PAT/bar protein monoclonal antibody to 0.5mg/mL by using a coating diluent, wherein the streaking concentration is 1 muL/cm; the distance between the line C and the line T is 4 mm.
And after the completion, placing the NC film in an oven with the humidity of less than 30% and the temperature of 45 ℃, drying for 24 hours, and then hermetically storing at 4-8 ℃ for 18 months as an effective period for later use.
(4) Assembling the test strip: the method comprises the steps of sequentially overlapping and sticking a sample pad, a combination pad, an NC membrane and an absorption pad from left to right on a PVC base plate, wherein the sample pad, the combination pad, the NC membrane and the absorption pad are overlapped and fixed on the PVC base plate, the sample pad, the combination pad, the NC membrane and the absorption pad are overlapped and fixed for 2mm, the tail end of the sample pad is connected with the initial end of the combination pad, the tail end of the combination pad is connected with the initial end of a reaction membrane, the tail end of the reaction membrane is connected with the initial end of the absorption pad, the initial end of the sample pad is aligned with the initial end of the base plate, the tail end.
Example 2
The present embodiment provides a test strip for detecting PAT/bar protein, which is substantially the same as the test strip described in embodiment 1, except that the working concentration of the first PAT/bar protein monoclonal antibody is 0.7mg/mL, and the working concentration of goat anti-rabbit IgG is 0.7 mg/mL.
Example 3
The present embodiment provides a test strip for detecting PAT/bar protein, which is substantially the same as the test strip described in embodiment 1, except that the working concentration of the first PAT/bar protein monoclonal antibody is 0.3mg/mL, and the working concentration of goat anti-rabbit IgG is 0.3 mg/mL.
Example 4
This example provides a test strip for detecting PAT/bar protein, which is substantially the same as the test strip described in example 1, except that the rabbit IgG has a mass of 0.05mg in the step (2) of preparing the conjugate pad.
Example 5
This example provides a test strip for detecting PAT/bar protein, which is substantially the same as the test strip described in example 1, except that the rabbit IgG has a mass of 0.15mg in the step (2) of preparing the conjugate pad.
Comparative example 1
This example provides a test strip for detecting PAT/bar protein, which is substantially the same as the test strip described in example 1, except that, in step (3), the distance between the T line and the lower edge of the nitrocellulose membrane is 10mm, and the distance between the C line and the T line is 5 mm.
Comparative example 2
This example provides a test strip for detecting PAT/bar protein, which is substantially the same as the test strip described in example 1, except that in step (3), the distance between the T line and the lower edge of the nitrocellulose membrane is 12mm, and the distance between the C line and the T line is 3 mm.
Comparative example 3
This example provides a test strip for detecting PAT/bar protein, which is substantially the same as the test strip described in example 1, except that in step (3), the distance between the line C and the upper edge of the nitrocellulose membrane is 9mm, the distance between the line T and the lower edge of the nitrocellulose membrane is 9mm, and the distance between the line C and the line T is 7 mm.
Comparative example 4
This example provides a test strip for detecting PAT/bar protein, which is substantially the same as the test strip described in example 1, except that, in step (3), the distance between the line C and the upper edge of the nitrocellulose membrane is 8mm, the distance between the line T and the lower edge of the nitrocellulose membrane is 8mm, and the distance between the line C and the line T is 9 mm.
Performance test 1
The test strip of embodiment 1 and the plastic shell of packing the test strip are combined into a detection card, the card shell upper cover of the plastic shell is provided with a sample adding hole and an observation window, and the sample adding hole is arranged above the sample pad and observes a detection line and a quality control line through the observation window. The detection card is used for detecting the trans Bar gene corn material and the non-transgenic corn material.
(1) Sample pretreatment
Grinding 1g of Bar-transgenic corn material and 1g of non-transgenic corn material respectively, then adding 5mL of purified water respectively, shaking up, and standing for 5 minutes to obtain a Bar-transgenic corn material sample and a non-transgenic corn material sample.
(2) Detection with a detection card
And dropwise adding the supernatant of the sample into the sample adding hole of the detection card, standing for 15 minutes, and detecting the fluorescence signal intensity of the detection line and the quality control line of the visual window by using a fluorescence immunoassay analyzer, wherein the detection results are shown in Table 2.
TABLE 2
The Bar gene-transferred corn material sample is positive in expression, and the non-transgenic corn material sample is negative in expression.
Diluting the Bar-transgenic corn material sample in the performance test 1 by 10 times (sample 1), 100 times (sample 2) and 1000 times (sample 3) to obtain 3 diluted Bar-transgenic corn material samples, detecting the 3 diluted samples by using the detection card prepared by the test strip provided in the embodiment 1 according to the method in the performance test 1, wherein each sample is detected twice, and the detection results are shown in Table 3.
