Multicolor joint detection test strip for detecting transgenic polyproteins

Abstract

The invention relates to a multicolor joint detection test strip for simultaneously detecting multiple proteins in transgenes as well as a preparation method and application of the multicolor joint detection test strip. According to the multi-color joint inspection test paper, a group of colored nano microspheres with different colors and compatible analysis conditions are screened as visual optical signal probes, BT-Cry1Ab / Ac, CP4-EPSPS and PAT / bar target antibodies are marked in a covalent bonding mode and are preset on a combination pad, different positions of a nitrocellulose membrane detection area are coated with transgenic target antibodies of different components, and the visual optical signal probes are used for detecting the target antibodies. And preparing the multicolor joint inspection test strip. According to the test paper, BT-Cry1Ab, BT-Cry1Ac, CP4EPSPS and PAT / bar protein can be detected at the same time, different colors are displayed at different positions of a nitrocellulose membrane detection area, the result is accurate, good specificity and stability and high sensitivity are achieved, the screening efficiency can be remarkably improved, and cross interference among all components is effectively reduced.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a multicolor test strip for detecting transgenic CP4EPSPS, BT-Cry1Ab / Ac and PAT / bar proteins. Background Technology

[0002] Due to the significant advantages of genetically modified (GM) crops in terms of insect resistance, stress resistance, and nutritional levels, GM breeding has gradually become an important method in bio-breeding, and various GM products are constantly emerging. According to ISAAA statistics, by 2019, the global planting area of ​​GM crops was 112 times that of 1996. However, GM crops still pose some concerns regarding gene contamination, nutritional safety, and unintended effects. Therefore, many countries and regions have enacted corresponding labeling systems for GM crops. Despite strict regulatory systems, illegal GM products still enter the market. Therefore, establishing rapid and convenient methods for detecting GM products is crucial for combating illegal GM products and protecting the intellectual property rights of researchers.

[0003] Ideal on-site rapid detection methods should meet the ASSURED criteria (Accurate, Specific, Sensitive, User-friendly, Rapid, Equipment-free, Deliverable to end users) stipulated by the World Health Organization (WHO). Test strips are a membrane-based analytical method, simple to operate, rapid in detection, and equipment-free, making them ideal for rapid on-site diagnosis. Colloidal gold-based transgenic test strips have been widely developed. However, most of these test strips can only detect one analyte. With the continuous development of transgenic technology in biotechnology, transgenic crops have evolved from single-trait to multi-trait crops. Therefore, single-target transgenic test strips cannot meet the needs of detecting complexes in multi-trait and single-trait transgenic crops. Currently, insect-resistant and herbicide-resistant transgenic crops are the two most important types of transgenic crops. They are mainly achieved by inserting exogenous Bt genes (derived from the soil bacterium Bacillus subtilis) along with glyphosate resistance genes CP4-epsps and glufosinate resistance genes Pat / bar into the plant genome. Therefore, it is necessary to develop detection technologies for transgenic crops that simultaneously detect insect resistance and herbicide resistance. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention aims to provide a multicolor strip for detecting transgenic CP4 EPSPS, BT-Cry1Ab / Ac, and PAT / bar proteins. This strip is used for the detection of four target proteins, enabling rapid and simultaneous detection of transgenic crops containing CP4 EPSPS, BT-Cry1Ab, BT-Cry1Ac, and PAT / bar. It significantly improves screening efficiency, effectively reduces cross-interference between components, reduces reliance on instruments and professional operators, and overcomes the limitations of single-target strips for detecting composite trait crops or complexes of several single-trait crops. This provides strong support for large-scale, rapid on-site screening of transgenic crops.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a multicolor test strip for detecting BT-Cry1Ab / Ac protein, CP4EPSPS protein and PAT / bar protein in transgenic organisms, comprising a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane) and an absorbent pad that are fixed on the base plate in sequence.

[0007] Among them, the conjugate pad is coated with a microsphere-protein first antibody complex, and the microsphere-protein first antibody complex is of three types: a first microsphere-CP4mAb1 complex, a second microsphere-BTmAb1 complex, and a third microsphere-PATmAb1 complex.

[0008] The first microsphere-CP4mAb1 complex is a complex formed by coupling the first nanosphere with the first antibody of CP4 EPSPS protein; the second microsphere-BTmAb1 complex is a complex formed by coupling the second nanosphere with the first antibody of BT-Cry1Ab / Ac protein; and the third microsphere-PATmAb1 complex is a complex formed by coupling the third nanosphere with the first antibody of PAT / bar protein.

[0009] Three protein secondary antibodies are coated in parallel at different positions near the binding pad end of the nitrocellulose membrane, forming detection lines 1 (T1), 2 (T2), and 3 (T3); the three protein secondary antibodies are CP4mAb2, BTmAb2, and PATmAb2, respectively; CP4mAb2, BTmAb2, and PATmAb2 are the CP4 EPSPS protein secondary antibody, BT-Cry1Ab / Ac protein secondary antibody, and PAT / bar protein secondary antibody 2, respectively; a quality control line is set on the nitrocellulose membrane, with goat anti-mouse IgG antibody coated on the nitrocellulose membrane near the absorbent pad end as the quality control line;

[0010] The first, second, and third nanospheres are nanospheres of different colors;

[0011] The CP4 EPSPS protein primary antibody and the CP4 EPSPS protein secondary antibody are different antibodies that specifically bind to the CP4 EPSPS protein; the BT-Cry1Ab / Ac protein primary antibody and the BT-Cry1Ab / Ac protein secondary antibody are different monoclonal antibodies that specifically bind to the BT-Cry1Ab / Ac protein; the PAT / bar protein primary antibody and the PAT / bar protein secondary antibody are different antibodies that specifically bind to the PAT / bar protein.

