A method for detecting transgenic events in plants

Abstract

The present invention provides a method for improving the efficiency of detecting genetically modified events contained in a sample. The method comprises labeling a first target antibody with a first fluorescent dye, labeling a second target antibody with a second fluorescent dye; judging the number of genetically modified events in the sample to be detected according to the observed fluorescence color after sample processing, paraffin embedding and slicing, paraffin slicing dewaxing to water, antigen repair, shielding autofluorescence processing, immunofluorescence staining and microscopic photography.

Description

Technical Field

[0001] The invention relates to a simple and intuitive detection method for transgenic crops, and specifically to a detection method for transgenic crops with complex traits, and belongs to the fields of biotechnology and agricultural science. Background Art

[0002] Since the large-scale planting of genetically modified crops in 1996, more and more transformation events have been applied to different crops to meet people's needs for food, feed, clothing and other daily necessities. However, these transformation events must be formally approved in accordance with local laws and regulations before they can be circulated in a country. In recent years, with the continuous emergence of new transformation events, there has been a trend of gene stacking of new transformation events with existing excellent transformation events. As of 2014, the planting area of ​​genetically modified crops with complex traits caused by gene stacking has accounted for 28.3% of the total planting area of ​​genetically modified crops in the world. According to the relevant laws and regulations on genetically modified crops management in my country, genetically modified crops with multiple transformation events obtained by hybridization and other means, even if the individual transformation events have been approved, still need to be re-evaluated for safety according to the new type of genetically modified products. At present, a considerable number of genetically modified crops with complex traits that have been commercially planted abroad have not been re-declared and approved in accordance with the relevant laws on genetically modified biosafety management in my country. The detection of genetically modified products with complex traits with gene stacking is an important part of the current genetically modified regulatory work in my country. How to simply and intuitively detect these illegal genetically modified crops with complex traits has posed new challenges to the safety management of genetically modified organisms in my country!

[0003] At present, the detection process of transgenic crops with complex traits is usually as follows: after mixing the crop seeds, they are initially screened using the universal transgenic elements, and then the specific transformation events of the positive samples are identified using transformation event-specific PCR. Finally, the genomic DNA of the single seeds is extracted for transformation event analysis to identify whether there are multiple transformation events in the single seeds. This analysis process currently generally uses fluorescent PCR and digital PCR to perform parallel detection of multiple transgenic transformation events on single seeds in a large number of mixed samples. Therefore, it requires cumbersome and complex operation steps and high reagent and labor costs, which is not an ideal analysis method. Since the purpose of gene stacking to produce complex transgenic products is to aggregate different transgenic exogenous proteins to obtain multiple traits, we can use different color fluorescent dyes (such as cy3 and FITC, etc.) to label different transgenic protein antibodies to establish a new immunofluorescence method for intuitive and simple detection of complex transgenic complex traits. Summary of the invention

[0004] The present invention provides a method, a kit and an application for detecting the number of transgenic events contained in a sample based on an immunofluorescence method.

[0005] method

[0006] On the one hand, the present invention provides a method for detecting genetically modified events contained in a sample, the method comprising labeling a first target antibody with a first fluorescent dye, labeling a second target antibody with a second fluorescent dye; after sample processing, paraffin embedding and slicing, paraffin slicing dewaxing to water, antigen repair, shielding autofluorescence treatment, immunofluorescence staining and microscopic photography, the number of genetically modified events in the sample to be tested is judged according to the observed fluorescence color.

[0007] The first target event and the second target event are different target events. Preferably, the first target event and the second target event are PAT / bar and CP4-EPSPS target antibodies, respectively.

[0008] The first fluorescent dye and the second fluorescent dye are different fluorescent dyes, and preferably the first fluorescent dye and the second fluorescent dye are FITC (red) and Cy3 (green), respectively.

[0009] When the first fluorescent dye and the second fluorescent dye are FITC (red) and Cy3 (green) respectively, when observing fluorescence, if there is no fluorescence, there is no target conversion event; if there is red or green fluorescence, there is a conversion event; if the observed fluorescence is yellow, it is produced by the superposition of FITC (red) and Cy3 (green), indicating that there are two conversion events in the sample.