TABLE 3
The detection results of the three samples are still positive, which indicates that the detection card provided by the embodiment of the invention has higher sensitivity.
100 parts of non-transgenic corn material and 100 parts of Bar-transgenic corn material are respectively ground and then added into purified water, the concentration of the non-transgenic corn material is 0.2g/mL, the concentration of the Bar-transgenic corn material is 0.2g/mL (25 parts), 0.02g/mL (25 parts), 0.002g/mL (25 parts) and 0.0002g/mL (25 parts), 200 parts of samples are detected by adopting the detection card prepared by the test paper strip provided by the embodiment 1 according to the method of the performance test 1, the detection result is shown in table 4, the negative coincidence rate is 100%, and the positive coincidence rate is 100%.
TABLE 4
A commercially available Bar transgenic rapid detection kit (Bar transgenic rapid detection kit of Shanghai Orifice) is adopted to respectively detect the 100 parts of non-transgenic corn material samples and the 100 parts of Bar transgenic corn material samples, and the detection results are used as a control group.
As can be seen from the above table, when the test strip provided by the invention is used for detecting a negative sample, the detection result is relatively accurate, no false positive result occurs, and the condition of misjudgment during detection is prevented.
Taking a non-transgenic corn strain and a transgenic corn strain with different transgenic components: bt176 (bar transgenic maize), Mon810 (Cry 1Ab transgenic insect-resistant maize), GA21 (mEPSPS transgenic maize), and T25 (pat transgenic maize).
Taking 1g of the corn strains respectively, grinding the corn strains, adding the ground corn strains into 5mL of purified water respectively, shaking up, standing for 5 minutes to obtain solutions to be detected of different corn strains, taking supernate, and detecting by adopting the detection cards prepared by the test strips provided by the embodiments 1-5 according to the method of the performance test 1, wherein the detection results are shown in Table 5.
TABLE 5
As can be seen from Table 5, in 5 samples, only the Bt176 (transgenic bar corn) test result is positive, and the test results of the other samples are negative, which indicates that the test strip provided by the invention has good specificity.
Performance test 5
Grinding 1g of Bar-transgenic corn material, adding 5mL of purified water, shaking up, and standing for 5 minutes to obtain a Bar-transgenic corn material sample. And taking the supernatant, and detecting by using the detection card prepared by the test paper strips provided in the comparative examples 1-4 according to the method described in the performance test 1, wherein the detection results are shown in Table 6.
TABLE 6
The standard deviation of T/C can be calculated to be 3.7% according to the detection result of Bar-transgenic corn material in Table 2.
As can be seen from table 6, the detection card made by the test strip provided in comparative example 4 is used for detection, the obtained result data has the largest discrete degree, and the detection result is the most unstable, the detection card made by the test strip provided in example 1 is used for detection, the obtained result data has the smallest discrete degree, and the detection result is the most stable, so that the distance between the detection line and the quality control line has a larger influence on the stability of the detection result of the test strip, when the distance between the detection line and the quality control line is within the range of 3-5 mm, the detection result is more stable, when the distance is increased and exceeds the range of 3-5 mm, the stability of the detection result is reduced more quickly, and the repetition rate is reduced.
Performance test 6
1.5g (sample 4), 2g (sample 5), 2.5g (sample 6) and 3g (sample 7) of Bar transgenic corn material were ground, and then 5mL of purified water was added, followed by shaking and standing for 5 minutes, to obtain Bar transgenic corn material samples. The Bar transgenic corn material sample in the performance test 1 is diluted by 20 times (sample 8), 40 times (sample 9) and 80 times (sample 10) to obtain 3 diluted Bar transgenic corn material samples, and the samples are detected by using the detection card prepared by the test paper strip provided in the embodiment 1 according to the method in the performance test 1, and the detection results are shown in table 7.
TABLE 7
| Sample(s) | T/C test value |
| Sample No. 4 | 8.168 |
| Sample No. 5 | 12.423 |
| Sample No. 6 | 12.786 |
| Sample 7 | 14.486 |
| |
2.8402 |
| Sample 9 | 2.6309 |
| |
2.4987 |
The data of the determination results in table 7 are combined with the T/C average value of the trans-Bar gene corn material in the performance test 1 and the T/C average values of the sample 1 and the sample 2 to perform linear fitting, as shown in fig. 2, R2 is 0.981, which indicates that the test strip provided by the embodiment of the present invention has a good linear relationship of the determination results, can quantitatively detect PAT/Bar protein in agricultural products such as plants and food, and the quantitative detection result is accurate.