[0012] Furthermore, the preparation method of the microsphere-protein first antibody complex is as follows:

[0013] The activated nanospheres were mixed with CP4mAb1, BTmAb1 or PATmAb1 protein antibodies and reacted at room temperature for 4 hours. After centrifugation (centrifugal force >15000) for 5 min, the supernatant was removed, and the nanospheres were washed once by centrifugation with 1.0 mL of blocking buffer. 480 μL of storage buffer was added, and the nanospheres were dispersed by sonication and stored at 2-8 °C.

[0014] Furthermore, the activated nanospheres coupled to CP4mAb1, BTmAb1, or PATmAb1 proteins are activated nanospheres of different colors.

[0015] Furthermore, the activated nanospheres were prepared by the following method:

[0016] Add 2-(N-morpholino)ethanesulfonic acid buffer to the solution of nanospheres and sonicate to disperse. After pre-cooling, add N-hydroxysuccinimide solution and mix well. Then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and mix well until the reaction is complete. Remove the supernatant and wash to obtain activated nanospheres.

[0017] Furthermore, the sealing solution comprises the following components: 0.050M HEPES (pH 8.0) and 1% BSA, and the preservation solution comprises the following components: 0.1M Tris-HCl (pH 8.0), 10% sucrose, and 1% BSA.

[0018] Preferably, in the sample pad, conjugation pad, nitrocellulose membrane and absorbent pad, there is an overlap area of ​​1-2 mm between adjacent components.

[0019] Preferably, the sample pad and the conjugate pad are both made of glass fiber, and the absorbent pad is made of cellulose fiber; the nitrocellulose membrane is a CN140 membrane, and the pore size of the nitrocellulose membrane is 8-15 μm.

[0020] In a second aspect, the present invention provides a method for preparing a multicolor test strip as described in the first aspect, the method comprising the following steps:

[0021] (1) Prepare the first microsphere-CP4mAb1 complex, the second microsphere-BTmAb1 complex and the third microsphere-PATmAb1 complex, and then prepare a mixture of the three microsphere-protein first antibody complexes. After mixing them in a volume ratio of 1:1:1, the mixture is sprayed into a membrane to obtain the conjugation pad.

[0022] (2) Dilute goat anti-rabbit IgG and streak it onto the nitrocellulose membrane to form a quality control line. Dilute the three protein secondary antibodies CP4mAb2, BTmAb2 and PATmAb2 and streak them onto the NC membrane to form the detection line.

[0023] (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are fixed sequentially on the base plate to obtain the test strip.

[0024] Preferably, in step (1), the spray volume of the spray film is 3 to 6 μL / cm; and in step (2), the concentration of the scribing is 0.5 to 1.5 μL / cm.

[0025] In any of the above schemes, it is preferred that, in step (2), the concentration of CP4mAb2 is 0.6mg / mL-1.2mg / mL, the concentration of BTmAb2 is 0.6mg / mL-1.2mg / mL, and the concentration of PATmAb2 is 0.6mg / mL-1.2mg / mL when the line is drawn.

[0026] In any of the above schemes, it is preferred that, in step (1), the concentration of the first microsphere-CP4mAb1 complex is 60 μg / mL-120 μg / mL, the concentration of the second microsphere-BTmAb1 complex is 30 μg / mL-100 μg / mL, and the concentration of the third microsphere-PATmAb1 complex is 10 μg / mL-100 μg / mL.

[0027] Thirdly, the present invention provides the use of a multicolor test strip as described in the first aspect, the test strip being used to detect insect-resistant and herbicide-resistant transgenic plants or to detect single or mixtures of CP4 EPSPS, BT-Cry1Ab / Ac and PAT / bar proteins.

[0028] Preferably, the insect-resistant transgenic plant is a plant transgenic with the BT-Cry1Ab or BT-Cry1Ac gene, and the herbicide-resistant transgenic plant is a plant transgenic with the PAT / bar gene or the CP4 EPSPS gene.

[0029] Fourthly, the present invention provides a method for detecting the presence of genetically modified organisms in plants, the method comprising the following steps:

[0030] S1) Obtain the multicolor test strip as described in the first aspect;

[0031] S2) The plant to be tested is crushed, extracted with solvent, and the supernatant is used as the test solution;

[0032] S3) Drop the test solution onto the sample pad of the multicolor test strip and wait for the test results;

[0033] S4) Based on the test results on the nitrocellulose membrane, if only one control line shows a colored band, it means that the plant does not contain transgenic proteins CP4 EPSPS, BT-Cry1Ab / Ac, and PAT / bar; if one or more test lines show color, it means that there are transgenic proteins corresponding to the detection limit; if the control line does not show color, the test has failed.

[0034] The detection principle and result interpretation process of this invention are as follows: When CP4 EPSPS, BT-Cry1Ab, BT-Cry1Ac, and PAT / bar are present in the sample, they can flow along the test strip through capillary action and encapsulate with the corresponding microsphere-mAb1 probe and capture mAb2 on the test strip, forming antigen-antibody immune complexes. The accumulated microspheres will show blue, gray, and red bands on T1, T2, and T3. When two or one CP4 EPSPS, BT-Cry1Ab / Ac, and PAT / bar proteins are present, two or one colored band will appear on the corresponding test line on the NC membrane. In the absence of exogenous proteins, microspheres will not accumulate on T1, T2, and T3, and therefore only one control line will show a colored band.