[0010] The shielding of the autofluorescence treatment refers to the treatment with a mixture of phosphate buffer and sodium borohydride and an anhydrous ethanol solution of Sudan Black B, which can significantly reduce the background fluorescence and improve the detection efficiency.

[0011] In one embodiment, the sample may be derived from one or more of animals, plants and microorganisms.

[0012] In one embodiment, the sample can be fresh tissue or processed tissue.

[0013] In one embodiment, the method further comprises labeling a third target antibody with a third fluorescent dye to facilitate detection of 0, 1, 2, or 3 transgenic events.

[0014] In one embodiment, the method further comprises labeling a third target antibody with a third fluorescent dye for detecting 0-3 transgenic events, wherein the third target antibody is a target antibody different from the first target antibody and the second target antibody, and the third fluorescent dye is a fluorescent dye different from the first fluorescent dye and the second fluorescent dye.

[0015] In one embodiment, the sample processing is to take any part of the plant, dry it, grind it into powder, fix it with 3% agar, and put it into FAA fixative. Preferably, the any part of the plant refers to seeds, and more preferably, rapeseed seeds.

[0016] In one embodiment, the paraffin-embedded sections are prepared by dehydrating the cut tissue in a dehydrator with gradient alcohol and soaking it in wax, and after the wax solidifies and cools, slicing, baking the slices, and storing them at room temperature for later use.

[0017] In one embodiment, the paraffin sections are dewaxed to water by sequentially treating the sections with xylene, anhydrous ethanol, alcohol, and distilled water.

[0018] In one embodiment, the antigen repair is performed by placing the tissue section in EDTA antigen repair buffer (pH 9.0) and PBS (PH 7.4). Preferably, the buffer and operating conditions can be adjusted according to the type of sample.

[0019] In one embodiment, the shielding of autofluorescence treatment refers to the sequential addition of autofluorescence quencher A and autofluorescence quencher B, and finally serum blocking; the autofluorescence quencher A is a mixture of a phosphate buffer solution of pH 7.4 and sodium borohydride; the autofluorescence quencher B is a mixture of Sudan Black B and anhydrous ethanol. Specifically, the preparation method of autofluorescence quencher A is as follows: weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4, respectively, add ultrapure water to dissolve and make up to 1000ml, and obtain a phosphate buffer solution of pH 7.4; weigh 10mg sodium borohydride and dissolve it in 10ml phosphate buffer solution of pH 7.4 to obtain autofluorescence quencher A. The preparation method of autofluorescence quencher B is as follows: weigh 0.5g Sudan Black B, dissolve it in 70% alcohol, and filter it for later use.

[0020] In one embodiment, the immunostaining refers to decolorizing the slide, labeling the target with a transgenic target antibody labeled with a fluorescent dye, and sealing the slide after decolorization.

[0021] Reagent test kit

[0022] On the other hand, the present invention also provides a kit for efficiently detecting the number of transgenic events contained in a sample.

[0023] The kit comprises a first fluorescent dye and a second fluorescent dye respectively designed for target antibodies in a sample.

[0024] Preferably, the kit also includes reagents and instruments required for fluorescent immunoassay, and preferably, also includes a solution required for shielding autofluorescence.

[0025] Preferably, the kit also includes reagents and instruments required for processing the sample.

[0026] Composition

[0027] In another aspect, the present invention also provides a composition for efficiently detecting the number of transgenic events contained in a sample.

[0028] The composition comprises a first fluorescent dye and a second fluorescent dye respectively designed for target antibodies in a sample.

[0029] Preferably, the composition also includes reagents and instruments required for fluorescent immunoassay, and preferably, also includes a solution required for shielding autofluorescence.

[0030] Preferably, the composition further comprises reagents required for processing the sample.

[0031] application

[0032] On the other hand, the present invention also provides the use of the above kit in detecting the number of transgenic events contained in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 .A: Green color observed for PAT / bar protein, B: Blue fluorescence of the cell nucleus (background fluorescence), C: Overlay of blue and green.

[0034] Figure 2 .A: Red color observed by CP4-EPSPS protein, B: Blue fluorescence of the cell nucleus (background fluorescence), C: Overlay of blue and red.