In conclusion, the test strip for detecting the PAT/bar protein provided by the invention can detect the PAT/bar protein, and after three repeated detections are carried out on a sample, the detection result is relatively stable and the repetition rate is good; after the bar-transgenic corn material sample is diluted by 1000 times, the detection result of the test strip is still positive, which indicates that the test strip has high sensitivity; when 100 negative samples are detected, the negative coincidence rate is 100%, which indicates that the test strip is not easy to have false positives, and can effectively prevent the situation of misjudgment during detection; the transgenic corn samples of different strains are detected, and the detection result of only the transgenic Bar corn strain is positive, which indicates that the test strip has good specificity; the test strip provided by the invention has good linear relation of results of quantitative determination of the trans-Bar gene corn sample, can be used for quantitatively detecting PAT/Bar protein in agricultural products such as plants and food, and has more accurate quantitative determination results.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The test strip for detecting the PAT/bar protein is characterized in that a reaction membrane of the test strip is provided with a detection line and a quality control line, the detection line is coated with a first PAT/bar protein monoclonal antibody, and the quality control line is coated with goat anti-rabbit IgG;
the binding pad of the test strip is coated with a second PAT/bar protein monoclonal antibody marked by quantum dots and rabbit IgG marked by quantum dots.
2. The test strip of claim 1,
the reaction membrane is a nitrocellulose membrane;
the distance between the detection line and the quality control line is 3-5 mm;
the working concentration of the first PAT/bar protein monoclonal antibody is 0.1 mg/mL-1 mg/mL;
the working concentration of the goat anti-rabbit IgG is 0.1 mg/mL-1 mg/mL;
and a solution used for diluting the first PAT/bar protein monoclonal antibody and the goat anti-rabbit IgG is a coating diluent, and the coating diluent comprises 0.005-0.015 g/mL of bovine serum albumin and 0.005-0.015 g/mL of NaCl.
3. The test strip of claim 1, wherein the test strip comprises a sample pad, a conjugate pad, a reaction membrane, and an absorbent pad, which are sequentially overlapped; the quality control line is positioned at one end of the reaction membrane close to the absorption pad, and the distance between the quality control line and the absorption pad is 7-10 mm; the detection line is positioned at one end of the reaction membrane close to the combination pad, and the distance between the detection line and the combination pad is 7-10 mm.
4. The test strip of claim 3, wherein the sample pad treatment solution used in the preparation of the sample pad comprises 0.005-0.015 g/mL BSA and 0.005-0.015 g/mL NaCl.
5. The test strip of claim 3, wherein the sample pad treatment solution used in the preparation of the sample pad is a Tris-HCl buffer solution containing 0.005-0.015 g/mL BSA and 0.005-0.015 g/mL NaCl;
the concentration of the Tris-HCl buffer solution is 40 mM-80 mM;
the pH value of the Tris-HCl buffer solution is 7.0-9.0.
6. The test strip of claim 1, wherein the preparation method of the second PAT/bar protein monoclonal antibody labeled by the quantum dots comprises the following steps:
and mixing the activated quantum dot microspheres with the second PAT/bar protein monoclonal antibody for reaction, and covalently coupling the quantum dot microspheres with the antibody to obtain the quantum dot labeled second PAT/bar protein monoclonal antibody.
7. A method for preparing the test strip of any one of claims 1 to 6, wherein the method comprises the following steps:
(1) preparing a second PAT/bar protein monoclonal antibody marked by the quantum dots and rabbit IgG marked by the quantum dots, mixing the marked antibodies, and spraying a film to obtain the binding pad;
(2) diluting the goat anti-rabbit IgG, then scribing the diluted goat anti-rabbit IgG onto the reaction membrane to form the quality control line, diluting the first PAT/bar protein monoclonal antibody, then scribing the diluted goat anti-rabbit IgG onto the reaction membrane to form the detection line;
(3) and sequentially fixing the sample pad, the combination pad, the reaction membrane and the absorption pad on a bottom plate to obtain the test strip.
8. A test card for testing PAT/bar protein, which comprises the test strip of any one of claims 1 to 6 and a plastic shell for packaging the test strip;
the upper cover of the clamping shell of the plastic shell is provided with a sample adding hole and an observation window, wherein the sample adding hole is arranged above the sample pad and passes through the observation window to observe the detection line and the quality control line.
9. Use of the strip of any one of claims 1 to 6 for the detection of herbicide-resistant transgenic plants.
10. Use of the strip of any one of claims 1 to 6 for the detection of PAT/bar protein.
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