[0035] Furthermore, in the detection of all the samples, various microspheres-mAb1 and goat anti-mouse IgG coated on the control line will produce an immune response, and a colored band will appear on the control line; otherwise, the result will be deemed invalid.

[0036] Compared with existing technologies, the present invention has the following advantages:

[0037] The multicolor test strips of this invention can be used for the rapid detection of genetically modified insect-resistant BT-Cry1Ab and BT-Cry1Ac, as well as complex crops with combined traits such as herbicide CP4-EPSPS and PAT / bar, and complexes composed of single-trait crops. The specificity and sensitivity of the strips for detecting BT protein, CP4-EPSPS protein, and PAT / bar, as well as the sensitivity of the strips for detecting actual samples (soybeans, corn, rapeseed, etc.), are significantly higher than those of existing technologies.

[0038] Further evaluation showed that the multicolor test strip, by utilizing large-particle nanospheres as visual probes, significantly improved the detection sensitivity of the multicolor test strip. The sensitivity for detecting CP4 EPSPS protein reached 7.8 ng / mL, and the sensitivity for detecting transgenic maize NK603 and MON87427 containing CP4 EPSPS protein reached 0.05% and 0.1%, respectively; the sensitivity for detecting BT Cry1Ab / Ac protein reached 2.5 ng / mL, and the sensitivity for transgenic maize MON810 containing BT Cry1Ab / Ac protein reached 0.1%; the sensitivity for detecting PAT / bar protein reached 2.5 ng / mL, and the sensitivity for transgenic rapeseed RF3 and rice BT63 containing PAT / bar protein reached 0.1%. The test strip provides accurate results, exhibits good specificity and stability, significantly improves screening efficiency and detection performance, effectively reduces cross-interference between components, and provides strong support for large-scale rapid field screening of transgenic organisms. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 This is a schematic diagram of the structure of the multicolor test strip in an embodiment of the present invention.

[0041] Figure 2 The figures show the detection specificity results of the multicolor strip test paper in this embodiment of the invention. Figures A and B show the specificity detection of proteins and transgenic crops, respectively. N1 is non-transgenic rapeseed; N2 is non-transgenic corn; N3 is non-transgenic soybean; N4 is non-transgenic rice; 1 is transgenic rapeseed RF3; 2 is transgenic rapeseed GT73; 3 is transgenic rapeseed RF3 + rapeseed GT73; 4 is transgenic corn T25; 5 is transgenic corn NK603 + transgenic corn MON810; 6 is transgenic soybean RRS; 7 is transgenic rice BT63. Figure C shows the detection of mixtures of different transgenic crop varieties. 1 is transgenic rice BT63; 2 is transgenic corn MON810; 3 is transgenic soybean RRS; 4 is rice BT63 + corn MON810 + soybean RRS.

[0042] Figure 3These are the detection sensitivity results of the multicolor strip test paper in this embodiment of the invention, where (A) is the detection result of CP4 EPSPS protein, 1-10: 10000, 1000, 500, 250, 125, 62, 31, 15, 7.8, 3.9 ng / mL, and N is the corresponding non-GMO crop as a negative control. (B) is the detection result of BT-Cry1Ab protein, 1-8: 1000, 500, 100, 50, 10, 5, 2.5, 1 ng / mL, and N is the corresponding non-GMO crop as a negative control. (C) is the detection result of BT-Cry1Ac protein, 1-8: 1000, 500, 100, 50, 10, 5, 2.5, 1 ng / mL, and N is the corresponding non-GMO crop as a negative control. (D) shows the detection results of PAT / bar protein, 1-10: 1000, 500, 250, 100, 50, 25, 10, 5, 2.5, 1 ng / mL, N is blank.

[0043] (E) Detection results of transgenic maize NK603, (F) maize MON87427, (G) rapeseed GT63, ​​(H) maize MON810, (I) rapeseed RF3, and (J) rice BT63; the numbers in (EJ) correspond to the concentration in ng / mL, and N is the corresponding non-transgenic crop as the negative control.

[0044] Figure 4 The results are the detection stability results of the multicolor test strips in this embodiment of the invention, in ng / mL.

[0045] Figure 5 The results of the multicolor strip test for detecting actual samples in this embodiment of the invention are as follows: (A) Analysis of the test strip on actual crops; P: Positive control (CP4 EPSPS+BT-Cry1Ab+PAT / bar); 1-4: 4 corn seed samples; N1: Non-GMO corn; 5-6: 2 rapeseed samples; N2: Non-GMO rapeseed; 7-9: 3 soybean seed samples; N3: Non-GMO soybean; 10-11: 2 rice seed samples; N4: Non-GMO rice; (BD) PCR results of actual crops; (B) Amplification of cp4-epsps, (C) Bt-cry1Ab / Ac, (D) bar gene; M: DL 2000; 1-2: Positive control group; 3-10: Negative control group; 11-18: 4 corn seed samples; 19-22: 2 rapeseed samples; 23-28: 3 soybean seed samples; 29-32: 2 rice seed samples. Detailed Implementation

[0046] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but the implementation of the present invention is not limited thereto.

[0047] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; the amounts of experimental reagents used are, unless otherwise specified, the amounts used in conventional experimental operations; and the experimental methods used are, unless otherwise specified, conventional methods.