[0035] Figure 3 .A: Green observed for PAT / bar protein, B: Red observed for CP4-EPSPS protein, C: Blue fluorescence of the cell nucleus (background fluorescence), D: Overlay of blue, red and green. DETAILED DESCRIPTION

[0036] Example 1 Detection of the number of transgenic events

[0037] Plant softening treatment

[0038] 1. Transfer the sample into a culture dish and dry it in a 37°C oven for 48 hours

[0039] 2. Place the sample in a tissue grinder and collect the tissue powder after grinding thoroughly

[0040] 3. Take 1g of powder sample, mix thoroughly, transfer to EP tube and wrap tissue powder with 3% agar. After agar cools and solidifies, put it into

[0041] Fix in FAA fixative (50% for young plants and 70% for old hard tissues) for 24 h.

[0042] 4. Take out and rinse with running water for 2-4 hours

[0043] 5. If long-term storage is required, transfer back to FAA fixative

[0044] Sample paraffin embedding and sectioning 1. Sampling: Fix fresh tissue in FAA fixative for more than 24 hours. Take the tissue out of the fixative and trim the target area with a scalpel in a fume hood. Place the trimmed tissue and the corresponding label in a dehydration box.

[0045] 2. Dehydration: Place the dehydration box in the hanging basket and dehydrate in the dehydrator with gradient alcohols. 75% alcohol for 4 hours - 85% alcohol for 2 hours - 90% alcohol for 2 hours - 95% alcohol for 1 hour - anhydrous ethanol I for 30 minutes - anhydrous ethanol II for 30 minutes - alcohol benzene for 5-10 minutes - xylene I for 5-10 minutes - xylene II for 5-10 minutes - wax I for 1 hour - wax II for 1 hour - wax III for 1 hour.

[0046] 3. Embedding: Embed the wax-soaked tissue in the embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool it in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.

[0047] 4. Slice: Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 4-6μm. Float the slices on the 40℃ warm water of the slicer to flatten the tissue, pick up the tissue with a slide, and bake it in a 60℃ oven. After the water is dried and the wax is melted, take it out and store it at room temperature for later use.

[0048] Dewaxing of paraffin sections

[0049] The sections were sequentially placed in xylene Ⅰ for 15 min - xylene Ⅱ for 15 min - anhydrous ethanol Ⅰ for 5 min - anhydrous ethanol Ⅱ for 5 min - 85% alcohol for 5 min - 75% alcohol for 5 min - and distilled water for washing.

[0050] Antigen retrieval

[0051] Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH 9.0) in a microwave oven for antigen repair. Boil over medium heat and then turn off the power for 10 minutes before boiling over medium-low heat. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After cooling naturally, place the slides in PBS (PH7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time.

[0052] Immunofluorescence staining

[0053] Add fluorescent labeled antibodies (cy3-bar antibody and FITC-EPSPS antibody): Gently shake off the blocking solution, add the primary antibody prepared in a certain proportion of PBS on the slice, and incubate the slice flat in a humidified box at 4°C overnight. (Add a small amount of water in the humidified box to prevent the antibody from evaporating);

[0054] DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 minutes each time. After the slices are slightly dried, add DAPI staining solution in the circle and incubate at room temperature for 10 minutes away from light;

[0055] Mounting: Place the slides in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 min each time. After the sections are slightly dried, mount them with anti-fluorescence quenching mounting media.

[0056] Microscope photography

[0057] The sections were observed and images were collected under a Nikon inverted fluorescence microscope.

[0058] Results Analysis

[0059] like Figure 1 .A The green color observed for PAT / bar protein, B The blue fluorescence of the cell nucleus (background fluorescence), C The overlay of blue and green. If the sample is observed as Figure 1 As shown in C, it means that there is only PAT / bar protein in the sample, which means that the sample contains a transformation event.

[0060] Figure 2 .A The red color of CP4-EPSPS protein, B the blue fluorescence of cell nuclei (background fluorescence), C the superposition of blue and red. If the sample is observed as Figure 2 As shown in C, it means that there is only CP4-EPSPS protein in the sample, which means that the sample contains a transformation event.

[0061] Figure 3 A PAT / bar protein observed in green, B CP4-EPSPS protein observed in red, C blue fluorescence of the cell nucleus (background fluorescence), superposition of blue, red and green. If the sample is observed as Figure 3 As shown in C, it means that the sample contains two transformation events.