[0048] This invention provides a multicolor assay strip capable of simultaneously detecting four proteins: CP4 EPSPS, BT-Cry1Ab / Ac, and PAT / bar. Figure 1 As shown in Figure A, the test strip consists of a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and an absorbent pad, all connected sequentially to the base plate. The NC membrane has three detection lines and a control line. The detection lines are coated with CP4mAb2, BTmAb2, and PATmAb2, forming detection line 1 (T1), detection line 2 (T2), and detection line 3 (T3), respectively. CP4mAb2, BTmAb2, and PATmAb2 are CP4 EPSPS protein antibody 2, BT-Cry1Ab / Ac protein antibody 2, and PAT / bar protein antibody 2, respectively. The control line is coated with goat anti-mouse IgG. The conjugate pad of the test strip is coated with a blue microsphere-CP4mAb1 complex, a gray microsphere-BTmAb1 complex, and a red microsphere-PATmAb1 complex. The blue microsphere-CP4mAb1 complex is composed of blue nanospheres and CP4... The complexes formed by the coupling of EPSPS protein antibody 1, the gray microsphere-BTmAb1 complex is a complex formed by the coupling of gray nanospheres and BT-Cry1Ab / Ac protein antibody 1, and the red microsphere-PATmAb1 complex is a complex formed by the coupling of red nanospheres and PAT / bar protein antibody 1.

[0049] In this embodiment of the invention, the detection principle and result interpretation process of the multicolor assay strip are as follows: When CP4 EPSPS, BT-Cry1Ab, BT-Cry1Ac, and PAT / bar are present in the sample, they can flow along the test strip through capillary action and encapsulate with the corresponding microsphere-mAb1 probe and capture mAb2 on the test strip, forming antigen-antibody immune complexes. The accumulated microspheres will show blue, gray, and red bands on T1, T2, and T3. Figure 1B). In the presence of two or one CP4 EPSPS, BT-Cry1Ab / Ac, and PAT / bar proteins, two or one colored band will appear on the corresponding test line on the NC membrane. Figure 1 In the absence of exogenous proteins, microspheres do not aggregate on T1, T2, and T3, thus only one control line shows a colored band. Figure 1 C).

[0050] Furthermore, in the detection of all the samples, various microspheres-mAb1 and goat anti-mouse IgG coated on the control line will produce an immune response, and a colored band will appear on the control line; otherwise, the result will be deemed invalid.

[0051] Furthermore, the method for preparing the nanospheres conjugated with CP4mAb1, BTmAb1, and PATmAb1 protein antibodies is as follows:

[0052] The activated blue, gray, and red nanospheres were mixed with CP4mAb1, BTmAb1, and PATmAb1 protein antibodies, respectively. After reacting at room temperature for 4 hours, the mixture was centrifuged (centrifugal force >15000) for 5 minutes. After removing the supernatant, the mixture was washed once with 1.0 mL of blocking buffer, and 480 μL of preservation solution was added. The mixture was then sonicated and stored at 2–8 °C.

[0053] Furthermore, in the method for preparing the nanosphere protein antibody complex, the blocking solution comprises the following components: 0.050M HEPES (pH 8.0) and 1% BSA, and the preservation solution comprises the following components: 0.1M Tris-HCl (pH 8.0), 10% sucrose, and 1% BSA.

[0054] For example, the preparation method of the nanospheres conjugated with CP4mAb1, BTmAb1 and PATmAb1 protein antibodies includes the following steps:

[0055] (1) Washing: Take 120 μL of microspheres, disperse them by sonication, centrifuge (centrifugal force > 15000) for 5 min, remove the supernatant, and wash once with 1.0 mL 0.050 M MES (pH 6.0) by centrifugation.

[0056] (2) Activation: Add 300 μL of 0.050 M MES (pH 6.0) to the microsphere solution in (1) and disperse by ultrasonication. After pre-cooling with crushed ice, add 40 μL of 10 mg / mL sulfo-NHS solution, mix well, add 20 μL of 10 mg / mL EDC solution, mix well, and react at room temperature for 20 min. Centrifuge (centrifugal force > 15000) for 5 min, remove the supernatant, and wash once with 1.0 mL of 0.050 M MES (pH 6.0) by centrifugation.

[0057] (3) Antibody conjugation: Add 480 μL of 0.050 M MES (pH 6.0) to the solution in (2) and disperse by sonication. Add an appropriate amount of antibody, mix well, react at room temperature for 4 h, centrifuge (centrifugal force >15000) for 5 min, and remove the supernatant.

[0058] (4) Blocking: Add 480 μL of blocking solution to the solution in (3), disperse by sonication, react at room temperature for 1 h, centrifuge (centrifugal force > 15000) for 5 min, remove the supernatant, and wash once by centrifugation with 1.0 mL of blocking solution.

[0059] (5) Preservation: Add 480 μL of preservation solution to the solution in (4), disperse by ultrasonication, and store at 2-8℃.

[0060] Furthermore, the MES solution is a 2-(N-morpholino)ethanesulfonic acid buffer.

[0061] Furthermore, the sulfo-NHS solution is an N-hydroxysuccinimide solution prepared using 0.050M MES (pH 6.0) solution.

[0062] Furthermore, the EDC solution is 1-ethyl-(3-dimethylaminopropyl)carbodiimide prepared with 0.050M MES (pH 6.0) solution.

[0063] Furthermore, the sealing solution comprises 0.050M HEPES (pH 8.0) and 1% BSA; the preservation solution comprises 0.1M Tris-HCl (pH 8.0), 10% sucrose, and 1% BSA.

[0064] Further, the concentration of the blue microspheres-CP4mAb1 is 60 μg / mL-120 μg / mL, preferably 100 mg / mL; the concentration of the gray microspheres-BTmAb1 is 30 μg / mL-100 μg / mL, preferably 80 μg / mL; and the concentration of the red microspheres-PATmAb1 is 10 μg / mL-100 μg / mL, preferably 80 μg / mL.

[0065] Further, the concentration of CP4mAb2 is 0.6 mg / mL-1.2 mg / mL, preferably 1 mg / mL; the concentration of BTmAb2 is 0.6 mg / mL-1.2 mg / mL, preferably 1 mg / mL; and the concentration of PATmAb2 is 0.6 mg / mL-1.2 mg / mL, preferably 1 mg / mL.

[0066] Secondly, embodiments of the present invention provide a method for preparing a test strip as described in the first aspect, the method comprising the following steps:

[0067] (1) Prepare the blue microsphere-CP4mAb1 complex, gray microsphere-BTmAb1 complex and red microsphere-PATmAb1 complex, and then mix the labeled antibodies in a volume ratio of 1:1:1 and spray them into a film to obtain the binding pad.

[0068] (2) The goat anti-rabbit IgG is diluted and streaked onto the NC membrane to form the control line, and the protein antibodies of CP4mAb2, BTmAb2 and PATmAb2 are diluted and streaked onto the NC membrane to form the detection line;

[0069] (3) The sample pad, conjugate pad, reaction membrane and absorbent pad are fixed to the base plate in sequence to obtain the test strip.

[0070] Further, in step (1), the spray volume of the blue microsphere-CP4mAb1 complex, gray microsphere-BTmAb1 complex and red microsphere-PATmAb1 complex spray film is 3 to 6 μL / cm, preferably 5 μL / cm.

[0071] Further, in step (2), the concentration of the indentation is 0.5 to 1.5 μL / cm, preferably 1 μL / cm.

[0072] For example, the preparation method of the test strip includes the following steps:

[0073] (1) The antibodies labeled with the prepared blue microsphere-CP4mAb1 complex, gray microsphere-BTmAb1 complex and red microsphere-PATmAb1 complex were mixed at a volume ratio of 1:1:1 and loaded onto a gold spraying machine. The mixture was sprayed at a spraying rate of 3-6 μL / cm to obtain the conjugate pad. The concentration of the blue microsphere-CP4mAb1 was 60 μg / mL-120 μg / mL, preferably 100 mg / mL. The concentration of the gray microsphere-BTmAb1 was 30 μg / mL-100 μg / mL, preferably 80 μg / mL. The concentration of the red microsphere-PATmAb1 was 10 μg / mL-100 μg / mL, preferably 80 μg / mL.

[0074] (2) The goat anti-mouse IgG is diluted and streaked onto the NC membrane to form a control line, wherein the concentration of the goat anti-mouse IgG is 0.1 mg / mL-1 mg / mL; the protein antibodies of CP4mAb2, BTmAb2 and PATmAb2 are diluted and streaked onto the reaction membrane to form the detection line, wherein the concentration of CP4mAb2 is 0.6 mg / mL-1.2 mg / mL, preferably 1 mg / mL; the concentration of BTmAb2 is 0.6 mg / mL-1.2 mg / mL, preferably 1 mg / mL; and the concentration of PATmAb2 is 0.6 mg / mL-1.2 mg / mL, preferably 1 mg / mL.

[0075] (3) The sample pad, conjugate pad, reaction membrane, and absorbent pad are sequentially fixed on the base plate, and then cut into several test strips with a width of 3-4 mm using a machine to obtain the test strips described in this embodiment of the invention. Preferably, there is a 1-2 mm overlap area between adjacent components of the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. Preferably, the sample pad and conjugate pad are made of glass fiber, and the absorbent pad is made of cellulose fiber; the nitrocellulose membrane is a CN140 membrane with a pore size of 8-15 μm.

[0076] Thirdly, the present invention provides the use of a test strip as described in the first aspect, the test strip being used to detect insect-resistant and herbicide-resistant genetically modified plants.

[0077] Preferably, the insect-resistant transgenic plant is a plant transgenic with the BT-Cry1Ab or BT-Cry1Ac gene, and the herbicide-resistant transgenic plant is a plant transgenic with the PAT / bar gene or the CP4 EPSPS gene.

[0078] Fourthly, the present invention provides the use of a test strip as described in the first aspect, wherein the multicolor test strip is used to detect single proteins or mixtures of several proteins, such as CP4 EPSPS, BT-Cry1Ab / Ac, and PAT / bar.

[0079] The present invention will be further described below with reference to specific embodiments.

[0080] Example 1: Preparation of Multicolor Detection Test Strips

[0081] (1) Blue microsphere-CP4mAb1 complex, gray microsphere-BTmAb1 complex, and red microsphere-PATmAb1 complex were prepared. The three microsphere-protein primary antibody complexes were then mixed at the same concentration and sprayed onto a membrane at a volume ratio of 1:1:1 to obtain a conjugation pad (the concentration of blue microsphere-CP4mAb1 was 100 mg / mL, the concentration of gray microsphere-BTmAb1 was 80 μg / mL, and the concentration of red microsphere-PATmAb1 was 80 μg / mL). The microsphere-protein primary antibody complex was prepared by the following method:

[0082] (1-1) Washing: Take 120 μL of microspheres, disperse them by sonication, centrifuge (centrifugal force >15000) for 5 min, remove the supernatant, and wash once with 1.0 mL of 0.050 M MES (pH 6.0) by centrifugation.