[0062] Example 2 Sensitivity Detection

[0063] According to the experimental method in Example 1, rapeseed A seeds containing only PAT / bar transgenic event, rapeseed B seeds containing only CP4-EPSPS transgenic event, and rapeseed C seeds containing both CP4-EPSPS and PAT / bar transgenic events were prepared. The three rapeseeds, ABC, were mixed with non-transgenic rapeseed seeds at a ratio of 0.1% and then tested.

[0064] The results showed that when the genetically modified seeds accounted for only 0.1%, the fluorescence immunoassay method could accurately identify the number of genetically modified organisms in the sample to be tested, with a detection sensitivity of 0.1%.

[0065] Example 3: Sensitivity detection after shielding autofluorescence

[0066] The experimental method in Example 1-2 was used for the operation. Specifically, after antigen repair, the operation of shielding autofluorescence was performed. The specific operation was as follows: autofluorescence quencher A and autofluorescence quencher B were added in sequence, and finally serum blocking was performed; the autofluorescence quencher A was a mixture of phosphate buffer with pH 7.4 and sodium borohydride; the autofluorescence quencher B was a mixture of Sudan Black B and anhydrous ethanol; autofluorescence quencher A was first added, incubated at room temperature for 10-20 minutes, and washed; then, autofluorescence quencher B was added, incubated at room temperature for 10-15 minutes, washed, and then immunofluorescence staining and observation were performed.

[0067] The three rapeseed varieties, ABC, were mixed with non-GMO seeds at a lower ratio and treated to shield the autofluorescence. The test results showed that after shielding the autofluorescence, a lower ratio of GMO seeds could be detected, and the sensitivity was improved; compared with the control without shielding the autofluorescence, the detection sensitivity could be increased by about 10 times.

[0068] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for detecting transgenic events in plants, the method comprising labeling a first target antibody with a first fluorescent dye, labeling a second target antibody with a second fluorescent dye; after sample processing, paraffin embedding and sectioning, paraffin section dewaxing to water, antigen repair, shielding autofluorescence treatment, immunofluorescence staining and microscopic examination and photography, judging the number of transgenic events in the sample to be tested according to the observed fluorescence color; The shielding of autofluorescence treatment refers to first adding autofluorescence quencher A, incubating at room temperature for 10-20 minutes, and washing; then adding autofluorescence quencher B, incubating at room temperature for 10-15 minutes, and washing; and finally performing serum blocking; The autofluorescence quencher A is a mixture of a phosphate buffer solution at pH 7.4 and sodium borohydride; The autofluorescence quencher B is a mixture of Sudan Black B and ethanol; The sample processing is to take any part of the plant, dry it, grind it into powder, and then fix it in FAA fixative with 3% agar. The any part of the plant is rapeseed seeds.

2. The method according to claim 1, characterized in that The first target antibody and the second target antibody are different target antibodies, and the first target antibody and the second target antibody are PAT / bar and CP4-EPSPS target antibodies respectively.

3. The method according to claim 1 or 2, characterized in that: The first fluorescent dye and the second fluorescent dye are different fluorescent dyes, and the first fluorescent dye and the second fluorescent dye are FITC and Cy3 respectively.

4. The method according to claim 1, characterized in that: The method further comprises labeling a third target antibody with a third fluorescent dye for detecting 0-3 transgenic events, wherein the third target antibody is a target antibody different from the first target antibody and the second target antibody, and the third fluorescent dye is a fluorescent dye different from the first fluorescent dye and the second fluorescent dye.

5. The method according to claim 1, characterized in that The paraffin-embedded section is to dehydrate and wax-immerse the cut tissue in a dehydrator in a gradient alcohol, wait for the wax to solidify and cool, slice, bake, and store at room temperature for later use.

6. The method according to claim 1, characterized in that The paraffin sections are dewaxed to water by sequentially treating the sections with xylene, anhydrous ethanol, alcohol, and distilled water.

7. The method according to claim 1, characterized in that The antigen repair is to place the tissue section in EDTA antigen repair buffer and PBS for treatment, and the buffer and operating conditions can be adjusted according to the type of sample.

8. The method according to claim 1, characterized in that The immunofluorescence staining refers to decolorizing the slide, labeling the target with a transgenic target antibody labeled with a fluorescent dye, and sealing the slide after decolorization.

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