[0083] (1-2) Activation: Add 300 μL of 0.050 M 2-(N-morpholino)ethanesulfonic acid buffer MES (pH 6.0) to the microsphere solution in (1-1) and sonicate. After pre-cooling with crushed ice, add 40 μL of 10 mg / mL sulfo-NHS solution (N-hydroxysuccinimide solution prepared with 0.050 M MES (pH 6.0) solution), mix well, and then add 20 μL of 10 mg / mL EDC solution (1-ethyl-(3-dimethylaminopropyl)carbodiimide prepared with 0.050 M MES (pH 6.0) solution). Mix well and react at room temperature for 20 min. Centrifuge (centrifugal force >15000) for 5 min, remove the supernatant, and wash once with 1.0 mL of 0.050 M MES (pH 6.0) by centrifugation. The sealing solution consists of 0.050M HEPES (pH 8.0) and 1% BSA; the preservation solution consists of 0.1M Tris-HCl (pH 8.0), 10% sucrose, and 1% BSA.

[0084] (1-3) Antibody conjugation: Add 480 μL of 0.050 M MES (pH 6.0) to the solution in (1-2) and sonicate. Add an appropriate amount of antibody, mix well, react at room temperature for 4 h, centrifuge (centrifugal force >15000) for 5 min, and remove the supernatant.

[0085] (1-4) Blocking: Add 480 μL of blocking solution to the solution in (1-3), disperse by sonication, react at room temperature for 1 h, centrifuge (centrifugal force > 15000) for 5 min, remove the supernatant, and wash once by centrifugation with 1.0 mL of blocking solution.

[0086] (1-5) Preservation: Add 480 μL of preservation solution to the solution in (1-4), disperse by ultrasonication, and store at 2-8℃.

[0087] (2) The goat anti-rabbit IgG was diluted and streaked onto the nitrocellulose membrane to form a quality control line. The three protein secondary antibodies CP4mAb2, BTmAb2 and PATmAb2 were diluted and streaked onto the NC membrane to form the detection line. The concentration of CP4mAb2 was 1 mg / mL. The concentration of BTmAb2 was 1 mg / mL. The concentration of PATmAb2 was 1 mg / mL.

[0088] (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are fixed sequentially on the base plate to obtain the test strip.

[0089] Example 2 Specificity Detection

[0090] The following two methods are used to perform specificity testing on multicolor test strips:

[0091] (1) Cry1Ab, CP4 EPSPS and PAT / bar proteins were mixed at a concentration of 1 μg / mL, and Tris-base (TB, pH 7.4) was used as a blank control.

[0092] (2) Using multicolor test strips, non-GMO rice, non-GMO corn, non-GMO soybean, non-GMO rapeseed, GMO corn T25, a mixture of 5% GMO corn NK603 and 10% GMO corn MON810, GMO soybean RRS, GMO rice BT63, GMO rapeseed RF3, RF73 and a mixture of the two were tested.

[0093] The seeds were ground into powder using a grinder, and then double-distilled water was added at a volume ratio of 1:1.5. After vigorous shaking for 5 minutes, the mixture was centrifuged at 8000 rpm for 5 minutes. 70 μL of the supernatant was used for test strip detection. The results are shown below. Figure 2In Figure B, N1 represents non-GMO rapeseed; N2 represents non-GMO corn; N3 represents non-GMO soybean; N4 represents non-GMO rice; 1 represents GMO rapeseed RF3; 2 represents GMO rapeseed GT73; 3 represents GMO rapeseed RF3 + rapeseed GT73; 4 represents GMO corn T25; 5 represents GMO corn NK603 + GMO corn MON810; 6 represents GMO soybean RRS; and 7 represents GMO rice BT63. In Figure C, 1 represents GMO rice BT63; 2 represents GMO corn MON810; 3 represents GMO soybean RRS; and 4 represents rice BT63 + corn MON810 + soybean RRS.

[0094] Figure 2 It can be seen that both pairwise and tri-protein mixtures of the three proteins showed obvious color reactions only at their respective test lines, indicating that the analytes in the multicolor triplet test strip of this invention do not interfere with each other. When transgenic rapeseed containing PAT / bar protein and CP4 EPSPS protein were mixed, and transgenic maize containing Cry1Ab protein and CP4 EPSPS protein were mixed and tested separately, it was found that the test strip only showed color reactions at their respective test lines. Furthermore, mixing transgenic crops containing the three proteins did not affect the test results, further demonstrating that the multicolor triplet test strip of this invention can specifically detect exogenous proteins in these crops. Using the test strip to test transgenic maize T25 containing the pat gene, the results showed that only the control line showed obvious color bands, indicating that the test strip can distinguish between PAT / pat protein and PAT / bar protein, with good specificity.

[0095] Example 2 Sensitivity Analysis

[0096] The sensitivity of the multicolor test strips was tested using the following two methods:

[0097] (1) Verification of the sensitivity of the test strip to proteins

[0098] Transgenic Cry1Ab, Cry1Ac, CP4 EPSPS, and PAT / bar proteins were diluted with Tris-base to different concentration gradients. The dilution concentrations for CP4-EPSPS protein were 10000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62 ng / mL, 31 ng / mL, 15 ng / mL, 7.8 ng / mL, and 3.9 ng / mL, respectively. The dilution concentrations for BT-Cry1Ab / Ac protein were 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 100 ng / mL, 100 ng / mL, 125 ng / mL, 62 ng / mL, 31 ng / mL, 15 ng / mL, 7.8 ng / mL, and 3.9 ng / mL, respectively. The dilution concentrations of PAT / bar protein at ng / mL, 5ng / mL, 2.5ng / mL, and 1ng / mL were 1000ng / mL, 500ng / mL, 250ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, and 1ng / mL, respectively, to determine the sensitivity of the prepared multicolor test strips. 70μL of each sample was added to the sample pad of the test strip, and the results were observed after 5-10 minutes. Tris-base (TB, pH 7.4) was used as a blank control.

[0099] (2) Verification of the sensitivity of test strips to genetically modified crops

[0100] Genetically modified (GM) crops were mixed with non-GM crops in specific proportions. The contents of GM maize MON810 (10%, 1%, 0.5%, 0.1%, 0.01%), GM maize NK603 (5%, 0.5%, 0.1%, 0.05%, 0.025%), GM rapeseed RF3, GM rice BT63, and GM maize MON87427 (100%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%) were tested. Non-GM rice, maize, and rapeseed served as blank controls, and the limit of detection was repeated three times.

[0101] The seeds were ground into powder using a grinder, and then double-distilled water was added at a volume ratio of 1:1.5. After vigorous shaking for 5 minutes, the mixture was centrifuged at 8000 rpm for 5 minutes. 70 μL of the supernatant was used for test strip detection. The results are shown below. Figure 3 The mixtures of genetically modified and non-genetically modified organisms (GMOs) in different proportions were obtained by gradient dilution of the GMO samples by adding different volumes of non-GMO supernatant.

[0102] Figure 3It can be seen that the detection sensitivity of the combined test strip for CP4-EPSPS protein is 7.8 ng / mL, for BT-Cry1Ab / Ac protein is 2.5 ng / mL, and for PAT / bar protein is 2.5 ng / mL.

[0103] Furthermore, the test strip can detect transgenic maize NK603 with 0.05% CP4 EPSPS protein content and transgenic maize MON87427 with 0.1% CP4 EPSPS protein content. Figure 3 ), MON810 transgenic maize with a BT-Cry1Ab / Ac content of 0.5% ( Figure 3 ), genetically modified rapeseed RF3 with a PAT / bar content of 0.1% and genetically modified rice BT63 with a PAT / bar content of 0.1% ( Figure 3 This indicates that the test strip can detect 0.1% CP4EPSPS protein, 0.5% BT-Cry1Ab / Ac protein, and 0.1% PAT / bar protein in the actual sample.

[0104] Example 3 Stability Analysis

[0105] After the multicolor test strips were placed at room temperature for 3 months or at 37°C for 24 hours, they were used to detect the same gradient dilutions of BT-Cry1Ab, BT-Cry1Ac, CP4 EPSPS, and PAT / bar proteins as in Example 2. The color changes on the test strips were observed and compared with the sensitivity before placement.

[0106] See results Figure 4 The multicolor test strip showed no abnormal results for the detection of four exogenous proteins after being stored at room temperature for 3 months or at 37°C for 24 hours, indicating good stability.

[0107] Example 4: Actual Sample Testing

[0108] To further verify the practicality and accuracy of the multicolor test strip of the present invention for actual crop detection, the test strip and conventional PCR methods were used to test four corn seeds, two rapeseed seeds, three soybean seeds and two rice seeds purchased from the local market.

[0109] See results Figure 5 All seed samples tested negative, consistent with the PCR results.

[0110] In summary, this invention develops a sensitive, specific, multi-component, multicolor lateral flow immunoassay for the on-site detection of CP4 EPSPS, BT-Cry1Ab, BT-Cry1Ac, and PAT / bar proteins in transgenic crops. By screening a group of large-diameter colored microspheres, the test strip can detect CP4 EPSPS, BT-CryAb1, BT-CryAc, and PAT / bar proteins at concentrations as low as 2.5 ng / mL, 2.5 ng / mL, and 7.8 ng / mL, and can detect 0.1% CP4 EPSPS protein, 0.5% BT-Cry1Ab / Ac protein, and 0.1% PAT / bar protein in actual samples. Compared with previously reported test strips, this test strip can simultaneously detect four proteins and distinguish different biomarkers by differentiating the colors of the test lines, avoiding confusion between analytes. Compared with methods based on PCR, isothermal amplification, and immunoassay, this test strip does not require complex nucleic acid extraction and can achieve a rapid full analysis process within 10 minutes. Therefore, the test strip has the advantages of high sensitivity, good specificity, rapid detection, strong practicality, and multiplex analysis.

[0111] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A multicolor strip for detecting BT-Cry1Ab / Ac protein, CP4EPSPS protein, and PAT / bar protein in transgenic organisms, comprising a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and an absorbent pad connected sequentially on the base plate; characterized in that, The pad is coated with a microsphere-protein first antibody complex, which is of three types: a first microsphere-CP4mAb1 complex, a second microsphere-BTmAb1 complex, and a third microsphere-PATmAb1 complex. The first microsphere-CP4mAb1 complex is a complex formed by coupling the first nanosphere with the first antibody of CP4 EPSPS protein; the second microsphere-BTmAb1 complex is a complex formed by coupling the second nanosphere with the first antibody of BT-Cry1Ab / Ac protein; and the third microsphere-PATmAb1 complex is a complex formed by coupling the third nanosphere with the first antibody of PAT / bar protein. Three protein secondary antibodies are coated in parallel at different positions near the binding pad end of the nitrocellulose membrane, forming detection lines 1 (T1), 2 (T2), and 3 (T3); the three protein secondary antibodies are CP4mAb2, BTmAb2, and PATmAb2, respectively; CP4mAb2, BTmAb2, and PATmAb2 are the CP4 EPSPS protein secondary antibody, BT-Cry1Ab / Ac protein secondary antibody, and PAT / bar protein secondary antibody 2, respectively; a quality control line is set on the nitrocellulose membrane, with goat anti-mouse IgG antibody coated on the nitrocellulose membrane near the absorbent pad end as the quality control line; The first, second, and third nanospheres are nanospheres of different colors; The CP4 EPSPS protein primary antibody and the CP4 EPSPS protein secondary antibody are different antibodies that specifically bind to the CP4 EPSPS protein; the BT-Cry1Ab / Ac protein primary antibody and the BT-Cry1Ab / Ac protein secondary antibody are different monoclonal antibodies that specifically bind to the BT-Cry1Ab / Ac protein; the PAT / bar protein primary antibody and the PAT / bar protein secondary antibody are different antibodies that specifically bind to the PAT / bar protein.

2. The multicolor test strip according to claim 1, characterized in that, The preparation method of the microsphere-protein primary antibody complex is as follows: The activated nanospheres were mixed with CP4mAb1, BTmAb1 or PATmAb1 protein antibodies. After the reaction was complete at room temperature, the mixture was centrifuged, the supernatant was removed, and the mixture was washed once with blocking buffer. Then, the mixture was added to the preservation buffer and sonicated to obtain the microsphere-protein antibody complex.

3. The multicolor test strip according to claim 1, characterized in that, Activated nanospheres coupled with CP4mAb1, BTmAb1, or PATmAb1 proteins are activated nanospheres of different colors.

4. The multicolor test strip according to claim 3, characterized in that, Activated nanospheres were prepared by the following method: Add 2-(N-morpholino)ethanesulfonic acid buffer to the solution of nanospheres and sonicate to disperse. After pre-cooling, add N-hydroxysuccinimide solution and mix well. Then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and mix well until the reaction is complete. Remove the supernatant and wash to obtain activated nanospheres.

5. The multicolor test strip according to claim 2, characterized in that, The sealing solution comprises the following components: 0.050M HEPES and 1% BSA, and the preservation solution comprises the following components: 0.1M Tris-HCl, 10% sucrose, and 1% BSA.

6. The multicolor test strip according to claim 1, characterized in that, The sample pad, conjugation pad, nitrocellulose membrane, and absorbent pad have an overlap area of ​​1-2 mm between adjacent components.

7. The multicolor test strip according to claim 1, characterized in that, The sample pad and the binding pad are both made of glass fiber, and the absorbent pad is made of cellulose fiber; the nitrocellulose membrane is a CN140 membrane with a pore size of 8-15 μm.

8. The method for preparing the multicolor test strip according to any one of claims 1-7, characterized in that, The preparation method includes the following steps: (1) Prepare the first microsphere-CP4mAb1 complex, the second microsphere-BTmAb1 complex and the third microsphere-PATmAb1 complex, and then prepare a mixture of the three microsphere-protein first antibody complexes. After mixing them in a volume ratio of 1:1:1, the mixture is sprayed into a membrane to obtain the conjugation pad. (2) Dilute goat anti-rabbit IgG and streak it onto the nitrocellulose membrane to form a quality control line. Dilute the three protein secondary antibodies CP4mAb2, BTmAb2 and PATmAb2 and streak them onto the NC membrane to form the detection line. (3) The sample pad, conjugation pad, nitrocellulose membrane and absorbent pad are fixed sequentially on the base plate to obtain the test strip; Preferably, in step (1), the spray volume of the spray film is 3 to 6 μL / cm; in step (2), the concentration of the scribing is 0.5 to 1.5 μL / cm. Preferably, the concentration of the first microsphere-CP4mAb1 complex is 60 μg / mL-120 μg / mL, the concentration of the second microsphere-BTmAb1 complex is 30 μg / mL-100 μg / mL, and the concentration of the third microsphere-PATmAb1 complex is 10 μg / mL-100 μg / mL. Preferably, in step (2), the concentration of CP4mAb2 is 0.6 mg / mL-1.2 mg / mL, the concentration of BTmAb2 is 0.6 mg / mL-1.2 mg / mL, and the concentration of PATmAb2 is 0.6 mg / mL-1.2 mg / mL when the line is drawn.

9. Use of the multicolor test strip according to any one of claims 1-7, wherein the multicolor test strip is used to detect insect-resistant and herbicide-resistant transgenic plants or to detect single or mixtures of CP4 EPSPS, BT-Cry1Ab / Ac and PAT / bar proteins. Preferably, the insect-resistant transgenic plant is a plant transgenic with the BT-Cry1Ab or BT-Cry1Ac gene, and the herbicide-resistant transgenic plant is a plant transgenic with the PAT / bar gene or the CP4 EPSPS gene.

10. A method for detecting the presence of transgenic substances in plants, characterized in that... The detection method includes the following steps: S1) Obtain the multicolor test strip as described in any one of claims 1-7; S2) The plant to be tested is crushed, extracted with solvent, and the supernatant is used as the test solution; S3) Drop the test solution onto the sample pad of the multicolor test strip and wait for the test results; S4) Based on the test results on the nitrocellulose membrane, if only one control line shows a colored band, it means that the plant does not contain transgenic proteins CP4 EPSPS, BT-Cry1Ab / Ac, and PAT / bar; if one or more test lines show color, it means that there are transgenic proteins corresponding to the detection limit; if the control line does not show color, the test has failed.

Images